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Effects of prolonged ingestion of epigallocatechin gallate on diabetes type 1-induced vascular modifications in the erectile tissue of rats

Abstract

Diabetes Mellitus type 1 is a metabolic disease that predisposes to erectile dysfunction, partly owing to structural and molecular changes in the corpus cavernosum (CC) vessels. The aim of this study was to determine the effects of early treatment with the antioxidant epigallocatechin gallate (EGCG) in cavernous diabetes-induced vascular modifications. Diabetes was induced in two groups of young Wistar rats; one group was treated with EGCG for 10 weeks. A reduction in smooth muscle content was observed in the CC of diabetic rats, which was significantly attenuated with EGCG consumption. No differences were observed among groups, neither in the expression of VEGF assayed by western blotting nor in the immunofluorescent labeling of vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2). VEGFR2 was restricted to the endothelium, whereas VEGF and VEGFR1 co-localized in the smooth muscle layer. With regard to the Angiopoietin/Tie-2 system, no quantitative differences in Angiopoietin 1 were observed among the experimental groups. Ang1 localization was restricted to the smooth muscle layer, and receptor Tie2 and Angiopoietin 2 were both expressed in the endothelium. In brief, our results suggest that EGCG consumption prevented diabetes-induced loss of cavernous smooth muscle but does not affect vascular growth factor expression in young rats.

Introduction

Diabetes Mellitus (DM) is a serious disease, characterized by a hyperglycemic state owing to impaired insulin secretion, insulin signaling, or both. Besides the direct metabolic disorder, the concern pertaining to DM relates to its long-term complications, such as cardiovascular disease and microvascular disturbances.

The risk of developing erectile dysfunction (ED), an early manifestation of systemic cardiovascular disease,1 is three times higher in diabetic men when compared with the general population.2 Distinction in ED prevalence between type 1 and type 2 DM is not clear,3 being described by some authors as being more frequent in type 1 DM, once it starts at younger age.4 The high rates of ED observed in DM can be attributed to several pathways, including the formation of advanced glycation end products (AGE) that preferentially form and accumulate in the CC,5 reducing the activity of endothelial nitric oxide synthase (eNOS) and NO synthesis.6 Furthermore, AGE strongly favors oxidative stress through increase in oxygen reactive species (ROS), which react with NO producing peroxynitrite.7

Thus, DM through AGE formation contributes to disturbance in corpus cavernosum (CC) smooth muscle relaxation, mainly due to endothelial dysfunction, caused by deterioration in NO synthesis and activity. Along with the endothelial functional disruption, the decreased bioavailability of NO and the increased production of ROS also lead to impairment in angiogenesis, a process responsible for repair and formation of new vessels from pre-existing ones.8

Vascular endothelial growth factor (VEGF) is the main angiogenic factor responsible for regulation of the proliferation, migration and vascular permeability of endothelial cells (ECs). It exerts its actions through two tyrosine kinase receptors: VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2). VEGFR1 can potentiate or inhibit VEGF actions, having a dual role in angiogenic processes. On the other hand, VEGFR2 only transduces pro-angiogenic signals.9 In DM the balance between VEGF and its receptors is disrupted, threatening the survival of damaged vessels. As VEGF stimulates eNOS activity,10 its imbalance may predispose to ED.11 However, the molecular mechanisms through which VEGF is affected in DM remain poorly understood.

Other participants in vascular remodeling are Angiopoietin (Ang)1 and 2, which compete for binding the Tie2 receptor. Ang1 acts exclusively as an agonist of Tie2, whose activation leads to NO production, EC survival and vessel maturation, complementing VEGF actions.12 Conversely, Ang2 could function as a partial agonist in the presence of endogenous VEGF, or otherwise as a promoter of vessel regression through inhibition of Tie2, being confined to vessel remodeling sites.13 Similarly to VEGF signaling, the Ang-Tie2 system is disrupted in DM, though further details are needed to elucidate its role in ED progression.

DM causes oxidative damage in tissues, and orally administered anti-oxidants have been proposed to ameliorate DM complications. Epigallocatechin-3-gallate (EGCG), a flavanoid that belongs to the family of polyphenolic anti-oxidants, is the most abundant catechin present in green tea, a universal beverage with known health benefits.14 EGCG promotes scavenging of ROS and increases anti-oxidant enzyme activity,15 and its administration leads to improvement in cardiac function through reduction in cholesterol circulating levels and in atherosclerotic plaques in DM patients.16 Further, in aged tissues that accumulate oxidized molecules, long-term EGCG consumption has been shown to have beneficial effects, as shown in the CC of aged rats.6, 17 Hence, we hypothesize that the early treatment of type 1 diabetic rats with EGCG mitigates the structural and molecular changes in the CC, preventing further ED. Such preventive effects might be due to the preservation of erectile tissue integrity and to modulation of angiogenic factor expression. To our knowledge, this is the first time the EGCG effect on cavernous tissue of type 1 DM animals is being studied.

Materials and methods

Animals

Male Wistar rats weighing 250–300 g, maintained at 22±2 °C and humidity of 55±5%, under a 12-h light–dark cycle with free access to food and water for 10 weeks, were divided in two groups. One group of 16 rats received an intraperitoneal (i.p.) injection of Streptozotocin (STZ) (60 mg kg−1, Sigma Aldrich, Barcelona, Spain) dissolved in 0.1m citrate buffer (pH 4.5) to induce DM type 1. The control group of rats (n=8) received an i.p. injection of the vehicle as previously described.18

After 3 days, blood glucose levels were measured from the tail vein with Accu Chek Sensor Comfort (Roche Diagnostics, Berlin, Germany). DM was considered for fasting glycemia levels higher than 270 mg dl−1.

A group of eight randomly selected diabetic rats were treated with an aqueous solution of ECCG (2 g l−1, Holliday & Company, Toronto, ON,Canada) as the exclusive beverage for 10 weeks, constituting the group STZ/EGCG. The remaining diabetic rats, belonging to the STZ/H2O group, were given tap water. The levels of glucose were assessed at the end of the 4th and 10th week of treatment.18

All experiments were undertaken according to the European Community Council directive 2010/63/EU and were approved by the local Ethics Committee.

At the end of the treatment, the rats were anesthetized with an i.p. injection of sodium pentobarbital (60 mg kg−1), and the penises were removed before killing the rats with transcardiac vascular perfusion with phosphate-buffered saline (PBS). Each penis was dissected from the crura and divided into two parts on its longitudinal axis, one frozen at −80 °C for molecular analysis and the other fixed with 10% buffered formaldehyde for immunofluorescence and immunohistochemical analysis.

Immunohistochemistry

The portion of the penis fixed in formaldehyde was embedded in paraffin, cut along its transversal axis in 5-μm-thick sections and placed in 0.1% (w/v) poly-l-lysine-coated microscopy slides.

Sections were deparaffinized and hydrated using a series of aqueous ethanol solutions with decreasing concentrations until water for 5 min at each incubation. Blocking of the endogenous peroxidase activity was carried out at room temperature, with a solution of 3% H2O2 (v/v) in methanol for 30 min.

After washing with PBS, the sections were incubated in 1 m HCl for 30 min for antigen recovery and neutralized in 0.1 m sodium tetraborate for 5 min. Further, sections were incubated with blocking buffer 2% (w/v) bovine serum albumine (BSA) and 10% (v/v) goat normal serum in PBS for 1 h. Subsequently, blocking buffer was replaced by a solution of monoclonal mouse anti-α-actin (a marker of smooth muscle cells, SMC) antibody (1/500 diluted, Chemicon International, Billerica, USA) and incubated overnight at 4 °C in a humid chamber.

The following day, the primary antibody solution was removed with PBS washings and the sections were first incubated with biotin-conjugated appropriated secondary antibody and then with streptavidin–peroxidase complex, both diluted 1/400 in PBS. Sections were then reacted with a solution of diaminobenzidine containing 0.1% (v/v) H2O2, washed under running water for 10 min, stained with hematoxilyn and observed in an optical microscope (Carl Zeiss MicroImaging GmbH, Gottingen, Germany). Morphometric analysis of α-actin content was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) software in images captured of all experimental animals (five images per animal). Primary antibody specificity was assessed by its omission in a control slide.

Immunofluorescence

Tissue sections of the penis were processed according to the previous description for immunohistochemistry, and after 1 h blocking with 2% (w/v) BSA in PBS they were incubated with a mixture of mouse anti-α-actin and goat anti-platelet endothelial cell adhesion molecule (PECAM)-1, an endothelium marker (Santa Cruz Biotechnology, Paso Robles, CA, USA) antibodies (1/300 and 1/100 diluted, respectively), goat anti-Ang1 or goat anti-Ang2 combined with rabbit anti-Tie2 polyclonal antibodies (1/100 diluted for anti-angiopoietins and 1/100 for anti-Tie2, Santa Cruz Biotechnology) or goat anti-VEGF (1/25 diluted, R&D Abingdon, UK) combined with either rabbit anti-VEGFR1 (1/300 diluted, Labvision Corporation, Fremont, CA, USA) or anti-VEGFR2 (1/200 diluted, Santa Cruz Biotechnology) polyclonal antibodies, at 4 °C overnight in a humid chamber. Sections were further incubated with the appropriated combination of the secondary antibodies conjugated with fluorocromes (Alexa 568 (red) anti-goat with Alexa 488 (green) anti-rabbit or Alexa 488 anti-mouse, Molecular Probes, Cambridge, UK).

For nuclear DNA staining, 4′,6-diamidino-2-phenylindole (DAPI) was applied to sections for 30 s, immediately before mounting in a glycerol solution in phosphate buffer (3:1). The sections were then observed under an Apotome Fluorescence Microscope (Carl Zeiss MicroImaging GmbH) and images were captured using AxionVision 3.0 program (Carl Zeiss MicroImaging). Autofluorescence and specificity of primary antibodies were assessed by total and primary antibody omission, respectively.

Western blotting

For molecular analysis, the fragments of penis were mechanically homogenized in a solution containing 50 mM Tris pH 7.2, 0.1 M NaCl, 5 mM ethylenediaminetetraacetic acid, 0.5% (v/v) Triton x-100, and 0.2% (v/v) protease inhibitor cocktail P8340 (Sigma Aldrich). Thirty micrograms of total protein per well was separated in sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) with 8% or 12% acrylamide resolving gels in an electrophoresis system (Bio-Rad, Hercule, CA, USA) for 1 h, applying 25 mA/gel, and further transferred to a nitrocellulose membrane (0.45 μm pores, Bio-Rad) in a Hoefer system (San Francisco, USA), using 48 mM Tris, 39 mM glycine, 0.037% (w/v) SDS and 20% (v/v) methanol transfer buffer, for 2 h at 25 V at room temperature. After transfer, the membranes were submerged in blocking solution of 5% (w/v) nonfat dried milk (Molico, Nestlé, Portugal) and 0.1% (v/v) Tween-20 in Tris buffer saline (TBS-T) for 30 min, and incubated with primary antibodies from goat anti-Ang1 (1/100) and mouse monoclonal anti-VEGF (R&D) over 2 days at 4 °C. Next day, after washing with TBS-T, the membranes were incubated with anti-goat or anti-mouse peroxidase-conjugated secondary antibodies (1/5000 or 1/1000 diluted, respectively, Santa Cruz) for 1 h at room temperature. The bands were quantified by densitometry using ScionImage software (Scion Corporation, National Institutes of Health, MN, USA) after covering the membranes with a chemiluminescent solution (Kit Supersignal, West Pico Chemiluminescent Substrate, Pierce Biotechnology, Rockford, IL, USA) for 5 min. Normalization of the studied proteins was done with α-tubulin (1/10 000 diluted, Sigma-Aldrich). The mean values of the studied proteins in each experimental group were calculated after individual quantification of each protein in all of the CC samples.

Statistical analysis

Statistical analysis was carried out with the Statistical Package for the Social Sciences (SPSS), version 18.0 for Windows (IBM, Armonk, New York, USA). Differences between the mean values of the three experimental groups were determined through one-way ANOVA test. A probability value of P<0.05 was considered statistically significant. Values are expressed throughout the text as mean±s.e.m.

Results

Morphometric evaluation of smooth muscle density

Figure 1 depicts the smooth muscle structure in the cavernous tissue of the different experimental groups after detection of α-actin by immunohistochemistry. The smooth muscle layer was restricted to the endothelium periphery in all analyzed samples. Morphometric study, shown in the graphic, demonstrated that STZ/H2O rats present a reduced vascular smooth muscle layer area fraction (12.8%) when compared with either those treated with EGCG (23.7%) or controls (26.2%) (P=0.036).

Figure 1
figure1

Immunohistochemical detection of the smooth muscle marker α-actin (arrows) in the corpus cavernosum of the control group (a), STZ/H2O group (b) and STZ/EGCG group (c). In all experimental groups, α-actin expression is restricted to the endothelial periphery surrounding the vascular spaces (VS). Vascular spaces are interconnected in a matrix of connective tissue—trabeculae (Tr). Morphometric analysis of smooth muscle content is displayed in the graph. STZ/H2O rats present a reduced vascular smooth muscle layer area (12.8±3.5) when compared with controls (26.2±3.5) or STZ/EGCG (23.7±5.5). (*P<0.05 relative to control and STZ/EGCG groups).

Dual-immunolabelling of PECAM-1/α-actin, VEGF/VEGFR1, VEGF/VEGFR2, Ang1/Tie2 and Ang2/Tie2

Dual-labeling immunofluorescence was performed to identify the distribution of endothelium and vascular smooth muscle layer in CC by using anti-PECAM-1 (red) and anti-α-actin (green) antibodies, respectively. Representative images of each experimental group are depicted in Figures 2a–c.

Figure 2
figure2

Dual immunolabeling of α-actin (green) and PECAM-1 (red) in erectile tissue of rats (a–c). α-actin, a specific marker of smooth muscle cells, was confined to the smooth muscle layer surrounding the vascular spaces (VS), whereas PECAM-1 was restricted to the endothelial layer (arrow). Staining for VEGF (red) and its receptors VEGFR1 and VEGFR2 (green) (d–i) detected VEGF expression both in the endothelium and in the vascular smooth muscle layer, co-localizing with VEGFR1 (yellow, d–f) but not with VEGFR2, which was identified only in the endothelial layer (g–i). The distribution of receptor Tie2 and its ligands Ang1 and 2 (j–o) identified Tie2 as being restricted to the endothelium and Ang1 mostly on smooth muscle, even though some co-localization can be seen in the endothelium of the control group (j). Regarding Ang2, co-localization with Tie2 was found in the endothelium, but it is barely detected, particularly in the STZ/H2O group. Staining for cell nucleus is blue (DAPI). A full color version of this figure is available at the International Journal of Impotence Research journal online.

With regard to VEGF (red) and its receptors VEGFR1 and VEGFR2 (green), VEGF is scarcely present in the endothelium and vascular smooth muscle, often co-localizing with VEGFR1 as evidenced by superposition of fluorescence (yellow) (Figures 2d–f). In contrast, receptor VEGFR2 was found restricted to the endothelium (Figures 2g–i).

The immunolocalization of Tie2 (green) and its ligands Ang1 and 2 (red) was also carried out (Figures 2j–o). In all experimental groups, Tie2 was exclusively present in the endothelium. Conversely, Ang1 was mainly detected in the SMC, even though there was some co-localization with Tie2 in the endothelium, mostly in the control group (Figure 2j). Ang2 was barely observed, especially in the STZ group (Figure 2n), but when present it was faintly localized in the endothelium, co-localizing with Tie2.

Western blotting semi-quantification of VEGF and Ang1

The expression of VEGF and Ang1 in the CC of the experimental groups was assessed by means of semi-quantitative western blotting. Single bands presenting 21kDa for VEGF and 61 kDa for Ang1 were identified in all samples of the cavernous tissue (Figure 3). Graphic representation of the mean values in arbitrary units for each protein in the experimental groups is also shown. No differences were observed among the groups. However, a tendency for decrease in VEGF expression was observed in both groups of diabetic rats, especially in those that drank tap water. Ang1 was also less abundant in both groups of diabetic rats comparared with controls.

Figure 3
figure3

Western blotting semi-quantification of Ang1 and VEGF in cavernous tissue in all experimental groups. (a) Representative bands of VEGF (21 kDa), Ang1 (61 kDa) and α-tubulin (55 kDa, used as loading control). Graphic representation of the mean VEGF (b) and Ang1 (c) levels in control, STZ/H2O and STZ/EGCG rats and the corresponding s.d. Results are shown in arbitrary units. No significant differences were detected among the experimental groups.

Discussion

ED is a known complication of DM with prevalence higher than 50% in diabetic patients.19 Phosphodiesterase-5 (PDE5) inhibitors constitute the first line of treatment for ED. However, diabetic patients present a low response to these drugs owing to downregulation of endothelium-derived NO synthesis.20, 21 The combination of anti-oxidant therapy with PDE5 inhibitors increases the rate of response among diabetic patients.22 However, as far as we know, its efficacy in the prevention of DM-induced damage remains to be elucidated. EGCG, because of its anti-oxidant and anti-angiogenic properties, emerges as a good candidate for preventing or retarding the vascular damage caused by DM.

In the present study, we intended to evaluate the effects of EGCG on structural organization and vascular growth factor expression in the erectile tissue of the diabetic rat. Previous studies demonstrated that rats with STZ-induced diabetes present an altered CC structure, with loss of vascular SMC, due to higher apoptotic indexes of endothelial and SMC and increase in collagen deposition, in comparison with non-diabetic rats.23, 24 In agreement with these reports, we found that diabetic rats presented a reduction in vascular smooth muscle layer, when compared with the other groups, thus suggesting that EGCG consumption prevented the smooth muscle loss induced by STZ. This effect can be explained by the EGCG ability to scavenge superoxide and peroxynitrite, recognized promoters of apoptotic processes and tissue injury, as well as other ROS.25, 26 In addition, EGCG at low doses can inhibit apoptosis through additional mechanisms.27

Angiogenesis is a complex process dependent on the balance between anti-angiogenic and pro-angiogenic factors such as VEGF and angiopoietins. VEGF is the most important factor in angiogenesis intervening at the beginning of the angiogenic cascade.28 However, in the absence of vascular stabilizing factors, VEGF-induced angiogenesis would give rise to leaky and disorganized vessels.29 Thus, endothelial cells interact with supporting cells and extracellular matrix components, in a process sustained by Ang1, which promotes vessel maturation.30 Conversely, Ang2 is able to induce vessel regression in the absence of VEGF.13 DM leads to a disruption in angiogenic factor balance in a tissue-dependent manner that is mainly documented for VEGF signaling.31 Regarding CC, partial recovery of erectile function with VEGF administration was reported in diabetic rats;11 however, the explanation for this change remains uncharted. In the same manner, the contribution of the angiopoietins-Tie2 system in the DM-related CC functional disturbance has not been elucidated yet.

As previously observed in other studies in rats and humans, VEGF is localized in the endothelium and in the SMC layer, likewise VEGFR1,17, 32, 33, 34 whereas VEGFR2 is restricted to the endothelium. In contrast, the present report does not show differences between groups in VEGF expression. Only a decreasing trend in diabetic rats compared with controls, which apparently was attenuated by EGCG consumption, was detected. Previous reports in aged rats where EGCG consumption lasted for 6 months led to a diminution in VEGF expression.17

One possible explanation for the discrepancy in our data compared with other studies could be the age of the rats or the duration of the treatment. Probably in young rats as those included in this study early vascular modifications are hard to detect. Longer treatments with EGCG could induce higher differences in VEGF expression regulation. Besides inhibition of pathologic angiogenesis, EGCG seems to be involved in the maintenance of physiological angiogenesis, which is otherwise downregulated in the CC of diabetic rats. Considering that NO mediates angiogenesis promoted by VEGF35 and that in DM VEGFR2 could be alternatively activated by ROS, which decreases the number of receptors at the cell surface, despairing angiogenesis,36 EGCG through increase in cavernous NO content and reduction in ROS could mediate the slight elevation verified in VEGF and ensuing signaling in treated animals.37 However, additional studies including VEGFR1, VEGFR2 and phospho-VEGFR2 quantification will be necessary to evaluate the EGCG effect in VEGF-mediated angiogenesis in CC.

With regard to the Angiopoietin-Tie2 system, the Tie2 receptor was restricted to the endothelium in the CC of all experimental groups, co-localizing with Ang2 when detectable. Consistent with its interference in vascular mural cells (SMCs and pericytes) interaction, Ang1 is expressed in small clusters in SMCs and the endothelium,30 agreeing with previous studies.32, 33, 38, 39 To our knowledge, this is the first characterization of the expression and localization of Ang1 and Ang2 in the CC of a rat model of type 1 DM. No differences in Ang1 expression were seen between groups, which agrees with the findings in the CC of rodent and human origin in metabolic syndrome and along aging.39, 40, 41, 42 The clarification of the Angiopoietin-Tie2 system needs further analysis of Ang2 and Tie2 expression, as well as phospho-Tie2 quantification.

In summary, our study shows for the first time that the consumption of EGCG since early establishment of type 1 DM can prevent loss of smooth muscle content of CC vessels. This study opens up new perspectives in the prevention of ED in diabetic patients through a low-risk dietary intervention.

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Acknowledgements

We thank Doctor Inês Tomada (Department of Experimental Biology, Faculty of Medicine, University of Porto) for her contribution in the CC morphometrical analysis. The study was funded by EU Project REDDSTAR (FP7-Health-2012.2.4.3-1) and by the grant IJUP/UNICER 2010.

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Correspondence to D Neves.

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Lombo, C., Morgado, C., Tavares, I. et al. Effects of prolonged ingestion of epigallocatechin gallate on diabetes type 1-induced vascular modifications in the erectile tissue of rats. Int J Impot Res 28, 133–138 (2016). https://doi.org/10.1038/ijir.2016.19

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