Rationale: There are conflicting data on the effects of vascular endothelial growth factor (VEGF) in vascular remodeling. Furthermore, there are species-specific differences in leukocyte and vascular cell biology and little is known about the role of VEGF in remodeling of human arteries. Objective: We sought to address the role of VEGF blockade on remodeling of human arteries in vivo. Methods and Results: We used an anti-VEGF antibody, bevacizumab, to study the effect of VEGF blockade on remodeling of human coronary artery transplants in severe combined immunodeficient mice. Bevacizumab ameliorated peripheral blood mononuclear cell–induced but not interferon-γ–induced neointimal formation. This inhibitory effect was associated with a reduction in graft T-cell accumulation without affecting T-cell activation. VEGF enhanced T-cell capture by activated endothelium under flow conditions. The VEGF effect could be recapitulated when a combination of recombinant intercellular adhesion molecule 1 and vascular cell adhesion molecule-1 rather than endothelial cells was used to capture T cells. A subpopulation of CD3+ T cells expressed VEGF receptor (VEGFR)-1 by immunostaining and FACS analysis. VEGFR-1 mRNA was also detectable in purified CD4+ T cells and Jurkat and HSB-2 T-cell lines. Stimulation of HSB-2 and T cells with VEGF triggered downstream ERK phosphorylation, demonstrating the functionality of VEGFR-1 in human T cells. Conclusions: VEGF contributes to vascular remodeling in human arteries through a direct effect on human T cells that enhances their recruitment to the vessel. These findings raise the possibility of novel therapeutic approaches to vascular remodeling based on inhibition of VEGF signaling.

Cha, Charles MD; Mulkeen, Abby L. MD; Yoo, Peter S. MD; Silva, Teresa BS; Schmitz, John C. PhD; Chu, Edward MD; Uchio, Edward MD Author Information

Hypothesis By using short interfering RNA (siRNA) to inhibit the in vitro expression of vascular endothelial growth factor (VEGF) A, we hope to further investigate the presence of an autocrine loop in colon cancer cells. We hypothesize that VEGF inhibition will result in decreased cellular proliferation. Design Human colon cancer cells were evaluated for the expression of VEGF and VEGF receptor 2 (VEGFR-2). In vitro assessments were then made of the ability of anti-VEGF siRNA to knock down expression of VEGF and the subsequent effect this decreased expression had on colon cancer cell proliferation. Setting Surgical oncology research laboratory. Interventions Human colon cancer cells from the RKO cell line were transfected with siRNA targeting the coding region of VEGF. Main Outcome Measures Enzyme-linked immunosorbent assay, Northern blot analysis, and real-time quantitative polymerase chain reaction were performed to establish the ability of siRNA to decrease VEGF production. Proliferation assays were run on transfected and wild-type cells to establish concomitant decrease in VEGF expression and cellular proliferation. Results The RKO colon cancer cells expressed both VEGF and VEGFR-2. Those cells transfected with siRNA targeting VEGF showed a 94% knockdown in VEGF expression and a 67% decrease in cellular proliferation. Conclusion Colon cancer cells expressing VEGF and VEGFR-2 may possess an autocrine growth pathway that can be effectively targeted using RNA interference as an antiangiogenic therapy.

Introduction: Vascular endothelial growth factor (VEGF) is the most potent angiogenic factor and is expressed by nearly all solid tumors. VEGF mRNA is intrinsically labile but is stabilized by the RNA-binding protein, HuR. Recent animal experiments have demonstrated that the expression of HuR is upregulated in colorectal cancer. We investigated the role of HuR in the regulation of VEGF expression in human colorectal cancer cells. Methods: Human colorectal carcinoma RKO cells were cultured and transiently transfected with siHuR, a short interfering RNA (siRNA) targeting the coding region of HuR mRNA. Untransfected cells and cells transfected with siGL2, a nontargeting functional control siRNA, were used as controls. VEGF expression was quantified by sandwich ELISA, Northern and Western blotting. Expression of HuR was measured by Western and Northern blot analysis. Tumor cell proliferation was measured by WST proliferation assay. Results: RKO colorectal cancer cells were observed to express both HuR and VEGF on Western blot analysis and ELISA. Cells transfected with siHuR demonstrated an 86% knockdown of HuR expression by Western blot analysis (Figure 1a). Additionally, after transient transfection with siHuR, the RKO cells demonstrated a statistically significant decrease in VEGF expression compared to untransfected (UT) cells and cells transfected with siGL2 (Figure 1b). Average decrease in VEGF was 53% (p <0.01) and 44.5% (p <0.02) at 48 and 72 hours following transfection, respectively. Conclusion: We conclude that HuR is an important post-transcriptional regulator of VEGF in colorectal carcinoma cells. Therapies targeting HuR and other translational regulators of VEGF may have important clinical applications.

Introduction. RNA interference (RNAi) allows for the specific targeting and silencing of genes, mediated by small interfering RNA (siRNA). VEGF is one of the most potent pro-angiogenic factors for tumor growth and is expressed in virtually all solid tumors. Furthermore, recent clinical trials have demonstrated efficacy of anti-VEGF therapy in colon cancer. We therefore investigated the effect of siRNA mediated silencing of VEGF in human colorectal cancer cells. Methods. siRNAs, designed in silico using a bioinformatics approach, were synthesized as double-stranded RNA and screened for VEGF gene silencing efficacy in human colon cancer cells. VEGF expression was quantified by sandwich ELISA analysis and confirmed by Northern and Western blot analysis. Tumor proliferation was measured by WST proliferation assay. Results. A series of VEGF-specific siRNAs were synthesized targeting the coding region of VEGF and four showed evidence of significant gene silencing in a variety of human colon cancer cell lines including RKO and HCT 116. Greater than 95% knockdown was seen on ELISA assay with some sequences, and gene silencing occurred in a dose-dependent manner. A significant effect on tumor proliferation was seen on WST assay with treatment with VEGF targeted siRNA compared to a scrambled control siRNA sequence, as seen in the figure (P = .033), and 0.1 nM siRNA was sufficient to show some effect. We are currently testing the efficacy of these siRNAs in inhibiting tumor growth in a mouse xenograph tumor model. Conclusion. SiRNA targeted therapy is effective in silencing VEGF gene expression leading to decreased tumor proliferation in human colorectal cancer cells. This approach represents a potentially effective therapeutic approach to the treatment of a broad range of malignancy.