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Christian G. Stief - One of the best experts on this subject based on the ideXlab platform.
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isolation of primary endothelial and stromal cell cultures of the Corpus Cavernosum Penis for basic research and tissue engineering
European Urology, 2005Co-Authors: Adrian Pilatz, Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Heike Mertsching, U. Jonas, Christian G. StiefAbstract:Abstract Objectives: Primary cell cultures derived from the Corpus Cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human Penis. Methods: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. Results: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0±0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. Conclusion: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.
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cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human Corpus Cavernosum Penis
Urology, 2004Co-Authors: Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Heike Mertsching, Michael Sohn, U. Jonas, A Pilatz, Christian G. StiefAbstract:Abstract Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1 ), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the Corpus Cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the Corpus Cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1 , 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1 . Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the Corpus Cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.
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Androgen and estrogen receptors in the human Corpus Cavernosum Penis: immunohistochemical and cell culture results
World Journal of Urology, 2003Co-Authors: Dirk Schultheiss, Rafael Badalyan, Adrian Pilatz, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Reinhard Wasielewski, Heike Mertsching, Michael Sohn, Christian G. StiefAbstract:Despite the central and peripheral effects of androgens on the nervous system, the local effects of androgens in the Corpus Cavernosum Penis and their importance for erectile function is still unclear. In this study Corpus Cavernosum biopsies of eight adult potent patients, aged 19–63 years, undergoing penile deviation surgery (group A) and 12 patients undergoing male-to-female transsexual surgery (group B) were immunostained for nuclear androgen and estrogen-alpha receptors. Additionally, primary Corpus Cavernosum endothelial cell cultures were obtained from six transsexual patients and exposed to testosterone, dihydrotestosterone, estradiol and progesterone likewise for 7 days. Total cell count was performed and cell metabolic activity was measured by a tetrazolium salt-based assay. Androgen and estrogen-alpha receptors were detected in stromal as well as in endothelial cells. Of all cell nuclei, 74.9% (SD 16.4) in group A and 63.5% (SD 17.1) in group B were positively stained for androgen receptors. The respective percentage of estrogen receptors was 11% (SD 9.5) and 21.2% (SD 12.6). An age-dependent difference in receptor distribution was not observed in either group. In the cell culture system only cultures exposed to testosterone and dihydrotestosterone showed a dose-dependent increase of cell metabolic activity compared to the cultures supplemented with estradiol and progesterone. The significant and age-independent high androgen and low estrogen-alpha receptor distribution found in both groups suggests a possible peripheral effect of androgens at the level of the Corpus Cavernosum Penis in adult humans. This is supported by the observed effect of testosterone and dihydrotestosterone on cell count and endothelial cell metabolism in our cell culture system. The role of estrogens remains unclear.
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gene expression of the phosphodiesterases 3a and 5a in human Corpus Cavernosum Penis
European Urology, 2000Co-Authors: Andrea Kuthe, Christian G. Stief, Hansjurgen Magert, Stefan Uckert, W G Forssmann, U. JonasAbstract:Objective: The following study was performed to evaluate the importance of phosphodiesterases 3A (PDE3A) and 5A (PDE5A) for the regulation of penile smooth muscle tone. Furthermore,
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functional electromyostimulation of the Corpus Cavernosum Penis preliminary results of a novel therapeutic option for erectile dysfunction
World Journal of Urology, 1995Co-Authors: Christian G. Stief, T. Noack, M. Djamilian, M. Meschi, M. Truss, Edgar Dr Weller, U. JonasAbstract:Transcutaneous application of low-frequency electric current in the treatment of partially or temporarily denervated striated muscles is widely used to prevent or treat muscular atrophy. Due to the high regenerative capacity of smooth-muscle cells, this approach should be beneficial in the treatment of diseases with smooth-muscle degeneration due to partial denervation. Our study was done to evaluate the possible beneficial effect of transcutaneously applied low-frequency electric current on the Corpus Cavernosum Penis in the treatment of erectile dysfunction. After a comprehensive workup, 21 patients with chronic erectile dysfunction (20/21 vasoactive nonresponders) received daily (3–5×20 min) transcutaneous functional electromyostimulation of the Corpus Cavernosum smooth muscles [FEMCC; zero-line symmetric impulses of trapezoid shape, 2-channel device with alternating stimulations; frequency (f), 10–20 Hz for channel I and 20–35 Hz for channel II; impulse duration (ti), 100–150 μs; approx. 12 mA; rise time, 0.5 s; stimulation time, 5 s/channel; pause between stimulations, 0.5 s. In all, 4/21 patients (19%) regained full spontaneous erections and another 3/21 (14%) responded to vasoactive drugs after FEMCC. Overall, 14/21 were FEMCC failures, including 2 who subjectively “improved.” In a similar group of patients who were evaluated during the same period but received no therapy, no spontaneous improvement in erectile function was observed. Our preliminary findings suggest that FEMCC is feasible and results in an improvement in erectile capacity in a significant number (33%) of patients. Further studies will be carried out to corroborate our results, to improve the stimulation parameters, and to evaluate the selection criteria for patients suitable for FEMCC.
U. Jonas - One of the best experts on this subject based on the ideXlab platform.
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isolation of primary endothelial and stromal cell cultures of the Corpus Cavernosum Penis for basic research and tissue engineering
European Urology, 2005Co-Authors: Adrian Pilatz, Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Heike Mertsching, U. Jonas, Christian G. StiefAbstract:Abstract Objectives: Primary cell cultures derived from the Corpus Cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human Penis. Methods: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. Results: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0±0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. Conclusion: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.
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cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human Corpus Cavernosum Penis
Urology, 2004Co-Authors: Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Heike Mertsching, Michael Sohn, U. Jonas, A Pilatz, Christian G. StiefAbstract:Abstract Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1 ), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the Corpus Cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the Corpus Cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1 , 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1 . Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the Corpus Cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.
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gene expression of the phosphodiesterases 3a and 5a in human Corpus Cavernosum Penis
European Urology, 2000Co-Authors: Andrea Kuthe, Christian G. Stief, Hansjurgen Magert, Stefan Uckert, W G Forssmann, U. JonasAbstract:Objective: The following study was performed to evaluate the importance of phosphodiesterases 3A (PDE3A) and 5A (PDE5A) for the regulation of penile smooth muscle tone. Furthermore,
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Functional electromyostimulation of the Corpus Cavernosum Penis — preliminary results of a novel therapeutic option for erectile dysfunction
World Journal of Urology, 1995Co-Authors: C. G. Stief, E. Weller, T. Noack, M. Djamilian, M. Meschi, M. Truss, U. JonasAbstract:Transcutaneous application of low-frequency electric current in the treatment of partially or temporarily denervated striated muscles is widely used to prevent or treat muscular atrophy. Due to the high regenerative capacity of smooth-muscle cells, this approach should be beneficial in the treatment of diseases with smooth-muscle degeneration due to partial denervation. Our study was done to evaluate the possible beneficial effect of transcutaneously applied low-frequency electric current on the Corpus Cavernosum Penis in the treatment of erectile dysfunction. After a comprehensive workup, 21 patients with chronic erectile dysfunction (20/21 vasoactive nonresponders) received daily (3–5×20 min) transcutaneous functional electromyostimulation of the Corpus Cavernosum smooth muscles [FEMCC; zero-line symmetric impulses of trapezoid shape, 2-channel device with alternating stimulations; frequency ( f ), 10–20 Hz for channel I and 20–35 Hz for channel II; impulse duration ( t _ i ), 100–150 μs; approx. 12 mA; rise time, 0.5 s; stimulation time, 5 s/channel; pause between stimulations, 0.5 s. In all, 4/21 patients (19%) regained full spontaneous erections and another 3/21 (14%) responded to vasoactive drugs after FEMCC. Overall, 14/21 were FEMCC failures, including 2 who subjectively “improved.” In a similar group of patients who were evaluated during the same period but received no therapy, no spontaneous improvement in erectile functïon was observed. Our preliminary findings suggest that FEMCC is feasible and results in an improvement in erectile capacity in a significant number (33%) of patients. Further studies will be carried out to corroborate our results, to improve the stimulation parameters, and to evaluate the selection criteria for patients suitable for FEMCC.
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functional electromyostimulation of the Corpus Cavernosum Penis preliminary results of a novel therapeutic option for erectile dysfunction
World Journal of Urology, 1995Co-Authors: Christian G. Stief, T. Noack, M. Djamilian, M. Meschi, M. Truss, Edgar Dr Weller, U. JonasAbstract:Transcutaneous application of low-frequency electric current in the treatment of partially or temporarily denervated striated muscles is widely used to prevent or treat muscular atrophy. Due to the high regenerative capacity of smooth-muscle cells, this approach should be beneficial in the treatment of diseases with smooth-muscle degeneration due to partial denervation. Our study was done to evaluate the possible beneficial effect of transcutaneously applied low-frequency electric current on the Corpus Cavernosum Penis in the treatment of erectile dysfunction. After a comprehensive workup, 21 patients with chronic erectile dysfunction (20/21 vasoactive nonresponders) received daily (3–5×20 min) transcutaneous functional electromyostimulation of the Corpus Cavernosum smooth muscles [FEMCC; zero-line symmetric impulses of trapezoid shape, 2-channel device with alternating stimulations; frequency (f), 10–20 Hz for channel I and 20–35 Hz for channel II; impulse duration (ti), 100–150 μs; approx. 12 mA; rise time, 0.5 s; stimulation time, 5 s/channel; pause between stimulations, 0.5 s. In all, 4/21 patients (19%) regained full spontaneous erections and another 3/21 (14%) responded to vasoactive drugs after FEMCC. Overall, 14/21 were FEMCC failures, including 2 who subjectively “improved.” In a similar group of patients who were evaluated during the same period but received no therapy, no spontaneous improvement in erectile function was observed. Our preliminary findings suggest that FEMCC is feasible and results in an improvement in erectile capacity in a significant number (33%) of patients. Further studies will be carried out to corroborate our results, to improve the stimulation parameters, and to evaluate the selection criteria for patients suitable for FEMCC.
Naoki Yasuda - One of the best experts on this subject based on the ideXlab platform.
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in vitro contraction of the canine Corpus Cavernosum Penis by direct perfusion with prolactin or growth hormone
The Journal of Urology, 1996Co-Authors: S Ra, Hikaru Aoki, Tomoaki Fujioka, Takashi Kubo, F Sato, Naoki YasudaAbstract:AbstractPurpose: It is well established that hyperprolactinemia, most typically seen in prolactinoma patients, causes hypogonadism and impotence. There seem to be a good possibility that hyperprolactinemia causes impotence, at least partially via some intrinsic property of prolactin (PRL), rather than through its suppressive effects on the hypothalamic-pituitary-gonadal testosterone dynamics. In the present investigation, we used an in vitro canine model to attempt to clarify whether direct action of PRL on the Corpus Cavernosum Penis may lead to erectile insufficiency. Growth hormone (GH) and placental lactogen (PL), both having close structural and functional homologies to PRL, were also studied.Materials and Methods: Isometric tension measurement with cavernous strips was performed in the presence or absence of 10 sup -5 to 10 sup -9 M. PRL, GH, or PL in the perfusion medium. The tension change induced by the test substances was normalized relative to that induced by 120 mEq KCl.Results: Both PRL and G...
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mechanisms of veno occlusion within and outside the canine Corpus Cavernosum Penis using a pressure flow technique and cavernoso venography
International Journal of Urology, 1996Co-Authors: Junichi Matsuzaka, Hikaru Aoki, Tomoaki Fujioka, Takashi Kubo, Naoki YasudaAbstract:Background: Physiological erection of the Penis requires multiple mechanisms causing an increase in the arterial blood influx into, and decrease in the venous drainage out of the cavernous space. Methods: We investigated the extent and location of the venous occlusion that occurs with penile erection within (intrinsic mechanism) and outside (extrinsic mechanism) the Corpus Cavernosum Penis, using 15 adult male mongrel dogs. Under controlled flows produced by a combination of aortic ligation and constant infusion of saline into the Corpus Cavernosum Penis, or into the deep dorsal vein, pressures within the cavernous space or deep dorsal vein were measured before and after electrical stimulation of the pelvic splanchnic (pelvic nerve), the hypogastric, and pudendal nerve. An increase in pressure following nerve stimulations represented an increase in outflow resistance due to occlusion of the venous system. Pre- and post-stimulation radiologic evaluations were performed to determine the site(s) of venous occlusion. Results: Unilateral stimulation of the pelvic nerve caused leftward shift of the corporeal pressure-flow curve. Bilateral stimulation of the pudendal nerve caused a marked rise in deep dorsal vein pressure. Conclusions: Both intrinsic and extrinsic venous occlusion mechanisms exist and that the former is activated primarily by unilateral stimulation of the pelvic nerve and the latter by bilateral stimulation of the pudendal nerve. The occlusion site for the extrinsic mechanism was localized to where the dorsal vein penetrates the muscles at the base of the pelvis, whereas the precise site for the intrinsic mechanism could not be determined.
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suppression by prolactin of the electrically induced erectile response through its direct effect on the Corpus Cavernosum Penis in the dog
The Journal of Urology, 1995Co-Authors: Hikaru Aoki, Tomoaki Fujioka, Junichi Matsuzaka, Takashi Kubo, Kazuyoshi Nakamura, Naoki YasudaAbstract:AbstractPurpose: Men become impotent when exposed to hyperprolactinemia. To clarify its mechanisms the effects of intracorporal infusion of prolactin on electrically induced penile erection were evaluated in 12 male dogs.Materials and Methods: Prolactin (10 microgram/ml.) or control saline was directly infused into the Corpus Cavernosum Penis 5 minutes before electrical pulse stimulation of the pelvic nerve and the intracorporal pressure was monitored.Results: In 8 dogs erection was markedly suppressed or completely abolished by prolactin. In the remaining 4, this effect of prolactin became manifest only when the ipsilateral internal pudenal artery was ligated. Saline infusion was without effect.Conclusions: An excess of prolactin directly inhibited the smooth muscle relaxation of Corpus Cavernosum Penis.
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participation of vasoactive intestinal polypeptide vip as a humoral mediator in the erectile response of canine Corpus Cavernosum Penis
Journal of Andrology, 1994Co-Authors: Hikaru Aoki, Tomoaki Fujioka, Junichi Matsuzaka, S Ra, F Sato, T Kubo, Tsutomu Ohhori, Naoki YasudaAbstract:: The ability of anti-vasoactive intestinal peptide (anti-VIP) serum to suppress the electrically induced relaxation of the Corpus Cavernosum was evaluated in vitro to define the role of VIP in penile erection. Strips of canine corpora cavernosa were placed in 5-ml organ chambers containing oxygenated Krebs-Ringer solution. They were stretched and fixed in place at both ends and pretreated with 2 × 10−7 M noradrenaline (NA). NA was given to produce an optimal state of isometric smooth muscle contraction so that subsequent electrical field stimulation (EFS) could induce a good range of measurable relaxation response. This response was deemed to be an in vitro representation of penile erection. After NA treatment the cavernous tension rose markedly by 2–2.5 g; it then declined by up to 1–1.2 g upon EFS. Anti-VIP serum (1:16) or atropine sulfate (10−6 M) was added at various time points between NA administration and EFS. When anti-VIP serum was administered, subsequent EFS-induced relaxation was attenuated by 20%–55% compared to the control EFS treatments. The degree of attenuation depended upon the frequency of EFS applied, being 20.6% ± 4.0% at 20 Hz and 54.7% ± 6.3% at 2 Hz. Atropine administered additionally following anti-VIP serum produced no further attenuation. However, atropine alone was capable of producing up to 23.7% ± 3.5% attenuation. When anti-VIP serum was administered following atropine, the degree of attenuation that ensured was the sum of the attenuations produced by each of the two substances independently. These results indicated that both VIP and acetylcholine were involved in the EFS-induced erectile response, the contribution of the former being greater than that of the latter. A considerable portion of the EFS-induced cavernous relaxation, however, appeared to be dependent on factors other than VIP and acetylcholine.
Dirk Schultheiss - One of the best experts on this subject based on the ideXlab platform.
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isolation of primary endothelial and stromal cell cultures of the Corpus Cavernosum Penis for basic research and tissue engineering
European Urology, 2005Co-Authors: Adrian Pilatz, Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Heike Mertsching, U. Jonas, Christian G. StiefAbstract:Abstract Objectives: Primary cell cultures derived from the Corpus Cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human Penis. Methods: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. Results: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0±0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. Conclusion: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.
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cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human Corpus Cavernosum Penis
Urology, 2004Co-Authors: Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Heike Mertsching, Michael Sohn, U. Jonas, A Pilatz, Christian G. StiefAbstract:Abstract Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1 ), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the Corpus Cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the Corpus Cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1 , 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1 . Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the Corpus Cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.
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Androgen and estrogen receptors in the human Corpus Cavernosum Penis: immunohistochemical and cell culture results
World Journal of Urology, 2003Co-Authors: Dirk Schultheiss, Rafael Badalyan, Adrian Pilatz, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Reinhard Wasielewski, Heike Mertsching, Michael Sohn, Christian G. StiefAbstract:Despite the central and peripheral effects of androgens on the nervous system, the local effects of androgens in the Corpus Cavernosum Penis and their importance for erectile function is still unclear. In this study Corpus Cavernosum biopsies of eight adult potent patients, aged 19–63 years, undergoing penile deviation surgery (group A) and 12 patients undergoing male-to-female transsexual surgery (group B) were immunostained for nuclear androgen and estrogen-alpha receptors. Additionally, primary Corpus Cavernosum endothelial cell cultures were obtained from six transsexual patients and exposed to testosterone, dihydrotestosterone, estradiol and progesterone likewise for 7 days. Total cell count was performed and cell metabolic activity was measured by a tetrazolium salt-based assay. Androgen and estrogen-alpha receptors were detected in stromal as well as in endothelial cells. Of all cell nuclei, 74.9% (SD 16.4) in group A and 63.5% (SD 17.1) in group B were positively stained for androgen receptors. The respective percentage of estrogen receptors was 11% (SD 9.5) and 21.2% (SD 12.6). An age-dependent difference in receptor distribution was not observed in either group. In the cell culture system only cultures exposed to testosterone and dihydrotestosterone showed a dose-dependent increase of cell metabolic activity compared to the cultures supplemented with estradiol and progesterone. The significant and age-independent high androgen and low estrogen-alpha receptor distribution found in both groups suggests a possible peripheral effect of androgens at the level of the Corpus Cavernosum Penis in adult humans. This is supported by the observed effect of testosterone and dihydrotestosterone on cell count and endothelial cell metabolism in our cell culture system. The role of estrogens remains unclear.
Norbert Schlote - One of the best experts on this subject based on the ideXlab platform.
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isolation of primary endothelial and stromal cell cultures of the Corpus Cavernosum Penis for basic research and tissue engineering
European Urology, 2005Co-Authors: Adrian Pilatz, Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Heike Mertsching, U. Jonas, Christian G. StiefAbstract:Abstract Objectives: Primary cell cultures derived from the Corpus Cavernosum are frequently used as in vitro models to define cellular mechanisms involved in erectile function. However, previous studies often lack detailed isolation protocols or a precise characterisation of the culture composition excluding especially contaminating fibroblasts. This study aimed at critically analysing and reproducing reported isolation methods, as well as establishing new procedures to receive highly pure and morphologically differentiated endothelial, smooth muscle and fibroblastic cells derived from the human Penis. Methods: We evaluated numerous isolation and enrichment techniques using cavernosal tissue from 57 patients. Assessment factors displayed the purity, cell yield, practicability and reproducibility. The purity in cultured cells was analysed using immunocytochemistry and Western blots. Results: An enzymatic protocol was established for the isolation and cultivation of cavernosal endothelial cells with an impressive purity of 98.0±0.8%. In contrast, already published nearly pure smooth muscle cell cultures were not reproducible in our laboratory. Meaningful evidence for an overwhelming presence of fibroblasts in these widely accepted pure smooth muscle cell cultures is presented. Conclusion: Endothelial cell cultures derived from human corpora cavernosa are reproducible and reliable to serve for cell culture-based investigations of the endothelial dysfunction. The discrepancy in the purity of smooth muscle cell cultures might reflect laboratory and tissue source factors, lacking an exclusion of fibroblasts in other studies or changes in stromal phenotype under culture conditions. Further research is necessary to clarify a possible plasticity between smooth muscle cells and (myo)fibroblasts and assess functional properties.
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cytotoxicity of different intracavernous vasoactive drugs on cultured endothelial cells of human Corpus Cavernosum Penis
Urology, 2004Co-Authors: Dirk Schultheiss, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Heike Mertsching, Michael Sohn, U. Jonas, A Pilatz, Christian G. StiefAbstract:Abstract Objectives To investigate the cytotoxic effect of prostaglandin E 1 (PGE 1 ), a standard combination of papaverine/phentolamine, and a triple mixture of these agents on human cavernosal endothelial cells using a cell culture model. The endothelial layer of the Corpus Cavernosum plays an important role in signal transduction of penile erection and is directly exposed to vasoactive agents after intracavernous injection for erectile dysfunction. Methods Primary endothelial cells were obtained from the Corpus Cavernosum of 13 potent patients undergoing penile surgery. Cultured cells were exposed for 30 minutes to physiologic dilutions of 20 μg PGE 1 , 30 mg papaverine/1 mg phentolamine, or the same dosages of the triple mixture of these agents, each dissolved in 5 to 50 mL sodium chloride. Lactate dehydrogenase release as a cytotoxicity marker was measured 6 hours after drug exposure, and the total cell metabolic activity was quantified after 48 hours with a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Additionally, the amount of viable cells was identified with a dual fluorescent staining procedure. Results The initial release of lactate dehydrogenase was elevated up to 3.2-fold in the concentrated papaverine/phentolamine and triple mixture group compared with PGE 1 and the control. After 48 hours, the papaverine-containing formulations led to a significant dose-dependent decrease in the viable cell count and metabolic activity of the cultures that was not noticed with PGE 1 . Conclusions These in vitro data strongly suggest an unfavorable effect of vasoactive agents containing papaverine on cavernosal endothelial cells. Before fibrotic changes of the smooth muscle stroma, the functionally important endothelium of the Corpus Cavernosum might suffer significantly from intracavernous injection therapy. Therefore, papaverine should no longer be used for this indication.
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Androgen and estrogen receptors in the human Corpus Cavernosum Penis: immunohistochemical and cell culture results
World Journal of Urology, 2003Co-Authors: Dirk Schultheiss, Rafael Badalyan, Adrian Pilatz, Alexander I. Gabouev, Norbert Schlote, Jörg Wefer, Reinhard Wasielewski, Heike Mertsching, Michael Sohn, Christian G. StiefAbstract:Despite the central and peripheral effects of androgens on the nervous system, the local effects of androgens in the Corpus Cavernosum Penis and their importance for erectile function is still unclear. In this study Corpus Cavernosum biopsies of eight adult potent patients, aged 19–63 years, undergoing penile deviation surgery (group A) and 12 patients undergoing male-to-female transsexual surgery (group B) were immunostained for nuclear androgen and estrogen-alpha receptors. Additionally, primary Corpus Cavernosum endothelial cell cultures were obtained from six transsexual patients and exposed to testosterone, dihydrotestosterone, estradiol and progesterone likewise for 7 days. Total cell count was performed and cell metabolic activity was measured by a tetrazolium salt-based assay. Androgen and estrogen-alpha receptors were detected in stromal as well as in endothelial cells. Of all cell nuclei, 74.9% (SD 16.4) in group A and 63.5% (SD 17.1) in group B were positively stained for androgen receptors. The respective percentage of estrogen receptors was 11% (SD 9.5) and 21.2% (SD 12.6). An age-dependent difference in receptor distribution was not observed in either group. In the cell culture system only cultures exposed to testosterone and dihydrotestosterone showed a dose-dependent increase of cell metabolic activity compared to the cultures supplemented with estradiol and progesterone. The significant and age-independent high androgen and low estrogen-alpha receptor distribution found in both groups suggests a possible peripheral effect of androgens at the level of the Corpus Cavernosum Penis in adult humans. This is supported by the observed effect of testosterone and dihydrotestosterone on cell count and endothelial cell metabolism in our cell culture system. The role of estrogens remains unclear.
