We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sari]-SP-sulfone, a stable and selective agonist for the tachykinin NK, receptor, and by prostaglandin El (PGE,), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N"-nitro-Larginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGEj.Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors, and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis. (J. Clin. Invest. 1994.94:2036-2044

By real-time RT-PCR and Western blot analysis, we found that phosphodiesterase type 5 (PDE5) mRNA and protein abundance was several fold higher in human male than in female reproductive tracts. The highest mRNA level (>1 x 10(7) molecules/microg total RNA) was detected in human corpora cavernosa (CC), where PDE5 protein was immunolocalized in both muscular and endothelial compartment. The possible role of androgens in regulating PDE5 expression was studied using a previously established rabbit model of hypogonadotropic hypogonadism. In this model, hypogonadism reduced, and testosterone (T) supplementation restored, CC PDE5 gene and protein expression. In addition, T supplementation completely rescued and even enhanced cyclic GMP conversion to metabolites, without changing IC(50) for sildenafil (IC(50) = 2.16 +/- 0.62 nm). In control CC strips, sildenafil dose-dependently increased relaxation induced by electrical field stimulation, with EC(50) = 3.42 +/- 1.7 nm. Hypogonadism reduced, and T increased, sildenafil effect on electrical field stimulation, again without changing their relative EC(50) values. CC sensitivity to the NO-donor NCX4040 was greater in hypogonadal rabbit strips than in control or T-treated counterparts. Moreover, sildenafil enhanced NCX4040 effect in eugonadal rabbit strips but not in hypogonadal ones. This suggests that androgens up-regulate PDE5 in rabbit penis. We also measured PDE5 gene expression and metabolic activity in human CC from male-to-female transsexual individuals, chronically treated with estrogens and cyproterone acetate. Comparing the observed values vs. eugonadal controls, PDE5 mRNA, protein, and functional activity were significantly reduced. In conclusion, we demonstrated, for the first time, that androgens positively regulate PDE5, thus providing a possible explanation about the highest abundance of this enzyme in male genital tract.

We recently demonstrated that nitric oxide (NO) significantly contributes to the mitogenic effect of vascular endothelial growth factor (VEGF), suggesting a role for the NO pathway in the signaling cascade following kinase-derivative receptor activation in vascular endothelium. The aim of this study was to investigate the intracellular pathways linked to VEGF/NO-induced endothelial cell proliferation. We assessed the activity of the mitogen-activated protein kinase (MAPK) that is specifically activated by growth factors, extracellularregulated kinase (ERK1 ⁄2 ), on cultured microvascular endothelium isolated from coronary postcapillary venules. ERK1 ⁄2 was immunoprecipitated, and its activity was assessed with an immunocomplex kinase assay. In endothelial cells exposed for 5 min to the NO donor drug sodium nitroprusside at a concentration of 100 M, ERK1 ⁄2 activity significantly increased. VEGF produced a time-and concentration-dependent activation of ERK1 ⁄2 . Maximal activity was obtained after 5 min of stimulation at a concentration of 10 ng/ml. The specific MAPK kinase inhibitor PD 98059 abolished ERK1 ⁄2 activation and endothelial cell proliferation in a concentration-dependent manner in response to VEGF and sodium nitroprusside. The NO synthase inhibitor N -monomethyl-Larginine, as well as the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, blocked the activation of ERK1 ⁄2 induced by VEGF, suggesting that NO and cGMP contributed to the VEGF-dependent ERK1 ⁄2 activation. These results demonstrate for the first time that kinase-derivative receptor activation triggers the NO synthase/guanylate cyclase pathway to activate the MAPK cascade and substantiates the hypothesis that the activation of ERK1 ⁄2 is necessary for VEGF-induced endothelial cell proliferation.

Vascular endothelial growth factor (VEGF) is a secreted protein that is a specific growth factor for endothelial cells. We have recently demonstrated that nitric oxide (NO) donors and vasoactive peptides promoting NO-mediated vasorelaxation induce angiogenesis in vivo as well as endothelial cell growth and motility in vitro; in contrast, inhibitors of NO synthase suppress angiogenesis. In this study we investigated the role of NO in mediating the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary postcapillary venules. VEGF induced a dose-dependent increase in cell proliferation and DNA synthesis. The role of NO was determined by monitoring proliferation or guanosine 3',5'-cyclic monophosphate (cGMP) levels in the presence and absence of NO synthase blockers. The proliferative effect evoked by VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. This effect was potentiated by superoxide dismutase addition and was abolished by NO synthase inhibitors. VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation.

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Bradykinin (BK) mediates inflammation and contributes to angiogenesis. We assessed the mechanisms for BK contribution to angiogenesis. Nanomolar concentrations of BK induced angiogenesis in rabbit corneas in absence of inflammation. The effect was dose‐dependent and mediated by the B1 receptor. B2 receptor stimulation failed to directly promote vascular growth unless inflammation was induced. Anti‐fibroblast growth factor‐2 (FGF‐2) antibody blocked the effect of BK or B1 receptor agonist. In postcapillary venular endothelial cells (CVEC), B2 receptor activation induced inositol phosphate turnover and calcium transients, whereas the B1 receptor was coupled to nitric oxide synthase (NOS) up‐regulation and activation and cGMP increase. Differential RT‐PCR and Western blot analysis revealed FGF‐ 2 up‐regulation in cells exposed to BK or to the selective B1 agonist, whereas the B2 agonist was without effect. Consistently, BK and the B1 but not the B2 agonist exerted a proliferative effect on CVEC, which was prevented by anti‐FGF‐2 antibody and by NOS inhibition. These results demonstrate that BK is angiogenic despite its proinflammatory activity and that the B1 receptor is involved. The B1 receptor is coupled to NOS activation and FGF‐2 up‐regulation, events not shared by the B2 receptor activation.

Research on penile smooth muscle physiology has increased the number of drugs available for treating erectile dysfunction (ED). Penile erection involves the relaxation of smooth muscle in the corpus cavernosum. The key mediator of smooth muscle relaxation is nitric oxide (NO), which acts by increasing the cellular level of cGMP. Another cyclic nucleotide, cAMP, is involved in smooth muscle cell relaxation; cAMP formation is stimulated by a number of compounds, such as alprostadil. An increase in cAMP and/or cGMP levels can also be induced by inhibition of phosphodiesterases (PDEs), the enzymes involved in cyclic nucleotide breakdown. Both papaverine and sildena®l are PDE inhibitors. Papaverine is a non-speci®c inhibitor of these enzymes; sildena®l is an orally active, potent and selective inhibitor of GMP-speci®c PDE5, the predominant isoenzyme metabolizing cGMP in the cells of the corpus cavernosum. Penile smooth muscle contraction, induced by adrenergic ®bers through a 1 adrenoceptors, produces detumescence, thus making a adrenoceptor antagonists suitable for maintenance of penile erection. The orally active drug yohimbine is a mixed a 1 -a 2 adrenoceptor antagonist that works by a dual mechanism; it facilitates sexual arousal by acting on a 2 adrenoceptors in the central nervous system and blocks adrenergic in¯uences at peripheral level.

We report for the first time that penile smooth muscle cells (SMC) not only respond to, but also synthesize, endothelin-1 (ET-1), one of the main regulators of SMC activity. Immunohistochemical studies indicated that, beside endothelial cells (EC), SMC of the human adult and fetal penis also express ET-1 and its converting enzyme, ECE-1. Accordingly, cultures of adult penile stromal cells express these genes. We also prepared and characterized penile SMC from human fetuses. These cells express SMC specific markers such as alpha smooth muscle actin and phosphodiesterase type 5A3 along with hallmarks of androgen-dependent cells (androgen receptor and 5alpha reductase type 2). Human fetal penile SMC (hfPSMC) are immunopositive for ET-1 and release ET-1. ET-1 expression in hfPSMC was strongly increased by several factors such as transforming growth factor-beta1 (TGF-beta1), interleukin-1alpha (IL-1alpha), ET-1 itself and prolonged (24 h) hypoxia. This latter condition not only affected ET-1 expression but also responsiveness. While at normal oxygen tension, hfPSMC responded to ET-1 with a decreased proliferation mediated by the endothelin-A receptors and TGF-beta1; however, during hypoxia, ET-1 stimulated cell growth. Accordingly, prolonged hypoxia up-regulated endothelin-B receptor mRNA expression. In conclusion, our results indicate that in penile tissues SMC produce ET-1 and that such production is modulated by factors involved in penile physiology and tissue remodelling. In addition, the hfPSMC we have characterized might be a useful model for studying biochemical aspects of the human erectile process in vitro.