ACE-inhibition ameliorates vascular reactivity and delays diabetes outcome in NODmice.

Recently, we have demonstrated a direct correlation among hyperglycaemia, vascular dysfunction and eNOS post-translational regulation in non non-obese diabetic mice (NOD). Here, we evaluate the impact of two ACE-inhibitors therapy, zofenopril and enalapril in NOD mice. Insulin-dependent diabetes mellitus (IDDM) development was monitored weekly through glycosuria measurement. Zofenopril and enalapril were dosed at 0.5 mg/kg/die orally. Animals were sacrificed at different points and aortas used for western blotting or for tissue bath experiments. Bovine aortic endothelial cells in high glucose medium are treated with zofenoprilat or enalaprilat. Cells and supernatant were utilised for western blot analysis and for nitrite/nitrate determination, respectively. In ex-vivo experiments chronic administration of both drugs restored PE-induced contraction but not Isop-induced vasodilatation, however only zofenopril reduced caveolin-1 expression. In vitro, both drugs inhibited caveolin-1 expression and increased NOx production. However, zofenopril caused inhibition of both parameters at a concentration 200 fold lower than enalalpril. In vivo, zofenopril delays the onset of diabetic conditions of about 50%, and ameliorates polyuria. In conclusion our data suggest that ACE-inhibitor therapy may be useful in IDDM, in particular sulphydrylated inhibitor would display a better efficacy especially if administered early on the development of diabetes

Hydrogen sulphide is involved in testosterone vascular effect

BACKGROUND: Testosterone (T) induces a rapid relaxation in vascular tissues of different species due to a nongenomic effect of this steroid on vessels. Different mechanisms have been proposed to explain T-induced vasodilatation but the effective mechanism(s) and the mediators involved are still a matter of debate. OBJECTIVES: We have evaluated if H(2)S pathway is involved in T vascular effects. DESIGN AND SETTING: Male Wistar rats were sacrificed and thoracic aorta was rapidly dissected and cleaned from fat and connective tissue. Rings of 2-3 mm length were cut and placed in organ baths filled with oxygenated Krebs solution at 37 degrees C and mounted to isometric force transducers. H(2)S determination was performed on thoracic aortic rings incubated with T or vehicle and in presence of inhibitors. H2S concentration was calculated against a calibration curve of NaHS (3-250 microM). Results were expressed as nmoles/mg protein. MEASUREMENTS: Vascular reactivity was evaluated by using isometric transducers. H(2)S determination was performed by using a cystathionine beta-synthetase (CBS) and cystathionine gamma lyase (CSE) activity assay. CSE and CBS protein levels were assessed by Western blot analysis. Statistical analysis was performed by using two-way ANOVA and unpaired Student's t-test where appropriate. RESULTS: T significantly increased conversion of L-cysteine to H(2)S. This effect was significantly reduced by PGG and BCA, two specific inhibitors of CSE. T (10 nM-10 microM) induced a concentration-dependent vasodilatation of rat aortic rings in vitro that was significantly and concentration-dependent inhibited by PGG, BCA, and glybenclamide. Incubation of aorta with T up to 1 h did not change CBS/CSE expression, suggesting that T modulates enzymatic activity. CONCLUSIONS: Here we demonstrate that T vasodilator effect involves H(2)S, a novel gaseous mediator. T modulates H(2)S levels by increasing the enzymatic conversion of L-cysteine to H(2)S

Peripheral relaxant activity of apomorphine and of a D1 selective receptor agonist on human corpus cavernosum strips

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Essential requirement for sphingosine kinase activity in eNOS-dependent NO release and vasorelaxation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular second messenger. Cellular levels of S1P are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the biological effects of receptor-mediated S1P. Here we have studied the possible interplay between intracellular SIP generation and the eNOS activation pathway. S1P causes an endothelium-dependent vasorelaxation in rat aorta that is PTX sensitive, inhibited by L-NAME that involves eNOS phosphorylation, and mainly dependent on hsp90. When rat aorta rings were incubated with the SPK inhibitor DL-threo-dihydrosphingosine (DTD), there was a concentration-dependent reduction of Ach-induced vasorelaxation, implying a consistent contribution of sphingolipid pathway through intracellular sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to S1P as well to BK or calcium ionophore A-23187. Interestingly, as opposite to A-23187, BK and S1P effect were significantly inhibited by pretreatment with the SPK inhibitor DTD. In conclusion, our data demonstrate that an interplay exists among eNOS, hsp90, and intracellularly generated S1P where eNOS coupling to hsp90 is a major determinant for NO release as confirmed by our functional and molecular studies

Antagonizing S1P3 receptor with cell-penetrating pepducins in skeletal muscle fibrosis

S1P is the final product of sphingolipid metabolism, which interacts with five widely expressed GPCRs (S1P1-5). Increasing numbers of studies have indicated the importance of S1P3 in various pathophysiological processes. Recently, we have identified a pepducin (compound KRX- 725-II) acting as an S1P3 receptor antagonist. Here, aiming to optimize the activity and selectivity profile of the described compound, we have synthesized a series of derivatives in which Tyr, in position 4, has been substituted with several natural aromatic and unnatural aromatic and nonaromatic amino acids. All the compounds were evaluated for their ability to inhibit vascular relaxation induced by KRX-725 (as S1P3 selective pepducin agonist) and KRX-722 (an S1P1-selective pepducin agonist). Those selective towards S1P3 (compounds V and VII) were also evaluated for their ability to inhibit skeletal muscle fibrosis. Finally, molecular dynamics simulations were performed to derive information on the preferred conformations of selective and unselective antagonists

Exploration of Long-Chain Vitamin E Metabolites for the Discovery of a Highly Potent, Orally Effective, and Metabolically Stable 5-LOX Inhibitor that Limits Inflammation.

Endogenous long-chain metabolites of vitamin E (LCMs) mediate immune functions by targeting 5-lipoxygenase (5-LOX) and increasing the systemic concentrations of resolvin E3, a specialized proresolving lipid mediator. SAR studies on semisynthesized analogues highlight α-amplexichromanol (27a), which allosterically inhibits 5-LOX, being considerably more potent than endogenous LCMs in human primary immune cells and blood. Other enzymes within lipid mediator biosynthesis were not substantially inhibited, except for microsomal prostaglandin E2 synthase-1. Compound 27a is metabolized by sulfation and β-oxidation in human liver-on-chips and exhibits superior metabolic stability in mice over LCMs. Pharmacokinetic studies show distribution of 27a from plasma to the inflamed peritoneal cavity and lung. In parallel, 5-LOX-derived leukotriene levels decrease, and the inflammatory reaction is suppressed in reconstructed human epidermis, murine peritonitis, and experimental asthma in mice. Our study highlights 27a as an orally active, LCM-inspired drug candidate that limits inflammation with superior potency and metabolic stability to the endogenous lead

In Vitro and In Vivo Antagonism of a G Protein-Coupled Receptor (S1P3) with a Novel Blocking Monoclonal Antibody

Background: S1P 3 is a lipid-activated G protein-couple receptor (GPCR) that has been implicated in the pathological processes of a number of diseases, including sepsis and cancer. Currently, there are no available high-affinity, subtypeselective drug compounds that can block activation of S1P3. We have developed a monoclonal antibody (7H9) that specifically recognizes S1P3 and acts as a functional antagonist. Methodology/Principal Findings: Specific binding of 7H9 was demonstrated by immunocytochemistry using cells that over-express individual members of the S1P receptor family. We show, in vitro, that 7H9 can inhibit the activation of S1P3mediated cellular processes, including arrestin translocation, receptor internalization, adenylate cyclase inhibiton, and calcium mobilization. We also demonstrate that 7H9 blocks activation of S1P3 in vivo, 1) by preventing lethality due to systemic inflammation, and 2) by altering the progression of breast tumor xenografts. Conclusions/Significance: We have developed the first-reported monoclonal antibody that selectively recognizes a lipidactivated GPCR and blocks functional activity. In addition to serving as a lead drug compound for the treatment of sepsi

Expression and Functional Roles of Angiopoietin-2 in Skeletal Muscles

Angiopoietin-1 (ANGPT1) and angiopoietin-2 (ANGPT2) are angiogenesis factors that modulate endothelial cell differentiation, survival and stability. Recent studies have suggested that skeletal muscle precursor cells constitutively express ANGPT1 and adhere to recombinant ANGPT1 and ANGPT2 proteins. It remains unclear whether or not they also express ANGPT2, or if ANGPT2 regulates the myogenesis program of muscle precursors. In this study, ANGPT2 regulatory factors and the effects of ANGPT2 on proliferation, migration, differentiation and survival were identified in cultured primary skeletal myoblasts. The cellular networks involved in the actions of ANGPT2 on skeletal muscle cells were also analyzed.Primary skeletal myoblasts were isolated from human and mouse muscles. Skeletal myoblast survival, proliferation, migration and differentiation were measured in-vitro in response to recombinant ANGPT2 protein and to enhanced ANGPT2 expression delivered with adenoviruses. Real-time PCR and ELISA measurements revealed the presence of constitutive ANGPT2 expression in these cells. This expression increased significantly during myoblast differentiation into myotubes. In human myoblasts, ANGPT2 expression was induced by H(2)O(2), but not by TNFα, IL1β or IL6. ANGPT2 significantly enhanced myoblast differentiation and survival, but had no influence on proliferation or migration. ANGPT2-induced survival was mediated through activation of the ERK1/2 and PI-3 kinase/AKT pathways. Microarray analysis revealed that ANGPT2 upregulates genes involved in the regulation of cell survival, protein synthesis, glucose uptake and free fatty oxidation.Skeletal muscle precursors constitutively express ANGPT2 and this expression is upregulated during differentiation into myotubes. Reactive oxygen species exert a strong stimulatory influence on muscle ANGPT2 expression while pro-inflammatory cytokines do not. ANGPT2 promotes skeletal myoblast survival and differentiation. These results suggest that muscle-derived ANGPT2 production may play a positive role in skeletal muscle fiber repair

Hypertension Is Associated with Marked Alterations in Sphingolipid Biology: A Potential Role for Ceramide

Background Hypertension is, amongst others, characterized by endothelial dysfunction and vascular remodeling. As sphingolipids have been implicated in both the regulation of vascular contractility and growth, we investigated whether sphingolipid biology is altered in hypertension and whether this is reflected in altered vascular function. Methods and Findings In isolated carotid arteries from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats, shifting the ceramide/S1P ratio towards ceramide dominance by administration of a sphingosine kinase inhibitor (dimethylsphingosine) or exogenous application of sphingomyelinase, induced marked endothelium-dependent contractions in SHR vessels (DMS: 1.4±0.4 and SMase: 2.1±0.1 mN/mm; n = 10), that were virtually absent in WKY vessels (DMS: 0.0±0.0 and SMase: 0.6±0.1 mN/mm; n = 9, p Conclusions Hypertension is associated with marked alterations in vascular sphingolipid biology such as elevated ceramide levels and signaling, that contribute to increased vascular tone