Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

Abstract

RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-α (TNF-α). siRNA was designed and synthesized targeting tumor necrosis factor-α receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5 % and SHP-1; 97.2 %) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-α expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-α expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

Hypoxia effects on proangiogenic factors in human umbilical vein endothelial cells: functional role of the peptide somatostatin

“…1a, the predicted length of each qPCR product was confirmed by agarose gel electrophoresis (148, 106, 92 and 60 bp which correspond to the predicted length of VEGF, VEGFR-1, VEGFR-2 and GAPDH mRNA, respectively). In agreement with previous results demonstrating that hypoxia is a potent stimulus for VEGF upregulation in HUVEC (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008), we found that hypoxia caused a drastic increase in the level of the VEGF mRNA (∼163%, **P < 0.01; Fig. 1b) while we also determined a significant decrease in the expression of both VEGFR-1 (∼58%, **P<0.01; Fig.…”

“…Each column represents the mean±SEM of data from three (d-f) or four (a-c) independent samples. *P<0.05, **P<0.01 and ***P<0.001 vs. the respective normoxic (ANOVA followed by Newman-Keuls multiple comparison post-test) expression is reduced by hypoxia is in agreement with previous studies which demonstrate that hypoxia downregulates VEGFR-2 mRNA in HUVEC (Cho et al 2008;Gerber et al 1997). However, there are also studies demonstrating that, in HUVEC, hypoxia either downregulates VEGFR-2 mRNA, but not protein (Avouac et al 2008), or upregulates VEGFR-2 protein, but not mRNA (Takata et al 2008;Waltenberger et al 1996).…”

“…Our study shows that hypoxia is a potent stimulus for VEGF expression and production, confirming previous findings in HUVEC (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008). Hypoxia has been proposed to play an important role in the regulation of VEGF receptor expression although previous studies have yielded conflicting results.…”

“…In particular, in HUVEC, nuclear factor kappa-B (NFkB) has been observed to be activated by hypoxia (Pearlstein et al 2002). In addition, hypoxia increases the expression of the cytokine tumour necrosis factor α (TNFα; Cho et al 2008), which is a NFkB target gene able to downregulate the expression of both VEGFR-1 and VEGFR-2 in HUVEC (Patterson et al 1996), indicating that pathways involving cytokine production may be involved in the regulation of VEGF receptor expression in these cells.…”

“…VEGF is one of the most potent proangiogenic factors which promotes the proliferation and migration of endothelial cells and increases vascular permeability. In human umbilical vein endothelial cells (HUVEC), which are widely used as an endothelial cell model in vitro, hypoxia increases VEGF expression and secretion (Avouac et al 2008;Cho et al 2008;Liu et al 2009;Nilsson et al 2004;Takata et al 2008;Zheng et al 2008).…”