Pedro Cuevas

Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo

Although it has been clearly established that basic fibroblast growth factor (FGF) is a potent mitogen for chondrocytes in vitro, there is little evidence that it can stimulate this cell type in vivo. In an effort to address this problem, we examined the effect of an intraarticular administration of basic FGF. Alzet osmotic pumps delivering the mitogen to the site of injury promotes the healing of intra-chondrial lesions by stimulating chondrocyte proliferation and the formation of extracellular matrix. The observation that chronic infusions of basic FGF can elicit a repair response at the site of injury suggests that this growth factor may have therapeutic applications that extend beyond its capacity to induce neovascularization. The results also suggest that one of the ways that the perichondrium mediates cartilage repair may be by the local production of FGF-like mitogens.

The phosphodiesterase inhibitory selectivity and the in vitro and in vivo potency of the new PDE5 inhibitor vardenafil

We investigated the potency and the selectivity profile of vardenafil on phosphodiesterase (PDEs) enzymes, its ability to modify cGMP metabolism and cause relaxation of penile smooth muscle and its effect on erections in vivo under conditions of exogenous nitric oxide (NO) stimulation. PDE isozymes were extracted and purified from human platelets (PDE5) or bovine sources (PDEs 1, 2, 3, 4 and 6). The inhibition of these PDEs and of human recombinant PDEs by vardenafil was determined. The ability to potentiate NO-mediated relaxation and influence cGMP levels in human corpus cavernosum strips was measured in vitro, and erection-inducing activity was demonstrated in conscious rabbits after oral administration together with intravenous doses of sodium nitroprusside (SNP). The effects of vardenafil were compared with those of the well-recognized PDE5 inhibitor, sildenafil (values for sildenafil in brackets). Vardenafil specifically inhibited the hydrolysis of cGMP by PDE5 with an IC50 of 0.7 nM (6.6 nM). In contrast, the IC50 of vardenafil for PDE1 was 180 nM; for PDE6, 11 nM; for PDE2, PDE3 and PDE4, more than 1000 nM. Relative to PDE5, the ratios of the IC50 for PDE1 were 257 (60), for PDE6 16 (7.4). Vardenafil significantly enhanced the SNP-induced relaxation of human trabecular smooth muscle at 3 nM (10 nM). Vardenafil also significantly potentiated both ACh-induced and transmural electrical stimulation-induced relaxation of trabecular smooth muscle. The minimum concentration of vardenafil that significantly potentiated SNP-induced cGMP accumulation was 3 nM (30 nM). In vivo studies in rabbits showed that orally administered vardenafil dose-dependently potentiated erectile responses to intravenously administered SNP. The minimal effective dose that significantly potentiated erection was 0.1 mg/kg (1 mg/kg). The selectivity for PDE5, the potentiation of NO-induced relaxation and cGMP accumulation in human trabecular smooth muscle and the ability to enhance NO-induced erection in vivo indicate that vardenafil has the appropriate properties to be a potential compound for the treatment of erectile dysfunction. Vardenafil was more potent and selective than sildenafil on its inhibitory activity on PDE5.

An in vivo model for study of the angiogenic effects of basic fibroblast growth factor

We have investigated the angiogenic effects of basic fibroblast growth factor following its implantation in slow release beads under the kidney capsule. The presence of basic fibroblast growth factor in the subcapsular space induced a marked angiogenic response maximal at 1 microgram dose per kidney. Histological examination at the site of treatment failed to reveal evidence of an inflammatory response, thus supporting the observation that basic fibroblast growth factor alone can stimulate in vivo neovascularization. Beads pretreated with saline or with human growth hormone had no angiogenic effect. Because of the readily accessible location in the retroperitoneal space, the ease of drug delivery, and the marked vascular proliferation seen in response to FGF, our results suggest that the kidney capsule is an excellent model for study of the physiological role played by FGF and related peptides in promoting angiogenesis in vivo.

Pericyte endothelial gap junctions in human cerebral capillaries.

SummaryHuman cerebral tissue has been ultrastructurally studied and gap junctions have been visualized between endothelial cells and pericytes that permit ion exchange. We propose that the functional interrelationship between endothelium and pericytes may play a role in the alteration of capillary diameter for the control of local cerebral blood flow.

Vascular response to basic fibroblast growth factor when infused onto the normal adventitia or into the injured media of the rat carotid artery.

Immunohistochemical techniques localize basic fibroblast growth factor (FGF) in endothelial and smooth muscle cells of the common carotid artery. Thus, we studied the effect in rats of basic FGF infused for 14 days onto the adventitia or into the media in vivo. In untreated rats, the adventitial layer is uniform, and few vessels are observed in cross sections (mean +/- SEM is 0.351 +/- 0.16 capillaries/field at a magnification of x 480). Whereas saline infusion increases the mean number of vasa vasorum to 2.73 +/- 0.011 capillaries/field (p less than 0.01), basic FGF (1 ng/microliter/hr) increases the capillary number to 13.4 +/- 0.67 capillaries/field. The effects are local and restricted to the site of delivery; no cell proliferation is observed even 2 mm from the site of infusion. There is also no evidence of the infiltration of macrophages and monocytes. In an effort to determine the effect of basic FGF in the media, a small longitudinal (1-mm) incision was made in the adventitia, and saline or basic FGF (1 ng/microliter/hr) was infused for 14 days into the arterial wall. Under these conditions, basic FGF is a potent inducer of smooth muscle cell proliferation in the vascular wall as well as of new capillaries. In these instances, however, the capillaries formed are thick-walled. The results support the hypothesis that basic FGF may be contributing to the growth and maintenance of the vasa vasorum and of vascular smooth muscle cells.

Bone marrow stromal cell implantation for peripheral nerve repair.

Abstract Cell therapy using bone marrow stromal cells is a new promising therapy for regenerative medicine. Previous studies demonstrated that local bone marrow stromal cells implantation in the distal stump of transected sciatic nerve of rats promotes early functional recovery. The purpose of this study was to expand on the preliminary research by investigating the long-term efficacy of bone marrow stromal cells using the same experimental setting. Functional test and histological studies demonstrate that bone marrow stromal cell-treated rats exhibit significant improvement on a walking tract test at day 180 after surgery compared with control rats. Taken together, these data suggest that bone marrow stromal cell therapy is a safe and effective strategy for peripheral nerve injuries.

Immunohistochemical detection of inhibin in the gonad

Antiserum to inhibin was produced in rabbits by immunization with a synthetic [Tyr30]alpha-chain(1-30)NH2 fragment of porcine inhibin coupled to bovine serum albumin, and the elicited antiserum was used in conjunction with the avidin-biotin immunoperoxidase procedure to localize inhibin-reactive cells in various rat tissue preparations. In the testes, only the Sertoli cells revealed immunoreactivity with the antiserum. Intense staining was also observed in ovarian follicular granulosa cells but not in the theca layer outside the basement membrane. In addition, the luteal cells in the corpus luteum were also stained by the antiserum. The positive staining in the gonadal tissues could be blocked completely by pre-adsorbing the serum with either the synthetic peptide or native inhibin. Immunostaining was not detected in brain, pituitary, thymus, stomach, pancreas, kidney and adrenal section, thus confirming that inhibin is a polypeptide originating only from specific cells of the gonad.

Diabetes Exacerbates the Functional Deficiency of NO/cGMP Pathway Associated with Erectile Dysfunction in Human Corpus Cavernosum and Penile Arteries

INTRODUCTION Diabetic men with erectile dysfunction (ED) are less responsive to therapy with type 5 phosphodiesterase (PDE5) inhibitors. Although an impairment of the nitric oxide (NO)/cyclic guanosin-monophosphate (cGMP) pathway has been shown in diabetic ED vs. non-diabetic ED, the functionality of NO/cGMP pathway in non-diabetic and diabetic ED patients with respect to non-ED patients has not been established. AIM The aim of this study is to evaluate the function of NO/cGMP signalling in human erectile tissues from ED patients exploring the added impact of diabetes. METHODS Corpus cavernosum strips (human corpus cavernosum [HCC]) and penile resistance arteries (HPRA) were collected from penile specimens from organ donors (OD) and from diabetic and non-diabetic men with ED undergoing penile prosthesis implantation. MAIN OUTCOME MEASURES Relaxations to acetylcholine, electrical field stimulation, sodium nitroprusside, and sildenafil were evaluated in phenylephrine-contracted HCC and norepinephrine-contracted HPRA. cGMP content in HCC was also determined. RESULTS The impairment of endothelium-dependent relaxation in HCC and HPRA from ED patients was exacerbated by diabetes (E(max) 76.1, 62.9, and 49.3% in HCC and 73.1, 59.8, and 46.0% in HPRA from OD, non-diabetic and diabetic ED, respectively). Hypertension, hypercholesterolemia, or aging did not exert a further impairment of endothelial relaxation among ED patients. Diabetes also causes a further impairment of neurogenic relaxation in HCC and HPRA. The basal and stimulated content of cGMP in HCC was significantly decreased in patients with ED, but specially reduced in diabetic patients. Diabetes clearly impaired PDE5 inhibitor-induced vasodilation of HPRA from ED patients. CONCLUSIONS ED is related to impaired vasodilation, reduced relaxant capacity, and diminished cGMP content in penile tissue. These alterations are more severe in diabetes and accompany reduced relaxant efficacy of PDE5 inhibition. Thus, an exacerbated reduction of nitric oxide/cGMP signaling could be responsible for ED in diabetic men and would explain their reduced response to treatment.

Differential effects of serotonin reuptake inhibitors on erectile responses, NO-production, and neuronal NO synthase expression in rat corpus cavernosum tissue

Increased incidence of impotence is associated with some selective serotonin‐reuptake‐inhibitors (SSRIs), but the pathophysiological mechanism is unknown. Paroxetine and citalopram are extensively used SSRIs, but only paroxetine has been shown to inhibit nitric oxide synthase (NOS) activity. NO is a key mediator of penile erection. Thus, the aim of this study was to determine the effects of paroxetine and citalopram on erectile function and NO production, in a rat model. Application of cavernosal nerve electrical stimulation produced frequency‐related intracavernosal pressure (ICP) increases, which were inhibited by the NOS inhibitor, NG‐nitro‐L‐arginine (0.3 mg kg−1). Acute or chronic (2 weeks) paroxetine‐treatment (10 mg kg−1) reduced ICP‐responses, while citalopram did not. Paroxetine, but not citalopram, significantly reduced nitrite+nitrate plasma levels by 61.4% and inhibited penile neuronal NOS (nNOS) protein expression by 31.2% after chronic treatment. The results show that paroxetine inhibits erectile responses in rats. We propose that this effect is due to reduced NO production and nNOS expression.

Regulation of human penile smooth muscle tone byprostanoid receptors

We have characterized the prostanoid receptors involved in the regulation of human penile arterial and trabecular smooth muscle tone. Arachidonic acid induced relaxation of human corpus cavernosum strips (HCCS) that was blocked by the cyclo‐oxygenase inhibitor, indomethacin, and augmented by the thromboxane receptor (TP) antagonist, SQ29548, suggesting that endogenous production of prostanoids regulates penile smooth muscle tone. TP‐receptors mediate contraction of HCCS and penile resistance arteries (HPRA), since the agonist of these receptors, U46619, potently contracted HCCS (EC50 8.3±2.8 nM) and HPRA (EC50 6.2±2.2 nM), and the contractions produced by prostaglandin F2α at high concentrations (EC50 6460±3220 nM in HCCS and 8900±6700 nM in HPRA) were inhibited by the selective TP‐receptor antagonist, SQ29548 (0.02 μM). EP‐receptors are responsible for prostanoid‐induced relaxant effects in HCCS because only prostaglandin E1 (PGE1), prostaglandin E2 and the EP2/EP4‐receptor agonist, butaprost, produced consistent relaxation of this tissue (EC50 93.8±31.5, 16.3±3.8 and 1820±1284 nM, respectively). In HPRA, both prostacyclin and PGE1 (EC50 60.1±18.4 and 109.0±30.9 nM, respectively) as well as the selective IP receptor agonist, cicaprost, and butaprost (EC50 25.2±15.2 and 7050±6020 nM, respectively) caused relaxation, suggesting co‐existence of IP‐ and EP‐receptors (EP2 and/or EP4). In summary, endogenous production of prostanoids may regulate penile smooth muscle contractility by way of specific receptors. TP‐receptors mediate contraction in HCCS and HPRA, while the relaxant effects of prostanoids are mediated by EP2‐ and/or EP4‐receptors in HCCS and by EP‐ and IP‐receptors in HPRA.