Erectile dysfunction is associated with defective L-cysteine/hydrogen sulfide pathway in human corpus cavernosum and penile arteries.

H2S signaling was proposed to participate in erectile physiology. L-cysteine (CYS)/H2S pathway stimulation causes cGMP-dependent relaxation of human corpus cavernosum (HCC) and penile arteries (HPRA). The aim was to evaluate the impact of ED on CYS/H2S pathway at functional and molecular level in human penile vascular tissues. NaHS- and CYS-induced responses were evaluated in HCC and HPRA from organ donors without ED (NoED, n = 29) and from ED patients undergoing penile prosthesis insertion (n = 45). cGMP accumulation and cystathionine ß-synthase and cystathionine γ-lyase expression were also determined. NaHS-induced relaxations were slightly but significantly impaired in HCC but not in HPRA from ED patients. In contrast, CYS-induced relaxations were markedly impaired in HCC (Emax 67.6 ± 4.9% vs 46.2 ± 4.6%, P < 0.01) and HPRA (Emax 80.8 ± 4.0% vs 48.1 ± 8.6%, P < 0.05) from men with ED. Impairment of CYS-induced responses was observed even after separating diabetic ED patients. In HPRA from ED patients, CYS- but not NaHS-induced vasodilation was significantly associated to endothelial function measured as vasodilatory capacity of acetylcholine (ACh) in these preparations (r2 = 0.481, P < 0.01). Impairment of CYS-induced relaxations was related to significant reduction in CYS-induced accumulation of cGMP in cavernosal tissue. Furthermore, the expression of H2S synthesizing enzymes was significantly reduced in HCC from ED patients with respect to NoED. This was confirmed by immunofluorescence in HCC and HPRA sections. ED involves impairment of CYS/H2S pathway in penile vascular tissues associated with decreased expression of H2S generating enzymes, CBS and CSE. These evidences support a therapeutic potential for modulation of CYS/H2S signaling in the management of ED.

Enhanced Contribution of Orai Channels to Contractility of Human Penile Smooth Muscle in Erectile Dysfunction.

BACKGROUND: Store-operated calcium entry and its key players, stromal interaction molecule (STIM) and Orai calcium channels, have been proposed as emergent therapeutic targets in cardiovascular pathophysiology. We hypothesize alteration of STIM/Orai signaling in erectile dysfunction (ED). AIM: To evaluate the contribution of STIM/Orai to human penile tissue contraction and to analyze the influence of ED on STIM/Orai signaling at functional and expression levels in human penile vascular tissues. METHODS: Human penile resistance arteries (HPRA) and human corpus cavernosum (HCC) were dissected from cavernosal specimens from 30 organ donors without history of ED (No ED) and from 48 patients with ED undergoing penile prosthesis insertion and functionally evaluated in wire myographs and organ chambers, respectively. Expression of STIM-1, Orai1, and Orai3 in HCC was localized and quantified by immunofluorescence. MAIN OUTCOME MEASURES: The main outcome measures are functional responses in HCC and HPRA and STIM/Orai channel protein expression in human cavernosal tissue. RESULTS: Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced norepinephrine-induced contractions in both HCC and HPRA from either No ED or ED subjects, but the effects were more marked in ED (-20.1 ± 5.9% vs -45.5 ± 13.2% and -15.9 ± 4.0% vs -31.4 ± 6.9% reduction in Emax to norepinephrine in HCC and HPRA, respectively). Thromboxane-induced contractions were reduced and neurogenic contractile and relaxant responses modulated by Orai inhibition in penile tissues from patients with ED. In fact, addition of YM-58483 concentration dependently relaxed precontracted HPRA and HCC. These relaxations were significantly more pronounced in tissues from patients with ED (EC50 7.5 vs 1.3 µM and 10.5 vs 1.3 µM, for HCC and HPRA, respectively). All HCC specimens displayed expression of STIM-1, Orai1, and Orai3. Significantly increased expression of Orai1 and Orai3 but not STIM-1 was observed in patients with ED. CLINICAL TRANSLATION: Inhibition of enhanced Orai activity in human penile vascular tissue could facilitate erectile responses, alleviating ED. STRENGTHS AND LIMITATIONS: Enhanced STIM/Orai activity contribution to penile smooth muscle tone in ED is demonstrated at functional and structural levels in human tissues from a representative sample of patients with ED and in comparison with healthy tissue. We cannot differentiate the specific contribution of risk factors associated with ED to hyperactivity of the Orai system. CONCLUSIONS: Orai channels significantly contribute to human penile smooth muscle contraction. Orai contribution to penile smooth muscle tone is functionally enhanced in ED accompanied by increased expression of Orai channels in cavernosal tissue. Orai inhibition could be a potential therapeutic strategy to reduce penile smooth muscle contraction in ED. Sevilleja-Ortiz A, El Assar M, García-Rojo E, et al. Enhanced Contribution of Orai Channels to Contractility of Human Penile Smooth Muscle in Erectile Dysfunction. J Sex Med 2020;17:881-891.

Inhibition of Nitro-Oxidative Stress Attenuates Pulmonary and Systemic Injury Induced by High-Tidal Volume Mechanical Ventilation.

Mechanisms contributing to pulmonary and systemic injury induced by high tidal volume (VT) mechanical ventilation are not well known. We tested the hypothesis that increased peroxynitrite formation is involved in organ injury and dysfunction induced by mechanical ventilation. Male Sprague-Dawley rats were subject to low- (VT, 9 mL/kg; positive end-expiratory pressure, 5 cmH2O) or high- (VT, 25 mL/kg; positive end-expiratory pressure, 0 cmH2O) VT mechanical ventilation for 120 min, and received 1 of 3 treatments: 3-aminobenzamide (3-AB, 10 mg/kg, intravenous, a poly adenosine diphosphate ribose polymerase [PARP] inhibitor), or the metalloporphyrin manganese(III) tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP, 5 mg/kg intravenous, a peroxynitrite scavenger), or no treatment (control group), 30 min before starting the mechanical ventilation protocol (n = 8 per group, 6 treatment groups). We measured mean arterial pressure, peak inspiratory airway pressure, blood chemistry, and gas exchange. Oxidation (fluorescence for oxidized dihydroethidium), protein nitration (immunofluorescence and Western blot for 3-nitrotyrosine), PARP protein (Western blot) and gene expression of the nitric oxide (NO) synthase (NOS) isoforms (quantitative real-time reverse transcription polymerase chain reaction) were measured in lung and vascular tissue. Lung injury was quantified by light microscopy. High-VT mechanical ventilation was associated with hypotension, increased peak inspiratory airway pressure, worsened oxygenation; oxidation and protein nitration in lung and aortic tissue; increased PARP protein in lung; up-regulation of NOS isoforms in lung tissue; signs of diffuse alveolar damage at histological examination. Treatment with 3AB or MnTMPyP attenuated the high-VT mechanical ventilation-induced changes in pulmonary and cardiovascular function; down-regulated the expression of NOS1, NOS2, and NOS3; decreased oxidation and nitration in lung and aortic tissue; and attenuated histological changes. Increased peroxynitrite formation is involved in mechanical ventilation-induced pulmonary and vascular dysfunction.

Differential effect of amylin on endothelial-dependent vasodilation in mesenteric arteries from control and insulin resistant rats.

Insulin resistance (IR) is frequently associated with endothelial dysfunction and has been proposed to play a major role in cardiovascular disease (CVD). On the other hand, amylin has long been related to IR. However the role of amylin in the vascular dysfunction associated to IR is not well addressed. Therefore, the aim of the study was to assess the effect of acute treatment with amylin on endothelium-dependent vasodilation of isolated mesenteric arteries from control (CR) and insulin resistant (IRR) rats and to evaluate the possible mechanisms involved. Five week-old male Wistar rats received 20% D-fructose dissolved in drinking water for 8 weeks and were compared with age-matched CR. Plasmatic levels of glucose, insulin and amylin were measured. Mesenteric microvessels were dissected and mounted in wire myographs to evaluate endothelium-dependent vasodilation to acetylcholine. IRR displayed a significant increase in plasmatic levels of glucose, insulin and amylin and reduced endothelium-dependent relaxation when compared to CR. Acute treatment of mesenteric arteries with r-amylin (40 pM) deteriorated endothelium-dependent responses in CR. Amylin-induced reduction of endothelial responses was unaffected by the H2O2 scavenger, catalase, but was prevented by the extracellular superoxide scavenger, superoxide dismutase (SOD) or the NADPH oxidase inhibitor (VAS2870). By opposite, amylin failed to further inhibit the impaired relaxation in mesenteric arteries of IRR. SOD, or VAS2870, but not catalase, ameliorated the impairment of endothelium-dependent relaxation in IRR. At concentrations present in insulin resistance conditions, amylin impairs endothelium-dependent vasodilation in mircrovessels from rats with preserved vascular function and low levels of endogenous amylin. In IRR with established endothelial dysfunction and elevated levels of amylin, additional exposure to this peptide has no effect on endothelial vasodilation. Increased superoxide generation through NADPH oxidase activity may be a common link involved in the endothelial dysfunction associated to insulin resistance and to amylin exposure in CR.

Preserved endothelial function in human obesity in the absence of insulin resistance.

BACKGROUND: Insulin resistance (IR) is frequently associated with endothelial dysfunction and has been proposed to play a major role in cardiovascular disease (CVD). On the other hand, obesity has long been related to IR and increased CVD. However it is not known if IR is a necessary condition for endothelial dysfunction in human obesity, allowing for preserved endothelial function in obese people when absent. Therefore, the purpose of the study was to assess the relationship between IR and endothelial dysfunction in human obesity and the mechanisms involved. METHODS: Twenty non-insulin resistant morbid obese (NIR-MO), 32 insulin resistant morbid obese (IR-MO), and 12 healthy subjects were included. Serum concentrations of glucose, insulin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), resistin and adiponectin were determined. IR was evaluated by HOMA-index. Endothelium-dependent relaxation to bradykinin (BK) in mesenteric microvessels was assessed in wire myograph. RESULTS: Serum IL-6, and TNF-α levels were elevated only in IR-MO patients while resistin was elevated and adiponectin reduced in all MO individuals. Mesenteric arteries from IR-MO, but not from NIR-MO subjects displayed blunted relaxation to BK. Vasodilatation was improved in IR-MO arteries by the superoxide scavenger, superoxide dismutase (SOD) or the mitochondrial-targeted SOD mimetic, mito-TEMPO. NADPH oxidase inhibitors (apocynin and VAS2870) and the nitric oxide synthase (NOS) cofactor, tetrahydrobiopterin failed to modify BK-induced vasodilatations. Superoxide generation was higher in vessels from IR-MO subjects and reduced by mito-TEMPO. Blockade of TNF-α with infliximab, but not inhibition of inducible NOS or cyclooxygenase, improved endothelial relaxation and decreased superoxide formation. CONCLUSIONS: Endothelial dysfunction is observed in human morbid obesity only when insulin resistance is present. Mechanisms involved include augmented mitochondrial superoxide generation, and increased systemic inflammation mediated by TNF-α. These findings may explain the different vascular risk of healthy vs unhealthy obesity.

Genetic predisposition to acute kidney injury induced by severe sepsis.

PURPOSE: The aim of this study was to demonstrate that candidate gene polymorphisms are associated with an increased risk of acute kidney injury (AKI). MATERIALS AND METHODS: Patients admitted to the intensive care unit with the diagnosis of severe sepsis and an expected intensive care unit length of stay more than 48 hours were included. Genetic polymorphisms studied included angiotensin-converting enzyme insertion/deletion (polymerase chain reaction); tumor necrosis factor α -376, - 308, and -238; interleukin-8 -251; vascular endothelial growth factor (VEGF) +405 and +936; and pre-B-cell colony-enhancing factor -1001 (TaqMan SNP genotyping assay, Life Technologies, Grand Island, NY). Acute kidney injury was defined as the risk, injury, and failure categories, as per the RIFLE (risk, injury, failure, loss, end-stage kidney disease) classification. RESULTS: One hundred thirty-nine patients were included, 65 of whom developed AKI. In univariate analysis, the VEGF +936 CC and the pre-B-cell colony-enhancing factor -1001 GG genotypes were associated with AKI. In multivariate analysis, Simplified Acute Physiology Score II score (odds ratio [95% confidence interval], 1.06 [1.03-1.09]), chronic arterial hypertension (3.15 [1.39-7.15]), and the presence of the VEGF +936 CC genotype (3.41 [1.19-9.79]) were associated with AKI. CONCLUSION: This is the first study demonstrating an association between the VEGF +936 CC genotype and the risk to develop AKI in patients with severe sepsis.

Genetic predisposition to acute respiratory distress syndrome in patients with severe sepsis.

OBJECTIVE: The objective of this study was to analyze the association between candidate gene polymorphisms and susceptibility to acute respiratory distress syndrome (ARDS) in patients with severe sepsis. METHODS: Patients older than 18 years admitted to the intensive care unit (ICU) with the diagnosis of severe sepsis were prospectively included. A blood sample was drawn on the first day of ICU admission, and DNA was extracted. We genotyped the insertion/deletion polymorphism of the angiotensin-converting enzyme (ACE) gene (polymerase chain reaction) and the following single-nucleotide polymorphisms (TaqMan SNP genotyping assay): tumor necrosis factor α -376 G/A, -308 G/A, and -238 G/A; interleukin 8 -251 T/A; pre-B cell colony-enhancing factor -1001 G/T; and vascular endothelial growth factor +405 C/G and +936 C/T. Polymorphisms were selected based on reports on their association with ARDS. Variables associated in univariate analysis (P < 0.1) with the diagnosis of ARDS were included in a multiple logistic regression analysis. RESULTS: We studied 149 patients, of whom 35 presented ARDS. Variables included in the maximal multivariate model were male sex, chronic alcoholism, use of ACE inhibitors or angiotensin-receptor blockers, Simplified Acute Physiology Score II score, serum glucose concentration at ICU admission, and the presence of the allele D of the ACE gene. After adjustment for those variables, the presence of the allele D of the ACE gene (odds ratio, 4.75; 95% confidence interval, 1.02-22.20; P = 0.048) was significantly associated with the diagnosis of ARDS. CONCLUSION: The presence of the allele D of the ACE gene is associated with ARDS in patients with severe sepsis.

Pathways responsible for apoptosis resulting from amadori-induced oxidative and nitrosative stress in human mesothelial cells.

BACKGROUND: Apoptosis and inflammatory/oxidative stress have been associated with hyperglycemia in human peritoneal mesothelial cells (HPMCs) and other cell types. We and others have highlighted the role of early products of non-enzymatic protein glycation in inducing proinflammatory conditions and increasing apoptotic rates in HPMCs. Loss of HPMCs seems to be a hallmark of complications associated with peritoneal membrane dysfunction. The aim of this work is to elucidate the mechanisms by which Amadori adducts may act upon HPMC apoptosis. METHODS: HPMCs isolated from different patients were exposed to different Amadori adducts, i.e. highly glycated hemoglobin (10 nM) and glycated bovine serum albumin (250 µg/ml), to study cell death and several proapoptotic markers by different experimental approaches. RESULTS: Amadori adducts, but not their respective controls, impaired cell proliferation and cell viability by means of apoptosis in a time-dependent manner. They regulated the intrinsic mitochondrial cell death signaling pathway and modulated activation of caspases, Bax, iNOS, p53, NF-κB, and mitogen-activated protein kinases (p38 and JNK) through different reactive oxygen and nitrosative species. CONCLUSIONS: Our data strongly support the idea that long-term hyperglycemia could act as an inducer of apoptosis in HPMCs through Amadori adducts, involving different oxidative and nitrosative reactive species.

Transcriptional profiling and genotyping of degraded nucleic acids from autopsy tissue samples after prolonged formalin fixation times.

BACKGROUND: Samples used for genotyping and transcription studies are obtained and conserved in very specific conditions. The possibility to use autopsy tissue samples, which contain nucleic acids of very poor quality, would open new possibilities for genetic studies. METHODS: We have used liver tissue samples from autopsy cases to (i) determine its quality; (ii) study gene expression of 13 genes involved in different cell processes, before and after cDNA pre-amplification (quantitative reverse transcriptase polymerase chain reaction); and (iii) analyze the presence of 2 common polymorphisms of relevance for illness (ACE I/D genotype by PCR amplification, and TNF-α promoter gene polymorphism, by DNA sequencing). RESULTS: Samples were grouped according to different buffered formalin fixation times (group 1, <15 days; group 2, 60-90 days; group 3, 150-180 days; group 4, 240-270 days). Nucleic acids showed a time-dependent degradation. The expression of 13 genes could be studied in all cases from groups 1 and 2, only 7 from group 3 and none from group 4. cDNA preamplification allowed the study of all genes in all samples. DNA genotyping for ACE and TNF-α promoter region was possible in all cases. CONCLUSIONS: We conclude that nucleic acids extracted from autopsy specimens after prolonged periods of time in formalin were of sufficient quality to study gene expression and genotyping using currently available methodology and cDNA pre-amplification.

Amylin and hypertension: association of an amylin -G132A gene mutation and hypertension in humans and amylin-induced endothelium dysfunction in rats.

CONTEXT: Amylin has been linked to the development of hypertension in several pathological states related to hypertension and insulin resistance, although there is scant data regarding its potential mechanisms of action. The -132 G/A mutation located within an activator domain of the amylin gene's promoter was first identified in a small cohort of Spanish patients with type 2 diabetes. OBJECTIVE: The objective of the study was to test the interference of amylin peptide with endothelium-dependent responses as an added potential mechanism for amylin-induced hypertension. DESIGN: A total of 384 patients with type 2 diabetes and 207 healthy controls were subjected to clinical analysis and genetic screening for the -132 G/A mutation of the amylin gene. The effect of amylin on endothelium-dependent responses was analyzed in aortic rings and mesenteric microvessels from nondiabetic rats. RESULTS: The prevalence of the mutation was 10.1 vs. 0.9% in the control population (P<0.001). Hypertension was higher in a diabetic population carrying the mutation than in diabetic noncarriers (74 vs. 57%; P<0.05). Diabetic carriers showed higher fasting amylin levels than diabetic noncarriers (11.4+/-7 vs. 8.2+/-3 pmol/liter; P<0.05). Preincubation with 20 pmol/liter amylin impaired the relaxant responses induced by acetylcholine in rat aorta and mesenteric microvessels. This effect was abolished in both vascular beds in the presence of 100 micromol/liter NG-nitro-L-arginine methyl ester. CONCLUSIONS: We propose that amylin levels and hypertension may be linked by a novel mechanism involving the capacity of amylin to induce endothelial dysfunction by interfering with nitric oxide-mediated responses.

Amadori adducts activate nuclear factor-kappaB-related proinflammatory genes in cultured human peritoneal mesothelial cells.

Diabetes mellitus leads to a high incidence of several so-called complications, sharing similar pathophysiological features in several territories. Previous reports points at early nonenzymatic glycosylation products (Amadori adducts) as mediators of diabetic vascular complications. In the present study, we analysed a possible role for Amadori adducts as stimulators of proinflammatory pathways in human peritoneal mesothelial cells (HPMCs). Cultured HPMCs isolated from 13 different patients (mean age 38.7+/-16 years) were exposed to different Amadori adducts, that is, highly glycated haemoglobin (10 nM) and glycated bovine serum albumin (0.25 mg ml(-1)), as well as to their respective low glycosylation controls. Amadori adducts, but not their respective controls, elicited a marked increase of NF-kappaB activation, as determined by electromobility shift assays and transient transfection experiments. Additionally, Amadori adducts significantly increased the production of NF-kappaB-related proinflammatory molecules, including cytokines, such as TNF-alpha, IL-1beta or IL-6, and enzymes, such as cyclooxygenase-2 and inducible nitric oxide (NO) synthase, this latter leading to the release of NO by HPMCs. The effects of Amadori adducts were mediated by different reactive oxygen and nitrosative species (e.g. superoxide anions, hydroxyl radicals, and peroxynitrite), as they were blunted by coincubation with the appropriate scavengers. Furthermore, NO generated upon exposure to Amadori adducts further stimulated NF-kappaB activation, either directly or after combination with superoxide anions to form peroxynitrite. We conclude that Amadori adducts can favour peritoneal inflammation by exacerbating changes in NO synthesis pathway and triggering NF-kappaB-related proinflammatory signals in human mesothelial cells.