Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine.

Inhibitors of the interaction between Neuropilin-1 (NRP-1) and Vascular Endothelial Growth Factor-A165 (VEGF-A165) hold significant promise as therapeutic and diagnostic agents directed against cancers overexpressing NRP-1. In our efforts in this field, a few series of strong and fairly stable peptide-like inhibitors of the general formula Lys(Har)1-Xaa2-Xaa3-Arg4 have been previously discovered. In the current work, we focused on Lys(Har)-Dap/Dab-Pro-Arg sequence. The aim was to examine whether replacing C-terminal Arg with its homologs and mimetics would yield more stable yet still potent inhibitors. Upon considering the results of modelling and other factors, ten novel analogues with Xaa4 = homoarginine (Har), 2-amino-4-guanidino-butyric acid (Agb), 2-amino-3-guanidino-propionic acid (Agp), citrulline (Cit), 4-aminomethyl-phenylalanine [Phe(4-CH2-NH2)] were designed, synthesized and evaluated. Two of the proposed modifications resulted in inhibitors with activity slightly lower [e.g. IC50 = 14.3 µM for Lys(Har)-Dab-Pro-Har and IC50 = 19.8 µM for Lys(Har)-Dab-Pro-Phe(4-CH2-NH2)] than the parent compounds [e.g. IC50 = 4.7 µM for Lys(Har)-Dab-Pro-Arg]. What was a surprise to us, the proteolytic stability depended more on position two of the sequence than on position four. The Dab2-analogues exhibited half-life times beyond 60 h. Our results build up the knowledge on the structural requirements that effective VEGF-A165/NRP-1 inhibitors should fulfil.

Gene Therapeutic Drug pCMV-VEGF165 Plasmid ('Neovasculgen') Promotes Gingiva Soft Tissue Augmentation in Rabbits.

Currently, an increasing number of patients are undergoing extensive surgeries to restore the mucosa of the gums in the area of recessions. The use of a connective tissue graft from the palate is the gold standard of such surgical treatment, but complications, especially in cases of extensive defects, have led to the development of approaches using xenogeneic collagen matrices and methods to stimulate their regenerative and vasculogenic potential. This study investigated the potential of a xenogeneic scaffold derived from porcine skin Mucoderm and injections of the pCMV-VEGF165 plasmid ('Neovasculgen') to enhance soft gingival tissue volume and vascularization in an experimental model in rabbits. In vitro studies demonstrated the biocompatibility of the matrix and plasmid with gingival mesenchymal stem cells, showing no toxic effects and supporting cell viability and metabolic activity. In the in vivo experiment, the combination of Mucoderm and the pCMV-VEGF165 plasmid (0.12 mg) synergistically promoted tissue proliferation and vascularization. The thickness of soft tissues at the implantation site significantly increased with the combined application (3257.8 ± 1093.5 µm). Meanwhile, in the control group, the thickness of the submucosa was 341.8 ± 65.6 µm, and after the implantation of only Mucoderm, the thickness of the submucosa was 2041.6 ± 496.8 µm. Furthermore, when using a combination of Mucoderm and the pCMV-VEGF165 plasmid, the density and diameter of blood vessels were notably augmented, with a mean value of 226.7 ± 45.9 per 1 mm2 of tissue, while in the control group, it was only 68.3 ± 17.2 per 1 mm2 of tissue. With the application of only Mucoderm, it was 131.7 ± 37.1 per 1 mm2 of tissue, and with only the pCMV-VEGF165 plasmid, it was 145 ± 37.82 per 1 mm2 of the sample. Thus, the use of the pCMV-VEGF165 plasmid ('Neovasculgen') in combination with the xenogeneic collagen matrix Mucoderm potentiated the pro-proliferative effect of the membrane and the pro-vascularization effect of the plasmid. These results indicate the promising potential of this innovative approach for clinical applications in regenerative medicine and dentistry.

A Splice Form of VEGF, a Potential Anti-Angiogenetic Form of Head and Neck Squamous Cell Cancer Inhibition.

Angiogenesis, primarily mediated by vascular endothelial growth factor (VEGF), is a fundamental step in the progression and metastasis of head and neck squamous cell carcinoma (HNSCC). Traditional anti-angiogenic therapies that target the VEGF pathway have shown promise but are often associated with significant side effects and variable efficacy due to the complexity of the angiogenic signaling pathway. This review highlights the potential of a specific VEGF splice form, VEGF165b, as an innovative therapeutic target for HNSCC. VEGF165b, unlike standard VEGF, is a natural inhibitor that binds to VEGF receptors without triggering pro-angiogenic signaling. Its distinct molecular structure and behavior suggest ways to modulate angiogenesis. This concept is particularly relevant when studying HNSCC, as introducing VEGF165b's anti-angiogenic properties offers a novel approach to understanding and potentially influencing the disease's dynamics. The review synthesizes experimental evidence suggesting the efficacy of VEGF165b in inhibiting tumor-induced angiogenesis and provides insight into a novel therapeutic strategy that could better manage HNSCC by selectively targeting aberrant vascular growth. This approach not only provides a potential pathway for more targeted and effective treatment options but also opens the door to a new paradigm in anti-angiogenic therapy with the possibility of reduced systemic toxicity. Our investigation is reshaping the future of HNSCC treatment by setting the stage for future research on VEGF splice variants as a tool for personalized medicine.

[Results of comprehensive conservative treatment of patients with no-option chronic limb-threatening ischaemia]. / Rezul'taty kompleksnogo konservativnogo lecheniia 'nerekonstruktabel'nykh' patsientov s ugrozhaiushchei khronicheskoi ishemiei nizhnikh konechnostei.

AIM: The study was aimed at comparing efficacy of conventional conservative therapy and comprehensive treatment including a plasmid VEGF-165-gene therapy drug in 'no-option' chronic limb-threatening ischaemia with different prevalence of trophic ulcers and infection during a 1-year follow-up period. PATIENTS AND METHODS: A total of 101 patients (54% being men and 46% women, mean age 69 years) with 'no-option' chronic limb-threatening ischaemia underwent comprehensive conservative treatment. They were subdivided into 4 groups according to the WIFI classification: WIFI 130 (n=38), 131 (n=23), 230 (n=16), 231 (n=24). The control group patients (n=58) received standard treatment using a PGE1 analogue (Vasaprostan) and the study group patients (n=43) underwent standard conservative treatment (SCT) in combination with gene therapy. The end points of the study were as follows: major amputation rate, amputation-free survival, total mortality, and ulcer healing rate during a 1-year of follow up. RESULTS: Major amputation rate in the control and study groups amounted to 35 and 28% (p=0.48), respectively, with amputation-free survival of 53 and 63% (p=0.35), total mortality of 21 and 12% (p=0.23), ulcer healing rate of 31 and 51% (p=0.04), respectively. The WIfI classification made it possible to single out a subgroup of patients (WIfI combination 130) yielding other statistically significant results: major amputation rate 27% and 0% (p=0.03), amputation-free survival 59 and 94% (p=0.025), ulcer healing rate 50 and 88% (p=0.016), respectively. CONCLUSION: Using plasmid-based VEGF-165 gene therapy in the subgroup with the WIfI combination 130 decreases the major amputation rate (p=0.03), increases amputation-free survival (p=0.025) and promotes ulcer healing (p=0.016) compared with the standard therapy during 1-year follow up. No significant differences in the compared groups were revealed by all endpoints of the study for other combinations analysed. The total mortality rate in patients with limb-threatening ischaemia does not depend on either the initial severity of ulcer or the selcted methods of conservative treatment.

Highly sensitive electrochemiluminescence biosensor for VEGF165 detection based on a g-C3N4/PDDA/CdSe nanocomposite.

In this work, an electrochemiluminescence (ECL) biosensor was fabricated for the selective detection of vascular endothelial growth factor (VEGF165). g-C3N4/PDDA/CdSe nanocomposites were used as the ECL substrate. Then, DNA labeled at the 5' end with amino groups (DNA1) was immobilized on the surface of g-C3N4/PDDA/CdSe nanocomposite-modified glassy carbon electrode (GCE) by amido linkage. AuNP-labeled target DNA (Au-DNA2) could hybridize with DNA1 to form a double strand. The ECL of the g-C3N4/PDDA/CdSe nanocomposite was efficiently quenched due to the resonance energy transfer between CdSe QDs and Au NPs. After VEGF165 was recognized and bound by Au-DNA2, the double helix was disrupted, and the energy transfer was broken. In this case, Au-DNA2 was released from the electrode surface, and the ECL intensity recovered to a higher level. Under optimal conditions, this ECL biosensor possesses excellent selectivity, accuracy, and stability for VEGF165 detection in a linear range of 2 pg mL-1 to 2 ng mL-1 with a detection limit of 0.68 pg mL-1. In addition, this assay has been successfully applied to the determination of VEGF165 in serum samples. Graphical abstract Schematic representation of the electrochemiluminescence sensor based on a g-C3N4/PDDA/CdSe nanocomposite, which can be determined in the concentration of vascular endothelial growth factor in serum.

Plasma Ephrin-A1 level in a cohort of diabetic retinopathy patients.

BACKGROUND: To determine plasma ephrin-A1 and VEGF165 levels in a cohort of diabetic retinopathy patients. METHODS: Plasma ephrin-A1 and VEGF165 levels in fifty-five subjects including 19 individuals without diabetes (non-DM), 16 patients with diabetes (DM) but without diabetic retinopathy, and 20 patients with diabetic retinopathy (DR), were determined by ELISA. Serum creatinine, total cholesterol, fasting blood glucose and HbA1c were also measured. One-way ANOVA, Kruskal-Wallis Test, Mann-Whitney U Test corrected by Bonferroni, Pearson Correlation Analysis and Spearman Correlation Coefficient Analysis were used for data analysis. RESULTS: Ephrin-A1 expression could be detected in human plasma with an average of 1.52 ± 0.43 (mean ± SEM) ng/ml. In DR subjects, the plasma ephrin-A1concentration was 3.63 ± 4.63 ng/ml, which was significantly higher than that of the other two groups (non-DM: 0.27 ± 0.13 ng/ml, DM: 0.35 ± 0.34 ng/ml). The expression of VEGF165 in human plasma was 34.00 ± 42.55 pg/ml, with no statistical difference among the three groups. There was no correlation between ephrin-A1 and VEGF165 in human plasma, but there was a correlation between plasma ephrin-A1 and duration of diabetes. CONCLUSIONS: Plasma ephrin-A1 was highly expressed in patients with diabetic retinopathy, and there was no difference of plasma VEGF165 expression in patients with diabetic retinopathy compared to the other two groups, suggesting that changes of plasma ephrin-A1 may be a more sensitive biomarker than plasma VEGF165 in detecting diabetic retinopathy.

Decreased Ratio of VEGF165b/VEGF in Aqueous Humor Predicts Progression of Diabetic Retinopathy.

BACKGROUND: Different splicing of vascular endothelial growth factor (VEGF) gene results in 2 families of VEGF, the proangiogenic isoforms (VEGFxxxa) and the antiangiogenic isoforms (VEGFxxxb). VEGF165b is the major antiangiogenic isoform of VEGF and the most studied member of the VEGFxxxb family so far. OBJECTIVES: To determine the concentration of VEGF165b and VEGF in the aqueous humor (AH) in diabetic eyes with or without diabetic retinopathy (DR) and to address the predictive value of VEGF165b/VEGF ratio for progression of DR. METHODS: AH samples from 20 eyes in healthy controls (CON group), 40 eyes in diabetic patients without DR (nDR group), and 30 eyes in diabetic patients with mild nonproliferative DR (DR group) were collected. All of the patients were followed up for at least 5 years. VEGF165b and VEGF levels of AH samples were measured by enzyme-linked immunosorbent assay (ELISA). The predictive value of the initial VEGF165b/VEGF ratio for progression of DR was studied. RESULTS: The mean concentration of VEGF165b significantly decreased in diabetic eyes vs. controls. The mean concentration of VEGF significantly increased in the DR group vs. the CON group. The VEGF165b/VEGF ratio was significantly lower in diabetic patients compared to the CON group. The VEGF165b/VEGF ratio was significantly lower in diabetic patients compared to the control group. The mean follow-up was 66.1months (range 60-71 months). The risk of DR progression was greater with a lower VEGF165b/VEGF ratio. CONCLUSION: The VEGF165b/VEGF ratio is lower in the AH of DR patients and the decreased ratio of VEGF165b/VEGF predicts DR progression.

Base amount-dependent fluorescence enhancement for the assay of vascular endothelial growth factor 165 in human serum using hairpin DNA-silver nanoclusters and oxidized carbon nanoparticles.

A base amount-dependent fluorescence enhancement-based strategy is put forward to determine vascular endothelial growth factor 165 (VEGF165) in human serum by the use of hairpin DNA-silver nanoclusters (hDNA-AgNCs) and oxidized carbon nanoparticles (CNPs). The hDNA-AgNCs aptasensing probe consists of AgNCs-contained hairpin loop (that generates a fluorescence signal), hairpin stem (that makes the structure stable), and the terminal aptamer 1 (that recognizes the target together with aptamer 2). It has been demonstrated that the fluorescence intensity of hDNA-AgNCs is ~ 3-fold stronger than that of single-stranded DNA-AgNCs (ssDNA-AgNCs), and hDNA-AgNCs have a strong dependence of fluorescence enhancement on the base amount in hairpin stem and loop. Upon the addition of oxidized CNPs, the terminal aptamer 1 of hDNA-AgNCs can adsorb onto the surface of oxidized CNPs via π-π stacking, and the fluorescence of hDNA-AgNCs (with excitation/emission maxima at 490/567 nm) is quenched via fluorescence resonance energy transfer (FRET). When aptamer 2 and VEGF165 are subsequently added, aptamer 1, VEGF165, and aptamer 2 reassemble into an intact tertiary structure, and the fluorescence is recovered because hDNA-AgNCs are far away from the surface of oxidized CNPs and the FRET efficiency decreases. Under the optimized conditions, the aptasensing probe can selectively assay VEGF165 with a detection limit of 14 pM. The results provide a label-free and sensitive method to monitor VEGF165 in human serum. Schematic representation of the strong dependence of fluorescence enhancement on base amount in stem and loop of hairpin DNA-silver nanoclusters. The probe can be used to assay vascular endothelial growth factor 165 (VEGF165) and give a judgment whether human serum VEGF165 is at a normal or abnormal level for clinical diagnosis.

Tuning the Polymorphism of the Anti-VEGF G-rich V7t1 Aptamer by Covalent Dimeric Constructs.

In the optimization process of nucleic acid aptamers, increased affinity and/or activity are generally searched by exploring structural analogues of the lead compound. In many cases, promising results have been obtained by dimerization of the starting aptamer. Here we studied a focused set of covalent dimers of the G-quadruplex (G4) forming anti-Vascular Endothelial Growth Factor (VEGF) V7t1 aptamer with the aim of identifying derivatives with improved properties. In the design of these covalent dimers, connecting linkers of different chemical nature, maintaining the same polarity along the strand or inverting it, have been introduced. These dimeric aptamers have been investigated using several biophysical techniques to disclose the conformational behavior, molecularity and thermal stability of the structures formed in different buffers. This in-depth biophysical characterization revealed the formation of stable G4 structures, however in some cases accompanied by alternative tridimensional arrangements. When tested for their VEGF165 binding and antiproliferative activity in comparison with V7t1, these covalent dimers showed slightly lower binding ability to the target protein but similar if not slightly higher antiproliferative activity on human breast adenocarcinoma MCF-7 cells. These results provide useful information for the design of improved dimeric aptamers based on further optimization of the linker joining the two consecutive V7t1 sequences.

Expression and characterization of recombinant human VEGF165 in the middle silk gland of transgenic silkworms.

Recombinant human vascular endothelial growth factor (rhVEGF) has important applications in therapeutic angiogenesis and inhibition of VEGF-mediated pathological angiogenesis. Previous studies have shown that rhVEGF can be produced in several expression systems, including Escherichia coli, yeasts, insect cells and mammalian cells. However, little is known regarding the effective production of this protein in organs of live organisms. Here, we report for the first time the expression and characterization of rhVEGF165 in the middle silk gland (MSG) of the transgenic silkworm line S1-V165. Our results confirmed that (1) rhVEGF165 was highly expressed in MSG cells and was secreted into the cocoon of S1-V165; (2) the dimeric form of rhVEGF165 could be easily dissolved from S1-V165 cocoons using an alkaline solution; (3) rhVEGF165 extracted from S1-V165 cocoons exhibited slightly better cell proliferative activity than the hVEGF165 standard in cultured human umbilical vein endothelial cells. This study provides an alternative strategy for the production of bioactive rhVEGF165 using the MSG of transgenic silkworms.

Roles of Sema3A and VEGF165 in cortical neurons and vascular endothelial cells during oxygen glucose deprivation stimulation.

To investigate the expressions and roles of semaphorin3A (Sema3A) and vascular endothelial growth factor 165 (VEGF165) in cultured rat cortical neurons and vascular endothelial cells after oxygen glucose deprivation (OGD) stimulation. Cultured cortical neurons (NC) and vascular endothelial cells (VEC) of Sprague Dawley (SD) rats (SPF grade) were randomly divided into control group and OGD treatment group. Western blot assay, immunofluorescent staining and immunohistochemical methods were used to determine the expressions of VEGF165, Sema3A and neuropilin-1 (Nrp-1) protein. Cell migration was determined by Transwell, while TUNEL assay was used to measure apoptosis. The expressions of Sema3A, Nrp-1 and VEGF165 in NC and VEC cells after OGD treatment were up-regulated, when compared with the control group. With transfection of Sema3A shRNA, apoptosis of neurons decreased significantly after 2 h of OGD treatment, but the apoptosis of VEC cells was not obvious. The migration rate of VEC cells in the treatment group was significantly increased, relative to that of the control group. Stimulation with OGD induces neuronal expression of VEGF165 and regulates the migration of vascular endothelial cells, thereby enhancing their participation in angiogenesis, which may involve Sema3A.

Triazolopeptides Inhibiting the Interaction between Neuropilin-1 and Vascular Endothelial Growth Factor-165.

Inhibiting the interaction of neuropilin-1 (NRP-1) with vascular endothelial growth factor (VEGF) has become an interesting mechanism for potential anticancer therapies. In our previous works, we have obtained several submicromolar inhibitors of this interaction, including branched pentapeptides of general structure Lys(Har)-Xxx-Xxx-Arg. With the intent to improve the proteolytic stability of our inhibitors, we turned our attention to 1,4-disubstituted 1,2,3-triazoles as peptide bond isosteres. In the present contribution, we report the synthesis of 23 novel triazolopeptides along with their inhibitory activity. The compounds were synthesized using typical peptide chemistry methods, but with a conversion of amine into azide completely on solid support. The inhibitory activity of the synthesized derivatives spans from 9.2% to 58.1% at 10 µM concentration (the best compound Lys(Har)-GlyΨ[Trl]GlyΨ[Trl]Arg, 3, IC50 = 8.39 µM). Synthesized peptidotriazoles were tested for stability in human plasma and showed remarkable resistance toward proteolysis, with half-life times far exceeding 48 h. In vitro cell survival test resulted in no significant impact on bone marrow derived murine cells 32D viability. By means of molecular dynamics, we were able to propose a binding mode for compound 3 and discuss the observed structure-activity relationships.

[Comprehensive treatment of a patient with Buerger's disease using genetically engineered complexes VEGF-165]. / Kompleksnoe lechenie patsienta s bolezn'iu Biurgera s primeneniem genno-inzhenernykh konstruktsii VEGF-165.

Buerger's disease, also known as thromboangiitis obliterans, is a severe invalidating systemic vascular disease. To one of the modern methods, which is distinguished by its radically new principles of action, as well as holding much promise for further study and application in treatment of patients with lower limb chronic ischaemia induced by thromboangiitis obliterans belongs the use of genetically engineered complexes based on vascular endothelial growth factor VEGF-165 ('Neovasculgen'). 'Neovasculgen' is a genetically engineered complex being a circular DNA (native plasmid on the CELO vector and Ad5), carrying the human VEGF-165 gene, encoding VEGF synthesis. Injection of this drug to the ischaemised tissues of lower extremities ensures long-term synthesis of vascular endothelial growth factor 165 leading to the development of an additional collateral vascular network and consequently to increased perfusion of tissues with oxygen and decreased degree of ischaemia. Presented herein is a clinical case report of a successful therapeutic outcome achieved in a patient suffering for a long time from thromboangiitis obliterans (Buerger's disease) and treated with genetically engineered complexes based on vascular endothelial growth factor ('Neovasculgen') used as a component of comprehensive conservative therapy.

[Conservative treatment of chronic lower limb ischemia in ambulatory practice (in Russian only)]. / Konservativnoe lechenie khronicheskoi ishemii nizhnikh konechnostei v praktike ambulatornogo khirurga.

Peripheral artery disease is still one of the most important surgical problems. General surgeons treat the majority of patients with chronic lower limb ischemia due to the lack of specialized surgical care. Current methods for risk factors adjustment, exercise therapy and the most common drugs for intermittent claudication management are reviewed in the article. The effect of these medicines on subjective (pain-free walking distance, maximal walking distance, etc.) and objective (ankle-brachial index) parameters and the incidence of complications are analyzed.

Intraocular Penetration of a vNAR: In Vivo and In Vitro VEGF165 Neutralization.

Variable new antigen receptor domain (vNAR) antibodies are novel, naturally occurring antibodies that can be isolated from naïve, immune or synthetic shark libraries. These molecules are very interesting to the biotechnology and pharmaceutical industries because of their unique characteristics related to size and tissue penetrability. There have been some approved anti-angiogenic therapies for ophthalmic conditions, not related to vNAR. This includes biologics and chimeric proteins that neutralize vascular endothelial growth factor (VEGF)165, which are injected intravitreal, causing discomfort and increasing the possibility of infection. In this paper, we present a vNAR antibody against human recombinant VEGF165 (rhVEGF165) that was isolated from an immunized Heterodontus francisci shark. A vNAR called V13, neutralizes VEGF165 cytokine starting at 75 µg/mL in an in vitro assay based on co-culture of normal human dermal fibroblasts (NHDFs) and green fluorescence protein (GFP)-labeled human umbilical vein endothelial cells (HUVECs) cells. In the oxygen-induced retinopathy model in C57BL/6:Hsd mice, we demonstrate an endothelial cell count decrease. Further, we demonstrate the intraocular penetration after topical administration of 0.1 µg/mL of vNAR V13 by its detection in aqueous humor in New Zealand rabbits with healthy eyes after 3 h of application. These findings demonstrate the potential of topical application of vNAR V13 as a possible new drug candidate for vascular eye diseases.

VEGF165 induces differentiation of hair follicle stem cells into endothelial cells and plays a role in in vivo angiogenesis.

Within the vascular endothelial growth factor (VEGF) family of five subtypes, VEGF165 secreted by endothelial cells has been identified to be the most active and widely distributed factor that plays a vital role in courses of angiogenesis, vascularization and mesenchymal cell differentiation. Hair follicle stem cells (HFSCs) can be harvested from the bulge region of the outer root sheath of the hair follicle and are adult stem cells that have multi-directional differentiation potential. Although the research on differentiation of stem cells (such as fat stem cells and bone marrow mesenchymal stem cells) to the endothelial cells has been extensive, but the various mechanisms and functional forms are unclear. In particular, study on HFSCs' directional differentiation into vascular endothelial cells using VEGF165 has not been reported. In this study, VEGF165 was used as induction factor to induce the differentiation from HFSCs into vascular endothelial cells, and the results showed that Notch signalling pathway might affect the differentiation efficiency of vascular endothelial cells. In addition, the in vivo transplantation experiment provided that HFSCs could promote angiogenesis, and the main function is to accelerate host-derived neovascularization. Therefore, HFSCs could be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischaemic diseases.

Structure-activity relationship study of a small cyclic peptide H-c[Lys-Pro-Glu]-Arg-OH: a potent inhibitor of Vascular Endothelial Growth Factor interaction with Neuropilin-1.

Inhibition of angiogenesis is one of the most promising approaches in anticancer therapy. It was recently suggested that Neuropilin-1 (NRP-1) in tumour cells may serve as a separate receptor for Vascular Endothelial Growth Factor-165 (VEGF165) which is one of the main pro-angiogenic agents in the organism. Therefore molecules inhibiting VEGF165 binding to NRP-1 could be potential candidates for new antiangiogenic and anticancer drugs. Here we present a structure-activity relationship study of the peptide H-c[Lys-Pro-Glu]-Arg-OH which showed high inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18µM) in our previous study. We report the design, synthesis, in vitro assays and docking analysis of four small cyclic peptides (14-,15-membered ring) and one bigger cyclic compound (30-membered ring). Our study shows that both the ring size and configuration of amino acid residues present in the structure are crucial for high inhibitory effect.

Conformational latitude - activity relationship of KPPR tetrapeptide analogues toward their ability to inhibit binding of vascular endothelial growth factor 165 to neuropilin-1.

Neuropilin-1 has been found to be overexpressed in several kinds of malignant tumors, and it is postulated that its interaction with the vascular endothelial growth factor 165 leads to progression of tumor vascularization and growth. Several analogues (KxxR) with various conformational latitudes have been synthesized and found as inhibitors of NRP-1. Detailed insight provided by molecular dynamics simulation allowed forming a clear relationship between flexibility of xx part of the molecule and its inhibitory activity.

Human gingival fibroblasts induced and differentiated into vascular endothelial-like cells.

A novel method for repair of vascular disease, mechanical damage, and tissue rebuilding is urgently required. Vascular endothelial cells (VECs) play an essential role in vascular rebuilding and vasotransplantation. In the present study, human gingival fibroblasts (HGFs) were cultured and induced into endothelial-like cells in vitro in order to confirm that HGFs with stem cell properties possessed the potential for differentiation into endothelial-like cells. The epithelium was extracted from normal human gingiva consisting of epithelium and connective tissue, which was isolated from patients. The identification of HGFs and induced endothelial-like cells were confirmed by flow cytometry, reverse transcription polymerase chain reaction (RT-PCR), immunocytochemical stain (ICS), and immunofluorescence stain (ISA). The morphology of human gingival fibroblasts with 8 ng/mL VEGF165 induced for different periods of days were observed by inverted microscope. Before induction, flow cytometry analysis showed that HGFs were positive for vimentin, but negative for CD31. RT-PCR, ICS, and ISA showed vimentin, S100A4, α-SMA, collagen III, and S100A4 were specifically expressed in these fibroblast cells. After induction, ICS showed induced vascular endothelial-like cells were positive for CD34 and CD31; ISA showed cells induced were positive for vWF and E-cadherin; RT-PCR results demonstrated that tie2 was specifically expressed in the cells induced. Flow cytometry analysis of the transformation efficiency from HGFs to endothelial-like cells. In conclusion, we found that HGFs possessed capacity for being induced and differentiated into vessel endothelial-like cells with typical and specific morphological, ultrastructural, and immunological characters of endothelial-like cells by induction with VEGF.

Design, synthesis and in vitro biological evaluation of a small cyclic peptide as inhibitor of vascular endothelial growth factor binding to neuropilin-1.

Neuropilin-1 (NRP-1) is a co-receptor of VEGFR (vascular endothelial growth factor receptor), but it is also suggested that NRP-1 in tumour cells may serve as a separate receptor for VEGF165. Therefore molecules interfering with VEGF165 binding to NRP-1 seem to be promising candidates as new anti-angiogenic and anti-tumour drugs. Here, we report the design, synthesis, biological evaluation and molecular modelling of the small cyclic peptide, which shows a good inhibitory effect on VEGF165/NRP-1 binding (IC50=0.18µM). The reported compound could be considered as one of the smallest cyclic peptides (MW=510) interfering with VEGF165/NRP-1 binding presented up to now.