Vascular Smooth Muscle Remodeling in Conductive and Resistance Arteries in Hypertension.

Cardiovascular disease is a leading cause of death worldwide and accounts for >17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 2030. 1 Cardiovascular death represents 31% of all global deaths 2 -with stroke, heart attack, and ruptured aneurysms predominantly contributing to these high mortality rates. A key risk factor for cardiovascular disease is hypertension. Although treatment or reduction in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially effective. A key pathological hallmark of hypertension is increased peripheral vascular resistance because of structural and functional changes in large (conductive) and small (resistance) arteries. In this review, we discuss the clinical implications of vascular remodeling, compare the differences between vascular smooth muscle cell remodeling in conductive and resistance arteries, discuss the genetic factors associated with vascular smooth muscle cell function in hypertensive patients, and provide a prospective assessment of current and future research and pharmacological targets for the treatment of hypertension.

The antioxidant glutathione protects against enteric neuron death in situ, but its depletion is protective during colitis.

Enteric glia play an important neuroprotective role in the enteric nervous system (ENS) by producing neuroprotective compounds such as the antioxidant reduced glutathione (GSH). The specific cellular pathways that regulate glial production of GSH and how these pathways are altered during, or contribute to, neuroinflammation in situ and in vivo are not fully understood. We investigated this issue using immunohistochemistry to localize GSH synthesis enzymes within the myenteric plexus and tested how the inhibition of GSH synthesis with the selective inhibitor l-buthionine sulfoximine impacts neuronal survival and inflammation. Both enteric glia and neurons express the cellular machinery necessary for GSH synthesis. Furthermore, glial GSH synthesis is necessary for neuronal survival in isolated preparations of myenteric plexus. In vivo depletion of GSH does not induce colitis but alters myenteric plexus neuronal phenotype and survival. Importantly, global depletion of glutathione is protective against some macroscopic and microscopic measures of colonic inflammation. Together, our data highlight the heterogeneous roles of GSH in the myenteric plexus of the ENS and during gastrointestinal inflammation. NEW & NOTEWORTHY Our results show that both enteric glia and neurons express the cellular machinery necessary for glutathione (GSH) synthesis and that glial GSH synthesis is necessary for neuronal survival in isolated enteric nervous system (ENS) preparations. In vivo depletion of GSH with the selective inhibitor l-buthionine sulfoximine is not sufficient to induce inflammation but does alter neuronal neurochemical composition and survival. Together, our data highlight novel heterogeneous roles for GSH in the ENS and during gastrointestinal inflammation.

Sirtuin-3 Is Expressed by Enteric Neurons but It Does not Play a Major Role in Their Regulation of Oxidative Stress.

Gut inflammation contributes to the development of gut motility disorders in part by disrupting the function and survival of enteric neurons through mechanisms that involve oxidative stress. How enteric neurons regulate oxidative stress is still poorly understood. Importantly, how neuron autonomous antioxidant mechanisms contribute to the susceptibility of enteric neurons to oxidative stress in disease is not known. Here, we discover that sirtuin-3 (Sirt3), a key regulator of oxidative stress and mitochondrial metabolism, is expressed by neurons in the enteric nervous system (ENS) of the mouse colon. Given the important role of Sirt3 in the regulation of neuronal oxidative stress in the central nervous system (CNS), we hypothesized that Sirt3 plays an important role in the cell autonomous regulation of oxidative stress by enteric neurons and that a loss of Sirt3 increases neuronal vulnerability during intestinal inflammation. We tested our hypothesis using a combination of traditional immunohistochemistry, oxidative stress measurements and in vivo and ex vivo measures of GI motility in healthy and inflamed wild-type (wt) and Sirt3 null (Sirt3 (-/-)) mice. Our results show that Sirt3 is widely expressed by neurons throughout the myenteric plexus of the mouse colon. However, the deletion of Sirt3 had surprisingly little effect on gut function and susceptibility to inflammation. Likewise, neither the genetic ablation of Sirt3 nor the inhibition of Sirt3 with antagonists had a significant effect on neuronal oxidative stress. Therefore, we conclude that Sirt3 contributes very little to the overall regulation of neuronal oxidative stress in the ENS. The functional relevance of Sirt3 in enteric neurons is still unclear but our data show that it is an unlikely candidate to explain neuronal vulnerability to oxidative stress during inflammation.

Enteric glia mediate neuron death in colitis through purinergic pathways that require connexin-43 and nitric oxide.

BACKGROUND AND AIMS: The concept of enteric glia as regulators of intestinal homeostasis is slowly gaining acceptance as a central concept in neurogastroenterology. Yet how glia contribute to intestinal disease is still poorly understood. Purines generated during inflammation drive enteric neuron death by activating neuronal P2X7 purine receptors (P2X7R), triggering ATP release via neuronal pannexin-1 channels that subsequently recruits intracellular calcium ([Ca2+]i) responses in the surrounding enteric glia. We tested the hypothesis that the activation of enteric glia contributes to neuron death during inflammation. METHODS: We studied neuroinflammation in vivo using the 2,4-dinitrobenzenesulfonic acid model of colitis and in situ using whole-mount preparations of human and mouse intestine. Transgenic mice with a targeted deletion of glial connexin-43 (Cx43) [GFAP∷CreERT2+/-/Cx43f/f ] were used to specifically disrupt glial signaling pathways. Mice deficient in inducible nitric oxide (NO) synthase (iNOS-/-) were used to study NO production. Protein expression and oxidative stress were measured using immunohistochemistry and in situ Ca2+ and NO imaging were used to monitor glial [Ca2+]i and [NO]i. RESULTS: Purinergic activation of enteric glia drove [Ca2+]i responses and enteric neuron death through a Cx43-dependent mechanism. Neurotoxic Cx43 activity, driven by NO production from glial iNOS, was required for neuron death. Glial Cx43 opening liberated ATP and Cx43-dependent ATP release was potentiated by NO. CONCLUSIONS: Our results show that the activation of glial cells in the context of neuroinflammation kills enteric neurons. Mediators of inflammation that include ATP and NO activate neurotoxic pathways that converge on glial Cx43 hemichannels. The glial response to inflammatory mediators might contribute to the development of motility disorders.

Novel diagnostic assays for heparin-induced thrombocytopenia.

Laboratory testing for heparin-induced thrombocytopenia (HIT) has important shortcomings. Immunoassays fail to discriminate platelet-activating from nonpathogenic antibodies. Specific functional assays are impracticable due to the need for platelets and radioisotope. We describe 2 assays that may overcome these limitations. The KKO-inhibition test (KKO-I) measures the effect of plasma on binding of the HIT-like monoclonal antibody KKO to platelet factor 4 (PF4)/heparin. DT40-luciferase (DT40-luc) is a functional test comprised of a B-cell line expressing FcγRIIa coupled to a luciferase reporter. We compared these assays to polyspecific and immunoglobulin (Ig)G-specific PF4/heparin enzyme-linked immunosorbent assays (ELISAs) in samples from 58 patients with suspected HIT and circulating anti-PF4/heparin antibodies. HIT was defined as a 4Ts score ≥ 4 and positive (14)C-serotonin release assay. HIT-positive plasma demonstrated greater mean inhibition of KKO binding than HIT-negative plasma (78.9% vs 26.0%; P < .0001) and induced greater luciferase activity (3.14-fold basal vs 0.96-fold basal; P < .0001). The area under the receiver-operating characteristic curve was greater for KKO-I (0.93) than for the polyspecific (0.82; P = .020) and IgG-specific ELISA (0.76; P = .0044) and for DT40-luc (0.89) than for the IgG-specific ELISA (P = .046). KKO-I and DT40-luc showed better discrimination than 2 commercially available immunoassays, are simple to perform, and hold promise for improving the specificity and feasibility of HIT laboratory testing.

Solubilization of a membrane protein by combinatorial supercharging.

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Uso do suporte ventilatório com pressäo positiva contínua em vias aéreas (CPAP) por meio de máscara nasofacial no tratamento da insuficiência respiratória aguda / Continuous positive airway pressure (CPAP) by face mask to treat acute respiratory failure

Objetivo. O objetivo dos autores foi avaliar o efeito da ventilaçäo com CPAP oferecida por meio de máscara nasofacial como método de suporte ventilatório em pacientes com insuficiência respiratória aguda com critérios de indicaçäo para intubaçäo traqueal. Casuística e Método. Foram estudados 11 pacientes com idade média de 41,3 anos em insuficiência respiratória aguda internados na Unidade Respiratória do Hospital Säo Paulo - Escola Paulista de Medicina. A admissäo, era colhida gasometria arterial em ar ambiente e monitorizava-se freqüência respiratória (f), freqüência cardíaca (FC) e pressäo arterial (PA). Os mesmos parâmetros eram avaliados após oxigenoterapia via máscara facial aberta e com máscara facial de CPAP usando PEEP de 5cm H2O. Resultados. Com o uso de CPAP através de máscara nasofacial, houve melhora significativa dos níveis de PaO2 e diminuiçäo da freqüência respiratória (<0,05), quando comparados aos valores em ar ambiemte e com máscara facial aberta. Conclusäo. Este trabalho permitiu concluir que a máscara facial de CPAP com 5cm H2O foi eficiente em melhorar a oxigenaçäo arterial e diminuir a freqüência respiratória dos pacientes com insuficiência respiratória aguda, proporcionando-lhes maior conforto, constituindo uma medida terapêutica capaz de evitar o suporte ventilatório invasivo.

[Continuous positive airway pressure (CPAP) with face mask to treat acute respiratory failure]. / Uso do suporte ventilatório com pressão positiva contínua em vias aéreas (CPAP) por meio de máscara nasofacial no tratamento da insuficiência respiratória aguda.

OBJECTIVE: To evaluate the efficacy of continuous positive airway pressure (CPAP) administered by face mask in patients with acute respiratory failure with indication of mechanical ventilation. MATERIALS AND METHOD: Eleven patients (mean age 41.3 years) in acute respiratory failure were admitted in the Respiratory Unit--Hospital São Paulo--Escola Paulista de Medicina. At the admission pH, PaO2, PaCO2, respiratory rate (f), arterial pressure and heart rate were measured. The measurements were repeated with administration of oxygen with a high flow face mask at ambient airway pressure and then with 5 cm H2O of CPAP by face mask. RESULTS: The use of CPAP by face mask significantly improved the arterial blood oxygenation and decreased the respiratory rate (p < 0.05). CONCLUSION: These data allow the conclusion that CPAP administered by face mask improves gas exchange and decreases respiratory rate in patients with acute respiratory failure and may reduce the need for invasive mechanical ventilation.