CN114903979A - Use of PEDF and polypeptides derived therefrom for treating pulmonary hypertension - Google Patents

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of PEDF and polypeptides derived from the PEDF in treatment of pulmonary hypertension. Aiming at the problems of high PH morbidity, high mortality and vascular remodeling, the invention discloses the application of PEDF in treating pulmonary hypertension, and screens PEDF source functional peptide with good functional advantages, which can also obviously inhibit pulmonary arterial structure remodeling, reduce vascular pressure, improve PH and right ventricular insufficiency caused by PH. Meanwhile, PEDF is a multifunctional protein naturally existing in a human body, the effect of the PEDF on the aspects of PH improvement and prognosis is discovered, and a small peptide segment combination with a targeted function is prepared, so that the application biosafety is higher compared with that of a chemical synthetic drug.

Description

Use of PEDF and polypeptides derived therefrom for treating pulmonary hypertension

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of PEDF and polypeptide derived from the PEDF in treatment of pulmonary hypertension.

Background

Pigment epithelial cell derived factor (PEDF), a secreted glycoprotein with a molecular weight of 50kD (NCBI accession No.: NP-002606.3), was first discovered and purified in culture of human Retinal Pigment Epithelial (RPE) cell secretion products. The human PEDF protein coding gene is positioned at the end of the short arm of the human chromosome 17, consists of 418 amino acids for coding polypeptide, and belongs to the members of the serine protease non-inhibitory factor superfamily. It is widely expressed in various tissues and organs of human eyes, brain, spinal cord, bone, liver, heart, lung, etc., and plays an extremely important role in the pathophysiological process of human body. PEDF is a pleiotropic protein with multiple binding sites, and can play multiple biological roles, including inhibiting endothelial cell migration and angiogenesis, resisting tumor, inflammation, oxidation, neurotrophic factors, cell necrosis and apoptosis. The heparin binding site and the collagen binding site of the PEDF can enable the PEDF to be deposited in the interstitial tissue. At present, there are many reports on the application of PEDF-derived polypeptides, and the functions mainly involved include: inhibiting and/or improving skin aging, treating ocular disease due to angiogenesis, treating osteoarthritis, alopecia and/or hair discoloration, inhibiting tumor angiogenesis, preventing and treating ischemic heart disease, treating liver cirrhosis, and promoting stem cell proliferation and wound healing. However, because the molecular weight of PEDF is too large, it is not easy to be absorbed and used in pharmacy, and because PEDF is a multifunctional protein, it has great difficulty in clinical application although it has the effect of treating pulmonary hypertension in various directions.

Pulmonary Hypertension (PH) is a disease in which the blood pressure of the Pulmonary arteries is abnormally elevated due to a variety of known or unknown causes. PH is clinically intractable, the prognosis is poor, patients die after symptoms appear due to uncontrollable right heart failure, and the prognosis is very poor. The pathological features of PH are mainly manifested by persistent constriction of the pulmonary artery and progressive vascular remodeling, which together lead to stenosis of the vascular lumen and persistent elevation of pulmonary artery pressure, a key factor driving disease progression. The current drugs for preventing and treating PH are very limited, and there is no effective treatment for vascular remodeling, and the common drugs can only be used for improving vasoconstriction, such as prostate alcohol, sildenafil, bosentan, and the like. While relieving pulmonary vasoconstriction can only improve clinical symptoms, but cannot prevent the progression of PH, because pulmonary vascular remodeling, a key factor driving disease development, cannot be effectively intervened, which is also the main reason for poor PH prognosis and high mortality. Therefore, there is a need to develop new PH therapeutics to address the problems of PH morbidity, mortality, and vascular remodeling.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the invention provides the use of PEDF and/or PEDF-derived polypeptides in the preparation of products for treating pulmonary hypertension. The PEDF and/or PEDF derived polypeptide can inhibit the generation and/or release of inflammatory factors, inhibit the accumulation of interstitial cells and reduce the pulmonary vascular remodeling, thereby achieving the treatment or health care effect of preventing the pulmonary hypertension.

It is a second object of the present invention to provide a medicament for the treatment of pulmonary hypertension.

The first object of the present invention is achieved by the following technical solutions:

the invention provides application of PEDF and/or PEDF source derivative polypeptides in preparing products for treating pulmonary hypertension.

Aiming at the problems of high PH morbidity and mortality and vascular remodeling, the research of the invention discovers that the PEDF can reduce the accumulation of interstitial cells in the wall of the pulmonary artery blood vessel and relieve the pulmonary vascular remodeling; when PH occurs, pulmonary artery pressure, right ventricular systolic pressure and diastolic pressure can be relieved, pulmonary blood perfusion is improved, right heart function is protected, and progress of disease is inhibited.

Preferably, the PEDF-derived polypeptides include polypeptides represented by SEQ ID NO.1(33mer) and SEQ ID NO.2(43 mer).

Further, the PEDF-derived polypeptide comprises a polypeptide (33mer) shown in SEQ ID NO. 1.

In order to further solve the problems that the molecular weight of PEDF is too large, and the PEDF is not easy to prepare medicines and absorb, PEDF source polypeptides, namely 33mer and 43mer, which can replace macromolecular protein PEDF are screened out. Wherein the sequence of the 33mer (positions 5-36aa in PEDF) is VLLLWTGALLGHGSSQNVPDSSQDSPAPDSTG. The sequence is located at the proximal end of full-length PEDF and is an important part of short peptides of PEDF, and a large number of studies prove that PEDF inhibits macular degeneration and other blinding diseases and is related to the sequence. The sequence of the 43mer (position 131-173aa in PEDF) is APEKNFKSASRIVFERKLRVKSSFVAPLEKS YGTRPRILTGNP. The sequence comprises an amino acid sequence of PEDF for binding to heparin receptor and an amino acid sequence for binding to hyaluronic acid receptor, and also comprises a part of an aminodextran binding site. The 43mer can exert various biological effects after binding to its receptor. The research shows that the 33mer and/or 43mer can also obviously inhibit the pulmonary artery structure remodeling, reduce the vascular pressure, improve the PH and the right ventricular insufficiency caused by the PH, and have obvious effect on treating the pulmonary hypertension. Besides, PEDF is a multifunctional protein naturally existing in human bodies, the effect of the PEDF on the aspect of PH improvement and prognosis is found, and the specific functional small peptide fragment combination is prepared, so that the application biosafety is higher compared with that of a chemical synthetic drug.

Preferably, the PEDF is a vector overexpressing PEDF.

Further, the vector comprises a liposome, a polycationic vector, a polyanionic vector, a targeting peptide, a plasmid vector, an adenoviral vector, an adeno-associated vector or a lentiviral vector.

Preferably, the treating pulmonary hypertension is inhibiting pulmonary vascular remodeling.

Preferably, the product comprises a medicament, a food and a health product.

The second object of the present invention is achieved by the following technical solutions:

the invention also provides a medicament for treating pulmonary hypertension, which comprises PEDF and/or PEDF-derived polypeptides.

Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

Further, the carrier includes solvents, diluents, suspending agents, emulsifiers, antioxidants, pharmaceutical preservatives, colorants, flavors, vehicles, oily bases, excipients.

Preferably, the dosage form of the medicament comprises but is not limited to injection, capsules, tablets, pills and granules.

Preferably, the administration method of the medicament comprises internal jugular vein injection, respiratory tract instillation, direct injection of lung tissue, systemic vein injection, local pulmonary artery injection, sublingual buccal administration or injection, intraperitoneal injection, nasal mucosa spray or injection, pulmonary atomization, rectal administration, intramuscular injection or oral administration.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses application of PEDF and polypeptide derived from the PEDF in treating pulmonary hypertension, and the following technical effects are achieved:

(1) the PEDF and the PEDF source functional peptide have good functional advantages, and have remarkable effects of reducing the accumulation of interstitial cells in the wall of pulmonary artery blood vessels, inhibiting the reconstruction of pulmonary vessels, reducing the pressure of pulmonary arteries, improving the perfusion of pulmonary blood flow, protecting the right heart function and the like.

(2) The PEDF and the PEDF source functional peptide have extremely high functional selectivity, only inhibit the apoptosis of activated endothelial cells, and inhibit the activation of the endothelial cells; in the acute initial stage of endothelial cell injury caused by hypoxia and inflammation stimulation, PEDF plays a role in stabilizing the connection of endothelial cells and inhibiting the activation of the endothelial cells, and cannot promote apoptosis; and when the condition of the patient continues to progress, and the hypoxia and inflammation further aggravate to cause the connection and the breakage of endothelial cells, the activation of the endothelial cells and the disintegration of the vascular structure, the PEDF plays a role in promoting the apoptosis of the endothelial cells and lightens the inflammatory reaction of tissues. Therefore, the curative effect on the pulmonary hypertension disease is higher, and the side effect is lower.

(3) The PEDF and the PEDF source functional peptide have different action targets, and the surfaces of vascular smooth muscle cells, fibroblasts and endothelial cells have different PEDF receptors, so that the PEDF and the PEDF source functional peptide can respectively play a role in protecting blood vessels in different aspects.

(4) PEDF is a multifunctional protein naturally existing in a human body, the effect of the PEDF on treating pulmonary hypertension is discovered, and a functional small peptide fragment composition is prepared in a targeted manner, so that the application biosafety is higher compared with a chemical synthetic drug.

(5) The PEDF functional peptide combination (single preparation or mixed preparation can be selected for use) is reasonably selected based on the pathological stage of pulmonary hypertension development, and compared with the whole process of using whole protein PEDF, the effect of improving the potency and reducing the side effect can be better.

Drawings

Figure 1 shows the PEDF protein expression in lung tissue of patients with PH (n-10, ^** P<0.01, vs Control group);

FIG. 2 shows the transfection condition of PEDF overexpression vector virus in PH animal model (A. PEDF virus transfection and PH model modeling process schematic diagram; B. virus vector self-carried fluorescence high expression in lung tissue; C and D.WB detection shows that PEDF protein expression in PEDF overexpression group is obviously increased. n is 4, ^** P<0.01, compared to Normal group; ^## P<0.01, compared to MCT group; ^△△ P<0.01, compared to SuHx group);

figure 3 shows the effect of PEDF on pulmonary vascular pressure and perfusion (a.mct-induced PH rat pulmonary artery angiography, performed using digital subtraction technique (DSA) and photographed, R representing the right side of the limb, the arrow pointing to the right pulmonary artery trunk, width measured at its root and recorded, triangle representing the catheter; B-d.mct-induced PH rat pulmonary artery filling time analysis statistical plot and right pulmonary artery root high density angiogram width analysis statistical plot; n is 10 per group, ^** P<0.01, vs Control group);

FIG. 4 shows the effect of PEDF on right heart function of PH animal model (cardiac ultrasound right heart function: A. Doppler ultrasound tricuspid valve annular side wall contraction velocity (RVS'), reflecting right ventricular systoleA function; B. tricuspid annular systolic displacement (TAPSE); C. two-dimensional right ventricular area change fraction (RV FAC) and right ventricular diastolic inner diameter: spindle (RVDd maj), middle chamber (RVDd mid), base (RVDd base); d and E, cardiac ultrasonic detection of right ventricular structure and function statistical map. n is equal to 10, and n is equal to 10, ^* P<0.05、 ^** P<0.01 compared to Control group);

figure 5 is a graph of the effect of PEDF on pulmonary vascular remodeling (a. HE staining of lung tissue sections; b. immunofluorescence staining of lung tissue sections; c.wb measures the expression levels of proteins CD31, VE-cadherin (vascular endothelial cadherin), α -SMA (α -smooth muscle actin), Vimentin (Vimentin) in lung tissue, and a statistical plot, n 10, ^** P<0.01 compared to Control group, NS stands for no statistical significance);

FIG. 6 shows the effect of PEDF on endothelial cell junctions (protein expression of PEDF observed in A-D.WB experiments, the effect of over/under expression of PEDF on total level and phosphorylation of VE-cadherin protein, as well as statistical plots; n-4, ^** P<0.01, compared to Normal group; ^## P<0.01, vs Control group);

FIG. 7 shows the results of screening for polypeptides capable of replacing the macromolecular protein PEDF (A-B, the effect of PEDF-derived peptides on pulmonary vascular pressure and blood perfusion, Digital Subtraction (DSA) was used for pulmonary angiography and pictures were taken, R represents the right side of the limb, the arrow points to the trunk of the right pulmonary artery, the width was measured and recorded at its root, the triangle represents the catheter, the time analysis statistical chart for MCT-induced and SuHx-induced filling of the pulmonary artery in PH rats and the width analysis statistical chart for high density vascular shadows at the root of the right pulmonary artery, n is 6 for each group of polypeptides, ^** P<0.01, in comparison with Control group; ^## P<0.01, compared to the PEDF group; C-D, HE staining of lung tissue sections, establishment of MCT-induced and SuHx-induced PH animal models, intervention with over-expressed lentiviruses of PEDF-derived peptides).

Detailed Description

The following further describes the embodiments of the present invention. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features involved in the respective embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

The experimental procedures in the following examples were carried out by conventional methods unless otherwise specified, and the test materials used in the following examples were commercially available by conventional methods unless otherwise specified.

All peptide sequences mentioned herein are written according to general convention with the N-terminal amino acid on the left and the C-terminal amino acid on the right. The short line between two amino acid residues indicates a peptide bond.

For convenience in describing the invention, conventional and non-conventional abbreviations for the various amino acid residues are used. These abbreviations are familiar to those skilled in the art, but are listed below for clarity:

asp ═ D ═ aspartic acid; ala ═ a ═ alanine; arg ═ R ═ arginine;

asn ═ N ═ asparagine; gly ═ G ═ glycine; glu ═ E ═ glutamic acid;

gln ═ Q ═ glutamine; his ═ H ═ histidine; ile ═ I ═ isoleucine;

leu ═ L ═ leucine; lys ═ K ═ lysine; met ═ M ═ methionine;

phe ═ F ═ phenylalanine; pro ═ P ═ proline; ser ═ S ═ serine;

thr ═ T ═ threonine; trp ═ W ═ tryptophan; tyr ═ Y ═ tyrosine;

val ═ V ═ valine; cys ═ C ═ cysteine.

Example 1 correlation study of PEDF with pulmonary arterial hypertension (PH)

Diagnostic PH criteria: according to the consensus of the European Heart Association PH guidelines and the Congren conference PH, the PH diagnostic criteria: the pH value is determined by heart ultrasonic when the tricuspid valve regurgitation speed is more than or equal to 25mmHg (1 mmHg-0.133 kPa). Meanwhile, according to the relevant diagnosis standard of Chinese expert consensus, according to the level of pulmonary arterial systolic pressure, mild PH is defined as: 30-40mmHg, moderate PH: 40-70mmHg, severity: > 70 mmHg. Inclusion criteria for PH patients related to this example: patients with PH meet the following four criteria: (1) included patients were all diagnosed with PH for the first time as determined by relevant criteria and guidelines. (2) The selected patients are the patients who are hospitalized for the first time in our hospital and are detected by related indexes such as serum Cys C, N-terminal pro-brain natriuretic peptide (NT-proBNP), high sensitive troponin T (hs-cTnT), hypersensitive C-reactive protein (hs-CRP), AST, ALT, Scr, blood potassium, leucocyte, blood platelet and the like. Meanwhile, each patient completes the heart color ultrasound examination by a special person, and measures the relevant indexes of the inner diameter of the ventricle. Exclusion conditions: patients with one or more of the following conditions were excluded (1) patients with non-CHD-induced PH. (2) Patients with malignant tumor, severe infection and severe cerebrovascular disease are combined. (3) Patients with serious liver and kidney insufficiency are combined. (4) Patients with thyroid disease and pulmonary artery stenosis. (5) A patient undergoing steroid related drug therapy. (6) Patients < 18 years of age.

Blood from patients with PH was collected, centrifuged and the supernatant plasma was collected and assayed for PEDF protein levels using enzyme linked immunosorbent assay (ELISA). Referring to fig. 1, PEDF protein expression was significantly reduced in lung tissue of patients with pulmonary arterial hypertension (PH) compared to normal controls, with significant statistical significance. The PEDF is proved to be extremely high in expression in lung tissues and is an important factor for regulating the lung internal environment and protecting blood vessels.

Example 2 exploration of the efficacy of PEDF in pulmonary hypertension treatment

1. Experimental materials and methods

1.1 delivery of PEDF over-expression vector Virus into pulmonary vasculature by smooth muscle cells

All animal experiments were performed according to the guidelines of the Chinese animal protection Committee. Fisher 344 rats, 21 days old, were purchased and euthanized.

1.1.1 culture of Primary smooth muscle cells

Preparing a culture solution: DMEM (Gibco, USA), Hepes (15mmol/L), penicillin (100U/mL), streptomycin (100mg/mL), fetal bovine serum (primary culture concentration 20%, subculture concentration 10%, Gibco, USA). A culture vessel: 25cm ² Plastic culture flask (Costar), cultureArea 25cm ² (ii) a Petri dishes with a diameter of 3.5 cm.

1.1.1.1 rat Vascular Smooth Muscle Cell (VSMC) culture

a. Drawing materials from arteries: rats were sacrificed by cervical dislocation, whole aorta aseptically isolated and immediately placed in sterile saline containing penicillin 100U/mL and streptomycin 100 mg/mL.

b. Primary culture by a wall attaching method: on the clean bench, remove the connective tissue and peel the adventitia. The blood vessel is cut open longitudinally, the inner membrane surface is scraped blunt with curved forceps to eliminate endothelial cells, and the residual blood vessel has thin, transparent and high toughness. Repeatedly flushing with physiological saline containing penicillin and streptomycin to remove impurities such as lipid drops, blood clots and the like and possible residual endothelial cells and adventitial fibroblasts, mixing the mesomembranous tissues of 2-3 blood vessels obtained at one time, and placing the mixed tissues in a sterile culture dish. A small amount of culture medium was added dropwise to keep the tissue moist, and the tissue was repeatedly cut into tissue blocks of 1mm × 1mm in size by means of ophthalmologic curved scissors. And uniformly placing the cut small tissue blocks at the bottom of the bottle, wherein the distance between the tissue blocks is 0.5 cm. And covering a bottle cover, slightly turning over the culture bottle, enabling the bottom of the bottle to be upward, injecting a proper amount of culture solution into the bottle, placing the bottle in an incubator at 37 ℃ for 2-4 hours to enable the tissue blocks to be dry and attached to the wall of the bottle, slowly turning over and horizontally placing the culture bottle to enable the tissue blocks to be completely immersed in the culture solution, and continuing to perform standing culture for 3-5 days. The fluid is changed after the cells have migrated from around the tissue mass.

1.1.1.2 subculture of Primary culture arterial VSMC

When most of the tissue mass develops a cell halo and comes into contact with the cell halo of the adjacent cell, it can be passaged. Adding prepared digestive juice (trypsin-EDTA (0.05%), Saimeifei, cat # 25300054) to cover the cell surface, standing at room temperature for about 1-2 min, rapidly turning the culture bottle to separate the cells from the digestive juice when cytoplasm retracts and cell gaps increase under an inverted microscope, removing the digestive juice, adding 3-4 mL of culture solution containing fetal calf serum to stop digestion, repeatedly blowing the cells on the bottle wall to separate the cells from the wall, and dispersing into single cell suspension. And (3) dividing the cell suspension into two parts, inoculating the two parts into a new culture bottle, and supplementing a culture solution (3-4 mL per bottle). The tissue mass that does not disappear is removed by changing the fluid and passing the cells.

1.1.1.3 identification of cultured cells

The isolated cells were identified as Vascular Smooth Muscle Cells (VSMCs) by morphological and immunohistochemical identification:

morphology: the growth form and the growth rule of the cells are observed by a phase contrast microscope, the cells grow in an adherent manner, are mostly in a triangular spindle shape or a square shape, and can observe hill and velley phenomena, hill peaks are formed at places with more cells, and velley valleys are formed at places with less cells.

Immunohistochemical identification: alpha-SMA is a marker molecule of vascular smooth muscle cells, after immunofluorescence staining by a specific rabbit anti-human alpha-smooth muscle actin polyclonal antibody (abcam, cat number: ab5694), the alpha-SMA is observed by using laser confocal focusing and arranged in cytoplasm in a myofilament shape along a longitudinal axis, the cytoplasm emits green fluorescence, the nucleus is blue-stained, and the purity of the cultured primary cells is higher if the positive cell rate reaches more than 95%.

1.1.2 delivery of pulmonary artery smooth muscle cells overexpressing PEDF

1.1.2.1 transfection of smooth muscle cells with PEDF Lentiviral vector PEDF-LVs (purchased from Kjekay, Shanghai, Ltd.)

24 hours before lentivirus transfection, cells were plated at 5X 10 ⁵ Density of/well was plated in 6-well plates, the next day the original medium was replaced with 2mL fresh medium containing 6 μ g/mL polybrene, appropriate amount of virus suspension was added, after incubation for 4 hours at 37 ℃ 2mL fresh medium was added to dilute polybrene, culture was continued for 24 hours, and the medium containing virus was replaced with fresh medium. The culture was continued and 72 hours after transfection was used as a jugular vein injection to rats.

1.1.2.2 rat jugular vein injection

Then digested with trypsin and aliquoted into 5X 10 fractions ⁵ Aliquots of individual cells. Cells were cultured between the fifth and ninth passages and then PEDF-LVs transfected (including over-expression) or empty vector transfected cells were injected into the internal jugular vein of recipient Fisher 344 rats at 6 to 8 weeks, and animals were fed for 24 hours before subsequent construction of the pH model.

The administration method can adopt the method used by the invention but is not limited to the method, and can also adopt the methods of respiratory tract dripping virus vector, direct injection of lung tissue, systemic intravenous injection, local pulmonary artery injection, sublingual buccal administration or injection, intraperitoneal injection, nasal mucosa spray or injection, pulmonary atomization, rectal administration, intramuscular injection or oral administration, and the like. PEDF overexpression vectors include, but are not limited to, liposomes, polycationic vectors, polyanionic vectors, targeting peptides, plasmid vectors, adenoviral vectors, adeno-associated vectors, or lentiviral vectors, and the like.

1.2 construction of PH animal model

PH animal models induced by Monocrotaline (MCT) or Sugen5416/hypoxia (SuHx) are respectively established 24h after the pulmonary artery vascular smooth muscle cells are injected into the jugular vein of a rat. Wherein the MCT induction method comprises the following steps: (1) fisher 344 rats were first acclimated for one week in a new environment at a temperature of 18-20 deg.C, a humidity of 40-50%, and light conditions of 12 hours light and dark cycles, and body weights were recorded weekly. (2) MCT was completely dissolved in 1-3 mL of 1N HCl and the pH was adjusted to 7.4 with 10N NaOH, and a 20mg/mL MCT solution was prepared by adding sterile PBS. (3) MCT (60mg/kg) was injected subcutaneously in the ventral thorax of rats at multiple sites to prevent skin necrosis. (4) Animals were kept for 4 weeks at 18-20 deg.C, 40-50% humidity, and 12 hours light-dark cycle, and weighed once a week to understand disease progression. The SuHx induction method comprises the following steps: (1) fisher 344 rats were weighed and prepared for Sugen5416 for injection, Sugen crystals were dissolved in DMSO to a concentration of 20mg/mL, and then the solution was diluted with PBS (DMSO: PBS ═ 1:3) and pH was adjusted to 7.2. (2) Sugen was injected subcutaneously into the abdomen of animals at 20mg/Kg once a week for three consecutive weeks using a 1mL syringe. (3) The animals were placed under normbaric hypoxia (10% O) ₂ ) The environment was maintained for 3 weeks, after which the animals were placed in normoxic (21% O) ₂ ) The rearing was carried out under conditions for more than one week, during the test period, the rearing room was opened every 3 days to clean the cages and to replenish the food and water supply. (4) Maintaining normoxic animals at 21% O ₂ For 4 weeks in a semi-sealed chamber. (5) During the hypoxic exposure, animals were examined daily for abnormal conditions of foreign bodies, such as hair loss, wasting, and dyspnea.

The method for establishing the animal model can adopt the method used by the invention but is not limited to the method, and can also adopt an anoxia method, a low-pressure method, a surgical shunting method, a mouse with a mutant protein receptor 2, a pulmonary lobe/partial division method, a pulmonary artery ligation method and the like, and various methods are combined for use.

1.3 isolation, culture and treatment of pulmonary artery endothelial cells

1.3.1, separation: 200-300g of healthy male rats, abdominal anesthesia (10% chloral hydrate 4mL/kg), and carotid bleeding; soaking the whole rat in 75% alcohol for 3s, cutting the thoracic cavity, cutting off the heart and lung, and placing into PBS or Hanks solution (containing penicillin 200u/mL and streptomycin 200 ug/mL); separating and cutting off the pulmonary artery trunk, flushing blood in blood vessels with PBS, longitudinally cutting off the pulmonary artery, flattening the pulmonary artery on a plastic tube, cutting into small artery pieces of 1.5mm multiplied by 1.5mm, and attaching the inner membrane surface to a sterilized culture dish; after the incubator was dried for 2 hours, 2mL of the culture medium was added, and the resulting mixture was placed in an incubator (37 ℃ C., 5% CO) ₂ Humidity 100%); after 72h, the cells can reach a certain density, the arterial disc is removed, and the liquid is changed once; and the liquid is changed every 2 days, 1/2 is changed every time, and the culture is continued for 6-10d to form a cell monolayer.

1.3.2, cell identification: centrifuging at 800r/min for 5min after digestion to remove culture solution, re-suspending with warm PBS, taking a little suspension, thinly coating on a slide, blotting with absorbent paper, fixing with methanol for 5-L0min, taking out, washing with PBS (0.0L mol/L pH7.2) for 3min × 3 times, adding dilution (50uL of 0.01mol/L pH7.2 PBS) of CD31 primary antibody (Abcam, Cat. Ex. ab 24690), sealing and incubating at 37 deg.C for 30min, washing with PBS for 3min × 3 times, adding blocking solution 1:100 to dilute fluorescent secondary antibody (goat anti-mouse IgG H)&L(Alexa

488) (ab150113), incubated sealed at 37 ℃ for 30min, washed 3min × 3 times with PBS, blotted, buffered glycerol mounted, and observed with a fluorescence microscope. The positive cell rate reaches more than 95 percent, which indicates that the purity of the cultured primary cells is higher.

1.3.3, cell transfection: 18-24 hours before lentivirus transfection, adherent cells were plated at 1X 10 ⁵ The/well was plated in 24-well plates. Number of cells at lentivirus transfectionIs 2 x 10 ⁵ About hole. The next day, the original medium was replaced with 2mL of fresh medium containing 6. mu.g/mL polybrene, a suitable amount of PEDF-overexpressing viral suspension or siRNA interfering (knockdown PEDF) viral suspension (both designed, packaged and supplied by Shanghai Jikai Genscience and technology Co., Ltd.) was added, incubation was carried out at 37 ℃ for 4 hours, 2mL of fresh medium was added to dilute the polybrene, culture was continued for 24 hours, and the medium containing the virus was replaced with fresh medium.

1.4 echocardiography cardiac function determination

Animals in each group were modeled 4 weeks later and pulmonary and aortic pressures were measured by doppler cardiac ultrasound. For right ventricle assessment, M-mode images were acquired in 4-chamber view of the apex to obtain tricuspid annulus systolic displacement (tase). Type B images were acquired on 4-chamber slices of the apex to obtain the diastolic right chamber diameter, the major axis (RVDd, maj), the middle chamber (RVDd, mid), the fundus (RVDd, base), and the right ventricular cross-sectional area change (RV FAC). The transverse tricuspid annular contraction velocity (RV S') was obtained in a 4-chamber view of the apex of the heart using doppler ultrasound imaging.

1.5 hemodynamics, method for measuring right ventricular hypertrophy and pulmonary arteriography

After the animals are raised for 4 weeks, right heart catheters are sequentially received for measuring blood pressure and DSA pulmonary artery radiography, and the influence of PEDF on pulmonary artery pressure and pulmonary vascular resistance is evaluated.

(1) Preparation in a laboratory: in an animal treatment room, a pulmonary artery manometry and DSA integrated laboratory is additionally provided with a small animal respirator and a right heart catheter manometry instrument.

(2) Personnel arrangement: the animal treatment room 3 is responsible for anesthesia, sacrifice, material drawing and recording; measuring the pressure of the pulmonary artery by 1 person, operating an instrument and recording; DSA chamber 4 persons, two responsible for right heart catheterization and endotracheal intubation and performing pulmonary angiography, one responsible for transporting animals, and one for storing data ex vivo.

(3) Animal pretreatment: rats were deprived of food and drink for 12 hours, pre-anesthetized with a mixture of 4% isoflurane and oxygen (0.5L/min) in a glass box with a lid in an animal treatment room, weighed after anesthesia, surgically prepared by shaving the chest and neck with a hair clipper, exposing the neck and precordial skin, and placing the animal on a warming pad throughout the procedure. Sending into DSA chamber, fixing on constant temperature pad (temperature 37 deg.C) of operation table, injecting pentobarbital sodium (60mg/kg) and isoflurane (4%) mixed oxygen to maintain anesthesia, connecting with PowerLab electrocardio data acquisition system, installing respiration rhythm receptor under abdomen, and observing respiration rhythm. The method comprises the following steps of placing an animal on a platform, sleeving the upper incisors of the animal with rubber bands, fixing limbs to the platform with adhesive tapes, straightening the head and the neck of the animal, disinfecting the neck with alcohol, paving a towel, making a 2-centimeter incision in a midline neck region, carrying out blunt separation on subcutaneous tissues and muscle layers, separating a right external jugular vein and an trachea, and observing the trachea through blunt dissection. A surgical thread is placed under the trachea, a small incision is made at the suture, the catheter is inserted into the trachea, and the catheter is fixed by cerclage of the surgical thread. The breathing machine system is connected with the catheter through the Y-shaped connector and is connected with the small animal breathing machine to control breathing (the frequency is 60-80 times/min, the tidal volume is 2mL/100g, when the chest is opened, 3mL/100g, after the chest is opened).

(3) Pulmonary artery pressure measurement: 1) the tip of the 1.9F (pulmonary artery) PV catheter was placed in saline solution for 30 minutes, the pressure-volume controller was turned on and the data acquisition software was run. 2) The PV catheter is calibrated with saline at body temperature, the sternum is cut and the chest is fully exposed, and the retractor is placed to keep the chest open. 3) The atraumatic needle is used to guide the PV catheter to penetrate into the apical area of the right ventricle, the needle is removed, and the PV catheter is advanced until the electrode at the proximal end completely enters the heart. 4) The PV catheter was moved up to the Pulmonary Artery (PA) until the pressure waveform changed, the catheter was allowed to stabilize in the new position for 3 minutes, the ventilator system was stopped and the recording of pulmonary artery diastolic and systolic pressures was started. 5) Data acquisition software was used to repeat the recordings 3-6 times for each parameter for each rat. 6) The catheter is removed and then the suture is used for hemostasis.

(4) DSA: keeping the state of the animal anesthesia belt machine, performing purse-string suture on the root of the pulmonary artery by using a non-invasive suture, cutting the center position of the purse-string, inserting an injection hose, tightening the purse-string for fixation, opening a drug delivery device to inject an iodine-containing contrast agent, and recording DSA (digital radiography) in real time.

(5) After the above experiment, the animals were transported to a treatment room, sacrificed with excess anesthetic, the right atrium was excised, the circulating blood was flushed by perfusing 10mL PBS through the left ventricle, the left atrium was excised, and the blood in the lungs was flushed by perfusing 10mL PBS through the right ventricle. Tissue weight was normalized to Tibial Length (TL) to assess pulmonary edema (lung weight/TL), hepatic congestion (liver weight/TL) and left ventricular hypertrophy (LV/TL). The ratio of RV to left ventricular septal (LV) body weight (RV/LV ratio) is determined as an indicator of long-term right ventricular hypertrophy.

1.6 protein extraction and Western blot method

(1) Weighing and homogenizing lung tissue: more than 100mg of lung tissue was placed in each EP tube and lysate was added at 100mg/1 mL. Lysate formulation (50. mu.L: 40. mu.L RIPA + 5. mu.L protease inhibitor + 5. mu.L phosphatase inhibitor + 0.5. mu.L PMSF). 2 50mL beakers, one clean saline for washing the homogenizer head; the other was used to hold organized EP tubes. Each time was homogenized for 2s (or 5-8 s).

(2) Centrifuging: after centrifugation at 14000rpm for 20min at 4 ℃, the supernatant was taken, 15-20 μ L of protein sample was left for protein quantification, and the remaining samples were stored in a refrigerator at-20 ℃ for further use.

(3) Protein quantification by BCA method:

taking 10 tubes, adding corresponding reagents according to the following standards (1-4 are standard tubes, 5-10 are samples), and each tube contains 2.4 mL:

test tube	1	2	3	4	5-10
						Double distilled water	2400μL	2380μL	2360μL	2340μL	2390μL
BSA	0	20μL	40μL	60μL	0
						Sample(s)	0	0	0	0	10μL

Adding 2mL of Folin A into each tube, mixing uniformly immediately, and carrying out 30min in a 30 ℃ water bath tank;

adding Folin B, 200 μ L per tube, shaking, mixing, and 30min in 30 deg.C water bath;

measuring the protein OD value by a spectrophotometer, and calculating the protein concentration;

(4) protein samples were mixed with SDS-PAGE protein loading buffer (5 ×) (Biyuntian Bio Inc., product number: P0015) in a 4: 1, boiling for 10min to fully denature the protein; naturally cooling, and storing at-20 deg.C for use.

(5) Preparing polyacrylamide gel:

separating glue:

1.5M Tris-HCl 2 mL; 2.67mL of Acr-Bis; 0.08mL of 10% SDS; 3.25mL of double distilled water. After mixing evenly, adding 0.04mL of 10% Ap; TEMED 0.004 mL. After shaking and mixing evenly, adding the mixture into a glass plate of an electrophoresis tank, and standing the mixture at room temperature for 30min until the glue is solidified.

Concentrating the glue:

1mL of 0.5M Tris-HCl; 0.53mL of Acr-Bis; 0.04mL of 10% SDS; 1.2mL of 40% Sucrose; 1.23mL of double distilled water; after mixing uniformly, adding 0.02mL of 10% Ap; TEMED 0.004 mL. After oscillating and mixing uniformly, adding the mixture into a glass plate of an electrophoresis tank, and standing for 40min at room temperature until the mixture is solidified by gelation.

The method comprises the following specific steps: the samples were first separated by SDS-PAGE protein electrophoresis (voltage: gel 80V, gel 100V; time about 120 min). Placing the blots in the order of an anode, three layers of Blot paper, a cellulose nitrate film, an electrophoretic adhesive, three layers of Blot paper and a cathode, wherein the wet-turning conditions are as follows: 12h and 15V. The membranes were placed in blocking solution (5% skim milk powder, 0.1% Tween-20 in PBS) and blocked for 1h at room temperature. Primary antibodies (PEDF, Boo-Sen, cat # bs-0731R; CD31, Abcam, cat # ab 24690; VE-cadherin, Boo-Sen, cat # bs-0878R; alpha-SMA, Abcam, cat # ab 5694; Vimentin, Abcam, cat # ab 31) were diluted separately with blocking solution for the purpose of detection, reacted overnight at 4 ℃ and washed three times with PBST (0.1% Tween20 in PBS), 5min each. Add blocking solution 1:100 to dilute the fluorescent secondary antibody (goat anti-mouse IgG H)&L(Alexa

488) (ab150113), donkey anti-Rabbit IgG H&L(Alexa

647) (ab150075)) at room temperature for 1h, PBST washed three times, 10min each time, protected from light. Scanning the result by using a far infrared imaging system, carrying out protein quantitative analysis by using ImageJ software, and carrying out statistical analysis by using the ratio of the gray value of the target protein to the gray value of an internal reference (GAPDH or beta-actin).

1.7 immunofluorescence and quantitative analysis

For cardiac tissue staining, frozen myocardial tissue was horizontally sliced into 6 μm slices and adsorbed onto glass slides. Fixed in 4% paraformaldehyde for 15 min, permeabilized with Triton X-100 (0.1%) and blocked with a solution containing 5% bovine serumBlocking, and then applying to the primary antibody, i.e., the sample, with the antibody anti-CD 31(Abcam Corp., cat # ab 24690; 1: 200); anti-fibrinogen specific factor 1(FSP1) (Abcam, cat # S100A 4; 1: 200); anti-VE-cadherin (Abcam Corp., cat # ab 33168; 1: 300) was incubated at 4 ℃ for 12 hours. Washed three times in PBS and incubated with donkey anti-rabbit IgG H at room temperature&L Secondary antibody (Alexa Fluor) ^TM 594 Life Technologies, Inc., cat # A21207; 1: 200) incubate for 1 hour and wash three times in PBS. Nuclei were stained with DAPI (KeyGen Biotech, Inc., Cat. KGA215-10) and washed three times in PBS. Finally, the piece was mounted with 50% glycerol. Ten fields were randomly selected using an Olympus fluorescence microscope with representative areas in 3 different samples (fixed magnification: 400 ×; 10 fields per sample); and capturing the image by using Olympus software.

1.8 statistical analysis

The data are measured as mean + -standard deviation

And (4) showing. Statistical analysis of the data was performed using SPSS 16.0 statistical software. The difference between groups is compared by variance analysis, the pairwise comparison is carried out by q test, and the difference with P less than or equal to 0.05 has statistical significance.

2. Results of the experiment

(1) Transfection condition of PEDF overexpression vector virus in PH animal model

Referring to fig. 2A, adult Fisher 344 rats were used to establish an animal model of pre-transfected lentiviruses inducing pulmonary vessels to overexpress PEDF protein, followed by MCT or SuHx induction of PH, after 4 weeks the model was successfully established and experiments were performed. See figure 2B for lentivirus expression efficiency (green fluorescence) of PEDF vector. After the animal is sacrificed, lung tissue is taken out and prepared into paraffin sections which are observed and photographed under a fluorescence microscope, and the whole process is protected from light. See figure 2C and D for protein expression levels and analytical statistics for PEDF in lung tissue. As can be seen from the above results, compared with the normal control group, the MCT-induced PH animal model lung tissue has significantly reduced PEDF protein expression level (P < 0.01), and the SuHx-induced PH animal model group also has significantly reduced PEDF protein expression level (P < 0.01); however, compared with the normal control group, the MCT + PEDF overexpression group and the SuHx + PEDF overexpression group have no significant difference in PEDF protein level; furthermore, PEDF protein levels were significantly elevated in the MCT + PEDF overexpression group when compared to the MCT group (P < 0.01); PEDF protein levels were also significantly elevated in the SuHx + PEDF overexpression group when compared to the SuHx group (P < 0.01). These results indicate that the viral transfection method with PEDF overexpression vectors used in the present invention is effective.

(2) Effect of PEDF on pulmonary vascular pressure and blood perfusion

Referring to fig. 3A, DSA pulmonary artery angiography showed that in the MCT-induced PH animal model, the pulmonary arteries of the control group were altered in the form of stub, the main trunk of the left and right branches were thickened, and the number of branches was reduced; the empty vector group (Vehicle) performed consistently with the control group; the PEDF overexpression group has more branches than slender pulmonary artery left and right branches. Referring to fig. 3B, the diameters of the right main pulmonary artery branch roots were significantly reduced in the PEDF-overexpressed group (P < 0.01) compared to the MCT-induced PH control group, while the empty vector group did not significantly change. Referring to fig. 3C, DSA pulmonary arteriography showed that in the SuHx-induced PH animal model, the pulmonary arteries of the control group were altered in the form of stub, the main trunk of the left and right branches were thickened, and the number of branches was reduced; the empty vector group and the control group showed consistent performance; the PEDF over-expression group has more slender branches in the left and right pulmonary arteries. Referring to fig. 3D, the diameters of the right main pulmonary artery branch roots were significantly reduced in the PEDF-overexpressed group (P < 0.01) compared to the SuHx-induced PH control group, while the empty vector group was not significantly changed. These results suggest that PEDF has a potent PH-modifying effect, which is reflected in vascular perfusion and vascular morphology.

(3) Effect of PEDF on right heart function in PH animal models

After the animals of each group are modeled for 4 weeks, the relevant indexes of the right ventricular function are detected by using heart color ultrasound, and pictures are taken of the important indexes. Referring to fig. 4A, doppler ultrasound detects tricuspid valve annulus lateral wall contraction velocity (RV S'), reflecting right ventricular systolic function; referring to fig. 4B, tricuspid annulus systolic displacement (tase); referring to fig. 4C, two-dimensional right ventricular area change fraction (RV FAC) and right ventricular diastolic inner diameter: spindle (RVDd maj), middle chamber (RVDd mid), base (RVDd base). See fig. 4D for a statistical analysis of right ventricular function-related indicators for MCT-induced or SuHx-induced PH models, respectively. In the MCT-induced PH model, PEDF was able to significantly improve right ventricular diastolic inner diameter major axis (RVDd maj) (P < 0.05), tricuspid annulus lateral wall contraction velocity (RV S') (P < 0.05), and tricuspid annulus systolic displacement (tase) (P < 0.01); however, PEDF had no statistical effect on the two-dimensional right ventricular area change fraction (RV FAC), right ventricular diastolic inner diameter median (RVDd mid), and right ventricular diastolic inner diameter base (RVDd base). PEDF had a significant effect on all right ventricular function-related indicators mentioned above (P < 0.05) in the SuHx-induced PH model compared to the control group, and these effects were beneficial for right ventricular function. The above results suggest that PEDF has a significant ameliorating effect on PH-induced right heart insufficiency. Studies have reported that abdominal subcutaneous injection of MCT not only induces PH but also has a direct damaging effect on the myocardium, which is involved in causing right heart insufficiency, and it is seen that PEDF can improve right heart insufficiency by relieving PH.

(4) Effect of PEDF on pulmonary vascular remodeling

The vascular remodeling is characterized by cell accumulation between pulmonary artery walls, and is a key factor of PH condition progression, and the vascular remodeling is evaluated by methods such as HE staining, immunofluorescence staining and WB quantitative interstitial specific markers. Referring to fig. 5A, after euthanasia of PH animal models induced by MCT or SuHx, lung tissue was removed and made into paraffin sections for HE staining, and the results showed: pulmonary artery structures in the control group and the empty vector group are remarkably remodeled, thickened blood vessel endothelium and a middle layer of a vessel wall accumulate a large amount of abnormally increased cells, and the vessel cavity is narrow; compared with the control group, the PEDF overexpression group has the advantages that the number of cells abnormally accumulated in the pulmonary artery is obviously reduced, the thickening of the tube wall is obviously less, and the stenosis of the tube cavity is obviously improved. Referring to fig. 5B, animal lung tissue sections were immunofluorescent stained, with the interstitial-specific marker Alpha-smooth muscle actin (Alpha-SM a) labeled red and the endothelial-specific marker Platelet-endothelial cell adhesion molecule (PECAM-1/CD 31) labeled green, as shown, in MCT-induced or SuHx-induced PH animal models, there was a significant increase in vascular interstitial markers and thickening of the vessel wall in the control group as compared to the empty vector group; compared with the control group, the PEDF group has obviously reduced vascular interstitial markers and thinned vascular walls. Referring to fig. 5C, the lung tissue intermediate mass markers (α -SM Α, Vimentin) and endothelial markers (CD31, VE-cadherin) were analyzed by immunoblotting for protein expression levels, all protein levels were compared to the internal reference β -actin, and the results were shown in analytical statistical figures, where the expression levels of the endothelial marker (CD31, VE-cadherin) proteins in the PEDF group were not significantly changed and were statistically insignificant when compared to the control group; however, the expression of the mesenchymal markers (α -SM Α, Vimentin) in the PEDF group was significantly reduced compared to the control group (P < 0.01). The results suggest that PEDF contributes to inhibition of PH-induced vascular remodeling.

(5) Effect of PEDF on endothelial cell junctions

Endothelial cell damage is closely related to the occurrence and development of PH, and when endothelial cell damage occurs, endothelial cell junctions are broken, and endothelial-cadherin (VE-cadherin) is phosphorylated and endocytosed. The inner surface of the pulmonary artery lumen is covered with a monolayer of endothelial cells, and VE-cadherin proteins on the cell membranes of adjacent cells are mutually connected through aggregation to form an adhesion connection structure, so that the continuous and stable endothelium is maintained; VE-cadherin is only expressed in endothelial cells, is an important specific marker, is important for the phenotype of the endothelial cells, and controls initial angioplasty, endothelial cell proliferation, cell polarity and the like; in contrast, VE-cadherin deletion results in disruption of the adhesive junctions and endothelial dysfunction, which is also the initiating link for endothelial injury. Research shows that the deletion of VE-cadherin is regulated and controlled by phosphorylation modification, and when endothelial cells are stimulated by hypoxia, inflammation and the like, VE-cadherin on cell membranes is phosphorylated and modified and then degraded by endocytosis. Referring to fig. 6A, a PH cell model was constructed, proteins were extracted after overexpression or knock-down of PEDF, Western blot verified PEDF protein expression, total VE-cadherin protein expression, and phosphorylated VE-cadherin protein expression, each protein was compared to internal reference GAPDH. Referring to fig. 6B, compared with the normal group, the PEDF protein expression of the PH control group was significantly reduced (P < 0.01), the PEDF protein expression level of the PEDF overexpression group was not statistically different, and the PEDF protein expression of the PEDF knockdown group and the PEDF empty vector group was significantly reduced (P < 0.01); when compared with the PH control group, the PEDF protein expression level of the PEDF overexpression group is obviously reduced (P is less than 0.01), and the PEDF protein expression levels of the PEDF knockdown group and the empty vector group are not obviously changed. Referring to fig. 6C, PEDF had no significant effect on total VE-cadherin protein expression, and there were no statistical differences between the groups. Referring to FIG. 6D, the level of phosphorylation of VE-cadherin protein was significantly increased in the PH control group (P < 0.01) compared to the normal group, whereas the level of phosphorylation of VE-cadherin protein was not significantly changed in the PEDF overexpression group, and further the level of phosphorylation of VE-cadherin protein was significantly increased in the PEDF knockdown group and the empty vector group (P < 0.01); compared with the PH control group, the phosphorylation level of the VE-cadherin protein of the PEDF overexpression group is obviously reduced (P is less than 0.01), and the phosphorylation level of the VE-cadherin protein of the PEDF knockdown group and the empty vector group is not obviously changed. The results suggest that PEDF is capable of inhibiting VE-cadherin phosphorylation.

Example 3 screening for Polypeptides capable of substituting for the macromolecular protein PEDF

Through analyzing the amino acid arrangement and the spatial structure of PEDF, the corresponding peptide segment for maintaining the vascular stability is screened from the functional peptide segments which are known to be capable of binding receptors in lung tissues, and a series of experiments verify the effect of the functional peptide segments on treating pulmonary hypertension, so that the optimal functional peptide segment capable of replacing PEDF is obtained. The design screening process is divided into two steps:

the first step is as follows: by integrating the results of previous studies on PEDF and analyzing and summarizing, functional polypeptides corresponding to the bound PEDF receptor in full-length PEDF are screened, and the screening results are as follows:

33mer sequence (positions in PEDF: 5-36 aa):

VLLLWTGALLGHGSSQNVPDSSQDSPAPDSTG(SEQ ID NO.1)；

43mer sequence (position in PEDF: 131-:

APEKNFKSASRIVFERKLRVKSSFVAPLEKSYGTRPRILTGNP(SEQ ID NO.2)。

the second step is that: the therapeutic effect of PEDF-derived peptides on vascular remodeling was verified by DSA detection and HE staining of lung tissue sections, respectively. Referring to fig. 7A, the filling time was also reduced in the 33mer group (P < 0.01) compared to the MCT-induced PH control group, whereas the filling time was not significantly changed in the 43mer group; when compared with the PEDF group, the filling time of the pulmonary artery of the 43mer group by the contrast agent is increased (P is less than 0.01), and the filling time of the pulmonary artery of the 33mer group by the contrast agent is not obviously changed. Furthermore, the pulmonary artery right main branch root diameter was significantly reduced in the 33mer group (P < 0.01) compared to the MCT-induced PH control group, while the pulmonary artery right main branch root diameter was not significantly changed in the 43mer group; when compared with the PEDF group, the root diameter of the right main pulmonary artery branch of the 33mer group is not obviously changed, and the root diameter of the right main pulmonary artery branch of the 43mer group is obviously increased (P < 0.01). Referring to FIG. 7B, both 33 mers were effective in reducing the time to filling of the pulmonary artery with contrast agent (P < 0.01) when compared to the SuHx-induced pH control, and 43 mers did not; when compared to the PEDF group, the filling time of the pulmonary artery of the 33mer group with the contrast agent did not change significantly, while the filling time of the pulmonary artery of the 43mer group with the contrast agent increased significantly (P < 0.01). In addition, 33mer significantly reduced the pulmonary artery right main branch root diameter (P < 0.01) when compared to the SuHx-induced PH control, and the 43mer group had no significant effect on the pulmonary artery right main branch root diameter; when compared with the PEDF group, the root diameter of the right main pulmonary artery branch of the 33mer group is not obviously changed, and the root diameter of the right main pulmonary artery branch of the 43mer group is obviously increased (P < 0.01). Meanwhile, the effect of PEDF-derived peptides on vascular remodeling was evaluated by HE staining. Referring to fig. 7C, after the PH animal model induced by MCT or SuHx was euthanized, lung tissues were taken out and made into paraffin sections for HE staining, the number of abnormally accumulated cells in the pulmonary artery of the 33mer group was significantly reduced, the wall of the vessel was significantly less thickened, and the luminal stenosis was significantly improved; the 43mer group was not significantly changed. These results suggest that 33 mers in PEDF-derived peptides can exert similar PH-modifying effects and contribute to inhibition of PH-induced vascular remodeling.

As can be seen from the comprehensive examples 1 to 3, aiming at the difficult problem of post-PH vascular remodeling therapy, the invention provides a drug capable of inhibiting vascular remodeling, improving pulmonary artery perfusion and protecting right ventricular function, and the drug screened by the invention is pigment epithelial cell derived factor (PEDF) protein and its derived polypeptide (33mer or/43 mer).

(1) The PEDF source functional peptide designed by the invention has good functional advantages, and can obviously inhibit pulmonary artery structure remodeling, reduce blood vessel pressure, and improve PH and right ventricular insufficiency caused by PH.

(2) PEDF is a multifunctional protein naturally existing in human bodies, the effect of the PEDF on the aspect of PH improvement and prognosis thereof is found, and the prepared combination of small peptide segments with targeted functions has higher application biosafety compared with a chemical synthetic drug.

The embodiments of the present invention have been described in detail, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, and the scope of protection is still within the scope of the invention.

Sequence listing

<110> Zhongshan university affiliated eighth Hospital (Shenzhen Futian)

<120> use of PEDF and polypeptides derived therefrom for treating pulmonary hypertension

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 32

<212> PRT

<213> 33mer(Artificial Sequence)

<400> 1

Val Leu Leu Leu Trp Thr Gly Ala Leu Leu Gly His Gly Ser Ser Gln

1 5 10 15

Asn Val Pro Asp Ser Ser Gln Asp Ser Pro Ala Pro Asp Ser Thr Gly

20 25 30

<210> 2

<211> 43

<212> PRT

<213> 43mer(Artificial Sequence)

<400> 2

Ala Pro Glu Lys Asn Phe Lys Ser Ala Ser Arg Ile Val Phe Glu Arg

1 5 10 15

Lys Leu Arg Val Lys Ser Ser Phe Val Ala Pro Leu Glu Lys Ser Tyr

20 25 30

Gly Thr Arg Pro Arg Ile Leu Thr Gly Asn Pro

35 40

Claims (10)

1. Use of PEDF and/or PEDF-derived polypeptides in the preparation of a product for treating pulmonary hypertension.
2. 2. Use according to claim 1, wherein the PEDF-derived polypeptide comprises the polypeptides shown in SEQ ID No.1 and SEQ ID No. 2.
3. 3. Use according to claim 2, wherein the PEDF-derived polypeptide comprises a polypeptide as set forth in SEQ ID No. 1.
4. 4. Use according to claim 1, wherein the PEDF is a vector overexpressing PEDF.
5. 5. The use of claim 4, wherein the vector comprises a liposome, a polycationic vector, a polyanionic vector, a targeting peptide, a plasmid vector, an adenoviral vector, an adeno-associated vector, or a lentiviral vector.
6. 6. The use of claim 1, wherein the treatment of pulmonary hypertension is the inhibition of pulmonary vascular remodeling.
7. 7. The use according to claim 1, wherein the product comprises a medicament, a food product, a nutraceutical.
8. 8. A medicament for the treatment of pulmonary hypertension, comprising PEDF and/or a polypeptide derived from a PEDF source.
9. 9. The medicament for treating pulmonary hypertension according to claim 8, further comprising a pharmaceutically acceptable carrier.
10. 10. The medicament for treating pulmonary hypertension according to claim 9, wherein the carrier includes solvents, diluents, suspending agents, emulsifiers, antioxidants, pharmaceutical preservatives, colorants, flavors, vehicles, oily bases, excipients.

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