Increased expression of endothelin-1 and its receptors in varicocele: an immunohistochemical study

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1 Increased expression of endothelin-1 and its receptors in varicocele: an immunohistochemical study We hypothesized that diminished endothelin 1 (ET-1) expression at the spermatic vein wall level might be responsible for the development of varicocele. However, immunohistochemical evaluation of spermatic and control vein samples from 55 patients with varicocele showed overexpression of ET-1 and its receptors ETA and ETB in varicose veins. (Fertil Steril Ò 2011;95: Ó2011 by American Society for Reproductive Medicine.) Key Words: Endothelin, receptors, immunohistochemistry, varicocele Varicocele is a common cause of male infertility (1). The venous dilatation of the pampiniform plexus is mainly attributed to absent or incompetent valves, either at the left renal vein junction or along the entire internal spermatic vein(s) (2). Recent studies, however, reveal that in up to 85% varicocele is bilateral (3, 4). Thus, previous theories (e.g., the nutcracker phenomenon, increased length of left spermatic vein) seem inadequate to explain the high prevalence of bilateral disease (5). A more unifying theory would address the varicocele as part of wide pattern of venous insufficiency. Recent studies suggest that varicocele may have a hereditary behavior, especially in first-degree relatives (6, 7). The presence of lower limb varicosis is also an independent risk factor for the presence of varicocele (8). Studies on chronic vein insufficiency of the lower limb have suggested that the primary etiology may lie in an inherent venous wall weakness and endothelial dysfunction, as shown by aberrant expression of endothelin-1 (ET-1) and its receptors ETA and ETB (9). We therefore hypothesized that the development of varicose spermatic veins is due to an inherent endothelial axis malfunction at the ET-1 production and ET receptor level in the venous wall of the dilated veins. During a period of 3 years (February 2007 March 2010), 55 male adults were operated with spermatic vein ligation, according to a report by Marmar et al. (10), for the presence of varicocele and concomitant impairment of sperm values and/or infertility. Kostis Gyftopoulos, M.D., Ph.D. a,b Christina Chondrogianni, M.Sc. b Helen Papadaki, M.D., Ph.D. b a Urology Clinic, Olympion Hospital, Patras, Greece b Department of Anatomy, School of Medicine, University of Patras, Rion, Greece Received December 9, 2010; revised April 5, 2011; accepted April 18, 2011; published online May 20, K.G. has nothing to disclose. C.C. has nothing to disclose. H.P. has nothing to disclose. Reprint requests: Kostis Gyftopoulos, M.D., Ph.D., Department of Anatomy, University of Patras Medical School, Rion, Greece ( kogyftop@yahoo.gr). Mean age of the patients was 32.5 years (0.832 SEM). Preoperatively, all patients gave full medical history, underwent physical examination and at least two consecutive semen analyses to confirm oligoasthenospermia. As a result of the physical examination, varicocele was classified according to Dubin et al. (11) into three grades (Supplemental Table 1, available online). After Institutional Review Board approval and patient informed consent, samples of the varicose spermatic veins were harvested at the time of operation. In addition, to overcome the ethical problem of control tissue sampling, subcutaneous veins in the course of the subinguinal incision were harvested as control veins. The samples were fixed in formalin, embedded in paraffin, and sectioned for further immunohistochemical procedure. Briefly, consecutive 4-mm sections of varicose and normal veins were stained for ET-1, ETA, and ETB (monoclonal mouse anti-et-1, 1:100; polyclonal rabbit anti-eta, 1:250; polyclonal rabbit anti- ETB, 1:250; Acris Antibodies) by overnight incubation in moist chambers at 4 C. Immunohistochemical expression was detected with ChemMate Dako Envision (Dako Cytomation). Immunoreactivity was assessed in a semiquantitative method, by developing a score (Histoscore/H-score) where both intensity and distribution of staining are taken into account. Distribution was graded on a scale of 0 3 (0: immunoreactivity in <10% of cells; 1: 10% 35% of cells; 2: 35% 70% of cells; 3: >70% of cells). Intensity of staining was scored as follows: 0, negative; 1, weak; 2, moderate; 3, strong staining. Statistical analyses were performed using the commercially available GraphPad PRISM 5.0 software. The Wilcoxon signed rank test and the Kruskal- Wallis test were used for comparisons between two or three groups accordingly. Nonparametric correlations were tested with the Spearman r 2 correlation coefficient test. A 5% significance level was used for all tests. Morphologically, the control vein wall was generally thin and regular, with the tunica media layer comprising most of the wall thickness. The lumen of the varicose veins was generally dilated, with thickening of the tunica intima, which resulted in narrowing of the lumen in several sections. Endothelin-1 was localized mainly at the tunica media of both varicose and control veins (Supplemental Figure 1, available online). When the H-score was compared between varicose spermatic and control veins, 2554 Fertility and Sterility â Vol. 95, No. 8, June 30, /$36.00 Copyright ª2011 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 FIGURE 1 Immunohistochemical expression of combined intensity and distribution (H-score) for endothelin 1 (ET-1), ETA, and ETB in varicose (V) versus control (c) veins. (Whiskers: minimum to maximum; P<.0001, Wilcoxon signed rank test.) a statistically significant difference was present (Wilcoxon signed rank test, P<.0001; Fig. 1). In a similar fashion, the receptors of endothelin ETA and ETB were expressed at the tunica media of both varicose and control veins. The intensity and distribution of both receptors were significantly higher in the varicose veins compared with controls (Wilcoxon signed rank test, P<.0001; Fig. 1). When the expression of ET-1, ETA, and ETB receptors were evaluated in varicoceles of different grade, no statistically significant differences were present between groups (Kruskal-Wallis test, P¼.029). The side of the varicocele in bilateral cases did not affect ET-1 and its receptor expressions (Wilcoxon signed rank test, P>.05 in all cases). No statistically significant correlation was present between ET-1, ETA, and ETB expression and the parameters of age, sperm count, and motility (Spearman r 2, P>.05 in all cases). According to our hypothesis, an inherent endothelial malfunction due to aberrant ETA and ETB expression might be responsible for the decreased contractility of the internal spermatic veins, leading to gradual dilatation of the veins and the vicious circle of varicose formation. However, our results were strikingly against this hypothesis. Endothelin-1 and both its receptors ETA and ETB were significantly increased and not decreased, as anticipated, in varicose veins compared with controls. These findings are in contrast with previous reports on varicose veins of the lower limb, where a diminished ET-1 binding and ET receptor availability suggest a local reduction in receptor content (9, 12). Barber et al. (12) have proposed a possible down-regulation of the receptors due to primary ET-1 increased production. However, our results make this hypothesis of a primary insult at the receptor level highly questionable. A possible explanation of ET-1, ETA, and ETB overexpression in varicose spermatic veins may lie at the effect of shear stress on the endothelial function. Studies on cultured endothelial cells have documented the regulation of ET-1 release by shear stress (13, 14). Along the same lines, studies on vein graft disease have shown increased expression of ET-1 and its receptors in response to shear stress and mechanical stretching (15, 16). The increased ET-1 and ETA/ETB receptor production by the endothelium in response to high intraluminal pressure could explain our findings, providing that we accept that this is a secondary effect. Yildiz et al. (17) have proposed that increased venous pressure in the spermatic vein may actually precede the development of varicocele. This is highly probable, especially on left-sided varicoceles (17, 18). In our study, no differences in the immunohistochemical expression of ET-1 and its receptors were present when veins from both sides were compared in bilateral varicoceles. Nevertheless, independently of the primary cause, increased intraluminal pressure and shear stress may be the initial insult in the spermatic veins, leading to a secondary ET-1 receptor axis activation. The effect that hypoxia exerts on the endothelium might further explain our findings. Previous studies have suggested the role of hypoxia-activated endothelial cells in venous disease (19). Takahashi et al. (20) have reported that chronic hypoxia is associated with an increase of coexpression of ET1 and ETA/ETB receptors in pulmonary veins. Interestingly, the localization of ET-1 and its receptors in immunohistochemistry corresponds to the site where media thickening occurs in response to hypoxia. It is possible that the endothelium of varicose spermatic veins is subject to hypoxic conditions, mainly because of venous stasis, resulting in secondary endothelin axis activation. The secondary up-regulation of the endothelin axis could also validate the morphological changes observed in varicose spermatic veins. Several studies have documented the mitogenic properties of ET-1 on vascular smooth muscle cells. The ET-1 overexpression stimulates a proliferative response of smooth muscle cells both at the media and intima tunica, by ETA receptor binding, resulting in neointima formation and wall thickening (21). Accordingly, in our study, the varicose vein morphology showed distinct changes compared with normal vein structure, with media and intima thickening, focal hypertrophy, and neointima formation. Our findings are in accordance with other studies on the remodeling of the varicose spermatic vein (22, 23). To the best of our knowledge this is the first study to evaluate the endothelial axis in varicocele patients with an in situ immunohistochemical study. However, the literature is extremely limited; only one study available suggests elevated plasma endothelin levels in varicose spermatic veins (24). Thus, the present hypotheses are extrapolated from studies in lower limb, pulmonary, or interposition graft veins and should be viewed with caution. Another limitation may be the use of subcutaneous veins as controls. Spermatic veins from healthy subjects might be more appropriate. However, in our opinion this was the only ethically acceptable alternative in control tissue harvesting, which additionally allows the same subject to serve as an internal control, eliminating possible selection bias. In conclusion, contrary to the hypothesis that an intrinsic underexpression of the ET-1 axis at the varicose vein wall level may play a role in the development of varicocele, we observed an increase in ET-1 and ETA and ETB receptors in varicose veins. This is likely a secondary event and may at least partly interpret the morphological changes that characterize the varicose spermatic veins. Fertility and Sterility â 2555

3 REFERENCES 1. Misseri R, Gershbein AB, Horowitz M, Glassberg KI. The adolescent varicocele. II: the incidence of hydrocele and delayed recurrent varicocele after varicocelectomy in a long-term follow-up. BJU Int 2001;87: Marmar JL. The pathophysiology of varicoceles in the light of current molecular and genetic information. Hum Reprod Update 2001;7: Fujisawa M, Ishikawa T, Takenaka A. The efficacy of bilateral varicocelectomy in patients with palpable bilateral varicoceles: comparative study with unilateral varicocele. Urol Res 2003;31: Gat Y, Zukerman ZV, Bachar GN, Feldberg DO, Gornish M. Adolescent varicocele: is it a unilateral disease? Urology 2003;62:742 6 [discussion: 746 7]. 5. Carmignani G, Tedde G, De Stefani S, Montella A, Pirozzi-Farina F, Maffezzini M. Experimental varicocele in the rat a new experimental model. I. Effect on testicular structure. Urol Res 1983;11: Raman JD, Walmsley K, Goldstein M. Inheritance of varicoceles. Urology 2005;65: G okçe A, Davarci M, Yalçinkaya FR, G uven EO, Kaya YS, Helvaci MR, et al. Hereditary behavior of varicocele. J Androl 2010;31: Kiliç S, Aksoy Y, Sincer I, Oguz F, Erdil N, Yetkin E. Cardiovascular evaluation of young patients with varicocele. Fertil Steril 2007;88: Agu O, Hamilton G, Baker DM, Dashwood MR. Endothelin receptors in the aetiology and pathophysiology of varicose veins. Eur J Vasc Endovasc Surg 2002;23: Marmar JL, DeBenedictis TJ, Praiss D. The management of varicoceles by microdissection of the spermatic cord at the external inguinal ring. Fertil Steril 1985;43: Dubin L, Amelar RD. Varicocelectomy: 986 cases in a twelve-year study. Urology 1977;10: Barber DA, Wang X, Gloviczki P, Miller VM. Characterization of endothelin receptors in human varicose veins. J Vasc Surg 1997;26: Kuchan MJ, Frangos JA. Shear stress regulates endothelin-1 release via protein kinase C and cgmp in cultured endothelial cells. Am J Physiol 1993;264: Morawietz H, Talanow R, Szibor M, Rueckschloss U, Schubert A, Bartling B, et al. Regulation of the endothelin system by shear stress in human endothelial cells. J Physiol 2000;525(Pt 3): Dashwood MR, Mehta D, Izzat MB, Timm M, Bryan AJ, Angelini GD, et al. Distribution of endothelin-1 (ET) receptors (ET(A) and ET(B)) and immunoreactive ET-1 in porcine saphenous vein carotid artery interposition grafts. Atherosclerosis 1998;137: Gan LM, Selin-Sj ogren L, Doroudi R, Jern S. Temporal regulation of endothelial ET-1 and enos expression in intact human conduit vessels exposed to different intraluminal pressure levels at physiological shear stress. Cardiovasc Res 2000;48: Yildiz O, Gul H, Ozgok Y, Onguru O, Kilciler M, Aydin A, et al. Increased vasoconstrictor reactivity and decreased endothelial function in high grade varicocele; functional and morphological study. Urol Res 2003;31: Graif M, Hauser R, Hirshebein A, Botchan A, Kessler A, Yabetz H. Varicocele and the testicularrenal venous route: hemodynamic Doppler sonographic investigation. J Ultrasound Med 2000;19: Michiels C, Arnould T, Janssens D, Bajou K, Geron I, Remacle J. Interactions between endothelial cells and smooth muscle cells after their activation by hypoxia. A possible etiology for venous disease. Int Angiol 1996;15: Takahashi H, Soma S, Muramatsu M, Oka M, Fukuchi Y. Upregulation of ET-1 and its receptors and remodeling in small pulmonary veins under hypoxic conditions. Am J Physiol Lung Cell Mol Physiol 2001;280:L Yu JC, Davenport AP. Secretion of endothelin-1 and endothelin-3 by human cultured vascular smooth muscle cells. Br J Pharmacol 1995;114: Tanji N, Fujiwara T, Kaji H, Nishio S, Yokoyama M. Histologic evaluation of spermatic veins in patients with varicocele. Int J Urol 1999;6: Tilki D, Kilic E, Tauber R, Pfeiffer D, Stief CG, Tauber R, et al. The complex structure of the smooth muscle layer of spermatic veins and its potential role in the development of varicocele testis. Eur Urol 2007;51: Xu QQ, Zhu JC, Jiang H, Wang XF, Hou SK, Liu QL. Studies on plasma endothelin changes in varicocele patients. Asian J Androl 1999;1: Gyftopoulos et al. Correspondence Vol. 95, No. 8, June 30, 2011

4 SUPPLEMENTAL FIGURE 1 (A) Control vein stained poorly for endothelin 1 (ET-1). (B) Increased immunostaining for ET-1 in varicose vein. Note intima and media tunica thickening. ETA immunostaining in control vein (C) and in varicose vein (D). Analogous low ETB expression in control vein (E) compared with varicose veins (F). All microphotographs, 20. Fertility and Sterility â 2556.e1

5 SUPPLEMENTAL TABLE 1 Patient characteristics and semen parameters. Continuous variables Mean 5th 95th percentile Age Adults (y) Children (y) Semen parameters Volume (ml) Concentration (10 6 /ml) Motility at 1 hour, progressive (%) Motility at 3 hours, progressive (%) Morphology (% normal) Cases total (n) 55 Unilateral left (n, %) % Unilateral right (n, %) 1 1.8% Bilateral (n, %) % Grade 1 (n, %) % Grade 2 (n, %) % Grade 3 (n, %) % Note: Sperm motility was estimated at 1- and 3-hour intervals, as the sum of rapid and medium progressive motile sperm (World Health Organization type A þ B) e2 Gyftopoulos et al. Correspondence Vol. 95, No. 8, June 30, 2011