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-alpha (TNF-alpha). siRNA was designed and synthesized targeting tumor necrosis factor-alpha 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-alpha expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-alpha 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.

Fig. 1

Silencing of target gene (TNFR-1 and SHP-1) expression by siRNA transfection. (A) RT-PCR of HUVECs for each target at 2 days after transfection with TNFR-1, SHP-1, or GFP siRNA. Quantitative real-time PCR of siRNA-transfected HUVECs (n=3) for (B) TNFR-1 and (C) SHP-1. The each gene expression in TNFR-1 or SHP-1 siRNA-transfected HUVECs was normalized to that in GFP siRNA-transfected HUVECs. TNFR-1 and SHP-1 siRNA delivery using Lipofectamine 2000 resulted in a significant and specific silencing of each molecule’s expression compared with GFP siRNA delivery (*; p<0.01, **; p<0.05, ***; p>0.05).

Fig. 2

Gene expression profiles in siRNA-transfected HUVECs under hypoxic (1% oxygen) and survival factor-deprived (EBM-2 basal medium with no serum and growth factors) conditions. (A) RT-PCR for various molecules of siRNA-transfected HUVECs retrieved at days 1 and 2 after culture. Quantitative real-time PCR for (B) TNF-α, (C) VEGF, (D) KDR/Flk-1, (E) eNOS, (F) Bcl-xL, and (G) Bax. The gene expression levels of TNFR-1 or SHP-1 siRNA-transfected HUVECs (n=4) at days 1 and 2 was normalized to that in GFP siRNA-transfected HUVECs (*; p<0.01, **; p<0.05, compared to GFP siRNA group).

Fig. 3

Apoptotic activity of siRNA-transfected HUVECs cultured under hypoxic (1% oxygen) and survival factor-deprived (EBM-2 basal medium with no serum and growth factors) conditions. (A) TUNEL staining of siRNA-transfected HUVECs at 2 days after culture (×100). Scale bar indicates 200 µm. (B) The percentage ratio of TUNEL-positive cells to total cells at days 1 and 2 after culture (n=4, *; p<0.01, compared to GFP siRNA group). (C) The viability of siRNA-transfected HUVECs at days 1 and 2 after culture (n=4, *; p<0.01, compared to GFP siRNA group).

Fig. 4

Capillary formation by siRNA-transfected HUVECs cultured for 2 days under hypoxic (1% oxygen) and survival factor-deprived (EBM-2 basal medium with no serum and growth factors) conditions. When cultured onto GFR-Matrigel, HUVECs transfected with siRNAs for (A) TNFR-1 and (B) SHP-1 showed more extensive capillary structure formation, compared to HUVECs transfected with (C) GFP siRNA (×100). Scale bar indicates 200 µm. The density of (D) lumens and (E) tubes in the vascular structures generated on GFR-Matrigel (*; p<0.01, **; p<0.05, compared to GFP-siRNA group).