Suppression of NHE1 by small interfering RNA inhibits HIF-1α-induced angiogenesis in vitro via modulation of calpain activity

• Published in: Microvascular research 2011-03, Vol.81 (2), p.160-168
• Main Authors: Mo, Xian-Gang, Chen, Qing-Wei, Li, Xing-Sheng, Zheng, Min-Ming, Ke, Da-Zhi, Deng, Wei, Li, Gui-Qiong, Jiang, Jin, Wu, Zhi-Qin, Wang, Li, Wang, Peng, Yang, Yan, Cao, Guang-Yi
• Format: Article
• Language: English
• Subjects: Angiogenesis; Calpain; Calpain - antagonists & inhibitors; Calpain - metabolism; Cation Transport Proteins - deficiency; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cell Movement - drug effects; Cell Movement - physiology; Cell Proliferation - drug effects; Cell Survival - genetics; Cell Survival - physiology; Cells, Cultured; Cysteine Proteinase Inhibitors - pharmacology; Cytoplasm - metabolism; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - metabolism; Gene Expression - genetics; Human umbilical vein endothelial cells (HUVECs); Humans; Hydrogen-Ion Concentration; Hypoxia-Inducible Factor 1, alpha Subunit - genetics; Hypoxia-Inducible Factor 1, alpha Subunit - metabolism; Hypoxia-inducible factor-1 (HIF-1); Intracellular pH; Migration; Na +/H + exchanger-1 (NHE1); Neovascularization, Physiologic - drug effects; Neovascularization, Physiologic - physiology; RNA, Small Interfering - genetics; Small interfering RNA (siRNA); Sodium-Hydrogen Exchanger 1; Sodium-Hydrogen Exchangers - genetics; Sodium-Hydrogen Exchangers - metabolism; Transduction, Genetic; Tube formation; Up-Regulation - genetics; Vascular endothelial growth factor (VEGF); Vascular Endothelial Growth Factor A - genetics; Vascular Endothelial Growth Factor A - metabolism; Vascular Endothelial Growth Factor A - pharmacology
• ISSN: 0026-2862; 1095-9319
• DOI: 10.1016/j.mvr.2010.12.004

Hypoxia-inducible factor-1 (HIF-1) orchestrates angiogenesis under hypoxic conditions mainly due to increased expression of such target genes as vascular endothelial growth factor (VEGF). Na+/H + exchanger-1 (NHE1), a potential HIF target gene product, plays a pivotal role in proliferation, survival, migration, adhesion and so on. However, it is unknown whether NHE1 is involved in HIF-1α-induced angiogenesis. This present study demonstrated that the expression of NHE1 was much higher in human umbilical vein endothelial cells (HUVECs) infected with adenovirus encoding HIF-1α (rAd-HIF) than with vacuum adenovirus (vAd). HIF-1α also increased the expression of VEGF, the expression and activity of calpains, and the intracellular pH. Moreover, small interfering RNA targeting NHE1 (NHE1 siRNA) dramatically decreased the expression of NHE1 and thus lowered the intracellular pH, and it also attenuated the protein expression of calpain-2 but not calpain-1, resulting in the lower calpain activity. Furthermore, HIF-1α enhanced the proliferation, migration and Matrigel tube formation, which were inhibited by NHE1 siRNA. Finally, the inhibitory effect of NHE1 siRNA was reversed by VEGF and the reversibility of the later was abrogated by the calpain inhibitor ALLM. In conclusion, the findings have revealed that NHE1 might participate in HIF-1-induced angiogenesis due, at least in part, to the alteration of the calpain activity, suggesting that NHE1 as well as calpains might represent a potential target of controlling angiogenesis in response to the hypoxic stress under various pathological conditions. [Display omitted] ►HIF-1α overexpression increased the expression of NHE1 and the expression and activity of calpains. ►Knockdown of NHE1 inhibited the expression of calpain-2, not calpain-1, and reduced the activity of calpains. ►Supression of NHE1 by small interfering RNA inhibited HUVECs proliferation/viability, migration, and matrigel tube formation.