US20230210858A1 - Podosomes as a therapeutic target for intracranial aneurysm - Google Patents

Podosomes as a therapeutic target for intracranial aneurysm Download PDF

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US20230210858A1
US20230210858A1 US17/824,631 US202217824631A US2023210858A1 US 20230210858 A1 US20230210858 A1 US 20230210858A1 US 202217824631 A US202217824631 A US 202217824631A US 2023210858 A1 US2023210858 A1 US 2023210858A1
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thsd1
compound
aneurysm
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podosome
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Dong H. Kim
Zhen Xu
Yanning Rui
John Hagan
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University of Texas System
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Definitions

  • Intracranial aneurysm is a cerebrovascular disease that predominantly occurs in the cerebral artery and is characterized by pathologic dilatation of blood vessels.
  • Each intracranial aneurysm (IA) is a weakened area in a cerebral artery wall that leads to abnormal dilatation and rupture causing subarachnoid hemorrhage (SAH), a major cause of hemorrhagic stroke.
  • SAH subarachnoid hemorrhage
  • SAH subarachnoid hemorrhage
  • the present disclosure demonstrates that endothelial podosomes are regulated by the transmembrane receptor protein THSD1.
  • THSD1 has been shown to have a pathological role in intracranial aneurysm disease.
  • the present disclosure provides that endothelial podosomes are novel therapeutic targets for the treatment, modulation, and prevention of aneurysm rupture. There is thus a need in the art for understanding the molecular mechanisms driving aneurysms. The present disclosure addresses this need.
  • FIG. 1 A illustrates simplified pedigrees of three IA families (NR4748, NR4931, and NR5704). Specifically, FIG. 1 A illustrates Intracranial Aneurysm (IA) affected (black), aortic aneurysm (AA) affected (half black), unaffected individuals >45 (white), and individuals with unknown/unclear status (gray symbols).
  • IA Intracranial Aneurysm
  • AA aortic aneurysm
  • unaffected individuals >45 white
  • individuals with unknown/unclear status gray symbols
  • FIG. 1 B depicts patient-identified THSD1 variants identified in the pedigrees of the families from FIG. 1 B .
  • the black bar represents the 853 amino acids of the WT version of the THSD1 gene.
  • the diagram lists 8 variants found in patients affected by IA and AA, namely LSF, R450X, R460W, E466G, G600E, P639L, T6531, S775P.
  • the back boxes illustrate the relative position of the signal peptide (SP), thrombospondin type 1 domain (TSP1), and transmembrane (TM) domains of THSD1.
  • SP signal peptide
  • TSP1 thrombospondin type 1 domain
  • TM transmembrane
  • FIG. 2 is a drawing depicting an intracranial aneurysm.
  • An intracranial aneurysm also known as a brain aneurysm, is a cerebrovascular disorder in which weakness in the wall of a cerebral artery or vein causes a localized dilation or ballooning of the blood vessel.
  • FIG. 3 ( FIG. 3 ) is a drawing depicting a model of SRC-mediated podosome formation and ECM degradation.
  • FIG. 3 schematic model of podosome illustrates an actin-enriched core surrounded by an adhesive ring.
  • FIG. 4 a through FIG. 4 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation.
  • FIG. 5 a through FIG. 5 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function.
  • FIG. 6 a through FIG. 6 b are experimental results depicting that loss-of-function of THSD1 increase MT1-MMP cell surface exposure.
  • FIG. 7 a through FIG. 7 b are experimental results depicting that loss-of-function of THSD1 activates MMP2.
  • FIG. 8 a through FIG. 8 b are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish.
  • FIG. 9 ( FIG. 9 ) are experimental results depicting that loss-of-function of Thsd1 promotes IA formation in circle of Willis in mice.
  • FIG. 10 a through FIG. 10 b are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice.
  • FIG. 11 is a protein alignment between human and zebrafish THSD1.
  • FIG. 12 is a sequence alignment illustrating three rare variants of thsd1 conserved between humans and fish.
  • FIG. 13 is a graph charting the results of treatment of a model organism engineered with conserved mutations demonstrating that SRC inhibitors rescued intracranial hemorrhage.
  • Three exemplary inhibitors disclosed on this chart include PP2, SU5565, and dasatinib.
  • SAH subarachnoid hemorrhage
  • THSD1 Thrombospondin-type 1 domain-containing protein 1
  • Thsd1 loss-of-function leads to brain hemorrhage and premature death in both zebrafish and mice. Further, Thsd1 heterozygous and null mice developed IA and suffered SAH.
  • THSD1 is highly expressed in endothelial cells of the cerebrovasculature, is important for cell adhesion, promotes nascent focal adhesion assembly via Talin interactions, and potentially regulates downstream signaling.
  • THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression.
  • the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or function.
  • the compound is a Src inhibitor, such as PP2.
  • the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.
  • the aneurysm is an intracranial aneurysm. In other instances the aneurysm is an aortic aneurysm.
  • the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • the variant can be in a coding region of the THSD1 gene, in a control sequence of a non-coding region of the THSD1 gene, or in any other suitable region.
  • the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele, such as the exemplary variants identified and described in FIG. 1 B , which include single codon substitutions at codon LSF, R460W, E466G, G600E, P639L, T6531, S775P.
  • the compound that modulates podosome formation or function can be administered systemically.
  • Systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal administration.
  • the compound that modulates podosome formation or function is administered locally.
  • Local administration can include, but is not limited to, (i) intracranial; (ii) intra-ocular; (iii) intra-nasal; (iv) intrathecal or (v) intra-vescular administration.
  • the subject is a human.
  • the disclosure provides an inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.
  • the disclosure provides an inhibitor of podosome function for use in a method of treating an aneurysm in a subject.
  • the disclosure provides a kit comprising an inhibitor of podosome formation or function described herein and instructions for use thereof.
  • Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to, and degradation of, the extracellular matrix.
  • a podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM ( FIG. 3 ).
  • the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9.
  • SRC actin regulators cortactin and (N)-WASP
  • Tks4 and Tks5 adaptor proteins
  • MT1-MMP metalloprotease MT1-MMP
  • the present disclosure contemplates that modulation of podosome formation and function can provide a target for treating an aneurysm.
  • MMPs matrix metalloproteinases
  • initiation of podosome formation has been shown to be dependent on the underlying matrix, both in terms of its nature (which ligands are present) and its geometry (whether the ligands are uniformly distributed or arranged in subcellular-sized islands).
  • Cells use different integrin receptors to detect the mechanical constraints of their environment and to decide accordingly whether a podosome should be initiated or not.
  • substrate stiffness continues to play a role in the lifespan of the podosome, with increased stiffness resulting in increased longevity and decreased distance between individual podosomes.
  • THSD1 knockdown in endothelial cells increases the expression of metalloprotease and evaluates the role of podosome formation and function in managing podosome formation in subjects, particularly in subjects afflicted with loss-of-function variants of THSD1 gene.
  • the present disclosure describes the identification and characterization of genes whose perturbation leads to intracranial aneurysms (IA) and/or vascular endothelial cell dysfunction. Further, the present disclosure provides a characterization of molecular pathways that can provide novel therapeutic targets for treatment of aneurysms, particularly IA.
  • the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or podosome function.
  • the compound is a Src inhibitor such as PP2.
  • the compound is SU6656.
  • SU6656 was initially identified as a Src kinase inhibitor by virtue of its ability to reverse an effect that an activated mutant form of Src (hu SRC Y530F) has on the actin cytoskeleton, namely the formation of podosome rosettes, otherwise known as invadopodia.
  • SU6656 was initially published as a Src family kinase inhibitor with selectivity relative to Platelet-derived growth factor receptor Tyrosine kinase. Subsequent studies have confirmed that SU6656 is relatively selective for Src family kinases.
  • KX2-391 also known as KX-01, is a first-in-class dual Src kinase and tubulin polymerization inhibitor that is currently only developed as a topical medicinal product.
  • Saracatinib is an experimental drug that acts as a dual kinase inhibitor, with selective actions as a Src inhibitor and a Bcr-Abl tyrosine-kinase inhibitor.
  • Bosutinib is a small molecule BCR-ABL and src tyrosine kinase inhibitor currently used for the treatment of chronic myelogenous leukemia.
  • the compound is dasatinib is a selective tyrosine kinase receptor inhibitor.
  • the compound is ponatinib.
  • Ponatinib also known as AP24534
  • AP24534 is a potent, orally active tyrosine kinase inhibitor.
  • the methods, compositions, and uses of this disclosure may comprise a treatment method to arrest, reverse, or ameliorate an aneurysm, e.g., an intracranial aneurysm.
  • the therapeutic effect is achieved by administrating a therapeutically-effective dose of a compound that modulates podosome formation and function.
  • exemplary compounds contemplated by the disclosure include SU6656, a selective SRC inhibitor, KX2-391 and saracatinib (currently in clinical trials), and three FDA-approved compounds including, bosutinib, dasatinib, and ponatinib.
  • the treatment may comprise treating a subject (e.g. a patient at risk of having an intracranial aneurysm due to the presence of a THSD1 genetic variant or an animal with a similar genetic variant).
  • the disease may be a weakness in a blood vessel in the brain that balloons and fills with blood, for example, a brain aneurysm (also called a cerebral aneurysm or an intracranial aneurysm) is a ballooning arising from a weakened area in the wall of a blood vessel in the brain.
  • the subject may be a human.
  • Treatment may be provided to the subject before clinical onset of disease. For instance, in specific cases, treatment may be provided upon the identification of a THSD1 variant in a subject, before the onset of a disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial.
  • treatment may be provided through the lifetime of a subject that is afflicted with a THSD1 variant that may lead to subarachnoid hemorrhage.
  • treatment will be prescribed to prevent IA in a subject that carries a THSD1 variant associated with IA.
  • a treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure.
  • a treatment can comprise modulating the levels of podosomes in vivo.
  • a treatment may comprise administering a suitable level of an autophagy inhibitor for reducing bulge's in the wall of a blood vessel and preventing Subarachnoid hemorrhage.
  • ADPKD autosomal dominant polycystic kidney disease
  • FIG. 1 A discloses a large family with variants ( FIG. 1 B ) in the THSD1 gene that have been causally linked to IA.
  • Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.
  • a subject can be of any age.
  • Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.
  • administering includes any method which is effective to result in delivery of an autophagy inhibitor to the subject.
  • aneurysm refers to broad classes of aneurysm, including aneurysms: abdominal aortic, thoracic aortic, and cerebral.
  • Cerebral aneurysm or “intracranial aneurysm” (also known as a brain aneurysm) is a weak or thin spot on an artery in the brain that balloons or bulges out and fills with blood.
  • the bulging aneurysm can put pressure on the nerves or brain tissue. It may also burst or rupture, spilling blood into the surrounding tissue (called a hemorrhage).
  • An unruptured aneurysm usually causes no symptoms.
  • a key symptom of a ruptured aneurysm is a sudden, severe headache.
  • Treatments for an unruptured aneurysm include medications to control blood pressure and procedures to prevent a future rupture.
  • AAA anterior aortic aneurysm
  • AAA anterior aortic aneurysm
  • thoracic aortic aneurysm is an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel.
  • a thoracic aortic aneurysm occurs in the part of the body's largest artery (the aorta) that passes through the chest.
  • the term “at risk” or more specifically a “subject at risk of developing an intracranial aneurysm” is a subject afflicted with a genetic variant, e.g., THSD1 variant, that causes the subarachnoid hemorrhage seen when an aneurysm ruptures.
  • control sequences refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell.
  • EMBODIMENT 1 A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation.
  • EMBODIMENT 2 A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome function.
  • EMBODIMENT 3 The method of any one of embodiments 1 or 2, wherein the compound is a Src inhibitor.
  • EMBODIMENT 4 The method of embodiment 3, wherein the Src inhibitor is PP2.
  • EMBODIMENT 5 The method of any one of claim 1 or 2 , wherein the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.
  • EMBODIMENT 6 The method of embodiment 5, wherein the compound is SU6656.
  • EMBODIMENT 7 The method of embodiment 5, wherein the compound is KX2-391.
  • EMBODIMENT 8 The method of embodiment 5, wherein the compound is saracatinib.
  • EMBODIMENT 9 The method of embodiment 5, wherein the compound is bosutinib.
  • EMBODIMENT 10 The method of embodiment 5, wherein the compound is dasatinib.
  • EMBODIMENT 11 The method of embodiment 5, wherein the compound is ponatinib.
  • EMBODIMENT 12 The method of any one of embodiments 1-5, wherein the aneurysm is an intracranial aneurysm.
  • EMBODIMENT 13 The method of any one of embodiments 1-5, wherein the aneurysm is an aortic aneurysm.
  • EMBODIMENT 14 The method of any one of embodiments 1-13, wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • THSD1 Thrombospondin Type 1 Domain Containing 1
  • EMBODIMENT 15 The method of embodiment 14, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • THSD1 Thrombospondin Type 1 Domain Containing 1
  • EMBODIMENT 16 The method of embodiment 14, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • THSD1 Thrombospondin Type 1 Domain Containing 1
  • EMBODIMENT 17 The method of embodiment 14, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.
  • EMBODIMENT 18 The method of embodiment 14, wherein the single codon substitution in the at least one THSD1 allele is at codon LSF, R460W, E466G, G600E, P639L, T653I, S775P.
  • EMBODIMENT 19 The method of any one of embodiments 1-18, wherein the compound that modulates podosome formation or function is administered systemically.
  • EMBODIMENT 20 The method of embodiment 19, wherein systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal.
  • EMBODIMENT 21 The method of embodiment 1, wherein the compound that modulates podosome formation or function is administered locally.
  • EMBODIMENT 22 The method of embodiment 21, wherein local administration includes, but is not limited to, (i) intracranial; (ii) intra-ocular (iii) intra-nasal; (iv) intrathecal or (v) intra-vascular.
  • EMBODIMENT 23 The method of embodiment 1, wherein the subject is a human.
  • EMBODIMENT 24 An inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.
  • EMBODIMENT 25 An inhibitor of podosome function for use in a method of treating an aneurysm in a subject.
  • EMBODIMENT 26 A kit comprising an inhibitor of podosome formation or function of any one of embodiments Error! Reference source not found.-Error! Reference source not found. and instructions for use thereof.
  • EXAMPLE 1 Molecular Dissection of THSD1 Function in Vascular Endothelial Cells from Global Transcriptomics
  • FIG. 1 A illustrates a large family pedigree studied as part of the present disclosure to identify novel candidate genes whose rare variants cause intracranial aneurysm. including NR4748, NR4931, NR5704.
  • THSD1 Deleterious Thrombospondin-type 1 domain-containing protein 1
  • THSD1 Rare variants cause disease in both familial and sporadic cases with supporting evidence from animal models. THSD1 is predominantly expressed in vascular endothelial cells.
  • the work identified deleterious variants in thrombospondin-type 1 domain-containing protein 1 (THSD1) that can cause IA and SAH.
  • Initial characterization of Thsd1 in two vertebrate models including zebrafish and mice lead to the discovery that THSD1 mediated cerebral hemorrhage is located in subarachnoid space in mice.
  • THSD1 mechanism of action utilized by the discovered THSD1 variants to drive disease remained elusive. Further, there was little information describing genes and pathways regulated by THSD1 using global transcriptomics that could be used to inform the mechanism of action of THSD1. Thus, on its own the identification of THSD1 in the context of AI was not sufficient to inform a therapeutic strategy.
  • THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression.
  • the present disclosure provides results from global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown that identifies THSD1-regulated specific genes and pathways that are critical for mediating its function, providing potential targets for therapeutic intervention in IA.
  • RNAseq experiments in two THSD1 knock-down endothelial cell lines.
  • the RNAseq results from both cell lines support the evidence that THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling.
  • FAK Focal Adhesion Kinase
  • HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 ⁇ g/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
  • Knockdown experiments in human vascular endothelial cells were performed using two distinct cell lines [HUVECs and Human brain microvascular endothelial cells(HBMECs)] using four siRNAs (two control siRNAs and two THSD1-specific siRNAs) to minimize erroneous findings due to off-target effects.
  • THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling) as well as TGF ⁇ signaling.
  • FAK Focal Adhesion Kinase
  • Table 3 lists genes differentially expressed in THSD1 knockdown HUVECs.
  • Table 4 lists genes differentially expressed in THSD1 knockdown HUVECs
  • Bioinformatic analyses highlighted a potential role for the Src Signaling pathway, and other pathways, in the pathology of IA. We evaluated the potential link to the Src signaling pathway below.
  • Intracranial aneurysm is a weakened area in the wall of cerebral artery that leads to a bulging in a brain blood vessel.
  • the rupture of IA causes aneurysmal subarachnoid hemorrhage (SAH), a devastating form of stroke. More than 30% will die due to SAH and more than half of survivors will never return to independent living.
  • SAH aneurysmal subarachnoid hemorrhage
  • Podosomes are subcellular organelles located at cell-ECM interaction sites in different cell types including a variety of endothelial cells.
  • a podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM ( FIG. 3 ).
  • the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9.
  • SRC a non-receptor tyrosine kinase.
  • SRC a non-receptor tyrosine kinase.
  • SRC phosphorylates a wide array of substrates. For instance, SRC phosphorylates cortactin and promotes actin assembly in podosomes. See, e.g., FIG. 3 .
  • the present disclosure considered the molecular pathways affected by the knock-down of THSD1 and investigated the role of podosome formation and function role in THSD1-mediated aneurysm development.
  • HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 ⁇ g/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
  • Cells were grown on glass coverslips in the DMEM medium for 16 h before fixed with 4% formaldehyde-phosphate-buffered saline (Electronic Microscopy Sciences, 15710; Fisher Scientific, BP399-500) for 10 min. Cells were incubated with primary antibodies against focal adhesion markers such as paxillin overnight at 4° C. after 1 h blocking by 5% goat serum. Secondary antibodies conjugated with Alexa Fluor 488 or 594 were used to visualize the localization. Image was taken with a Leica Confocal Microscope and processed by ImageJ and GraphPad 7 software for statistical analysis.
  • Anti-sense morpholinos against thsd1, atg5 or talin was injected into one-cell stage zebrafish singly or in combination to silence the gene accordingly.
  • embryos will be checked under regular optical microscope. Embryos with intracranial hemorrhages will be counted and imaged by Olympus microscope.
  • MicroFil (Flow Tech, Inc. Carver, Mass.) casting solution according to the manufacturer's instruction: Mix 5 ml of MV diluent with 4 ml of filtered MV-112 compound (yellow). Add 450 ⁇ l (5%) of catalyst (MV curing agent). Use 10 ml syringe to inject MicroFil mixture into the left ventricle after the blood was flushed out by saline. Inject MicroFil mixture slowly into the left ventricle at approximately 3 ml/min.
  • tissue samples were washed in phosphate buffered saline (PBS), blocked with 5% goat serum in PBST (0.1% Triton-X in PBS), then incubated with primary antibodies followed by the appropriate fluorescent-labeled secondary antibodies.
  • PBS phosphate buffered saline
  • FIGS. 4 a - 4 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation in THSD1-deficient HBMECs.
  • FIG. 4 a illustrates podosomes such as puncta (arrowheads) or rosette (arrow) were assessed by co-immunostaining of cortactin and actin in control or THSD1-deficient HBMECs.
  • FIGS. 5 a - 5 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function.
  • FIG. 5 a shows representative images for Alexa 488-conjugated Collagen-IV (Co-IV) degradation in control or THSD1-deficient HBMECs.
  • FIGS. 6 a - 6 b are experimental results depicting that loss-of-function of THSD1 increases MT1-MMP cell surface exposure.
  • FIG. 6 a shows a Western Blot illustrating that biotinylated cell-surface proteins were pull down by streptavidin beads from control and THSD1-deficient HBMECs. The level of MT1-MMP were revealed by Western blot and quantified in FIG. 6 b .
  • TCL total cell lysates; GAPDH as loading control; **p ⁇ 0.01 by student t-test.
  • FIGS. 7 a - 7 b are experimental results depicting that MMP2 is activated upon THSD1 knockdown in HBMECs.
  • FIG. 7 a illustrates cell extracts and supernatants from control and THSD1-deficient HBMECs were analyzed by gelatin zymography. Loading was normalized by total protein concentration. Quantification was calculated by a fold change of active MMP2 expression. See FIG. 7 b . ***p ⁇ 0.001 by student t-test.
  • Intracranial aneurysm and hemorrhage are tightly associated with compromised cerebrovascular integrity.
  • zebrafish have gained additional popularity as a vertebrate model organism for studying the cerebrovasculature. Since zebrafish embryos are transparent, intracranial hemorrhage can be directly observed using a standard microscope. Furthermore, zebrafish fecundity and rapid development permits rapid phenotypic evaluation as intracranial hemorrhage in zebrafish fry are detectable as early as 2-3 days post fertilization. Importantly, gain-of-function and loss-of-function approaches are well established in zebrafish that include morpholino and mRNA injections and more recently, through applications of CRISPR/Cas9 technology.
  • FIGS. 8 a - 8 b are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish, and treatment with a PP2 SRC inhibitor prevents intracranial hemorrhage in Thsd1-deficient zebrafish.
  • FIG. 8 a is a picture illustrating a prominent cerebral hemorrhage phenotype was observed in thsd1 knockdown zebrafish.
  • FIG. 8 b is a chart quantifying the number of embryos with intracranial hemorrhage (%).
  • PP2 treatment SRC inhibitor
  • DMSO treatment did not (comparing grey bars in b).
  • n>120 embryos were analyzed. *p ⁇ 0.05.
  • FIGS. 10 a - 10 b are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice.
  • Student t-test was used for statistical difference. **p ⁇ 0.01. Scale bar: 10 ⁇ M.
  • FIG. 10 a illustrates representative images of the staining and
  • FIG. 10 b is a chart representation quantitation of the same.
  • EXAMPLE 4 Genetic Engineering of Non-Human Animal Models Encoding Distinct THSD1 Rare Variants for In-Vivo Assessment of Efficacy of Various Autophagy Inhibitors
  • FIG. 11 illustrates the result of the alignment with black color indicating identical amino acids and the grey color indicating similar amino acids between humans and zebra fish.
  • the alignment underscored that three rare variants of THSD1 identified in the aforementioned examples were conserved between human and zebrafish.
  • the conserved amino acids include R450, R460 and T653. See, e.g., FIG. 12 .
  • the conserved amino acids were used as a rational for generating animal models of the human condition. The following constructs were generated:
  • Mutations R449X, R459W and T6651 were found to be conserved between human and zebrafish. Subsequently, the nucleic acid constructs encoding these sequences were introduced into zebrafish thsd1 by PCR mutagenesis. Specifically, the thsd1 variant or the WT sequence was subcloned into pCS2+ zebrafish vector for expression. These constructs were used as tools to study cerebrovascular integrity mediated by thsd1 WT and rare variants in the zebrafish animal model selected to model a human condition.
  • the data demonstrates a body of work that first identified and studied a large number of human families with a disease. After significant effort in contacting individuals and sequencing the families shown in FIG. 1 A , the present disclosure first identified THSD1 as a gene associated with sub-cranial hemorrhage. In specific cases, a single codon substitution was identified as responsible for the phenotype LSF, R460W, E466G, G600E, P639L, T6531, or S775P. The inventors developed a plethora of nucleic acid constructs, cell lines, and transgenic animal models to study the condition. Subsequently, a detailed characterization of molecular pathways whose transcription was disturbed in the disease condition was used to identify potential druggable targets and pathways for treatment.
  • podosome formation and function Three distinct SRC inhibitors were tested including PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and its ability to treat subjects, particularly its ability to treat subjects susceptible to an aneurism rupture was tested by counting the hemorrhagic incidence under the microscope on an animal model. The results illustrate that podosome modulation provides a treatment for modulating the rupture of an aneurysm in vivo.
  • kits comprising podosome modulators such as the podosome modulators selected from the group consisting of PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and instructions for use of the same in the treatment of a condition disclosure herein.
  • podosome modulators such as the podosome modulators selected from the group consisting of PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and instructions for use of the same in the treatment of a condition disclosure herein.
  • the disclosure contemplates use of these drugs in treating an intracranial aneurysm.

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Abstract

Disclosed here are uses of anti-podosome therapies for treating a subject at risk of suffering from an aneurysm. The present disclosure demonstrates that endothelial podosomes plays a role in THSD1-mediated focal adhesion stability and aneurysm formation and characterizes molecular targets for therapeutic intervention.

Description

    CROSS-REFERENCE
  • The present application claims priority to U.S. Provisional Application Ser. No. 63/296,817, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,820, filed Jan. 5, 2022; U.S. Provisional Application Ser. No. 63/296,821, filed Jan. 5, 2022; and U.S. Provisional Application Ser. No. 63/296,825, filed Jan. 5, 2022, the contents of each being hereby incorporated by reference in their entirety.
    • FIELD OF THE INVENTION
  • The present disclosure relates to the characterization of molecular targets for treating intracranial aneurysms (IA). Intracranial aneurysm (IA) is a cerebrovascular disease that predominantly occurs in the cerebral artery and is characterized by pathologic dilatation of blood vessels. Each intracranial aneurysm (IA) is a weakened area in a cerebral artery wall that leads to abnormal dilatation and rupture causing subarachnoid hemorrhage (SAH), a major cause of hemorrhagic stroke. A rupture of IA induces a subarachnoid hemorrhage (SAH), a type of hemorrhagic stroke that frequently leads to death or severe disability. Due to early age of onset and high mortality, SAH accounts for >25% of years lost for all stroke victims under the age of 65 years. Despite treatment advances, SAH mortality rate is 40% and only half of survivors return to independent life.
  • There is a critical unmet need for understanding the genetic and molecular basis for IA to improve clinical outcomes through early therapeutic intervention.
    • SUMMARY
  • This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
  • In some aspects, the present disclosure demonstrates that endothelial podosomes are regulated by the transmembrane receptor protein THSD1. THSD1 has been shown to have a pathological role in intracranial aneurysm disease. Briefly, the present disclosure provides that endothelial podosomes are novel therapeutic targets for the treatment, modulation, and prevention of aneurysm rupture. There is thus a need in the art for understanding the molecular mechanisms driving aneurysms. The present disclosure addresses this need.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • The foregoing and other features and advantages of the present invention will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings in which:
  • FIG. 1A (FIG. 1A) illustrates simplified pedigrees of three IA families (NR4748, NR4931, and NR5704). Specifically, FIG. 1A illustrates Intracranial Aneurysm (IA) affected (black), aortic aneurysm (AA) affected (half black), unaffected individuals >45 (white), and individuals with unknown/unclear status (gray symbols).
  • FIG. 1B (FIG. 1B) depicts patient-identified THSD1 variants identified in the pedigrees of the families from FIG. 1B. The black bar represents the 853 amino acids of the WT version of the THSD1 gene. The diagram lists 8 variants found in patients affected by IA and AA, namely LSF, R450X, R460W, E466G, G600E, P639L, T6531, S775P. The back boxes illustrate the relative position of the signal peptide (SP), thrombospondin type 1 domain (TSP1), and transmembrane (TM) domains of THSD1.
  • FIG. 2 (FIG. 2 ) is a drawing depicting an intracranial aneurysm. An intracranial aneurysm, also known as a brain aneurysm, is a cerebrovascular disorder in which weakness in the wall of a cerebral artery or vein causes a localized dilation or ballooning of the blood vessel.
  • FIG. 3 (FIG. 3 ) is a drawing depicting a model of SRC-mediated podosome formation and ECM degradation. FIG. 3 schematic model of podosome illustrates an actin-enriched core surrounded by an adhesive ring.
  • FIG. 4 a through FIG. 4 b (FIGS. 4 a-4 b ) are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation.
  • FIG. 5 a through FIG. 5 b (FIGS. 5 a-5 b ) are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function.
  • FIG. 6 a through FIG. 6 b (FIGS. 6 a-6 b ) are experimental results depicting that loss-of-function of THSD1 increase MT1-MMP cell surface exposure.
  • FIG. 7 a through FIG. 7 b (FIGS. 7 a-7 b ) are experimental results depicting that loss-of-function of THSD1 activates MMP2.
  • FIG. 8 a through FIG. 8 b (FIGS. 8 a-8 b ) are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish.
  • FIG. 9 (FIG. 9 ) are experimental results depicting that loss-of-function of Thsd1 promotes IA formation in circle of Willis in mice.
  • FIG. 10 a through FIG. 10 b (FIGS. 10 a-10 b ) are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice.
  • FIG. 11 is a protein alignment between human and zebrafish THSD1.
  • FIG. 12 is a sequence alignment illustrating three rare variants of thsd1 conserved between humans and fish.
  • FIG. 13 is a graph charting the results of treatment of a model organism engineered with conserved mutations demonstrating that SRC inhibitors rescued intracranial hemorrhage. Three exemplary inhibitors disclosed on this chart include PP2, SU5565, and dasatinib.
    •
  • It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.
    • INCORPORATION BY REFERENCE
  • All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
    • DETAILED DESCRIPTIONS
  • In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention. The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art.
  • The rupture of an intracranial aneurysm frequently causes a subarachnoid hemorrhage (SAH), a type of stroke characterized by high morbidity and mortality. Specifically, the present disclosure demonstrates with data from three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status.
  • Previously, it has been reported that deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants caused disease in both familial and sporadic cases with supporting evidence from animal models. Of note, whole exome sequencing of large IA families identified (some members of the affected family are shown in the pedigree of FIG. 1A) a THSD1 nonsense mutation that segregated in all nine (9) affected and was absent in 13 unaffected family members. It was further discovered that eight (8) THSD1 rare missense variants in 507 unrelated patients/probands where each perturbed THSD1 cell adhesion activity.
  • These rare variants were highly enriched in case-control studies in comparison to ethnically matched controls. It was found that Thsd1 loss-of-function leads to brain hemorrhage and premature death in both zebrafish and mice. Further, Thsd1 heterozygous and null mice developed IA and suffered SAH. The study further demonstrated that THSD1 is highly expressed in endothelial cells of the cerebrovasculature, is important for cell adhesion, promotes nascent focal adhesion assembly via Talin interactions, and potentially regulates downstream signaling. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference in their entireties). However the study did not provide any insights on the THSD1 molecular pathways.
  • To further study the role of THSD1 in IA/SAH, additional analysis of whole exome sequencing of the IA families described in FIG. 1 was conducted. The detailed analysis identified a THSD1 nonsense mutation that segregated in all 9 affected and was absent in 13 unaffected family members. Notably, the exome sequencing analysis uncovered multiple signaling pathways that appeared to be affected by THSD1 expression: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling as well as TGFβ signaling (see Table 1, discussed in Example 1). The present disclosure contemplates that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression.
  • In some aspects, the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or function. In many instances the compound is a Src inhibitor, such as PP2. In some instances, the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib. In some instances the aneurysm is an intracranial aneurysm. In other instances the aneurysm is an aortic aneurysm. In many instances the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene. The variant can be in a coding region of the THSD1 gene, in a control sequence of a non-coding region of the THSD1 gene, or in any other suitable region. In some instances, the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele, such as the exemplary variants identified and described in FIG. 1B, which include single codon substitutions at codon LSF, R460W, E466G, G600E, P639L, T6531, S775P.
  • In many instances, the compound that modulates podosome formation or function can be administered systemically. Systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal administration. In many instances the compound that modulates podosome formation or function is administered locally. Local administration can include, but is not limited to, (i) intracranial; (ii) intra-ocular; (iii) intra-nasal; (iv) intrathecal or (v) intra-vescular administration. In many instances the subject is a human.
  • In some aspects the disclosure provides an inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.
  • In some aspects the disclosure provides an inhibitor of podosome function for use in a method of treating an aneurysm in a subject.
  • In other aspects the disclosure provides a kit comprising an inhibitor of podosome formation or function described herein and instructions for use thereof.
    • Podosomes
  • Podosomes and invadopodia are actin-based dynamic protrusions of the plasma membrane of metazoan cells that represent sites of attachment to, and degradation of, the extracellular matrix. A podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM (FIG. 3 ). Unlike other adhesive structures such as focal adhesion complexes that tether the endothelium to the ECM, the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9. Key proteins in these structures include the actin regulators cortactin and (N)-WASP, the adaptor proteins Tks4 and Tks5, and the metalloprotease MT1-MMP. Among all the upstream events that occur during podosome formation, the best characterized is the activation of SRC, a non-receptor tyrosine kinase. When SRC is enriched and activated in pre-podosome structures, it phosphorylates a wide array of substrates. For instance, SRC phosphorylates cortactin and promotes actin assembly in podosomes. Many cell types elaborate these structures, including vascular smooth muscle and endothelial cells. The present disclosure demonstrates that podosomes are also present in aneurysms.
  • Since intracellular matrix degradation is a hallmark of aneurysm development the present disclosure contemplates that modulation of podosome formation and function can provide a target for treating an aneurysm.
  • Podosomes are intimately involved in cell motility, with podosomes specifically implicated in cell invasion. Invasiveness is achieved through the secretion of matrix metalloproteinases (MMPs) from the core of podosomes, which degrade the extracellular matrix (ECM). This promotes motile behaviors that aid a range of processes including migration of aortic endothelial cells for arterial vessel remodeling. In some aspects, the
  • In addition, the initiation of podosome formation has been shown to be dependent on the underlying matrix, both in terms of its nature (which ligands are present) and its geometry (whether the ligands are uniformly distributed or arranged in subcellular-sized islands). Cells use different integrin receptors to detect the mechanical constraints of their environment and to decide accordingly whether a podosome should be initiated or not. Following initiation, substrate stiffness continues to play a role in the lifespan of the podosome, with increased stiffness resulting in increased longevity and decreased distance between individual podosomes. The present disclosure demonstrates that THSD1 knockdown in endothelial cells increases the expression of metalloprotease and evaluates the role of podosome formation and function in managing podosome formation in subjects, particularly in subjects afflicted with loss-of-function variants of THSD1 gene.
    • Podosome Inhibitors
  • The present disclosure describes the identification and characterization of genes whose perturbation leads to intracranial aneurysms (IA) and/or vascular endothelial cell dysfunction. Further, the present disclosure provides a characterization of molecular pathways that can provide novel therapeutic targets for treatment of aneurysms, particularly IA.
  • In some aspects the disclosure provides a method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation and/or podosome function.
  • In some instances the compound is a Src inhibitor such as PP2.
  • In other instances the compound is SU6656. SU6656 was initially identified as a Src kinase inhibitor by virtue of its ability to reverse an effect that an activated mutant form of Src (hu SRC Y530F) has on the actin cytoskeleton, namely the formation of podosome rosettes, otherwise known as invadopodia. SU6656 was initially published as a Src family kinase inhibitor with selectivity relative to Platelet-derived growth factor receptor Tyrosine kinase. Subsequent studies have confirmed that SU6656 is relatively selective for Src family kinases.
  • In other instances the compound is KX2-391. KX2-391, also known as KX-01, is a first-in-class dual Src kinase and tubulin polymerization inhibitor that is currently only developed as a topical medicinal product.
  • In other instances the compound is saracatinib. Saracatinib is an experimental drug that acts as a dual kinase inhibitor, with selective actions as a Src inhibitor and a Bcr-Abl tyrosine-kinase inhibitor.
  • In other instances the compound is bosutinib. Bosutinib is a small molecule BCR-ABL and src tyrosine kinase inhibitor currently used for the treatment of chronic myelogenous leukemia.
  • In other instances the compound is dasatinib is a selective tyrosine kinase receptor inhibitor.
  • In other instances the compound is ponatinib. Ponatinib (also known as AP24534) is a potent, orally active tyrosine kinase inhibitor.
    • Methods of Treating Aneurysms
  • The methods, compositions, and uses of this disclosure may comprise a treatment method to arrest, reverse, or ameliorate an aneurysm, e.g., an intracranial aneurysm. In some cases, the therapeutic effect is achieved by administrating a therapeutically-effective dose of a compound that modulates podosome formation and function. Exemplary compounds contemplated by the disclosure include SU6656, a selective SRC inhibitor, KX2-391 and saracatinib (currently in clinical trials), and three FDA-approved compounds including, bosutinib, dasatinib, and ponatinib.
  • The treatment may comprise treating a subject (e.g. a patient at risk of having an intracranial aneurysm due to the presence of a THSD1 genetic variant or an animal with a similar genetic variant). The disease may be a weakness in a blood vessel in the brain that balloons and fills with blood, for example, a brain aneurysm (also called a cerebral aneurysm or an intracranial aneurysm) is a ballooning arising from a weakened area in the wall of a blood vessel in the brain. The subject may be a human.
  • Treatment may be provided to the subject before clinical onset of disease. For instance, in specific cases, treatment may be provided upon the identification of a THSD1 variant in a subject, before the onset of a disease. Treatment may be provided to the subject after clinical onset of disease. Treatment may be provided to the subject after 1 day, 1 week, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may be provided to the subject for more than 1 day, 1 week, 1 month, 6 months, 12 months, 2 years or more after clinical onset of disease. Treatment may be provided to the subject for less than 1 day, 1 week, 1 month, 6 months, 12 months, or 2 years after clinical onset of the disease. Treatment may also include treating a human in a clinical trial. Because of the genetic aspect of IA, treatment may be provided through the lifetime of a subject that is afflicted with a THSD1 variant that may lead to subarachnoid hemorrhage. In some aspects, treatment will be prescribed to prevent IA in a subject that carries a THSD1 variant associated with IA.
  • A treatment can comprise administering to a subject a pharmaceutical composition, such as one or more of the pharmaceutical compositions described throughout the disclosure. A treatment can comprise modulating the levels of podosomes in vivo. A treatment may comprise administering a suitable level of an autophagy inhibitor for reducing bulge's in the wall of a blood vessel and preventing Subarachnoid hemorrhage.
  • Further, there are many risk factors for the development of intracranial aneurysms, both inherited and acquired. Females are more prone to aneurysm rupture, with SAH times more common in women. The prevalence of aneurysms is increased in certain genetic diseases; the classic example is autosomal dominant polycystic kidney disease (ADPKD), but other diseases such as Ehlers-Danlos syndrome, neurofibromatosis, al-antitrypsin deficiency also demonstrate a link.
  • In ADPKD, 10% to 15% of patients develop intracranial aneurysms. Marfan's Syndrome was once thought to be linked to intracranial aneurysm formation, but recent evidence suggests that this may not be true. Aneurysms also run in families in the absence of an identified genetic disorder, with a prevalence of 7% to 20% in first or second degree relatives of patients who have suffered a SAH. FIG. 1A discloses a large family with variants (FIG. 1B) in the THSD1 gene that have been causally linked to IA.
    • Definitions
  • All of the functionalities described in connection with one embodiment of the methods, devices or instruments described herein are intended to be applicable to the additional embodiments of the methods, devices and instruments described herein except where expressly stated or where the feature or function is incompatible with the additional embodiments. For example, where a given feature or function is expressly described in connection with one embodiment but not expressly mentioned in connection with an alternative embodiment, it should be understood that the feature or function may be deployed, utilized, or implemented in connection with the alternative embodiment unless the feature or function is incompatible with the alternative embodiment.
  • Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” refers to one or more cells, and reference to “the system” includes reference to equivalent steps, methods and devices known to those skilled in the art, and so forth. Furthermore, terms such as “first,” “second,” “third,” etc., merely identify one of a number of steps, components, functions, and/or points of reference as disclosed herein, and likewise do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.
  • Subjects can be humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. A subject can be of any age. Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants.
  • As used in the specification and claims of this application, the term “administering” includes any method which is effective to result in delivery of an autophagy inhibitor to the subject.
  • As used in this specification, the term “aneurysm” refers to broad classes of aneurysm, including aneurysms: abdominal aortic, thoracic aortic, and cerebral.
  • As used in this specification, the term “cerebral aneurysm” or “intracranial aneurysm” (also known as a brain aneurysm) is a weak or thin spot on an artery in the brain that balloons or bulges out and fills with blood. The bulging aneurysm can put pressure on the nerves or brain tissue. It may also burst or rupture, spilling blood into the surrounding tissue (called a hemorrhage). An unruptured aneurysm usually causes no symptoms. A key symptom of a ruptured aneurysm is a sudden, severe headache. Treatments for an unruptured aneurysm include medications to control blood pressure and procedures to prevent a future rupture.
  • As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.
  • As used in this specification, the term “abdominal aortic” aneurysm (AAA) is a bulge or swelling in the aorta, the main blood vessel that runs from the heart down through the chest and tummy. An AAA can be dangerous if it is not spotted early on. It can get bigger over time and could burst (rupture), causing life-threatening bleeding.
  • As used in this specification, the term “thoracic aortic” aneurysm is an abnormal widening or ballooning of a portion of an artery due to weakness in the wall of the blood vessel. A thoracic aortic aneurysm occurs in the part of the body's largest artery (the aorta) that passes through the chest.
  • As used in the specification and claims of this application, the term “at risk” or more specifically a “subject at risk of developing an intracranial aneurysm” is a subject afflicted with a genetic variant, e.g., THSD1 variant, that causes the subarachnoid hemorrhage seen when an aneurysm ruptures.
  • The term DNA “control sequences” refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell.
  • Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
    • EMBODIMENTS
  • EMBODIMENT 1. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation.
  • EMBODIMENT 2. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome function.
  • EMBODIMENT 3. The method of any one of embodiments 1 or 2, wherein the compound is a Src inhibitor.
  • EMBODIMENT 4. The method of embodiment 3, wherein the Src inhibitor is PP2.
  • EMBODIMENT 5. The method of any one of claim 1 or 2, wherein the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.
  • EMBODIMENT 6. The method of embodiment 5, wherein the compound is SU6656.
  • EMBODIMENT 7. The method of embodiment 5, wherein the compound is KX2-391.
  • EMBODIMENT 8. The method of embodiment 5, wherein the compound is saracatinib.
  • EMBODIMENT 9. The method of embodiment 5, wherein the compound is bosutinib.
  • EMBODIMENT 10. The method of embodiment 5, wherein the compound is dasatinib.
  • EMBODIMENT 11. The method of embodiment 5, wherein the compound is ponatinib.
  • EMBODIMENT 12. The method of any one of embodiments 1-5, wherein the aneurysm is an intracranial aneurysm.
  • EMBODIMENT 13. The method of any one of embodiments 1-5, wherein the aneurysm is an aortic aneurysm.
  • EMBODIMENT 14. The method of any one of embodiments 1-13, wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • EMBODIMENT 15. The method of embodiment 14, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • EMBODIMENT 16. The method of embodiment 14, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
  • EMBODIMENT 17. The method of embodiment 14, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.
  • EMBODIMENT 18. The method of embodiment 14, wherein the single codon substitution in the at least one THSD1 allele is at codon LSF, R460W, E466G, G600E, P639L, T653I, S775P.
  • EMBODIMENT 19. The method of any one of embodiments 1-18, wherein the compound that modulates podosome formation or function is administered systemically.
  • EMBODIMENT 20. The method of embodiment 19, wherein systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal.
  • EMBODIMENT 21. The method of embodiment 1, wherein the compound that modulates podosome formation or function is administered locally.
  • EMBODIMENT 22. The method of embodiment 21, wherein local administration includes, but is not limited to, (i) intracranial; (ii) intra-ocular (iii) intra-nasal; (iv) intrathecal or (v) intra-vascular.
  • EMBODIMENT 23. The method of embodiment 1, wherein the subject is a human.
  • EMBODIMENT 24. An inhibitor of podosome formation for use in a method of treating an aneurysm in a subject.
  • EMBODIMENT 25. An inhibitor of podosome function for use in a method of treating an aneurysm in a subject.
  • EMBODIMENT 26. A kit comprising an inhibitor of podosome formation or function of any one of embodiments Error! Reference source not found.-Error! Reference source not found. and instructions for use thereof.
    • EXAMPLES
  • The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.
  • The practice of some molecular techniques described herein may employ, unless otherwise indicated, techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry, and genetic engineering technology, which are within the skill of those who practice in the art. Such techniques and descriptions can be found in standard laboratory manuals such as Westerfield, M. (2000). The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). 4th ed., Univ. of Oregon Press, Eugene.; all of which are herein incorporated in their entirety by reference for all purposes.
    • EXAMPLE 1: Molecular Dissection of THSD1 Function in Vascular Endothelial Cells from Global Transcriptomics
  • Genetic factors play a significant role in IA pathogenesis as illustrated by family studies and several IA predisposing syndromes. 7%-20% of all patients have a known family history and a family history is the strongest risk factor for disease. Excluding syndromes that account for less than 1% of all IA cases, candidate IA genes have been primarily identified by genome-wide association studies and more recently, by whole exome sequencing in affected families. Yet, little is known about the genetic causes of IA providing minimum insight for the understanding and development of therapeutic targets that could treat the disease.
  • Single-family genetic studies are a powerful tool to identify candidate high-risk genetic variants. FIG. 1A illustrates a large family pedigree studied as part of the present disclosure to identify novel candidate genes whose rare variants cause intracranial aneurysm. including NR4748, NR4931, NR5704.
  • The subject highlighted participated in the sequencing studies described herein. We studied three large IA/SAH families with at least 4 affected individuals where whole exome sequencing has been performed to identify rare variants that segregate with disease. For each family, whole exome sequencing has been performed on at least 15 family members, irrespective of their IA status. Specifically, 21 individuals provided samples for sequencing for family NR4748. 32 individuals provided samples for sequencing for family NR4931. 16 individuals provided samples for sequencing for family NR5704.
  • Deleterious Thrombospondin-type 1 domain-containing protein 1 (THSD1) rare variants cause disease in both familial and sporadic cases with supporting evidence from animal models. THSD1 is predominantly expressed in vascular endothelial cells. The work identified deleterious variants in thrombospondin-type 1 domain-containing protein 1 (THSD1) that can cause IA and SAH. Initial characterization of Thsd1 in two vertebrate models including zebrafish and mice lead to the discovery that THSD1 mediated cerebral hemorrhage is located in subarachnoid space in mice. For further description of this work see. Z. Xu, D. Kim, et al., NeuroMolecular Medicine (2019) 21:325-343; T. Santiago-Sim, D. Kim, et al., Stroke. 2016; 47:3005-3013. DOI: 10.1161/STROKEAHA.116.014161); Yan-Ning Rui and D. Kim, et al., Cell Physiol Biochem 2017; 43:2200-2211; each of which incorporated by reference).
  • However, the mechanism of action utilized by the discovered THSD1 variants to drive disease remained elusive. Further, there was little information describing genes and pathways regulated by THSD1 using global transcriptomics that could be used to inform the mechanism of action of THSD1. Thus, on its own the identification of THSD1 in the context of AI was not sufficient to inform a therapeutic strategy.
  • The present disclosure contemplated that THSD1 regulated genes may contribute to IA pathogenesis and that modulating their function may be beneficial as an IA treatment or in other diseases with aberrant THSD1 expression. The present disclosure provides results from global transcriptome profiling in human vascular endothelial cells upon THSD1 knockdown that identifies THSD1-regulated specific genes and pathways that are critical for mediating its function, providing potential targets for therapeutic intervention in IA.
  • The instant disclosure provides RNAseq experiments in two THSD1 knock-down endothelial cell lines. The RNAseq results from both cell lines support the evidence that THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling. A few of these pathways were selected for further analysis and characterization.
  • Materials and Methods
  • Cell Culture
  • HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
  • Knock-Down Experiments
  • Knockdown experiments in human vascular endothelial cells were performed using two distinct cell lines [HUVECs and Human brain microvascular endothelial cells(HBMECs)] using four siRNAs (two control siRNAs and two THSD1-specific siRNAs) to minimize erroneous findings due to off-target effects.
  • Transcriptome Profiling
  • Bioinformatic analyses of the global transcriptome were performed on rRNA-depleted RNA samples by RNA-Seq. Table 1 illustrates results of the analysis. As shown on Table 1, THSD1 regulates multiple signaling pathways: Integrin, Src, PI3/AKT/mTor, and Rho signaling that are functionally linked to Focal Adhesion Kinase (FAK) signaling) as well as TGFβ signaling.
  • TABLE 1
    Statistically Significant THSD1-Regulated Pathways
    (p-value < 0.005; Efron-Tibshirani's GSA test)
    Description
    Integrin Signaling
    PI3K/Akt/mTOR Signaling
    Src Signaling
    Rho Signaling
    Tgfβ
  • We identified a number of genes that are affected by the lack of THSD1 in the knock-down cell lines and are likely regulated by THSD1. A subset of these genes likely contributes to disease pathobiology and may be targets for therapeutic intervention. Table 2. Describes genes differentially expressed in THSD1 knockdowns.
  • TABLE 2
    Genes differentially expressed in THSD1 knockdowns; human
    brain microvascular endothelial cells and HUVECs, combined
    Description Genes
    Differential Expression ALOX5AP, ARL17B, CTSS, GUK1,
    upon THSD1 Loss IFNAR2, LAMC2, NHLRC4, NISCH,
    Upregulated genes OLFML3, PSMD11, PUM2, SLC37A1,
    SMAP1, SNORA27, TMEM50B, UBAP2,
    ZNF780A
    Differential Expression ABCG2, ADAMTS1, AQP1, BCAP31,
    upon THSD1 Loss CDC45, CHCHD7, DMD, ENOX2,
    Downregulated genes FAM219A, FGF2, FGFR1OP2, G0S2, GJA4,
    IGFBP3, INAFM1, MAP2, MGP, NOL3,
    NPR1, NVL, PPHLN1, SERPINB2, SETBP1,
    (THSD1), TMEM107, ZNF185
  • Table 3. lists genes differentially expressed in THSD1 knockdown HUVECs.
  • TABLE 3
    Differentially Expressed Genes in THSD1 Knockdown HUVECs
    Ensembl Gene Gene Name logFC logCPM LR PValue FDR
    ENSG00000136114 THSD1 −2.07 4.85 141.12 1.52E−32 2.15E−28
    ENSG00000265107 GJA5 −3.81 3.42 137.13 1.13E−31 8.01E−28
    ENSG00000130066 SAT1 −1.74 8.37 128.15 1.04E−29 4.92E−26
    ENSG00000111341 MGP −1.46 6.50 112.09 3.42E−26 1.21E−22
    ENSG00000187513 GJA4 −2.29 4.32 102.47 4.39E−24 1.25E−20
    ENSG00000284057 AP001273.2 8.67 −0.99 71.34 3.00E−17 7.11E−14
    ENSG00000170323 FABP4 −2.09 6.36 66.29 3.90E−16 7.91E−13
    ENSG00000125266 EFNB2 −1.28 7.77 63.28 1.79E−15 3.18E−12
    ENSG00000113389 NPR3 −3.16 1.25 56.87 4.65E−14 7.35E−11
    ENSG00000134668 SPOCD1 1.16 5.45 54.03 1.97E−13 2.80E−10
    ENSG00000118777 ABCG2 −2.38 4.92 51.45 7.36E−13 9.50E−10
    ENSG00000203812 HIST2H2AA3 4.21 0.20 50.59 1.14E−12 1.35E−09
    ENSG00000272921 AC005832.4 −8.85 −0.72 48.91 2.69E−12 2.93E−09
    ENSG00000078018 MAP2 −1.06 4.84 48.51 3.29E−12 3.34E−09
    ENSG00000115884 SDC1 −1.53 3.23 48.33 3.61E−12 3.42E−09
    ENSG00000139278 GLIPR1 1.13 6.53 47.73 4.89E−12 4.34E−09
    ENSG00000101335 MYL9 1.09 4.76 47.07 6.83E−12 5.71E−09
    ENSG00000181634 TNFSF15 1.64 5.10 45.62 1.43E−11 1.13E−08
    ENSG00000122861 PLAU 1.03 5.58 43.85 3.55E−11 2.65E−08
    ENSG00000107551 RASSF4 −1.10 5.07 43.68 3.86E−11 2.74E−08
    ENSG00000274611 TBC1D3 −7.97 −1.52 43.13 5.12E−11 3.46E−08
    ENSG00000149591 TAGLN 1.81 2.54 42.71 6.35E−11 4.10E−08
    ENSG00000171435 KSR2 1.18 3.90 38.39 5.80E−10 3.58E−07
    ENSG00000153165 RGPD3 4.26 −1.48 37.47 9.26E−10 5.48E−07
    ENSG00000251569 AC093899.2 7.32 −2.20 34.85 3.55E−09 2.02E−06
    ENSG00000163083 INHBB −2.57 2.18 34.75 3.75E−09 2.05E−06
    ENSG00000272949 AC093668.2 −7.24 −2.15 33.67 6.51E−09 3.43E−06
    ENSG00000152217 SETBP1 −1.78 3.75 33.17 8.42E−09 4.27E−06
    ENSG00000119900 OGFRL1 0.85 6.59 32.79 1.03E−08 5.02E−06
    ENSG00000082684 SEMA5B −4.57 −1.62 32.23 1.37E−08 6.26E−06
    ENSG00000169604 ANTXR1 0.98 5.91 32.24 1.36E−08 6.26E−06
    ENSG00000137573 SULF1 −1.27 5.03 31.88 1.64E−08 7.29E−06
    ENSG00000115232 ITGA4 1.35 3.29 30.82 2.83E−08 1.22E−05
    ENSG00000058085 LAMC2 1.06 5.71 30.05 4.21E−08 1.76E−05
    ENSG00000022567 SLC45A4 −1.35 3.07 29.70 5.04E−08 1.99E−05
    ENSG00000172602 RND1 −1.74 1.64 29.71 5.03E−08 1.99E−05
    ENSG00000103426 CORO7-PAM16 4.38 −1.48 29.02 7.16E−08 2.75E−05
    ENSG00000120217 CD274 1.35 4.21 28.77 8.14E−08 3.04E−05
    ENSG00000116667 C1orf21 −0.99 4.42 27.88 1.29E−07 4.70E−05
    ENSG00000169129 AFAP1L2 −2.16 3.16 27.67 1.44E−07 5.10E−05
    ENSG00000138646 HERC5 −1.85 1.47 27.51 1.56E−07 5.29E−05
    ENSG00000197646 PDCD1LG2 1.07 3.58 27.55 1.53E−07 5.29E−05
    ENSG00000064651 SLC12A2 0.73 7.05 26.91 2.13E−07 7.03E−05
    ENSG00000171388 APLN 0.85 9.85 26.02 3.39E−07 0.000109
    ENSG00000175426 PCSK1 1.51 2.68 25.80 3.79E−07 0.00012
    ENSG00000057019 DCBLD2 0.91 6.78 25.67 4.05E−07 0.000125
    ENSG00000008517 IL32 0.69 6.11 25.52 4.37E−07 0.000132
    ENSG00000138685 FGF2 −1.20 5.72 25.21 5.15E−07 0.000152
    ENSG00000065308 TRAM2 0.67 8.10 25.04 5.60E−07 0.000162
    ENSG00000137507 LRRC32 −0.87 7.22 24.74 6.57E−07 0.000187
    ENSG00000129116 PALLD 0.69 6.27 24.56 7.22E−07 0.000201
    ENSG00000129521 EGLN3 −1.85 0.09 24.44 7.66E−07 0.000209
    ENSG00000125954 CHURC1-FNTB −6.59 −2.67 24.25 8.46E−07 0.000227
    ENSG00000152402 GUCY1A2 1.90 1.31 24.20 8.68E−07 0.000228
    ENSG00000169429 CXCL8 1.22 4.62 23.66 1.15E−06 0.000297
    ENSG00000164683 HEY1 −1.25 3.49 23.30 1.38E−06 0.000345
    ENSG00000176907 TCIM 0.95 3.77 23.31 1.38E−06 0.000345
    ENSG00000082126 MPP4 0.76 4.76 23.08 1.56E−06 0.000379
    ENSG00000163637 PRICKLE2 −0.99 3.76 23.06 1.57E−06 0.000379
    ENSG00000148143 ZNF462 0.72 4.95 22.92 1.69E−06 0.0004
    ENSG00000139289 PHLDA1 0.63 7.00 22.22 2.43E−06 0.000566
    ENSG00000028137 TNFRSF1B −0.99 6.14 22.02 2.69E−06 0.000617
    ENSG00000139629 GALNT6 0.66 5.98 21.94 2.82E−06 0.000635
    ENSG00000117586 TNFSF4 0.66 7.43 21.80 3.03E−06 0.000672
    ENSG00000256514 AP003419.1 1.68 0.91 21.47 3.60E−06 0.000787
    ENSG00000240583 AQP1 −1.84 3.43 21.27 3.99E−06 0.000858
    ENSG00000115252 PDE1A −2.04 0.57 21.17 4.20E−06 0.00089
    ENSG00000178695 KCTD12 −0.71 8.94 20.99 4.62E−06 0.000965
    ENSG00000173535 TNFRSF10C −0.65 6.69 20.89 4.87E−06 0.001002
    ENSG00000156298 TSPAN7 −1.50 3.22 20.81 5.08E−06 0.001031
    ENSG00000037280 FLT4 −0.95 6.22 20.66 5.48E−06 0.001066
    ENSG00000118515 SGK1 1.02 6.19 20.70 5.37E−06 0.001066
    ENSG00000171105 INSR −0.82 5.20 20.67 5.47E−06 0.001066
    ENSG00000128917 DLL4 −1.06 6.99 20.55 5.81E−06 0.001116
    ENSG00000164284 GRPEL2 0.71 5.83 20.28 6.70E−06 0.001268
    ENSG00000130449 ZSWIM6 0.84 6.22 20.20 6.96E−06 0.0013
    ENSG00000165029 ABCA1 −1.20 4.86 20.18 7.06E−06 0.001303
    ENSG00000143127 ITGA10 −0.95 4.84 20.08 7.43E−06 0.001336
    ENSG00000187720 THSD4 0.77 5.68 20.08 7.43E−06 0.001336
    ENSG00000073008 PVR 0.69 7.34 19.90 8.14E−06 0.001422
    ENSG00000107731 UNC5B −1.58 3.90 19.93 8.03E−06 0.001422
    ENSG00000122786 CALD1 0.71 9.31 19.89 8.21E−06 0.001422
    ENSG00000110092 CCND1 0.77 8.26 19.76 8.76E−06 0.0015
    ENSG00000154734 ADAMTS1 −1.32 3.65 19.66 9.23E−06 0.001561
    ENSG00000142178 SIK1 1.16 2.11 19.62 9.46E−06 0.001581
    ENSG00000168843 FSTL5 1.55 0.35 19.53 9.92E−06 0.001638
    ENSG00000121858 TNFSF10 −0.94 4.92 19.41 1.05E−05 0.001722
    ENSG00000169213 RAB3B 0.71 5.47 19.35 1.09E−05 0.001754
    ENSG00000166670 MMP10 1.61 6.00 19.29 1.12E−05 0.00179
    ENSG00000138347 MYPN 2.35 −1.12 19.03 1.29E−05 0.002029
    ENSG00000145247 OCIAD2 0.76 5.62 18.90 1.38E−05 0.002126
    ENSG00000160801 PTH1R −2.70 −1.67 18.89 1.38E−05 0.002126
    ENSG00000184113 CLDN5 −0.91 7.73 18.88 1.39E−05 0.002126
    ENSG00000152518 ZFP36L2 −0.62 6.87 18.76 1.48E−05 0.002235
    ENSG00000171617 ENC1 0.87 6.92 18.56 1.64E−05 0.002457
    ENSG00000166833 NAV2 0.61 5.37 18.42 1.77E−05 0.00262
    ENSG00000177076 ACER2 −1.05 3.11 18.28 1.91E−05 0.002793
    ENSG00000154928 EPHB1 1.07 2.28 18.16 2.03E−05 0.002915
    ENSG00000162407 PLPP3 −0.99 5.19 18.16 2.03E−05 0.002915
    ENSG00000099204 ABLIM1 −0.73 8.31 18.11 2.09E−05 0.002957
    ENSG00000177606 JUN 0.68 7.59 18.09 2.10E−05 0.002957
    ENSG00000170006 TMEM154 0.74 5.48 18.03 2.17E−05 0.003026
    ENSG00000118762 PKD2 −0.61 5.46 17.91 2.32E−05 0.003197
    ENSG00000108387 ‘SEPT4 −1.64 −0.22 17.45 2.96E−05 0.004038
    ENSG00000170891 CYTL1 0.91 3.83 17.25 3.28E−05 0.004439
    ENSG00000175040 CHST2 0.88 4.58 17.17 3.42E−05 0.004583
    ENSG00000167037 SGSM1 −1.41 1.33 17.15 3.45E−05 0.004587
    ENSG00000185070 FLRT2 0.70 7.60 17.09 3.57E−05 0.004694
    ENSG00000180304 OAZ2 −0.56 6.82 16.78 4.20E−05 0.005473
    ENSG00000257093 KIAA1147 −0.59 7.24 16.71 4.36E−05 0.005628
    ENSG00000184897 H1FX −0.58 6.28 16.64 4.53E−05 0.005794
    ENSG00000075426 FOSL2 0.57 6.66 16.52 4.80E−05 0.006094
    ENSG00000105825 TFPI2 0.57 7.14 16.40 5.13E−05 0.006449
    ENSG00000135318 NT5E 0.61 7.62 16.38 5.18E−05 0.00645
    ENSG00000127533 F2RL3 1.23 1.06 16.34 5.30E−05 0.006492
    ENSG00000205683 DPF3 1.21 2.98 16.34 5.30E−05 0.006492
    ENSG00000105499 PLA2G4C 0.76 3.68 16.29 5.44E−05 0.006599
    ENSG00000167984 NLRC3 0.99 2.82 16.02 6.26E−05 0.007507
    ENSG00000168685 IL7R 1.30 1.79 16.00 6.32E−05 0.007507
    ENSG00000172985 SH3RF3 0.64 5.05 16.00 6.34E−05 0.007507
    ENSG00000108551 RASD1 1.21 2.55 15.95 6.49E−05 0.00762
    ENSG00000081087 OSTM1 0.67 5.66 15.81 6.99E−05 0.008139
    ENSG00000128849 CGNL1 −0.78 6.46 15.61 7.78E−05 0.008985
    ENSG00000133816 MICAL2 0.69 7.58 15.59 7.87E−05 0.009013
    ENSG00000132965 ALOX5AP 2.25 −1.61 15.55 8.04E−05 0.009132
    ENSG00000084734 GCKR −2.84 −1.71 15.36 8.89E−05 0.009794
    ENSG00000108854 SMURF2 0.53 7.94 15.38 8.81E−05 0.009794
    ENSG00000111859 NEDD9 0.52 6.27 15.36 8.89E−05 0.009794
    ENSG00000154678 PDE1C 0.89 3.99 15.37 8.84E−05 0.009794
    ENSG00000033867 SLC4A7 0.58 7.03 15.33 9.05E−05 0.009889
    ENSG00000152207 CYSLTR2 −2.45 −0.56 15.25 9.42E−05 0.010156
    ENSG00000188452 CERKL 1.26 0.91 15.25 9.44E−05 0.010156
    ENSG00000105357 MYH14 1.43 0.19 15.20 9.66E−05 0.010323
    ENSG00000185432 METTL7A −1.01 3.66 15.17 9.83E−05 0.010418
    ENSG00000004799 PDK4 −1.18 2.61 15.12 0.000101 0.010639
    ENSG00000122694 GLIPR2 0.65 4.88 15.10 0.000102 0.010655
    ENSG00000213402 PTPRCAP −3.20 −2.40 15.08 0.000103 0.010665
    ENSG00000115902 SLC1A4 −0.69 4.85 14.96 0.00011 0.011119
    ENSG00000169248 CXCL11 1.35 0.90 14.97 0.000109 0.011119
    ENSG00000189367 KIAA0408 1.83 −0.79 14.96 0.00011 0.011119
    ENSG00000102755 FLT1 −0.77 8.53 14.90 0.000114 0.011449
    ENSG00000135905 DOCK10 0.62 5.30 14.86 0.000116 0.01158
    ENSG00000079102 RUNX1T1 −0.82 4.50 14.83 0.000118 0.011588
    ENSG00000153208 MERTK 0.58 5.53 14.83 0.000118 0.011588
    ENSG00000198435 NRARP −0.63 5.05 14.82 0.000118 0.011588
    ENSG00000141682 PMAIP1 1.02 4.25 14.80 0.00012 0.01165
    ENSG00000011347 SYT7 −1.86 −0.09 14.71 0.000126 0.012144
    ENSG00000135842 FAM129A −1.24 1.66 14.52 0.000139 0.013339
    ENSG00000020577 SAMD4A 0.62 5.94 14.47 0.000143 0.013504
    ENSG00000143153 ATP1B1 0.74 5.54 14.47 0.000142 0.013504
    ENSG00000177666 PNPLA2 −0.55 6.36 14.43 0.000145 0.013671
    ENSG00000085276 MECOM −0.53 7.33 14.39 0.000148 0.013802
    ENSG00000111145 ELK3 −0.64 8.64 14.39 0.000149 0.013802
    ENSG00000140675 SLC5A2 −5.88 −3.18 14.34 0.000152 0.014056
    ENSG00000005108 THSD7A −0.61 5.99 14.27 0.000159 0.014543
    ENSG00000157613 CREB3L1 1.04 1.76 14.23 0.000162 0.014743
    ENSG00000163584 RPL22L1 −0.61 5.30 14.18 0.000166 0.015011
    ENSG00000169418 NPR1 −1.45 3.01 14.12 0.000171 0.015403
    ENSG00000131016 AKAP12 0.59 10.29 14.07 0.000176 0.015751
    ENSG00000141298 SSH2 0.55 6.75 13.98 0.000184 0.016299
    ENSG00000163131 CTSS 0.96 2.75 13.98 0.000185 0.016299
    ENSG00000026508 CD44 0.69 5.54 13.94 0.000189 0.016335
    ENSG00000136404 TM6SF1 0.69 4.25 13.92 0.00019 0.016335
    ENSG00000144583 ‘MARCH4 0.82 5.53 13.95 0.000188 0.016335
    ENSG00000175899 A2M −1.56 2.41 13.92 0.000191 0.016335
    ENSG00000176749 CDK5R1 1.01 2.89 13.94 0.000189 0.016335
    ENSG00000258984 UBE2F-SCLY 3.28 −1.94 13.88 0.000194 0.016538
    ENSG00000136960 ENPP2 −1.14 1.35 13.86 0.000197 0.016639
    ENSG00000140416 TPM1 0.61 8.65 13.81 0.000202 0.016977
    ENSG00000112541 PDE10A −0.92 3.09 13.76 0.000208 0.017288
    ENSG00000198286 CARD11 0.59 5.90 13.76 0.000208 0.017288
    ENSG00000108825 PTGES3L-AARSD1 1.90 −0.91 13.67 0.000218 0.017886
    ENSG00000185650 ZFP36L1 0.51 6.82 13.66 0.000219 0.017886
    ENSG00000188290 HES4 −1.30 2.20 13.66 0.000219 0.017886
    ENSG00000114631 PODXL2 0.66 4.05 13.62 0.000224 0.018189
    ENSG00000106852 LHX6 0.94 4.20 13.59 0.000227 0.018332
    ENSG00000171115 GIMAP8 −0.63 7.66 13.56 0.000231 0.018514
    ENSG00000189120 SP6 1.04 1.77 13.54 0.000234 0.018568
    ENSG00000265972 TXNIP −1.35 3.74 13.54 0.000234 0.018568
    ENSG00000154639 CXADR 0.57 4.98 13.51 0.000237 0.018606
    ENSG00000169242 EFNA1 −0.75 6.23 13.52 0.000237 0.018606
    ENSG00000204304 PBX2 −0.72 5.80 13.49 0.00024 0.018755
    ENSG00000157557 ETS2 0.56 6.59 13.45 0.000244 0.018975
    ENSG00000105855 ITGB8 1.01 3.67 13.44 0.000246 0.018999
    ENSG00000144802 NFKBIZ 0.88 4.33 13.37 0.000255 0.019615
    ENSG00000204262 COL5A2 0.51 8.22 13.33 0.000262 0.019978
    ENSG00000163092 XIRP2 2.56 −0.54 13.31 0.000264 0.020085
    ENSG00000049540 ELN −2.37 −1.72 13.23 0.000275 0.020704
    ENSG00000166341 DCHS1 0.50 7.06 13.24 0.000274 0.020704
    ENSG00000115414 FN1 0.69 12.30 13.17 0.000284 0.021259
    ENSG00000094880 CDC23 −0.63 5.49 13.10 0.000295 0.021903
    ENSG00000164647 STEAP1 −0.67 4.33 13.10 0.000296 0.021903
    ENSG00000119681 LTBP2 0.48 9.54 13.03 0.000306 0.022511
    ENSG00000107562 CXCL12 2.40 −1.46 12.98 0.000314 0.022927
    ENSG00000138448 ITGAV 0.55 8.76 12.96 0.000318 0.022927
    ENSG00000160223 ICOSLG −0.72 3.61 12.95 0.00032 0.022927
    ENSG00000163644 PPM1K −0.64 3.93 12.96 0.000318 0.022927
    ENSG00000186575 NF2 0.58 6.53 12.95 0.000319 0.022927
    ENSG00000109906 ZBTB16 −1.08 1.12 12.94 0.000322 0.022986
    ENSG00000144476 ACKR3 −0.77 4.06 12.89 0.00033 0.023468
    ENSG00000136011 STAB2 −3.99 −2.80 12.86 0.000335 0.023698
    ENSG00000074181 NOTCH3 −1.01 1.47 12.84 0.000338 0.023798
    ENSG00000168916 ZNF608 −0.63 5.15 12.72 0.000362 0.025298
    ENSG00000076351 SLC46A1 0.82 2.77 12.71 0.000364 0.025315
    ENSG00000151474 FRMD4A 0.53 7.31 12.66 0.000374 0.025951
    ENSG00000139508 SLC46A3 −1.45 4.08 12.58 0.00039 0.026876
    ENSG00000131386 GALNT15 −1.70 0.16 12.53 0.000401 0.027499
    ENSG00000101017 CD40 −0.65 4.42 12.49 0.000409 0.027854
    ENSG00000270316 BORCS7-ASMT −6.14 −3.00 12.49 0.00041 0.027854
    ENSG00000141668 CBLN2 2.75 0.19 12.48 0.000412 0.027879
    ENSG00000146072 TNFRSF21 0.47 6.64 12.44 0.000419 0.028234
    ENSG00000183287 CCBE1 1.06 1.39 12.39 0.000432 0.028931
    ENSG00000029534 ANK1 −1.37 0.01 12.37 0.000436 0.029068
    ENSG00000124593 AL365205.1 −0.90 3.18 12.35 0.00044 0.029134
    ENSG00000281028 AC104662.2 −6.09 −3.04 12.35 0.000441 0.029134
    ENSG00000187942 LDLRAD2 −0.76 3.41 12.33 0.000446 0.029332
    ENSG00000071246 VASH1 −0.66 7.76 12.30 0.000453 0.029655
    ENSG00000269307 AC010463.1 −6.06 −3.06 12.27 0.000461 0.030027
    ENSG00000050405 LIMA1 0.47 7.26 12.26 0.000463 0.030037
    ENSG00000259171 AL163636.2 −1.37 1.58 12.25 0.000465 0.030049
    ENSG00000048740 CELF2 0.49 6.23 12.18 0.000483 0.030936
    ENSG00000116774 OLFML3 0.99 1.45 12.18 0.000482 0.030936
    ENSG00000182240 BACE2 −0.56 7.52 12.17 0.000486 0.030971
    ENSG00000204767 FAM196B 0.77 3.39 12.15 0.00049 0.031102
    ENSG00000211448 DIO2 2.26 −1.84 12.14 0.000494 0.031198
    ENSG00000151468 CCDC3 −1.17 0.72 12.11 0.000501 0.031481
    ENSG00000196498 NCOR2 0.52 7.49 12.09 0.000506 0.031651
    ENSG00000049130 KITLG 0.70 5.16 12.07 0.000512 0.031901
    ENSG00000137033 IL33 −1.94 4.40 12.06 0.000516 0.031997
    ENSG00000130635 COL5A1 0.52 8.58 12.03 0.000524 0.032215
    ENSG00000133056 PIK3C2B −0.67 5.90 12.03 0.000522 0.032215
    ENSG00000116741 RGS2 0.79 3.02 12.02 0.000528 0.0323
    ENSG00000129757 CDKN1C −0.95 2.05 11.98 0.000536 0.032564
    ENSG00000179195 ZNF664 −0.66 6.99 11.99 0.000535 0.032564
    ENSG00000185924 RTN4RL1 −2.85 −2.54 11.97 0.000542 0.032704
    ENSG00000189060 H1F0 −0.50 6.44 11.96 0.000543 0.032704
    ENSG00000255767 AC108488.2 5.64 −3.35 11.95 0.000548 0.032834
    ENSG00000108691 CCL2 0.56 5.05 11.89 0.000566 0.033765
    ENSG00000106069 CHN2 0.83 2.52 11.87 0.000571 0.033914
    ENSG00000132702 HAPLN2 1.07 3.28 11.85 0.000575 0.033914
    ENSG00000183691 NOG 1.96 0.87 11.85 0.000575 0.033914
    ENSG00000105738 SIPA1L3 0.53 5.37 11.84 0.000578 0.033924
    ENSG00000154096 THY1 −1.73 0.37 11.84 0.00058 0.033924
    ENSG00000140937 CDH11 0.58 5.50 11.80 0.000592 0.034495
    ENSG00000127241 MASP1 −1.60 −0.50 11.77 0.000601 0.034815
    ENSG00000164574 GALNT10 0.47 7.18 11.77 0.000603 0.034815
    ENSG00000259112 NDUFC2-KCTD14 5.59 −3.38 11.75 0.000609 0.035044
    ENSG00000109046 WSB1 −0.57 8.59 11.72 0.000619 0.035055
    ENSG00000130054 FAM155B 1.73 −1.28 11.72 0.000619 0.035055
    ENSG00000133401 PDZD2 −0.68 3.72 11.72 0.000618 0.035055
    ENSG00000284041 AC073111.3 −5.87 −3.18 11.72 0.000619 0.035055
    ENSG00000115008 IL1A 1.56 1.13 11.67 0.000634 0.035722
    ENSG00000185737 NRG3 −0.86 2.50 11.66 0.000638 0.035834
    ENSG00000196923 PDLIM7 0.57 7.15 11.64 0.000644 0.036026
    ENSG00000187583 PLEKHN1 1.60 −1.07 11.62 0.000652 0.036337
    ENSG00000135324 MRAP2 −0.97 3.21 11.57 0.000672 0.037286
    ENSG00000158186 MRAS 0.56 4.27 11.55 0.000678 0.037503
    ENSG00000092969 TGFB2 1.40 3.41 11.54 0.000682 0.037523
    ENSG00000158373 HIST1H2BD 1.95 0.56 11.53 0.000684 0.037523
    ENSG00000118946 PCDH17 1.00 3.06 11.49 0.0007 0.038238
    ENSG00000067798 NAV3 0.64 5.24 11.48 0.000705 0.038357
    ENSG00000143344 RGL1 0.81 6.48 11.46 0.00071 0.03848
    ENSG00000188042 ARL4C 0.65 3.97 11.43 0.000723 0.039053
    ENSG00000162772 ATF3 0.98 1.96 11.41 0.000731 0.039331
    ENSG00000164104 HMGB2 −0.50 7.51 11.38 0.000742 0.039771
    ENSG00000138411 HECW2 0.47 7.31 11.36 0.000749 0.03998
    ENSG00000142627 EPHA2 0.49 7.57 11.33 0.000763 0.040464
    ENSG00000258947 TUBB3 0.62 7.55 11.33 0.000761 0.040464
    ENSG00000183775 KCTD16 1.30 −0.12 11.30 0.000773 0.040837
    ENSG00000114948 ADAM23 0.55 5.15 11.26 0.000793 0.041703
    ENSG00000114315 HES1 −0.49 5.56 11.22 0.000811 0.042315
    ENSG00000138772 ANXA3 0.65 5.85 11.22 0.000808 0.042315
    ENSG00000270276 HIST2H4B −1.49 −0.13 11.21 0.000813 0.042315
    ENSG00000213694 S1PR3 0.71 4.78 11.16 0.000835 0.043284
    ENSG00000150687 PRSS23 0.44 9.70 11.15 0.000841 0.043462
    ENSG00000095303 PTGS1 0.62 4.28 11.12 0.000852 0.043854
    ENSG00000146674 IGFBP3 −0.93 1.59 11.11 0.00086 0.04411
    ENSG00000109436 TBC1D9 0.45 6.66 11.09 0.000868 0.044376
    ENSG00000274933 TBC1D3I 3.01 −2.32 11.07 0.000879 0.044738
    ENSG00000116678 LEPR −0.62 4.60 11.04 0.000893 0.044947
    ENSG00000156642 NPTN −0.65 6.37 11.04 0.000892 0.044947
    ENSG00000198720 ANKRD13B 0.53 4.45 11.03 0.000895 0.044947
    ENSG00000228144 AC078927.1 5.57 −3.50 11.04 0.000891 0.044947
    ENSG00000256966 AL513165.2 2.50 −2.24 11.01 0.000907 0.045379
    ENSG00000156920 ADGRG4 3.17 −2.89 10.97 0.000926 0.046138
    ENSG00000171877 FRMD5 0.66 4.15 10.92 0.00095 0.047183
    ENSG00000164946 FREM1 1.21 0.17 10.87 0.000975 0.048254
    ENSG00000159640 ACE −0.99 5.64 10.86 0.000982 0.048417
    ENSG00000176771 NCKAP5 1.30 0.36 10.84 0.000993 0.048822
    ENSG00000198513 ATL1 1.00 3.22 10.83 0.000999 0.048924
    ENSG00000100234 TIMP3 −0.98 3.13 10.82 0.001006 0.049108
  • Table 4 lists genes differentially expressed in THSD1 knockdown HUVECs
  • TABLE 4
    Differentially Expressed Genes in THSD1 Knockdown HBMECs
    Ensembl Gene Gene Symbol logFC logCPM LR PValue FDR
    ENSG00000117152 RGS4 −1.59 6.07 88.46 5.18E−21 7.37E−17
    ENSG00000272949 AC093668.2 8.74 −0.80 64.63 9.02E−16 6.40E−12
    ENSG00000283088 AC010487.3 −8.71 −0.85 60.76 6.45E−15 3.05E−11
    ENSG00000136114 THSD1 −1.51 4.73 58.41 2.13E−14 7.56E−11
    ENSG00000146674 IGFBP3 −2.59 2.50 54.84 1.31E−13 3.72E−10
    ENSG00000284057 AP001273.2 8.19 −1.30 52.15 5.15E−13 1.22E−09
    ENSG00000133101 CCNA1 −1.31 4.76 45.04 1.93E−11 3.92E−08
    ENSG00000078018 MAP2 −1.31 4.98 43.56 4.11E−11 7.29E−08
    ENSG00000240583 AQP1 −2.37 3.93 41.78 1.02E−10 1.61E−07
    ENSG00000008517 IL32 1.06 6.45 39.73 2.92E−10 4.15E−07
    ENSG00000154734 ADAMTS1 −1.55 3.82 38.76 4.78E−10 6.18E−07
    ENSG00000068489 PRR11 −1.00 6.19 37.14 1.10E−09 1.30E−06
    ENSG00000118777 ABCG2 −1.75 3.44 36.14 1.84E−09 1.87E−06
    ENSG00000168542 COL3A1 2.68 1.64 36.27 1.72E−09 1.87E−06
    ENSG00000138180 CEP55 −0.99 6.19 35.71 2.30E−09 2.17E−06
    ENSG00000272414 FAM47E-STBD1 4.42 −1.05 35.52 2.52E−09 2.24E−06
    ENSG00000264187 AC055811.2 7.52 −1.89 35.39 2.70E−09 2.26E−06
    ENSG00000072571 HMMR −1.09 5.70 34.95 3.39E−09 2.67E−06
    ENSG00000112984 KIF20A −1.03 6.21 34.59 4.07E−09 3.05E−06
    ENSG00000126787 DLGAP5 −0.96 6.86 34.15 5.09E−09 3.62E−06
    ENSG00000081087 OSTM1 1.10 5.56 33.06 8.93E−09 6.04E−06
    ENSG00000117399 CDC20 −1.02 6.40 32.40 1.25E−08 8.08E−06
    ENSG00000100292 HMOX1 0.94 6.83 31.53 1.96E−08 1.21E−05
    ENSG00000134057 CCNB1 −0.94 6.95 31.40 2.10E−08 1.24E−05
    ENSG00000143228 NUF2 −1.06 5.01 30.90 2.71E−08 1.48E−05
    ENSG00000166851 PLK1 −1.03 6.17 30.91 2.70E−08 1.48E−05
    ENSG00000108691 CCL2 1.38 5.33 29.45 5.74E−08 2.86E−05
    ENSG00000132470 ITGB4 −1.94 3.69 29.41 5.84E−08 2.86E−05
    ENSG00000164104 HMGB2 −0.86 7.35 29.54 5.47E−08 2.86E−05
    ENSG00000163661 PTX3 −1.01 7.71 29.11 6.85E−08 3.24E−05
    ENSG00000145386 CCNA2 −1.01 6.45 29.03 7.12E−08 3.26E−05
    ENSG00000142945 KIF2C −0.95 5.36 28.79 8.07E−08 3.58E−05
    ENSG00000146678 IGFBP1 −2.51 0.45 28.44 9.65E−08 4.15E−05
    ENSG00000137812 KNL1 −1.09 5.81 28.26 1.06E−07 4.44E−05
    ENSG00000138182 KIF20B −1.01 5.81 28.10 1.15E−07 4.67E−05
    ENSG00000080986 NDC80 −1.05 5.15 27.56 1.52E−07 5.90E−05
    ENSG00000136928 GABBR2 −0.95 6.03 27.44 1.62E−07 5.90E−05
    ENSG00000137804 NUSAP1 −0.95 6.08 27.46 1.60E−07 5.90E−05
    ENSG00000273294 C1QTNF3-AMACR 7.05 −2.28 27.45 1.62E−07 5.90E−05
    ENSG00000131747 TOP2A −0.97 8.09 26.97 2.07E−07 7.35E−05
    ENSG00000087586 AURKA −0.86 5.67 26.90 2.14E−07 7.42E−05
    ENSG00000094880 CDC23 −0.93 5.49 26.82 2.24E−07 7.43E−05
    ENSG00000161888 SPC24 −1.08 4.35 26.81 2.25E−07 7.43E−05
    ENSG00000066279 ASPM −1.18 6.89 26.71 2.36E−07 7.62E−05
    ENSG00000198901 PRC1 −0.91 6.85 26.11 3.23E−07 0.000102
    ENSG00000088325 TPX2 −0.86 7.28 25.96 3.49E−07 0.000108
    ENSG00000138778 CENPE −1.03 6.40 25.44 4.58E−07 0.000136
    ENSG00000140525 FANCI −0.85 5.87 25.43 4.60E−07 0.000136
    ENSG00000111206 FOXM1 −0.80 6.61 25.29 4.94E−07 0.000143
    ENSG00000076382 SPAG5 −0.83 5.81 25.10 5.46E−07 0.000155
    ENSG00000170312 CDK1 −0.95 6.03 25.05 5.59E−07 0.000156
    ENSG00000161800 RACGAP1 −0.88 5.47 24.90 6.04E−07 0.000162
    ENSG00000168078 PBK −0.93 5.37 24.91 6.02E−07 0.000162
    ENSG00000146072 TNFRSF21 0.88 6.42 24.77 6.45E−07 0.00017
    ENSG00000134690 CDCA8 −1.05 5.25 24.59 7.11E−07 0.000184
    ENSG00000148773 MKI67 −1.17 7.99 24.06 9.33E−07 0.000237
    ENSG00000102575 ACP5 −3.55 2.81 24.03 9.50E−07 0.000237
    ENSG00000024526 DEPDC1 −0.99 5.61 23.90 1.02E−06 0.000249
    ENSG00000099937 SERPIND1 −1.85 2.87 23.80 1.07E−06 0.000258
    ENSG00000276612 FP565260.2 −6.67 −2.61 23.74 1.10E−06 0.000261
    ENSG00000118193 KIF14 −1.04 5.04 23.64 1.16E−06 0.000266
    ENSG00000175063 UBE2C −1.03 5.18 23.65 1.16E−06 0.000266
    ENSG00000123485 HJURP −0.92 4.98 23.46 1.28E−06 0.000286
    ENSG00000157456 CCNB2 −0.84 5.61 23.41 1.31E−06 0.000286
    ENSG00000163584 RPL22L1 −0.90 5.95 23.41 1.31E−06 0.000286
    ENSG00000117724 CENPF −0.96 7.44 22.28 2.35E−06 0.000506
    ENSG00000100297 MCM5 −0.86 6.30 22.12 2.56E−06 0.000535
    ENSG00000105357 MYH14 4.21 −0.61 22.15 2.53E−06 0.000535
    ENSG00000138160 KIF11 −0.95 6.34 22.01 2.71E−06 0.000558
    ENSG00000093009 CDC45 −1.00 4.55 21.97 2.77E−06 0.000562
    ENSG00000185070 FLRT2 0.76 8.29 21.87 2.92E−06 0.000584
    ENSG00000116774 OLFML3 1.52 2.30 21.78 3.06E−06 0.000603
    ENSG00000123689 G0S2 −1.97 2.29 21.72 3.15E−06 0.000613
    ENSG00000167900 TK1 −0.76 5.68 21.56 3.43E−06 0.000655
    ENSG00000184661 CDCA2 −0.93 4.87 21.54 3.46E−06 0.000655
    ENSG00000013810 TACC3 −0.76 6.36 21.52 3.50E−06 0.000655
    ENSG00000101057 MYBL2 −0.78 6.00 21.42 3.70E−06 0.000682
    ENSG00000112742 TTK −1.03 5.14 21.29 3.95E−06 0.000719
    ENSG00000163808 KIF15 −1.04 4.74 21.00 4.59E−06 0.000826
    ENSG00000011426 ANLN −1.01 7.23 20.77 5.19E−06 0.00091
    ENSG00000265107 GJA5 −6.45 −2.77 20.77 5.19E−06 0.00091
    ENSG00000121152 NCAPH −0.96 4.79 20.73 5.28E−06 0.000915
    ENSG00000114631 PODXL2 1.26 3.20 20.67 5.44E−06 0.000932
    ENSG00000129173 E2F8 −1.04 4.57 20.50 5.97E−06 0.00101
    ENSG00000101335 MYL9 0.82 5.36 20.46 6.10E−06 0.00102
    ENSG00000164109 MAD2L1 −0.92 5.54 20.32 6.55E−06 0.001082
    ENSG00000104738 MCM4 −0.92 6.63 20.27 6.74E−06 0.0011
    ENSG00000169679 BUB1 −0.99 5.93 20.11 7.32E−06 0.001182
    ENSG00000073111 MCM2 −0.82 5.76 19.94 7.98E−06 0.00126
    ENSG00000165480 SKA3 −0.99 4.57 19.95 7.95E−06 0.00126
    ENSG00000075218 GTSE1 −0.89 5.40 19.81 8.54E−06 0.001333
    ENSG00000267618 AC004223.3 6.30 −2.86 19.78 8.70E−06 0.001344
    ENSG00000163131 CTSS 1.44 3.29 19.60 9.54E−06 0.001458
    ENSG00000089685 BIRC5 −0.77 6.29 19.52 9.98E−06 0.001508
    ENSG00000178999 AURKB −0.98 4.85 19.36 1.08E−05 0.001618
    ENSG00000186193 SAPCD2 −0.87 4.80 19.32 1.10E−05 0.001632
    ENSG00000071539 TRIP13 −0.84 5.48 19.22 1.17E−05 0.001707
    ENSG00000156504 FAM122B −0.84 4.71 19.18 1.19E−05 0.001722
    ENSG00000165092 ALDH1A1 0.69 6.16 19.14 1.21E−05 0.001741
    ENSG00000123975 CKS2 −0.80 5.71 19.02 1.29E−05 0.001764
    ENSG00000135476 ESPL1 −0.97 4.76 19.09 1.25E−05 0.001764
    ENSG00000173597 SULT1B1 −0.97 7.01 19.02 1.29E−05 0.001764
    ENSG00000189431 RASSF10 −3.28 −1.94 19.03 1.29E−05 0.001764
    ENSG00000237649 KIFC1 −0.93 5.31 19.05 1.27E−05 0.001764
    ENSG00000122966 CIT −0.81 5.46 18.93 1.36E−05 0.001836
    ENSG00000117650 NEK2 −0.85 4.48 18.90 1.38E−05 0.001846
    ENSG00000100526 CDKN3 −0.93 4.10 18.77 1.47E−05 0.001954
    ENSG00000156970 BUB1B −0.93 5.84 18.68 1.55E−05 0.002039
    ENSG00000284041 AC073111.3 −6.19 −2.96 18.66 1.57E−05 0.002041
    ENSG00000115163 CENPA −1.06 3.87 18.25 1.94E−05 0.002507
    ENSG00000269891 ARHGAP19-SLIT1 6.14 −2.97 18.05 2.15E−05 0.002751
    ENSG00000065328 MCM10 −1.01 4.18 17.99 2.22E−05 0.002822
    ENSG00000125378 BMP4 0.74 6.11 17.97 2.25E−05 0.002823
    ENSG00000079616 KIF22 −0.75 5.65 17.89 2.35E−05 0.002915
    ENSG00000151640 DPYSL4 0.98 4.09 17.87 2.36E−05 0.002915
    ENSG00000053747 LAMA3 −0.87 4.23 17.67 2.62E−05 0.003214
    ENSG00000154175 ABI3BP −0.84 5.18 17.65 2.65E−05 0.003219
    ENSG00000183856 IQGAP3 −0.81 5.10 17.63 2.68E−05 0.003226
    ENSG00000198826 ARHGAP11A −0.92 6.08 17.48 2.91E−05 0.003475
    ENSG00000228716 DHFR −0.74 6.16 17.44 2.97E−05 0.003512
    ENSG00000076003 MCM6 −0.84 6.10 17.39 3.05E−05 0.003576
    ENSG00000173166 RAPH1 −0.94 5.58 17.28 3.22E−05 0.003754
    ENSG00000123473 STIL −0.90 4.92 17.23 3.31E−05 0.003819
    ENSG00000117595 IRF6 1.52 3.34 17.10 3.55E−05 0.004062
    ENSG00000146918 NCAPG2 −0.77 5.94 17.06 3.63E−05 0.004089
    ENSG00000164647 STEAP1 −0.83 5.07 17.07 3.61E−05 0.004089
    ENSG00000196878 LAMB3 −0.85 4.47 17.03 3.67E−05 0.004108
    ENSG00000171241 SHCBP1 −0.86 5.58 17.01 3.72E−05 0.004126
    ENSG00000150630 VEGFC −0.96 4.78 16.89 3.95E−05 0.004353
    ENSG00000090889 KIF4A −0.77 5.75 16.62 4.56E−05 0.004974
    ENSG00000121621 KIF18A −0.92 4.27 16.61 4.59E−05 0.004974
    ENSG00000101447 FAM83D −0.88 5.40 16.54 4.77E−05 0.005136
    ENSG00000169604 ANTXR1 1.03 4.62 16.47 4.93E−05 0.005267
    ENSG00000096060 FKBP5 −0.68 6.90 16.31 5.38E−05 0.005672
    ENSG00000167434 CA4 −4.19 −2.69 16.31 5.39E−05 0.005672
    ENSG00000163092 XIRP2 1.93 1.44 16.29 5.43E−05 0.005675
    ENSG00000104147 OIP5 −1.14 2.91 16.25 5.56E−05 0.005762
    ENSG00000119403 PHF19 −0.64 6.01 16.19 5.74E−05 0.005868
    ENSG00000211448 DIO2 1.64 0.55 16.20 5.71E−05 0.005868
    ENSG00000185432 METTL7A −0.90 5.20 16.11 5.99E−05 0.006078
    ENSG00000105499 PLA2G4C 1.03 3.47 16.09 6.05E−05 0.006091
    ENSG00000139734 DIAPH3 −0.80 5.47 16.01 6.30E−05 0.0063
    ENSG00000163453 IGFBP7 0.68 7.65 15.92 6.60E−05 0.006561
    ENSG00000140416 TPM1 0.64 8.27 15.87 6.77E−05 0.006677
    ENSG00000109805 NCAPG −0.77 6.26 15.73 7.30E−05 0.007153
    ENSG00000137807 KIF23 −0.78 5.95 15.66 7.59E−05 0.007335
    ENSG00000179195 ZNF664 −0.88 6.90 15.66 7.58E−05 0.007335
    ENSG00000144554 FANCD2 −0.87 4.59 15.57 7.95E−05 0.007632
    ENSG00000101003 GINS1 −0.83 4.79 15.51 8.19E−05 0.007811
    ENSG00000137310 TCF19 −0.81 4.92 15.35 8.92E−05 0.008445
    ENSG00000115008 IL1A 1.97 0.90 15.32 9.06E−05 0.008525
    ENSG00000163751 CPA3 1.34 1.70 15.30 9.18E−05 0.008576
    ENSG00000197632 SERPINB2 −1.60 3.06 15.21 9.61E−05 0.008924
    ENSG00000133119 RFC3 −0.80 4.52 15.16 9.87E−05 0.009103
    ENSG00000140545 MFGE8 0.73 5.90 15.12 0.00010086 0.009244
    ENSG00000101188 NTSR1 −1.16 2.79 15.08 0.00010312 0.009391
    ENSG00000171848 RRM2 −1.18 6.87 15.06 0.00010394 0.009404
    ENSG00000185480 PARPBP −0.79 4.44 15.05 0.00010467 0.009411
    ENSG00000140675 SLC5A2 4.89 −2.99 14.82 0.00011808 0.010484
    ENSG00000151725 CENPU −0.74 4.67 14.83 0.00011777 0.010484
    ENSG00000168243 GNG4 2.60 −1.74 14.78 0.00012062 0.010643
    ENSG00000173207 CKS1B −0.73 5.38 14.77 0.0001214 0.010646
    ENSG00000010292 NCAPD2 −0.62 7.04 14.64 0.00012988 0.011319
    ENSG00000173281 PPP1R3B 0.64 6.98 14.61 0.0001319 0.011425
    ENSG00000164611 PTTG1 −0.64 5.60 14.58 0.00013445 0.011569
    ENSG00000167261 DPEP2 2.79 −2.01 14.57 0.00013518 0.011569
    ENSG00000184445 KNTC1 −0.70 5.37 14.55 0.00013641 0.011604
    ENSG00000012048 BRCA1 −0.90 4.77 14.49 0.00014056 0.011791
    ENSG00000092853 CLSPN −0.87 4.95 14.49 0.0001411 0.011791
    ENSG00000203668 CHML −0.70 5.13 14.49 0.00014087 0.011791
    ENSG00000101868 POLA1 −0.84 4.68 14.45 0.00014375 0.011893
    ENSG00000122694 GLIPR2 0.70 5.12 14.45 0.000144 0.011893
    ENSG00000149591 TAGLN 1.24 1.91 14.37 0.00015051 0.012359
    ENSG00000280537 AC068946.1 2.14 −1.15 14.35 0.00015184 0.012397
    ENSG00000105011 ASF1B −0.91 4.72 14.28 0.00015721 0.012762
    ENSG00000105889 STEAP1B −0.83 4.64 14.20 0.00016442 0.013271
    ENSG00000124721 DNAH8 1.61 3.82 14.17 0.00016717 0.013341
    ENSG00000130816 DNMT1 −0.60 7.04 14.17 0.00016671 0.013341
    ENSG00000162645 GBP2 0.60 6.15 14.14 0.00016943 0.013447
    ENSG00000058804 NDC1 −0.71 5.97 14.03 0.00018026 0.014227
    ENSG00000145604 SKP2 −0.67 5.33 14.00 0.0001825 0.014324
    ENSG00000123219 CENPK −0.71 4.81 13.98 0.00018513 0.014341
    ENSG00000136824 SMC2 −0.81 6.08 13.97 0.00018575 0.014341
    ENSG00000188517 COL25A1 2.38 −1.61 13.97 0.00018564 0.014341
    ENSG00000107984 DKK1 −0.63 5.85 13.95 0.00018762 0.01437
    ENSG00000204262 COL5A2 0.61 8.73 13.95 0.00018814 0.01437
    ENSG00000162063 CCNF −0.67 5.31 13.89 0.00019352 0.014701
    ENSG00000258064 AC073612.1 −6.56 −2.69 13.76 0.00020816 0.01573
    ENSG00000120802 TMPO −0.66 7.44 13.64 0.00022161 0.016569
    ENSG00000163507 CIP2A −0.82 5.14 13.64 0.0002212 0.016569
    ENSG00000186185 KIF18B −0.86 4.35 13.61 0.00022501 0.016735
    ENSG00000149503 INCENP −0.86 5.22 13.41 0.00025051 0.018535
    ENSG00000167601 AXL −0.55 7.33 13.28 0.00026806 0.019731
    ENSG00000139618 BRCA2 −0.91 4.49 13.19 0.00028185 0.020639
    ENSG00000163554 SPTA1 2.18 −0.67 13.16 0.00028663 0.020881
    ENSG00000100311 PDGFB 0.82 6.70 13.08 0.00029782 0.021586
    ENSG00000106069 CHN2 0.94 3.27 12.98 0.00031547 0.022542
    ENSG00000239389 PCDHA13 6.39 −2.80 12.99 0.00031302 0.022542
    ENSG00000258947 TUBB3 0.59 7.10 12.97 0.00031577 0.022542
    ENSG00000144354 CDCA7 −0.74 4.99 12.94 0.0003212 0.022815
    ENSG00000123080 CDKN2C −0.81 4.00 12.93 0.00032414 0.022909
    ENSG00000103489 XYLT1 1.13 2.69 12.83 0.00034094 0.023859
    ENSG00000178538 CA8 −1.71 0.71 12.83 0.00034088 0.023859
    ENSG00000163535 SGO2 −0.80 5.30 12.81 0.00034481 0.024012
    ENSG00000133110 POSTN −1.11 3.05 12.78 0.00035075 0.024306
    ENSG00000035499 DEPDC1B −0.85 4.24 12.75 0.00035616 0.024325
    ENSG00000150540 HNMT 0.80 4.05 12.75 0.0003557 0.024325
    ENSG00000176890 TYMS −0.61 6.99 12.75 0.00035522 0.024325
    ENSG00000261459 AC002310.5 −6.20 −2.95 12.71 0.00036311 0.024681
    ENSG00000117593 DARS2 −0.67 5.54 12.68 0.00036955 0.024999
    ENSG00000111341 MGP −0.57 6.67 12.65 0.00037599 0.025314
    ENSG00000058085 LAMC2 0.75 4.73 12.57 0.00039222 0.026282
    ENSG00000103257 SLC7A5 −0.71 4.74 12.49 0.00040895 0.027275
    ENSG00000214357 NEURL1B −1.41 0.86 12.46 0.0004151 0.027556
    ENSG00000258555 SPECC1L-ADORA2A 6.16 −2.96 12.37 0.00043612 0.028683
    ENSG00000268643 AC006486.1 −6.10 −3.02 12.37 0.00043584 0.028683
    ENSG00000186871 ERCC6L −0.97 3.80 12.31 0.00045156 0.029562
    ENSG00000128944 KNSTRN −0.64 5.27 12.26 0.00046273 0.029611
    ENSG00000134222 PSRC1 −0.89 3.87 12.28 0.00045803 0.029611
    ENSG00000142731 PLK4 −0.89 4.91 12.27 0.00046122 0.029611
    ENSG00000147536 GINS4 −0.85 4.14 12.28 0.00045741 0.029611
    ENSG00000267022 AC067968.1 −2.73 −1.12 12.26 0.00046177 0.029611
    ENSG00000155093 PTPRN2 1.40 0.73 12.23 0.00047029 0.029846
    ENSG00000188229 TUBB4B −0.69 8.39 12.23 0.00047061 0.029846
    ENSG00000215252 GOLGA8B 0.68 4.46 12.21 0.00047536 0.030013
    ENSG00000149573 MPZL2 −0.67 5.40 12.20 0.00047877 0.030095
    ENSG00000129195 PIMREG −0.79 4.07 12.18 0.00048388 0.030282
    ENSG00000152104 PTPN14 −0.60 7.17 12.16 0.00048777 0.030391
    ENSG00000197457 STMN3 2.81 3.31 12.14 0.00049373 0.030629
    ENSG00000168874 ATOH8 0.84 3.70 12.13 0.00049593 0.030631
    ENSG00000255073 ZFP91-CNTF −5.43 −3.45 12.10 0.00050547 0.031085
    ENSG00000188486 H2AFX −0.63 6.27 12.02 0.00052646 0.032209
    ENSG00000213297 ZNF625-ZNF20 1.41 0.93 12.01 0.00052828 0.032209
    ENSG00000102007 PLP2 −0.55 6.27 11.97 0.00053967 0.032496
    ENSG00000135842 FAM129A −1.02 3.29 11.97 0.00053985 0.032496
    ENSG00000168843 FSTL5 1.09 3.04 11.98 0.00053834 0.032496
    ENSG00000178878 APOLD1 −0.89 3.58 11.94 0.00055082 0.033017
    ENSG00000113368 LMNB1 −0.66 6.70 11.92 0.00055512 0.033135
    ENSG00000137473 TTC29 1.72 −0.20 11.88 0.0005664 0.033666
    ENSG00000148848 ADAM12 1.25 1.79 11.86 0.00057272 0.0339
    ENSG00000114346 ECT2 −0.68 5.97 11.84 0.00058063 0.034226
    ENSG00000196739 COL27A1 0.57 5.97 11.77 0.00060245 0.03522
    ENSG00000258677 AC022826.2 −1.79 −0.91 11.77 0.00060175 0.03522
    ENSG00000129810 SGO1 −0.89 3.52 11.75 0.00060846 0.035425
    ENSG00000198830 HMGN2 −0.53 8.42 11.73 0.00061337 0.035566
    ENSG00000174371 EXO1 −0.93 3.73 11.69 0.00062871 0.036307
    ENSG00000127564 PKMYT1 −0.90 3.52 11.66 0.00063706 0.03664
    ENSG00000100368 CSF2RB 0.55 5.77 11.64 0.00064394 0.036738
    ENSG00000135451 TROAP −0.73 4.10 11.64 0.00064385 0.036738
    ENSG00000092470 WDR76 −0.72 4.40 11.60 0.00065967 0.037485
    ENSG00000143401 ANP32E −0.56 6.78 11.58 0.00066731 0.037768
    ENSG00000257411 AC034102.2 1.93 0.75 11.54 0.0006809 0.038384
    ENSG00000146670 CDCA5 −0.72 5.02 11.50 0.00069768 0.039175
    ENSG00000167325 RRM1 −0.57 6.89 11.47 0.00070846 0.039623
    ENSG00000152253 SPC25 −0.89 3.82 11.46 0.00071221 0.039677
    ENSG00000122952 ZWINT −0.71 5.53 11.43 0.00072353 0.04015
    ENSG00000121966 CXCR4 1.02 5.44 11.40 0.00073256 0.040493
    ENSG00000101224 CDC25B −0.56 6.33 11.38 0.00074161 0.040707
    ENSG00000136490 LIMD2 0.60 5.23 11.38 0.00074216 0.040707
    ENSG00000127528 KLF2 −0.82 4.46 11.35 0.00075267 0.041125
    ENSG00000151388 ADAMTS12 0.85 3.45 11.34 0.00075879 0.041163
    ENSG00000154839 SKA1 −0.87 4.20 11.34 0.00075917 0.041163
    ENSG00000119969 HELLS −0.65 4.77 11.30 0.00077592 0.041911
    ENSG00000108055 SMC3 −0.53 6.68 11.28 0.00078172 0.042065
    ENSG00000125695 AC046185.1 5.80 −3.20 11.24 0.00079891 0.042828
    ENSG00000146411 SLC2A12 1.26 2.09 11.23 0.00080506 0.042888
    ENSG00000159167 STC1 −0.93 4.54 11.23 0.00080607 0.042888
    ENSG00000181634 TNFSF15 0.78 5.81 11.19 0.00082212 0.043579
    ENSG00000111665 CDCA3 −0.78 3.97 11.16 0.00083518 0.044106
    ENSG00000067141 NEO1 −2.49 −2.06 11.10 0.00086297 0.045237
    ENSG00000099282 TSPAN15 0.66 6.21 11.10 0.00086289 0.045237
    ENSG00000120256 LRP11 0.57 5.86 10.97 0.00092765 0.048365
    ENSG00000145990 GFOD1 −0.59 5.47 10.96 0.00093285 0.048365
    ENSG00000166250 CLMP −1.02 2.47 10.96 0.00093028 0.048365
    ENSG00000072501 SMC1A −0.59 6.94 10.94 0.00093894 0.048504
    ENSG00000112378 PERP −0.59 7.18 10.92 0.00095053 0.048925
    ENSG00000077152 UBE2T −0.67 4.49 10.90 0.00096365 0.049243
    ENSG00000198553 KCNRG 1.57 −0.55 10.90 0.00096042 0.049243
    ENSG00000120337 TNFSF18 0.87 4.84 10.87 0.00097964 0.049881
  • Bioinformatic analyses highlighted a potential role for the Src Signaling pathway, and other pathways, in the pathology of IA. We evaluated the potential link to the Src signaling pathway below.
    • EXAMPLE 2: THSD1 Suppresses Podosome Formation and Function, and this Mechanism Compromises Cerebrovascular Integrity and Promotes IA Development
  • Intracranial aneurysm (IA) is a weakened area in the wall of cerebral artery that leads to a bulging in a brain blood vessel. The rupture of IA causes aneurysmal subarachnoid hemorrhage (SAH), a devastating form of stroke. More than 30% will die due to SAH and more than half of survivors will never return to independent living. Unfortunately, there are no treatments for IA except open or endovascular surgery. Attempts to find new therapeutic avenues are greatly hindered by the lack of knowledge of the gene(s) and pathways responsible for IA development and growth.
  • Podosomes are subcellular organelles located at cell-ECM interaction sites in different cell types including a variety of endothelial cells. A podosome contains a core of enriched F-actin and cortactin surrounded by a ring structure, in which an integrin complex resides and adheres the podosome to the ECM (FIG. 3 ). Unlike other adhesive structures such as focal adhesion complexes that tether the endothelium to the ECM, the function of podosome is to degrade ECM through activation of a cascade of metalloproteases including MT1-MMP, MMP2 and MMP9. Among all the upstream events that occur during podosome formation, the best characterized is the activation of SRC, a non-receptor tyrosine kinase. When SRC is enriched and activated in pre-podosome structures, it phosphorylates a wide array of substrates. For instance, SRC phosphorylates cortactin and promotes actin assembly in podosomes. See, e.g., FIG. 3 .
  • The present disclosure considered the molecular pathways affected by the knock-down of THSD1 and investigated the role of podosome formation and function role in THSD1-mediated aneurysm development.
  • Materials and Methods
  • Cell Culture
  • HEK293T cells were maintained in DMEM medium (Corning, 10-013-CV) containing 10% fetal bovine serum (Invitrogen, 10082147), 100 IU penicillin, and 100 μg/ml streptomycin. Transfections of small interfering RNAs and plasmid DNA were performed using lipofectamine 2000 (Life Technologies, 11668027) according to the manufacturer's instructions. Alternatively, for cells such as endothelial cells that are hard to transfect, we will utilize lentiviral system to generate stable cell lines.
  • Western Bot
  • Cells were lysed in 1% Triton lysis buffer and sonicated briefly before centrifuged at 18506 g for 30 min at 4° C. Total cell lysates were added by 2X SDS sample buffer and then subjected to discontinuous SDS-PAGE analysis. Proteins were transferred to nitro-cellulose membranes using a Bio-Rad (Hercules, Calif.) mini transfer apparatus followed by blocking with 5% nonfat milk. Primary antibodies and secondary antibodies were used usually at 1:1000 and 1:10000 dilutions respectively before using an Odyssey system to detect the fluorescence signal.
  • Immunofluorescence
  • Cells were grown on glass coverslips in the DMEM medium for 16 h before fixed with 4% formaldehyde-phosphate-buffered saline (Electronic Microscopy Sciences, 15710; Fisher Scientific, BP399-500) for 10 min. Cells were incubated with primary antibodies against focal adhesion markers such as paxillin overnight at 4° C. after 1 h blocking by 5% goat serum. Secondary antibodies conjugated with Alexa Fluor 488 or 594 were used to visualize the localization. Image was taken with a Leica Confocal Microscope and processed by ImageJ and GraphPad 7 software for statistical analysis.
  • Intracranial Hemorrhage in Zebrafish
  • Anti-sense morpholinos against thsd1, atg5 or talin was injected into one-cell stage zebrafish singly or in combination to silence the gene accordingly. At day 2 to day 3, embryos will be checked under regular optical microscope. Embryos with intracranial hemorrhages will be counted and imaged by Olympus microscope.
  • MicroFil Injection in Mice
  • Prepare MicroFil (Flow Tech, Inc. Carver, Mass.) casting solution according to the manufacturer's instruction: Mix 5 ml of MV diluent with 4 ml of filtered MV-112 compound (yellow). Add 450 μl (5%) of catalyst (MV curing agent). Use 10 ml syringe to inject MicroFil mixture into the left ventricle after the blood was flushed out by saline. Inject MicroFil mixture slowly into the left ventricle at approximately 3 ml/min.
  • En Face Immunostaining of Circle of Willis in Mice
  • For en face immunostaining, isolated intracerebral arteries were dissected out from the brain with micro-needle and micro-scissors under the stereomicroscope. For immunofluorescent staining, tissue samples were washed in phosphate buffered saline (PBS), blocked with 5% goat serum in PBST (0.1% Triton-X in PBS), then incubated with primary antibodies followed by the appropriate fluorescent-labeled secondary antibodies.
  • Loss-of-Function of THSD1 Induces Endothelial Podosome Formation and Function.
  • Formation:
  • FIGS. 4 a-4 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome formation in THSD1-deficient HBMECs. FIG. 4 a illustrates podosomes such as puncta (arrowheads) or rosette (arrow) were assessed by co-immunostaining of cortactin and actin in control or THSD1-deficient HBMECs. FIG. 4 b illustrates the percentage in HBMECs (n=3 independent experiments) and student t-test was used for statistical difference. *p<0.05, Scale bar: 10 μm.
  • Function:
  • FIGS. 5 a-5 b are experimental results depicting that loss-of-function of THSD1 induces endothelial podosome function. FIG. 5 a shows representative images for Alexa 488-conjugated Collagen-IV (Co-IV) degradation in control or THSD1-deficient HBMECs. FIG. 5 b shows a tabulation of the degradation index (degraded area per cellular profile) calculated from at least 9 different fields (10X objective) in HBMECs (n=3 independent experiments), and student t-test was used for statistical difference. *p<0.05, Scale bar: 10 μm.
  • Loss-of-Function of THSD1 Increase MT1-MMP Cell Surface Exposure.
  • FIGS. 6 a-6 b are experimental results depicting that loss-of-function of THSD1 increases MT1-MMP cell surface exposure. FIG. 6 a shows a Western Blot illustrating that biotinylated cell-surface proteins were pull down by streptavidin beads from control and THSD1-deficient HBMECs. The level of MT1-MMP were revealed by Western blot and quantified in FIG. 6 b . TCL: total cell lysates; GAPDH as loading control; **p<0.01 by student t-test.
  • FIGS. 7 a-7 b are experimental results depicting that MMP2 is activated upon THSD1 knockdown in HBMECs. FIG. 7 a illustrates cell extracts and supernatants from control and THSD1-deficient HBMECs were analyzed by gelatin zymography. Loading was normalized by total protein concentration. Quantification was calculated by a fold change of active MMP2 expression. See FIG. 7 b . ***p<0.001 by student t-test.
    • EXAMPLE 3: In-Vivo SRC Inhibition Rescues Cerebrovascular Integrity Defects
  • In-Vivo Zebrafish Model
  • Intracranial aneurysm and hemorrhage are tightly associated with compromised cerebrovascular integrity. Recently, zebrafish have gained additional popularity as a vertebrate model organism for studying the cerebrovasculature. Since zebrafish embryos are transparent, intracranial hemorrhage can be directly observed using a standard microscope. Furthermore, zebrafish fecundity and rapid development permits rapid phenotypic evaluation as intracranial hemorrhage in zebrafish fry are detectable as early as 2-3 days post fertilization. Importantly, gain-of-function and loss-of-function approaches are well established in zebrafish that include morpholino and mRNA injections and more recently, through applications of CRISPR/Cas9 technology.
  • FIGS. 8 a-8 b are experimental results depicting that loss-of-function of Thsd1 induces SRC-dependent intracranial hemorrhage in zebrafish, and treatment with a PP2 SRC inhibitor prevents intracranial hemorrhage in Thsd1-deficient zebrafish. FIG. 8 a is a picture illustrating a prominent cerebral hemorrhage phenotype was observed in thsd1 knockdown zebrafish. FIG. 8 b is a chart quantifying the number of embryos with intracranial hemorrhage (%). As shown in FIG. 8 b PP2 treatment (SRC inhibitor) ameliorated Thsd1-dependent hemorrhage incidents, while DMSO treatment (control) did not (comparing grey bars in b). n>120 embryos were analyzed. *p<0.05.
  • FIG. 9 (FIG. 9 ) are experimental results depicting that loss-of-function of Thsd1 promotes IA formation in circle of Willis in mice. IAs were visualized by Microfil including a PCA (top panel), a left Pcom (middle panel), and a right ICA (bottom panel) as indicated by arrows. n=10
  • FIGS. 10 a-10 b are experimental results depicting that loss-of-function of Thsd1 promotes podosome formation in intimal endothelial cells in mice. Podosomes were revealed by en face immunostaining against cortactin (green) and VE-cadherin (red) in CoW of Thsd1+/+(WT, n=9) vs. Thsd1−/− (KO, n=10) mice. Student t-test was used for statistical difference. **p<0.01. Scale bar: 10 μM. FIG. 10 a illustrates representative images of the staining and FIG. 10 b is a chart representation quantitation of the same.
    • EXAMPLE 4: Genetic Engineering of Non-Human Animal Models Encoding Distinct THSD1 Rare Variants for In-Vivo Assessment of Efficacy of Various Autophagy Inhibitors
  • Identified Conserved Sequences Suitable for Assessing Cross Species Efficacies of Treatments:
  • A protein alignment between human and zebrafish THSD1 was performed. See FIG. 11 illustrates the result of the alignment with black color indicating identical amino acids and the grey color indicating similar amino acids between humans and zebra fish. The alignment underscored that three rare variants of THSD1 identified in the aforementioned examples were conserved between human and zebrafish. The conserved amino acids include R450, R460 and T653. See, e.g., FIG. 12 . The conserved amino acids were used as a rational for generating animal models of the human condition. The following constructs were generated:
  • TABLE 5
    1 pCS2 + zf-thsd1-WT-CT2FLAG (for full rescue, positive control)
    2 pCS2 + zf-thsd1-R449X-CT2FLAG (corresponding to R450X
    in human)
    3 pCS2 + zf-thsd1-R459W-CT2FLAG (corresponding to R460W
    in human)
    4 pCS2 + zf-thsd1-T665I-CT2FLAG (corresponding to T653I
    in human)
  • Mutations R449X, R459W and T6651 were found to be conserved between human and zebrafish. Subsequently, the nucleic acid constructs encoding these sequences were introduced into zebrafish thsd1 by PCR mutagenesis. Specifically, the thsd1 variant or the WT sequence was subcloned into pCS2+ zebrafish vector for expression. These constructs were used as tools to study cerebrovascular integrity mediated by thsd1 WT and rare variants in the zebrafish animal model selected to model a human condition.
  • In-Vivo Zebrafish Model
  • To test the hypothesis that podosome modulation via SRC inhibition could treat intracranial hemorrhage in-vivo 80-120 different zebrafish animals genetically engineered as described in Table 5 were treated with three distinct SRC inhibitors, namely, PP2, SU5565, or dasatinib. Control (DMSO) or other known SRC inhibitors including PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) were added to embryo water at 1 dpf for 24 hours. All embryos were injected by thsd1-MO and the hemorrhagic incidence was counted under microscope. For each injection, around 80-120 embryos were injected. *p<0.05, **p<0.01. See FIG. 13 .
  • The data demonstrates a body of work that first identified and studied a large number of human families with a disease. After significant effort in contacting individuals and sequencing the families shown in FIG. 1A, the present disclosure first identified THSD1 as a gene associated with sub-cranial hemorrhage. In specific cases, a single codon substitution was identified as responsible for the phenotype LSF, R460W, E466G, G600E, P639L, T6531, or S775P. The inventors developed a plethora of nucleic acid constructs, cell lines, and transgenic animal models to study the condition. Subsequently, a detailed characterization of molecular pathways whose transcription was disturbed in the disease condition was used to identify potential druggable targets and pathways for treatment. One such pathway was podosome formation and function. Three distinct SRC inhibitors were tested including PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and its ability to treat subjects, particularly its ability to treat subjects susceptible to an aneurism rupture was tested by counting the hemorrhagic incidence under the microscope on an animal model. The results illustrate that podosome modulation provides a treatment for modulating the rupture of an aneurysm in vivo.
    • EXAMPLE 6: Kits
  • The disclosure contemplates kits comprising podosome modulators such as the podosome modulators selected from the group consisting of PP2 (10 uM), SU5565 (1 uM), and dasatinib (100 nM) and instructions for use of the same in the treatment of a condition disclosure herein. Specifically, the disclosure contemplates use of these drugs in treating an intracranial aneurysm.
  • It should be understood that the drawings are not necessarily to scale, and that like reference numbers refer to like features.
  • While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is understood that the present disclosure is to be considered as exemplary of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Numerous variations may be made by persons skilled in the art without departure from the spirit of the invention. The scope of the invention will be measured by the appended claims and their equivalents. The abstract and the title are not to be construed as limiting the scope of the present invention, as their purpose is to enable the appropriate authorities, as well as the general public, to quickly determine the general nature of the invention. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112, 6.

Claims (23)

1. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome formation.
2. A method to treat a subject at risk of suffering from an aneurysm comprised of administering to said subject a therapeutically effective dose of a compound that modulates podosome function.
3. The method of any one of claim 1 or 2, wherein the compound is a Src inhibitor.
4. The method of claim 3, wherein the Src inhibitor is PP2.
5. The method of any one of claim 1 or 2, wherein the compound is SU6656, KX2-391, saracatinib, bosutinib, dasatinib, or ponatinib.
6. The method of claim 5, wherein the compound is SU6656.
7. The method of claim 5, wherein the compound is KX2-391.
8. The method of claim 5, wherein the compound is saracatinib.
9. The method of claim 5, wherein the compound is bosutinib.
10. The method of claim 5, wherein the compound is dasatinib.
11. The method of claim 5, wherein the compound is ponatinib.
12. The method of any one of claims 1-5, wherein the aneurysm is an intracranial aneurysm.
13. The method of any one of claims 1-5, wherein the aneurysm is an aortic aneurysm.
14. The method of any one of claims 1-13, wherein the subject carries a variant affecting the expression of a Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
15. The method of claim 14, wherein the variant is in a coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
16. The method of claim 14, wherein the variant is in a control sequence of a non-coding region of the Thrombospondin Type 1 Domain Containing 1 (THSD1) gene.
17. The method of claim 14, wherein the variant in the THSD1 gene is a single codon substitution in at least one THSD1 allele.
18. The method of claim 14, wherein the single codon substitution in the at least one THSD1 allele is at codon LSF, R460W, E466G, G600E, P639L, T6531, S775P.
19. The method of claim 1, wherein the compound that modulates podosome formation or function is administered systemically.
20. The method of claim 19, wherein systemic administration includes, but is not limited to: (i) intravenous; (ii) intra-arterial; (iii) subcutaneous; or (iv) intraperitoneal.
21. The method of claim 1, wherein the compound that modulates podosome formation or function is administered locally.
22. The method of claim 21, wherein local administration includes, but is not limited to, (i) intracranial; (ii) intra-ocular; (iii) intra-nasal; (iv) intrathecal (v) vescular.
23. The method of claim 1, wherein the subject is a human.
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