Fluid shear stress-induced nitric oxide production in human cavernosal endothelial cells: inhibition by hyperglycaemia.

OBJECTIVE: To investigate whether fluid shear stress (FSS) induces endothelial nitric oxide synthase (eNOS) activity and NO production in isolated human corpus cavernosal endothelial cells (HCCECs), and whether this response is altered during hyperglycaemia in vitro, as haemodynamic signalling during penile erection induces eNOS-mediated NO production in vivo. MATERIALS AND METHODS: ECs were cultured from HCC and characterized by the uptake of acetylated low-density lipoprotein and the expression of von Willebrand factor, VE-cadherin, CD31 and eNOS. HCCECs were exposed to FSS (1.2 Pa (12 dynes/cm2), 5 min) using a cone-and-plate viscometer in the presence or absence of high glucose (30 mm, 48 h). The phosphorylation of ser1177 on eNOS and total eNOS protein expression after FSS was examined by Western blot. NO in the conditioned media was assessed by measuring nitrate and nitrite levels. RESULTS: Compared to static conditions, FSS induced a significant increase in the phosphorylation of eNOS on ser1177 in HCCECs, and the release of NO to the conditioned media. Treatment of HCCECs with high glucose levels did not alter the ratio FSS-induced phosphorylated eNOS/total eNOS, but did result in the down-regulation of total eNOS and significantly attenuated FSS-induced NO release. CONCLUSION: These in vitro data suggest that FSS contributes to eNOS activation and NO release in HCCECs, and supports in vivo reports suggesting a role for haemodynamic signalling in the erectile response. Treatment with high glucose levels prevented FSS-induced NO release, suggesting a mechanism that may contribute to decreased erectile function associated with diabetes.

Microarray analysis reveals novel gene expression changes associated with erectile dysfunction in diabetic rats.

To investigate the full range of molecular changes associated with erectile dysfunction (ED) in Type 1 diabetes, we examined alterations in penile gene expression in streptozotocin-induced diabetic rats and littermate controls. With the use of Affymetrix GeneChip arrays and statistical filtering, 529 genes/transcripts were considered to be differentially expressed in the diabetic rat cavernosum compared with control. Gene Ontology (GO) classification indicated that there was a decrease in numerous extracellular matrix genes (e.g., collagen and elastin related) and an increase in oxidative stress-associated genes in the diabetic rat cavernosum. In addition, PubMatrix literature mining identified differentially expressed genes previously shown to mediate vascular dysfunction [e.g., ceruloplasmin (Cp), lipoprotein lipase, and Cd36] as well as genes involved in the modulation of the smooth muscle phenotype (e.g., Kruppel-like factor 5 and chemokine C-X3-C motif ligand 1). Real-time PCR was used to confirm changes in expression for 23 relevant genes. Further validation of Cp expression in the diabetic rat cavernosum demonstrated increased mRNA levels of the secreted and anchored splice variants of Cp. CP protein levels showed a 1.9-fold increase in tissues from diabetic rats versus controls. Immunohistochemistry demonstrated localization of CP protein in cavernosal sinusoids of control and diabetic animals, including endothelial and smooth muscle layers. Overall, this study broadens the scope of candidate genes and pathways that may be relevant to the pathophysiology of diabetes-induced ED as well as highlights the potential complexity of this disorder.