Reduced caveolae density in arteries of SHR contributes to endothelial dysfunction and ROS production

Caveolae are plasma membrane invaginations enriched with high cholesterol and sphingolipid content; they also contain caveolin proteins in their structure. Endothelial nitric oxide synthase (eNOS), an enzyme that synthesizes nitric oxide (NO) by converting L-arginine to L-citrulline, is highly concentrated in plasma membrane caveolae. Hypertension is associated with decreased NO production and impaired endothelium-dependent relaxation. Understanding the molecular mechanisms that follow hypertension is important. For this study, we hypothesized that spontaneously hypertensive rat (SHR) vessels should have a smaller number of caveolae, and that the caveolae structure should be disrupted in these vessels. This should impair the eNOS function and diminish NO bioavailability. Therefore, we aimed to investigate caveolae integrity and density in SHR aortas and mesenteric arteries and the role played by caveolae in endothelium-dependent relaxation. We have been able to show the presence of caveolae-like structures in SHR aortas and mesenteric arteries. Increased phenylephrine-induced contractile response after treatment with dextrin was related to lower NO release. In addition, impaired acetylcholine-induced endothelium-dependent relaxation could be related to decreased caveolae density in SHR vessels. The most important finding of this study was that cholesterol depletion with dextrin induced eNOS phosphorylation at Serine1177 (Ser1177) and boosted reactive oxygen species (ROS) production in normotensive rat and SHR vessels, which suggested eNOS uncoupling. Dextrin plus L-NAME or BH4 decreased ROS production in aorta and mesenteric arteries supernatant's of both SHR and normotensive groups. Human umbilical vein endothelial cells (HUVECs) treated with dextrin confirmed eNOS uncoupling, as verified by the reduced eNOS dimer/monomer ratio. BH4, L-arginine, or BH4 plus L-arginine inhibited eNOS monomerization. All these results showed that caveolae structure and integrity are essential for endothelium-dependent relaxation. Additionally, a smaller number of caveolae is associated with hypertension. Finally, caveolae disruption promotes eNOS uncoupling in normotensive and hypertensive rat vessels and in HUVECs.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2016/22180-9, 2016/21239-0, 2017/14797-9]; Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [400.164/2014-0, 304.137/2014-6]Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

The Glycocalyx and Pressure-Dependent Transcellular Albumin Transport

Purpose Acute increases in hydrostatic pressure activate endothelial signaling pathways that modulate barrier function and vascular permeability. We investigated the role the glycocalyx and established mechanotransduction pathways in pressure-induced albumin transport across rat lung microvascular endothelial cells. Methods Rat lung microvascular endothelial cells (RLMEC) were cultured on Costar Snapwell chambers. Cell morphology was assessed using silver nitrate staining. RLMEC were exposed to zero pressure (Control) or 30 cmH(2)O (Pressure) for 30 or 60 min. Intracellular albumin uptake and transcellular albumin transport was quantified. Transcellular transport was reported as solute flux (J(s)) and an effective permeability coefficient (P-e). The removal of cell surface heparan sulfates (heparinase), inhibition of NOS (L-NAME) and reactive oxygen species (apocynin, Apo) was investigated. Results Acute increase in hydrostatic pressure augmented albumin uptake by 30-40% at 60 min and J(s)and P(e)both increased significantly. Heparinase increased albumin uptake but attenuated transcellular transport while L-NAME attenuated both pressure-dependent albumin uptake and transport. Apo interrupted albumin uptake under both control and pressure conditions, leading to a near total lack of transcellular transport, suggesting a different mechanism and/or site of action. Conclusion Pressure-dependent albumin uptake and transcellular transport is another component of endothelial mechanotransduction and associated regulation of solute flux. This novel albumin uptake and transport pathway is regulated by heparan sulfates and eNOS. Albumin uptake is sensitive to ROS. The physiological and clinical implications of this albumin transport are discussed.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Norepinephrine Induces Lung Microvascular Endothelial Cell Death by NADPH Oxidase-Dependent Activation of Caspase-3

Norepinephrine (NE) is the naturally occurring adrenergic agonist that is released in response to hypotension, and it is routinely administered in clinical settings to treat moderate to severe hypotension that may occur during general anesthesia and shock states. Although NE has incontestable beneficial effects on blood pressure maintenance during hypotensive conditions, deleterious effects of NE on endothelial cell function may occur. In particular, the role of reactive oxygen species (ROS) and NADPH oxidase (Nox) on the deleterious effects of NE on endothelial cell function have not been fully elucidated. Therefore, we investigated the effects of NE on ROS production in rat lung microvascular endothelial cells (RLMEC) and its contribution to cell death. RLMEC were treated with NE (5 ng/mL) for 24 hours and ROS production was assessed by CellROX and DCFDA fluorescence. Nox activity was assessed by NADPH-stimulated ROS production in isolated membranes and phosphorylation of p47phox; cell death was assessed by flow cytometry and DNA fragmentation. Caspase activation was assessed by fluorescent microscopy. Nox1, Nox2, and Nox4 mRNA expression was assessed by real-time PCR. NE increased ROS production, Nox activity, p47phox phosphorylation, Nox2 and Nox4 mRNA content, caspase-3 activation, and RLMEC death. Phentolamine, an α1-adrenoreceptor antagonist, inhibited NE-induced ROS production and Nox activity and partly inhibited cell death while β-blockade had no effect. Apocynin and PEGSOD inhibited NE-induced caspase-3 activation and cell death while direct inhibition of caspase-3 abrogated NE-induced cell death. PEG-CAT inhibited NE-induced cell death but not caspase-3 activation. Collectively, these results indicate that NE induces RLMEC death via activation of Nox by α-adrenergic signaling and caspase-3-dependent pathways. NE has deleterious effects on RLMECs that may be important to its long-term therapeutic use

Testosterone induces leucocyte migration by NADPH oxidase-driven ROS- and COX2-dependent mechanisms

The mechanisms whereby testosterone increases cardiovascular risk are not clarified. However, oxidative stress and inflammation seem to be determinants. Herein, we sought to determine whether exogenous testosterone, at physiological levels, induces leucocyte migration, a central feature in immune and inflammatory responses and the mediating mechanisms. We hypothesized that testosterone induces leucocyte migration via NADPH oxidase (NADPHox)-driven reactive oxygen species (ROS) and cyclooxygenase (COX)-dependent mechanisms. Sixteen-week-old Wistar rats received an intraperitoneal injection (5 ml) of either testosterone (10−7 mol/l) or saline. Rats were pre-treated with 5 ml of sodium salicylate (SS, non-selective COX inhibitor, 1.25×10−3 mol/l, 1 h prior to testosterone or saline), flutamide (androgen receptor antagonist, 10−5 mol/l), apocynin (NADPHox inhibitor, 3×10−4 mol/l), N-[2-Cyclohexyloxy-4-nitrophenyl]methanesulfonamide (NS398, COX2 inhibitor, 10−4 mol/l) or saline, 4 h before testosterone or saline administration. Leucocyte migration was assessed 24 h after testosterone administration by intravital microscopy of the mesenteric bed. Serum levels of testosterone were measured by radioimmunoassay. NADPHox activity was assessed in membrane fractions of the mesenteric bed by dihydroethidium (DHE) fluorescence and in isolated vascular smooth muscle cells (VSMC) by HPLC. NADPHox subunits and VCAM (vascular cell adhesion molecule) expression were determined by immunoblotting. Testosterone administration did not change serum levels of endogenous testosterone, but increased venular leucocyte migration to the adventia, NADPHox activity and expression (P<0.05). These effects were blocked by flutamide. SS inhibited testosterone-induced leucocyte migration (P<0.05). Apocynin and NS398 abolished testosterone-induced leucocyte migration and NADPHox activity (P<0.05). Testosterone induces leucocyte migration via NADPHox- and COX2-dependent mechanisms and may contribute to inflammatory processes and oxidative stress in the vasculature potentially increasing cardiovascular risk

The Glycocalyx and Trauma: A Review

In the United States trauma is the leading cause of mortality among those under the age of 45, claiming approximately 192,000 lives each year. Significant personal disability, lost productivity, and long-term healthcare needs are common and contribute 580 billion dollars in economic impact each year. Improving resuscitation strategies and the early acute care of trauma patients has the potential to reduce the pathological sequelae of combined exuberant inflammation and immune suppression that can co-exist, or occur temporally, and adversely affect outcomes. The endothelial and epithelial glycocalyx has emerged as an important participant in both inflammation and immunomodulation. Constituents of the glycocalyx have been used as biomarkers of injury severity and have the potential to be target(s) for therapeutic interventions aimed at immune modulation. In this review, we provide a contemporary understanding of the physiologic structure and function of the glycocalyx and its role in traumatic injury with a particular emphasis on lung injury.Wo

Ropivacaine attenuates endotoxin plus hyperinflation-mediated acute lung injury via inhibition of early-onset Src-dependent signaling

BACKGROUND: Acute lung injury (ALI) is associated with high mortality due to the lack of effective therapeutic strategies. Mechanical ventilation itself can cause ventilator-induced lung injury. Pulmonary vascular barrier function, regulated in part by Src kinase-dependent phosphorylation of caveolin-1 and intercellular adhesion molecule-1 (ICAM-1), plays a crucial role in the development of protein-/neutrophil-rich pulmonary edema, the hallmark of ALI. Amide-linked local anesthetics, such as ropivacaine, have anti-inflammatory properties in experimental ALI. We hypothesized ropivacaine may attenuate inflammation in a "double-hit" model of ALI triggered by bacterial endotoxin plus hyperinflation via inhibition of Src-dependent signaling. METHODS: C57BL/6 (WT) and ICAM-1 (-/-) mice were exposed to either nebulized normal saline (NS) or lipopolysaccharide (LPS, 10 mg) for 1 hour. An intravenous bolus of 0.33 mg/kg ropivacaine or vehicle was followed by mechanical ventilation with normal (7 ml/kg, NTV) or high tidal volume (28 ml/kg, HTV) for 2 hours. Measures of ALI (excess lung water (ELW), extravascular plasma equivalents, permeability index, myeloperoxidase activity) were assessed and lungs were homogenized for Western blot analysis of phosphorylated and total Src, ICAM-1 and caveolin-1. Additional experiments evaluated effects of ropivacaine on LPS-induced phosphorylation/expression of Src, ICAM-1 and caveolin-1 in human lung microvascular endothelial cells (HLMVEC). RESULTS: WT mice treated with LPS alone showed a 49% increase in ELW compared to control animals (p = 0.001), which was attenuated by ropivacaine (p = 0.001). HTV ventilation alone increased measures of ALI even more than LPS, an effect which was not altered by ropivacaine. LPS plus hyperinflation ("double-hit") increased all ALI parameters (ELW, EVPE, permeability index, MPO activity) by 3-4 fold compared to control, which were again decreased by ropivacaine. Western blot analyses of lung homogenates as well as HLMVEC treated in culture with LPS alone showed a reduction in Src activation/expression, as well as ICAM-1 expression and caveolin-1 phosphorylation. In ICAM-1 (-/-) mice, neither addition of LPS to HTV ventilation alone nor ropivacaine had an effect on the development of ALI. CONCLUSIONS: Ropivacaine may be a promising therapeutic agent for treating the cause of pulmonary edema by blocking inflammatory Src signaling, ICAM-1 expression, leukocyte infiltration, and vascular hyperpermeability

Testosterone induces vascular smooth muscle cell migration by NADPH oxidase and c-Src-dependent pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10−7 mol/L, 0–120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase–derived ROS and c-Src–dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs