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EXPRESSION OF TGF-beta-1 mRNA AND ULTRASTRUCTURAL ALTERATIONS IN PHARMACOLOGICALLY INDUCED PROLONGED PENILE ERECTION IN A CANINE MODEL
0022-5347/98/1606-2263$03.00/0 THE JOURNAL OF UROLOGY Copyright 0 1998 by AMERICAN UROLOCICAL ASSOCIATION, INC
Val. 160, 2263-2266, December 1998 Printed in U S A .
EXPRESSION OF TGF-P-1 mRNA AND ULTRASTRUCTURAL ALTERATIONS IN PHARMACOLOGICALLY INDUCED PROLONGED PENILE ERECTION IN A CANINE MODEL MAQSOOD U L - W A N , AHMED I. EL-SAKKA, CELESTE LEE, TIEN-SZE B. YEN, RAJVIR DAHrYA AND TOM F. LUE* From the Department of Urology, Uniuersity of California, San Francisco, CA 94143-0738
ABSTRACT
Pupose: Transforming growth factor beta (TGF-P) is known to induce fibrosis. Our objective was to study the role of TGF-P as a possible mediator of fibrosis that may follow prolonged penile erection. Materials and Methods: Prolonged penile erection was induced in seven adult male mongrel dogs by intracavernosal injection of papaverine into one of the corpora cavernosa while the other was used as a control. Intracavernosal pressure measurements were carried out prior to administration of papaverine and a t the end of the procedure. Penile tissue was collected from anesthetized animals prior to euthanasia for histological and electron microscopic (EM) studies. RT-PCR was carried out for detection of mRNA on same tissue samples. Results: The light microscopy showed stasis of blood in the cavernosal sinusoids. EM studies revealed sporadic endothelial defects, loss of plasma membrane integrity and cytoplasmic condensation. There was expression of TGF-P1 mRNA in 66.7% of the experimental group compared with 16.7% of the control group. Conclusions: Pharmacologically induced low flow prolonged penile erection in canine models is associated with histomorphological changes in relatively short periods of time, suggesting that early therapeutic intervention is desirable. The gene expression for TGF-P1 may be a mediator of fibrosis; therefore the use of anti-TGF-p agents presents a possible tool for therapeutic intervention. KEY WOR~S:penile erection, fibrosis, growth factors, TGF-B Priapism is a sustained erection due to various disorders, including hematological disorders such as sickle cell disease, antidepressants such as trazodone, antipsychotics such as phenothiazines, and antihypertensives such as hydralazine and pra~ocine.l-~ Perineal or genital trauma may cause priapism as a result of blockage of venous drainage due to thrombosis, hemorrhage or edema (low flow priapism), or laceration of the cavernosal artery (high flow Intracavernosal administration of vasoactive drugs has also emerged as an important cause of p r i a p i ~ mThe . ~ underlying cause is not known in one third of patients and is considered to be related to adrenergic neurotransmitter dysfunction. Based on predominant hemodynamic pattern, which also reflects clinical outcome, priapism is categorized as low flow (ischemic) or high flow (nonischemic) priapism.' If ischemic priapism is allowed to persist, it results in trabecular edema and fibrosis, resulting in erectile dysfun~tion.~ The mediator of fibrosis is not known in priapism. Transforming growth factor beta (TGF-p1) is associated with several fibrotic conditions. We investigated its role as a possible mediator. TGF-pl was found to be upregulated at the transcriptional level. MATERIALS AND METHODS
Experimental animals. Penile erection was induced in seven adult mongrel dogs under endotracheal anesthesia lasting six to eight hours. The corpora cavernosa were exposed by midline incision proximally and a 21 gauge needle was inserted under vision for injecting papaverine and tak-
ing pressure measurements which were camed out in three animals. Forty-five milligrams of papaverine (King Pharmaceuticals, Inc., Bristol, TN) was injected into one of the corpora cavernosa of each animal while the other was used as a control. Systolic blood pressure was taken fifteen minutes before and fifteen minutes following intracavernosal injection. Penile tissue was collected just prior to sacrificing animals. Cavernosal tissue was examined histologically using eosin and hemotoxylin stain in addition to electron microscopic (EM) study. TGF-p1 gene expression was studied using RT-PCR technique. Transmission electron microscopy. The samples were immersion-fixed with 2.5% glutaraldehyde and 2.5% paraformaldedyde in 0.15 M sodium cacodylate buffer (pH 7.4). After post-fixation in 2% osmium tetroxide, the tissue was dehydrated in graded ethanol and propylene oxide, and subsequently embedded in Epon 812. Thick sections were cut on a Sorvall MT2-p microtome and stained with 1%toluidine blue. Thin sections (approximately 900 #A) were mounted on a 200 mesh copper grid and stained with uranyl acetate and lead citrate as a contrasting agent. Ultrastructure examination was performed with a Philips 400 transmission electron microscope. TGF-pl mRNA expression by RT-PC. To examine the expression of TGF-p1, total RNA was extracted from experimental and control cavernosal tissue as described earlier.l o In a 50 ml. reaction, 1 mg. of total RNA was precipitated and dried in a DEPC-treated thin-walled PCR reaction tube (Perkin Elmer, Foster City, CA) and resuspended in DEPCtreated water. The RNA was heated to 75C for 3 minutes and immediately placed on ice to remove secondary structure formation. The reverse transcription reaction was carried out
Accepted for publication June 12, 1998. * Requests for reprints:Department of Urology, University of California, San Francisco, CA 94143-0738. 2263
2264
TGF-P AND PENILE FIBROSIS
by adding 160 ng. random hexamer primers (Life Technologies, Gaithersurg, MD), 0.1 mM dNTP mixture (equal amounts dATP, dCTP, dGTP, dTTP, Perkin Elmer), 40 units RNasin ribonuclease inhibitor (Promega Corp. Madison, WI), and 200 units Superscript 11reverse transcriptase (Life Technologies) in 1X PCR buffer (10 mM Tris-HC1, 1.5 mM MgCt? and 50 mM KCI, pH 8.3, Perkin Elmer) as described earlier. PCR amplification. When all primers were added at the beginning of the reaction, amplification was linear over 35 cycles for TGF-P1 genes. The RT reaction (5 pl.) was amplified in a 50 p1. reaction using a thin-walled PCR reaction tube (Perkin Elmer) with 1X PCR Buffer, (10 mM Tris-HC1, 1.5 mM MgC12,50 mM KCI, pH 8.3, Perkin Elmer), using 2.5 units AmpliTaq polymerase (Perkin Elmer), 0.55 pg. TaqStart antibody (Clontech, Palo Alto, CA), 0.2 niM sense and antisense primers, 0.4 mM dNTP mixture (equal amounts of dATP, dGTP, dTTP, Perkin Elmer) and 1 u Ci(a32P) dCPT. PCR products were electrophoresed on a 4% polyacrylamide sequencing gel. The gel was dried and bands were visualized by exposing to x-ray film as described earlier.'" RESULTS
Pressure measurement studies. Preinjection systemic systolic blood pressure ranged from 105 to 112 mm. Hg with a mean of 109 mm. Hg. Fall in blood pressure was measured fifteen minutes after administration of intracavernosal papaverine injection. There was a fall in pressure from 7 to 10 mm. Hg (mean 9.6 mm. Hg). Systolic pressure at the end of procedure ranged from 90 t o 105 with a mean of 98.5 mm. Hg. Intracavernosal pressure measurements before injecting papaverine and at the end of procedure were 7.23 2 0.12, 14.84 +- 0.62, 11.52 +- 1.68 cm. water and 47.81 t 1.06, 73.81 5 4.95, 88.43 +- 4.84 cm. water respectively (intracavernous pressure was obtained in 3 dogs only). Histopathology and ultrastructural changes. Light microscopy showed stasis of blood in the cavernosal tissue (fig. 1, A, B).EM studies revealed sporadic endothelial defects, loss of plasma membrane integrity, and cytoplasmic condensation (fig. 2, A, B).The deposits on the endothelium and smooth muscle were possibly fibrin-like deposits. RT-PCR. TGF-P1 mRNA was expressed in 66.76 of the experimental group compared with 16.78 of the control group (fig. 3)
FIG. 1. A, corpus cavernosum. Control. Ma ification X100. B . corpus cavernosum. Priapism. Note stasis of good. Magnification x 100.
activity in the chicken, termed TGF-P4, is encoded by a gene homologous to mammalian TGF-Pl."" The first three subtypes are mammalian. TGF-PI, which corresponds to a classical form of TGF-p purified from human placenta, was charDISCUSSION acterized as a 2340 25-kDa protein composed of two In low flow priapism there is very little drainage of blood polypeptide chains held together by interchain disulfide linkTGF-p gene is located on human chromosome 19, a t from emissary veins and arterial flow is very low, with resultant ischemia which becomes painful if it lasts for four to region 19 q13.1-q13.3."' The action ofTGF-p on cell growth is six hours. Priapism has been shown to result in trabecular bifunctional. In general TGF-P stimulates proliferation of edema and fibrosis and various ultrastructural changes de- mesenchyme-derived cells but inhibits growth of epithelial pending on the degree and duration of ischemia.". l 3Priapism and endothelial cells. TGF-/3 plays an important role in tissue in our experimental model induced stagnation of blood and repair by transiently attracting fibroblasts to the wound site sporadic endothelial defects, loss of plasma membrane integ- and by stimulating synthesis of matrix molecules such as rity, and cytoplasmic condensation, suggesting that early fibronectin and collagen, ultimately leading to the incorporatherapeutic intervention is warranted. Smooth muscle cells tion of these compounds into the extracellular matrix." -:30 remained essentially normal following short term ischemia The integrins are receptors that mediate cell adhesion to the but priapism lasting for a longer time has been shown to extracellular matrix and basement membrane, to other cells, result in more pronounced ultrastuctural changes: the trans- and to plasma proteins. TGF-P increases expression of inteformation of smooth muscle cells into fibroblast-like cells and grins facilitating adhesion of cells to matrix."' Degradation of thrombus extracellular matrix is mediated by specific proteases, this TGFs were first detected in cells transformed in vitro by process being counterbalanced by protease inhibitors. TGF-P murine and feline sarcoma viruses and were named sarcoma decreases synthesis of proteases and increases levels of progrowth factor (SGF).14.'" It was shown later that SGF activ- tease inhibitor^.^',^^ ity is composed of two distinct factors, TGF-a and TGF-p.16 Ischemia is associated with TGF-P expression. In a rat TGF-P is present in normal and transformed cells and has model of focal brain ischemia, a significant increase of TGF-P been purified from human placenta, human platelets, bovine was observed in ischemic cortex and the TGF-B mRNA Drofile kidney, and other source^.^^-^^ TGF-P activity is represented paralleled the increased formation of extracellular m a t r i ~ . : ' ~ by a family of closely related peptides that are encoded by Global brain ischemia in adult rats and ischemic cardiovasduplicate genes.'2323Four distinct subtypes of TGF beta have cular accidents in humans are associated with TGF-P expresbeen described: TGF-131. TGF-132. TGF-B3. and T G F - B ~ ~An ; .- ~- -~ sion,35,36TGF-P upregulation is related t o several conditions ~~
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2265
TGF-P AND PENILE FIBROSIS
within nephritic glomeruli.”0 Decorin is particularly attractive because it is a natural substance produced in the human body and can be manufactured using recombinant gene technology. In the first three animals, intracavernous pressure was measured (and data given) every hour via a heparinized scalp vein needle placed in the corpus cavernosum. We found a gradual decrease of intracavernous pressure and a reduction of turgidity every time we let out some blood from the corpus cavernosum to measure pressure. A significant pressure drop was noted from the 6th to 8th hours i n two of the three dogs (85 to 48 and 90 to 74 cm. water respectively). Therefore, in the 4 remaining dogs, we decided to confirm priapism by palpation instead of measuring pressure by needle puncture. All 4 dogs seemed to remain turgid at 6 hours by palpation. However, some dogs may have lost some degree of erection at 6 hours which might explain why there was no TGF-P1 mRNA expression in lanes 7 and 10 in fig. 3. Regarding whether this is a true priapism, obviously the pressure and turgidity did not return to baseline and by definition, it is a prolonged erection. We are not sure of the pressure criterion of priapism in the canine corpus cavernosum. From this study, we conclude that a pharmacologicallyinduced low-flow prolonged erection in a canine model is associated with histomorphological changes in relatively short periods of time, suggesting that early therapeutic intervention is desirable. There is also gene expression for TGF-P, which may be a mediator of fibrosis. Anti-TGF-p agents may be a possible tool for therapeutic intervention. FIG.2. A, corpus cavernosum, endothelial lining. Control. Note normal endothelium. Magnification X 15100. B , corpus cavernosum, endothelial lining. Priapism. Note endothelial defects (arrows), loss of plasma membrane integrity, and cytoplasmic condensation. Magnification ~ 2 2 5 0 0 .
1. Tarry, W. F., Duckett, J. W., Jr. and Synder, H., 111: Urological
TGF-61 161 bp
J. Urol., 138 592, 1987. 2. Saenz de Tejada, I., Ware, J. C., Blanco, R., Pittard, J. T., Nadig, P. W. and Azadzoi, K. M.: Pathophysiology of prolonged penile erection is associated with trazodone use. J. Urol., 1 4 5 601,
1
2
3
4 5 6 7 8
910111213
1-6 Control; 7-12:prolonged penile erection; 13: TGB- p1 negative standard FIG. 3. RT-PCR showed expression of TGF-Pl in four out of six dogs in priapism and one out of six dogs in control.
with abnormal fibrosis: immune mediated mesangioproliferative glomerulonephritis induced by antithymocyte serum (ATS-GN) and chronic glomerulosclerosis in a rat model, alcoholic liver diseases and active cirrhosis of various origins, idiopathic pulmonary fibrosis, and Peyronie’s disease i n humans,:37-43 Azadzoi e t al have shown increased deposition of connective tissue in the corpora cavernosa following experimentally induced atherosclerosis, leading to cavernosal ischemia associated with abnormal veno-occlusive function. They have also demonstrated elevated levels of TGF-P1 and prostaglandins in cavernosal tissue as a result of ischemia due to the atherosclerosis modulating nitric oxide pathway i n a n animal model.““.”“ The ability of TGF-P to induce its own production may be the key to the development of scarring and fibrosis into chronic, progressive conditions.46 Although TGF-P plays a n essential role in several physiologic functions including wound healing, regulatory dysfunction or excessive action is undesirable. Therefore i t i s the target of therapeutic intervention with various modalities. TGF-P neutralizing antibodies successfully suppressed excessive extracellular matrix accumulation in vivo in t h e kidney, in the skin, and in central nervous system Decorin, a proteoglycan was as effective a s a neutralizing antibody in preventing extracellular matrix deposition
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TGF-j3 AND PENILE FIBROSIS
sarcoma virus-transformed cells. Roc. Natl. Acad. Sci. USA, 75 4001, 1978. 16. Anzano. M. A,. Robert. A. B.. Smith, J . M., Sporn, M. B. and _. DeLarco, J. E.: Sarcoma growth factor from conditioned medium of virally transformed cells is composed of both type alpha and type beta transforming growth factor. Proc. Natl. Acad. Sci. USA, 80: 6264, 1983. 17. Kryceve-Martinerie, C., Lawrence, D. A,, Crochet, J., Jullien, P.and Vgier, P.: Further study of TGF betas released by virally transformed and non-transformed cells. Int. J . Cancer, 35: 553, 1985. 18. Frolik, C. A., Dart, L. L., Meyers, C. A., Smith, D. M. and Sporn, M. B.: Purification and initial characterization of a type beta transforming growth factor from human placenta. Proc. Natl. Acad. Sci. USA, 80: 3676, 1983. 19. Roberts, A. B., Anzano, M. A,, Meyers, C. A., Wideman, J., Blacher, R., Pan, Y., Stein, C. E., Lehrman, S. R., Smith, L. M., Lamb, L. C. and Sporn, B. M.: Purification and properties of a type beta transforming growth factor from bovine kidney. Biochemistry, 2 2 5692, 1983. 20. Assoian, P. K., Komoria, A,, Meyers, C. A,, Miller, D. M. and Sporn, M. B.: Transforming growth factor beta in human platelets: identification of major site, purification and characterization. J . Biol. Chem., 3 3 95, 1987. 21. Keski-Oja, J., Leof, E. B., Lyons, R. M., Coffey, R. J., J r . and Moses, H. L.: Transforming growth factors and control of neoplastic growth. J . Cell. Biochem., 33:95, 1987. 22. Massague, J.: The transforming growth factor-/3 family. Ann. Rev. Cell. Biol., 6 597, 1990. 23. Massague J.: The TGF-/3 family of growth and differentiating factors. Cell, 4 9 437, 1987. 24. Burt, D. W. and Paton, I. R.: Evolutionary origin of transforming growth factor beta family. DNA Cell. Biol., 11: 497, 1992. 25. Jakowlew, S. B., Dillard, P. G., Sporn, M. B. and Roberts, A. B.: Complementary deoxyribonucleic acid cloning of a messenger ribonucleic acid encoding transforming growth factor beta-4 from chick embryo chondrocyte. Mol. Endocrinol., 2 1186, 1988. 26. Sporn, M. B., Robert, A. B., Wakefield, L. M. and Assoian, R. K.: Transforming growth factor beta biological function and chemical structure. Science, 233: 532, 1986. 27. Fujii, D., Brissenden, J. E., Derynck, R. and Francke, U.: Transforming growth factor beta gene maps to human chromosome 19 long arm and to mouse chromosome 7. Somatic Cell. Mol. Genet., 12 281, 1986. 28. Leof, E. B., Proper, J . A., Gets, M. J . and Moses, H. L.: Transforming growth factor type beta regulation of actin mRNA. J. Cell. Physiol., 127:83, 1986. 29. Ignotz, R. A. and Massague, J.: Transforming growth factor beta stimulates the expression of fibronectin and collagen and their incorporation into the extracellular matrix. J . Biol. Chem., 261: 4337, 1986. 30. Penntinen, R. P., Kobayashi, S. and Bornstein, P.: Transforming growth factor beta increase mRNA for matrix proteins in the presence and the absence of mRNA stability. Proc. Natl. Acad. Sci. USA, 8 5 1105, 1988,. 31. Ignotz, R. A. and Massague, J.: Cell adhesion protein receptors as target for transforming growth factor beta action. Cell, 51: 189, 1987. 32. Edward, D. R. G., Murphy, J. J., Renalds, S. E., Withham, A. J. P., Docherty, P. A. and Heath, J . K.: Transforminggrowth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. J. Eur. Mol. Biol. Organ., 6 1899, 1987. 33. Laiho, M. O., Saksela, K-0. J.: Transforming growth factor beta induction of type 1 plasminogen activator inhibitor. J . Biol. ~~
Chem., 262 17467,1987. 34. Transforming growth factor beta 1exhibits delayed gene expression following focal cerebral ischemia. Brain Res. Bull., 36: 607, 1995. 35. Wiessner, C., Gerhmann, J., Lindholm, D., Topper, R.. Kreurtzberg. G. W. and Hossman, K. A.: Expression of transforming growth factor beta 1 and interleukin 1 niRNA in rat brain following transient forebrain ischemia. Acta Neuropathol., 86: 439, 1993. 36. b p i n s k i , J., Kumar, P., Kumar, S. and Maluza, J . : Increased expression of transforming growth factor beta 1in brain tissue after ischemic stroke. Stroke, 27: 852, 1996. 37. Okuda, S., Languino, L. R., Ruoslahti. E. and Border, W. A,: Elevated expression of transforming growth factor beta and proteoglycan production in experimental glomerulonephritis. J. Clin. Invest., 86: 453, 1990. 38. Yamamoto, Y. and Wilson, C. B.: Qualitative and quantitative studies of antibody induced mesangial cell damage in the rat. Kidney Int., 3 2 514, 1987. 39. Yamamoto, T., Noble, N. A,, Miller, D. E. and Border, W. A,: Sustained expression of TGF beta 1 underlies development of progressive kidney fibrosis. Kidney Int., 4 5 916, 1994. 40. Annoni, G., Weiner, S. and Zern, M. A,: Increased transforming growth factor beta 1 expression in human liver disease. J . Hepatol., 14: 259, 1992. 41. Broekelmann, T. J., Limper, A. H., Colby, T. V. and McDonald, J. A,, Transforming growth factor beta 1 is present a t sites of extracellular matrix gene expression in human pulmonary fibrosis. Proc. Natl. Acad. Sci. USA, 88: 6642, 1991. 42. JShalil, N., OConnor, R. N., Unruh, H. W., Warren, P. W., Flanders, K. C., Kamp, A., Bereznay, 0. H. and Greenberg, A. H.: Increased production and immunohistochemical localization of transforming growth factor beta 1 in idiopathic pulmonary fibrosis. Am. J. Respir. Cell. Mol. Biol., 5 155, 1991. 43. El-Sakka, A. I., Hassoba, H. M., F’illarisetty, R. J., Nunes, L., Dahiya, R. and Lue, T. F.: Peyronie’s disease is associated with an increase in transforming growth factor beta protein expression. J . Urol. (Suppl.), 157: 361, 1997. 44. Azadzoi, K. M., Park, K., Goldstein, I. and Siroky, M. B.: Relationship between cavernosal ischemia and corporal venoocclusive dysfunction in an animal model. J . Urol., 157: 1011, 1997. 45. Azadzoi, K. M., Krane, R. J., Goldstein, I. and Siroky. M. B.: Ischemia induced alteration in cavernosal eicosanoids and TGF-P1 impairs nitric oxide pathway and relaxation of cavernosal tissue. Int. J . Impotence Res., 8 107, 1996. 46. Border, W. A. and Rouslahti, E.: Transforming growth factor beta in diseases: the dark side of tissue repair. J. Clin. Invcst.. 90.1, 1992. 47. Border, W, A., Okuda, S., Languino, L. R., Sporn, M. B. and Rouslahti, E.: Suppression of experimental glomcruloncphritis by antiserum against transforming growth factor beta 1. Nature (Lond. ), 346 371, 1990. 48. Shah, M., Foerman, D. M. and Ferguson, W. J . : Control of scarring in adult wounds by neutralizing antibody to transforming growth factor beta. Lancet, 339 213, 1992. 49. Logan, A., Berry, M., Gonzales, A. M., Frautschy, S. A,, Sporn, M. B. and Baird, A.: Effects of transforming growth factor beta 1 on scar production in the central nervous system of the rat. Eur. J. Neurosci., 6: 355, 1994. 50. Boarder, W. A., Noble, N. A., Yamamoto, T., Harper, J. R., Yamaguchi, Y., Pierschbacher, M. D. and Rouslahti, E.: Natural inhibitor of transforming growth factor beta protects against scarring in experimentalkidney disease. Nature, 360: 361, 1992.
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Val. 160, 2263-2266, December 1998 Printed in U S A .
EXPRESSION OF TGF-P-1 mRNA AND ULTRASTRUCTURAL ALTERATIONS IN PHARMACOLOGICALLY INDUCED PROLONGED PENILE ERECTION IN A CANINE MODEL MAQSOOD U L - W A N , AHMED I. EL-SAKKA, CELESTE LEE, TIEN-SZE B. YEN, RAJVIR DAHrYA AND TOM F. LUE* From the Department of Urology, Uniuersity of California, San Francisco, CA 94143-0738
ABSTRACT
Pupose: Transforming growth factor beta (TGF-P) is known to induce fibrosis. Our objective was to study the role of TGF-P as a possible mediator of fibrosis that may follow prolonged penile erection. Materials and Methods: Prolonged penile erection was induced in seven adult male mongrel dogs by intracavernosal injection of papaverine into one of the corpora cavernosa while the other was used as a control. Intracavernosal pressure measurements were carried out prior to administration of papaverine and a t the end of the procedure. Penile tissue was collected from anesthetized animals prior to euthanasia for histological and electron microscopic (EM) studies. RT-PCR was carried out for detection of mRNA on same tissue samples. Results: The light microscopy showed stasis of blood in the cavernosal sinusoids. EM studies revealed sporadic endothelial defects, loss of plasma membrane integrity and cytoplasmic condensation. There was expression of TGF-P1 mRNA in 66.7% of the experimental group compared with 16.7% of the control group. Conclusions: Pharmacologically induced low flow prolonged penile erection in canine models is associated with histomorphological changes in relatively short periods of time, suggesting that early therapeutic intervention is desirable. The gene expression for TGF-P1 may be a mediator of fibrosis; therefore the use of anti-TGF-p agents presents a possible tool for therapeutic intervention. KEY WOR~S:penile erection, fibrosis, growth factors, TGF-B Priapism is a sustained erection due to various disorders, including hematological disorders such as sickle cell disease, antidepressants such as trazodone, antipsychotics such as phenothiazines, and antihypertensives such as hydralazine and pra~ocine.l-~ Perineal or genital trauma may cause priapism as a result of blockage of venous drainage due to thrombosis, hemorrhage or edema (low flow priapism), or laceration of the cavernosal artery (high flow Intracavernosal administration of vasoactive drugs has also emerged as an important cause of p r i a p i ~ mThe . ~ underlying cause is not known in one third of patients and is considered to be related to adrenergic neurotransmitter dysfunction. Based on predominant hemodynamic pattern, which also reflects clinical outcome, priapism is categorized as low flow (ischemic) or high flow (nonischemic) priapism.' If ischemic priapism is allowed to persist, it results in trabecular edema and fibrosis, resulting in erectile dysfun~tion.~ The mediator of fibrosis is not known in priapism. Transforming growth factor beta (TGF-p1) is associated with several fibrotic conditions. We investigated its role as a possible mediator. TGF-pl was found to be upregulated at the transcriptional level. MATERIALS AND METHODS
Experimental animals. Penile erection was induced in seven adult mongrel dogs under endotracheal anesthesia lasting six to eight hours. The corpora cavernosa were exposed by midline incision proximally and a 21 gauge needle was inserted under vision for injecting papaverine and tak-
ing pressure measurements which were camed out in three animals. Forty-five milligrams of papaverine (King Pharmaceuticals, Inc., Bristol, TN) was injected into one of the corpora cavernosa of each animal while the other was used as a control. Systolic blood pressure was taken fifteen minutes before and fifteen minutes following intracavernosal injection. Penile tissue was collected just prior to sacrificing animals. Cavernosal tissue was examined histologically using eosin and hemotoxylin stain in addition to electron microscopic (EM) study. TGF-p1 gene expression was studied using RT-PCR technique. Transmission electron microscopy. The samples were immersion-fixed with 2.5% glutaraldehyde and 2.5% paraformaldedyde in 0.15 M sodium cacodylate buffer (pH 7.4). After post-fixation in 2% osmium tetroxide, the tissue was dehydrated in graded ethanol and propylene oxide, and subsequently embedded in Epon 812. Thick sections were cut on a Sorvall MT2-p microtome and stained with 1%toluidine blue. Thin sections (approximately 900 #A) were mounted on a 200 mesh copper grid and stained with uranyl acetate and lead citrate as a contrasting agent. Ultrastructure examination was performed with a Philips 400 transmission electron microscope. TGF-pl mRNA expression by RT-PC. To examine the expression of TGF-p1, total RNA was extracted from experimental and control cavernosal tissue as described earlier.l o In a 50 ml. reaction, 1 mg. of total RNA was precipitated and dried in a DEPC-treated thin-walled PCR reaction tube (Perkin Elmer, Foster City, CA) and resuspended in DEPCtreated water. The RNA was heated to 75C for 3 minutes and immediately placed on ice to remove secondary structure formation. The reverse transcription reaction was carried out
Accepted for publication June 12, 1998. * Requests for reprints:Department of Urology, University of California, San Francisco, CA 94143-0738. 2263
2264
TGF-P AND PENILE FIBROSIS
by adding 160 ng. random hexamer primers (Life Technologies, Gaithersurg, MD), 0.1 mM dNTP mixture (equal amounts dATP, dCTP, dGTP, dTTP, Perkin Elmer), 40 units RNasin ribonuclease inhibitor (Promega Corp. Madison, WI), and 200 units Superscript 11reverse transcriptase (Life Technologies) in 1X PCR buffer (10 mM Tris-HC1, 1.5 mM MgCt? and 50 mM KCI, pH 8.3, Perkin Elmer) as described earlier. PCR amplification. When all primers were added at the beginning of the reaction, amplification was linear over 35 cycles for TGF-P1 genes. The RT reaction (5 pl.) was amplified in a 50 p1. reaction using a thin-walled PCR reaction tube (Perkin Elmer) with 1X PCR Buffer, (10 mM Tris-HC1, 1.5 mM MgC12,50 mM KCI, pH 8.3, Perkin Elmer), using 2.5 units AmpliTaq polymerase (Perkin Elmer), 0.55 pg. TaqStart antibody (Clontech, Palo Alto, CA), 0.2 niM sense and antisense primers, 0.4 mM dNTP mixture (equal amounts of dATP, dGTP, dTTP, Perkin Elmer) and 1 u Ci(a32P) dCPT. PCR products were electrophoresed on a 4% polyacrylamide sequencing gel. The gel was dried and bands were visualized by exposing to x-ray film as described earlier.'" RESULTS
Pressure measurement studies. Preinjection systemic systolic blood pressure ranged from 105 to 112 mm. Hg with a mean of 109 mm. Hg. Fall in blood pressure was measured fifteen minutes after administration of intracavernosal papaverine injection. There was a fall in pressure from 7 to 10 mm. Hg (mean 9.6 mm. Hg). Systolic pressure at the end of procedure ranged from 90 t o 105 with a mean of 98.5 mm. Hg. Intracavernosal pressure measurements before injecting papaverine and at the end of procedure were 7.23 2 0.12, 14.84 +- 0.62, 11.52 +- 1.68 cm. water and 47.81 t 1.06, 73.81 5 4.95, 88.43 +- 4.84 cm. water respectively (intracavernous pressure was obtained in 3 dogs only). Histopathology and ultrastructural changes. Light microscopy showed stasis of blood in the cavernosal tissue (fig. 1, A, B).EM studies revealed sporadic endothelial defects, loss of plasma membrane integrity, and cytoplasmic condensation (fig. 2, A, B).The deposits on the endothelium and smooth muscle were possibly fibrin-like deposits. RT-PCR. TGF-P1 mRNA was expressed in 66.76 of the experimental group compared with 16.78 of the control group (fig. 3)
FIG. 1. A, corpus cavernosum. Control. Ma ification X100. B . corpus cavernosum. Priapism. Note stasis of good. Magnification x 100.
activity in the chicken, termed TGF-P4, is encoded by a gene homologous to mammalian TGF-Pl."" The first three subtypes are mammalian. TGF-PI, which corresponds to a classical form of TGF-p purified from human placenta, was charDISCUSSION acterized as a 2340 25-kDa protein composed of two In low flow priapism there is very little drainage of blood polypeptide chains held together by interchain disulfide linkTGF-p gene is located on human chromosome 19, a t from emissary veins and arterial flow is very low, with resultant ischemia which becomes painful if it lasts for four to region 19 q13.1-q13.3."' The action ofTGF-p on cell growth is six hours. Priapism has been shown to result in trabecular bifunctional. In general TGF-P stimulates proliferation of edema and fibrosis and various ultrastructural changes de- mesenchyme-derived cells but inhibits growth of epithelial pending on the degree and duration of ischemia.". l 3Priapism and endothelial cells. TGF-/3 plays an important role in tissue in our experimental model induced stagnation of blood and repair by transiently attracting fibroblasts to the wound site sporadic endothelial defects, loss of plasma membrane integ- and by stimulating synthesis of matrix molecules such as rity, and cytoplasmic condensation, suggesting that early fibronectin and collagen, ultimately leading to the incorporatherapeutic intervention is warranted. Smooth muscle cells tion of these compounds into the extracellular matrix." -:30 remained essentially normal following short term ischemia The integrins are receptors that mediate cell adhesion to the but priapism lasting for a longer time has been shown to extracellular matrix and basement membrane, to other cells, result in more pronounced ultrastuctural changes: the trans- and to plasma proteins. TGF-P increases expression of inteformation of smooth muscle cells into fibroblast-like cells and grins facilitating adhesion of cells to matrix."' Degradation of thrombus extracellular matrix is mediated by specific proteases, this TGFs were first detected in cells transformed in vitro by process being counterbalanced by protease inhibitors. TGF-P murine and feline sarcoma viruses and were named sarcoma decreases synthesis of proteases and increases levels of progrowth factor (SGF).14.'" It was shown later that SGF activ- tease inhibitor^.^',^^ ity is composed of two distinct factors, TGF-a and TGF-p.16 Ischemia is associated with TGF-P expression. In a rat TGF-P is present in normal and transformed cells and has model of focal brain ischemia, a significant increase of TGF-P been purified from human placenta, human platelets, bovine was observed in ischemic cortex and the TGF-B mRNA Drofile kidney, and other source^.^^-^^ TGF-P activity is represented paralleled the increased formation of extracellular m a t r i ~ . : ' ~ by a family of closely related peptides that are encoded by Global brain ischemia in adult rats and ischemic cardiovasduplicate genes.'2323Four distinct subtypes of TGF beta have cular accidents in humans are associated with TGF-P expresbeen described: TGF-131. TGF-132. TGF-B3. and T G F - B ~ ~An ; .- ~- -~ sion,35,36TGF-P upregulation is related t o several conditions ~~
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2265
TGF-P AND PENILE FIBROSIS
within nephritic glomeruli.”0 Decorin is particularly attractive because it is a natural substance produced in the human body and can be manufactured using recombinant gene technology. In the first three animals, intracavernous pressure was measured (and data given) every hour via a heparinized scalp vein needle placed in the corpus cavernosum. We found a gradual decrease of intracavernous pressure and a reduction of turgidity every time we let out some blood from the corpus cavernosum to measure pressure. A significant pressure drop was noted from the 6th to 8th hours i n two of the three dogs (85 to 48 and 90 to 74 cm. water respectively). Therefore, in the 4 remaining dogs, we decided to confirm priapism by palpation instead of measuring pressure by needle puncture. All 4 dogs seemed to remain turgid at 6 hours by palpation. However, some dogs may have lost some degree of erection at 6 hours which might explain why there was no TGF-P1 mRNA expression in lanes 7 and 10 in fig. 3. Regarding whether this is a true priapism, obviously the pressure and turgidity did not return to baseline and by definition, it is a prolonged erection. We are not sure of the pressure criterion of priapism in the canine corpus cavernosum. From this study, we conclude that a pharmacologicallyinduced low-flow prolonged erection in a canine model is associated with histomorphological changes in relatively short periods of time, suggesting that early therapeutic intervention is desirable. There is also gene expression for TGF-P, which may be a mediator of fibrosis. Anti-TGF-p agents may be a possible tool for therapeutic intervention. FIG.2. A, corpus cavernosum, endothelial lining. Control. Note normal endothelium. Magnification X 15100. B , corpus cavernosum, endothelial lining. Priapism. Note endothelial defects (arrows), loss of plasma membrane integrity, and cytoplasmic condensation. Magnification ~ 2 2 5 0 0 .
1. Tarry, W. F., Duckett, J. W., Jr. and Synder, H., 111: Urological
TGF-61 161 bp
J. Urol., 138 592, 1987. 2. Saenz de Tejada, I., Ware, J. C., Blanco, R., Pittard, J. T., Nadig, P. W. and Azadzoi, K. M.: Pathophysiology of prolonged penile erection is associated with trazodone use. J. Urol., 1 4 5 601,
1
2
3
4 5 6 7 8
910111213
1-6 Control; 7-12:prolonged penile erection; 13: TGB- p1 negative standard FIG. 3. RT-PCR showed expression of TGF-Pl in four out of six dogs in priapism and one out of six dogs in control.
with abnormal fibrosis: immune mediated mesangioproliferative glomerulonephritis induced by antithymocyte serum (ATS-GN) and chronic glomerulosclerosis in a rat model, alcoholic liver diseases and active cirrhosis of various origins, idiopathic pulmonary fibrosis, and Peyronie’s disease i n humans,:37-43 Azadzoi e t al have shown increased deposition of connective tissue in the corpora cavernosa following experimentally induced atherosclerosis, leading to cavernosal ischemia associated with abnormal veno-occlusive function. They have also demonstrated elevated levels of TGF-P1 and prostaglandins in cavernosal tissue as a result of ischemia due to the atherosclerosis modulating nitric oxide pathway i n a n animal model.““.”“ The ability of TGF-P to induce its own production may be the key to the development of scarring and fibrosis into chronic, progressive conditions.46 Although TGF-P plays a n essential role in several physiologic functions including wound healing, regulatory dysfunction or excessive action is undesirable. Therefore i t i s the target of therapeutic intervention with various modalities. TGF-P neutralizing antibodies successfully suppressed excessive extracellular matrix accumulation in vivo in t h e kidney, in the skin, and in central nervous system Decorin, a proteoglycan was as effective a s a neutralizing antibody in preventing extracellular matrix deposition
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