An Angiotensin I-Converting Enzyme Mutation (Y465D) Causes a Dramatic Increase in Blood ACE via Accelerated ACE Shedding

Angiotensin I-converting enzyme (ACE) metabolizes a range of peptidic substrates and plays a key role in blood pressure regulation and vascular remodeling. Thus, elevated ACE levels may be associated with an increased risk for different cardiovascular or respiratory diseases. Previously, a striking familial elevation in blood ACE was explained by mutations in the ACE juxtamembrane region that enhanced the cleavage-secretion process. Recently, we found a family whose affected members had a 6-fold increase in blood ACE and a Tyr465Asp (Y465D) substitution, distal to the stalk region, in the N domain of ACE.HEK and CHO cells expressing mutant (Tyr465Asp) ACE demonstrate a 3- and 8-fold increase, respectively, in the rate of ACE shedding compared to wild-type ACE. Conformational fingerprinting of mutant ACE demonstrated dramatic changes in ACE conformation in several different epitopes of ACE. Cell ELISA carried out on CHO-ACE cells also demonstrated significant changes in local ACE conformation, particularly proximal to the stalk region. However, the cleavage site of the mutant ACE--between Arg1203 and Ser1204--was the same as that of WT ACE. The Y465D substitution is localized in the interface of the N-domain dimer (from the crystal structure) and abolishes a hydrogen bond between Tyr465 in one monomer and Asp462 in another.The Y465D substitution results in dramatic increase in the rate of ACE shedding and is associated with significant local conformational changes in ACE. These changes could result in increased ACE dimerization and accessibility of the stalk region or the entire sACE, thus increasing the rate of cleavage by the putative ACE secretase (sheddase)

Role of Caveolae Mediated Transport for Abraxane Uptake in vivo

An in vivo study was conducted to determine whether the chemotherapeutic drug Abraxane is taken up by tissues and tumors via caveolae-mediated endocytosis and transcytosis. Cancer is one of the leading causes of death worldwide and a global health concern due to the increase in global death rates reported each year. A major focus of basic and clinical research is to find new drug delivery systems that increase the therapeutic efficacy of chemotherapeutic agents while reducing the associated toxicity in normal tissues. In the present study, we focused on the mechanism of tissue uptake of the chemotherapeutic drug Abraxane, a newly developed drug currently used for treatment for breast cancer. Abraxane is an albumin-bound paclitaxel conjugate specifically designed to replace the commonly used paclitaxel formulation, Taxol, which is delivered in a cremophor-ethanol solution. Taxol requires long infusion cycles and is associated with highly toxic side effects that have been associated with this drug formulation. The creators of Abraxane proposed internalization into the tissue and tumors is via a caveolin-1 mediated transport pathway. Our experimental design included the use of murine models, wild-type B6/129 female mice as compared to female caveolin-1 knockout mice, which were intravenously injected with either paclitaxel suspended in 1% albumin-saline solution or Abraxane suspended in saline. Blood and tissues were collected and processed for analysis of paclitaxel by liquid chromatography and mass spectrometry. The results obtained showed a higher accumulation of Abraxane in the liver, kidney, and breast tissues while paclitaxel uptake was greater in the lung and liver tissues. Plasma levels were higher for Abraxane, suggesting it has a longer circulation time in the blood. Both Abraxane and paclitaxel accumulated at low levels in the brain suggesting neither is able to cross the blood-brain barrier. Finally, there were no significant differences in the accumulation of either paclitaxel formulation between wild type and knockout mice. We conclude from this study that the uptake and transport of Abraxane in normal tissues is not mediated by caveolae-dependent mechanisms and that the beneficial effects of the albumin-paclitaxel conjugation of Abraxane may have more to do with its greater bioavailability

Volatile anesthetics improve survival after cecal ligation and puncture

BACKGROUND: Sepsis remains a leading cause of death in intensive care units. There is growing evidence that volatile anesthetics have beneficial immunomodulatory effects on complex inflammation-mediated conditions. The authors investigated the effect of volatile anesthetics on the overall survival of mice in a sepsis model of cecal ligation and puncture (CLP). METHODS: Mice (N = 12 per treatment group) were exposed to anesthetic concentrations of desflurane, isoflurane, and sevoflurane either during induction of sepsis or when the mice showed pronounced symptoms of inflammation. Overall survival, as well as organ function and inflammation was compared with the CLP group without intervention. RESULTS: With desflurane and sevoflurane conditioning (1.2 minimal alveolar concentration for 2 h immediately after induction of CLP) overall survival was improved to 58% and 83%, respectively, compared with 17% in the untreated CLP group. Isoflurane did not significantly affect outcome. Application of sevoflurane 24 h after sepsis induction significantly improved overall survival to 66%. CONCLUSIONS: Administration of the volatile anesthetics desflurane and sevoflurane reduced CLP-induced mortality. Anesthesia may be a critical confounder when comparing study data where different anesthesia protocols were used

Hypoxia‐induced pulmonary hypertension upregulates eNOS and TGF‐β contributing to sex‐linked differences in BMPR2+/R899X mutant mice

Abstract Dysfunctional bone morphogenetic protein receptor 2 (BMPR2) and endothelial nitric oxide synthase (eNOS) have been largely implicated in the pathogenesis of pulmonary arterial hypertension (PAH); a life‐threatening cardiopulmonary disease. Although the incident of PAH is about three times higher in females, males with PAH usually have a worse prognosis, which seems to be dependent on estrogen‐associated cardiac and vascular protection. Here, we evaluated whether hypoxia‐induced pulmonary hypertension (PH) in humanized BMPR2+/R899X loss‐of‐function mutant mice contributes to sex‐associated differences observed in PAH by altering eNOS expression and inducing expansion of hyperactivated TGF‐β‐producing pulmonary myofibroblasts. To test this hypothesis, male and female wild‐type (WT) and BMPR2+/R899X mutant mice were kept under hypoxic or normoxic conditions for 4 weeks, and then right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were measured. Chronic hypoxia exposure elevated RVSP, inducing RVH in both groups, with a greater effect in BMPR2+/R899X female mice. Lung histology revealed no differences in vessel thickness/area between sexes, suggesting RVSP differences in this model are unlikely to be in response to sex‐dependent vascular narrowing. On the other hand, hypoxia exposure increased vascular collagen deposition, the number of TGF‐β‐associated α‐SMA‐positive microvessels, and eNOS expression, whereas it also reduced caveolin‐1 expression in the lungs of BMPR2+/R899X females compared to males. Taken together, this brief report reveals elevated myofibroblast‐derived TGF‐β and eNOS‐derived oxidants contribute to pulmonary microvascular muscularization and sex‐linked differences in incidence, severity, and outcome of PAH

Role of Caveolin-1 Expression in the Pathogenesis of Pulmonary Edema in Ventilator-Induced Lung Injury

Caveolin-1 is a key regulator of pulmonary endothelial barrier function. Here, we tested the hypothesis that caveolin-1 expression is required for ventilator-induced lung injury (VILI). Caveolin-1 gene-disrupted (Cav-1(-/-)) and age-, sex-, and strain-matched wild-type (WT) control mice were ventilated using two protocols: volume-controlled with protective (8 mL/kg) versus injurious (21 mL/Kg) tidal volume for up to 6 hours; and pressure-controlled with protective (airway pressure = 12 cm H(2)O) versus injurious (30 cm H(2)O) ventilation to induce lung injury. Lung microvascular permeability (whole-lung (125)I-albumin accumulation, lung capillary filtration coefficient [K(f, c)]) and inflammatory markers (bronchoalveolar lavage [BAL] cytokine levels and neutrophil counts) were measured. We also evaluated histologic sections from lungs, and the time course of Src kinase activation and caveolin-1 phosphorylation. VILI induced a 1.7-fold increase in lung (125)I-albumin accumulation, fourfold increase in K(f, c), significantly increased levels of cytokines CXCL1 and interleukin-6, and promoted BAL neutrophilia in WT mice. Lung injury by these criteria was significantly reduced in Cav-1(-/-) mice but fully restored by i.v. injection of liposome/Cav-1 cDNA complexes that rescued expression of Cav-1 in lung microvessels. As thrombin is known to play a significant role in mediating stretch-induced vascular injury, we observed in cultured mouse lung microvascular endothelial cells (MLECs) thrombin-induced albumin hyperpermeability and phosphorylation of p44/42 MAP kinase in WT but not in Cav-1(-/-) MLECs. Thus, caveolin-1 expression is required for mechanical stretch-induced lung inflammation and endothelial hyperpermeability in vitro and in vivo

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

Survival analysis.

<p>Seven day survival of CLP mice, CLP mice receiving HFIP immediately after CLP induction (CLP+HFIP, N = 12) (CLP vs. CLP+HFIP, p = 0.037) and CLP animals receiving HFIP 24 hours after sepsis induction (CLP+HFIP postcond., 24 h, N = 12) (CLP vs. CLP+HFIP postcond., 24 h, ns) CLP: cecal ligation and puncture. HFIP: hexafluoroisopropanol. ns: non –significant.</p

Organ damage and Inflammation.

<p>Plasma levels of organ damage markers (blood urea nitrogen (BUN), alanine aminotransferase (ALT) and aspartate aminotransferase (AST)) (<b>A</b>) and inflammatory mediators (interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1) and high mobility group box protein-1 (HMGB-1)) (<b>B</b>) for the experimental groups (N = 6) 24 hours after sepsis induction. CLP: cecal ligation and puncture. HFIP: hexafluoroisopropanol. IL-6: * denotes p<0.05 compared to SHAM.</p

Intravenous application of a primary sevoflurane metabolite improves outcome in murine septic peritonitis: first results

Volatile anesthetics are known to have immunomodulatory effects in conditions of organ injury. A recent study in an experimental sepsis model has shown remarkably improved survival when mice were exposed to volatile anesthetics. In the present study, we show that hexafluoroisopropanol - a water-soluble primary sevoflurane metabolite - has beneficial effects on the overall survival in a murine model of cecal ligation and puncture. Seven-day survival as well as tissue damage markers including transaminases and high mobility group box protein-1 were assessed as measures of end organ damage. In animals undergoing cecal ligation and puncture procedure hexafluoroisopropanol conditioning - but not late postconditioning 24 hours after sepsis induction - significantly increased survival rate (17% vs. 77%, p = 0.037) and attenuated secretion of organ damage markers. This study shows survival benefits by administration of the metabolite of a volatile anesthetic. If successfully translated, hexafluoroisopropanol might offer interesting therapeutic opportunities in the future treatment of abdominal sepsis