Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways.

OBJECTIVE: • To investigate the role that oxidative stress plays in the development of diabetic cystopathy. MATERIALS AND METHODS: • Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month- old diabetic rats was carried out using microarray analysis. • Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. • The activity of protein degradation pathways was assessed using Western blot analysis. RESULTS: • Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10(-10)). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. • It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. • This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. CONCLUSION: • Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function.

Angiogenesis of the frozen-thawed human fetal ovarian tissue at the early stage after xenotransplantation and the positive effect of Salviae miltiorrhizae.

Cryopreserving ovarian tissue followed by transplantation has been suggested to preserve fertility for young cancer survivors. However, ischemia in the early stage after transplantation causes massive follicle loss. The aim was to investigate the histological and ultrastructural characteristics of the frozen-thawed human fetal ovarian tissue after xenotransplantation and the effects of Salviae miltiorrhizae (SM) on the angiogenesis. The human fetal ovarian tissues were frozen-thawed, xenografted into the immunodeficient nu/nu mice, and then collected 2, 7, and 28 days after transplantation. SM was administered. Compared with that of the frozen-thawed ovarian tissue, the total follicle number of the grafts was greatly reduced. Nearly half of the primordial follicles were damaged at different levels on day 2. Moreover, edema was prevalent in the stroma during the first week after the graft, especially on day 2. The microvessel density of the grafts was increased on day 2, reached a peak on day 7, and then declined on day 28. Both healthy primordial follicle proportion and the total healthy primordial follicles pool in the SM group were significantly higher than those of the control group (P = 0.003 and P = 0.001). We found a statistically significant difference of microvessel density between the two groups on day 2 (P < 0.001). In the frozen-thawed fetal ovarian grafts, angiogenesis has been begun on day 2, and the first week is the critical time for the grafts to regain their function, in which SM can facilitate graft vascularization and improve the preservation of primordial follicles.

The mechanism of opiorphin-induced experimental priapism in rats involves activation of the polyamine synthetic pathway.

Intracorporal injection of plasmids encoding opiorphins into retired breeder rats can result in animals developing a priapic-like condition. Microarray analysis demonstrated that following intracorporal gene transfer of plasmids expressing opiorphins the most significantly upregulated gene in corporal tissue was the ornithine decarboxylase gene (ODC). Quantitative RT-PCR confirmed the upregulation of ODC, as well as other genes involved in polyamine synthesis, such as arginase-I and -II, polyamine oxidase, spermidine synthase, spermidine acetyltransferase (SAT), and S-adenosylmethionine decarboxylase. Western blot analysis demonstrated upregulation of arginase-I and -II, ODC, and SAT at the protein level. Levels of the polyamine putrescine were upregulated in animals treated with opiorphin-expressing plasmids compared with controls. A direct role for the upregulation of polyamine synthesis in the development of the priapic-like condition was supported by the observation that the ODC inhibitor 1,3-diaminopropane, when added to the drinking water of animals treated with plasmids expressing opiorphins, prevented experimental priapism. We also demonstrate that in sickle cell mice, another model of priapism, there is increased expression of the mouse opiorphin homologue in corporal tissue compared with the background strain at a life stage prior to evidence of priapism. At a life stage when there is onset of priapism, there is increased expression of the enzymes involved in polyamine synthesis (ODC and arginase-I and -II). Our results suggest that the upregulation of enzymes involved in the polyamine synthetic pathway may play a role in the development of experimental priapism and represent a target for the prevention of priapism.

[The effect of danshen on the angiogenesis of the frozen-thawed human fetal ovarian tissue after transplantation].

AIM: (1) To investigate the mRNA expression of the key angiogenic growth factors in the grafts after transplantation. (2) To investigate the potential impact of danshen (Chinese traditional medicine) administration on grafts angiogenesis. METHODS: The frozen-thawed ovarian tissue from aborted fetus were xenografted into the renal capsule of the nude mice, recovered 48 h, 7 d and 28 d after respectively. Either danshen or saline (as the control) was administered after transplantation. RESULTS: The mRNA levels of VEGF showed a temporary raise in 48 h after transplantation, then decreased in one week, and no significant difference was fund between the control group and danshen group. Ang-2 was increased in 48 h after transplantation, when Danshen group was significantly higher than the control group (P < 0.05). The microvessel density significantly increased in all the tissues after transplantation. The control group peaked on day 7 after transplantation, while danshen group peaked in 48 h and kept correspondingly steady after that. CONCLUSION: Early angiogenesis began within 48 h after transplantation of the thawed human fetal ovarian tissue, and its microvessel density peaked within the first week after transplantation. Our results also suggested that the use of danshen injection in conjunction with transplantation could facilitate revascularization of the grafts.

Transcription of G-protein coupled receptors in corporeal smooth muscle is regulated by the endogenous neutral endopeptidase inhibitor sialorphin.

PURPOSE: Several reports suggest that the rat Vcsa1 gene is down-regulated in models of erectile dysfunction. The Vcsa protein product sialorphin is an endogenous neutral endopeptidase inhibitor and its down-regulation could result in prolonged activation of G-protein activated signaling pathways by their peptide agonists. We investigated whether Vcsa1 down-regulation could result in an adaptive change in GPCR (G-protein coupled receptor) expression. MATERIALS AND METHODS: Gene expression in cultured rat corporeal smooth muscle cells following treatment with siRNA directed against Vcsa1 or the neutral endopeptidase gene was analyzed using microarray and quantitative reverse transcriptase-polymerase chain reaction. In rats Vcsa1 is one of the most down-regulated genes following bilateral transection of the cavernous nerves. In that animal model we also investigated whether Vcsa1 down-regulation was accompanied by similar changes in gene expression in corporeal smooth muscle cells in which Vcsa1 was knocked down in vitro. RESULTS: Microarray analysis and quantitative reverse transcriptase-polymerase chain reaction demonstrated that corporeal smooth muscle cells treated in vitro with siRNA against Vcsa1 resulted in GPCR up-regulation as a functional group. In contrast, treatment of corporeal smooth muscle cells that lowered neutral endopeptidase activity resulted in decreased GPCR expression. These results suggest that the peptide product of Vcsa1, sialorphin, can effect GPCR expression by acting on neutral endopeptidase. In animals with bilaterally transected cavernous nerves the decreased Vcsa1 expression is accompanied by increased GPCR expression in cavernous tissue. CONCLUSIONS: These experiments suggest that the mechanism by which Vcsa1 modulates erectile function is partly mediated through changes in GPCR expression.

hSMR3A as a marker for patients with erectile dysfunction.

PURPOSE: We recently reported that Vcsa1 is one of the most down-regulated genes in the corpora of rats in 3 distinct models of erectile dysfunction. Since gene transfer of plasmids expressing Vcsa1 or intracorporeal injection of its mature peptide product sialorphin into the corpora of aging rats was shown to restore erectile function, we proposed that the Vcsa1 gene has a direct role in erectile function. To determine if similar changes in gene expression occur in the corpora of human subjects with erectile dysfunction we identified a human homologue of Vcsa1 (hSMR3A) and determined the level of expression of hSMR3A in patients. MATERIALS AND METHODS: hSMR3A was identified as a homologue of Vcsa1 by searching protein databases for proteins with similarity. hSMR3A cDNA was generated and subcloned into the plasmid pVAX to generate pVAX-hSMR3A. pVAX-hSMR3A (25 or 100 microg) was intracorporeally injected into aging rats. The effect on erectile physiology was compared histologically and by measuring intracorporeal pressure/blood pressure with controls treated with the empty plasmid pVAX. Total RNA was extracted from human corporeal tissue obtained from patients undergoing previously scheduled penile surgery. Patients were grouped according to normal erectile function (3), erectile dysfunction and diabetes (5) and patients without diabetes but with erectile dysfunction (5). Quantitative reverse-transcriptase polymerase chain reaction was used to determine the hSMR3A expression level. RESULTS: Intracorporeal injection of 25 microg pVAX-hSMR3A was able to significantly increase the intracorporeal pressure-to-blood pressure ratio in aging rats compared to age matched controls. Higher amounts (100 microg) of gene transfer of the plasmid caused less of an improvement in the intracorporeal pressure-to-blood pressure ratio compared to controls, although there was histological and visual evidence that the animals were post-priapitic. These physiological effects were similar to previously reported effects of intracorporeal injection of pVAX-Vcsa1 into the corpora of aging rats, establishing hSMR3A as a functional homologue of Vcsa1. More than 10-fold down-regulation in hSMR3A transcript expression was observed in the corpora of patients with vs without erectile dysfunction. In patients with diabetes associated and nondiabetes associated erectile dysfunction hSMR3A expression was found to be down-regulated. CONCLUSIONS: These results suggest that hSMR3A can act as a marker for erectile dysfunction associated with diabetic and nondiabetic etiologies. Given that our previous studies demonstrated that gene transfer of the Vcsa1 gene and intracorporeal injection of its protein product in rats can restore erectile function, these results suggest that therapies that increase the hSMR3A gene and product expression could potentially have a positive impact on erectile function.

Monitoring in real time with a microelectrode the release of reactive oxygen and nitrogen species by a single macrophage stimulated by its membrane mechanical depolarization.

Macrophages are key cells of the immune system. During phagocytosis, the macrophage engulfs a foreign bacterium, virus, or particle into a vacuole, the phagosome, wherein oxidants are produced to neutralize and decompose the threatening element. These oxidants derive from in situ production of superoxide and nitric oxide by specific enzymes. However, the chemical nature and sequence of release of these compounds is far from being completely determined. The aim of the present work was to study the fundamental mechanism of oxidant release by macrophages at the level of a single cell, in real time and quantitatively. The tip of a microelectrode was positioned at a micrometric distance from a macrophage in a culture to measure oxidative-burst release by the cell when it was submitted to physical stimulation. The ensuing release of electroactive reactive oxygen and nitrogen species was detected by amperometry and the exact nature of the compounds was characterized through comparison with in vitro electrochemical oxidation of H2O2, ONOO-, NO*, and NO2(-) solutions. These results enabled the calculation of time variations of emission flux for each species and the reconstruction of the original flux of production of primary species, O2*- and NO*, by the macrophage.