Application of pigment epithelium-derived factor in synergistically enhancing hepatic cell growth factor in resisting pulmonary arterial hypertension

Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a pigment epithelium-derived factor (PEDF) in synergistically enhancing a hepatocyte growth factor (HGF) in resisting pulmonary arterial hypertension (PH). Research finds that the HGF can improve pulmonary artery remodeling in PH, but can aggravate vascular leakage through a VEGF / VEGFR2 pathway. The PEDF can be combined with VEGFR2 (vascular endothelial growth factor receptor 2) to inhibit VEGF-induced endothelial cell activation and permeability increase. Meanwhile, a specific region of the PEDF is combined with the VEGFR2 under the action of a hydrogen bond and the like, and a derivative peptide fragment of the PEDF can block a VEGF / VEGFR2 signal. Compared with single HGF, cotransfection of the PEDF and the HGF can better inhibit angiogenesis and leakage and delay PH progress. Researches prove that the PEDF counteracts the leakage promoting side effect of the HGF through specific binding with the VEGFR2, and a theoretical basis is provided for developing a PH treatment strategy of the HGF combined with the PEDF derived peptide.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the use of pigment epithelial-derived factor to synergistically enhance hepatocyte growth factor in the treatment of pulmonary hypertension. Background Technology

[0002] Pulmonary hypertension (PH) is a fatal disease characterized by progressive distal pulmonary arterial remodeling and obstruction leading to elevated pulmonary artery pressure. Current clinical treatment for PH is limited, primarily focusing on vasodilators (such as phosphodiesterase inhibitors and prostacyclin analogs), with no drugs specifically targeting vascular remodeling. Therefore, patients with advanced PH often progress to right heart failure and die. Lung transplantation or heart-lung transplantation is considered the only effective treatment for advanced PH, but donor shortages and immune rejection limit its implementation. Therefore, finding drugs that target pulmonary artery remodeling will slow the progression of PH and effectively improve patient prognosis.

[0003] Hepatocyte growth factor (HGF) is a double-edged sword in treating pulmonary arterial remodeling and pulmonary vascular leakage in patients with pulmonary embolism (PH). HGF is a pleiotropic cytokine with pulmonary nutritional functions, primarily exerting its effects by binding to the transmembrane tyrosine kinase receptor c-Met. Previous research in our group showed that HGF expression was significantly reduced in the lungs of PH patients and experimental PH animal models. Exogenous HGF supplementation reduced distal pulmonary arterial smooth muscle proliferation, medial remodeling, and delayed PH progression; the corresponding mechanism may be related to the downregulation of endothelin-1 and transforming growth factor-β mediated by the HGF / c-Met pathway. However, HGF can also increase vascular endothelial growth factor (VEGF)-mediated immature angiogenesis and endothelial cell leakage, reducing effective perfusion of the pulmonary vascular bed, thus affecting the therapeutic effect of HGF on PH. Furthermore, as an expression product of the proto-oncogene MET, HGF / c-Met's pro-cancer effects have also been reported to be closely related to vascular endothelial regeneration and leakage. Therefore, if the adverse effects of HGF on endothelial cells can be blocked while retaining its beneficial effects on vascular smooth muscle cells, the therapeutic efficacy and safety of HGF will be greatly improved. Summary of the Invention

[0004] To overcome the shortcomings of the prior art, this invention provides the use of PEDF (NCBI accession number for protein sequence: NP_002606.3) to synergistically enhance HGF (Gene ID: 3082) in the treatment of pulmonary arterial hypertension, providing a theoretical basis for developing PH treatment strategies combining HGF and PEDF-derived peptides, and offering new insights into clinical PH treatment strategies.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides the use of PEDF-derived peptides and HGF in the preparation of drugs for treating pulmonary hypertension.

[0006] Preferably, the PEDF-derived peptide is a PEDF-derived polypeptide capable of binding to VEGFR2 and preventing leakage, including the 52mer-derived peptide shown in SEQ ID No: 2 and the 62mer-derived peptide shown in SEQ ID No: 3.

[0007] More preferably, the PEDF-derived peptide is the 52mer-derived peptide shown in SEQ ID No: 2.

[0008] A second aspect of the present invention provides an anti-pulmonary hypertension drug, the drug comprising PEDF-derived peptide and HGF.

[0009] Preferably, to enrich the application forms of the drug and make it applicable to different ranges, the drug also includes pharmaceutically acceptable excipients.

[0010] More preferably, the excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, integrators, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, antioxidants, adsorbents, filter aids, and release inhibitors.

[0011] More preferably, to improve the form of drug use, the dosage form of the drug includes tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories. The drug formulation can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or locally). If certain drugs are unstable under gastric conditions, they can be formulated as enteric-coated tablets.

[0012] Compared with the prior art, the beneficial effects of the present invention are: This invention focuses on the promoting effect of PEDF on HGF in the treatment of pulmonary hypertension (PH). HGF can improve pulmonary artery remodeling in PH, but it exacerbates vascular leakage through the VEGF / VEGFR2 pathway. PEDF can competitively bind to VEGFR2, inhibiting VEGF-induced endothelial cell activation and increased permeability. Molecular docking and mutation experiments showed that the Glu331-Ser71-Glu291 region of PEDF binds to VEGFR2 through hydrogen bonds and electrostatic interactions. Among them, the 52mer (aa280-331) and other derived peptides can effectively block VEGF / VEGFR2 signaling and stabilize endothelial junctions. Co-transfection of PEDF and HGF genes into a PH rat model showed that it inhibited angiogenesis and leakage more effectively than HGF alone, and enhanced the delaying effect on PH progression. The study confirms that PEDF counteracts the pro-angiogenic and permeable side effects of HGF by specifically binding to VEGFR2, providing a theoretical basis for developing a PH treatment strategy combining HGF and PEDF-derived peptides, and offering new insights into clinical PH treatment strategies. Attached Figure Description

[0013] Figure 1 To demonstrate that simultaneous administration of hepatocyte growth factor (HGF) and pigment epithelial-derived factor (PEDF) better alleviates the development of pulmonary hypertension (PH) than administration of either alone; (A) Experimental strategy for inducing a rat PH model with monoclonal antibody (MCT): MCT (60 mg / kg) was subcutaneously injected into four square points on the abdomen of rats to establish a PH model. Two weeks later, recombinant adenovirus (Ad) carrying an HGF overexpression plasmid (Ad-HGF) or Ad-PEDF was infused into the rat trachea. Subsequent detection and tissue collection were performed two weeks after Ad injection; (B and C) Representative immunohistochemical staining of rat lung tissue to confirm the overexpression of PEDF and HGF after (B) Ad delivery. Scale bar: 50 mm; (D) Quantitative analysis of PEDF mRNA levels in lung tissue of rats in the control group, HGF group, PEDF group, and HGF+PEDF group by qRT-PCR; (E) Representative immunoblotting and quantitative analysis of PEDF protein expression in rat lung tissue (n=4 rats per group); (F) Hemodynamic measurements of systolic, diastolic, and mean PA pressures in the pulmonary artery (PA) of sham rats and MCT-induced PH rats; (G) Measurement of the ratio of heart weight to body weight; (H) The ratio of right ventricle (RV) to left ventricle (LV) plus interventricular septum weight [RV / (LV1S)] was also determined in the sham group and MCT-induced PH group (5-6 rats per group); *P, 0.05 and **P, 0.01; BW = body weight; dPAP = diastolic pressure; HW = heart weight; mPAP = mean PA pressure; sPAP = systolic pressure.

[0014] Figure 2RV function was significantly improved in PH rats treated with Ad-HGF and Ad-PEDF; (A) Representative tissue and Doppler echocardiographic images (upper part of each image) assessing RV systolic function by lateral tricuspid annular systolic velocity S9 (S9 wave velocity) and M-mode assessment images (lower part of each image) of tricuspid annular plane systolic deviation (TAPSE) in the sham group, control group, HGF group, PEDF group, and HGF1PEDF group; (B) Representative echocardiographic images of RV area change (FAC) and RV diastolic size (RVDds) assessed by B-mode; (CH) Echocardiograms of sham rats and MCT-induced PH rats (n=5-6 per group), respectively (C) S9 wave velocity, (D) TAPSE, (E) RV Quantitative analysis of FAC, (F)RVDd principal axis dimensions, (G)RVDd mid-cavity dimensions and (H)RVDd base dimensions; *P, 0.05 and **P, 0.01; Base = base dimension; Maj = principal axis dimension; Mid = mid-cavity dimension; RV = right ventricle.

[0015] Figure 3 To demonstrate that PEDF delivery reduces immature angiogenesis and promotes vascular perfusion in PH lungs with high HGF expression; (A) Representative immunostaining of CD31 (endothelial cell [EC] marker) and α-smooth muscle actin (α-SMA; smooth muscle cell marker) in rat lung tissue vessels, with DAPI dye used to label cell nuclei, scale bar 50 mm; (BD) Quantitative analysis of the density of (B) CD311 vessels and (C) CD311 α-SMA1 vessels per high power field (hpf) in the sham group, control group, HGF group, and HGF+PEDF group, and (D) the ratio of CD311 α-SMA1 vessels to total CD311 vessels; (E) Representative staining and quantitative assessment of vascular endothelial growth factor (VEGF) in MCT-induced PH rat lung tissue, scale bar 50 mm; (F) Representative staining and quantitative assessment of lectin-FITC labeled effective perfusion vessels per hpf, scale bar 50 mm.

[0016] Figure 4To demonstrate that PEDF can improve HGF-induced vascular leakage and VE-cadherin linkage breakdown in rat PH lung tissue; (A) Representative staining of α-SMA and fibrinogen in the sham group, control group, HGF group, and PEDF group, and quantification (percentage) of extravascular fibrinogen exudation per hpf, scale bar at 50 mm, arrows indicating fibrinogen deposition sites; (B) Measurement of Evans blue dye concentration in lung homogenate; (C) Representative staining of VE-cadherin and CD31 in rat lung tissue, scale line at 10 mm; (D and E) Quantitative assessment of (D) VE-cadherin area at cell-cell junctions and (E) total VE-cadherin area per hpf (percentage). (F) Representative immunoblots of VE-cadherin (cytoplasm and cell membrane), ZO-1, and occludin in lung tissues of rats in the control group, PEDF group, HGF group, and HGF+PEDF group; (GJ) Quantitative analysis of related proteins in PH lung tissue, where (G) is the relative content of VE-cadherin in the cytoplasm, (H) is the proportion of VE-cadherin on the cell membrane, (I) is the relative expression level of ZO-1, and (J) is the relative expression level of occludin protein. Four rats were used in each group (n=4); *P, 0.05 and **P, 0.01, RFU = relative fluorescence units.

[0017] Figure 5To investigate whether the effect of PEDF on the function of rat PH lung ECs under HGF treatment is regulated by tyrosine (Y) phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 and Y1175 sites; (A) Representative immunostaining of Ki67 in rat lung ECs of sham group, control group, HGF group, HGF+PEDF group, lentivirus group carrying interfering RNA, siRNA group targeting ATGL (lipoglycerol triglyceride lipase) gene (si-ATGL group), si-lamin receptor group or si-VEGFR2 treated HGF+PEDF group, with scale bars at 100 mm; (B) Representative staining of VE-cadherin and CD31 in isolated rat lung ECs and quantitative analysis of VE-cadherin at cell-cell junctions, with scale bars at 10 mm. mm; (C) Representative immunoblots of VEGFR2 phosphorylation at Y951 (pY951), pY1175, pY1214, Met, and Met pY1234 / 5 in lung ECs induced by sham or MCT in PH rats; (DG) Quantitative analysis of VEGFR2 at (D) pY951, (E) pY1175, (F) pY1214, and (G) Met pY1234 / 5 in lung ECs (4 independent experiments per group); *P, 0.05 and **P, 0.01; LR = laminin receptor.

[0018] Figure 6PEDF inactivates VEGFR2 Y951 and Y1175 in pulmonary ECs from patients with idiopathic phlebitis (PH); (A) Representative staining of CD31 and α-SMA in PA lung tissue from patients with idiopathic phlebitis (PH), scale bar 100 mm, asterisks indicate bronchi, right side magnified image; (B) Representative immunohistochemical staining of HGF and PEDF in lung tissue from patients with PH, asterisks indicate remodeled vessels, scale line 100 mm; (C) Determination of HGF and PEDF concentrations in serum of healthy individuals and PH patients by enzyme-linked immunosorbent assay (ELISA) (6-7 individuals per group); (D) Experimental strategy for pulmonary ECs from patients with idiopathic phlebitis (PH): pulmonary ECs were isolated and continuously exposed to a hypoxic environment for 24 hours, then treated with Ad-HGF and Ad-PEDF for 24 hours, and then the cells were harvested for further study; (E) Representative immunoblots of HGF, PEDF, Met, and VEGFR2 in human PH lung ECs control group, PEDF group, HGF group, and HGF1PEDF group; (F) Representative images of lung EC proliferation in control group, PEDF group, HGF group, and HGF+PEDF group, scale bar 100 mm; (G) Representative immunostaining of VE-cadherin and CD31 in human PH lung ECs and intercellular localization and quantification of VE-cadherin, scale bar 10 mm; (H) Representative immunoblots of VEGFR2, VEGFR2 pY951, pY1175, and pY1214 in PH lung ECs; (IK) Quantification of VEGFR2 (I) pY951, (J) pY1175, and (K) pY1214 in human lung ECs (n=4, independent experiments for each group); *P, 0.05 and **P, 0.01.

[0019] Figure 7To promote the stability of the VEGFR2 / VEPTP / VE-cadherin complex and the inactivation of Akt and Erk1 / 2 by targeting VEGFR2 with PEDF; (A) Formation of the VEGFR2 / VEPTP / VE-cadherin complex in human PH lung ECs overexpressing PEDF, and representative immunoblots of PEDF, VEGFR2, T cell-specific aptamers, VEPTP, and VE-cadherin with VEGFR2, with normal rabbit IgG as the negative control; (B) Representative immunoblots of Src, Src pY418, VE-cadherin, VE-cadherin pY685, Akt, Akt pS473, Erk1 / 2, and Erk1 / 2 pT202 / Y204 in total cell lysate; (CF) Immunoprecipitation analysis of (C) PEDF / VEGFR2, (D) TSAd / VEGFR2, and (E) VEPTP / VEGFR2. Quantification of the ratio of (F) VE-cadherin / VEGFR2; Quantification of the ratios of (G) Src pY418 / Src, (H) VE-cadherin pY685 / VE-cadherin, (I) Akt pS473 / Akt, and (J) Erk1 / 2 pT202 / Y204 / Erk1 / 2 in immunoblotting analysis (n=4, independent experiments for each group); **P, 0.01. IB = immunoblotting; IP = immunoprecipitation; TSAd = T cell-specific aptamers.

[0020] Figure 8 The values ​​of RMSD(a) and Rg(b) of the VEGFR2-PEDF system during the simulation process vary with simulation time.

[0021] Figure 9 The flexible distribution of the protein (a: RMSF distribution of Cα atoms in VEGFR2 during the simulation; b: RMSF distribution of Cα atoms in PEDF during the simulation; c: Location of regions of greater protein flexibility in the complex structure).

[0022] Figure 10 Statistics on the number of hydrogen bonds between VEGFR2 and PEDF during the simulation process (a) and changes in binding energy (b).

[0023] Figure 11 The interaction between the hydrophilic and hydrophobic surfaces of VEGFR2 and PEDF proteins (blue and orange surfaces represent the hydrophilic and hydrophobic regions of the protein surface, respectively).

[0024] Figure 12This is a binding model for the docking of VEGFR2 and PEDF proteins; the cyan limon cartoon represents VEGFR2, the gray represents PEDF, the purple and blue represent the amino acid residues involved in the interaction, and the green dashed line represents hydrogen bonding.

[0025] Figure 13 The distribution of stable hydrogen bonding in the interaction between VEGFR2 and PEDF on the protein surface (blue and red surfaces represent the positive and negative electrostatic potentials of the PEDF protein surface, respectively, and yellow sticks represent amino acid residues in VEGFR2). Figure 14 Figure 1 shows the results of Western blot (WB) and intraepithelial immunoassay (IP) experiments, as well as functional validation experiments. These results were used to investigate the effects of the peptide on the vascular endothelial growth factor receptor 2 (VEGFR2) pathway and endothelial function under hypoxic conditions, providing a basis for studying its therapeutic potential for hypoxia. A: IP binding to WB results, used to verify the binding of the peptide to VEGFR2, detecting VEGFR2, His tag, VEGF, and other proteins to clarify the peptide's binding to VEGFR2 and its effect on VEGF-VEGFR2 binding. B: WB results, detecting phosphorylated VE-cadherin (p-VE-cadherin) and the internal reference β-actin, exploring the effect of the peptide on VE-cadherin phosphorylation. C: Statistical results of endothelial permeability testing (based on the dextran-rhodamine transwell assay), reflecting changes in endothelial permeability under different treatments. Detailed Implementation

[0026] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0027] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0028] Example 1: PEDF blocks the adverse effects of HGF on PH pulmonary artery endothelial cells and improves PH Pigment epithelium-derived factor (PEDF) is an endogenous, secreted, multifunctional glycoprotein belonging to the serine protease inhibitor gene family. PEDF does not inhibit either serine or cysteine ​​proteases, but it exerts various physiological activities through its receptor, including angiogenesis, neurotrophic effects, cardioprotection, regulation of glucose and lipid metabolism, antioxidant effects, and anti-inflammatory and anti-tumor effects. Preliminary experimental results from this invention suggest that PEDF competitively inhibits the binding of VEGF to VEGFR2. Specifically, when pulmonary artery endothelial cells overexpressed PEDF, an immunoprecipitation assay was performed on extracted protein. Upon pulling down PEDF, an increased binding of PEDF to VEGFR2 was detected, while the binding of VEGF to VEGFR2 decreased.

[0029] The specific research content is as follows: PEDF is a highly conserved, endogenous, secreted pleofunctional glycoprotein whose function is closely related to angiogenesis and development. Our previous research group discovered that PEDF can inhibit the activation, proliferation, and endothelial leakage of myocardial endothelial cells in the circulatory system, thereby improving blood perfusion after acute myocardial infarction and reducing the occurrence of no-reflow after interventional therapy. To investigate the effect of PEDF on myocardial infarction (PH), we used monoclonal induced PH rats and endothelial cells isolated from rat and human PH lung tissues to evaluate PH progression, right ventricular function, and lung perfusion in PH rats treated with HGF and / or PEDF. Furthermore, we investigated the receptors and mechanisms by which PEDF plays a role in HGF-based PH treatment. We found that HGF and PEDF can synergistically prevent the occurrence of PH and improve right ventricular function in PH rats. Furthermore, PEDF can increase lung perfusion in pulmonary hysterosalpingitis (PH) lungs and inhibit HGF-induced immature angiogenesis and vascular endothelial (VE)-cadherin linkage breakdown without affecting the therapeutic inhibitory effect of HGF on pulmonary vascular remodeling. Mechanistically, PEDF targets VEGFR2, inhibiting its phosphorylation at Y951 and Y1175. Finally, the formation of the VEGFR2 / VE protein tyrosine phosphatase / VE-cadherin complex and the inactivation of Akt and Erk1 / 2 were observed in rat and human PH lung endothelial cells. These results indicate that PEDF combined with HGF has great potential for the treatment of PH.

[0030] The application of PEDF can enhance the ameliorative effect of HGF on the progression of PH. To investigate the role of PEDF in HGF-based PH treatment, an experimental PH model was established in rats via MCT injection, followed by intratracheal instillation of recombinant adenovirus (Ad) carrying human HGF (Ad-HGF) and / or PEDF (Ad-PEDF) overexpression plasmids (Ad-PEDF). Figure 1 A). Immunohistochemical staining confirmed the successful overexpression of HGF and PEDF. Figure 1 B and 1C). Notably, Ad-HGF administration had no effect on PEDF expression in PH lung tissue, either endogenously or after Ad-PEDF perfusion (B and C). Figure 1 (D and 1E). Furthermore, hemodynamic assessments and the Fulton index also indicated that, compared to HGF or PEDF alone, HGF+PEDF significantly reduced PA pressure, the heart weight-to-body weight ratio, and the right ventricle (RV) to left ventricle plus interventricular septum weight ratio. Figure 1 F-1H).

[0031] Based on the confirmed effect that the combination of HGF and PEDF improves right ventricular function and dilation in MCT-induced PH rats, we subsequently evaluated the role of PEDF in right ventricular function in HGF-treated PH rats. Figure 2 As shown in A and 2B, although overexpression of HGF or PEDF alone alleviated right ventricular systolic dysfunction and dilation to some extent (manifested as increased S9 wave velocity of the lateral tricuspid annulus systolic velocity, increased tricuspid annulus plane systolic deviation, and decreased right ventricular diastolic long axis size), combined therapy significantly improved almost all cardiac function parameters. Figure 2 (C-2H). In summary, these results indicate that PEDF can enhance the efficacy of HGF in inhibiting the progression of PH and right heart failure.

[0032] PEDF inhibited HGF-induced angiogenesis and improved vascular perfusion in pH-treated lungs of pH-treated rats. Considering the important role of PEDF in promoting EC apoptosis, we next investigated the effect of PEDF on angiogenesis induced by HGF-based pH therapy. Immunofluorescence staining of blood vessels in rat lung tissue showed that HGF delivery significantly increased total vascular density, which was reversed by PEDF without altering the density of the microcardiomyocytes. Figure 3 Interestingly, this anti-angiogenic effect of PPEDF ultimately led to a sharp increase in the proportion of microcardiae in pH lungs treated with HGF (A-3C). Figure 3 D). Additionally, PEDF treatment can alleviate intravascular obstruction and the increase in VEGF levels (D). Figure 3A and 3E). The lectin perfusion assay showed that even though HGF partially increased pulmonary vascular perfusion, an improvement in insufficient vascular perfusion was also observed in the HGF+PEDF group ( Figure 3 In summary, these findings suggest that PEDF improves poor lung perfusion without impairing the primary therapeutic effect of HGF in MCT-induced pH rats.

[0033] PEDF treatment inhibited HGF-induced vascular leakage and improved the intercellular localization of VE-cadherin. We then sought to determine the role of PEDF in vascular function (e.g., vascular leakage) in HGF-treated PH lungs. Figure 4 As shown in A and 4B, significant vascular leakage was observed in both the control group and the HGF group, manifested as increased extravascular fibrinogen exudation. After PEDF treatment, the vascular leakage was significantly reversed as measured by Evans blue dye. VE-cadherin, as a component of the EC-cell adhesion junction, showed reduced localization at the cell junction in the HGF group. Figure 4 C and 4D), although the total content of VE-cadherin was increased compared with the control group ( Figure 4 E). Regardless of HGF treatment, administration of PEDF significantly improved intercellular localization and total VE-cadherin levels (E). Figure 4 F-4H). Furthermore, PEDF did not affect the expression of occludin (ZO-1 and occludin) in MCT-induced rat PH lung tissue. Figure 4 (I and 4J). These results indicate that in HGF-treated PH lungs, the application of PEDF reduces angiogenesis and vascular leakage, and enhances the barrier function of ECs and the intercellular localization of VE-cadherin.

[0034] The regulatory effect of PEDF on EC function may be related to the inactivation of VEGFR2 pY951 and pY1175. For example... Figure 5 As shown in A and 5B, the HGF group promoted EC proliferation and the disintegration of VE-cadherin linkages, while PEDF overexpression inhibited these processes. VEGFR2 was involved in these processes, but ATGL and LR were not. Furthermore, in ECs isolated from MCT-induced PH lungs (but not pseudolungs), VEGFR2 pY951, pY1175, and pY1214 were all increased, while PEDF significantly inhibited the increase of VEGFR2 pY951 and pY1175 in HGF-treated ECs. Figure 5 C-5F). Importantly, PEDF treatment had no effect on HGF-induced upregulation of Met pY1234 / 5 (C-5F). Figure 5 G).

[0035] We subsequently obtained lung tissue from human idiopathic PH patients, confirming significant thickening and muscularization of the pulmonary vascular walls. Figure 6 A), and HGF and PEDF levels were low ( Figure 6 B and 6C), further confirming the role of PEDF overexpression in the progression of human PH. ECs were isolated from human PH lung tissue, and the cells were maintained in a hypoxic environment before being treated with Ad-HGF and Ad-PEDF to overexpress HGF and PEDF (B and 6C). Figure 6 D and 6E). Corresponding to the in vitro experimental results, the in vitro experimental results showed that EC proliferation and VE-cadherin linkage disintegration were reduced in the PEDF group and the HGF+PEDF group (D and 6E). Figure 6 PEDF can also inactivate VEGFR2pY951 and pY1175, but not pY1214, regardless of whether HGF is involved. Figure 6 (H-6K). Therefore, the regulatory effect of PEDF on EC function and VE-cadherin degradation may be related to the inhibition of VEGFR2 pY951 and pY1175 in rat and human PH lung ECs.

[0036] PEDF interacts with vascular endothelial growth factor receptor 2 (VEGF2) to maintain the stability of the VEGF2 / VEGF protein tyrosine phosphatase / VE-Cadherin complex and interferes with the activation of Akt and Erk1 / 2 in human PH lung ECs. The Y951 phosphorylation site in VEGF2 is a specific binding site for a T-cell-specific aptamer that regulates the VEGF2-dependent Src pathway at the EC junction, which is involved in VEGF-induced vascular permeability. Furthermore, pY1175 can also bind phospholipase Cg and plays an important role in the activation of endothelial Akt and Erk1 / 2 signaling. Therefore, we next investigated the molecular mechanism by which PEDF affects these VEGFR2 phosphate sites in human PH lung ECs using immunoprecipitation and Western blotting experiments. Figure 7 A and 7B). We observed increased VEGF-induced VEGFR2 / TSAd complex formation in both the control and HGF groups ( Figure 7 C) Overexpression of PEDF leads to excessive binding to VEGFR2 and restricts TSAd anchoring ( Figure 7D). Since vascular endothelial protein tyrosine phosphatase (VEPTP) also acts on VEGFR2 and VE-cadherin at the endothelial junction, inhibiting VEGFR2 phosphorylation, we evaluated the effect of PEDF on the binding of VEPTP and VE-cadherin to VEGFR2. Regardless of HGF treatment, PEDF significantly promoted the binding of the VEGFR2 / VEPTP complex ( Figure 7 E) and VEGFR2 / VE-cadherin complex ( Figure 7 The formation of F). Furthermore, overexpression of PEDF also inhibited the phosphorylation of Src (pY418), VE-cadherin (pY685), Akt (pS473), and Erk1 / 2 (pT202 / Y204). Figure 7 (G-7J). In summary, these results indicate that PEDF promotes the formation of the VEGFR2 / VEPTP / VE-cadherin complex and the inactivation of the Akt and Erk1 / 2 pathways in human PH lung ECs.

[0037] However, given the unique pharmacological and physiological functions of HGF and PEDF, simultaneously introducing both large protein molecules into the body may pose unexpected therapeutic risks and exhibit stronger immunogenicity compared to introducing a single protein drug. Therefore, replacing the large protein molecules with equivalent peptides is an effective strategy to mitigate these risks. Currently, HGF is believed to have only one c-Met receptor, while PEDF, in addition to interacting with the VEGFR2 receptor, can also bind to adipose triglyceride lipase (ATGL), laminin receptor (LR), heparin, and type I collagen to exert other functions. Furthermore, PEDF-derived 34-mer (aa24-57) and 44-mer (aa78-121) peptides have been reported to exert anti-angiogenic and cardiomyocyte / neuronal protective effects comparable to PEDF through receptors such as ATGL, but PEDF-derived peptides that bind to VEGFR2 to exert anti-vascular leakage functions have not yet been designed. Therefore, designing and customizing PEDF peptides that specifically bind to VEGFR2 will maximize the efficacy and safety of HGF in the treatment of PH.

[0038] Example 2: Molecular docking analysis for screening PEDF peptides 1. Simulation Method 1.1 System Preparation To identify functional PEDF-derived peptides that bind to VEGFR2, this experiment first employed protein-protein docking analysis to conduct molecular simulations of PEDF and VEGFR2, exploring the driving forces behind their recognition and binding. Currently, the binding site between VEGFR2 and PEDF is unknown, but information on the binding site between VEGFR2 and its ligand VEGF is available; therefore, it was considered that the binding site of PEDF is near VEGF. Then, ZDOCK 3.0.2 was used to dock PEDF to the ligand-binding region of the VEGFR2 protein, and molecular dynamics (MD) simulations were employed to study the structure of the complex and obtain a stable VEGFR2-PEDF binding conformation.

[0039] 2. Protein-protein docking To investigate the binding mode of VEGFR2 and PEDF, this study used the ZDOCK 3.0.2 program to dock PEDF to the ligand-binding region of the VEGFR2 protein. ZDOCK is a protein-protein docking method based on fast Fourier transform correlation technology, which does not change the conformation of the protein during the calculation, only its spatial position. Finally, the conformation with the highest score was selected using the ZDOCK Score function. The ZDOCK Score can comprehensively evaluate the shape complementarity, electrostatic potential, and energy matching between proteins.

[0040] MD simulation To obtain a stable binding conformation of VEGFR2-PEDF, this study employed molecular dynamics (MD) simulations to study the complex structure. The initial structure used in the MD simulation was the docked VEGFR2-PEDF complex. The simulation was conducted using the Amber 18 software package, with the force field set to Amber14SB. All force field parameters of the protein were fitted based on experimental values. The simulation temperature was 300 K, the pH was 7.0, and the TIP3P water model was used as the solvent. A 1.0 nm cubic water box was first added around the solute, followed by the filling with water solvent.

[0041] Before the MD simulation, the system underwent two energy optimizations: first, the solute was constrained, and the aqueous solvent was optimized for 5000 steps using both the steepest descent method and the conjugate gradient method; then, the constraint was removed, and the entire system was optimized for 5000 steps using both methods. The MD simulation was also performed in two steps: first, a 100 ps constrained solute MD simulation was conducted, with the system temperature gradually increased from 0 K to 300 K; then, a 50 ns unconstrained isothermal MD simulation was performed. During the simulation, the SHAKE algorithm was used to constrain bond lengths, with an integration step size of 2 fs. Conformations were collected every 10 ps, ​​for a total of 5000 conformations. The VMD software package was used to monitor the dynamic process of the MD simulation.

[0042] Results and Analysis 3.1 Analysis of Convergence Parameters in MD Simulation The root mean square deviation (RMSD) represents the sum of the atomic deviations of the conformation at a certain moment and the target conformation, and is an important criterion for measuring the stability of a system. Figure 8 Figure a shows the change in RMSD of Cα atoms in the VEGFR2-PEDF system over time. It can be seen from the figure that the system becomes essentially stationary after 30 ns, with an RMSD value of 0.418 ± 0.032 nm. The radius of gyration (Rg) can represent the looseness of the system and is also one of the criteria for measuring the stability of the system. Figure 8 Figure b shows the variation of the Rg value of the complex system during the simulation. As can be seen from the figure, the radius of gyration of the system fluctuates around 2.75 nm, and the average Rg value during the MD simulation is 2.756 ± 0.014 nm. Overall, the VEGFR2-PEDF complex system reached a stable state during the MD simulation.

[0043] Protein flexibility analysis Root mean square fluctuation (RMSF) represents the flexibility of amino acid residues in a protein; a higher RMSF value indicates greater flexibility of the corresponding amino acid residue. The amino acid residue flexibility distribution in VEGFR2 and PEDF proteins is shown in the figure. Figure 9 As shown in the figure, the overall flexibility of the two proteins is relatively low, with only three amino acid regions exhibiting greater flexibility: C200~Q210 in VEGFR2, and K220~K225, G370, and A380 in PEDF. Figure 9 a and Figure 9 b). Figure 9Figure c shows the positions of three relatively flexible regions in the three-dimensional structure. As can be seen from the figure, all three regions are located on the periphery of the protein structure and have a section of random coil structure. They are subjected to relatively strong solvent forces during the MD simulation, hence their high flexibility.

[0044] Molecular recognition of VEGFR2 and PEDF The structure and function of proteins are closely related to hydrogen bonding between amino acids. Hydrogen bonds are crucial for the formation of protein secondary structure and are also an important form of protein-protein interaction. Therefore, given the important role of hydrogen bonds, this study statistically analyzed the number of hydrogen bonds in VEGFR2 and PEDF during the simulation process. Figure 10 As shown in figure a, after 30 ns, the number of hydrogen bonds between VEGFR2 and PEDF stabilizes at around 6. Table 1 shows the distribution of relatively stable hydrogen bonds between VEGFR2 and PEDF during MD simulation. As can be seen from the table, most of the hydrogen bonds are distributed between charged amino acids, such as Glu331 of PEDF and Arg275 of VEGFR2.

[0045] Binding energy can represent the magnitude of intermolecular affinity. In MD simulations, the binding energy between VEGFR2 and PEDF is as follows: Figure 10 As shown in figure b, the binding energy between the two molecules stabilizes after 30 ns, with an average value of -2472.05 ± 194.16 kJ / mol. Analysis of hydrogen bonds and binding energies indicates that VEGFR2 and PEDF can stably bind during MD simulations, with hydrogen bonds likely being one of the key driving forces for their binding.

[0046] No Acceptor Donor Distance (Å) Angle (°) Frequency 1 PEDF-Ser71-O VEGFR2-Asp276-NH 2.88 162.32 59.64% 2 PEDF-Glu291-OE1 VEGFR2-Gln280-NE2-HE22 2.82 161.97 49.15% 3 PEDF-Glu331-OE2 VEGFR2-Arg275-NH2-HH22 2.80 158.04 46.75% 4 PEDF-Glu331-OE1 VEGFR2-Arg275-NH1-HH12 2.79 161.34 44.66% 5 PEDF-Glu331-OE2 VEGFR2-Arg275-NH1-HH12 2.79 159.82 41.06% 6 PEDF-Glu291-OE2 VEGFR2-Gln280-NE2-HE22 2.81 162.55 35.56% 7 PEDF-Glu331-OE1 VEGFR2-Arg275-NH2-HH22 2.81 159.63 35.06% 4.4. Combination Mode of VEGFR2 and PEDF To further investigate the molecular recognition pattern between VEGFR2 and PEDF, Figure 11 The binding mode of VEGFR2 to the hydrophilic and hydrophobic surfaces of the PEDF protein after MD simulation is presented. As can be seen from the figure, there are both hydrophilic and hydrophobic regions at the interface where they bind, indicating that both hydrophilic and hydrophobic interactions play important roles in the binding process.

[0047] To further investigate the binding mode of VEGFR2 and PEDF protein, Figure 12 The amino acid residue distribution at the binding interface between the two is shown. As can be seen from the figure, The amino acid residues involved in the binding of PEDF to these two substances mainly include Arg69, Ser71, Thr72, Pro74, Thr75, Asp201, Glu202, Tyr232, Glu235, Glu236, Arg237, Leu255, Lys262, Leu280, Lys281, Val283, Thr284, Asn285, Thr287, Glu291, Lys327, Glu331, and Asp387. The amino acid residues involved in VEGFR2 binding are mainly His133, Tyr137, Arg164, Tyr165, Pro166, Ser193, Tyr194, Ala195, Met213, Ile215, Val216, Val217, Val218, Asn253, Ile256, Asp257, Gln268, Lys271, Val273, Asn274, Arg275, Asp276, Lys278, Gln280, Lys286, Phe288, and Leu313. The distribution of amino acid residues at the binding interface shows a high number of charged amino acid residues (such as Arg, Lys, Asp, and Glu) at the interface, suggesting that hydrogen bonding and electrostatic interactions are the key driving forces behind the recognition between VEGFR2 and PEDF molecules. In addition, the presence of numerous hydrophobic amino acids (such as Leu and Val) at the interface can further enhance the affinity between the two.

[0048] Figure 13 The distribution of relatively stable hydrogen bonds on the protein surface during MD simulations is presented. As can be seen from the figure, the relatively stable hydrogen bonds Glu291-Gln280, Ser71-Asp276, and Glu331-Arg275 in the interaction are all present in the same region on the PEDF protein surface. This region exhibits a strong negative electrostatic potential, while the positively charged Arg275 in VEGFR2 can form a strong electrostatic attraction with this region. Therefore, based on the MD simulation results and the analysis of the binding mode, it can be inferred that the strongly negatively charged region formed by Ser71-Glu291-Glu331 in PEDF is the key region for the binding of the two proteins.

[0049] in conclusion This paper constructs a three-dimensional model of the VEGFR2-PEDF complex using a protein-protein docking method, and verifies the model's stability in aqueous solvents using molecular dynamics (MD) simulations. The results show that the docked VEGFR2-PEDF complex remains stable during the simulation. Analysis of hydrogen bonds, binding energies, and binding modes during the MD simulation indicates that hydrogen bonding and hydrophobic interactions are crucial forces for the molecular recognition of VEGFR2 and PEDF, and the negatively charged region of PEDF, Ser71-Glu291-Glu331, plays a vital role in their binding. The simulation results provide theoretical guidance for subsequent research on molecular recognition based on VEGFR2 and PEDF.

[0050] Example 3: PEDF-derived peptides bind to VEGFR2 PEDF in brief: PEDF is a secretory glycoprotein with a molecular weight of 50 kDa, first discovered and purified from the secretory products of cultured human retinal pigment epithelium (RPE) cells. The human PEDF protein-coding gene is located at the end of the short arm of human chromosome 17, and consists of a 418-amino acid polypeptide, belonging to the serine protease non-inhibitor superfamily. It is widely expressed in many tissues and organs in the human body, including the eye, brain, spinal cord, bone, liver, heart, and lungs, playing a crucial role in pathophysiology. PEDF is a multi-binding, pleiotropic protein capable of various biological functions, including inhibiting endothelial cell migration and angiogenesis, anti-tumor, anti-inflammatory, antioxidant, neurotrophic, anti-necrosis, and anti-apoptotic effects. PEDF's heparin-binding and collagen-binding sites allow it to deposit in the interstitial tissue.

[0051] This invention demonstrates that PEDF is highly expressed in lung tissue and is an important factor in regulating the pulmonary environment and protecting blood vessels. Our previous studies have also proven that PEDF has a potent protective effect against pulmonary hypertension. If it could be used in pharmaceuticals, it would undoubtedly be a boon to many patients with pulmonary hypertension. However, due to its large molecular weight, PEDF is difficult to manufacture and absorb. Furthermore, as a multifunctional protein, although PEDF has multiple therapeutic effects on pulmonary hypertension, its clinical application remains challenging. Protein-derived peptide drugs, with their advantages of small molecular weight, high specificity, and low immunogenicity, are widely used.

[0052] To identify PEDF-derived peptides that can bind to VEGFR2 and prevent leakage, we synthesized three peptides based on the key and minor binding sites of PEDF-VEGFR2. Following the principle of minimizing amino acid sequence length while including as many key and minor sites as possible, all binding amino acid sites were divided into three enrichment regions: aa69-75, aa201-262, and aa280-331. Therefore, we synthesized three peptides: 7mer (aa69-75), 62mer (aa201-262), and 52mer (aa280-331).

[0053] The research in this invention involves 7mer, 52mer, and 62mer as follows: 1) 7mer: Sequence (aa 69-75: RSSMSPT, SEQ ID No: 1). This peptide includes the key site Ser71 and participates in binding sites Arg69, Thr72, Pro74, and Thr75. This peptide is present in the 34mer (aa44-77) peptide, which is a known peptide first proposed in 1999. It has been subsequently shown to bind to the membrane receptor LR and exert anti-inflammatory and anti-angiogenic effects; in addition, it has been reported to promote endothelial cell apoptosis, inhibit endothelial cell migration activity, and inhibit endothelial cell tube formation. Recently, our research group (Zhang H, Li Z, Quan X, et al. Strategies to attenuate myocardial infarction and no-reflow through preservation of vascular integrity by pigment epithelium-derived factor[J]. Human Gene Therapy, 2022, 33(5-6): 330-345.) discovered that in an acute myocardial infarction rat model, PEDF binds to and activates LR receptors, which helps improve ischemia-induced vascular leakage. Furthermore, by inhibiting Src and VE-cadherin phosphorylation, it helps improve hypoxia-induced vascular leakage, suggesting that 34mer may have an anti-vascular leakage effect.

[0054] 2) 52mer: Sequence (aa280-331: MFVFLPDDVTTNTTLLEESLTAEFVQDLSMA LQPARVSLTLPVLKLSYSIDLLS, SEQ ID No: 2). This peptide includes key binding sites Glu291 and Glu331, and sites involved in their binding: Leu280, Lys281, Val283, Thr284, Asn285, Thr287, and Lys327. This peptide has not been previously reported. A few amino acid residues it contains have been reported to be associated with type I collagen binding, such as 290, 291, 296, 299, and 300; in addition, site 285 has been reported to be associated with PEDF secretion activity.

[0055] 3) 62mer: Sequence (aa 201-262: FLTNGLPRIAGINAISAAYFKGGWVTRFSQ GGVQQDFQVENGAPVRVPMMQQDNYPVKMGVD, SEQ ID No: 3). This peptide includes binding sites Asp201, Glu202, Tyr232, Glu235, Glu236, Arg237, Leu255, and Lys262. This peptide has not been previously reported. Amino acid residues 212 and 214 play important roles in the binding of PEDF to glycosaminoglycans, and phosphorylation at site 227 participates in the anti-angiogenic effect of PEDF.

[0056] To verify whether these peptides could bind to VEGFR2, we added four synthesized peptides at different concentrations to HPAEC medium. After hypoxia intervention, the extracted total cell protein was used for immunoprecipitation experiments. The results showed that hypoxia significantly increased the binding of VEGFR2 and VEGF. Furthermore, 7-mer, 52-mer, and 62-mer peptides could all bind to VEGFR2. When the binding of peptides to VEGFR2 increased, the binding of VEGF-VEGFR2 decreased, with 52-mer showing the strongest effect. Figure 14 A). To further investigate the effects of peptides on VEGF-induced phosphorylation activation of VEGFR2 and VE-cadherin, and the disruption of endothelial cell junctions, we used Western blotting to detect VE-cadherin phosphorylation expression (A). Figure 14 B), endothelial permeability was detected by the dextran-rhodamine transwell assay across a single endothelium. Figure 14 (C). The results showed that 52mer significantly inhibited VEGFR2 and VE-cadherin phosphorylation and suppressed leakage. This suggests that 52mer may have potential for PH treatment.

[0057] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. Application of PEDF-derived peptides and HGF in the preparation of drugs for pulmonary hypertension.

2. The application according to claim 1, characterized in that, The PEDF-derived peptides are PEDF-derived polypeptides that can bind to VEGFR2 and resist leakage, including the 52mer-derived peptide shown in SEQ ID No: 2 and the 62mer-derived peptide shown in SEQ ID No:

3.

3. The application according to claim 2, characterized in that, The PEDF-derived peptide is the 52mer-derived peptide shown in SEQ ID No:

2.

4. A drug for treating pulmonary hypertension, characterized in that, The drug includes PEDF-derived peptides and HGF.

5. The anti-pulmonary hypertension drug according to claim 4, characterized in that, The drug also includes pharmaceutically acceptable excipients.

6. The anti-pulmonary hypertension drug according to claim 5, characterized in that, The excipients include at least one of the following: excipients, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, binding agents, penetration promoters, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculation agents, antioxidants, adsorbents, filter aids, and release inhibitors.

7. The anti-pulmonary hypertension drug according to claim 5, characterized in that, The dosage forms of the drug include tablets, capsules, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal preparations, or suppositories.

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