AU2020100423A4 - Angiogenesis agonist polypeptide and application thereof - Google Patents

Abstract

The present invention relates to the field of medicine, and specifically relates to an angiogenesis agonist polypeptide, a polypeptide that can treat ischemic disease. In particular, a sequence of the polypeptide is a new sequence, having an effect of promoting angiogenesis. It is used for treatment of ischemic stroke. The beneficial effect of the present invention is that the sequence of the present invention is a novel small-molecule polypeptide truncated from GPR124 cell membrane surface receptor sequence protein and modified to specifically link integrin receptor with angiogenesis activity. The sequence of the polypeptide can specifically bind to integrin and has the ability to regulate cells in adhesion and migration during angiogenesis, thereby playing a role in the treatment of ischemic diseases.

Description

ANGIOGENESIS AGONIST POLYPEPTIDE AND APPLICATION THEREOF

TECHNICAL FIELD The present invention relates to the field of medicine, and specifically relates to an angiogenesis agonist polypeptide for treating cardiovascular and cerebrovascular diseases.

BACKGROUND Stroke is a vascular-related disease of the central nervous system (CNS), which has the characteristics of high incidence rate and high disability rate. Stroke is divided into ischemic stroke and hemorrhagic stroke. Ischemic stroke, also known as cerebral infarction, is the most common in cerebrovascular diseases, accounting for about 75%, with an average mortality rate of 10% to 15% and an extremely high disability rate. At present, strategies for clinical intervention of ischemic stroke are very limited. Anticoagulant in the therapeutic window of an acute period has a cure rate of only 5%; and neuroprotective drugs for stroke treatment have failed during the preclinical research. Studies have reported that angiogenesis can reduce brain injury after ischemic stroke in a variety of approaches. Firstly, new vessels are beneficial to the formation of new collateral circulation, improvement of tissue perfusion around the ischemic region, and promotion oxygen and nutrients into the tissue; secondly, growth factors, such as fibroblast growth factor (FGF2), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF) and other neurotrophic factors, can promote the survival of endothelial cells, glial and nerve cells in brain injury areas, and inhibit neuronal apoptosis; thirdly, new vessels are involved in removing injured tissue; and finally, the pro-angiogenic microenvironment can provide a "vascular niche" for neural stem cell reform and migration. Therefore, pro-angiogenesis can be benefit for recovery after ischemic stroke, which is an important approach for early treatment of ischemic stroke. G protein-coupled receptor 124 (GPR124), as a novel cell membrane surface receptor, is discovered in 2001. Initially, GPR124 was identified as a tumor endothelial marker (TEM), so it is also known as TEM5. GPR124 has been identified as an adhesion-type G protein-coupled receptor because of its long extracellular region and GPCR proteolytic site (GPS) of 7 transmembrane regions. In recent years, in vivo research has found that GPR124 plays a vital role in regulating CNS angiogenesis. Firstly, GPR124 is specifically expressed in CNS endothelial cells and pericytes, and this characteristic indicates that GPR124 has CNS vascular specificity; secondly, GPR124 promotes specific CNS angiogenesis by regulating sprouting angiogenesis and cell migration, indicating that GPR124 not only has the effect of angiogenesis, but also has effects on the formation of mature vessels in the late stage of angiogenesis; and thirdly, GPR124 promotes the establishment of the blood-brain barrier. Therefore, GPR124 is a novel endothelial cell receptor that specifically mediates CNS angiogenesis and can be a new target for the treatment of CNS vascular-related diseases. Research finds that GPR124 can act on angiogenesis: soluble TEM5 (sTEM5) is proteolysised from GPR124 by proteases on extracellular matrix (ECM), such as thrombin, matrix metalloproteinase 2 (MMP2), and matrix metalloproteinase 9 (MMP9). It specifically binds to integrin avp3, regulates cell adhesion and migration during angiogenesis, and differentiates endothelial cells into tubules. At the same time, proteolytically processed sTEM5 promotes endothelial cells migration and adhesion to pericyte, and form specific CNS vessels. However, currently, there is no report that sTEM5 is used in the treatment of ischemic stroke. Further sTEM5 with a large molecular weight, does not easily enter the blood-brain barrier, and cannot play a therapeutic role in the brain. Therefore, it is desired to find a polypeptide that has the function of sTEM5 promoting angiogenesis and has a small molecular weight, which can be used to treat CNS vascular-related diseases, especially, ischemic stroke.

SUMMARY OF THE INVENTION The objective of the present invention is to design an angiogenesis agonist polypeptide, which has an effect of promoting angiogenesis and can be used to treat ischemic stroke. It has a small molecular weight and high specificity. The technical solution of the present invention is to provide an angiogenesis agonist polypeptide with a sequence of FRWPRKGD which is a new sequence. It has an effect of promoting angiogenesis and is used for ischemic stroke treatment. The polypeptide is prepared by chemical synthesis. The principle of the present invention is that: different functional regions of GPR124 cell membrane surface receptor have been analyzed; according to different functional regions, a plurality of polypeptide sequences are preliminarily truncated from the GPR124 sequence protein and further modified to make sure that polypeptide sequences can specifically bind to the functional integrin receptor; then angiogenesis experiments are conducted and novel small-molecule peptides with pro-angiogenesis activity are identified; and finally, in vivo animal disease model tests are performed to verify the role of the polypeptide of the present invention in promoting angiogenesis and treating ischemic diseases, especially stroke.

Beneficial effect: The beneficial effect of the present invention is that, a sequence FRWPRKGD of the present invention is an angiogenesis agonist polypeptide, which is a new small-molecule polypeptide with pro-angiogenesis activity, obtained by truncated from GPR124 cell membrane surface receptor sequence protein and modified to specifically link integrin receptor. The present application has verified the function of the polypeptide in the present application by various experiments, and the results show that: the angiogenesis agonist polypeptide of the present application can specifically bind to integrin to promote human vascular endothelial cell migration with the cell migration rate of up to 63.47%; not affect on HUVEC cell proliferation, that is, no cytotoxicity; can promote formation of tube-like structures; and can promote angiogenesis in rat arterial rings. The results of in vivo experiments prove that, when a dose of the polypeptide of the present application is 5 to 40 mg/Kg, it can significantly improve the neurobehavioral scores and reduce the area of cerebral infarction in middle cerebral artery occlusion (MCAO) model rats. That is, the polypeptide of the present application has a significant therapeutic effect on ischemic stroke, and can be used as an effective candidate drug for ischemic stroke.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the mass spectrometry results of an angiogenesis agonist polypeptide. FIG. 2 shows a high performance liquid chromatography chromatogram result of an angiogenesis agonist polypeptide. FIG. 3 shows a molecular docking simulation diagram of an angiogenesis agonist polypeptide, wherein (A) shows a molecular docking diagram, inside the white box is the angiogenesis agonist polypeptide; (B) shows a molecular docking active site diagram, indicating amino acids residues that bind the receptor molecule to the angiogenesis agonist polypeptide.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Embodiment 1 Chemical synthesis of polypeptide The polypeptide was synthesized by solid-phase chemical synthesis (synthesized by Karebay Biochem), and purified by high-performance liquid chromatography. Mass spectrometry was used, as shown in FIG. 1, its amino acid sequence is SEQ ID NO: 1. RP-HPLC was used to measure purity of the polypeptide. The results show that the purity of the polypeptide obtained in Embodiment 1 is 95.12%, which meets the experiment requirements. The results of the chromatogram are shown in FIG. 2. Embodiment 2 Result of docking of polypeptide with receptor molecule The molecular docking function module of molecular docking software was used to evaluate the degree of docking of polypeptide with integrin receptor molecules. The method includes: (1) receptor preparation: the integrin receptor with the PDB number (PDB: 1L5G) was extracted from Brookheaven protein crystal structure database (http://www.rcsb.org/pdb/); (2) ligand preparation: the amino acid sequence of SEQ ID NO: 1 was used as a ligand structure; and (3) molecular docking: selected the C-score option, ran docking, and performed scoring function. The evaluation and scoring results show that the total-score value of the polypeptide of Embodiment 1 binding to the specific receptor is 10.2526, which meets the inclusion criteria of the specific binding (The total-score value is greater than 4). Therefore, the polypeptide of Embodiment 1 can specifically bind to the target receptor. The docking simulation diagram is shown in FIG. 3.

Embodiment 3 A scratch wound healing assay was used to evaluate the effect of the polypeptide on the migration of vascular endothelial cells. First, a marker pen was used to draw horizontal lines evenly on the back of a 24-well plate by using a ruler, at intervals of about 0.5 cm, crossing the wells. Each well was crossed by three lines. HUVEC cells in logarithm growth phase were added to a 24-well culture plate at 2.0x10 5 cells/well and cultured for 24 hour. On the next day, a 10 tl pipette was used to make wounds perpendicular to the horizontal line at the back by using a ruler, and the intersection of the wound and the horizontal line at the back was used as a fixed observation point. The experimental wells and positive control wells were respectively added with different concentrations of the angiogenesis agonist polypeptide and positive control polypeptide stem. The blank group was added with the same volume of vehicle. Five duplicate wells were set for each group. All wells were put into a 37°C, 5% CO 2 incubator for culturing. They were imaged at 0, 12, 24 hours. Wound distances were measured at 0, 12, 24 hours. At different time points, the change in wound distance at fixed positions per well was recorded, which is the cell migration distance. Migration distance was calculated according to the following equation: migration distance = wound distance at 0 th hour of experiment - wound distance at nth hour of experiment, and migration rate % (MR) = (migration distance of experimental group - migration distance of blank group) x 100% / migration distance of blank group. T-test was used to compare the differences in migration distance between the experimental group and the blank group, where p< 0 .0 5 indicated a significant difference, and p< 0 .0 1 indicated an extremely significant difference. Table 1 Effect of polypeptide of Embodiment 1 on human vascular endothelial cell migration

Migration distance Migration rate(%) Medicine Dose 1 2 Ihhour 2 4 Ihhour 1 2 Ihhour 2 4 Ihhour

Blank group - 249.07±116.67 446.89±121.96 --

Positive 40 sTEM5 394.13±114.00* 608.22±151.33* 58.24 36.10 control group pg/ml

Polypeptide of 5 tg/m 334.68±108.72 586.02±80.93* 34.37 31.13 Embodiment 1

Polypeptide of 10 339.24±80.28 580.50±165.46 36.20 29.90 Experimental Embodiment1 [pg/ml

group Polypeptide of 20 316.24±121.11 530.18±209.50 26.97 18.64 Embodiment [pg/ml

Polypeptide of 40 407.17±135.89* 647.02±185.47* 63.47 44.78 Embodiment [tg/ml

*p<0.05, ** p<0.01 compared with the blank group The results are shown in Table 1. The angiogenesis agonist polypeptide can promote the migration of human vascular endothelial cells. The migration rate at 40tg/ml at 12 hours is the highest, which is 63.47%. Embodiment 4 Effect of the angiogenesis agonist polypeptide on human vascular endothelial cell proliferation MTT assay was used to determine the inhibitory effect of the angiogenesis agonist polypeptide on human vascular endothelial cell proliferation, and to evaluate the effect of polypeptide on human vascular endothelial cell toxicity. HUVEC cells in logarithmic growth phase were added to a 96-well culture plate at 8.0x10 4 cells/well and cultured for 24 hours. The experimental wells and positive control wells were respectively added with different concentrations of the angiogenesis agonist polypeptide and positive control polypeptide sTEM5. The blank group was added with the same volume of vehicle. Five duplicate wells were set for each group. After 48 hour- culture, MTT was added to each well. After 4 hours, DMSO was added and incubation was performed for 10 minutes. The absorbance (A) value was measured at 570nm on a microplate reader, and cellular proliferation inhibition rate = (1 - absorbance of experimental group / absorbance of control group) x 100%. The results are shown in Table 2. The angiogenesis agonist polypeptide has no cell proliferation inhibition effect on HUVEC cells, that is, has no cytotoxicity. Table 2 Effect of polypeptide of Embodiment 1 on human vascular endothelial cell proliferationinhibition

Results

Medicine Dose Absorbance Proliferation inhibition

(A) rate

% Blank group - 0.782±0.083

Positive control 40 sTEM5 0.756±0.083 3.68 group pg/ml

Polypeptide of Embodiment 5 tg/ml 0.719±0.035 7.99 1

Polypeptide of Embodiment 10 0.758±0.034 3.01 Experimental 1 pg/ml

group Polypeptide of Embodiment 20 0.787±0.019 -0.70 1 pg/ml

Polypeptide of Embodiment 40 0.755±0.053 3.43 1 [tg/ml

*p<0.05, **p<0.01 compared with the blank group Embodiment 5 Effect of the angiogenesis agonist polypeptide on the tube formation of vascular endothelial cells The vascular endothelial cell tube formation test was used to evaluate the effect of polypeptide on the formation of tube-like structures of vascular endothelial cells. 10 mg/ml Matrigel was added to a 96-well plate, 40 tl per well, and polymerized in a 37°C incubator for 1 hour. HUVEC cells in logarithmic growth phase were added to a culture plate at 4000 cells/100tl/well. At the same time, the experimental wells and the positive control wells were added with different concentrations of the angiogenesis agonist polypeptide and positive control polypeptide stem, respectively. The blank group was added with the same volume of vehicle. Three duplicate wells were set for each group for culture at 37°C in serum-free medium. The formation of the tube-like structures in each group was observed under the microscope at 6 hours, 9 hours, and 12 hour, respectively, and the number of tubes was photographed and recorded. Table 3 Effect of polypeptide of Embodiment 1 on tube formation test

Number of tubules Number of tubules Number of tubules Medicine Dose formed (6 hhour) formed (9 h hour) formed (1 2 hhour)

Blank group - 239.3±52.1 145.0±25.2 108.0±12.5

Positive sTEM5 40 tg/ml 266.3±39.9 173.7±39.1 157.3±21.6* control group

Polypeptide of 1 tg/ml 271.3±67.9 179±37 146.7±26.1 Embodiment 1

Polypeptide of 5 tg/m 263.7±48.6 170.3±31.0 152.7±25.3 Embodiment 1

Experimental Polypeptide of 10 tg/ml 261.7±62.9 208.6±52.4 196.0±58.4 group Embodiment 1

Polypeptide of 20 tg/ml 262±47.1 190.0±25.5 185.0±43.9* Embodiment 1

Polypeptide of 40 tg/ml 243.3±34.4 175.7±12.7 157.0±6.2* Embodiment 1

*p<0.05, ** p<0.01 compared with the blank group The results are shown in Table 3. The angiogenesis agonist polypeptide can promote the tube formation of human vascular endothelial cells. At 12 hours, the doses between 5 and 40 pg/ml, the number of tubes was more than that of the blank group. In particular, at the doses of 20 pg/ml, 40 pg/ml, there was statistical significance (p<0.05 ). Embodiment 6 Effect of the angiogenesis agonist polypeptide on angiogenesis in rat arterial rings assay 6 SD male rats of 6-8 weeks were sacrificed. The arteries were taken and washed by a PBS solution containing antibiotics, and the extravascular fibers and adipose tissue were removed with ophthalmic surgical scissors and forceps. 0.5 to 1 mm long arterial rings were cut and washed several times with PBS solution containing antibiotics. In a pre-cooled 96-well cell culture plate, each well was added with 60 tl of 5 mg/ml Matrigel. After the arterial rings were embed in Matrigel, each well was added with 100 tl of DMEM culture medium(containing 10% FBS, VEGF 5 ng/ml), and the experimental wells and the positive control wells were added with different concentrations of the angiogenesis agonist polypeptide and positive control polypeptide sTEM5, respectively. The blank group was added with the same volume of vehicle, three duplicate wells were set for each group, and it was changed with fresh culture medium and peptides were added the next day. On the 7th and 8th days, outgrowth from the rat aortic rings was observed under an inverted microscope, and the number of microvascular-like structures in each circle of the arterial rings was recorded. The recorded results were used to calculate mean ±SD, and a statistical T-test was performed. *P<0.05 indicates a significant difference, and **P <0.01 indicates an extremely significant difference. Table 4 Effect of Polypeptide of Embodiment 1 on angiogenesis in rat arterial rings assay

Number of vascularized structures Medicine Dose 7 thday 8 1h day

Blank group -- 12.1±18.7 34.0±30.8

Positive control sTEM5 40 tg/ml 60.4±35.5* 78.5±34.4* group

Polypeptide of Embodiment 1 5 tg/ml 0.0±0.0 4.0±4.0 Experimental Polypeptide of Embodiment 1 10 tg/ml 88.2±62.4* 109.5±96.0* group Polypeptide of Embodiment 1 20 tg/ml 54.8±76.4 74.0±106.8.5

*p<0.05, ** p<0.01 compared with the blank group The results are shown in Table 4. The polypeptide of Embodiment 1 can promote the angiogenesis of rat arterial rings, the effect is the strongest at a dose of 10 pg/ml, compared with the blank group, there is a statistical difference (p<0.05). Embodiment 7 Effect of angiogenesis agonist polypeptide on recovery after ischemic stroke Middle cerebral artery occlusion (MCAO) model was used to observe the effect of angiogenesis agonist polypeptide on recovery after ischemic stroke. SD male rats of 8-12 weeks were anesthetized. The right external carotid artery (ECA) and internal carotid artery (ICA) were separated. The ECA branch was separated, ligated and cut off at 1.5 cm at the distal end. The carotid sheath was not open, and the pterygopalatine artery (PPA) was not separated and ligated. The ligated stump of the ECA was lifted. The ECA was temporarily closed by a microvascular clip clamping at CCA. ECA was ligated between the clip and the stump of the ECA by using a surgical line without tightening. A 0.2 to 0.3mm nylon line was entered from the stump of the ECA. The clip was loosened, and the nylon line was pushed into the ICA. The nylon line was advanced until a slight resistance was felt at about 1.8 to 1.9 cm from the intersection of the ICA and the ECA, which indicated that the nylon line had crossed the middle cerebral artery (MCA) and reached the beginning of the anterior cerebral artery (ACA). Then, the start time of embolization was recorded, and the surgical line was fastened on the ECA. After 2 hour-embolization, the nylon line was pulled out. After the model was established, 60 MCAO model rats were divided into 6 groups, the model group, the treatment groups and the positive control groups. The day that the nylon line was pulled out was as the first day. On the 4 th day, each group started to be subcutaneously injected the corresponding peptides 2 times per day for 10 days. The treatment groups were received with the polypeptide of Embodiment 1 at doses of 40, 20, 10, and 5 mg/Kg, respectively, the model group were received with normal saline and the positive control group was treated with urokinase with a dose of 5000 U/Kg. On the 1 4th day, the neurologic deficit score was used to evaluate the degree of brain impairment, which is described as: 0 points-no neurological deficit symptoms, the two forelimbs straighten to the ground when the rat is lifted by its tail; 1 point-slight neurological deficit, when the rat is lifted by its tail, the contralateral forelimbs of the lesion show flexion, elevation, shoulder adduction, and elbow joint extension; 2 points-moderate focal neurological deficit with signs of rotation towards the paralyzed side; 3 points-severe focal neurological deficit with signs of fall towards the opposite side of the lesion; and 4 points-no spontaneous activity and cognitive decline. At the same time, brain tissues were taken. After sectioning, 2% TTC staining, incubation, and fixation, pictures were taken and the Image ProPlus 6.0 image analysis system was used to calculate the infarct area and area of the brain slice, and the percentage of cerebral infarction area was calculated according to the equation: the percentage of cerebral infarction area=

(the infarct area of the brain slice / area of the brain slice) x100%. Table 5 Effect of polypeptide of Embodiment 1 in MCAO model

Percentage of Number of Neurologic Medicine Dose cerebral infarction in animals deficit score rats(%)

Model group 10 2.78±0.64 53.56±5.47

Positive urokinase 5000 U/Kg 10 1.47±0.68** 21.43±3.63** control group

polypeptide of 40 mg/Kg 10 1.04±0.57** 15.86±3.47** Embodiment 1

polypeptide of 20 mg/Kg 10 1.35±0.30** 20.12±4.58** Experimental Embodiment 1

group polypeptide of 10 mg/Kg 10 1.26±0.25* 25.76±4.48** Embodiment 1

polypeptide of 5 mg/Kg 10 1.69±0.75 31.53±6.56* Embodiment 1

*p<0.05, ** p<0.01 compared with the blank group The results of the polypeptide of Embodiment 1 in MCAO rat model are shown in Table 5. Compared with the model group, the polypeptide of embodiment 1 can improve the neurologic deficit score at a dose of 5 to 40 mg/Kg with statistical difference. In addition, the polypeptide of Embodiment 1 can significantly reduce the cerebral infarct area, and there is statistically different compared with the model group. Conclusion: the polypeptide of Embodiment 1 has obvious effect on improvement of the neurobehavioral scores and reduction of the area of cerebral infarction in MCAO model rats ,

and has the therapic effect on ischemic stroke.

SEQUENCE LISTING

<110> Nanjing Medical University Affiliated Brain Hospital

<120> ANGIOGENESIS AGONIST POLYPEPTIDE AND APPLICATION THEREOF

<130>

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 8

<212> PRT

<213> Artificial sequence

<400> 1

Phe Arg Trp Pro Arg Lys Gly Asp

1 5

Claims (5)

What is claimed is:

1. An angiogenesis agonist polypeptide, characterized in that a sequence of the polypeptide is SEQ ID NO: 1.

2. The polypeptide according to claim 1, wherein the polypeptide has a function of promoting angiogenesis.

3. The polypeptide according to claim 1, wherein the polypeptide has an effect of treating an ischemic disease.

4. The polypeptide according to claim 3, wherein the ischemic disease is ischemic stroke.

5. The polypeptide according to any one of claims 1 to 4, wherein the polypeptide can be prepared by recombinant expression or chemical synthesis.