Exercise training causes a partial improvement through increasing testosterone and eNOS for erectile function in middle-aged rats.

PURPOSE: Aging changes the balance of sex hormones and causes endothelial dysfunction in the penis, both of which are important determinants of erectile dysfunction (ED). The purpose of this study was to evaluate whether exercise training could protect against erectile dysfunction by increasing serum testosterone and penile eNOS levels in aging rats. METHODS: A total of 14 young (2-month-old) and 14 middle-aged (18-month-old) Sprague Dawley rats were randomly assigned to either untrained control (young control, [YC], middle-aged control, [MC]) or endurance exercise-trained (young exercise, [YE], middle-aged exercise, [ME]) groups with seven rats per group. The exercise groups trained with treadmill running for 6 weeks. Body composition parameters (body weight, heart mass, liver mass, and testicular mass), serum sex hormone levels (testosterone, luteinizing hormone, follicle-stimulating hormone, and prolactin), endothelial function-related parameters in the penis (endothelial nitric oxide synthase [eNOS], CD31, alpha smooth muscle actin [α-SMA]), and maximal intracavernous pressure measure (ICP) and total ICP were analyzed in middle-aged rats. RESULTS: The middle-aged groups showed increased body weight, as compared with the young groups, but exercise training attenuated the aging-induced increase in body weight. The middle-aged groups had lower testicular mass compared with the young groups, but exercise training attenuated aging-induced decreases in testicular mass. Exercise training increased serum testosterone levels in both the young and middle-aged groups. However, there were no changes in the levels of luteinizing hormone, follicle-stimulating hormone, and prolactin among the groups. MC group showed decreased protein levels of p-eNOS, as compared with the YC group. However, exercise training protected against aging-induced decrease in eNOS and p-eNOS protein levels in the penis. Interestingly, exercise training also increased protein levels of α-SMA and maximal ICP in the middle-aged group. CONCLUSIONS: Exercise training has beneficial effects on erectile function in aged rats through increased testosterone production from the testis and strengthening of the cavernous endothelium with activation of eNOS. Therefore, exercise training may be a therapeutic modality for improving erectile dysfunction associated with aging.

Glucocorticoid receptor positively regulates transcription of FNDC5 in the liver.

Irisin is secreted by skeletal muscle during exercise and influences energy and metabolic homeostasis. This hormone is a cleaved and secreted fragment of fibronectin type III domain-containing 5 (FNDC5). Elucidation of the FNDC5 gene regulation mechanism is necessary to clarify the function of irisin as a potential therapeutic target in human metabolic diseases. Thus, we investigated the genetic and epigenetic mechanisms that regulate expression of the FNDC5 gene. FNDC5 mRNA was strong expressed in major energy-dependent human tissues, including heart, brain, liver, and skeletal muscle. Promoter analysis of the FNDC5 gene revealed that the core promoter region of the FNDC5 gene contained one CpG island that was located just upstream of the transcriptional start site for variants 2 and 3. Treatment with the histone deacetylase inhibitor sodium butyrate and the demethylating agent 5-azacytidine increased mRNA expression of FNDC5 in Huh7 cells. Prediction of transcription factor binding sites suggested that the glucocorticoid receptor was involved in the regulation of FNDC5 expression, and indeed, cortisol treatment increased mRNA expression of FNDC5 in Huh7 cells. Collectively, these findings offer insight into the genetic and epigenetic regulation of FNDC5, providing the initial steps required for understanding the role of irisin in the metabolic homeostasis.

Voluntary stand-up physical activity enhances endurance exercise capacity in rats.

Involuntary physical activity induced by the avoidance of electrical shock leads to improved endurance exercise capacity in animals. However, it remains unknown whether voluntary stand-up physical activity (SPA) without forced simulating factors improves endurance exercise capacity in animals. We examined the eff ects of SPA on body weight, cardiac function, and endurance exercise capacity for 12 weeks. Twelve male Sprague-Dawley rats (aged 8 weeks, n=6 per group) were randomly assigned to a control group (CON) or a voluntary SPA group. The rats were induced to perform voluntary SPA (lifting a load equal to their body weight), while the food height (18.0 cm) in cages was increased progressively by 3.5 every 4 weeks until it reached 28.5 cm for 12 weeks. The SPA group showed a lower body weight compared to the CON group, but voluntary SPA did not affect the skeletal muscle and heart weights, food intake, and echocardiography results. Although the SPA group showed higher grip strength, running time, and distance compared to the CON group, the level of irisin, corticosterone, genetic expression of mitochondrial biogenesis, and nuclei numbers were not affected. These findings show that voluntary SPA without any forced stimuli in rats can eff ectively reduce body weight and enhance endurance exercise capacity, suggesting that it may be an important alternative strategy to enhance endurance exercise capacity.

Mechanical stretch enhances the expression and activity of osteopontin and MMP-2 via the Akt1/AP-1 pathways in VSMC.

Hemodynamic forces causing mechanical stretch (MS) of vascular smooth muscle cells (VSMC) play an important role in vascular remodeling, but the underlying mechanism involved is not fully understood. Thus, this study investigated whether osteopontin (OPN) expression in VSMC was induced by MS, and identified the intracellular signaling pathways involved in OPN production. The plasma level of OPN and its expression in aortic tissue were increased in various animal models of hypertension including spontaneous hypertensive rats and hypertensive mice induced by angiotensin II or L-NAME. When aortic VSMC was stimulated with MS, OPN production was increased, which was markedly attenuated in VSMC treated with PI3K/Akt inhibitor as well as in Akt1-depleted cells, but not in Akt2-depleted cells, suggesting a pivotal role of Akt1 isoform in OPN expression in VSMC. In the promoter assay, MS increased OPN promoter activity, which was attenuated when the region harboring AP-1 binding sites was mutated. The MS-enhanced promoter activity and OPN expression were also decreased in cells treated with AP-1 siRNA or inhibitor. Moreover, MS-induced MMP-2 production was attenuated in cells treated with OPN siRNA or anti-OPN antibody as well as in OPN-deficient VSMC cultured from aorta of OPN deficient mice. In in vivo experiments, the expressions of OPN and MMP-2 were increased in the aortic tissues from hypertensive mice, but these increases were markedly attenuated in OPN-deficient mice with hypertension. In conclusion, these results suggested that OPN expression in the hypertensive vasculature was increased via signaling pathways that involve Akt1/AP-1, leading to vascular remodeling by increasing the production of MMP-2.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role in monocyte adhesion to vascular endothelium.

Toll-like receptor 4 (TLR4) is known to mediate monocyte adhesion to endothelial cells, however, its role on the expression of monocyte adhesion molecules is unclear. In the present study, we investigated the role of TLR4 on the expression of monocyte adhesion molecules, and determined the functional role of TLR4-induced adhesion molecules on monocyte adhesion to endothelial cells. When THP-1 monocytes were stimulated with Kdo2-Lipid A (KLA), a specific TLR4 agonist, Mac-1 expression was markedly increased in association with an increased adhesion of monocytes to endothelial cells. These were attenuated by anti-Mac-1 antibody, suggesting a functional role of TLR4-induced Mac-1 on monocyte adhesion to endothelial cells. In monocytes treated with MK886, a 5-lipoxygenase (LO) inhibitor, both Mac-1 expression and monocyte adhesion to endothelial cells induced by KLA were markedly attenuated. Moreover, KLA increased the expression of mRNA and protein of 5-LO, suggesting a pivotal role of 5-LO on these processes. In in vivo studies, KLA increased monocyte adhesion to aortic endothelium of wild-type (WT) mice, which was attenuated in WT mice treated with anti-Mac-1 antibody as well as in TLR4-deficient mice. Taken together, TLR4-mediated expression of Mac-1 in monocytes plays a pivotal role on monocyte adhesion to vascular endothelium, leading to increased foam cell formation in the development of atherosclerosis.

Resistin derived from diabetic perivascular adipose tissue up-regulates vascular expression of osteopontin via the AP-1 signalling pathway.

Perivascular adipose tissue (PVAT) is implicated in the development of vascular diseases; however, the roles of PVAT on OPN expression in diabetic vasculature remain to be determined. This study investigated the role of adipokines derived from diabetic PVAT in regulating the vascular expression of OPN and explored the mechanisms involved. Aortic sections of ob/ob and high-fat diet (HFD)-induced obese (DIO) mice showed an increased expression of OPN, which was paralleled by increased amounts of PVAT characterized by enlargement of adipocytes. In the earlier phase of HFD feeding, macrophage infiltration was mainly localized to the area of PVAT at which adipocytes were enlarged, suggesting a potential link of activated adipocytes to macrophage infiltration. PVAT sections of ob/ob and DIO mice revealed a significantly greater number of macrophages with increased expression of adipokines, including resistin and visfatin. The distribution of resistin in PVAT mostly co-localized with macrophages, while visfatin was expressed in macrophages and other cells. In in vitro studies, OPN expression in vascular smooth muscle cells (VSMCs) co-cultured with PVAT of DIO mice was significantly increased, which was attenuated by a resistin-neutralizing antibody. Likewise, resistin up-regulated expression of OPN mRNA and protein in cultured VSMCs and the pivotal role of AP-1 in resistin-induced OPN transcription was demonstrated. Resistin produced by PVAT plays a pivotal role in the up-regulated expression of OPN in the diabetic vasculature via a signalling pathway that involves activation of AP-1.

Mechanical stretch increases MMP-2 production in vascular smooth muscle cells via activation of PDGFR-ß/Akt signaling pathway.

Increased blood pressure, leading to mechanical stress on vascular smooth muscle cells (VSMC), is a known risk factor for vascular remodeling via increased activity of matrix metalloproteinase (MMP) within the vascular wall. This study aimed to identify cell surface mechanoreceptors and intracellular signaling pathways that influence VSMC to produce MMP in response to mechanical stretch (MS). When VSMC was stimulated with MS (0-10% strain, 60 cycles/min), both production and gelatinolytic activity of MMP-2, but not MMP-9, were increased in a force-dependent manner. MS-enhanced MMP-2 expression and activity were inhibited by molecular inhibition of Akt using Akt siRNA as well as by PI3K/Akt inhibitors, LY293002 and AI, but not by MAPK inhibitors such as PD98059, SP600125 and SB203580. MS also increased Akt phosphorylation in VSMC, which was attenuated by AG1295, a PDGF receptor (PDGFR) inhibitor, but not by inhibitors for other receptor tyrosine kinase including EGF, IGF, and FGF receptors. Although MS activated PDGFR-α as well as PDGFR-ß in VSMC, MS-induced Akt phosphorylation was inhibited by molecular deletion of PDGFR-ß using siRNA, but not by inhibition of PDGFR-α. Collectively, our data indicate that MS induces MMP-2 production in VSMC via activation of Akt pathway, that is mediated by activation of PDGFR-ß signaling pathways.

5-Lipoxygenase plays a pivotal role in endothelial adhesion of monocytes via an increased expression of Mac-1.

AIMS: 5-Lipoxygenase (5-LO) is known to participate in the pathogenesis of atherosclerosis; however, the underlying mechanisms are unclear. Thus, this study investigated the molecular mechanisms responsible for 5-LO expression in monocytes as well as the role of 5-LO in monocyte adhesion to the vascular endothelium, which is a key early event in macrophage foam cell formation. METHODS AND RESULTS: An en face immunohistochemistry of endothelial surfaces revealed a marked increase in monocyte adhesion to the aortic endothelium in wild-type (WT) mice treated with lipopolysaccharide (LPS), which was significantly attenuated in 5-LO((-/-)) mice. Likewise, the adhesion capacity of primary monocytes isolated from LPS-treated WT mice was higher than those of monocytes from 5-LO((-/-)) mice. In in vitro study, LPS increased monocyte adhesion to endothelial cells with an enhanced Mac-1 expression. These were attenuated by a 5-LO inhibitor, MK886, as well as by molecular depletion of 5-LO in monocytes. Furthermore, LPS-induced Mac-1 expression on monocytes was significantly inhibited by pre-treatment with U-75302, a BLT1-receptor antagonist, suggesting a pivotal role of 5-LO-derived leukotrienes. In promoter activity analysis and chromatin immunoprecipitation assays to identify transcription factors involved in 5-LO expression, both NF-κB and Sp1 played central roles to increase 5-LO expression in LPS-treated monocytes. CONCLUSION: 5-LO expression in monocytes is modulated via NF-κB and Sp1 signalling pathways, and 5-LO plays a pivotal role in LPS-mediated monocyte adhesion to the vascular endothelium through an increased expression of Mac-1 on monocytes.

Gomisin J from Schisandra chinensis induces vascular relaxation via activation of endothelial nitric oxide synthase.

Gomisin J (GJ) is a lignan contained in Schisandra chinensis (SC) which is a well-known medicinal herb for improvement of cardiovascular symptoms in Korean. Thus, the present study examined the vascular effects of GJ, and also determined the mechanisms involved. Exposure of rat thoracic aorta to GJ (1-30µg/ml) resulted in a concentration-dependent vasorelaxation, which was more prominent in the endothelium (ED)-intact aorta. ED-dependent relaxation induced by GJ was markedly attenuated by pretreatment with L-NAME, a nitric oxide synthase (NOS) inhibitor. In the intact endothelial cells of rat thoracic aorta, GJ also enhanced nitric oxide (NO) production. In studies using human coronary artery endothelial cells, GJ enhanced phosphorylation of endothelial NOS (eNOS) at Ser(1177) with increased cytosolic translocation of eNOS, and subsequently increased NO production. These effects of GJ were attenuated not only by calcium chelators including EGTA and BAPTA-AM, but also by LY294002, a PI3K/Akt inhibitor, indicating calcium- and PI3K/Akt-dependent activation of eNOS by GJ. Moreover, the levels of intracellular calcium were increased immediately after GJ administration, but Akt phosphorylation was started to increase at 20min of GJ treatment. Based on these results with the facts that ED-dependent relaxation occurred rapidly after GJ treatment, it was suggested that the ED-dependent vasorelaxant effects of GJ were mediated mainly by calcium-dependent activation of eNOS with subsequent production of endothelial NO.

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways.

Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways led to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways.

Cilostazol Attenuates 4-hydroxynonenal-enhanced CD36 Expression on Murine Macrophages via Inhibition of NADPH Oxidase-derived Reactive Oxygen Species Production.

Although anti-atherogenic effects of cilostazol have been suggested, its effects on the expression of SR in macrophages are unclear. This study investigated the role of cilostazol on CD36 expression of murine macrophages enhanced by HNE, a byproduct of lipid peroxidation. The stimulation of macrophages with HNE led to an increased expression of CD36, which was significantly attenuated by NAC, an antioxidant. Moreover, the increased production of ROS by HNE was completely abolished by NADPH oxidase inhibitors, DPI and apocynin, as well as by the 5-LO inhibitor, MK886, but not by inhibitors for other oxidases. This suggested that NADPH-oxidase and 5-LO were major sources of ROS induced by HNE. In addition, HNE-enhanced expression of CD36 was reduced by these inhibitors, which indicated a role for NADPH oxidase and 5-LO on CD36 expression. In our present study, cilostazol was a significant inhibitor of ROS production, as well as CD36 expression induced by HNE. An increase in NADPH oxidase activity by HNE was significantly attenuated by cilostazol, however cilostazol had no effect on HNE-enhanced 5-LO activity. Together, these results suggest that cilostazol attenuates HNE-enhanced CD36 expression on murine macrophages thorough inhibition of NADPH oxidase-derived ROS generation.

Gomisin A from Schisandra chinensis induces endothelium-dependent and direct relaxation in rat thoracic aorta.

Schisandra chinensis (SC), a member of the Magnoliaceae family, has been used to improve the vascular health for postmenopausal women in Korea. In order to provide some scientific rationales for such effectiveness, this study investigated the vascular effects of gomisin A (GA) from SC. In the endothelium (ED)-intact rings of rat thoracic aorta, GA (1 x 10 (-6) to 3 x 10 (-4) M) caused a concentration-dependent relaxation which was markedly attenuated not only by removal of ED but also by pretreatment with N(G)-nitro- L-arginine (10 (-4) M) or 1 H-[1,2,4]oxadiazol[4,3- a]quinoxalin-1-one (3 x 10 (-5) M). Direct measurement of nitrite, a metabolite of nitric oxide (NO), confirmed that NO production in isolated aorta was increased by GA. In the ED-denuded specimens, the relaxation by GA was not abolished but reduced significantly. The relaxation by GA in ED-denuded aortic rings were clearly inhibited by calyculin A (3 x 10 (-8) M), an inhibitor of MLC phosphatase. Furthermore, the phenylephrine-enhanced phosphorylation ratio of MLC was significantly attenuated by GA. Based on these results, it is suggested that GA induced vascular relaxation by partially activating ED-dependent NO pathway, and partially dephosphorylation of MLC.