CN115569195A - Application of plasma kallikrein in preparation of medicine for preventing and/or treating cardiovascular and cerebrovascular diseases - Google Patents

Application of plasma kallikrein in preparation of medicine for preventing and/or treating cardiovascular and cerebrovascular diseases Download PDF

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CN115569195A
CN115569195A CN202110684160.2A CN202110684160A CN115569195A CN 115569195 A CN115569195 A CN 115569195A CN 202110684160 A CN202110684160 A CN 202110684160A CN 115569195 A CN115569195 A CN 115569195A
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plasma kallikrein
ldlr
protein
thr
ser
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宋保亮
王金凯
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Wuhan University WHU
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Abstract

The invention provides a new application of Plasma Kallikrein (PK) and plasma kallikrein (PKa), wherein the Plasma Kallikrein (PK) is used for preparing a medicament for treating hyperlipidemia and cardiovascular and cerebrovascular diseases, particularly preventing and/or treating atherosclerotic cardiovascular and cerebrovascular diseases.

Description

Application of plasma kallikrein in preparation of medicine for preventing and/or treating cardiovascular and cerebrovascular diseases
Technical Field
The invention relates to the field of medicines, in particular to new application of Plasma Kallikrein (PK) and plasma kallikrein (PKa).
Background
Atherosclerotic cardio-cerebral vascular disease (ASCVD) is currently the leading cause of death in the global population (1, 2). As a chronic disease, the underlying cause of ASCVD (atheroclerosis) is a long asymptomatic progression until a sudden rupture of an Atherosclerotic plaque causes a thrombus (atheroclerotic plaque rupture-induced thrombosis) to block the normal flow of blood, causing acute cardiovascular and cerebrovascular events such as acute myocardial infarction or stroke, which severely threatens human life safety (3). Lipoprotein particles containing apolipoprotein-B (apolipoprotein B) in the blood circulation are important risk factors for inducing atherosclerosis, especially low-density lipoprotein particles (LDL-c) rich in cholesterol. When the level of LDL-c in the blood is too high, it will sink in the arterial vessel wall, with the migration of monocytes under the arterial endothelium and differentiation into macrophages that engulf these excessive lipoprotein particles, a widely accepted initiating event of atherosclerosis (4,5). Lowering LDL-c levels in the blood is an effective way to prevent and treat ASCVD. The low-density-lipoprotein receptor (LDLR) mediated endocytic pathway of LDL-c is the main way of clearing LDL-c from blood, and LDLR is highly expressed mainly in liver, an important organ for cholesterol metabolism regulation (6, 7). The currently clinically used lipid-lowering drugs, statin and Proprotein Convertase Subtilisin/kexin Type 9 (PCSK 9), achieve the purpose of lowering LDL-c by up-regulating LDLR on the cell surface (8, 9). However, in addition to being driven by LDLs, there are many factors involved in the lengthy development of ASCVD, such as inflammation or thrombosis (10-12). Atherosclerotic plaques are rich in a range of procoagulant substances such as polyphosphate, DNA and RNA from necrotic cells, collagen and tissue factor, among others. These substances are released into the blood in the event of plaque rupture, provoking the coagulation system to form a thrombus, leading to the final cardiovascular and cerebrovascular events (10).
The coagulation cascade response is initiated by two activation pathways, the extrinsic coagulation pathway initiated by tissue factor and the intrinsic coagulation initiated by the contact system, and finally merges into a common pathway (fig. 6A) (13, 14). Plasma kallikrein (PKa) is a serine protease that plays an important regulatory role in the endogenous pathway. Human PK is a peptide bond converted from plasma kallikrein (PK, abbreviated as PK, also known as Fletcher factor or plasma prekallikrein) encoded by KLKB1 gene (NM-000892.5) by cleavage of R371-L372 by activated factor 12 (FXIIa), and the amino acid sequence of Plasma Kallikrein (PK) is shown in SEQ ID NO. 1. At the same time, PKa can activate factor 12 (FXII) to produce more FXIIa, and this mutually activated form rapidly amplifies the endogenous coagulation initiation signal and produces more downstream activated factor 10 (FXa) (15, 16). In the extrinsic coagulation pathway, tissue factor (tissue factor) is exposed to blood and FX is activated by factor 7 (FVII). The activated FXa activates prothrombin (prothrombin) to generate thrombin (thrombin), which ultimately leads to the generation of a thrombus of interwoven fibrin and platelets, etc. (17).
Disclosure of Invention
The invention provides a new application of Plasma Kallikrein (PK) and plasma kallikrein (PKa), wherein the Plasma Kallikrein (PK) is used for preparing a medicament for treating hyperlipidemia and cardiovascular and cerebrovascular diseases, particularly preventing and/or treating atherosclerotic cardiovascular and cerebrovascular diseases.
According to one aspect of the present invention, there is provided use of plasma kallikrein in the preparation of a medicament for the prevention and/or treatment of cardiovascular and cerebrovascular diseases. The prevention and/or treatment of cardiovascular and cerebrovascular diseases is to reduce cholesterol levels by inhibiting or reducing the expression level of the plasma kallikrein protein, thereby reducing or blocking the interaction of PK and LDLR.
The application of the invention, wherein the amino acid sequence of the plasma kallikrein is shown in SEQ ID NO. 1.
Preferably, the inhibition or reduction of the expression level of the plasma kallikrein protein according to the invention can be selected from, but not limited to, the following methods:
1) Knock out or gene editing of the inactivated Klkb1 gene;
2) Blocking the binding of plasma kallikrein and LDLR to each other with a neutralizing antibody against plasma kallikrein or a competing polypeptide;
3) Inhibiting expression of plasma kallikrein protein using a method selected from the group consisting of gene interference, antisense nucleic acid, dCas 9-mediated gene silencing, and Cas13 a-mediated RNA cleavage;
4) Blocking the binding of plasma kallikrein and LDLR to each other and/or inhibiting the activity of plasma kallikrein with a compound that binds plasma kallikrein and/or plasma kallikrein.
The cardiovascular and cerebrovascular diseases are selected from hyperlipidemia, atherosclerotic cardiovascular and cerebrovascular diseases and thrombotic diseases. More preferably, wherein the cardiovascular and cerebrovascular disease is atherosclerotic cardiovascular and cerebrovascular disease.
According to another aspect of the present invention, there is provided an isolated neutralizing antibody that binds to plasma kallikrein protein comprising SEQ ID No.1 (amino acid sequence of human PK or PKa protein) or SEQ ID No.2 (amino acid sequence of murine PK or PKa protein), wherein the neutralizing antibody blocks the binding of plasma kallikrein to LDLR
As smaller binding units, amino acids from the 601-630 segment of PK (SEQ ID NO.3 (murine) or SEQ ID NO.4 (human)) are necessary for the interaction of PK with LDLR and for inducing degradation of LDLR, and therefore, the isolated neutralizing antigen-binding proteins of the invention also include proteins that can bind to amino acids from this segment.
The invention also provides an isolated neutralizing antibody that binds to a protein comprising the amino acid sequence set forth in SEQ ID No.3 or SEQ ID No.4, wherein the neutralizing antibody blocks the binding of plasma kallikrein to LDLR.
The antibody or the antigen binding protein can be used for preparing medicines for treating cardiovascular and cerebrovascular diseases related to high serum cholesterol level and preventing and treating diseases related to thrombosis.
The invention discovers for the first time that the blood coagulation factor plasma kallikrein binds to a low density lipoprotein receptor and promotes the degradation of the receptor through a lysosomal pathway. The expression of the plasma kallikrein protein is reduced or blocked through gene knockout or antibody neutralization, the level of a liver low-density lipoprotein receptor can be obviously increased, and the blood cholesterol is reduced. Meanwhile, in population research, the concentration of low-density lipoprotein cholesterol in blood and the protein level of plasma kallikrein exhibit close positive correlation. As a blood coagulation factor, the absence of plasma kallikrein is effective in inhibiting thrombus caused by plaque rupture without detecting the risk of bleeding and growth and development defects. In conclusion, the inhibition of plasma kallikrein can simultaneously achieve the effects of reducing blood cholesterol and resisting thrombus, and has no bleeding risk, so that the composition is a very good way for preventing and treating cardiovascular and cerebrovascular diseases.
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FIG. 1PK promotes LDLR degradation and reduces LDL endocytosis
(A) Co-immunoprecipitation experiments for PK and LDLR. The transfection experiment of liver cancer Huh7 cells was performed according to the protocol shown in the figure. And (3) specifically combining and purifying LDLR-MYC protein from cell lysate by using agarose gel coupled with an anti-MYC-label antibody, and finally verifying the interaction between PK and LDLR by using a protein immunoblotting experiment.
(B) Overexpression of the PK protein in Huh7 cells promotes LDLR degradation. Doxycycline (DOX) -inducible PK-stably transfected Huh7 cells, with or without lovastatin treatment, were collected after 24 hours of DOX-induced PK expression, lysed, and then subjected to immunoblot assay to detect LDLR, PK, and CHC.
(C) Blocking PK by the lysosomal inhibitor NH4Cl induces LDLR degradation. Doxycycline (DOX) inducible PK stably transfected Huh7 cells were treated with DOX, NH4Cl or MG-132 for 18 hours under the experimental protocol shown in the figure, followed by analysis in immunoblotting experiments.
(D) The purified PK protein can induce LDLR degradation. After 12 hours of cholesterol starvation treatment of the Huh7 cells, PK or PCSK9 protein shown in the scheme shown in the figure is respectively added into a culture medium, immunoblot experiment analysis is carried out after 8 hours of treatment, cell lysate detects LDLR and Actin, and the culture medium detects Flag tag protein.
(E) Inhibition of PK protein expression increases cellular LDLR protein levels. The Huh7 cells were transfected with sirnas targeting ido and KLKB1, respectively, and cultured for 48 hours before collection and immunoblotting.
(F-G) inhibition of PK expression promotes LDL uptake by cells. Huh7 cells were transfected with siRNA against KLKB1, after 36 hours of culture, the cells were further subjected to cholesterol starvation for 12 hours, followed by 10. Mu.g ml -1 Is incubated for one hour. Representative confocal fluorescence microscopy pictures are shown in (F), and 60 cells from each group were individually selected for fluorescence quantification (G). The data presentation mode is mean + -s.e.m. statistical differences were determined using unpaired two-tailed student t-tests.
(H-K) correlation of serum PK protein with TC (H), TG (I), LDL-c (J) and HDL-c (K) was analyzed by partial correlation analysis, and sex and age were used as control variables. (n =198, double-tailed probability) in FIG.2, the liver LDLR protein level was higher and the blood cholesterol level was lower after deletion of the Klkb1 gene from hamster, rat and mouse
(A) Schematic diagram of making a Klkb1 gene knockout hamster systemically using CRISPR/Cas9 technology.
(B-L) five month-sized male hamsters (n =7 per group) were fed High Cholesterol Diet (HCD) for three weeks and analyzed for hamster body weight (B) and daily food intake (C), expression of LDLR and CHC in different tissues (D, the lowest band is the result of genotyping PCR), total plasma cholesterol (E), plasma triglycerides (F), cholesterol (G) and triglycerides (H) content in different lipoproteins separated by flash protein liquid chromatography, total liver cholesterol (I), liver triglycerides (J), AST (K) and ALT (L) activity levels in plasma.
(M-Q) five-month old female hamsters (N =12 per group) were fed high cholesterol diet for 4 weeks and analyzed for LDLR and CHC expression (M) in the liver, total cholesterol (N) and triglyceride (O) levels in plasma, and cholesterol (P) triglyceride (Q) levels in different lipoproteins separated by flash protein liquid chromatography. Data presentation was mean ± s.e.m., statistical differences were measured using unpaired two-tailed student t-test (B, C, E, F, I-L, N, O).
FIG. 3 neutralizing antibodies against PK blocks the interaction of PK and LDLR and elevates liver LDLR protein levels and lowers plasma cholesterol levels in mice
(A) Schematic representation of a series of truncation mutants of the PK protein.
(B) Amino acids 601-630 of the PK protein are necessary for the interaction of PK and LDLR. Huh7 cells were transfected with the expression plasmids of the experimental protocols shown in the figures, cultured for 48 hours, then the cells were harvested, proteins were extracted using PBS containing 1% NP-40 as cell lysate, and co-immunoprecipitation experiments were carried out using anti-MYC agarose gel as a matrix.
(C) anti-PK antibodies block the interaction of PK and LDLR extracellular domain proteins. Mu.g of purified PK protein with Flag tag and 5. Mu.g of anti-PK protein antibody are mixed in 50. Mu.l of binding buffer solution, and then the mixture is kept still for 30 minutes on ice, and then purified 8. Mu.g of LDLR extracellular domain protein is supplemented, mixed evenly and kept still for 1 hour on ice. Then 30. Mu.l of anti-Flag Sepharose beads was added to the treated protein mixture and the mixture was tumbled and mixed at 4 ℃ for 2 hours. And finally, fully washing the gel beads after binding the protein by using a binding buffer solution, eluting the bound protein by using a Flag polypeptide solution, and analyzing by using an immunoblotting experiment.
(D) Schematic representation of the procedure for treating mice with anti-PK antibody. Ldlr of 14 weeks old +/- Mice were first fed with high cholesterol feed for 1 week, after which they were treated intraperitoneally with control IgG or anti-PK 2H5-IgG at a dose and frequency of 30mg per kg per 2 days three times in a continuous manner and after one week, they were analyzed (E-L).
(E) Immunoblot analysis experiments to detect LDLR and CHC in liver, PK in plasma.
(F) After plasma lipoproteins are separated by a rapid protein liquid chromatography technology, the content of cholesterol in different lipoproteins is detected.
(G) Plasma total cholesterol concentration.
(H) Plasma triglyceride concentration.
(I) Total cholesterol content in liver.
(J) Visceral triglyceride content.
(K) Plasma AST enzyme activity level.
(L) plasma ALT enzyme activity level.
The data presentation mode is mean + -s.e.m., the statistical difference adopts unpaired two-tailed student t-test (G-L)
FIG. 4 protective resistance to atherosclerosis after mouse Klkb1 deletion
Eight weeks old male Klkb1 +/+ Apoe -/- And Klkb1 -/- Apoe -/- Mice (n =12 per group) were fed high cholesterol diet for a period of 8 weeks. Collecting samples after mice are sacrificed, analyzing the expression conditions (A) of liver LDLR, CHC and PK by immunoblotting experiment, detecting the concentration (B) of serum total cholesterol and the sweetness of serumTriglyceride concentration (C), total cholesterol (D) and triglyceride (E) levels in the liver. The distribution of plaques was observed by lipid staining of the entire aorta, representative pictures (F) were taken and the percentage of plaque area over the entire arterial area (G) was determined, aortic root sections were stained with oil red-O, plaque size (H) was observed by photographing, and the absolute area of plaques was measured (I, n =6 per group). Data presentation was mean ± s.e.m., statistical differences were measured using unpaired two-tailed student t-test (B-E, G, I)
FIG. 5 inhibition of FeCl after PK Elimination 3 Thrombus formation induced by induction and plaque rupture
3-month-old Male Klkb1 +/+ And Klkb1 -/- Mice, under basal feed feeding conditions, were used for activated partial thromboplastin time assays (a), prothrombin time assays (B), and tail tip bleeding time assays (C).
(D-E) Male Klkb1 of 3 months size +/+ And Klkb1 -/- Mice were fed basal diet followed by surgical development of FeCl 3 Experiment for inducing carotid artery thrombosis. After isolation and exposure of mouse carotid artery, 7.5% FeCl 3 The treatment was carried out for 1 minute and the formation of thrombus was monitored under a microscope. (D) Representative FeCl 3 Fluorescence picture of induced carotid thrombus, (E) fluorescence signal quantification reflecting thrombus size (n =6 per group)
(F-I) eight week size Klkb1 +/+ Apoe -/- And Klkb1 -/- Apoe -/- Mice (n =8 groups) were fed high cholesterol diet for 16 weeks before being used for plaque rupture induced thrombosis assays. (F) Graphical representation of ultrasound-induced plaque rupture, (G) fluorescence pictures of representative thrombi as monitored by fluorescence microscopy over time after ultrasound-induced plaque rupture, (H) quantification of fluorescence signals of thrombi at different time points. The thrombus fluorescence signal was recorded every 3 seconds, and the strongest fluorescence signal of the same thrombus during the observation period was defined as 100%. (I) Quantification of absolute fluorescence signal intensity of thrombus at the indicated time points after plaque rupture. (J) PK pattern of regulation of blood cholesterol levels and thrombus formation after plaque rupture. In wild type animals, PK binds LDLR, promotingIts lysosomal pathway is degraded, thus raising blood LDL-c levels, promoting the development of atherosclerotic plaques, and if atherosclerotic plaques rupture, PK and FXII activate each other, accelerating thrombosis. When PK is knocked out, hepatic LDLR protein levels rise, accelerating LDL clearance, thus lowering blood cholesterol levels. In addition, deletion of PK slows FXII activation, inhibiting thrombosis.
FIG. 6 identification of PK binding to LDLR
(A) Cartoon illustration shows the role of PK in the coagulation pathway. The coagulation cascade is divided into three pathways: endogenous pathways, exogenous pathways and common pathways. In the endogenous pathway, like nucleic acids or other negatively charged surfaces, activate FXII, activated FXIIa activates PK to produce PKa, which in turn cleaves FXII to produce FXIIa, which rapidly amplifies the coagulation signal, ultimately producing activated coagulation factor FXa. In the extrinsic pathway, exposed tissue factor activates coagulation factor FX via coagulation factor FVII. In the common pathway, activated FXa activates prothrombin to thrombin, which then cleaves fibrinogen to form fibrin and forms a thrombus with platelets and other blood cells.
(B) Protein binding to LDLR was identified in the delipidated plasma using mass spectrometry. LDLR-Flag protein was purified from HEK293T cells and bound to anti-Flag antibody-conjugated sepharose beads, which were subsequently incubated with delipidated plasma. After extensive washing, the bound proteins on the gel beads were eluted with Flag polypeptide solution and subjected to mass spectrometry. The top ranked portion of the candidate proteins is displayed in the graph.
(C) Co-immunoprecipitation experiments of PK and LDLR. Huh7 cells were transfected according to the protocol shown in the figure. After cell lysis, co-immunoprecipitation experiments were performed using anti-Flag sepharose beads.
(D) And (3) detecting the siRNA interference efficiency by real-time quantitative fluorescence PCR. Cells were the same batch as FIG. 1E.
FIG. 7 hamster correlation features
(A) Protein tissue expression profile of male hamster LDLR.
(B) Quantitative fluorescence analysis of expression of the relevant genes in hamster liver. The samples selected were from hamsters used in the fig.2d experiments.
(C) H & E and lipid oil red-O staining of liver sections. Samples were from hamsters used in fig.2d experiments.
FIG. 8 shows LDLR +/- In mice, after PK deficiency, the protein level of liver LDLR is increased, and the blood cholesterol concentration is reduced
(A) A strategy diagram of Klkb1 full-body knockout mice was prepared.
(B-I) Male Klkb1 of 6 months size +/+ Ldlr +/- And Klkb1 -/- Lldl +/- Mice were fed high cholesterol diet for 4 weeks, (B) immunoblot analysis of LDLR, CHC and PK in the liver, (C) total cholesterol concentration in plasma, (D) determination of cholesterol content of each fraction after separation of lipoproteins in plasma by flash liquid chromatography, (E) total cholesterol level in plasma, (F) total cholesterol content and (G) triglyceride content in liver, (H) AST and (I) ALT level in plasma.
FIG. 9 reduction of blood cholesterol levels using AAV-shRNA knock-down of rat PK protein expression
8-week-old SD rats (n =5 groups) were fed with basal diet and injected with AAV-sh-control and AAV-sh-Klkb1 viruses into caudal vein at a dose of 3X 10 12 Viral genome copy number. The feed is fed for two weeks under the condition of free food and drinking water.
(A) Immunoblot experiments detected the protein levels of LDLR and CHC in the liver and PK in the blood.
(B) Quantification of the LDLR band signals in (a) and calibration of the results with the Actin band signals.
(C) The total cholesterol concentration in serum was measured.
(D) And (3) measuring the content of cholesterol in each component after separating serum lipoprotein by using the rapid liquid chromatography. FIG. 10 production of rat monoclonal antibodies to PK
(A) Schematic representation of different truncations of PK.
(B) Effect of PK different truncations on LDLR protein stability. Huh7 cells were transfected with the plasmids shown in the figure, and after 48 hours of culture, the cells were lysed for immunoblotting.
(C) A simplified schematic of the production of rat monoclonal antibodies to PK. Synthesizing a polypeptide sequence corresponding to 601-630 amino acid of PK, coupling with a KLH immunized rat to prepare a hybridoma cell strain, screening a cell strain for specifically identifying PK by using the secreted antibody, and preparing the antibody on a large scale.
(D) The rat monoclonal antibody 2H5-IgG specifically recognizes PK protein in serum by taking the serum of a wild-type and PK-deleted mouse as a sample through a protein immunoblotting experiment.
(E) Purified PK and LDLR-ECD proteins were analyzed by Coomassie blue staining. Plasmid vectors expressing full-length PK protein with Flag tag and LDLR-ECD (extracellular segment 1-788 amino acid) protein with HIS tag are respectively transfected into HEK293T cells, and the protein is purified from the culture medium by using agarose gel beads resisting Flag and nickel ion agarose beads respectively. The purified protein was analyzed for purity using Coomassie Brilliant blue.
FIG. 11 measurement of coagulation index of PK deleted hamster and rat
(A-B) 5-month-old Male Klkb1 +/+ And Klkb1 -/- Hamsters (n =9 per group) were tested for (a) activated partial thromboplastin time and (B) prothrombin time in the case of basal feed feeding.
(C-D) Male Klkb1 of 6 months old +/+ Ldlr +/- And Klkb1 -/- Lldl +/- Mice (n =8 per group) were fed with high cholesterol diet for 4 weeks and tested for (C) activated partial thromboplastin time and (D) prothrombin time.
Detailed Description
The present invention will be described in detail below by way of a description of preferred embodiments of the invention, but the present invention is not meant to be limited. All experimental materials were purchased commercially, except where otherwise noted.
1. Test materials and methods
1.1 animals
Klkb1 Whole body knockout Syria golden hamster and C57BL/6j mouse respectively Shijiazhuang Yiweiwa Biotech limited company and Nanjing Jiejicaokang Biotech limited companyThe chip is prepared by using CRISPR/Cas9 technology. Briefly, a pair of sgRNA and Cas9 proteins targeting sequences inside the 3 exon of hamster Klkb1 or on both sides of the 3 exon of mouse Klkb1 are co-injected into fertilized eggs, and the target sequences are edited to result in 83 bases deletion of the 3 exon of hamster, complete deletion of the 3 exon of mouse, and early termination of the protein encoded by the Klkb1 gene. Wild type and PK whole body deficient hamsters of 5 months old littermates were fed high cholesterol diet (dietresearch, D12109C) for 3-4 weeks and then evaluated for the effect on blood lipid levels following PK deficiency. Ldlr Whole body knockout (Ldlr) -/- ) And apolipoprotein E systemic knockout (Apoe) -/- ) The mice were purchased from Nanjing Jicui Yaokang Biotech Co. Klkb1 -/- Mouse homo-Apoe -/- Mouse and Ldlr -/- The mice were mated and Klkb1 was obtained +/+ Apoe -/- ,Klkb1 -/- Apoe -/- And Klkb1 +/+ Ldlr +/- ,Klkb1 -/- Ldlr +/- A mouse. Male Ldlr -/- Mating the mouse with wild female mouse to obtain Ldlr directly +/- A mouse.
Male Sprague-Dawley rats of 8 weeks of age were purchased from Hubei disease control center, fed normal basal diet (Dietrexearch, D10001) after randomized groups for one week, and then injected with AAV-sh-control or AAV-sh-Klkb1 virus in caudal vein at a dose of 3X 10 for each injection 12 Viral gene copy number. Rats were then fed basal diet with free access to water for 2 weeks, and finally euthanized and sampled for analysis of blood lipid levels and hepatic LDLR expression.
After all animals subjected to CRISPR/Cas9 technology gene editing obtain F1 generation males, the F1 generation males and the wild type females are backcrossed for at least two generations, and then various experiments are carried out. All animals were housed in independent ventilated cages in SPF-rated animal houses at room temperature 21-23 deg.C, humidity 50-60%,12 hours light and 12 hours dark day and night cycles. Outside of the specific experiment, all animals had free access to water. In experiments analyzing LDLR expression and blood lipid levels, animals were deprived of food during the day (8: 00-20.
All animals were kept and used in compliance with the institutional animal care and ethics committee guidelines of wuhan university.
1.2 materials
Cell culture media was purchased from Thermo (DMEM, C11995500 BT) and fetal bovine serum was purchased from Gibco (FBS, 10099141). NH4Cl (A9434), sodium mevalonate (41288), rhodamine 6G (R4217), feCl 3 (31232) Paraformaldehyde (P6148), sudan IV (198102) and the like were purchased from Sigma. Doxycycline hydrochloride (DOX, a 600889) was purchased from a foundry. Phenylmethylsulfonyl fluoride (PMSF, HY-B0496) and MG132 (HY-13259) were purchased from MCE. ALLN (208719) and pepstatin A (516481-M) were purchased from Calbiochem. Dithiothreitol (DTT, T5370) was purchased from Targetmol. Rat control serum IgG was supplied by denham biotechnology limited (wuhan). De-lipoprotein serum (LPPS, density greater than 1.215g ml) -1 ) Was obtained from our laboratory by removing lipoproteins from FBS using an ultracentrifugation method.
1.3 plasmids
Various expression plasmids are constructed according to the molecular cloning technology. Briefly, the coding sequences (excluding stop codons) of Klkb1 (NM _ 008455.3), kng1 (NM _ 001102416.3), ldlr (NM _ 010700.3), PCSK9 (NM _ 174936.4) genes were amplified from cDNA by PCR and then integrated into the expression vector pCMV-14-3 xflag vector. The pCMV-LDLR-MYC plasmid expresses LDLR protein of a mouse and fuses a 5X MYC label at the C end, the pCMV-LDLR-ECD-HIS expresses amino acids at 1-788 sites of the LDLR protein extracellular segment of the mouse and fuses a 6X HIS label at the C end, and pLVX-Tet-on-PK encodes PK protein of the full length of the mouse. Various truncated variants of PK were obtained by rapid point mutation PCR technique.
1.4 cell culture
Huh7 hepatoma cells and HEK293T fetal kidney cells were cultured in DMEM medium containing 10% fbs and supplemented with 100 units per ml penicillin and 100 micrograms per ml streptomycin (mediumA). Cells grow in a culture dish attached to a monolayer on the bottom of the dish, and are cultured in an incubator with the temperature maintained at 37 ℃ and the carbon dioxide concentration of 5.5%. The cholesterol-depleted medium was DMEM-based supplemented with 5% LPPS and with 1 micromole per liter Lovastatin (Lovastatin) and 50 micromole per liter sodium mevalonate.
1.5 Western blot assay
The treated cells or animal tissues were prepared by running protein SDS-PAGE on RIPA lysates. The formula of RIPA lysate is as follows: 50mM Tris-HCl, pH8.0, 150mM NaCl,2mM MgCl 2 1.5% NP-40,0.1% SDS, and 0.5% sodium deoxyholate, used as supplemented with a protease inhibitor, 1mM phenyl methyl sulfonyl fluoride, 10. Mu.M MG132, 10. Mu.g ml -1 leupeptin, 5μg ml -1 pepstatin,25μg ml - 1 ALLN and 1mM dithiothreitol. The protein concentration in the prepared protein lysate was determined using the BCA protein kit (Thermo). In the same set of experiments, the protein concentration of each sample was adjusted to the same level with the lysis solution, supplemented with 4 × Loading solution (150 mM Tris-HCl, pH 6.8,12% SDS,30% glycerol,6%2-mercaptoethanol and 0.02% bromophenol blue), mixed and boiled at 95 ℃ for 10 minutes. SDS-PAGE gels were run at 40. Mu.g total protein per well to separate proteins of different sizes, which were then electrophoretically transferred to PVDF membrane. The protein-bound membrane bands were incubated with the specified primary antibody at 4 ℃ for 12-16 hours after 1 hour of closed incubation in TBST solution containing 5% skim milk (25mM Tris,137mM NaCl,2.7 mM KCl,0.075% Tween-20) at room temperature, the primary antibody being formulated in TBST solution and supplemented with 5% by mass volume Bovine Serum Albumin (BSA). After washing well with TBST solution, the membranes were incubated with secondary antibodies coupled to horseradish peroxidase (HRP) formulated in TBST solution supplemented with 5% by mass skim milk for 1-2 hours at room temperature. After subsequent extensive washing of the membrane with TBST solution at least three times, visual band-visualisation recordings were made with Pierce ECL Plus Western blot substrate (Thermo). Primary antibodies used for western blotting were as follows: monoclonal antibody (M2 clonal cell line) recognizing Flag tag was purchased from Sigma (F1804, 0.5. Mu.g ml) -1 ) (ii) a Murine monoclonal antibody (9E10, 0.5. Mu.g ml) recognizing the MYC tag -1 ) Purifying from culture supernatant of hybridoma cell strain (ATCC); rabbit-derived polyclonal antibody recognizing LDLR (0.5. Mu.g ml) -1 ) Preparation of antigen Immunity from our laboratoryPurifying from serum after the blue white rabbit; goat-derived cloned antibody recognizing PK was purchased from R&D(AF2498,0.5μg ml -1 ) (ii) a Murine monoclonal antibodies recognizing CHC were purchased from BD Transduction Laboratories (610500, 0.2. Mu.g ml) -1 ) (ii) a Murine monoclonal antibody recognizing beta-Actin was purchased from Sigma (A1978, 0.1. Mu.g ml) -1 ) (ii) a Rabbit-derived polyclonal antibodies recognizing GAPDH were purchased from Proteitech (10494, 0.1. Mu.g ml) -1 ). The secondary antibodies used were as follows: HRP-coupled secondary antibody recognizing mouse IgG (115-035-003, 0.1. Mu.g ml) was purchased from Jackson Immuno Research Laboratories -1 ) And a secondary antibody recognizing IgG of rabbit (111-035-144, 0.1. Mu.g ml -1 ) (ii) a IgG secondary antibody recognizing sheep was purchased from Santa Cruz Biotechnology (sc-2354, 0.1. Mu.g ml) -1 ). The rat-derived monoclonal antibody 2H5-IgG for PK is prepared by Wuhandaian company, and the antigen polypeptide sequence is
GCARKDQPGVYTKVSEYMDWILEKTQSSDV(SEQ ID NO.3)。
1.6 identification of LDLR binding proteins in lipoprotein-depleted plasma
HEK293T cells were seeded at a density of 8000K per dish in 15cm dishes in DMEM containing 10% FBS. After the cells were fully adherent, 20. Mu.g of plasmid of pCMV-LDLR-Flag was transfected into the cells. After 24 hours of culture, the medium was removed, the cells were washed twice with PBS, and then the culture was changed to DMEM medium containing 5% LPPS. After further incubation for 24 hours, the medium was removed and the cells washed multiple times with pre-cooled PBS, 1ml of lysis buffer per dish of cells (PBS, 1% -1 leupeptin,5μg ml -1 pepstatin,25μg ml -1 ALLN) were resuspended and lysed and the cells were lysed thoroughly by aspiration and blowing 15 times through a 22G syringe. The cell lysate was centrifuged at 13200rpm for 10 minutes at 4 deg.C, the supernatant collected and incubated with protein A/G sepharose beads for 2 hours at 4 deg.C to remove non-specifically bound proteins. The cell lysate and gel beads were then separated at 2000g and the supernatant cell lysate collected and incubated with anti-Flag sepharose beads (Sigma) for 8 hours at 4 degrees celsius, allowing LDLR protein to bind to the sepharose beads. The gel beads bound to the protein were sufficiently lysedAfter washing, a Tris solution (20 mmol) at pH =8 was added -1 Tris,100mmol -1 NaCl,0.5 mmol -1 CaCl 2 pH = 8.0) and pH =5 sodium acetate solution (56 mmol) -1 sodium acetate,100 mmol -1 NaCl,0.5mmol -1 CaCl 2 pH = 5.0) were washed in two rounds (experimental group). The cells of the control group were transfected with pCDNA 3-unloaded plasmid, and the remaining operations were completely identical to those of the experimental group.
Fresh mouse plasma was collected, and the plasma was centrifuged using an ultrafiltration column (Millipore, MWCO,100,000) having a filter cut-off molecular weight of 100kDa, and a plasma filtrate from which lipoproteins were removed was collected. The plasma filtrate was then incubated with anti-Flag sepharose beads to remove non-specifically bound gel bead proteins. The treated plasma filtrate was then incubated with LDLR-bound gel beads or control gel beads overnight at 4 ℃. After washing well, the protein bound to the gel beads was washed with Flag polypeptide solution (0.5 mg ml) -1 in PBS) was eluted. Proteins in the sample are identified using mass spectrometry techniques.
1.7 Co-immunoprecipitation experiments
The treated cells were lysed with 1ml of cell lysate on ice for 20 minutes, and the lysate was collected in a 1.5ml centrifuge tube and vortexed thoroughly for 20 seconds to completely lyse the cells. The lysate was centrifuged at 13200rpm for 10 minutes at 4 degrees Celsius and the supernatant collected. Mu.l of the supernatant was added to 30. Mu.l of a 4 × loading solution (150 mmol l) -1 Tris-HCl, pH 6.8,12% SDS,30% glycerol,6%2-mercaptoethanol and 0.02% bromophenol blue) as input samples. Mu.l of the supernatant was mixed with 30. Mu.l of anti-MYC or anti-Flag agarose gel beads and incubated at 4 ℃ for 2-4 hours. After washing the gel beads of the binding protein thoroughly 5 times with cell lysis solution, they were mixed with 120. Mu.l of 1 × loading solution and boiled in a metal bath at 95 ℃ for 10 minutes, and after centrifugation, the supernatant was collected as pellet sample. Finally, 20. Mu.l of the sample was used for Western blotting.
1.8 doxycycline induces PK expression in Huh7 cells
HEK293T cells are used for packaging PK protein Tet-on induced expression type lentiviruses with blast resistance to integrate and express virus particles. Package (I)Good viral particles infected Huh7 cells for 16 hours. Then use 30. Mu.g ml -1 Screening for PK-integrated positive cells. The positive cells obtained were 3X 10 5 The inoculation density per well was inoculated into 6-well plates, and after 24 hours of incubation, 2. Mu.g ml was added -1 DOX of (1). After 24 hours of induction, cells were collected for immunoblotting experiments.
1.9 eukaryotic purified recombinant proteins
HEK293T cells at 8X 10 6 The density of each plate was inoculated into a 15 cm-diameter petri dish, and 24 hours later, 20. Mu.g of expression plasmid was transfected into cells (plasmid-encoded proteins: PK-3 XFlag, PCSK9-3 XFlag, LDLR-ECD, respectively) 1-788 -6 × HIS). After 24 hours of transfection, the medium was changed to DMEM and the culture was continued for another 24 hours. The cell culture medium was collected and centrifuged at 2000g for 10 minutes to remove cells and cell debris. For the culture supernatant containing PK-3 XFlag or PCSK9-3 XFlag protein, it was slowly drained through an anti-Flag Sepharose bead-packed column at four degrees to allow the protein to bind to the gel beads, followed by washing the packing well with PBS at an increased flow rate to remove non-specifically bound protein. Finally, the proteins bound to the gel beads were eluted by competition with 3 XFlag polypeptide in PBS. For the LDLR-ECD-containing 1-788 -6 × HIS protein, the LDLR protein was purified by first replacing the solution by centrifugation on an ultrafiltration column (50 kDa-cutt-off, millipore) with PBS containing 1 mmol-1 CaCl2, followed by nickel-ion Sepharose beads (Qiagen). All eluted proteins are removed with small molecular weight impurities such as polypeptides or imidazole using an ultrafiltration column with a molecular cut-off of 10kDa and the solution of the proteins is replaced with the solution required for the experiment. The protein concentration of the purified protein was determined by BCA method, and the purity of the protein was analyzed by Coomassie blue staining after SDS-PAGE gel electrophoresis. And (3) packaging the protein into small parts, freezing and storing at-80 ℃ to avoid repeated freezing and thawing.
1.10PK or PCSK9 proteins induce LDLR degradation
Huh7 cells at 5X 10 5 The density of each well was inoculated into a 6-well plate, and after 24 hours of culture, the medium was replaced with a medium from which cholesterol was removed, and the culture was carried out for 16 hours. After that, the cells were washed twice with PBS and the cells were added with the same contents as shown in the figureDMEM medium (containing 10. Mu.g ml) containing PK or PCSK9 protein at indicated concentrations -1 Leupeptin) at 37 ℃ for 8 hours. Cells and media were subsequently collected for immunoblotting experiments. The cells were lysed with RIPA lysate to prepare samples. After the cells or fragments of the culture medium are removed by centrifugation, 4 × loading solution is directly added in proportion, and the culture medium is boiled at 95 ℃ for 10 minutes to denature proteins.
1.11RNA interference assay
siRNA was synthesized by lebr biotechnology limited, guangzhou. The sequence information is as follows: IDOL targeted was 5 'GACTTTAGCCCAATTAATTA-3' (SEQ ID NO. 5); two targeting KLKB1 are 5 'and 5' respectively GTGTAAGTGTTTCTTAAGA-3 '(SEQ ID NO. 6) and CCCAGAAAGACTGTAAGGAA-3' (SEQ ID NO. 7); the sequence of the control siRNA was 5-. siRNA was transferred into Huh7 cells using lipofectamine RNAiMAX (Invitrogen), and after 48 hours of culture, the cells were collected and examined.
1.12 preparation of adeno-associated Virus (AAV)
Three plasmids packaging AAV (AAV-U6-shRNAvector, delta F6 helper plasmid, rev cap 2/9 vector) were transfected into HEK293T cells to generate viral particles. After 60 hours of incubation following transfection, cells were harvested, resuspended in Tris cell lysate (150mM NaCl,20mM Tris, pH 8.0), and then freeze-thawed three times repeatedly to disrupt cells and release viral particles. Free nucleic acid fragments in solution were digested away with nuclease (Sigma, E8263). The virus particles in the cell lysate are separated by using iodixanol density gradient solution (17% -25% -40% -60%) and centrifuging at the speed of 53000rpm for 160 minutes at the temperature of 14 ℃, a liquid layer with the concentration of 40% of the virus particles is collected, and then the virus particle solution is replaced by PBS by centrifuging through an ultrafiltration column intercepted by 100 KDa. And finally, quantifying the copy number of the virus vector gene by using a real-time fluorescent quantitative PCR technology.
1.13 in vitro binding assays for PK and LDLR-ECD
Mu.g of PK-Flag protein mixed with 5. Mu.g of 2H5-IgG in 50ul of binding solution (PBS, pH =7.4, 5% ethanol, 0.1% Tween-20,1mM CaCl 2 ) In (1), the mixture was left standing on ice for 30 minutes. Subsequently, 8. Mu.g L of the PK protein solution or PK/2H5-IgG mixture was addedDLR-ECD protein, mixing, and standing on ice for 1 hr. LDLR protein was added directly to the binding solution as a negative control. Next, 800ul of the binding buffer solution was added to the above system, and after mixing, a portion was left as an input sample, and the remaining solution was incubated with 30. Mu.l of Flag-resistant gel beads at 4 ℃ for 2 hours. After the agarose gel after binding the protein was washed sufficiently with the binding buffer, the bound protein was eluted with a Flag polypeptide solution. Finally, the obtained sample is analyzed by western blotting experiment.
1.14LDL endocytosis assay
A portion of the human blood sample was collected and LDL was separated by density gradient centrifugation, followed by 12ul of 6mgml -1 With 1ml of 0.5mgml of DiI dye (Invitrogen) -1 The mixed LDL of (2) was incubated at 37 ℃ for 3 hours, thereby labeling LDL. The DiI-LDL solution was filtered through a 0.45 μm filter and used. Huh-7 cells grown on glass slides were first cultured in cholesterol-deprived medium for 12 hours after siRNA transfection, and then contained 10. Mu.g ml -1 DMEM with DiI-LDL was incubated at 37 deg.C for 1 hour. After washing the cells three times with pre-cooled PBS, they were fixed with 4% PFA solution in dark for 25 min. After mounting, fluorescence signals were detected by confocal fluorescence microscopy.
1.15 detection of serum PK protein content in human population
This study was assisted by the first subsidiary hospital of the university of medical Xinjiang. All participants provided blood samples signed informed consent before the experiment progressed. These participating volunteers were all adult han university students (n =198, 86 men, 112 women, age 17-25 years). The absolute concentration of PK protein in serum was determined using an ELISA kit for human PK (Novus Biologicals).
1.16 mouse tip bleeding time determination
Mice were anesthetized with chloral hydrate intraperitoneal injection at a dose of 400mg kg -1 And then placed on a hot plate at 37 degrees celsius to maintain body temperature. The tail tip was cut into about 5mm pieces with a sharp scalpel blade, and the cut tail tip was immediately immersed in pre-heated saline at 37 deg.C (15 ml in a transparent centrifuge)Tube), observing bleeding, and recording bleeding time (stop bleeding for at least 30 seconds).
1.17 coagulation indices APTT and PT determination
The partially activated thrombin time (APTT) and Prothrombin Time (PT) were determined using the kit according to its instructions. APTT (Shanghai Jianglai, JL TC 0305) kit and PT kit (Shanghai Taiyang biology, E103).
1.18FeCl 3 Experiment on induced carotid thrombosis
3-month-old C57BL/6j Male Klkb1 +/+ And Klkb1 -/- The mice are fed with normal basal feed, and after rhodamine 6G normal saline solution is injected through tail vein (the concentration of the rhodamine 6G solution is 0.5 mgml) -1 The injection dosage is 5ulkg -1 Body weight), mice were anesthetized with chloral hydrate and mounted ventrally up on a hot plate. The skin and fascia were cut open and the carotid artery, which was hidden under the muscle and soft tissue, was isolated, taking care not to injure the blood vessel during the procedure. A small piece of dark blue rubber membrane was placed under the carotid artery to reduce background fluorescence signal interference. 7.5% of small pieces of FeCl 3 The filter paper soaked in the aqueous solution was attached to the exposed carotid artery for 1 minute, and immediately after the removal of the filter paper, the carotid artery was washed with physiological saline and observed under a fluorescence microscope to record the formation of thrombus at the damaged site.
1.19 plaque rupture-induced atherosclerotic Thrombus test
8 week old Klkb1 +/+ Apoe -/- And Klkb1 -/- Apoe -/- Mice were fed high cholesterol Diet (Research Diet, D12109C) for 16 weeks. The rhodamine 6G dye was injected subcutaneously 20 minutes before mice were anesthetized, and the exposed carotid artery was then isolated as before. Under a stereomicroscope, an ultrasonic probe having a diameter of 1mm was pushed against the side portion of the plaque, and then treated with an ultrasonic instrument (TOKIMA, ST-250) at a power of 100W for 30 seconds to rupture the plaque. Thrombogenesis was recorded by horse examination under a fluorescent microscope (Olympus CCD camera, DP-74) and recorded for at least 10 minutes. The size of the thrombus was reflected by the fluorescence signal intensity and quantified by analysis with ImageJ software.
1.20 analysis of atherosclerotic plaques
8 week old Klkb1 +/+ Apoe -/- And Klkb1 -/- Apoe -/- Mice were fed high cholesterol Diet (Research Diet, D12109C) for 8 weeks. After blood collection, the mice were euthanized by cervical dislocation. Residual blood in the body was then removed by left ventricular perfusion with approximately 20ml of saline. Liver tissues were collected, snap frozen in liquid nitrogen and stored at-80 ℃. The heart, along with the entire aorta, was carefully separated, soaked in 4% PFA fixative and allowed to stand at 4 degrees celsius for 24 hours. After stripping away some of the adherent adipose tissue and small arterial branches, the aorta was detached (aortic arch to the lumbar iliac artery branches) and cut from the side, then fixed on a black rubber plate, stained with sudan IV solution for 15 minutes for lipids, followed by rinsing with 70 ethanol solution for 3 minutes. Finally, the arteries were soaked in PBS solution and recorded for plaque staining by observation with a stereomicroscope (Olympus SZX 16). After the heart is dehydrated and precipitated in 15% and 30% sucrose solutions step by step, the heart is embedded and fixed by OCT embedding medium. After a series of 8 μm serial cryosections were obtained, sections of the same part of each heart were selected for oil red-O staining. Plaque size was quantitatively assessed using ImageJ software.
1.21 blood Collection
After animals were anesthetized with isoflurane, blood was collected from the venous plexus of the eyeball or from the heart. In determining the coagulation index, blood was collected mixed with 0.109M sodium citrate at a volume ratio of 9. After blood collection, the supernatant was collected by centrifugation at 2000g for 30 minutes at 4 ℃ to obtain plasma or serum.
1.22 Fast Protein Liquid Chromatography (FPLC)
In the same experiment, different groups of samples of the same volume were injected separately into a composite gel column (GE Healthcare, superdex 75/300 GL) and then separated using an FPLC system (AKTA, GE Healthcare). Each component sample contained 5mmol of -1 The elution separation was carried out with a PBS solution of EDTA at a rate of 0.3ml per minute, and 300. Mu.l of eluate was collected for each fraction. A40. Mu.l portion of each fraction was subjected to lipid quantification. The distribution curves of cholesterol and triglyceride are generated by analyzing the measurement results of each component by the graphPad software.
1.23 blood and liver chemistry analysis
Approximately 60mg of liver tissue was homogenized with 1.2ml of chloroform-methanol (volume ratio 2. The organic liquid in the presence of lipids was dried in a fume hood at 50 degrees celsius and the lipids were then dissolved in ethanol. In each experiment, equal volumes of plasma, serum or liver lipid extract were taken and lipid levels were determined using kits (Cola Total Cholesterol kit and triglyceride kit). AST and ALT in plasma were determined according to the kit instructions (Nanjing Okinawa, AST-C010-2-1, ALT-C009-2-1).
1.24 real-time quantitative fluorescent PCR
RNA in the tissues or cells was extracted using TriZol reagent (Invitrogen). Mu.g of RNA was mixed with 0.1. Mu.g of random primer to synthesize cDNA in a 25. Mu.l Reverse transcription system using M-MLV Reverse Transcriptase (Promega). The q-PCR reaction was formulated with the Hieff qPCR SYBR Green Master Mix (Yeasen Biotech) of an assist saint organism and the program detection assay was run in a Bio-Rad CFX96 model PCR instrument.
2. Results of the experiment
2.1PK promotes LDLR degradation
In order to search for new LDLR-affecting proteins in blood circulation, a full-length and Flag-tagged LDLR protein was expressed in HEK293T cells, and the recombinant protein was used as a bait protein to capture proteins interacting with the LDLR protein from mouse delipidated plasma. By mass spectrometry, a series of candidate interacting proteins were identified, with the PK protein encoded by the KLKB1 gene ranked top (fig. 6B). In the co-immunoprecipitation experiment, it was confirmed that PK actually interacted with LDLR in the case of using blood factor bradykinin zymogen (Kininogen 1) as a negative control and PCSK9 as a positive control (fig. 1A, 6C). In order to further explore the influence of PK on LDLR, doxycycline (DOX) is constructed to induce overexpression type PK to stably integrate and transfect a liver cancer Huh-7 cell strain, the DOX is utilized to induce high PK expression, the protein level of intracellular LDLR can be effectively reduced, and even under the condition that statin drugs promote the increase of the expression level of LDLR, the effect of PK degradation on LDLR is still obvious (figure 1B). Lysosomal inhibitor NH 4 Cl effectively inhibits PK degradation LDLR, while proteasome inhibitor MG-132 did not affect PK degradation LDLR, indicating that PK degrades LDLR via the lysosomal pathway (fig. 1C). Like PCSK9 (18), PK is predominantly expressed in the liver and then secreted into the blood (19). The purified PK protein was added to the cell culture medium and the effect of degrading cellular LDLR comparable to PCSK9 capacity was detected (figure 1D). A significant increase in LDLR protein levels was detected using small interfering RNA (siRNA) to inhibit protein expression of intracellular PK (fig. 1E). The positive control IDOL selected was ubiquitin ligase (20, 21) which induced degradation of the LDLR ubiquitination pathway. In DiI-LDL endocytosis experiments, inhibition of PK followed by elevated LDLR protein effectively promoted endocytosis of LDL, suggesting that functional activity of LDLR endocytosis of LDL was unaffected (fig. 1F and fig. 1G).
Further collection and determination of the PK protein concentration in the serum of 198 college student volunteers revealed that the PK protein concentration has a significant positive correlation with LDL-c, total Cholesterol (TC) and Triglyceride (TG), but has no significant correlation with high-density lipoprotein cholesterol (HDL-c) (FIG. 1H-K). These data indicate that PK binds to LDLR, affecting the stability of LDLR, thereby modulating LDL-c levels in the blood.
2.2 in hamster, mouse and rat, the LDLR protein level in liver was significantly increased after PK deletion and the LDL-c level in blood was significantly decreased
Syrian golden hamster is closer to human than mouse in blood lipid metabolism, and especially hamster has a blood lipid profile similar to human (22), has higher levels of LDL and Very Low Density Lipoprotein (VLDL), and is easily induced to hyperlipidemia with high cholesterol feed (23), and LDLR has high liver expression level in hamster (fig. 7A). Knock-out hamster Klkb1 gene using CRISPR/Cas9 technology (FIG. 2A), resulting in a homozygous hamster with systemic deletion of PK protein (Klkb 1) -/- ) Therefore, the influence of PK on blood lipid at an animal level is studied. Klkb1 in contrast to wild type hamster -/- Hamsters were indistinguishable in appearance, behavior, reproductive ability, weight, and food intake from wild hamsters (fig. 2b, 2c). However, klkb1 -/- The levels of LDLR protein were significantly increased in the liver and other tissues of hamster (fig. 2D), and mRNA of LDLR and cholesterol metabolism-related genes corresponding thereto was not presentSignificant changes (fig. 7B), demonstrating that PK affects LDLR by degrading LDLR protein (fig. 1). In Klkb1 -/- In hamster plasma, total Cholesterol (TC) and Triglycerides (TG) decreased by 46.3% and 43.6%, respectively (fig. 2e, f), and flash protein liquid chromatography analysis showed that the decrease in blood lipids was predominantly in the form of a decrease in LDL (fig. 2g, h). In addition, in wild type and Klkb1 -/- In other comparisons of hamsters, the liver TC and TG contents were similar (FIG. 2I, J), the blood levels of glutamic-oxaloacetic transaminase and glutamic-pyruvic transaminase were not significantly different (FIG. 2K, L), and the liver sections H&No morphological differences were observed with E staining, nor was significant lipid accumulation seen with oil red-O staining (fig. 7C), which indicates that deletion of Klkb1 did not cause liver damage. Consistent with male hamsters, female Klkb1 -/- Hamster liver LDLR protein levels were significantly elevated (FIG. 2M), TC and TG were also significantly reduced in plasma, and the reduction in blood lipids was primarily due to a reduction in LDL and VLDL levels (FIG. 2, P, Q). Taken together, PK inhibition increases hepatic LDLR protein levels, accelerating LDL-c clearance in plasma.
The low level of LDL-c in the blood of mice is different from the high level of LDL-c in the blood of human beings, probably because the level of LDLR protein in the liver of mice is higher than the background level (24, 25). Therefore, we are at Ldlr +/- Effect of PK deletion on cholesterol metabolism was studied in background mice (FIG. 8A), and the results were almost in agreement with hamster data, klkb1 -/- Ldlr +/- Mouse homo-Klkb 1 +/+ Ldlr +/- In comparison to mice, hepatic LDLR protein levels were significantly increased (fig. 8B), cholesterol levels in plasma were decreased by 42% (fig. 8C), and were mainly contributed by decreased LDL-C levels (fig. 8D), while TG levels in blood were slightly decreased and TC and TG levels in liver were increased (fig. 8, e-G), indicating that high levels of LDLR in liver absorbed more cholesterol in blood circulation into hepatocytes after PK deletion. In this experiment, no difference in the enzymatic activities of AST and ALT, markers representative of liver damage, was observed in the blood of two groups of mice (FIG. 8, H, I).
We also used adeno-associated virus as a vector to deliver shRNA into rats to inhibit PK protein expression. Compared with the control group AAV-sh-control, the AAV-sh-Klkb1 serum PK protein level is obviously reduced, the liver LDLR protein level is obviously increased (figure 9, A and B), the TC level in the serum is reduced by about 21 percent (figure 9C), and FPLC analysis shows that the blood fat is reduced mainly due to the reduction of LDL-C (figure 9D).
2.3 neutralizing antibody against PK treatment of mice, the LDLR protein level of mice liver was significantly increased and the blood cholesterol level was significantly decreased
Using molecular cloning techniques and experiments in cellular molecular biology, the stability effect of different truncations of PK on LDLR was studied and attempts were made to find key segments of PK mediating the interaction between the two. Full-length forms of PK (FL), truncation B (Δ 20-390) and d (Δ 20-390 and 631-638) showed strong interaction with LDLR and strong reduction of LDLR protein levels in co-immunoprecipitation experiments (fig. 3, a, B), indicating that these truncations contain the critical segment of PK mediated LDLR degradation. In contrast, the PK truncates a (Δ 391-638) and c (Δ 21-390 &601-638) did not show interaction with LDLR and were unable to induce LDLR degradation (FIG. 3B). Removal of the PK segment 601-638 completely abolished the activity of PK in degrading LDLR protein, however, PK did not affect its LDLR degrading activity after deletion of 631-638 segment (fig. 10, a, b). Taken together, it can be concluded that the amino acids of the 601-630 segment of PK (SEQ ID No. 4) are essential for the interaction of PK and LDLR and for inducing LDLR degradation.
A polypeptide corresponding to amino acids 601 to 630 of PK (SEQ ID NO. 3) was synthesized, and after coupling to KLH, rats were immunized to obtain rat monoclonal antibodies (2H 5-IgG) against sections 601 to 630 of PK (FIG. 10C). Immunoblot experiments showed that 2H5-IgG specifically recognized PK protein in serum (fig. 10D) and was able to effectively block the interaction of LDLR extracellular domain protein LDLR-ECD with PK in vitro binding experiments (fig. 3C, fig. 10E). Subsequently, it was investigated at the mouse level whether the neutralizing antibody 2H5-IgG to PK had the effect of lowering blood cholesterol. After one week of feeding the mice with the high cholesterol diet, the mice were injected intraperitoneally with either the control antibody or the PK neutralizing antibody every two days, each injection being measured at 30mg/kg, and the assay was performed one week later (fig. 3D). The 2H5-IgG treated group showed a significant increase in mouse liver LDLR protein levels compared to the control group (FIG. 3E), with a 32% decrease in blood TC, mainly due to the contribution of LDL-c decrease (FIG. 3, F, G). No changes were detected in plasma TG levels (FIG. 3H), and TC and TG levels in the liver tended to rise, but there were no statistical differences (FIG. 3, I, J). There were no elevations in AST and ALT levels in plasma representing hepatocyte injury following 2H5-IgG treatment (FIG. 3, K, L). The above results indicate that blocking PK and LDLR interactions with neutralizing antibodies can increase hepatic LDLR protein levels, promote LDL-c clearance, and thereby reduce blood cholesterol levels.
2.4PK deletion protected inhibition of Atherosclerosis progression
To explore the effect on the development of atherosclerosis following PK deficiency. We compared PK and apolipoproteinE double knockout mice (Klkb 1 kb) -/- Apoe -/- ) And control group apolipoproteinE single knockout mice (Klkb 1) +/+ Apoe -/- ) Plaque development. After feeding high-cholesterol feed for 8 weeks, the same control group Klkb1 +/+ Apoe -/- Compared with mice, the experimental group Klkb1 -/- Apoe -/- The level of LDLR protein in the mouse liver is increased, and the level of cholesterol in the blood is obviously reduced. Blood TG levels were not significantly different in the two groups (fig. 4, a, b and C), with TC and TG in the liver being slightly elevated in the experimental group (fig. 4, d, e). On the basis of a decrease in blood cholesterol, klkb1 -/- Apoe -/- The whole arterial plaque area and aortic root plaque area of the mice are relative to the litter group Klkb1 +/+ Apoe -/- Mice decreased 50% and 40%, respectively (FIG. 4, F-I). Indicating that inhibition of PK can effectively block the development of atherosclerosis.
2.5PK deficiency inhibits thrombosis
PK regulates thrombosis by amplifying activation of the endogenous coagulation pathway initiation factor FXII (fig. 6A). In the test of blood coagulation function, the experimental group Klkb1 -/- Mouse and littermate control group Klkb1 +/+ In comparison, the index reflecting the intrinsic coagulation pathway Activated Partial Thromboplastin Time (APTT) was significantly prolonged in mice (fig. 5A). The Prothrombin Time (PT) and the tail tip bleeding time, which are indexes reflecting extrinsic coagulation pathways, were found in two groups of miceThere was no significant difference in (fig. 5b, c). This is also consistent with previous reports that the contact system in which the PK is located is not important for hemostasis (26). Subsequently, we also verified PK in FeCl 3 Effect in induced carotid thrombosis, control group Klkb1 +/+ Mouse carotid artery in FeCl 3 After the treatment, thrombus blocking the blood vessel was formed in about 6 minutes, and the experimental group mice almost completely inhibited the thrombus formation blocking the blood vessel in the observation process of 20 minutes (FIG. 5D), which was consistent with the previous study report (27-30). We also performed in Klkb1 -/- Hamster and Klkb1 -/- Ldlr +/- A significant prolongation of APTT was detected in mice, whereas PT was unchanged (FIG. 11, A-D).
Despite FeCl 3 The induced thrombus model is widely researched and applied, but the thrombus induced by the artificially-caused vascular injury is greatly different from the thrombus formed by plaque rupture under pathological conditions. Plaque rupture and formation of a common plaque thrombus (31) generally occurs under the induction of vigorous physical activity or hypertension. We fed Klkb1 +/+ Apoe -/- Mouse and Klkb1 -/- Apoe -/- The mice are fed with high-cholesterol feed for 16 weeks, so that atherosclerotic plaques are fully developed, and the difference of the sizes of carotid plaques of the two groups of mice is reduced. The plaque was then ruptured by ultrasound treatment and thrombus formation was monitored by fluorescence microscopy (32, 33) (FIG. 5F). We found that the control group Klkb1 was found to be comparable in carotid plaque size in two groups of mice +/+ Apoe -/- The carotid plaque of the mice reached maximal extent at around 60 seconds after rupture, and remained maximal after rupture, with no decay over the observation period (fig. 5G). While the experimental group Klkb1 -/- Apoe -/- The plaque thrombus reached a maximum in about 60 seconds in the mouse, and the intensity of the fluorescence signal representing the size of the thrombus was not significantly different from that of the control group at this time, but the signal rapidly decayed after the thrombus reached the maximum value (FIG. 5, G-I). This indicates that after sonication the plaque ruptures and platelets first activate to form a thrombus at the site of injury, followed by activation of thrombin by the coagulation pathway amplified by FXII initiation, producing fibrin to stabilize the thrombus. Thus, in two groupsIn mice, the initial thrombus formation after sonication did not differ during the first phase of platelet activation diffusion, but PK deficiency had a significant effect on the second phase of thrombus growth and stabilization. Taken together, these results indicate that PK inhibition is a good antithrombotic strategy and there is no bleeding risk.
Our experimental results show that elimination of PK promotes clearance of LDL, thereby resisting the development of atherosclerosis while inhibiting the formation of arterial plaque thrombus. PK in blood circulation binds to LDLR, promoting its lysosomal pathway to degrade rather than to recirculate to the cell surface, thus increasing LDL-c levels in the blood and accelerating plaque development. Once the lipid rich plaque is ruptured, the thrombus can be provoked. After PK activation amplifies FXII initiated coagulation signals, larger, more stable thrombi will form, blocking the vessels causing serious cardiovascular events. Low levels of LDL-c prevent the development of atherosclerotic plaques in the absence of PK, and are less prone to thrombosis in the case of plaque rupture. Thus, PK may be a very desirable target for treatment of cardiovascular and cerebrovascular diseases (fig. 5J).
3. Discussion of the related Art
In this study, we found two new functions of coagulation factor PK: induce LDLR degradation and have important effects in the process of inducing thrombosis by atherosclerotic plaque rupture. By gene knockout or PK inhibition through antibody neutralization, the increase of the LDLR level of the liver and the reduction of the blood fat level are detected in various animal models. The PK neutralizing antibody has similar effect with that of the PCSK9 neutralizing antibody which is a hypolipidemic drug which is already put into clinical use at present. In addition to the hypolipidemic effect, PK inhibition was effective in avoiding plaque rupture inducing large and firm thrombi without bleeding risk (figure 5). PK regulates both lipoprotein metabolism and thrombosis, which also demonstrates the precise intrinsic relationship between these two disparate systems. In line with this, previous studies have reported that high levels of blood LDL or blood cholesterol promote platelet aggregation and the progression of the clotting cascade (34).
There is countless evidence that high levels of LDL-c in the blood accelerate the development of atherosclerosis, and that lowering blood cholesterol is an effective treatment and prevention of atherosclerosis (7). There are a number of drugs that regulate blood cholesterol levels by different routes. For example, statins inhibit cholesterol synthesis and increase LDLR expression levels. Ezetimibe inhibits cholesterol absorption in the small intestine, while PCSK9 inhibitors directly raise LDLR protein levels to promote LDL-c clearance from the blood. However, merely controlling cholesterol levels is far from adequate for atherosclerotic cardiovascular and cerebrovascular diseases. In addition to cholesterol, there are many other risk factors that play a major role in the development of cardiovascular and cerebrovascular diseases, such as clonal inflammation, hematopoiesis, hypertension, air pollution, sleep disorders, and the like (11, 35). After myocardial infarction patients have clinical symptoms for the first time, even if cholesterol is controlled at a good level, at least 20 percent of patients still have recurrent myocardial infarction (36) within 3 years, which indicates that in addition to reducing blood fat, other methods are urgently needed to further reduce the death rate of cardiovascular and cerebrovascular diseases or diseases related to the cardiovascular and cerebrovascular diseases.
Atherosclerotic plaque is a chronic, progressive process. Plaque begins with phagocytosis of excess cholesterol by monocytes or macrophages invaginated into the vessel wall (37). At the same time, the plaque continues to undergo small lesions such as plaque rupture or plaque erosion, which subsequently provoke thrombi and repair the damaged site, a process that is generally clinically asymptomatic, but this small-lesion repair cycle accelerates the plaque development process and narrows the vessel more. Eventually this large plaque ruptures causing a vessel blockage, resulting in a catastrophic cardiovascular event. Thus, antithrombotic agents may not only play a role in acute cardiovascular events, but may delay the development of atherosclerotic plaques (38, 39).
Unfortunately, current anti-thrombotic drugs used clinically, including anti-platelet drugs or anti-coagulation factor drugs, have significant hemorrhagic side effects (13). Aspirin and P2Y12 receptor inhibitors, such as clopidogrel, are antiplatelet drugs that have been used for many years in the treatment of cardiovascular and cerebrovascular diseases. Although they are effective in reducing cardiovascular and cerebrovascular events, many people have bleeding problems or are not sensitive to aspirin. Anticoagulant drugs such as thrombin inhibitors or coagulation factor FX inhibitors, while reducing the incidence of atherosclerotic cardiovascular and cerebrovascular disease, also pose a serious bleeding risk and are ultimately discontinued. The bleeding risk of the current antithrombotic drugs greatly limits the application of the antithrombotic drugs in atherosclerotic cardiovascular and cerebrovascular diseases.
Current research suggests that the extrinsic coagulation pathway is a rapid process and plays a major role in hemostasis, while the intrinsic coagulation pathway responds relatively slowly and its contact system is not critical for hemostasis. Recently emerging inhibitors of antisense strand RNA and neutralizing antibodies against FXI or FXII show a strong potential for treating thrombi without significant risk of bleeding (14). PK is upstream of the intrinsic coagulation pathway, and participates in the coagulation process by activating coagulation factor FXII, similar to coagulation factors FXI or FXII, and we and other studies have revealed that it is not essential for hemostasis (27, 28). Indeed, the phenotype of PK deficiency in the human population was first discovered to show a significant prolongation of APTT, but no haemostatic disorders (40). Further PK deletion cases in the population reported subsequently did not show bleeding risk (41). Whales, during evolution, lose PK proteins to reduce the risk of thrombus induction by ascending after deep submergence (42). All of these information indicate significant inhibition of thrombus without affecting hemostasis after PK deletion, and reduction of PK is safe. The targeted inhibition of PK can achieve the beneficial effect of reducing the blood cholesterol level by increasing the LDLR level besides resisting thrombus. In summary, our studies suggest that PK inhibition is a novel approach for the treatment of hyperlipidemia, atherosclerotic cardiovascular and cerebrovascular diseases, and other thrombotic disorders.
Sequences referred to in the specification:
SEQ ID NO.1: amino acid sequence of human PK or PKa protein:
MILFKQATYFISLFATVSCGCLTQLYENAFFRGGDVASMYTPNAQYCQMRCTF HPRCLLFSFLPASSINDMEKRFGCFLK
DSVTGTLPKVHRTGAVSGHSLKQCGHQISACHRDIYKGVDMRGVNFNVSKV SSVEECQKRCTNNIRCQFFSYATQTFHKA
EYRNNCLLKYSPGGTPTAIKVLSNVESGFSLKPCALSEIGCHMNIFQHLAFSD VDVARVLTPDAFVCRTICTYHPNCLFF
TFYTNVWKIESQRNVCLLKTSESGTPSSSTPQENTISGYSLLTCKRTLPEPCHS KIYPGVDFGGEELNVTFVKGVNVCQE
TCTKMIRCQFFTYSLLPEDCKEEKCKCFLRLSMDGSPTRIAYGTQGSSGYSLR LCNTGDNSVCTTKTSTRIVGGTNSSWG
EWPWQVSLQVKLTAQRHLCGGSLIGHQWVLTAAHCFDGLPLQDVWRIYSGI LNLSDITKDTPFSQIKEIIIHQNYKVSEG
NHDIALIKLQAPLNYTEFQKPICLPSKGDTSTIYTNCWVTGWGFSKEKGEIQNI LQKVNIPLVTNEECQKRYQDYKITQR
MVCAGYKEGGKDACKGDSGGPLVCKHNGMWRLVGITSWGEGCARREQPG VYTKVAEYMDWILEKTQSSDGKAQMQSPA
SEQ ID NO.2: murine PK or PKa protein amino acid sequence:
MILFNRVGYFVSLFATVSCGCMTQLYKNTFFRGGDLAAIYTPDAQYCQKMCT FHPRCLLFSFLAVTPPKETNKRFGCFMK
ESITGTLPRIHRTGAISGHSLKQCGHQISACHRDIYKGLDMRGSNFNISKTDNI EECQKLCTNNFHCQFFTYATSAFYRP
EYRKKCLLKHSASGTPTSIKSADNLVSGFSLKSCALSEIGCPMDIFQHSAFADL NVSQVITPDAFVCRTICTFHPNCLFF
TFYTNEWETESQRNVCFLKTSKSGRPSPPIPQENAISGYSLLTCRKTRPEPCHS KIYSGVDFEGEELNVTFVQGADVCQE
TCTKTIRCQFFIYSLLPQDCKEEGCKCSLRLSTDGSPTRITYGMQGSSGYSLRL CKLVDSPDCTTKINARIVGGTNASLG
EWPWQVSLQVKLVSQTHLCGGSIIGRQWVLTAAHCFDGIPYPDVWRIYGGIL SLSEITKETPSSRIKELIIHQEYKVSEG
NYDIALIKLQTPLNYTEFQKPICLPSKADTNTIYTNCWVTGWGYTKEQGETQ NILQKATIPLVPNEECQKKYRDYVINKQ
MICAGYKEGGTDACKGDSGGPLVCKHSGRWQLVGITSWGEGCARKDQPGV YTKVSEYMDWILEKTQSSDVRALETSSA
SEQ ID NO.3: GCARKDQPGVYTKVSEYMDWILEKTQSDV (murine, antigen sequence for antibody preparation)
SEQ ID NO.4
GCARREQPGVYTKVAEYMDWILEKTQSDG (human, the sequence of the human and the sequence of the murine antigen SEQ ID NO.3 have high homology)
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SEQUENCE LISTING
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<120> application of plasma kallikrein in preparing medicine for preventing and/or treating cardiovascular and cerebrovascular diseases
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Glu Trp Pro Trp Gln Val Ser Leu Gln Val Lys Leu Val Ser Gln Thr
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Claims (9)

1. The application of the plasma kallikrein in preparing medicine for preventing and/or treating cardiac and cerebral vascular diseases.
2. The use according to claim 1, wherein the expression level of the plasma kallikrein protein and/or the plasma kallikrein activity is inhibited or decreased, or
By blocking the binding of the plasma kallikrein protein to the Low Density Lipoprotein Receptor (LDLR).
3. The use of claim 1, wherein the amino acid sequence of plasma kallikrein is shown in SEQ ID No. 1.
4. The use of claim 2, wherein the inhibiting or reducing the expression level of plasma kallikrein protein and/or plasma kallikrein activity, or by blocking the binding of the plasma kallikrein protein to a low density lipoprotein receptor is selected from the group consisting of:
1) Knock out or gene editing of the inactivated Klkb1 gene;
2) Blocking the binding of plasma kallikrein and LDLR to each other with a neutralizing antibody against plasma kallikrein or a competing polypeptide;
3) Inhibiting expression of plasma kallikrein protein using a method selected from the group consisting of gene interference, antisense nucleic acid, dCas 9-mediated gene silencing, and Cas13 a-mediated RNA cleavage;
4) Blocking the binding of plasma kallikrein and LDLR to each other and/or inhibiting the activity of plasma kallikrein with a compound that binds to plasma kallikrein and/or plasma kallikrein.
5. The use according to claim 1, wherein the cardiovascular and cerebrovascular diseases are selected from hyperlipidemia, atherosclerotic cardiovascular and cerebrovascular diseases, and thrombotic diseases.
6. An isolated neutralizing antibody that binds to plasma kallikrein protein comprising SEQ ID No.1 or SEQ ID No.2, wherein the neutralizing antibody blocks the binding of plasma kallikrein to LDLR.
7. An isolated neutralizing antibody that binds to a protein comprising the amino acid sequence set forth in SEQ ID No.3 or SEQ ID No.4, wherein the neutralizing antibody blocks the binding of plasma kallikrein to LDLR.
8. Use of the antibody of claim 6 or 7 for the manufacture of a medicament for the prevention and/or treatment of cardiovascular and cerebrovascular diseases.
9. Use of the antibody of claim 6 or 7 in the manufacture of a medicament for reducing blood cholesterol and/or thrombosis.
CN202110684160.2A 2021-06-21 2021-06-21 Application of plasma kallikrein in preparation of medicine for preventing and/or treating cardiovascular and cerebrovascular diseases Pending CN115569195A (en)

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