Peptidomimetic inhibitors of the VEGF-A165/NRP-1 complex obtained by modification of the C-terminal arginine
Ontology highlight
ABSTRACT:
SUBMITTER: Tymecka D
PROVIDER: S-EPMC11344719 | biostudies-literature | 2024 Jan
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Vascular endothelial growth factor (VEGF) is an indispensable element in many physiological processes, while alterations in its level in the circulating system are signs of pathology-associated diseases. Therefore, its precise and selective detection is critical for clinical applications to monitor the progression of the pathology. In this study, an optical immunoassay biosensor was developed as a model study for detecting recombinant VEGF165. The VEGF165 sample was purified from recombinant Kluyveromyces lactis GG799 yeast cells. Indirect ELISA was used during the detection, wherein iron oxide nanoparticles (FeNPs) were utilized to obtain optical signals. The FeNPs were synthesized in the presence of lactose p-amino benzoic acid (LpAB). VEGF165 antibody was conjugated to the LpAB-FeNPs through EDC/NHS chemistry to convert the iron oxide nanoparticles into VEGF165 specific probes. The specificity of the prepared system was tested in the presence of potential serum-based interferents (i.e., glucose, urea, insulin, C-reactive protein, and serum amyloid A), and validation studies were performed in a simulated serum sample. The proposed immunoassay showed a wide detection range (0.5 to 100 ng/mL) with a detection limit of 0.29 ng/mL. These results show that the developed assay could offer a sensitive, simple, specific, reliable, and high-throughput detection platform that can be used in the clinical diagnostics of VEGF.
| S-EPMC10867064 | biostudies-literature
Project description:BackgroundInhibition of angiogenesis using monoclonal antibodies is an effective strategy in cancer therapy. However, they could not penetrate sufficiently into solid tumors. Antibody fragments have solved this issue. However, they suffer from short in vivo half-life. In the current study, a tandem bivalent strategy was used to enhance the pharmacokinetic parameters of an anti-VEGF165 nanobody.MethodsHomology modeling and MD simulation were used to check the stability of protein. The cDNA was cloned into pHEN6C vector and the expression was investigated in WK6 Escherichia coli (E. coli) cells by SDS-PAGE and western blot. After purification, the size distribution of tandem bivalent nanobody was investigated by dynamic light scattering. Moreover, in vitro antiproliferative activity and pharmacokinetic study were studied in HUVECs and Balb/c mice, respectively.ResultsRMSD analysis revealed the tandem bivalent nanobody had good structural stability after 50 ns of simulation. A hinge region of llama IgG2 was used to fuse the domains. The expression was induced by 1 mM IPTG at 25°C for overnight. A 30 kDa band in 12% polyacrylamide gel and nitrocellulose paper has confirmed the expression. The protein was successfully purified using metal affinity chromatography. MTT assay revealed there is no significant difference between the antiproliferative activity of tandem bivalent nanobody and the native protein. The hydrodynamic radius and terminal half-life of tandem bivalent nanobody increased approximately 2-fold by multivalency compared to the native protein.ConclusionOur data revealed that the physicochemical as well as in vivo pharmacokinetic parameters of tandem bivalent nanobody was significantly improved.
| S-EPMC8112138 | biostudies-literature
Project description:Vascular endothelial growth factor-A (VEGF-A), a secreted homodimeric glycoprotein, is a critical regulator of angiogenesis in normal and pathological states. The binding of heparin (HE) to VEGF165 (the major form of VEGF-A) modulates the angiogenesis-related cascade, but the mechanism of the observed changes at the structural level is still insufficiently explored. In the present study, we examined the effect of HE on the structural and physicochemical properties of recombinant human VEGF165 (rhVEGF165). The HE binding results in an increase of hydrophobic surface exposure in rhVEGF165 without changes in its secondary structure. Differential scanning calorimetry measurements for intact and HE-bound rhVEGF165 reveals the absence of any pronounced thermally induced transitions in the protein in the temperature range from 20 to 100 °C. The apolar area increase during the heparin binding explains the pronounced HE-induced oligomerization/aggregation of rhVEGF165, as studied by chemical glutaraldehyde cross-linking and dynamic light scattering. Molecular modeling and docking techniques were used to model the full structure of dimeric VEGF165 and to reveal putative molecular mechanisms underlying the function of the VEGF165/HE system. In general, the results obtained can be a basis for explaining the modulating effect of HE on the biological activity of VEGF-A.
| S-EPMC9856036 | biostudies-literature
Project description:VEGF165 and its isoform VEGF165b have the same length but opposite functions in cancer. Some studies have indicated the important role of VEGF165 in osteosarcoma (OS); however, VEGF165b has not been taken into consideration. This study aims to clarify the roles of the two isoforms in OS and the mechanism controlling their formation from an alternative splicing perspective. By in vivo and in vitro experiments, we assessed the expression and function of VEGF165 and VEGF165b, screened the underlying splicing factors, and verified the regulatory function of splicing factor YBX1 on the two isoforms and its role in OS. The results showed that in OS, VEGF165 was upregulated but VEGF165b was downregulated. VEGF165 promoted the proliferation, migration and invasion of OS cells and induced angiogenesis in OS tumours; however, VEGF165b showed the opposite function. Of the four screened splicing factors, YBX1 was upregulated in OS tissues. It was positively correlated with VEGF165 but negatively correlated with VEGF165b. Further study indicated that YBX1 could upregulate VEGF165 but downregulate VEGF165b. Moreover, YBX1 promoted the proliferation, migration and invasion of OS cells and induced angiogenesis in OS tumours. OS patients with higher YBX1 had a poor prognosis within five years, but this difference disappeared in a longer follow-up. In conclusion, VEGF165b was antineoplastic and downregulated in OS, in contrast to VEGF165. YBX1 was found to be an important splicing factor that increased VEGF165 but decreased VEGF165b. Targeting YBX1 could endogenously alter the levels of VEGF165 and VEGF165b simultaneously.
| S-EPMC10405001 | biostudies-literature
Project description:Vascular endothelial growth factor 165 (VEGF165) is a prominent isoform of the VEGF-A protein that plays a crucial role in various angiogenesis-related diseases. It is homodimeric, and each of its monomers is composed of two domains connected by a flexible linker. DNA aptamers, which have emerged as potent therapeutic molecules for many proteins with high specificity and affinity, can also work for VEGF165. A DNA aptamer heterodimer composed of monomers of V7t1 and del5-1 connected by a flexible linker (V7t1:del5-1) exhibits a greater binding affinity with VEGF165 compared to either of the two monomers alone. Although the structure of the complex formed between the aptamer heterodimer and VEGF165 is unknown due to the highly flexible linkers, gaining structural information will still be valuable for future developments. Toward this end of accessing structural information, we adopt an ensemble docking approach here. We first obtain an ensemble of structures for both VEGF165 and the aptamer heterodimer by considering both small- and large-scale motions. We then proceed through an extraction process based on ensemble docking, molecular dynamics simulations, and binding free energy calculations to predict the structures of the VEGF165/V7t1:del5-1 complex. Through the same procedures, we reach a new aptamer heterodimer that bears a locked nucleic acid-modified counterpart of V7t1, namely RNV66:del5-1, which also binds well with VEGF165. We apply the same protocol to the monomeric units V7t1, RNV66, and del5-1 to target VEGF165. We observe that V7t1:del5-1 and RNV66:del5-1 show higher binding affinities with VEGF165 than any of the monomers, consistent with experiments that support the notion that aptamer heterodimers are more effective anti-VEGF165 aptamers than monomeric aptamers. Among the five different aptamers studied here, the newly designed RNV66:del5-1 shows the highest binding affinity with VEGF165. We expect that our ensemble docking approach can help in de novo designs of homo/heterodimeric anti-angiogenic drugs to target the homodimeric VEGF165.
| S-EPMC11012306 | biostudies-literature
Project description:BackgroundAtherosclerotic occlusions decrease blood flow to the lower limbs, causing ischemia and tissue loss in patients with peripheral artery disease (PAD). No effective medical therapies are currently available to induce angiogenesis and promote perfusion recovery in patients with severe PAD. Clinical trials aimed at inducing vascular endothelial growth factor (VEGF)-A levels, a potent proangiogenic growth factor to induce angiogenesis, and perfusion recovery were not successful. Alternate splicing in the exon-8 of VEGF-A results in the formation of VEGFxxxa (VEGF165a) and VEGFxxxb (VEGF165b) isoforms with existing literature focusing on VEGF165b's role in inhibiting vascular endothelial growth factor receptor 2-dependent angiogenesis. However, we have recently shown that VEGF165b blocks VEGF-A-induced endothelial vascular endothelial growth factor receptor 1 (VEGFR1) activation in ischemic muscle to impair perfusion recovery. Because macrophage-secreted VEGF165b has been shown to decrease angiogenesis in peripheral artery disease, and macrophages were well known to play important roles in regulating ischemic muscle vascular remodeling, we examined the role of VEGF165b in regulating macrophage function in PAD.MethodsFemoral artery ligation and resection were used as an in vivo preclinical PAD model, and hypoxia serum starvation was used as an in vitro model for PAD. Experiments including laser-Doppler perfusion imaging, adoptive cell transfer to ischemic muscle, immunoblot analysis, ELISAs, immunostainings, flow cytometry, quantitative polymerase chain reaction analysis, and RNA sequencing were performed to determine a role of VEGF165b in regulating macrophage phenotype and function in PAD.ResultsFirst, we found increased VEGF165b expression with increased M1-like macrophages in PAD versus non-PAD (controls) muscle biopsies. Next, using in vitro hypoxia serum starvation, in vivo pre clinical PAD models, and adoptive transfer of VEGF165b-expressing bone marrow-derived macrophages or VEGFR1+/- bone marrow-derived macrophages (M1-like phenotype), we demonstrate that VEGF165b inhibits VEGFR1 activation to induce an M1-like phenotype that impairs ischemic muscle neovascularization. Subsequently, we found S100A8/S100A9 as VEGFR1 downstream regulators of macrophage polarization by RNA-Seq analysis of hypoxia serum starvation-VEGFR1+/+ versus hypoxia serum starvation-VEGFR1+/- bone marrow-derived macrophages.ConclusionsIn our current study, we demonstrate that increased VEGF165b expression in macrophages induces an antiangiogenic M1-like phenotype that directly impairs angiogenesis. VEGFR1 inhibition by VEGF165b results in S100A8/S100A9-mediated calcium influx to induce an M1-like phenotype that impairs ischemic muscle revascularization and perfusion recovery.
| S-EPMC6322929 | biostudies-literature
Project description:Sensitive detection of vascular endothelial growth factor (VEGF165) is important for early cancer disease diagnosis in the clinic. A sensitive fluorescent sensing platform for VEGF165 detection is developed in this work. It is based on a target-triggered hybridization chain reaction (HCR) and graphene oxide (GO) selective fluorescence quenching. In this assay, in the presence of the VEGF165, the hairpin structure of Hp opens up and the initiation sequence will be exposed to Hp1 to open its hairpin structure. Then the opened Hp1 hybridizes with Hp2 to expose the complementary sequence of Hp1 which hybridizes with Hp1 again by HCR. Thus HCR would be initiated, generating super-long dsDNA. After the HCR, the double strands of the HCR product cannot be adsorbed on the GO surface. As a result, the HCR product gives a strong fluorescence signal which is dependent on the concentration of VEGF165. By using VEGF165 as a model analyte, the assay provides a highly sensitive fluorescence detection method for VEGF165 with a detection limit down to 20 pg mL-1. The proposed aptasensing strategy based on target-triggered HCR amplification can thus be realized. It was successfully applied to the determination of VEGF165 in spiked human serum, urine and saliva. Therefore, it can easily have wide applications in the diagnosis of vital diseases.
| S-EPMC9086580 | biostudies-literature
Project description:Neuropilin-1 (NRP-1), the major co-receptor of vascular endothelial growth factor receptor-2 (VEGFR-2), may also independently act with VEGF-A165 to stimulate tumour growth and metastasis. Therefore, there is great interest in compounds that can block VEGF-A165/NRP-1 interaction. Peptidomimetic type inhibitors represent a promising strategy in the treatment of NRP-1-related disorders. Here, we present the synthesis, affinity, enzymatic stability, molecular modeling and in vitro binding evaluation of the branched urea-peptide hybrids, based on our previously reported Lys(hArg)-Dab-Oic-Arg active sequence, where the Lys(hArg) branching has been modified by introducing urea units to replace the peptide bond at various positions. One of the resulting hybrids increased the affinity of the compound for NRP-1 more than 10-fold, while simultaneously improving resistance for proteolytic stability in serum. In addition, ligand binding to NRP-1 induced rapid protein stock exocytotic trafficking to the plasma membrane in breast cancer cells. Examined properties characterize this compound as a good candidate for further development of VEGF165/NRP-1 inhibitors.
| S-EPMC7793531 | biostudies-literature
Project description:Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652.
| S-EPMC5622042 | biostudies-literature
Project description:Vascular endothelial growth factor (VEGF) is associated with tumour growth and metastasis. Because VEGF is the major player in both angiogenesis and vascular permeability and the most explored factor in angio-inhibitory therapies, many expression procedures have been developed to produce functional VEGF165 in convenient yield. In this study, recombinant human VEGF165 was cloned and expressed in Escherichia coli (BL21)-DE3 cells and large scale production was performed by fermentation. A high yield of active soluble protein was obtained after protein extraction employing both lysozyme and sonication treatment. Inclusion bodies were also isolated from the cell lysate and subjected to a simple protocol of solubilisation and refolding. Single-step purification was performed using nickel affinity chromatography and the purified proteins were able to recognize monoclonal Anti-poly-His antibody. The biological activity of the VEGF165 was successfully tested using the Chicken chorioallantoic membrane assay, wound-healing migration and proliferation assay on human umbilical vein endothelial cells (HUVEC).
| S-EPMC5293700 | biostudies-literature