Fibroblast-derived matrix (FDM) as a novel vascular endothelial growth factor delivery platform.

Delivery of vascular endothelial growth factor (VEGF) to sham ovarian tissue autografts in non-human primates: optimal method of delivery and dose

Vascular Endothelial Growth Factor Localization in the Adult

In a previous study, we showed that in situ injection of glycosaminoglycan mimetics called RGTAs (ReGeneraTing Agents) enhanced neovascularization after skeletal muscular ischemia (Desgranges, P., Barbaud, C., Caruelle, J. P., Barritault, D., and Gautron, J. (1999) FASEB J. 13, 761-766). In the present study, we showed that the RGTA OTR4120 modulated angiogenesis in the chicken embryo chorioallantoic membrane assay, in a dose-dependent manner. We therefore investigated the effect of OTR4120 on one of the most specific angiogenesis-regulating heparin-binding growth factors, vascular endothelial growth factor 165 (VEGF165). OTR4120 showed high affinity binding to VEGF165 (Kd = 2.2 nm), as compared with heparin (Kd = 15 nm), and potentiated the affinity of VEGF165 for VEGF receptor-1 and -2 and for neuropilin-1. In vitro, OTR4120 potentiated VEGF165-induced proliferation and migration of human umbilical vein endothelial cells. In the in vivo Matrigel plug angiogenesis assay, OTR4120 in a concentration as low as 3 ng/ml caused a 6-fold increase in VEGF165-induced angiogenesis. Immunohistochemical staining showed a larger number of well differentiated VEGFR-2-expressing-cells in Matrigel sections of OTR4120-treated plug than in control sections. These findings indicate that OTR4120 enhances the VEGF165-induced angiogenesis and therefore may hold promise for treating disorders characterized by deficient angiogenesis.

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

Vascular endothelial growth factor (VEGF) is essential for many angiogenic processes both in normal conditions and in pathological conditions. However, the signaling pathways involved in VEGF-induced angiogenesis are not well defined. Protein kinase D (PKD), a newly described serine/threonine protein kinase, has been implicated in many signal transduction pathways and in cell proliferation. We hypothesized that PKD would mediate VEGF signaling and function in endothelial cells. Here we found that VEGF rapidly and strongly stimulated PKD phosphorylation and activation in endothelial cells via VEGF receptor 2 (VEGFR2). The pharmacological inhibitors for phospholipase Cgamma (PLCgamma) and protein kinase C (PKC) significantly inhibited VEGF-induced PKD activation, suggesting the involvement of the PLCgamma/PKC pathway. In particular, PKCalpha was critical for VEGF-induced PKD activation since both overexpression of adenovirus PKCalpha dominant negative mutant and reduction of PKCalpha expression by small interfering RNA markedly inhibited VEGF-induced PKD activation. Importantly, we found that small interfering RNA knockdown of PKD and PKCalpha expression significantly attenuated ERK activation and DNA synthesis in endothelial cells by VEGF. Taken together, our results demonstrated for the first time that VEGF activates PKD via the VEGFR2/PLCgamma/PKCalpha pathway and revealed a critical role of PKD in VEGF-induced ERK signaling and endothelial cell proliferation.

Role of Soluble Vascular Endothelial Growth Factor Receptor-1 in the Vitreous in Proliferative Diabetic Retinopathy

In vitro culture of endothelial cells to form capillary-like networks is essential in tissue engineering. Vascular endothelial growth factor (VEGF) is one of the primary signal proteins stimulating blood vessel formation. This growth factor can be soluble in the medium or protein-bound to the substrate. However, less attention has been paid to distinguishing the specific stimulations by soluble and bound VEGF. We conducted a series of experiments to explore the respective effects of these two VEGF forms. An in-house synthesized biogel comprising a definite concentration of collagen and fibronectin was designed to cultivate human umbilical vein endothelial cells to form the capillary-like network. Collagen served as the primary substrate for cell attachment. Fibronectin provided the surface to bind soluble VEGF in the culture medium to create the bound VEGF. The experiment of adding VEGF-blocking-peptide was conducted to prevent the formation of VEGF bound to the fibronectin domains, to distinguish the respective effects of the soluble and bound VEGF. With the in-house biogel of definite components, we were able to clarify the different roles of soluble and bound VEGF. The results indicated that the soluble VEGF promptly induced the cells to change from round to elongated shape, which contributed to forming network cords. Simultaneously, the bound VEGF provided long-term stimulation, causing the cells to migrate and differentiate into the final capillary-like network.

Sustained Improvement in Perfusion and Flow Reserve After Temporally Separated Delivery of Vascular Endothelial Growth Factor and Angiopoietin-1 Plasmid Deoxyribonucleic Acid

Event Abstract Back to Event Enhanced vascularization of VEGF synergizing with 2-N,6-O-sulfated chitosan Yuanman Yu1, 2, Rui Chen1, 2, Xiang Peng2, Jing Wang1, 2, 3 and Changsheng Liu1, 2, 3 1 East China University of Science and Technology, The State Key Laboratory of Bioreactor Engineering, China 2 East China University of Science and Technology, Engineering Research Center for Biomedical Materials of Ministry of Education, China 3 East China University of Science and Technology, Key Laboratory for Ultrafine Materials of Ministry of Education, China Introduction: Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications[1],[2]. While vascular endothelial growth factor (VEGF) which regards as an angiogenic regulator holds greatest potential to promote angiogenesis[3]. However, soluble VEGF will be quickly degraded by enzymes in vivo and clinical applications with high dose of VEGF are often associated with severe side effects[4],[5]. Thus, reducing the dosage of VEGF and enhancing its activity becomes particularly important. 2-N,6-O-sulfated chitosan (26SCS) as a synthetic sulfated polysaccharide has been demonstrated that it could efficiently associate with VEGF to promote angiogenesis in vitro during our previous work[6]. Nevertheless, the mechanism of the interaction between 26SCS and VEGF is not clear. This study aimed at the synergy of 26SCS with VEGF as well as its pro-angiogenesis in vitro and in vivo. Materials and Methods: Human umbilical vein endothelial cells (HUVECs) which purchased from the American Type Culture Collection was used as a cell model. A series concentrations of 26SCS was used to characterize the interaction with VEGF (2 ng/mL) on cell viability. Subsequently, two experimental groups which named as 40 SCS/VEGF (26SCS, 40 ng/mL) and 160 SCS/VEGF (26SCS, 160 ng/mL) respectively were chosen to investigate the cell migration, sprouting and qRT-PCR analysis in vitro. Furthermore, we employed the corresponding concentration ratio of 26SCS and VEGF to further investigate the pro-angiogenesis activity in vivo. Subcutaneous implantation of mice (VEGF, 100 ng) and chicken chorioallantoic membrane (CAM) assay (VEGF, 20 ng) were used as experimental models, respectively. Results and Discussion: The pro-angiogenesis of 26SCS synergizing with VEGF was found and mainly studied. As shown in Figure 1, 26SCS synergizing with VEGF could directly improve HUVECs cell proliferation through dose-dependently regulating the expression of relevant angiogenic genes, and the group of 40 SCS/VEGF exhibited optimal results. Moreover, the repaired area of 40 SCS/VEGF was almost 1.5 folds compared to negative control in scratch wound healing assay, and 40 SCS/VEGF generated more branch points and forming tubes in capillary-like tube formation assay. In addition, using gelatin sponge loaded with VEGF and different content of 26SCS as samples implanted in subcutaneous model of mice, we investigated the influence of 26SCS and VEGF facilitate vascularization in vivo at day 7 (Figure 2A). The results of histological analysis showed that 26SCS and VEGF could significantly promote the repair rate of vascular network at the defect site (Figure 2B). Through the CAM assay by implanting filter paper containing different ratios of 26SCS and VEGF compounds into fertilized eggs (Figure 2C), the results further revealed that 26SCS synergizing VEGF could signally promote angiogenesis. Conclusions: In this study, 26SCS was demonstrated that synergizing with VEGF could significantly promote angiogenesis in vitro and in vivo. This findings evidence that 26SCS have the potential to improve the VEGF activity and will be promising prospects as a synergistic factor of VEGF for vascularization in clinical applications. The National Basic Research Program of China (973 Program, No. 2012CB933600); The National Natural Science Foundation of China (Grant Nos. 31271011, 31330028, and 31470923); The New Century Excellent Talents in University (Grant No. NCET-12-0856); The National Science and Technology Support Program (Grant No. 2012BAI17B02)

BackgroundMaintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels.MethodsWe utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling.ResultsWestern blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs.ConclusionsThis study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

To investigate the mechanism(s) that prostaglandin E1 (PGE1) promotes human umbilical vein endothelial cell (HUVEC)proliferation and migration. Western blot, enzyme linked immunosorbent assay, cell proliferation and cell migration tests, and tube formation were used for analyzing the roles and mechanisms of PGE1 on HUVEC; Western blot was used for analyzing the effects of PGE1 on the expression of vascular endothelial growth factor (VEGF) in rat aortic vascular smooth muscle cells (VSMC). PGE1 significantly increased VEGF expression of HUVEC in time and a dose dependent manner with concomitantly increased HUVEC proliferation; treatment of HUVEC with Bevacizumab apparently suppressed PGE1-stimulated VEGF expression, which led to decreased tube formation, reduced cell proliferation and migration by 41% and 38%, respectively, compared with PGE1 treatment alone; PGE1 time-dependently induced both phosphorylation of ERK and p38 in HUVEC, whereas ERK inhibitor, PD98059, or p38 inhibitor, SB203580, blocked PGE1-induced VEGF expression of HUVEC, resulting in dramatically suppression of HUVEC proliferation and migration compared with PGE1 treatment alone (60% and 55% by PD98059, 62% and 51% by SB203580, respectively); in addition, cAMP-dependent protein kinase A inhibitor, H89 or Rp-cAMP blocked PGE1-induced VEGF expression in VSMC. PGE1 promotion of proliferation, migration and tube formation of HUVEC via VEGF further provides a novel theoretical support in efficacy of PGE1 treatment of critical limb ischemia and other related diseases.

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.

Vascular endothelial growth factor (VEGF) is essential for normal and pathological angiogenesis. However, the signaling pathways linked to gene regulation in VEGF-induced angiogenesis are not fully understood. Here we demonstrate a critical role of protein kinase D (PKD) and histone deacetylase 5 (HDAC5) in VEGF-induced gene expression and angiogenesis. We found that VEGF stimulated HDAC5 phosphorylation and nuclear export in endothelial cells through a VEGF receptor 2-phospholipase Cgamma-protein kinase C-PKD-dependent pathway. We further showed that the PKD-HDAC5 pathway mediated myocyte enhancer factor-2 transcriptional activation and a specific subset of gene expression in response to VEGF, including NR4A1, an orphan nuclear receptor involved in angiogenesis. Specifically, inhibition of PKD by overexpression of the PKD kinase-negative mutant prevents VEGF-induced HDAC5 phosphorylation and nuclear export as well as NR4A1 induction. Moreover, a mutant of HDAC5 specifically deficient in PKD-dependent phosphorylation inhibited VEGF-mediated NR4A1 expression, endothelial cell migration, and in vitro angiogenesis. These findings suggest that the PKD-HDAC5 pathway plays an important role in VEGF regulation of gene transcription and angiogenesis.

Endothelial cell migration is a crucial step in the process of new blood vessel formation—a necessary process to maintain cell viability inside thick tissue constructs. Here, we report a new method for maintaining cell viability and inducing cell migration using a perfused microfluidic platform based on collagen gel and a gradient hydrogel sheet. Due to the helpful role of the extracellular matrix components in cell viability, we developed a hydrogel sheet from decellularized tissue (DT) of the bovine heart and chitosan (CS). The results showed that hydrogel sheets with an optimum weight ratio of CS/DT = 2 possess a porosity of around 75%, a mechanical strength of 23 kPa, and display cell viability up to 78%. Then, we immobilized a radial gradient of vascular endothelial growth factor (VEGF) on the hydrogel sheet to promote human umbilical vein endothelial cell migration. Finally, we incorporated the whole system as an entirety on the top of the microfluidic platform and studied cell migration through the hydrogel sheet in the presence of soluble and immobilized VEGF. The results demonstrated that immobilized VEGF stimulated cell migration in the hydrogel sheet at all depths compared with soluble VEGF. The results also showed that applying a VEGF gradient in both soluble and immobilized states had a significant effect on cell migration at limited depths (<100 μm). The main finding of this study is a significant improvement in cell migration using an in vivo imitating, cost-efficient and highly reproducible platform, which may open up a new perspective for tissue engineering applications.