Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

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Vascular endothelial cell growth factor (VEGF) is a potent mitogen and permogen that increases in the plasma and decreases in the alveolar space in respiratory diseases such as acute respiratory distress syndrome (ARDS). This observation has led to controversy over the role of this potent molecule in lung physiology and disease. We hypothesized that some of the VEGF previously detected in normal lung may be of the anti-angiogenic family (VEGFxxxb) with significant potential effects on VEGF bioactivity. VEGFxxxb protein expression was assessed by indirect immunohistochemistry in normal and ARDS tissue. Expression of VEGFxxxb was also detected by immunoblotting in normal lung tissue, primary human alveolar type II (ATII) cells, and bronchoalveolar lavage (BAL) fluid in normal subjects and by ELISA in normal, “at risk,” and ARDS subjects. The effect of VEGF165 and VEGF165b on both human primary endothelial cells and alveolar epithelial cell proliferation was assessed by [3H]thymidine uptake. We found that VEGF165b was widely expressed in normal healthy lung tissue but is reduced in ARDS lung. VEGF121b and VEGF165b were present in whole lung, BAL, and ATII lysate. The proliferative effect of VEGF165 on both human primary endothelial cells and human alveolar epithelial cells was significantly inhibited by VEGF165b (P < 0.01). These data demonstrate that the novel VEGFxxxb family members are expressed in normal lung and are reduced in ARDS. A specific functional effect on primary human endothelial and alveolar epithelial cells has also been shown. These data suggest that the VEGFxxxb family may have a role in repair after lung injury.

Fenofibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, used as a normolipidemic agent, is thought to offer additional beneficial effects in atherosclerosis. Since angiogenesis is involved in plaque progression, hemorrhage, and instability, the main causes of ischemic events, this study was designed to evaluate the action of fenofibrate on angiogenesis. Our results show that fenofibrate (i) inhibits endothelial cell proliferation induced by angiogenic factors, followed at high concentrations by an increase in apoptosis, (ii) inhibits endothelial cell migration in a healing wound model, (iii) inhibits capillary tube formation in vitro, and (iv) inhibits angiogenesis in vivo. Concerning the mechanism of action, the inhibition of endothelial cell migration by fenofibrate can be explained by a disorganization of the actin cytoskeleton. At the molecular level, fenofibrate markedly decreased basic fibroblast growth factor-induced Akt activation and cyclooxygenase 2 gene expression. This inhibition of angiogenesis could participate in the beneficial effect of fenofibrate in atherosclerosis.

Pre-eclampsia is a pregnancy-related condition characterized by hypertension, proteinuria and endothelial dysfunction. VEGF165b, formed by alternative splicing of VEGF (vascular endothelial growth factor) pre-mRNA, inhibits VEGF165-mediated vasodilation and angiogenesis, but has not been quantified in pregnancy. ELISAs were used to measure means±S.E.M. plasma VEGF165b, sEng (soluble endoglin) and sFlt-1 (soluble fms-like tyrosine kinase-1). At 12 weeks of gestation, the plasma VEGF165b concentration was significantly up-regulated in plasma from women who maintained normal blood pressure throughout their pregnancy (normotensive group, 4.90±1.6 ng/ml; P<0.01, as determined using a Mann-Whitney U test) compared with non-pregnant women (0.40±0.22 ng/ml). In contrast, in patients who later developed pre-eclampsia, VEGF165b levels were lower than in the normotensive group (0.467±0.209 ng/ml), but were no greater than non-pregnant women. At term, plasma VEGF165b concentrations were greater than normal in both pre-eclamptic (3.75±2.24 ng/ml) and normotensive (10.58 ng/ml±3.74 ng/ml; P>0.1 compared with pre-eclampsia) pregnancies. Patients with a lower than median plasma VEGF165b at 12 weeks had elevated sFlt-1 and sEng pre-delivery. Concentrations of sFlt-1 (1.20±0.07 and 1.27±0.18 ng/ml) and sEng (4.4±0.18 and 4.1±0.5 ng/ml) were similar at 12 weeks of gestation in the normotensive and pre-eclamptic groups respectively. Plasma VEGF165b levels were elevated in pregnancy, but this increase is delayed in women that subsequently develop pre-eclampsia. In conclusion, low VEGF165b may therefore be a clinically useful first trimester plasma marker for increased risk of pre-eclampsia.

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