Recent evidence points to the involvement of vascular endothelial growth factor (VEGF) in mammalian reproductive physiology. Transgenic mice expressing VEGF (121 isoform) under the control of the polyepithelial mucin-1 (muc-1) promoter showed a reduction in male fertility due to impaired spermiogenesis, and aberrant placentation leading to preferential rejection of male embryos. A skew in the sex ratio of the litters was seen (three females to two males), independently of whether the transgene was carried by the male or female parent. In-situ hybridization permitted distinction of expression of the human VEGF transgene from endogenous mouse VEGF, and confirmed expression of the transgene in a wide range of epithelial tissues. Expression of the transgene in spermatocytes and in the embryonic portion of placenta is thought to be responsible for the reduced fertility and embryonic resorptions respectively. Males showed either complete sperm maturation arrest or various gradations of partial fertility. Abnormally high or low VEGF in human semen has been reported to be correlated with a lack of pregnancy success following IVF. The muc1-VEGF (121 isoform) transgenic mouse provides an animal model with which to further study this VEGF-induced pathology.
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.
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