Volume 126, Issue 3, Pages 886-894 (March 2004) Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice  Mercedes Fernandez, Francesco Vizzutti, Juan Carlos Garcia-Pagan, Juan Rodes, Jaime Bosch  Gastroenterology  Volume 126, Issue 3, Pages 886-894 (March 2004) DOI: 10.1053/j.gastro.2003.12.012

Figure 1 Development of portal-systemic collateral vessels and portal hypertension in mice. C57BL/6J mice were subjected to partial portal vein ligation (PPVL) to induce portal hypertension, or to a sham operation in the control group. At days 1, 2, 3, 4, 5, 7, and 12 after partial portal vein ligation, we measured (A) the formation of portal-systemic collateral vessels (%; mean ± SEM) and (B) the portal venous pressure (mm Hg; mean ± SEM). The formation of portal-systemic collateral vessels was minimal in sham-operated mice (average, 0.25 ± 0.1%), while it increased progressively after partial portal vein ligation, reaching values of 91.5 ± 6% by day 12. Discontinuous line in panel B represents the average value of portal pressure in sham-operated mice (8.1 ± 1.3 mm Hg). Partial portal vein-ligated mice had significant increases in portal pressure at each time point. ∗P < 0.05 compared with day 1. ∗∗ P < 0.01 compared with day 1. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)

Figure 2 Overexpression of VEGF in portal hypertensive mice. (A) VEGF protein expression in splanchnic organs (duodenum, intestine, and mesentery) from partial portal vein-ligated (PPVL) mice and sham-operated (SO) control animals, 7 days after the initial surgery. Representative Western blots for VEGF and the housekeeping protein α-tubulin are shown at the top, and densitometric quantification of VEGF protein expression normalized to α-tubulin (arbitrary units; mean ± SEM of 4 different experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels in sham-operated mice. (B) Intestinal VEGF protein expression at days 1, 5, and 7 after the induction of portal hypertension by partial portal vein ligation in mice. Representative Western blots are shown at the top, and quantification of protein signals normalized to α-tubulin (arbitrary units; mean ± SEM of 4 separate experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels at day 1. VEGF expression also increased, in a time-dependent manner, in the duodenum and mesentery of portal hypertensive mice. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)

Figure 3 Overexpression of VEGF receptor-2 (VEGFR-2) in portal hypertensive mice. (A) VEGF receptor-2 protein expression in splanchnic organs (duodenum, intestine, and mesentery) from mice subjected to partial portal vein ligation (PPVL) and from sham-operated (SO) control animals, 7 days after the initial surgery. Representative Western blots for VEGFR-2 and α-tubulin are shown at the top, and densitometric quantification of VEGFR-2 protein expression normalized to α-tubulin (arbitrary units; mean ± SEM of 4 different experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels in sham-operated mice. (B) Intestinal protein expression of VEGF receptor-2 at days 1, 5, and 7 after partial portal vein ligation in mice. Representative Western blots are shown at the top, and quantification of protein signals normalized to α-tubulin (arbitrary units; mean ± SEM of 4 separate experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels at day 1. VEGF receptor-2 expression also increased, in a time-dependent manner, in the duodenum and mesentery of portal hypertensive mice. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)

Figure 4 Increased vascularization in portal hypertensive mice. (A) CD31 protein expression in splanchnic organs (duodenum, intestine, and mesentery) from partial portal vein-ligated (PPVL) mice and sham-operated (SO) control animals, 7 days after the initial surgery. Representative Western blots for CD31 and the control α-tubulin protein are shown at the top, and densitometric quantification of CD31 protein expression normalized to α-tubulin (arbitrary units; mean ± SEM of 4 different experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels in sham-operated mice. (B) CD31 protein levels in mouse intestines at days 1, 5, and 7 after partial portal vein ligation. Representative Western blots are shown at the top, and quantification of protein signals normalized to α-tubulin (arbitrary units; mean ± SEM of 4 separate experiments) is shown at the bottom. ∗P < 0.05 compared with protein levels at day 1. CD31 expression also increased, in a time-dependent manner, in the duodenum and mesentery of portal hypertensive mice. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)

Figure 5 Effects of DC101 on the extent of collaterals formation and portal pressure. (A) Treatment of partial portal vein-ligated mice with a monoclonal antibody directed against VEGF receptor-2 (DC101 antibody) during 5, 7, or 12 days resulted in a significant inhibition of the formation of portal-systemic collateral vessels, compared with IgG-treated mice. ∗P < 0.05 compared with IgG-treated mice. ∗∗P < 0.01 compared with IgG-treated mice. (B) Administration of DC101 to partial portal vein-ligated mice did not modify significantly portal pressure, compared with IgG-treated mice. Not significant (NS) compared with IgG-treated mice. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)

Figure 6 Effects of DC101 on intestinal vascularization and VEGF receptor-2 (VEGFR-2) up-regulation. Protein expression of CD31 (A) and VEGF receptor-2 (B) in the intestines of partial portal vein-ligated mice treated, during 5 or 7 days, with either anti-VEGF receptor-2 monoclonal antibodies (DC101) or control IgG. In both panels, representative Western blots are shown at the top, and quantification of protein signals normalized to α-tubulin (arbitrary units; mean ± SEM of 4 separate experiments) is shown at the bottom. ∗P < 0.05 compared with IgG-treated mice. Gastroenterology 2004 126, 886-894DOI: (10.1053/j.gastro.2003.12.012)