Integrin‐linked kinase regulates endothelial cell nitric oxide synthase expression in hepatic sinusoidal endothelial cells

Abstract

Background Portal hypertension results from endothelial dysfunction after liver injury caused in part by abnormal production of endothelial cell derived nitric oxide synthase (eNOS). Here, we have postulated that endothelial mechanosensing pathways involving integrin linked kinase (ILK) may play a critical role in portal hypertension, eNOS expression and function. Aims In this study, we investigated the role of ILK and the small GTP-binding protein, Rho, in sinusoidal endothelial cell eNOS regulation and function. Methods Primary liver sinusoidal endothelial cells (SECs) were isolated using standard techniques. Liver injury was induced by performing bile duct ligation (BDL). To examine the expression of Rho and ILK in vivo during wound healing, SECs were infected with constitutively active Rho (V14), a dominant negative Rho (N19) and constructs encoding ILK and a short hairpin-inhibiting ILK. Results ILK expression was increased in SECs after liver injury; endothelin-1, vascular endothelial growth factor, and transforming growth factor beta-1 stimulated ILK expression in SECs. ILK expression in turn led to eNOS upregulation and to enhanced eNOS phosphorylation and NO production. ILK knockdown or ILK (kinase) inhibition reduced eNOS mRNA expression, promoter activity, eNOS expression, and ultimately NO production. In contrast, ILK over-expression had the opposite effect. Inhibition of ILK activity also disrupted the actin cytoskeleton in isolated SECs. Rho overexpression suppressed phosphorylation of the serinethreonine kinase, Akt, and inhibited eNOS phosphorylation. Finally, inhibition of Rho function with the RGS domain of the p115-Rho-specific GEF (p115-RGS) significantly increased eNOS phosphorylation. Conclusions Our data suggest a potential role for ILK, the cytoskeleton, and ILK signaling partners including Rho in regulating intrahepatic SEC eNOS expression and function.

A New Treatment for Portal Hypertension?

“…Rho in turn has widespread effects on the cytoskeleton, activates a number of nuclear transcription factors such as nuclear factor-kappa B, and affects eNOS function. 12 These data are consistent with the broad effects of statins in the cardiovascular and other systems. For example, statins reduce platelet aggregation, have direct effects on coagulation factors V and VII, have antiinflammatory effects in the vessel wall, and stimulate NO production.…”

Role and regulation of autophagy and apoptosis by nitric oxide in hepatic stellate cells during acute liver failure

“…A recent study showed that nitric oxide (NO) promotes HSC apoptosis by generating ROS . NO, a gaseous substance with potent vasodilator effects, can be produced by hepatocytes, Kupffer cells, HSCs and hepatic sinusoidal endothelial cells . NO is synthesized by NO synthase (NOS) through a series of redox reactions.…”

“…9 NO, a gaseous substance with potent vasodilator effects, can be produced by hepatocytes, Kupffer cells, HSCs and hepatic sinusoidal endothelial cells. 10 NO is synthesized by NO synthase (NOS) through a series of redox reactions. In mammals, 3 distinct genes encode the nitric oxide synthase (NOS) isoenzyme: neuronal (nNOS or NOS-1), inducible (iNOS or NOS-2), and endothelial (eNOS or NOS-3).…”

iNOS-Derived Nitric Oxide Induces Integrin-Linked Kinase Endocytic Lysosome-Mediated Degradation in the Vascular Endothelium

“…6,7,20 Thus, we tested superoxide levels in MAEC isolated from WT and iNOS knockout mice, finding an increase in superoxide production in TNF-α-treated WT MAEC, which was not observed in iNOS knockout mice ( Figure 2B). This increase could also be detected in WT MAEC treated with 2 different NO donors ( Figure 2C), which differ in the timing of NO release.…”

“…6 In addition, ILK can regulate eNOS expression. 7 ILK upregulation promotes a broadly cardioprotective role. 8 In the vasculature, ILK regulates angiogenesis, 9 endothelial survival, 10 apoptosis, 11 inflammation, 12 vasomotor tone, 6 vascular remodeling, 13 and cardiac remodeling.…”