VEGF-induced Endothelial Cell Research Articles

DPMA, a deoxypodophyllotoxin derivative, induces apoptosis and anti-angiogenesis in non-small cell lung cancer A549 cells

We found that the deoxypodophyllotoxin derivative, 2,6-dimethoxy-4-(6-oxo-(5R,5aR,6,8,8aR,9-hexahydrofuro[3′,4′:6,7]naphtho[2,3-d][1,3]dioxol-5-yl)phenyl ((R)-1-amino-4-(methylthio)-1-oxobutan-2-yl)carbamate (DPMA), exhibited superior cytotoxicity compared with etoposide. In this study, we investigated the mechanism of action of DPMA. DPMA exhibited anti-proliferative activity and induced apoptosis in A549 cells in a dose- and time-dependant manner. DPMA inhibited microtubule formation and induced expression of Bax, cleaved caspase-3, p53 and ROS, and inhibited Bcl-2 expression. DPMA also affected cyclinB1, cdc2 and p-cdc2 expression, inducing cell cycle arrest. DPMA also inhibited tube formation of VEGF-induced human umbilical vein endothelial cells. These studies demonstrate that DPMA inhibits p53/cdc2/Bax signaling, thereby inhibiting cell growth/angiogenesis and inducing apoptosis.

Thrombospondin-1 modulates VEGF signaling via CD36 by recruiting SHP-1 to VEGFR2 complex in microvascular endothelial cells

AbstractThrombospondin-1 (TSP-1) inhibits growth factor signaling at the receptor level in microvascular endothelial cells (MVEC), and CD36 has been suggested to be involved in this inhibition, but the mechanisms are not known. We hypothesized that CD36-TSP-1 interaction recruits Src homology 2 domain–containing protein tyrosine phosphatase (SHP)-1 to the vascular endothelial growth factor receptor 2 (VEGFR2) signaling complex and attenuates vascular endothelial growth factor (VEGF) signaling. Western blots of anti-CD36 and anti-VEGFR2 immunoprecipitates from VEGF-treated MVEC showed that exposure of the cells to a recombinant protein containing the CD36 binding domain of thrombospondin-1 (known as the TSR domain) induced association of SHP-1 with the VEGFR2/CD36 signaling complex and thereby suppressed VEGFR2 phosphorylation. SHP-1 phosphatase activity was increased in immunoprecipitated VEGFR2 complexes from TSR-treated cells. Silencing CD36 expression in MVEC by small interfering RNA (siRNA) or genetic deletion of cd36 in mice showed that TSR-induced SHP-1/VEGFR2 complex formation required CD36 in vitro and in vivo. Silencing SHP-1 expression in MVEC by siRNA abrogated TSR-mediated inhibition of VEGFR2 phosphorylation as well as TSR-mediated inhibition of VEGF-induced endothelial cell migration and tube formation. These studies reveal a SHP-1–mediated antiangiogenic pathway induced by CD36-TSP-1 interaction that inhibits VEGFR2 signaling and they provide a novel target to modulate angiogenesis therapeutically.

The antiangiogenic activity of a soluble fragment of the VEGFR extracellular domain

Vascular endothelial growth factor (VEGF) is a key regulator of pathological angiogenesis and vascular permeability and overexpressed by most solid tumors. VEGF receptor-2 (VEGFR-2 or kinase-insert domain-containing receptor as it is called in human, KDR) is a specific receptor of VEGF with a high binding affinity. A solube recombinant extracellular domain 1-3 of human VEGFR-2 (rKDR1-3) was expressed in Escherichia coli (E. Coli) and purified from the bacterial periplasmic extracts by immobilized metal affinity chromatography and anion exchange chromatography to inhibit the VEGF-induced angiogenesis. A surface plasmon resonance (SPR) technology was adopted to analyze the affinity and kinetics constant between rKDR1-3 and VEGF165. Under the given experimental conditions, the association rate constant Ka was 1.06×105M−1 S−1, the dissociation rate Kd was 6.09×10−3 S−1, the dissociation constant KD was 5.74×10−8M. The effect of rKDR1-3 on VEGF-induced endothelial cell proliferation was studied using MTT assay, scratch-wound healing assay and chorioallantoic membrane (CAM) assay. The results showed that rKDR1-3 could inhibit neovascularization and serve as a useful drug candidate in research, diagnostics and therapy of cancer.

Cathepsin proteases promote angiogenic sprouting and laser-induced choroidal neovascularisation in mice

Cysteine cathepsins are a family of proteases involved in intracellular protein turnover and extracellular matrix degradation. Cathepsin B (Ctsb) and cathepsin Z (Ctsz) promote tumorigenesis and Ctsb is a known modulator of tumor angiogenesis. We therefore investigated the angiomodulatory function of these cathepsins in vitro as well as in a mouse model of laser-induced choroidal neovascularization (laser-CNV). Ctsb−/−, Ctsz−/−, Ctsb/Ctsz double-knockout (Ctsb/z DKO), and wild type (WT) mice underwent argon laser treatment to induce choroidal neovascularization (CNV). The neovascularized area was quantified individually for each lesion at 14 days after laser coagulation. In vitro the effects of cathepsin inhibitors on angiogenesis were analysed by endothelial cell (EC) spheroid sprouting and EC invadosome assays. Retinas from cathepsin KO mice did not show gross morphological abnormalities. In the laser CNV model, however, Ctsb/z DKO mice displayed a significantly reduced neovascularized area compared to WT (0.027 mm2 vs. 0.052 mm2; p = 0.012), while single knockouts did not differ significantly from WT. In line, VEGF-induced EC spheroid sprouting and invadosome formation were not significantly altered by a specific cathepsin B inhibitor alone, but significantly suppressed when more than one cathepsin was inhibited. Our results demonstrate that laser-CNV formation is significantly reduced in Ctsb/z DKO mice. In line, EC sprouting and invadosome formation are blunted when more than one cathepsin is inhibited in vitro. These results reveal an angiomodulatory potential of cathepsins with partial functional redundancies between different cathepsin family members.

Response gene to complement 32 deficiency causes impaired placental angiogenesis in mice

The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in the placental angiogenesis during pregnancy and explore the underlying mechanisms. RGC-32-deficient (RGC32(-/-)) mice were generated from C57BL/6 embryonic stem cells with deletion of exon 2 and 3 of the RGC-32 gene. Most of the RGC32(-/-) mice can survive. However, their body sizes were much smaller compared with their wild-type littermates when they were born. By examining the embryo development and placentas at 16.5 days post-coitum, we found that RGC32(-/-) embryos and foetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32(-/-) placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF) were significantly down-regulated in RGC32(-/-) placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placental angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. RGC-32 appeared to regulate VEGFR2 expression via activation of NF-kB. Moreover, RGC-32 regulated trophoblasts proliferation via control of PlGF expression. Absence of RGC-32 caused foetal growth restriction through interrupting the placental angiogenesis, which was due to the decrease in VEGFR2 expression through the NF-kB-dependent pathway in endothelial cells and PlGF expression in trophoblasts.

Abstract 545: Response Gene to Complement 32-deficiency Causes Impaired Placental Angiogenesis in Mice

Aims The objectives of this study are to determine the role of response gene to complement 32 (RGC-32) in the placental angiogenesis during pregnancy and explore the underlying mechanisms. Methods and Results RGC-32-deficient (RGC32-/-) mice were generated from C57BL/6 embryonic stem cells with deletion of Exon 2 and 3 of RGC-32 gene. Most of the RGC32-/- mice can survive. However, their body sizes were much smaller compared to their wild-type littermates when they were born. By examining the embryo development and placentas at 16.5 days post-coitum, we found that RGC32-/- embryos and fetal placentas were significantly smaller than the wild-type. Further analysis showed that the labyrinth zone of RGC32-/- fetal placenta was smaller with defective angiogenesis. Mechanistically, vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) and placental growth factor (PlGF), but not other angiogenic factors, were significantly downregulated in RGC32-/- placentas, suggesting that VEGFR2 and PlGF may mediate RGC-32 function in placenta angiogenesis. Indeed, knockdown of RGC-32 by shRNA inhibited VEGF-induced endothelial cell proliferation, migration, and tube formation while blocking VEGFR2 expression. Knockdown of RGC-32 also inhibited trophoblast proliferation while blocking the expression of PlGF. Conclusion Absence of RGC-32 caused intrauterine growth restriction through interrupting the placental angiogenesis, which was due to the decreased expressions of VEGFR2 in endothelial cells and PlGF in trophoblast cells.

Vascular endothelial growth factor stimulates endothelial differentiation from mesenchymal stem cells via Rho/myocardin-related transcription factor-A signaling pathway

Mesenchymal stem cells (MSCs) are pluripotent progenitors that can differentiate into a variety of cell types. Vascular endothelial growth factor (VEGF) is one of the major factors of initiating and regulating angiogenesis. It has been reported that VEGF can induce MSCs differentiated into endothelial cells (ECs). However, the mechanism that VEGF-induced MSC differentiation is not completely understood. Here, we showed that VEGF induced human and rat bone marrow-derived MSCs differentiation to ECs. Rho family plays an important role in VEGF-induced endothelial cell migration and angiogenesis. Our results indicated that in MSCs, VEGF activated Rho/ROCK signaling pathway and promoted nuclear translocation of myocardin-related transcription factor-A (MRTF-A), which is controlled by Rho/ROCK signaling. In addition, Rho inhibitor C3 transferase, ROCK inhibitor Y27632 or depletion of endogenous MRTF-A abolished the VEGF-induced differentiation of MSCs into ECs. Furthermore, VEGF also enhanced the expression levels of CYR61/CCN1, as a regulator of vascular development and angiogenesis, and knockdown of endogenous MRTF-A reduced VEGF-induced the upregulation of CYR61/CCN1. Report assays with site-direct mutation analysis of CYR61/CCN1 promoter demonstrated that MRTF-A transactivated CYR61/CCN1 promoter mainly depending on CArG box. In this study, we identify the Rho/MRTF-A signaling pathway as a main actor in controlling VEGF-induced differentiation of human and rat bone marrow-derived MSCs into endothelial cells.

Abstract 2063: Synergistic anticancer activity of Sorafenib in combination with HS-173, a novel PI3K inhibitor against pancreatic cancer cells.

Abstract The K-RAS mutation, resulting in activation of the Raf/MEK/ERK pathway, has been implicated in pancreatic cancer development. And the PI3K/Akt pathway is highly activated in pancreatic cancer and considered to be a cancer hallmark. The principal objective of this study was to assess the synergic effect between Sorafenib (a Raf inhibitor) and HS-173 (a novel PI3K inhibitor) along with the underlying mechanism to gain insight into novel therapeutic strategies for treating pancreatic cancer. We first investigated the cytotoxic effect of co-treatment with Sorafenib and HS-173 using the Calcusyn program. Combined treatment with the two drugs synergistically inhibited the viability of Panc-1 cells (combination index < 1). Concomitantly, co-treatment with Sorafenib and HS-173 induced G2/M arrest and increased apoptosis with the loss of mitochondria potential. Apoptosis resulting from co-treatment with Sorafenib and HS-173 was accompanied by increased levels of cleaved caspase-3 and PARP as well as greater numbers of TUNEL-positive apoptotic cells compared to treatment with either drug alone. Furthermore, combined treatment with these drugs decreased the expression HIF-1α and VEGF, two factors that play an important role in angiogenesis. The anti-angiogenic effect of simultaneous treatment with Sorafenib and HS-173 was confirmed by observing the suppressed tube formation of VEGF-induced human umbilical vein endothelial cells and inhibition of blood vessel formation in a Matrigel plug assay in mice. Taken together, our study demonstrated that combined treatment with Sorafenib and HS-173 has a synergistic anticancer effect on pancreatic cancer cells, indicating that simultaneously targeting the Raf/MEK and PI3K/Akt pathways can induce a synergistic inhibitory effect on pancreatic cancers in which both pathways are activated. Based on the observations from our study, we suggest that the combined administration of these two drugs may be considered to be a new therapeutic regimen for treating pancreatic cancer. Citation Format: Sun-Mi Yun, Kyung Hee Jung, Hyunseung Lee, Byung Hee Park, Hong Hua Yan, Soon-Sun Hong. Synergistic anticancer activity of Sorafenib in combination with HS-173, a novel PI3K inhibitor against pancreatic cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2063. doi:10.1158/1538-7445.AM2013-2063

Abstract 3873: MMP9 processing of HSBP1 regulates tumor metastases.

Abstract Matrix metalloproteinases regulate pathophysiological events by processing matrix proteins and secreted proteins. Previously, we demonstrated that soluble heat shock protein B1 (HSPB1) is released primarily from endothelial cells (ECs) and regulates angiogenesis via direct interaction with vascular endothelial growth factor (VEGF). Here we report that MMP9 can cleave HSPB1 and release anti-angiogenic fragments, which play a key role in tumor metastasis. We mapped the cleavage sites and explored their physiological relevance during these processing events. HSPB1 cleavage by MMP9 inhibited VEGF-induced ECs activation and the C-terminal HSPB1 fragment exhibited more interaction with VEGF than did full-length HSPB1. HSPB1 cleavage occurs during B16F10 lung metastases in wild-type mice and is decreased in B16F10 lung metastases of MMP9-null mice. Finally, we confirmed that secretion of C-terminal HSPB1 fragment was significantly inhibited lung metastases of B6F10 melanoma cells, compared to full-length HSPB1. These data suggest that in vivo MMP9-mediated processing of HSPB1 acts to maintain an angiogenic balance against tumor metastases. Moreover, these findings potentially explain an anti-target effect for the failure of MMP inhibitors in clinical trials, suggesting that MMP inhibitors may have pro-tumorigenic effects by reducing HSPB1 fragmentation. Citation Format: Seo-Hyun Choi, Hae-June Lee, Yeung Bae Jin, Minyoung Lee, Joon Kim, Sam S. Yoon, Yun-Sil Lee, Yoon-Jin Lee. MMP9 processing of HSBP1 regulates tumor metastases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3873. doi:10.1158/1538-7445.AM2013-3873

Thioredoxin-Interacting Protein Mediates Sustained VEGFR2 Signaling in Endothelial Cells Required for Angiogenesis

Thioredoxin-interacting protein (TXNIP) is an α-arrestin protein whose function is important for the regulation of vascular endothelial growth factor receptor 2 (VEGFR2) signaling and endothelial cell survival. Because VEGFR2 is critical for angiogenesis, we explored the role of TXNIP in VEGF-induced angiogenesis. TXNIP knockdown inhibited VEGF-induced endothelial cell tube formation and proliferation in cultured human umbilical vein endothelial cell. To elucidate the mechanism by which TXNIP altered VEGFR2 signaling in human umbilical vein endothelial cell, we studied phosphorylation of VEGFR2, phospholipase C gamma-1 (PLCγ1), endothelial NO synthase, and Akt (known as protein kinase B). TXNIP knockdown significantly decreased phosphorylation of VEGFR2 and PLCγ1 at times >5 minutes, but phosphorylation was unchanged at 2 minutes, as was Akt and endothelial NO synthase phosphorylation. Cell-surface biotinylation assay showed that TXNIP knockdown significantly attenuated VEGFR2 internalization. These results suggested that TXNIP was required for sustained VEGFR2 signaling, which is mediated largely by internalized VEGFR2. Rab5 knockdown to inhibit the trafficking and fusion of early endosomes significantly blocked VEGF-induced VEGFR2 internalization and phosphorylation of VEGFR2 and PLCγ1. Immunofluorescence and coimmunoprecipitation showed that TXNIP was part of a complex that included Rab5 and VEGFR2. Finally, TXNIP knockdown prevented the association of VEGFR2 and Rab5. Our results show that TXNIP is essential for VEGFR2 internalization in Rab5 positive endosomes, which is required for endothelial cell growth and angiogenesis.

Synergistic anticancer activity of HS-173, a novel PI3K inhibitor in combination with Sorafenib against pancreatic cancer cells

AbstractThe RAF/MEK/ERK and PI3K/AKT pathways are highly implicated in the development of pancreatic cancer. The principal objective of this study was to assess the synergic effect between Sorafenib (a RAF inhibitor) and HS-173 (a novel PI3K inhibitor) to gain insight into novel therapeutic strategies for treating pancreatic cancer. We first investigated the cytotoxic effect of co-treatment with Sorafenib and HS-173 using the Calcusyn program. Combined treatment of the two drugs synergistically inhibited the viability of Panc-1 cells (combination index<1). Concomitantly, the co-treatment induced G2/M arrest and increased apoptosis with the loss of mitochondrial membrane potential. Apoptosis resulting from the co-treatment was accompanied by increased levels of cleaved caspase-3 and PARP as well as greater numbers of TUNEL-positive apoptotic cells compared to treatment with either drug alone. Furthermore, combined treatment with these drugs decreased the expression of HIF-1α and VEGF which play an important role in angiogenesis. This anti-angiogenic effect was confirmed by the suppressed tube formation of VEGF-induced human umbilical vein endothelial cells and inhibition of blood vessel formation in a Matrigel plug assay in mice. Taken together, our study demonstrates that combined treatment with Sorafenib and HS-173 has a synergistic anti-cancer effect on pancreatic cancer cells, indicating that simultaneously targeting the RAF/MEK and PI3K/AKT pathways can induce a synergistic inhibitory effect on pancreatic cancers in which both pathways are activated. Based on the observations from our study, we suggest that the combined administration of these two drugs may be considered to be a new therapeutic regimen for treating pancreatic cancer.

Targeting of Integrin-Linked Kinase with Small Interfering RNA Inhibits VEGF-Induced Angiogenesis in Retinal Endothelial Cells

Background: The pathological angiogenesis in the retina is a major cause of vision loss at all ages. Vascular endothelial growth factor (VEGF) has been reported as the most potent inducer of retinal neovascularization. We previously demonstrated that integrin-linked kinase (ILK) regulates retinal vascular endothelial proliferation, migration and tube formation. However, little is known about the existence of cross-talk between ILK and VEGF signaling in retinal vascular endothelial cells and the probable regulatory role of ILK during VEGF-induced retinal endothelial cell migration. The purpose of this work was to investigate the role of ILK in VEGF-induced retinal neovascularization. Methods: Cultured retinal endothelial cells (RF/6A) were knocked down for ILK using a small interfering RNA (siRNA). For this, cellular ILK expression was quantified by real-time quantitative PCR, Western blot analysis and immunocytochemical assay, and cytotoxicity of transfection was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. ILK siRNA-transfected RF/6A cells were induced by VEGF, and cell proliferation was determined by the MTT assay, cell migration was measured by cell counting in modified Boyden chambers and cell spreading and tube formation assays were performed. Furthermore, the impact of ILK-specific siRNA on VEGF-induced VEGF receptor 2 (VEGFR-2) phosphorylation and activation of downstream signal pathways were tested by Western blot analysis. Results: Both ILK mRNA and protein levels were virtually undetectable after transfection with ILK siRNA, and blocking the expression of ILK by siRNA significantly inhibited VEGF-induced retinal endothelial cell proliferation, attachment, spreading, migration and tube formation. Knockdown of ILK effectively suppressed VEGF-induced p38 mitogen-activated protein kinase (MAPK) and Akt phosphorylation, but had no effects on VEGFR-2, extracellular signal-regulated protein kinase and Jun N terminus kinase phosphorylation. Conclusion: We conclude that knockdown of ILK with siRNA effectively inhibited VEGF-induced retinal endothelial cell attachment, spreading, migration and tube formation. p38 MAPK and Akt are downstream signaling pathways of the ILK that regulated VEGF-induced retinal neovascularization. Targeting ILK may be a potentially useful therapeutic approach for treating ocular neovascularization.

Cross-talk between leukemic and endothelial cells promotes angiogenesis by VEGF activation of the Notch/Dll4 pathway

Angiogenesis is suggested to be important for leukemogenesis and chemosensitivity in acute myeloid leukemia (AML). The vascular endothelial growth factor (VEGF) and Notch/Dll4 pathways have been identified as critical in the regulation of embryonic vascular development and tumor angiogenesis. However, the potential role of the Notch/Dll4 pathway in leukemia-endothelium cross-talk and its functional link with VEGF remains obscure. This study assessed the expression of VEGF and Notch/Dll4 pathway molecules in primary AML and investigated their biological function in the coculture of endothelial cells with AML cells. The results demonstrated that bone marrow vascularity in the newly diagnosed AML patients was increased and correlated with high VEGF and Dll4 expression. Patients with untreated AML expressed higher levels of VEGFR2, Notch1, Dll4 and Hes1 than healthy controls. Moreover, the activation of the Notch/Dll4 pathway is associated with poor prognosis in AML. In addition, AML cells were shown to increase endothelial cell proliferation in Transwell coculture. This was associated with concomitant activation of the Notch/Dll4 pathway and upregulation of its downstream genes, such as matrix metalloproteinases, resulting in the enhancement of endothelial cell migration and tube formation. Our study also showed that upregulation of Dll4 expression in AML cells by cDNA transfection suppressed VEGF-induced endothelial cell proliferation and angiogenesis in direct contact coculture. These results elucidate a novel mechanism by which the interplay between AML and endothelial cells promotes angiogenesis through the Notch/Dll4 pathway. Modulation of this pathway may, therefore, hold promise as a novel antiangiogenic strategy for the treatment of AML.

Nox4- and Nox2-dependent oxidant production is required for VEGF-induced SERCA cysteine-674 S-glutathiolation and endothelial cell migration

Endothelial cell (EC) migration in response to vascular endothelial growth factor (VEGF) is a critical step in both physiological and pathological angiogenesis. Although VEGF signaling has been extensively studied, the mechanisms by which VEGF-dependent reactive oxygen species (ROS) production affects EC signaling are not well understood. The aim of this study was to elucidate the involvement of Nox2- and Nox4-dependent ROS in VEGF-mediated EC Ca2+ regulation and migration. VEGF induced migration of human aortic ECs into a scratch wound over 6 h, which was inhibited by overexpression of either catalase or superoxide dismutase (SOD). EC stimulation by micromolar concentrations of H2O2 was inhibited by catalase, but also unexpectedly by SOD. Both VEGF and H2O2 increased S-glutathiolation of SERCA2b and increased Ca2+ influx into EC, and these events could be blocked by overexpression of catalase or overexpression of SERCA2b in which the reactive cysteine-674 was mutated to a serine. In determining the source of VEGF-mediated ROS production, our studies show that specific knockdown of either Nox2 or Nox4 inhibited VEGF-induced S-glutathiolation of SERCA, Ca2+ influx, and EC migration. Treatment with H2O2 induced S-glutathiolation of SERCA and EC Ca2+ influx, overcoming the knockdown of Nox4, but not Nox2, and Amplex red measurements indicated that Nox4 is the source of H2O2. These results demonstrate that VEGF stimulates EC migration through increased S-glutathiolation of SERCA and Ca2+ influx in a Nox4- and H2O2-dependent manner, requiring Nox2 downstream.

β2-Glycoprotein I inhibits VEGF-induced endothelial cell growth and migration via suppressing phosphorylation of VEGFR2, ERK1/2, and Akt

β(2)-glycoprotein I (β(2)-GPI) is a plasma glycoprotein with diverse functions, but the impact and molecular effects of β(2)-GPI on vascular biology are as yet unclear. Based on the limited information available on the contribution of β(2)-GPI to endothelial cells, we investigated the effect of β(2)-GPI on cell growth and migration in human aortic endothelial cells (HAECs). The regulation of β(2)-GPI as part of intracellular signaling in HAECs was also examined. Vascular endothelial growth factor (VEGF) is a pro-angiogenic factor that may regulate endothelial functions. We found that β(2)-GPI dose-dependently inhibited VEGF-induced endothelial cell growth using the 3-(4,5-dimethylthiazol-2-yl)-2,5-dipenyl tetrazolium bromide assay and cell counts. Using wound healing and Boyden chamber assays, β(2)-GPI remarkably reduced VEGF-increased cell migration at the physiological concentration. Furthermore, β(2)-GPI suppressed VEGF-induced phosphorylation of VEGF receptor 2 (VEGFR2), extracellular signal-regulated kinase 1/2 (ERK1/2), and Akt. These results suggest that β(2)-GPI plays an essential role in the down-regulation of VEGF-induced endothelial responses and may be a useful component for anti-angiogenic therapy.

MiR-20a represses endothelial cell migration by targeting MKK3 and inhibiting p38 MAP kinase activation in response to VEGF

Endothelial cell migration induced in response to vascular endothelial growth factor (VEGF) is a crucial step of angiogenesis and it depends on the activation of the p38 MAP-kinase pathway downstream of VEGFR2. In this study, we investigated the role of microRNAs (miRNAs) in regulating these processes. We found that the VEGF-induced p38 activation and cell migration are modulated by overexpression of Argonaute 2, a key protein in the functioning of miRNAs. Thereafter, we found that miR-20a expression is increased by VEGF and that its ectopic expression inhibits VEGF-induced actin remodeling and cell migration. Moreover, the expression of miR-20a impairs the formation of branched capillaries in a tissue-engineered model of angiogenesis. In addition, the lentivirus-mediated expression of miR-20a precursor (pmiR-20a) is associated with a decrease in the VEGF-induced activation of p38. In contrast, these processes are increased by inhibiting miR-20a with a specific antagomir. Interestingly, miR-20a does not modulate VEGFR2 or p38 protein expression level. miR-20a does not affect either the expression of other known actors of the p38 MAP kinase pathway except MKK3. Indeed, by using quantitative PCR and Western Blot analysis, we found that pmiR-20a decreases the expression of MKK3 and we obtained evidence indicating that miR-20a specifically binds to the 3'UTR region of MKK3 mRNA. In accordance, the VEGF-induced activation of p38 and cell migration are impaired when the MKK3 expression is knocked down by siRNA. We conclude that miR-20a acts in a feedback loop to repress the expression of MKK3 and to negatively regulate the p38 pathway-mediated VEGF-induced endothelial cell migration and angiogenesis.

Redox Regulation of SERCA2 Is Required for Vascular Endothelial Growth Factor-Induced Signaling and Endothelial Cell Migration

Vascular endothelial growth factor (VEGF) increases angiogenesis by stimulating endothelial cell (EC) migration. VEGF-induced nitric oxide ((•)NO) release from (•)NO synthase plays a critical role, but the proteins and signaling pathways that may be redox-regulated are poorly understood. The aim of this work was to define the role of (•)NO-mediated redox regulation of the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) in VEGF-induced signaling and EC migration. VEGF-induced EC migration was prevented by the (•)NO synthase inhibitor, N (G)-nitro-L-arginine methyl ester (LNAME). Either VEGF or (•)NO stimulated endoplasmic reticulum (ER) (45)Ca(2+) uptake, a measure of SERCA activity, and knockdown of SERCA2 prevented VEGF-induced EC migration and (45)Ca(2+) uptake. S-glutathione adducts on SERCA2b, identified immunochemically, were increased by VEGF, and were prevented by LNAME or overexpression of glutaredoxin-1 (Glrx-1). Furthermore, VEGF failed to stimulate migration of ECs overexpressing Glrx-1. VEGF or (•)NO increased SERCA S-glutathiolation and stimulated migration of ECs in which wild-type (WT) SERCA2b was overexpressed with an adenovirus, but did neither in those overexpressing a C674S SERCA2b mutant, in which the reactive cysteine-674 was mutated to a serine. Increased EC Ca(2+) influx caused by VEGF or (•)NO was abrogated by overexpression of Glrx-1 or the C674S SERCA2b mutant. ER store-emptying through the ryanodine receptor (RyR) and Ca(2+) entry through Orai1 were also required for VEGF- and (•)NO-induced EC Ca(2+) influx. These results demonstrate that (•)NO-mediated activation of SERCA2b via S-glutathiolation of cysteine-674 is required for VEGF-induced EC Ca(2+) influx and migration, and establish redox regulation of SERCA2b as a key component in angiogenic signaling.

Prevention of VEGF-induced growth and tube formation in human retinal endothelial cells by aldose reductase inhibition

ObjectiveSince diabetes-induced vascular endothelial growth factor (VEGF) is implicated in retinal angiogenesis, we aimed to examine the role of aldose reductase (AR) in VEGF-induced human retinal endothelial cells (HREC) growth and tube formation. Materials and MethodsHRECs were stimulated with VEGF and cell-growth was determined by MTT assay. AR inhibitor, fidarestat, to block the enzyme activity and AR siRNA to ablate AR gene expression in HREC were used to investigate the role of AR in neovascularization using cell-migration and tube formation assays. Various signaling intermediates and angiogenesis markers were assessed by Western blot analysis. Immuno-histochemical analysis of diabetic rat eyes was performed to examine VEGF expression in the retinal layer. ResultsStimulation of primary HREC with VEGF caused increased cell growth and migration, and AR inhibition with fidarestat or ablation with siRNA significantly prevented it. VEGF-induced tube formation in HREC was also significantly prevented by fidarestat. Treatment of HREC with VEGF also increased the expression of VCAM, AR, and phosphorylation and activation of Akt and p38-MAP kinase, which were prevented by fidarestat. VEGF-induced expression of VEGFRII in HREC was also prevented by AR inhibition or ablation. ConclusionsOur results indicate that inhibition of AR in HREC prevents tube formation by inhibiting the VEGF-induced activation of the Akt and p38-MAPK pathway and suggest a mediatory role of AR in ocular neovascularization generally implicated in retinopathy and AMD.

Angiopoietin-1 and Vascular Endothelial Growth Factor Regulation of Leukocyte Adhesion to Endothelial Cells

Vascular endothelial growth factor (VEGF) promotes leukocyte adhesion to endothelial cells (ECs). Angiopoietin-1 (Ang-1) inhibits this response. Nuclear receptor-77 (Nur77) is a proangiogenic nuclear receptor. In the present study, we assessed the influence of Ang-1 and VEGF on Nur77 expression in ECs, and evaluated its role in Ang-1/VEGF-mediated leukocyte adhesion. Expression of Nur77 was evaluated with real-time polymerase chain reaction and immunoblotting. Adhesion of leukocytes to ECs was monitored with inverted microscopy. Nur77 expression or activity was inhibited using adenoviruses expressing dominant-negative form of Nur77, retroviruses expressing Nur77 in the antisense direction, and small interfering RNA oligos. Both Ang-1 and VEGF induce Nur77 expression, by >5- and 30-fold, respectively. When combined, Ang-1 potentiates VEGF-induced Nur77 expression. Ang-1 induces Nur77 through the phosphoinositide 3-kinase and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces Nur77 expression through the protein kinase D/histone deacetylase 7/myocyte enhancer factor 2 and extracellular signal-regulated protein kinase 1/2 pathways. VEGF induces nuclear factor-kappaB transcription factor, vascular cell adhesion molecule-1, and E-selectin expressions, and promotes leukocyte adhesion to ECs. Ang-1 inhibits these responses. This inhibitory effect of Ang-1 disappears when Nur77 expression is disrupted, restoring the inductive effects of VEGF on adhesion molecule expression, and increased leukocyte adhesion to ECs. Nur77 promotes anti-inflammatory effects of Ang-1, and functions as a negative feedback inhibitor of VEGF-induced EC activation.

Role of osteoprotegerin (OPG) in angiogenesis

Background: Angiogenesis, the development of blood vessels from an already existing microvascular network, is not only of fundamental importance to many physiological processes, but has also been associated with various pathologies, ranging from cancer and chronic inflammatory diseases to cardiovascular pathologies, including atherosclerosis and ischemic heart disease. Heme oxygenase-1 (HO-1) is the cytoprotective enzyme which catalyzes the rate-limiting step in the oxidative degradation of heme into its products carbon monoxide (CO), free iron (Fe), and biliverdin, which is rapidly converted to bilirubin. Complex anti-inflammatory, antiapoptotic, and anti-oxidanteffectshavebeen identified in thevasculature forHO-1 and its products. In addition, increasing evidence suggests a role for HO-1 in angiogenesis. However, the precise mechanisms through which HO-1 exerts its effects on angiogenesis remain elusive. Results: Herein, we show that inhibition of HO-1 either by synthetic inhibitor (ZnPP) or specific siRNA alters the angiogenic process at various levels.HO-1 depletion significantly reduced vascular endothelial growth factor A (VEGF)-mediated human endothelial cell (EC) proliferation and significantly inhibited capillary-like formation on a 2D-Matrigel in vitro. Further, we demonstrate that VEGF-induced EC cell cycle progression is inhibited by HO-1 siRNA; an observation associated with decreased expression of the cell cycle regulators cyclin A1 and cyclin E1. In contrast, HO-1 depletion did not alter susceptibility of endothelial cells to serum-starvation induced cell death and HO-1deficient cells were still protected from apoptosis by VEGF, most likely through induction of the anti-apoptotic genes Bcl-2 and A1. Interestingly,HO-1depletion alsonegatively affecteddirectionalmigrationof EC towards a VEGF gradient; a phenotype reversed by HO-1 overexpression using an adenoviral vector. Conclusion: Importantly, on-going experiments will utilise a proteomics approach and targeted microarrays to identify novel downstream targets of HO-1 involved in the regulation of angiogenesis. This may in turn reveal potential therapeutic targets for the manipulation of angiogenesis at sites of ischemia or wound healing, or conversely to inhibit angiogenesis associatedwith atherosclerosis or tumourogenesis.