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Introduction: Galectin-3 is a pro-fibrotic β-galactoside binding lectin highly expressed in fibrotic liver and implicated in hepatic fibrosis. Selvigaltin (previously known as GB1211) is a novel orally active galectin-3 small molecule inhibitor that has high affinity for galectin-3 (human KD = 25 nM; rabbit KD = 12 nM) and high oral bioavailability in rabbits and man. In this study the efficacy of selvigaltin was investigated in a high fat diet (HFD) rabbit model of metabolic-associated steatohepatitis (MASH). Methods: Male New Zealand White rabbits were individually caged under standard conditions in a temperature and humidity-controlled room on a 12 h light/darkness cycle. After 1 week of regular diet (RD), rabbits were randomly assigned for 8 or 12 weeks to different groups: RD/vehicle, RD/selvigaltin, HFD (8 weeks), HFD/vehicle and HFD/selvigaltin (0.3, 1.0, 5.0 or 30 mg/kg selvigaltin with vehicle/selvigaltin p.o. dosed therapeutically q.d. 5 days per week from week 9 or 12). Liver inflammation, steatosis, ballooning, and fibrosis was measured via blood metabolic markers, histomorphological evaluation [Oil Red O, Giemsa, Masson's trichome, picrosirius red (PSR) and second harmonic generation (SHG)], and mRNA and protein expression. Results: Steatosis, inflammation, ballooning, and fibrosis were all increased from RD to HFD/vehicle groups. Selvigaltin demonstrated target engagement by significantly decreasing galectin-3 levels in the liver as measured via immunohistochemistry and mRNA analysis. Selvigaltin dose-dependently reduced biomarkers of liver function (AST, ALT, bilirubin), inflammation (cells foci), and fibrosis (PSR, SHG), as well as decreasing the mRNA and protein expression of several key inflammation and fibrosis biomarkers (e.g., IL6, TGFβ3, SNAI2, collagen). Doses of 1.0 or 5.0 mg/kg demonstrated consistent efficacy across most biological endpoints supporting the current clinical doses of selvigaltin being investigated in liver disease. Discussion: Selvigaltin significantly reduced hepatic inflammation and fibrosis in an HFD rabbit model of MASH following therapeutic dosing for 4 weeks in a dosedependent manner. These data support the human selvigaltin dose of 100 mg b.i.d. that has been shown to reduce key liver biomarkers during a clinical study in liver cirrhosis. [ABSTRACT FROM AUTHOR]

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers. hvSMCs were isolated from vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated mRNA expression of several steroidogenic enzymes and sex steroid receptors using real time reverse transcription-polymerase chain reaction. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LC-MS/MS). In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α, whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of proandrogenic steroidogenic enzymes (HSD3β1/β2, HSD17β3/β5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short-term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone and DHT secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of genitourinary syndrome in menopause. [ABSTRACT FROM AUTHOR]

Erectile dysfunction (ED) is a frequent complication of obesity. The aim of this review is to critically analyze the framework of obesity and ED, dissecting the connections between the two pathological entities. Current clinical evidence shows that obesity, and in particular central obesity, is associated with both arteriogenic ED and reduced testosterone (T) levels. It is conceivable that obesity-associated hypogonadism and increased cardiovascular risk might partially justify the higher prevalence of ED in overweight and obese individuals. Conversely, the psychological disturbances related to obesity do not seem to play a major role in the pathogenesis of obesity-related ED. However, both clinical and preclinical data show that the association between ED and visceral fat accumulation is independent from known obesity-associated comorbidities. Therefore, how visceral fat could impair penile microcirculation still remains unknown. This point is particularly relevant since central obesity in ED subjects categorizes individuals at high cardiovascular risk, especially in the youngest ones. The presence of ED in obese subjects might help healthcare professionals in convincing them to initiate a virtuous cycle, where the correction of sexual dysfunction will be the reward for improved lifestyle behavior. Unsatisfying sexual activity represents a meaningful, straightforward motivation for consulting healthcare professionals, who, in turn, should take advantage of the opportunity to encourage obese patients to treat, besides ED, the underlying unfavorable conditions, thus not only restoring erectile function, but also overall health. [ABSTRACT FROM AUTHOR]

The article presents a study which explores the characterization of metabolic syndrome-related morpho-functional changes in the lung and the role of testosterone treatment in an animal model. It mentions that metabolic syndrome (MetS), a cluster of metabolic and cardiovascular risk factors strictly linked to unhealthy lifestyle, dietary habit and physical inactivity.

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of α-adrenoceptors (α1-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O2) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce atrophic effect in human endothelial cells through stimulation of the α1D-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective α1D-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that α1D-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by NG-nitro-L-arginine methyl ester. Conversely, the α1A-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the α1B-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of α1D-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of α1D-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury. [ABSTRACT FROM AUTHOR]

Objective: The influence of hypoxia and endothelial loss on the responsiveness of vascular smooth muscle cells (VSMCs) to vascular endothelial growth factor (VEGF-A) was tested. Methods and results: Exposure to hypoxia induced a potentiation of cultured cell proliferation in response to either the agonist for the VEGF receptor 1 (flt-1) placental growth factor (PlGF-1) or to VEGF-A. This effect was mediated by the mitogen activated protein kinase (MAPK) cascade, since it was inhibited by the MAPK kinase inhibitor PD98059 and by the farnesyl transferase inhibitor II. Accordingly, PlGF-1 activated extracellular signal-regulated kinase1/2. In rat aortic rings deprived of endothelium and cultured in three-dimensional fibrin gels, an increased sprouting of tubular structures in response to VEGF-A was observed only under hypoxia. Studies on rat aorta preparations revealed an endothelium-dependent vasorelaxation in response to either VEGF-A or PlGF1, which was reversed to a contractile response in endothelium-deprived preparations exposed to hypoxia. Western blot and immunohistochemistry of endothelium-deprived preparations exposed to hypoxia showed flt-1 receptor expression in all medial cells. Conversely, flt-1 mRNA, of endothelium-deprived aortic preparations and of tubular structures, was unchanged by hypoxia. Conclusion: These findings demonstrate that experimental conditions mimicking pathological vascular injury can make VSMCs responsive to VEGF-A through the induction of functional flt-1 receptors. [Copyright &y& Elsevier]

Metabolic Syndrome (MetS) is a cluster of clinical conditions, associated to an increased cardiovascular (CV) and metabolic risk along with hypogonadism (HG). Lifestyle modifications (including physical exercise, PhyEx) are well-known treatments for this condition [1]. We previously established a rabbit model of MetS that recapitulates the human phenotype, including HG [2]. We now report studies on the effects of PhyEx on hypothalamus-pituitary-testis (HPT) axis. MetS was induced in adult male rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet were used as controls (RD). RD and HFD rabbits were exercise-trained to run on a treadmill for 12 weeks (RD + PhyEx and HFD+ PhyEx). HFD rabbits showed typical metabolic and CV features of MetS along with hypogonadotropic HG (reduced testosterone and LH plasma levels). Within the hypothalamus (preoptic region) a significant reduction of GnRH- and KISS1R-positive neurons, along with the increase of genes related to inflammation (COX2, IL6, CD68), glucose metabolism (GLUT1, GLUT4, IRS-1) and estrogen action (ERb, GPR30) was detected in HFD rabbit, as compared to RD group. Immunohistochemistry analysis confirmed the HFD-induced hypothalamic inflammation. Interestingly, genes encoding for inhibitory factors for GnRH, such as NPY, were also increased in HFD hypothalamus. Within the testis, HFD down-regulated LH receptor and all the steroidogenic enzymes leading to T synthesis. PhyEx completely restored T and LH plasma levels and GnRH/KISS1R immunostaining. All the aforementioned HDF-induced increase of inflammatory markers were significantly reduced in HFD+ PhyEx, with the exception of IL6. Moreover, at hypothalamic level, PhyEx decreased orexigenic and GnRH-inhibiting factors (dinorphin and its receptors OPRD1 and OPRK1), whereas increased anorexigenic ones (POMC). Within the testis, genes related to T formation (17ßHSD3) and metabolism (5a-reductase 1) were increased by PhyEx. In conclusion, in this experimental model, endurance training (PhyEx) completely reverted MetS-induced hypogonadotropic hypogonadism, exerting beneficial effects on the HPT axis. Particularly, in the hypothalamus PhysEX reduced HFD-induced inflammation. Hence, aerobic exercise training can be regarded an interesting strategy to combat MetS-associated alterations of the HPT axis. [ABSTRACT FROM AUTHOR]

Metabolic Syndrome (MetS) is a cluster of clinical conditions, associated to an increased cardiovascular risk, as well as to hypogonadism in males. Lifestyle modification (including physical exercise, PhyEx) may be beneficial for the condition. Skeletal muscles (SkM) are some of the most highly plastic tissues, able of remodeling in response to use, disuse and disease. In particular, transformations of fiber type may occur in response to physiological milieu to induce functional adaptations. This study is aimed at investigating in experimental MetS, high fat diet-induced in male rabbits [1], the effect of PhyEx on hormonal and metabolic parameters, as well as on SkM composition. Control and MetS rabbits were exercise-trained to run on a treadmill for 12 weeks. Quadriceps femoris samples were collected for histomorphological and gene expression analyses. We found that exercise resistance was significantly reduced in MetS rabbits, as demonstrated by the significant reduction of both running time and distance, compared to control group. MetS rabbits also exhibited the lowest quadriceps mass. Fiber typing by PAS-staining showed a pronounced shift from slower type I to faster type II fibers in MetS group in response to PhysEx, suggesting that MetS condition addressed SkM function towards anaerobic metabolism. Accordingly, extracellular lactate levels were significantly increased and mitochondrial respiration-related genes reduced in SkM of MetS rabbits respect to controls. Interestingly, PhyEx significantly counteracted MetS-related testosterone deficiency and hypercholesterolemia. In conclusion, our results indicate that dysmetabolic milieu induces a reduced proportion of fatigue-resistant type I fibers in response to PhysEx, which however resulted beneficial for MetS condition.cilium. [ABSTRACT FROM AUTHOR]

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

Nitric oxide (NO) is known to promote vascular endothelial growth factor (VEGF)-stimulated permeability and angiogenesis. However, effector molecules that operate downstream of NO in this pathway remain poorly characterized. Herein, we determined the effect of soluble guanylyl cyclase (sGC) inhibition on VEGF responses in vitro and in vivo. Treatment of endothelial cells (EC) with VEGF stimulated eNOS phosphorylation and cGMP accumulation; pretreatment with the sGC inhibitor 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS-2028) blunted cGMP levels without affecting VEGF-receptor phosphorylation. Incubation of cells with NS-2028 blocked the mitogenic effects of VEGF. In addition, cells in which sGC was inhibited exhibited no migration and sprouting in response to VEGF. To study the mechanisms through which NS-2028 inhibits EC migration, we determined the effects of alterations in cGMP levels on p38 MAPK. Initially, we observed that inhibition of sGC attenuated VEGF-stimulated activation of p38. In contrast, the addition of 8-Br-cGMP to EC stimulated p38 phosphorylation. The addition of cGMP elevating agents (BAY 41-2272, DETA NO and YC-1) enhanced EC migration. To test whether sGC also mediated the angiogenic effects of VEGF in vivo, we used the rabbit cornea assay. Animals receiving NS-2028 orally displayed a reduced angiogenic response to VEGF. As increased vascular permeability occurs prior to new blood vessel formation, we determined the effect of NS-2028 in vascular leakage. Using a modified Miles assay, we observed that NS-2028 attenuated VEGF-induced permeability. Overall, we provide evidence that sGC mediates the angiogenic and permeability-promoting activities of VEGF, indicating the significance of sGC as a downstream effector of VEGF-triggered responses.

Human bladder contraction mainly depends on Ca2+ influx via L-type voltage-gated Ca2+ channels and on RhoA/Rho kinase contractile signaling, which is upregulated in overactive bladder (OAB). Elocalcitol is a vitamin D receptor agonist inhibiting RhoA/Rho kinase signaling in rat and human bladder. Since in the normal bladder from Sprague-Dawley rats elocalcitol treatment delayed the carbachol-induced contraction without changing maximal responsiveness and increased sensitivity to the L-type Ca2+ channel antagonist isradipine, we investigated whether elocalcitol upregulated L-type Ca2+ channels in human bladder smooth muscle cells (hBCs). In hBCs, elocalcitol induced a rapid increase in intracellular [Ca2+], which was abrogated by the L-type Ca2+ channel antagonist verapamil. Moreover, hBCs exhibited L-type voltage-activated Ca2+ currents (I Ca), which were selectively blocked by isradipine and verapamil and enhanced by the selective L-type agonist BAY K 8644. Addition of elocalcitol (10(-7) M) increased L-type I Ca size and specific conductance by inducing faster activation and inactivation kinetics than control and BAY K 8644, while determining a significant negative shift of the activation and inactivation curves, comparable to BAY K 8644. These effects were strengthened in long-term treated hBCs with elocalcitol (10(-8) M, 48 h), which also showed increased mRNA and protein expression of pore-forming L-type alpha(1C)-subunit. In the bladder from Sprague-Dawley rats, BAY K 8644 induced a dose-dependent increase in tension, which was significantly enhanced by elocalcitol treatment (30 microg.kg(-1).day(-1), 2 wk). In conclusion, elocalcitol upregulated Ca2+ entry through L-type Ca2+ channels in hBCs, thus balancing its inhibitory effect on RhoA/Rho kinase signaling and suggesting its possible efficacy for the modulation of bladder contractile mechanisms.

Spontaneously hypertensive rats (SHR) are characterized by impaired erectile function and overactivity of the procontractile RhoA/Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) pathway, as compared with their normotensive counterpart, Wistar-Kyoto rats. By measuring the intracavernous pressure:mean arterial pressure (ICP:MAP) ratio after electrostimulation of the cavernous nerve, we confirmed these findings and showed that responsiveness to sildenafil (25 mg/kg by oral gavage) also is hampered in SHR. A 2-week treatment with atorvastatin (5 and 30 mg/kg) improved the sildenafil-induced ICP:MAP increase and normalized RhoA and ROCK2 overexpression in SHR corpora cavernosa (CC). Conversely, other genes, neuronal nitric oxide synthase (NOS), endothelial NOS, and phosphodiesterase 5, were unaffected. In human fetal smooth muscle cells derived from CC (hfPSMC), atorvastatin inhibited RhoA membrane translocation and ROCK activity, as well as RhoA-dependent biologic functions like cell migration and cell proliferation. Atorvastatin's effect on migration was rescued in a dose-dependent manner by geranylgeranyl pyrophosphate, suggesting the involvement of RhoA geranylgeranylation. In hfPSMC, atorvastatin decreased the expression of RhoA-dependent genes such as ROCK2, desmin, alpha-smooth muscle actin, SM22alpha, and myocardin. In contrast to atorvastatin, elocalcitol, a vitamin D analog that also interferes with RhoA activation in SHR bladder, was unable to restore penile responsiveness to sildenafil. In conclusion, atorvastatin, but not elocalcitol, ameliorates sildenafil-induced penile erections in SHR, likely by interfering with RhoA/ROCK signaling within the penis.

Catecholamines have been shown to be involved in vascular remodeling through the stimulation of alpha(1)-adrenoceptors (alpha(1)-ARs). Recently, it has been demonstrated that catecholamines can stimulate angiogenesis in pathological conditions, even if the mechanisms and the AR subtypes involved still remain unclear. We investigated the influence of hypoxia (3% O(2)) on the ability of picomolar concentrations of phenylephrine (PHE), which are unable to induce any vascular contraction, to induce a trophic effect in human endothelial cells through stimulation of the alpha(1D)-subtype ARs. PHE, at picomolar concentrations, significantly promoted pseudocapillary formation from fragments of human mature vessels in vitro. Exposure to hypoxia significantly potentiated this effect, which was inhibited by the selective alpha(1D)-AR antagonist BMY-7378 and by the nitric oxide synthase inhibitor L-NAME, suggesting that alpha(1D)-ARs were involved in this effect through activation of the nitric oxide pathway. Proliferation and migration of HUVEC were also affected by picomolar PHE concentrations. Again, these effects were significantly potentiated in cells exposed to hypoxia and were inhibited by BMY-7378 and by N(G)-nitro-L-arginine methyl ester. Conversely, the alpha(1A)-AR-selective antagonist (S)-(+)-niguldipine hydrochloride and the alpha(1B)-AR antagonist chloroethylclonidine dihydrochloride did not modify endothelial cell migration and proliferation in response to PHE. These results demonstrate that the stimulation of alpha(1D)-ARs, triggered by picomolar PHE concentrations devoid of any contractile vascular effects, induces a proangiogenic phenotype in human endothelial cells that is enhanced in a hypoxic environment. The role of alpha(1D)-ARs may become more prominent in the adaptive responses to hypoxic vasculature injury.

Vessel wall remodeling is a complex phenomenon in which the loss of differentiation of vascular smooth muscle cells (VSMCs) occurs. We investigated the role of rat macrophage chemoattractant protein (MCP)-1 on rat VSMC proliferation and migration to identify the mechanism(s) involved in this kind of activity. Exposure to very low concentrations (1-100 pg/ml) of rat MCP-1 induced a significant proliferation of cultured rat VSMCs assessed as cell duplication by the counting of total cells after exposure to test substances. MCP-1 stimulated VSMC proliferation and migration in a two-dimensional lateral sheet migration of adherent cells in culture. Endogenous vascular endothelial growth factor-A (VEGF-A) was responsible for the mitogenic activity of MCP-1, because neutralizing anti-VEGF-A antibody inhibited cell proliferation in response to MCP-1. On the contrary, neutralizing anti-fibroblast growth factor-2 and anti-platelet-derived growth factor-bb antibodies did not affect VSMC proliferation induced by MCP-1. RT-PCR and Western blot analyses showed an increased expression of either mRNA or VEGF-A protein after MCP-1 activation (10-100 pg/ml), whereas no fms-like tyrosine kinase (Flt)-1 receptor upregulation was observed. Because we have previously demonstrated that hypoxia (3% O2) can enhance VSMC proliferation induced by VEGF-A through Flt-1 receptor upregulation, the effects of hypoxia on the response of VSMCs to MCP-1 were investigated. Severe hypoxia (3% O2) potentiated the growth-promoting effect of MCP-1, which was able to significantly induce cell proliferation even at a concentration as low as 0.1 pg/ml. These findings demonstrate that low concentrations of rat MCP-1 can directly promote rat VSMC proliferation and migration through the autocrine production of VEGF-A.