The Unmet Needs for Studying Chronic Pelvic/Visceral Pain Using Animal Models.

The different definitions of chronic pelvic/visceral pain used by international societies have changed over the years. These differences have a great impact on the way researchers study chronic pelvic/visceral pain. Recently, the role of systemic changes, including the role of the central nervous system, in the perpetuation and chronification of pelvic/visceral pain has gained weight. Consequently, researchers are using animal models that resemble those systemic changes rather than using models that are organ- or tissue-specific. In this review, we discuss the advantages and disadvantages of using bladder-centric and systemic models, enumerating some of the central nervous system changes and pain-related behaviors occurring in each model. We also present some drawbacks when using animal models and pain-related behavior tests and raise questions about possible, yet to be demonstrated, investigator-related bias. We also suggest new approaches to study chronic pelvic/visceral pain by refining existing animal models or using new ones.

Metformin Prevents Endothelial Dysfunction in Endometriosis through Downregulation of ET-1 and Upregulation of eNOS.

This study aimed to evaluate if the treatment with metformin affects the morphologic structure, endothelial function, angiogenesis, inflammation and oxidation-responsive pathways in the heart of mice with surgically induced endometriosis. B6CBA/F1 mice (n = 37) were divided into four groups; Sham (S), Metformin (M), Endometriosis (E) and Metformin/Endometriosis (ME). The cross-sectional area of cardiomyocytes was assessed after Hematoxylin-Eosin staining and fibrosis after Picrosirius-Red staining. ET-1, nitric oxide synthases-iNOS and eNOS, and VEGF and VEGFR-2 were detected by immunofluorescence. Semi-quantification of ET-1, eNOS, VEGF, NF-kB, Ikßα and KEAP-1 was performed by Western blotting. MIR199a, MIR16-1, MIR18a, MIR20a, MIR155, MIR200a, MIR342, MIR24-1 and MIR320a were quantified by Real-Time qPCR. The interaction of endometriosis and metformin effects was assessed by a two-way ANOVA test. Compared with the other groups, M-treated mice presented a higher cross-sectional area of cardiomyocytes. Heart fibrosis increased with endometriosis. Treatment of endometriosis with metformin in the ME group downregulates ET-1 and upregulates eNOS expression comparatively with the E group. However, metformin failed to mitigate NF-kB expression significantly incremented by endometriosis. The expression of MIR199a, MIR16-1 and MIR18a decreased with endometriosis, whereas MIR20a showed an equivalent trend, altogether reducing cardioprotection. In summary, metformin diminished endometriosis-associated endothelial dysfunction but did not mitigate the increase in NF-kB expression and cardiac fibrosis in mice with endometriosis.

Anti-oxidant effect of metformin through AMPK/SIRT1/PGC-1α/SIRT3- independent GPx1 expression in the heart of mice with endometriosis.

OBJECTIVES: Endometriosis is a gynecological disease associated with an imbalance between oxidative species production and anti-oxidative defenses. In women, endometriosis has been reported to associate with increased incidence of cardiovascular events. As such, this study aimed to analyze the oxidation-responsive AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart of a mouse model of endometriosis. The effect of metformin, an insulin-sensitizing and anti-oxidative drug with already shown positive results in endometriotic tissue was studied. METHODS: Thirty-six female B6CBA/F1 mice were divided into 4 groups (Control-C, Surgery-induced Endometriosis and Metformin-EM (50 mg/kg/day orally administrated for 3 months), Endometriosis-E and Metformin-M). Immunofluorescent labelling of SIRT1 and SIRT3 was performed in the heart tissue. Assessment of expression of AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPx1 was performed by Western Blotting. The quantification of microRNA(miR)-34a, miR-195, miR-217, miR-155 and miR-421, involved in the regulation of expression of SIRT1 and SIRT3, was performed by Real-Time PCR. RESULTS: Data showed an increase in phospho-AMPKα and in GPx1 expression in the EM group when compared to the C group, but not in the total AMPK, SIRT1, PGC-1α, SIRT3 and SOD2, suggesting a GPx1 expression increase independently of the AMPK/SIRT1/PGC-1α/SIRT3 pathway. MicroRNAs, excepting miR-217, showed a consistent trend of increase in the M group. CONCLUSIONS: Our study showed that endometriosis does not significantly affect the expression of the components of the AMPK/SIRT1/PGC-1α/SIRT3 pathway in the heart. However, it indicates that an oxidative condition underlying endometriosis is required for metformin to evidence an increment in the expression of the anti-oxidative enzyme GPx1.

Impact of curcumin, quercetin, or resveratrol on the pathophysiology of endometriosis: A systematic review.

Endometriosis, a gynecological disease that affects reproductive age women is difficultly controlled in the long term by currently available treatments, prompting patients to adopt self-controlled interventions including dietary changes. The aim of this review is to provide evidence of how curcumin, quercetin, and resveratrol can act as natural interventions to control endometriosis. The review followed PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. A comprehensive search was carried out in PubMed, Scopus, and Web of Science to gather together all the articles that study the specific actions of curcumin, resveratrol, or quercetin in endometriosis pathophysiology. All types of study designs including experimental data were considered. Thirty articles, including a clinical trial, were included. For the assessment of the quality of the selected studies that globally have "good quality", the GRADE (Grading of Recommendations Assessment, Development and Evaluation) and the SYRCLE ROB tool criteria were used. By acting on mechanisms of inflammation, oxidative stress, cell proliferation, invasion and adhesion, apoptosis, angiogenesis and glucose and lipid metabolism, curcumin, quercetin, and resveratrol showed to have beneficial effects, evidencing their potential application in the endometriosis treatment. However, future clinical studies are necessary to determine the real efficacy of these compounds in human endometriosis.

Characterization of α-MSH browning effect in diverse mice white adipose tissue depots.

White adipose tissue (WAT) browning is a potent mechanism to dissipate energy as heat and, thus, its activation constitutes a promise therapeutic approach to obesity. We previously reported the melanocortin α-melanocyte stimulating hormone (α-MSH) ability to increase the number of beige cells in subcutaneous inguinal WAT (ingWAT) in high fat diet (HFD)-fed mice. The current study examined the browning effect of intraperitoneally administered α-MSH on diverse fat depots from mice fed with HFD or standard rodent diet (SD). For this, mRNA expression of browning hallmark genes was quantified concomitantly with histological examination of the adipose tissue samples (epidydimal (eWAT), mesenteric (mWAT), retroperitoneal (rpWAT) or ingWAT). As well, α-MSH impact on body weight, serum profile, WAT mass and lipolytic rates were evaluated. In the visceral depots mWAT, eWAT and rpWAT from HFD-fed mice, α-MSH was not able to induce a browning mechanism. Surprisingly, in SD-fed mice, α-MSH decreased the expression of several beige-specific genes in rpWAT and promoted an increase of the size of lipid droplets. No browning effect was observed in ingWAT from SD-fed mice. We also verified that HFD ingestion per se stimulated the browning mechanisms in rpWAT, but not in mWAT and eWAT. In conclusion, the fat depots from diverse anatomical locations respond differently to α-MSH treatment when exposed to different diets.

Cumulative Effect of Cardiovascular Risk Factors on Regulation of AMPK/SIRT1-PGC-1α-SIRT3 Pathway in the Human Erectile Tissue.

Cardiovascular disease risk factors (CVDRF), especially diabetes mellitus (DM), disrupt oxidative stress response. This condition underlies endothelial dysfunction, early manifested in men as erectile dysfunction. The current study is aimed at elucidating the impact of CVDRF in the oxidation responsive AMPK/SIRT1-PGC-1α-SIRT3 pathway and related miRNAs in the human corpus cavernosum. Human penile tissue fragments from individuals submitted to programmed urological surgeries (n = 27), aged 43-63 years, were clustered depending on the presence of CVDRF; the control group included samples from patients without CVDRF, and groups A and B included samples from patients with DM and additional CVDRF, totalizing ≤2 CVDRF (group A) and ≥3 CVDRF (group B). Dual-immunolabelling of SIRT3, SOD2, or GPX1 with α-actin in tissue sections was carried out. The assessment of expression levels of NOX1, phospho-AMPKα, total AMPKα, SIRT1, PGC-1α, SIRT3, SOD2, and GPX1 was performed by western blotting and of miR-200a, miR-34a, miR-421, and miR-206 by real-time PCR. Phospho-AMPKα and SIRT3 expression was found significantly increased in group B relative to other groups, suggesting a marked influence of CVDRF, additional to DM, in the regulation of these enzymes. NOX1 was also increased in group B relative to controls. Only an increasing tendency was observed in the phospho-AMPKα/total AMPKα ratio, SIRT1, and PGC-1α expression in groups A and B when compared with controls. Concerning antioxidant enzymes, GPX1 expression was found incremented in group A, but SOD2 expression was decreased in groups A and B, comparative with controls. Group B presented significantly diminished levels of miR-421 and miR-200a, but only a decreasing trend on miR-34 and miR-206 expression was observed. Taken together, our findings demonstrated that besides DM, additional CVDRF presented a cumulative effect in the cellular response to oxidative unbalance, contributing to AMPK/SIRT1-PGC-1α-SIRT3 pathway activation. SOD2, a major mitochondrial antioxidant defence, did not follow the same variation.

Peripherally administered melanocortins induce mice fat browning and prevent obesity.

BACKGROUND/OBJECTIVES: The browning of white adipose tissue (WAT) has been in the spotlight during the last years, becoming an attractive approach to combat obesity. Melanocortin neuropeptides, such as α-melanocyte-stimulating hormone (α-MSH), are well-known regulators of appetite at the central nervous system, but its role in adipocyte metabolism is poorly elucidated. This study sought to verify if α-MSH can induce transdifferentiation of white to brown/beige adipocytes and to determine whether it can ameliorate the obesity phenotype. METHODS: The browning effect of α-MSH was determined in isolated adipocytes using the 3T3-L1 cell line and in inguinal subcutaneous adipose tissue (ingWAT) of diet-induced obese (DIO) mice by quantifying the expression of browning hallmark genes, oxygen consumption, and mitochondrial biogenesis. α-MSH protection from diet-induced obesity was evaluated by analyzing mice body weight, fat mass, and lipid and glucose serum profiles. RESULTS: Here, we report that α-MSH activates a thermogenic gene program and increases the mitochondrial respiratory rate in 3T3-L1 adipocytes and ingWAT of DIO mice. Without affecting food intake, peripheral administration of α-MSH decreases body weight and ingWAT mass, promoting a significant rise in the number of smaller adipocytes, whereas it lowered the larger ones. Additionally, there was an increase in the mass of brown adipose tissue. Browning activation occurs concomitantly with improvement on serum lipid profile, insulin resistance, and glucose homeostasis. CONCLUSIONS: This study highlights the anti-obesity properties of melanocortins by promoting ingWAT browning and provides new perspectives for future designing of more effective therapeutic strategies.

Natural mineral-rich water ingestion by ovariectomized fructose-fed Sprague-Dawley rats: effects on sirtuin 1 and glucocorticoid signaling pathways.

OBJECTIVE: Prevention or induction of metabolic disorders and obesity depend on estrogen signaling and/or exogenous factors, such as mineral content in diet. The protective effects of a Portuguese natural mineral-rich water against the induction of metabolic syndrome in fructose-fed male Sprague-Dawley rats have been reported. The present study was designed to assess the impact of this mineral-rich water on fructose-fed estrogen-deficient female Sprague-Dawley rats. METHODS: Ovariectomized rats had access to tap (TWO) or mineral-rich (MWO) waters, with and without 10% fructose (10-wk treatment). A sham-operated (tap water supplied) group was included and each of the five groups included six rats. Plasma biochemical and metabolic parameters were evaluated by routine clinical measurements. Western blotting was used to assess hepatic protein expression of sirtuins (Sirt) 1 and 3, phosphorylated AMP-activated protein kinase-α (p-AMPKα), peroxisome proliferator-activated receptor gamma coactivator-1-α (PGC1α), glucocorticoid receptor, and 11beta-hydroxysteroid dehydrogenase type 1 (11ßHSD1). RESULTS: Ovariectomy increased plasma total cholesterol (46%/P < 0.05), but had no significant effects on hepatic protein expression. Fructose intake by ovariectomized rats increased PGC1α and 11ßHSD1 (fructose in tap water [TWFO] vs TWO: 65%/P < 0.05 and 38%/P = 0.05, respectively) as well as glucocorticoid receptor (TWFO and fructose in natural mineral-rich water [MWFO] vs TWO and MWO: 107%/P = 0.05 and 182%/P < 0.05, respectively). Mineral-rich water ingestion exerted an increasing shape on Sirt1 (MWO vs TWO: 76%/P < 0.05; MWFO vs TWFO: 76%/P = 0.06), PGC1α (MWO vs TWO: 77%/P < 0.01), p-AMPKα (MWO vs TWO: 152%/P = 0.01; MWFO vs TWFO: 107%/P = 0.01), and 11ßHSD1 (MWO vs TWO: 91%/P = 0.05; MWFO vs TWFO: 47%/P = 0.05). CONCLUSIONS: Mineral-rich water ingestion may have a prime role on the activation of Sirt1 signaling and the modulation of glucocorticoid signaling in the postmenopause.

Characterization of TGF-ß expression and signaling profile in the adipose tissue of rats fed with high-fat and energy-restricted diets.

Transforming growth factor beta (TGF-ß) plays an important role in the pathogenesis of obesity, influencing the release of inflammation mediators and promoting remodeling and collagen deposition in the adipose tissue (AT). In this context, this work aims to elucidate whether TGF-ß and Smad-dependent or Smad-independent signaling pathways contribute to regional differentiation of AT in high-fat diet (HFD) and energy-restricted (ER) rat models. For this, TGF-ß, TGF-ß receptors I and II, PAI-1 and GLUT4 mRNA levels were quantified by real-time PCR, and western blotting assays allowed the semiquantification of TGF-ß and proteins from Smad3, ERK1/2 and Akt signaling pathways in subcutaneous and visceral (epididymal, retroperitoneal and mesenteric) fat depots from control, HFD and ER-treated rats. HFD was associated to increased levels of TGF-ß and PAI-1 mRNA in epididymal and retroperitoneal visceral fat depots, while ER diet induced a reduction of TGF-ß mRNA levels in mesenteric, but surprisingly an increase in retroperitoneal fat. Regarding the different signaling pathways, contrarily to what was found for Smad3, activation of ERK1/2 and Akt in response to HFD was detected in all the visceral but not in subcutaneous fat depots. ER-treated rats presented a more heterogeneous signaling response, as well as decreased TGF-ß receptors expression, in the different visceral fat depots. In conclusion, subcutaneous and visceral AT respond differently to distinct diet patterns regarding TGF-ß expression and activated signaling pathways. Furthermore, the present study points that visceral AT should not be understood as a homogeneous entity since that response also varied in the different fat depots.

Effects of Aging and Cardiovascular Disease Risk Factors on the Expression of Sirtuins in the Human Corpus Cavernosum.

INTRODUCTION: Sirtuin (SIRT)1 was recently identified in human corpus cavernosum (CC). We hypothesized that other sirtuins could also be expressed in the CC. Expression of these enzymes in tissues is affected by aging, the main independent risk factor for erectile dysfunction besides other cardiovascular disease risk factors (CVDRF), such as diabetes or obesity. AIM: The aim of this study was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC relatively to age and CVDRF. METHODS: Samples of CC collected from patients submitted to programmed surgeries or organ donors were divided in three groups according to age and presence of CVDRF. Expression of SIRT1-3 and SIRT5-7 mRNAs was assessed by real-time polymerase chain reaction. Cellular localization and semi-quantification of sirtuins proteins were performed by immunofluorescence and Western blotting (WB), respectively. Nuclear factor kappa B (NFkB)-p65, inducible (iNOS) and endothelial nitric oxide synthase (eNOS) levels were also assayed by WB. MAIN OUTCOME MEASURES: The main outcome measure was to characterize the expression of SIRT1-3 and SIRT5-7 in human CC. RESULTS: SIRT1-3 and SIRT5-7 mRNAs were detected in all individuals, without statistical differences among groups, excepting SIRT7 that decreased four times in aged groups relatively to young (P = 0.013). WB analysis demonstrated that aged individuals with CVDRF presented higher levels of SIRT7 protein relatively to young (P = 0.0495) and lower levels of SIRT3 protein relatively to healthy aged (P = 0.0077). Expression of NFkB-p65 and iNOS were higher in aged than in young individuals (P = 0.0185; P = 0.004, respectively). No differences in other sirtuins or total eNOS were seen among groups although phospho eNOS Ser(1177) levels decreased in groups of aged men relatively to young (P = 0.0043; P = 0.0099). CONCLUSIONS: Our results demonstrate for the first time expression of SIRT2-3 and SIRT5-7 in the human CC. Aged individuals with CVDRF presented an increase in SIRT7 protein levels and a decrease in mitochondrial SIRT3. This finding suggests that CVDRF induces the loss of antioxidant defense mechanisms leading to endothelial injury.

Natural mineral-rich water ingestion improves hepatic and fat glucocorticoid-signaling and increases sirtuin 1 in an animal model of metabolic syndrome.

BACKGROUND: We recently reported protection against metabolic syndrome (MetSyn) induction and endothelial dysfunction by natural mineral-rich water intake in fructose-fed Sprague-Dawley rats. As glucocorticoids are critical to MetSyn development, we aimed to further characterize the beneficial effects of mineral-rich water intake in that animal model, by assessing relevant effectors in glucocorticoid-signaling in liver and subcutaneous (SCAT) and visceral (VAT) adipose tissues, sites with a central role in metabolic (dys)regulation. MATERIALS AND METHODS: Adult male rats had free access to standard diet and different drinking solutions (8 weeks): a) tap water (CONT), b) 10% fructose/tap water (FRUCT) or c) 10% fructose/mineral-rich water (FRUCTMIN). 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), glucocorticoid receptor (GR), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1-α) and sirtuin 1 (Sirt1) tissue protein expressions were evaluated by Western blot. Plasma corticosterone (ELISA) and non-esterified fatty acids (NEFA) levels were quantified spectrophotometrically. RESULTS: Expectedly, Sirt1 and PGC1-α significantly decreased in liver, 11ß-HSD1 tended to increase in VAT and tended to decrease in liver and SCAT, and plasma corticosterone tended to increase in FRUCT vs. CONT. Mineral-rich water showed a trend towards a reduction of these fructose effects and significantly increased hepatic Sirt1 vs. CONT and FRUCT. GR significantly increased in VAT and plasma NEFA strongly tended to increase in FRUCTMIN vs. CONT and FRUCT. CONCLUSIONS: Glucocorticoid-signaling was different among SCAT and VAT and also in liver. Mineral-rich water modulation of fructose effects on glucocorticoid-signaling and Sirt1 underlines the better metabolic profile found earlier.

Effect of aging and cardiovascular risk factors on receptor Tie1 expression in human erectile tissue.

INTRODUCTION: Erectile dysfunction is highly prevalent in patients with advanced age or cardiovascular disease risk factors (CVDRFs). These conditions interfere on expression of vascular growth factors and respective receptors causing disturbance in endothelial function. AIM: This study aims to assess the effect of aging and CVDRF on the expression of tyrosine kinase with immunoglobulin-like and EGF-like domains (Tie) 1 in human corpus cavernosum (CC). METHODS: CC fragments obtained from programmed surgeries or organ donors were divided into three groups: young, healthy aged, and aged with CVDRF. Angiopoietin (Ang) 1, Ang2, Tie1, and Tie2 mRNA and protein levels were assessed by real-time polymerase chain reaction and Western blotting, respectively. Dual-immunolabeling of Tie1 with specific markers of endothelium and smooth muscle and Ang1 and Ang2 was performed. MAIN OUTCOME MEASURES: To characterize the expression of Tie1 in human CC and elucidate its potential inhibitory effect in Ang-Tie2 system. RESULTS: Analysis of mRNAs demonstrated a decrease in Tie1 expression in CVDRF individuals compared with aged or young healthy individuals. No variation for Tie2, Ang1, or Ang2 expression was observed among the studied groups. In all analyzed CC fragments, a 125 kDa band, Tie1, was detected. This protein presented a significant age-related decrease, specially in individuals with CVDRF. Immunofluorescence study revealed Tie1 expression in the endothelium of samples of all experimental groups. CONCLUSIONS: Employing different methodological approaches, we show for the first time that Tie1 is expressed in human CC endothelium, and its level of expression diminishes in aged individuals, particularly those with CVDRF. This finding reinforces the view that delivery of Ang1 to the CC of erectile dysfunction affected CVDRF patients is able to activate a beneficial Tie2 response.

Effects of natural mineral-rich water consumption on the expression of sirtuin 1 and angiogenic factors in the erectile tissue of rats with fructose-induced metabolic syndrome.

Consuming a high-fructose diet induces metabolic syndrome (MS)-like features, including endothelial dysfunction. Erectile dysfunction is an early manifestation of endothelial dysfunction and systemic vascular disease. Because mineral deficiency intensifies the deleterious effects of fructose consumption and mineral ingestion is protective against MS, we aimed to characterize the effects of 8 weeks of natural mineral-rich water consumption on the structural organization and expression of vascular growth factors and receptors on the corpus cavernosum (CC) in 10% fructose-fed Sprague-Dawley rats (FRUCT). Differences were not observed in the organization of the CC either on the expression of vascular endothelial growth factor (VEGF) or the components of the angiopoietins/Tie2 system. However, opposing expression patterns were observed for VEGF receptors (an increase and a decrease for VEGFR1 and VEGFR2, respectively) in FRUCT animals, with these patterns being strengthened by mineral-rich water ingestion. Mineral-rich water ingestion (FRUCTMIN) increased the proportion of smooth muscle cells compared with FRUCT rats and induced an upregulatory tendency of sirtuin 1 expression compared with the control and FRUCT groups. Western blot results were consistent with the dual immunofluorescence evaluation. Plasma oxidized low-density lipoprotein and plasma testosterone levels were similar among the experimental groups, although a tendency for an increase in the former was observed in the FRUCTMIN group. The mineral-rich water-treated rats presented changes similar to those observed in rats treated with MS-protective polyphenol-rich beverages or subjected to energy restriction, which led us to hypothesize that the effects of mineral-rich water consumption may be more vast than those directly observed in this study.

Energy restriction ameliorates metabolic syndrome-induced cavernous tissue structural modifications in aged rats.

High-fat (HF) diet regular intake along life highly contributes to vascular dysfunction and to an increment in prevalence of metabolic syndrome (MetS) and erectile dysfunction (ED), a surrogate symptom of occult vascular disease, in the elderly. However, little is known about the effects of energy restriction (ER) alone/or after an HF-feeding period. We show here that in male Sprague-Dawley rats, 16 months of HF-diet consumption led to an increase in body adiposity, blood pressure, lipidemia, C-reactive protein, and insulin resistance and to hypoadiponectinemia, conditions that cluster in MetS. In addition, this treatment strongly favored collagen deposition in cavernous tissue and myocardium. Conversely, for the same time period, the ingestion of 75 % of ad libitum energy intake by controls (ER) extensively counteracted these outcomes. The impact of 6-month ER after 10-month HF period was also analyzed, and despite the decrease in body weight, adiposity, blood pressure, lipidemia, and C-reactive protein and improvement of insulin sensitivity, no differences were observed either in adiponectin blood levels or in retroperitoneal fat pad mass. Moreover, this treatment led to a reduction in cavernous tissue collagen deposition, but not in the myocardium, and evidenced differential mobilization of adipose tissue accretions. The data show the ability of HF diet to cause MetS and produce unwanted effects on myocardium and corpora vascular structure. They also indicate that these consequences are preventable upon ER diet starting early, but not later, in life.

Androgen depletion in humans leads to cavernous tissue reorganization and upregulation of Sirt1-eNOS axis.

Aging and physiological androgen decay leads to structural changes in corpus cavernosum (CC) that associate with erectile function impairment. There is evidence that such changes relate to nitric oxide (NO) bioavailability, an endothelial compound produced by the action of endothelial NO synthase (eNOS), and is regulated by sirtuin-1 (Sirt1), a NAD(+)-dependent protein deacetylase. Taking into account the reduced NO synthesis observed in aging and erectile dysfunction, we aimed to characterize human CC of androgen-deprived, young, and aged individuals postulating that androgen deprivation induces modifications similar to those observed in aging. Human penile fragments were collected from young individuals submitted to male-to-female sex reassignment procedure, who undergone an androgen deprivation chemical regimen, from young organ donors and from aged patients submitted to penile deviation surgery. They were processed for histomorphometric analysis of smooth muscle (SM) and connective tissues (CT), and dual-immunofluorescence of alpha-actin/vWf or Sirt1, and endothelin-1/eNOS. Estrogen receptors were analyzed by immunohistochemistry and semiquantification of Sirt1, eNOS, and phospho-Akt was assayed by Western blotting. Androgen withdrawal, similarly to aging, leads to a noteworthy reduction of SM-to-CT ratio in CC. However, in contrast to young and aged, a significant increase in penile Sirt1 expression accompanied by an increase in total eNOS expression was observed in androgen-depleted individuals. No changes were evidenced in phospho-Akt system and estrogen receptors were undetectable. These findings indicate that Sirt1 regulates the expression of eNOS in human CC employing mechanisms influenced by androgen depletion.

Energy restriction and exercise modulate angiopoietins and vascular endothelial growth factor expression in the cavernous tissue of high-fat diet-fed rats.

The purpose of the current study was to evaluate the effect of a high-fat (HF) diet, energy restriction and exercise on the expression of vascular endothelial growth factor (VEGF), angiopoietin (Ang) 1 and 2, and their receptors in rat corpus cavernosum (CC). Male Wistar rats were fed ad libitum with an HF diet for 8 or 16 weeks. After 8 weeks of the HF diet, a group of rats was subjected to energy restriction with or without exercise for 8 weeks. Control animals had free access to standard diet for the same period. After euthanasia, blood was collected and the penises removed for immunofluorescence assays (VEGF, VEGF receptor (VEGFR) 1 and 2, Ang1, Ang2 and Tie2) and semiquantification of VEGF, VEGFR1, VEGFR2, Ang1, Ang2, Tie2, endothelial nitric oxide synthase (eNOS) and Akt/phospho-Akt by Western blotting. HF diet-fed rats exhibited lower high-density lipoprotein cholesterol (HDL-c) levels, higher systolic blood pressure and an increased atherogenic index. A significant increase in Ang2 expression in the CC was verified and coupled to a decrease in VEGF and VEGFRs. The Akt pathway was activated by the HF diet. Energy restriction and exercise increased eNOS expression and restored most HF diet-induced modifications except for VEGFR2 expression. These results emphasize the role of diet on vascular function regulation, demonstrating that cavernous imbalance of VEGF/VEGFRs and Angs/Tie2 systems occurs before serum lipid changes and obesity onset, antedating structural atherosclerotic features.

Real-time PCR study of Ang1, Ang2, Tie-2, VEGF, and KDR expression in human erectile tissue during aging.

INTRODUCTION: Aging is a recognized risk factor for erectile dysfunction (ED), contributing independently to vascular damage of penile tissue. Vascular maintenance depends on angiogenic balance in tissues. Vascular endothelial growth factor (VEGF) is a modulator of endothelial cells functions, after engagement to specific receptor kinase domain region (KDR). Other factors, such as angiopoietins, cross talk with VEGF, modulating its effects. Angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) compete for binding to Tie-2 and, while Ang1 promotes vascular stabilization, Ang2 acts as a partial agonist or antagonist of Ang1 signaling, depending on VEGF bioavailability. AIMS: To quantify the expression of Ang1, Ang2, Tie-2, VEGF, and KDR by real-time polymerase chain reaction (PCR) in human corpus cavernosum (CC) from young and aged healthy individuals. METHODS: Human CC fragments were obtained from organ donors without known risk factors to ED and divided in two groups: young (16-35 years) and aged (59-74 years). RNA was extracted and converted to cDNA. Real-time PCR reactions employed appropriate primers. KDR, Tie-2, Akt, and phospho-Akt protein levels were also assessed by Western blotting (WB). Computer-assisted evaluation of vascular areas was performed. MAIN OUTCOME MEASURES: Study of angiopoietins-Tie-2 and VEGF-KDR systems in human CC during aging by real-time PCR and WB. The ratios Ang1/Tie-2 and VEGF/KDR and Akt levels were also determined. RESULTS: Real-time PCR results showed a sixfold significant reduction in the Ang1/Tie-2 ratio during aging. Ang2, VEGF, and KDR expression results were highly variable. Nevertheless, the ratio VEGF/KDR was significantly higher in the aged individuals. Akt and phospho-Akt levels were similar in both groups. Immunohistological evaluation revealed a significant decrease in vascular areas and endothelial surface in CC with aging, despite no differences found in vessel number. CONCLUSIONS: The obtained results suggest an aging-associated downregulation of angiopoietins/Tie-2 system and an apparent compensatory upregulation of the VEGF/KDR system.

Characterization of the expression of Ang1, Ang2, and Tie2 in the Corpus Cavernosum of the rat during aging.

Aging is the single most significant risk factor for erectile dysfunction (ED), leading to structural modification of cavernous tissue and altering expression of vascular growth factors. The angiopoietin/Tie2 system has been recently considered as a potential target for therapy of vascular disorders, including ED. Hence, the aim of this study was to analyze expression of angiopoietin1 (Ang1), angiopoietin2 (Ang2), and their receptor Tie2 in corpus cavernosum (CC) of rat during aging (6, 12, 18, and 24 months). The expression of Ang1, Ang2, and Tie2 was studied by immunohistochemistry and immunofluorescence, followed by semiquantification after Western blotting. Both Ang1 and Ang2 were localized mainly in perivascular smooth muscle and endothelial cells, while Tie2 was strictly detected at the vascular endothelium. A significant decrease in Ang2's expression was observed at 12 months when compared with 6-month-old rats, a tendency that reverses in older animals. No significant differences were demonstrated for Ang1 or Tie2, which is consistent with their constitutive expression in CC. The ratios Ang1/Tie2 and Ang2/Tie2 were also calculated and both decrease during aging, while no marked variation was observed for Ang1/Ang2. Our results suggest that the angiopoietin/Tie2 system participate in the vascular maintenance and remodeling of the CC during aging.

Effects of chronic red wine consumption on the expression of vascular endothelial growth factor, angiopoietin 1, angiopoietin 2, and its receptors in rat erectile tissue.

Long-term consumption of red wine (RW) apparently confers some protection against cardiovascular diseases due to antiatherosclerotic properties of polyphenols and ethanol (EtOH). There is some evidence indicating that they do so by regulating angiogenesis, but the mechanism and the modulator factors involved are largely unknown. The aim of this study was to evaluate the effects of chronic ingestion of RW in vascular structure and in the pattern of expression of vascular growth factors in the rat corpus cavernosum. Male Wistar rats aged 6 mo were treated with RW or an equivalent EtOH solution, as the only liquid source for 6 mo. Expression of vascular endothelial growth factor (VEGF), angiopoietin 1 and angiopoietin 2, and their receptors (VEGFR1, VEGFR2, and Tie2) in cavernous tissue was assayed by immunofluorescence and Western blotting. A reduction of VEGF and VEGFR2 expression, respectively, in smooth muscle and endothelial cells was observed in RW-treated animals, which was balanced by an increase in angiopoietins/Tie2 expression. In EtOH rats, only a decrease in expression of the receptors VEGFR2 and Tie2 was observed. These results, taken together, suggest that antioxidants present in RW activate selected mechanisms for the maintenance of cavernous tissue vascularization. However, functional studies will be necessary to elucidate if RW is of benefit in the prevention of deleterious vascular events associated with ED.