Nuno Tomada

Characterization of VEGF and Angiopoietins Expression in Human Corpus Cavernosum during Aging

INTRODUCTION AND OBJECTIVES Erectile dysfunction (ED) is a highly prevalent and age-related disease, caused by endothelial dysfunction and impaired cavernous angiogenesis. However, cellular and molecular changes involved in erectile pathophysiology in aging male remain to be elucidated. AIM To characterize the vascular organization, concomitantly with analysis of the expression of vascular endothelial growth factor (VEGF), Angiopoietin 1 (Ang1) and Angiopoietin 2 (Ang2) in young and aged human corpus cavernosum. METHODS Human penile fragments were removed from patients submitted to penile deviation surgery (11 cases; 58-70 years) and from potential organ donors (four cases; 18-28 years) without ED or risk factors for ED. Smooth muscle and connective tissue were assessed by Massons trichrome staining and computer-assisted histomorphometry. Dual immunostaining for specific markers of endothelium (von Willebrand factor) and smooth muscle cell (alpha-actin), VEGF, Ang1 and Ang2 was assayed by fluorescence microscopy. Semi-quantification of expression of angiogenic factors was performed by Western blotting. MAIN OUTCOME MEASURES Expression of VEGF and Angiopoietins in human corpus cavernosum, using a combination of histologic stainings, and molecular biology tools in order to achieve a better understanding of cavernosal tissue remodeling with aging. RESULTS Aged human corpus cavernosum presented wider sinusoidal spaces, loss of muscle cell bundles, and increased connective tissue content. Ang1 was scarcely expressed in small clusters in smooth muscle cell cytoplasm with identical localization in both studied groups. VEGF expression was abundant in smooth muscle cell and its expression markedly decreased in aged tissue, contrasting with the expression of angiopoietins that increased in the aged corpus cavernosum. CONCLUSIONS Immunoflourescent studies of cellular markers and growth factors help clarifying vascular organization and angiogenesis mechanisms in erectile tissue. Our findings demonstrate that the organization pattern of vascular endothelium and smooth muscle components of cavernosal tissue modifies during aging. Ang1 and Ang2 upregulation in human-aged penile tissue suggest a VEGF-independent vascular remodeling mechanism.

Aging and Orchidectomy Modulate Expression of VEGF Receptors (Flt‐1 and Flk‐1) on Corpus Cavernosum of the Rat

Abstract:  Aging and hypogonadic states are known risk factors for erectile dysfunction (ED), contributing together to vascular damage of penile tissue. In the present study, VEGF‐specific membrane receptor (VEGFR‐1/Flt‐1 and VEGFR‐2/Flk‐1) expression was studied by confocal immunofluorescence in the corpus cavernosum of control rats, rats aged 12 and 18 months, and orchidectomized Wistar rats (90 days of bilateral orchidectomy). Immunocytochemical results demonstrated VEGFR‐2 expression restricted to the endothelium in both control and orchidectomized rats. Aged animals (12 and 18 months) presented enlarged vessels with intense VEGFR‐2 endothelial staining. On the other hand, VEGFR‐1 was demonstrated in smooth muscle fibers, particularly in those that surround vessel endothelium, the endothelial expression being very low in control and orchidectomized rats. However, in the aged rats, a shift resulting in a VEGFR‐1 and VEGFR‐2 co‐localization in the endothelial cell was observed. The findings suggest an upregulation of VEGFR‐1 in the corpora cavernosa during aging in the rat, which is evident from an increased expression by endothelial cells.

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.

Are All Metabolic Syndrome Components Responsible for Penile Hemodynamics Impairment in Patients with Erectile Dysfunction? The Role of Body Fat Mass Assessment

INTRODUCTION Erectile dysfunction (ED) is a common disease that is mostly vasculogenic in nature. ED correlates with cardiovascular risk factors, with endothelial dysfunction being the common link. Hypertension (HTA) and insulin resistance are the most important determinants of arteriogenic ED, and are also components of the metabolic syndrome (MetS), which supports a strong association between MetS and ED. However, MetS and, specifically, obesity interference on penile hemodynamics is still controversial. AIM To evaluate the impact of independent MetS criteria and obesity on penile duplex Doppler ultrasound (PDDU) parameters in men with ED. METHODS Consecutive patients (n = 212) referred to a unit of PDDU were evaluated for cardiovascular risk factors and MetS (ATP III criteria). Body mass index and body fat percentage (BF%) were calculated. Each patient underwent a PDDU by the same investigator. Data are expressed as mean ± standard deviation, and statistical significance was considered at P level < 0.05. Statistical analysis of clinical, laboratory, and PDDU parameters was performed with SPSS® software. MAIN OUTCOME MEASURES To evaluate the individual power of MetS clusters and obesity as predictive factors for penile hemodynamic changes namely mean peak systolic velocity (mPSV). RESULTS MetS was present in 24.8% of men, and 80.8% of them presented penile hemodynamics alterations, with mPSV significantly lower comparatively to no MetS patients (29.0 vs. 35.4 cm/s, P = 0.004). Multivariate analysis demonstrated that, considering all MetS parameters, only HTA was significantly associated with diminished mPSV. However, after further adjustment for all cardiovascular risk factors, BF% remained the sole independent clinical factor for penile hemodynamics impairment. CONCLUSIONS There is a strong association between MetS and ED, but within MetS criteria, only HTA was independently associated with the deterioration of penile hemodynamics parameters. Although the classical methods of evaluating obesity in MetS were not individually associated with PDDU impairment, BF% represented by itself an excellent predictor of vascular ED.

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.

Expression of vascular endothelial growth factor and angiopoietins in human corpus cavernosum.

To evaluate the expression of the angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietins (Ang) 1 and 2, in normal human penile erectile tissue.

Penile corporoplasty with Yachia's technique for Peyronie's disease: Single center experience with 117 patients.

Introduction: Peyronies disease is an acquired penile deformity with a variety of presentations, caused by the formation of fibrous plaques within the tunica albuginea, leading to bio-mechanical and vascular abnormalities. The objective is to investigate the 18 years outcome of patients with Peyronies disease treated with penile corporoplasty (Yachia technique) in our department. Materials and Methods: One hundred and seventeen patients underwent surgical treatment for PD between 1991 and 2009 and were retrospectively evaluated. We used the Levine and Lentings algorithm for surgical treatment. Data was obtained from medical records, clinical evaluation, and telephone interview. Post-operative follow-up was at 6 weeks and 12 months. The mean time of follow-up was 14 months (12-19 months). Main Outcome Measures: Patient demographic, co-morbidities, erectile function, penile curvature, and surgical intervention were documented. The main outcome measures of this study are postoperative complications, surgical purpose, and patients and partners satisfaction rates. Results: Surgical aim was obtained in 106 patients (success rate of 94.6%). Complications occurred in 4.5% of patients, but most of these were mild. At 6 weeks, complete straightening of the penis was achieved in 57 patients (50.9%), and partial straightening which allow sexual intercourse in 49 patients (43.7%). Nine patients report gland hypoesthesia and almost all report subjective perception of penis shortening (0.5 cm to 5 cm). Twenty-two patients developed recurrent deformity at 12 months follow-up, with compromise of sexual intercourse in 7 patients. Patients’ responses to our questionnaire showed that overall 88.4% of the patients and partners were satisfied with the surgical results. Conclusion: According to the results of this long-term, retrospective study, surgical correction, using the Yachia technique, is an excellent option for patients with functional impairment from their Peyronies disease, especially.

Endothelial function in patients with metabolic syndrome and erectile dysfunction : a question of Angiopoietin imbalance?

Erectile dysfunction (ED) is a highly prevalent disease whose aetiology is mostly vasculogenic. It is nowadays considered a marker of future cardiovascular events reflecting the underlying endothelial dysfunction, the common link with the metabolic syndrome (MetS). The recent association between MetS, endothelial dysfunction and peripheral artery disease, but not with coronary artery disease (CAD), suggests that the pathophysiologies of CAD and peripheral artery disease may be distinct. Moreover, several recent studies support an emerging role for an imbalance of angiogenic growth factor levels like Angiopoietin 1 and 2 in cardiovascular disease, considering its intimate association with chronic low‐grade inflammation. We aim to find a correlation between Angiopoietins levels and systemic and local endothelial function in MetS and ED patients. Forty‐five MetS patients with ED were enrolled. ED severity was assessed by International Index of Erectile Function questionnaire (IIEF5) and penile duplex Doppler ultrasound (PDDU). Endothelial function was assessed by Peripheral arterial tonometry (PAT), and serum asymmetric dimethylarginine (ADMA), Ang1 and Ang2 levels. Obesity and hypertension were the most frequent MetS parameters (91.1 and 88.9% respectively). Severe ED was present in 35.6% of patients. Penile haemodynamic was impaired in 77.5%. Endothelial dysfunction (PAT criteria) was present in 40.9% of patients. Ang2 levels were significantly higher in men with abdominal obesity. We observed an inverse correlation between Ang1 and peak systolic velocity, and in patients with penile arterial dysfunction, Ang1 levels were significantly higher and Ang2/Ang1 ratio significantly lower, than patients with normal arterial function. Neither ADMA nor PAT parameters were correlated with ED severity evaluated by IIEF5 or PDDU exam. In conclusion, there is an imbalance of angiopoietins in MetS and ED patients. The absence of correlation with PAT or ADMA levels suggests that angiopoietins may be early markers of endothelial dysfunction in this population of higher cardiovascular risk.

Comparative ultrastructural study of human corpus cavernosum during ageing

Erectile Dysfunction (ED), the inability to achieve or maintain an erection of sufficient rigidity for completion of sexual act, is a common condition affecting more than 150 million of men worldwide. This disorder is highly associated with aging, however concomitant pathologies such as hyperlipidemia, hypertension, and diabetes also contribute to ED progression. In the Massachusetts Male Aging Study, age was considered an independent variable strongly associated with ED, showing that the prevalence of this disease increased with age from 38% in the youngest group of men (mean age 40 y.) to almost 70% in the oldest group of men examined (mean age 70 y.). It is well demonstrated that aging leads to changes in the cardiovascular system, which results in a decrease in elasticity due to fibrosis and an increase in stiffness of the arterial system, independently of the effects of concurrent pathologies. Vasculogenic ED is the most prevalent condition, affecting nearly 80% of patients with organic etiology. Small vessels of the penis are very sensitive to structural and functional changes, and small disturbances can conduce to ED. ED is now considered by some authors as synonymous to endothelial dysfunction and an early manifestation of atherosclerosis, being a precursor of systemic vascular disease. Human cavernous tissue is mainly constituted by smooth muscle fibers that surround sinusoid vessels. Corpus cavernosum structural elements act in concert, allowing increase of intra-cavernous arterial flow and smooth muscle relaxation processes which are fundamental to penile erection. The aim of this study was to compare the ultrastructural anatomy of the young and aged human corpus cavernosum, in the absence of additional risk factors.

Hormonal Modulation in Aging Patients with Erectile Dysfunction and Metabolic Syndrome

Erectile dysfunction (ED), metabolic syndrome (MetS), and hypogonadism are closely related, often coexisting in the aging male. Obesity was shown to raise the risk of ED and hypogonadism, as well as other endocrinological disturbances with impact on erectile function. We selected 179 patients referred for ED to our andrology unit, aiming to evaluate gonadotropins and estradiol interplay in context of ED, MetS, and hypogonadism. Patients were stratified into groups in accordance with the presence (or not) of MetS and/or hypogonadism. Noticeable differences in total testosterone (TT) and free testosterone (FT) levels were found between patients with and without MetS. Men with MetS evidenced lower TT circulating levels with an increasing number of MetS parameters, for which hypertriglyceridemia and waist circumference strongly contributed. Regarding the hypothalamic-pituitary-gonadal axis, patients with hypogonadism did not exhibit raised LH levels. Interestingly, among those with higher LH levels, estradiol values were also increased. Possible explanations for this unexpected profile of estradiol may be the age-related adiposity, other estrogen-raising pathways, or even unknown mechanisms. Estradiol is possibly a molecule with further interactions beyond the currently described. Our results further enlighten this still unclear multidisciplinary and complex subject, raising new investigational opportunities.