Artur Chernoguz

VEGF Blockade Enables Oncolytic Cancer Virotherapy in Part by Modulating Intratumoral Myeloid Cells

Understanding the host response to oncolytic viruses is important to maximize their antitumor efficacy. Despite robust cytotoxicity and high virus production of an oncolytic herpes simplex virus (oHSV) in cultured human sarcoma cells, intratumoral (ITu) virus injection resulted in only mild antitumor effects in some xenograft models, prompting us to characterize the host inflammatory response. Virotherapy induced an acute neutrophilic infiltrate, a relative decrease of ITu macrophages, and a myeloid cell-dependent upregulation of host-derived vascular endothelial growth factor (VEGF). Anti-VEGF antibodies, bevacizumab and r84, the latter of which binds VEGF and selectively inhibits binding to VEGF receptor-2 (VEGFR2) but not VEGFR1, enhanced the antitumor effects of virotherapy, in part due to decreased angiogenesis but not increased virus production. Neither antibody affected neutrophilic infiltration but both partially mitigated virus-induced depletion of macrophages. Enhancement of virotherapy-mediated antitumor effects by anti-VEGF antibodies could largely be recapitulated by systemic depletion of CD11b(+) cells. These data suggest the combined effect of oHSV virotherapy and anti-VEGF antibodies is in part due to modulation of a host inflammatory reaction to virus. Our data provide strong preclinical support for combined oHSV and anti-VEGF antibody therapy and suggest that understanding and counteracting the innate host response may help enable the full antitumor potential of oncolytic virotherapy.

Pretreatment with anti-VEGF therapy may exacerbate inflammation in experimental acute colitis

AIM Our previous investigations of angiogenesis in inflammatory bowel disease showed that vascular endothelial growth factor (VEGF) blockade reduced colonic neovascularization and inflammation. We hypothesized that pretreatment with bevacizumab, a monoclonal anti-VEGF antibody, would attenuate the severity of angiogenesis and inflammation in a murine model of colitis. METHODS C57BL/6 mice were treated with intraperitoneal injections of bevacizumab (250 μg/dose) before induction of colitis with dextran sulfate sodium (DSS). The colons were examined at predetermined time points. Colonic inflammation and microvessel density were assessed microscopically. RESULTS All mice acutely developed melena and weight loss (18.8% ± 1.1% control vs 20.2% ± 1.1% treated, P = .37) and regained a similar weight percentage after the recovery (26.5% ± 4.0% vs 20.9% ± 4.4%, P = .37). Microvessel density acutely increased in both groups in response to DSS, with a trend toward inhibited angiogenesis in the treated group at the conclusion of the acute phase (194,100 ± 14,240 vs 149,400 ± 17,590 μm(2), P = .11). Bevacizumab-treated mice exhibited significantly increased inflammation after the acute phase (8.3 ± 0.8 vs 13.0 ± 2.0, P = .05), but were similar to control after the recovery (7.3 ± 1.5 vs 5.5 ± 1.0, P = .27). CONCLUSIONS Preemptive VEGF inhibition does not significantly attenuate angiogenesis and, in fact, worsens inflammation in a model of acute colitis. Preventive VEGF blockade may disrupt healing and exacerbate injury via alternative angiogenic or inflammatory pathways.

EGFR inhibition fails to suppress vascular proliferation and tumor growth in a Ewing's sarcoma model.

BACKGROUND Expression of epidermal growth factor receptor (EGFR), a potent regulator of cellular homeostasis, is associated with aggressive tumor behavior. The mechanism by which EGFR inhibition functions is unclear, with controversial results demonstrating an effect on the tumor cells, endothelial cells, or pericytes. EGFR activation has been linked to the expression of vascular endothelial growth factor (VEGF), a known mitogen of angiogenesis, but the relationship between these factors and their effect on tumor vessel development is vague. We hypothesized that using an EGFR inhibitor on a human Ewings sarcoma model would inhibit tumor growth by suppressing vessel proliferation. METHODS A cell proliferation assay was performed on the Ewings sarcoma (SK-NEP-1) cell line. Tumor cells were implanted intrarenally in athymic mice. Animals received daily gavage with vehicle or gefitinib 1 wk following implantation. Mice (n = 12/cohort) were euthanized 6 wk following implantation. Remaining mice were maintained without treatment for 2 wk. Vascular changes were assessed by angiography and immunohistochemically. EGFR and vascular endothelial growth factor (VEGF) expression were quantified using quantitative polymerase chain reaction (qPCR). RESULTS Gefitinib suppressed in vitro cell growth with an IC(50) = 1.36 μM. Minimal tumor growth suppression was noted at 6 wk (6.01 ± 1.2 g in control versus 4.61 ± 0.9 g treated, P = 0.36). After cessation of gefitinib, tumor growth was increased in both groups (7.37 ± 1.62 g versus 6.77 ± 1.53 g, P = 0.79). Microvessel density was unchanged despite EGFR inhibition (161,000 ± 16,000 pixels versus 135,000 ± 18,000 pixels, P = 0.31). At 6 wk, the vascular maturity index was similar in both groups (3.63 ± 1.12 versus 4.09 ± 1.71, P = 0.83). A downward trend in EGFR expression (49% of control) and an upward trend in VEGF levels (50% of control) occurred in the treated group. CONCLUSIONS EGFR expression was suppressed in cultured cells and xenograft tumors. Despite a cytotoxic effect on cell lines, gefitinib had little effect on tumor growth. No effects on the tumor vasculature were noted in the setting of EGFR suppression, suggesting that angiogenesis induced by SK-NEP-1 cells is refractory to EGFR inhibition. Interestingly, the resulting increase in VEGF expression following EGFR blockade, provides an alternative pro-angiogenic pathway promoting tumor survival.

Vascular endothelial growth factor receptor-2 inhibition in experimental murine colitis

BACKGROUND In the setting of inflammatory bowel disease, inflammation is associated with a simultaneous increase in angiogenesis; moreover, elevated vascular endothelial growth factor (VEGF) levels implicate angiogenesis as a pathologic contributor to disease severity. We hypothesize that selectively inhibiting vascular endothelial growth factor receptor-2 (VEGFR2) in a model of murine colitis will reduce angiogenesis, resulting in decreased inflammation and disease severity, providing mechanistic insight into the role of pathologic angiogenesis in IBD. MATERIALS AND METHODS In a dextran sodium sulfate model of murine colitis, anti-VEGFR2 monoclonal antibody (DC101) or placebo was administered. Clinical assessments followed by histologic and molecular tissue analysis were performed to quantify inflammation, microvessel density (MVD), VEGF and VEGFR2 gene expression, and phosphorylated mitogen-activated protein kinase protein expression. RESULTS Weight loss began after d 6 with the treatment group demonstrating a more favorable percent weight change. Inflammation and MVD were similar between cohorts, both increasing in parallel toward a plateau. VEGF and VEGFR2 messenger RNA expression were not significantly different, but phosphorylated mitogen-activated protein kinase was elevated in the DC101 cohort (P = 0.03). CONCLUSIONS Despite a more favorable weight change profile in the treated group, no difference was observed between cohorts regarding clinical disease severity. However, a parallel rise in inflammation and MVD was observed coinciding with weight loss, suggesting their relationship in IBD. VEGFR2 downstream signaling was significantly elevated in the treated cohort, possibly by VEGF-independent signal transduction. Early and effective inhibition of angiogenesis by limiting downstream VEGF signaling or targeting multiple angiogenic pathways may block angiogenesis, thereby reducing disease severity and provide evidence toward the mechanism and clinical benefit of antiangiogenics in the setting of IBD.

Angiogenesis and Vascular Endothelial Growth Factor-A Expression Associated with Inflammation in Pediatric Crohn’s Disease

Background and AimsAngiogenesis is a component of chronic inflammatory diseases including inflammatory bowel disease. Some studies describe increased angiogenesis associated with acute disease in adult Crohn’s disease and ulcerative colitis, while animal models aid investigations of mechanism and pathophysiology of angiogenesis. We aim to explore the role of angiogenesis and its pathways in pediatric Crohn’s disease.MethodsSurgical specimens were obtained from pediatric Crohn’s disease (both inflamed and non-inflamed regions of ileum) and control patients. Samples were examined for inflammation, microvessel density, and molecular expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, angiopoietin-1, and angiopoietin-2.ResultsAngiogenesis and inflammation were increased in parallel in Crohn’s disease compared to controls. We also discovered increased angiogenesis in Crohn’s disease tissue that was relatively free of inflammatory disease. Vascular endothelial growth factor-A gene expression (P = 0.034) was elevated in Crohn’s disease over controls, while gene expression of platelet-derived growth factor-β (P = 0.069), angiopoietin-1 (P = 0.206), and angiopoietin-2 (P = 0.082) was not significantly elevated.ConclusionsWe confirm that inflammation-associated angiogenesis is upregulated in pediatric Crohn’s disease. This population also exhibits elevated mucosal angiogenesis at the surgical margin with limited inflammation. This suggests that angiogenesis is an additional pathologic characteristic to potentially identify normal mucosa and margins of surgical resection that are uninvolved with disease and, furthermore, may have implications for monitoring complete disease remission. We further identify the vascular endothelial growth factor-A pathway involvement in the disease process, which may serve as a future molecular target for anti-angiogenic therapy in inflammatory bowel disease.