ABSTRACT
The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
ABSTRACT
The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.
Subject(s)
Animals , Humans , Male , Rats , Apoptosis , Diet , Endothelial Cells , Erectile Dysfunction/prevention & control , Fibrosis , Hyperhomocysteinemia/complications , Methionine , Oxidative Stress , Penis/pathology , Rats, Sprague-Dawley , Rats, Transgenic , Signal Transduction/genetics , Tissue Kallikreins/geneticsABSTRACT
AIM: To investigate the effects of adenovirus-mediated human tissue kallikerin (Ad-hKLK1) gene delivery on the proliferation, migration of VSMC_(SHR) induced by platelet derived growth factor-BB (PDGF-BB). METHODS: The VSMC_(SHR) proliferation induced by PDGF-BB was accessed by cell counting and methyl thiazolyl tetrazoliuin (MTT). The migration was assessed by modified Boyden chamber assay. Western blotting was used to determine the expressions of the cycle-independent kinase inhibitors p27~(Kip1) and p21~(Cip1).RESULTS: Proliferation of VSMC_(SHR) induced by PDGF-BB was inhibited after transfection of Ad-hKLK1 (20-100 MOI) in a MOI-dependent manner. The peak inhibition titer of Ad-hKLK1 fell on 100 MOI, with the peak inhibition rate of 39.3% (cell counting, n=3, P<0.01), 30.2% (MTT, n=3, P<0.01), peak stunning rate of cell-cycle in phase G0/G1 at 36.4%. The inhibitory effects of proliferation and cell-cycle caused by hKLK1 gene delivery were significantly abolished by Hoe140, a bradykinin B2 receptor antagonist. Migration of VSMC_(SHR) induced by PDGF-BB was inhibited after hKLK1 gene delivery, with the peak inhibitory rate of 34.6% (n=6, P<0.01). However the inhibitory effects of migration were not blocked by Hoe140. The protein expression of p27~(Kip1) and p21~(Cip1) increased significantly after the hKLK1 gene delivery, whereas Hoe140 nearly completely blocked these effects (n=3, P<0.01, respectively).CONCLUSION: The hKLK1 gene delivery may inhibit the proliferation and migration of VSMC_(SHR) induced by PDGF-BB. Bradykinin B2 receptor probably mediates the up-regulating expression of p27~(Kip1) and p21~(Cip1) that contributes to the inhibitory effects of proliferation of hKLK1. However, the inhibitory effects of migration by hKLK1 gene delivery may not be mediated by bradykinin B2 receptor.