199 research outputs found
Complementary effects of extracellular nucleotides and platelet-derived extracts on angiogenesis of vasa vasorum endothelial cells in vitro and subcutaneous Matrigel plugs in vivo
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Stable isotope metabolomics of pulmonary artery smooth muscle and endothelial cells in pulmonary hypertension and with TGF-beta treatment.
Altered metabolism in pulmonary artery smooth muscle cells (PASMCs) and endothelial cells (PAECs) contributes to the pathology of pulmonary hypertension (PH), but changes in substrate uptake and how substrates are utilized have not been fully characterized. We hypothesized stable isotope metabolomics would identify increased glucose, glutamine and fatty acid uptake and utilization in human PASMCs and PAECs from PH versus control specimens, and that TGF-Ξ² treatment would phenocopy these metabolic changes. We used 13C-labeled glucose, glutamine or a long-chain fatty acid mixture added to cell culture media, and mass spectrometry-based metabolomics to detect and quantify 13C-labeled metabolites. We found PH PASMCs had increased glucose uptake and utilization by glycolysis and the pentose shunt, but no changes in glutamine or fatty acid uptake or utilization. Diseased PAECs had increased proximate glycolysis pathway intermediates, less pentose shunt flux, increased anaplerosis from glutamine, and decreased fatty acid Ξ²-oxidation. TGF-Ξ² treatment increased glycolysis in PASMCs, but did not recapitulate the PAEC disease phenotype. In TGF-Ξ²-treated PASMCs, glucose, glutamine and fatty acids all contributed carbons to the TCA cycle. In conclusion, PASMCs and PAECs collected from PH subjects have significant changes in metabolite uptake and utilization, partially recapitulated by TGF-Ξ² treatment
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The Pulmonary Vascular Research Institute celebrates its first decade.
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Adenosine A1 Receptors Promote Vasa Vasorum Endothelial Cell Barrier Integrity via G and Akt-Dependent Actin Cytoskeleton Remodeling
Background: In a neonatal model of hypoxic pulmonary hypertension, a dramatic pulmonary artery adventitial thickening, accumulation of inflammatory cells in the adventitial compartment, and angiogenic expansion of the vasa vasorum microcirculatory network are observed. These pathophysiological responses suggest that rapidly proliferating vasa vasorum endothelial cells (VVEC) may exhibit increased permeability for circulating blood cells and macromolecules. However, the molecular mechanisms underlying these observations remain unexplored. Some reports implicated extracellular adenosine in the regulation of vascular permeability under hypoxic and inflammatory conditions. Thus, we aimed to determine the role of adenosine in barrier regulation of VVEC isolated from the pulmonary arteries of normoxic (VVEC-Co) or chronically hypoxic (VVEC-Hyp) neonatal calves. Principal Findings We demonstrate via a transendothelial electrical resistance measurement that exogenous adenosine significantly enhanced the barrier function in VVEC-Co and, to a lesser extent, in VVEC-Hyp. Our data from a quantitative reverse transcription polymerase chain reaction show that both VVEC-Co and VVEC-Hyp express all four adenosine receptors (A1, A2A, A2B, and A3), with the highest expression level of A1 receptors (A1Rs). However, A1R expression was significantly lower in VVEC-Hyp compared to VVEC-Co. By using an A1R-specific agonist/antagonist and siRNA, we demonstrate that A1Rs are mostly responsible for adenosine-induced enhancement in barrier function. Adenosine-induced barrier integrity enhancement was attenuated by pretreatment of VVEC with pertussis toxin and GSK690693 or LY294002, suggesting the involvement of G proteins and the PI3K-Akt pathway. Moreover, we reveal a critical role of actin cytoskeleton in VVEC barrier regulation by using specific inhibitors of actin and microtubule polymerization. Further, we show that adenosine pretreatment blocked the tumor necrosis factor alpha (TNF-Ξ±)-induced permeability in VVEC-Co, validating its anti-inflammatory effects. Conclusions: We demonstrate for the first time that stimulation of A1Rs enhances the barrier function in VVEC by activation of the G/PI3K/Akt pathway and remodeling of actin microfilament
Xanthine Oxidase-Derived ROS Upregulate Egr-1 via ERK1/2 in PA Smooth Muscle Cells; Model to Test Impact of Extracellular ROS in Chronic Hypoxia
Exposure of newborn calves to chronic hypoxia causes pulmonary artery (PA) hypertension and remodeling. Previous studies showed that the redox-sensitive transcription factor, early growth response-1 (Egr-1), is upregulated in the PA of chronically hypoxic calves and regulates cell proliferation. Furthermore, we established in mice a correlation between hypoxic induction of Egr-1 and reduced activity of extracellular superoxide dismutase (EC-SOD), an antioxidant that scavenges extracellular superoxide. We now hypothesize that loss of EC-SOD in chronically hypoxic calves leads to extracellular superoxide-mediated upregulation of Egr-1. To validate our hypothesis and identify the signaling pathways involved, we utilized PA tissue from normoxic and chronically hypoxic calves and cultured calf and human PA smooth muscle cells (PASMC). Total SOD activity was low in the PA tissue, and only the extracellular SOD component decreased with hypoxia. PA tissue of hypoxic calves showed increased oxidative stress and increased Egr-1 mRNA. To mimic the in vivo hypoxia-induced extracellular oxidant imbalance, cultured calf PASMC were treated with xanthine oxidase (XO), which generates extracellular superoxide and hydrogen peroxide. We found that 1) XO increased Egr-1 mRNA and protein, 2) XO induced the phosphorylation of ERK1/2 and, 3) pretreatment with an ERK1/2 inhibitor prevented induction of Egr-1 by XO. siRNA knock-down of EC-SOD in human PASMC also upregulated Egr-1 mRNA and protein, activated ERK1/2, and enhanced SMC proliferation and reduced apoptosis. We conclude that an oxidant/antioxidant imbalance arising from loss of EC-SOD in the PA with chronic hypoxia induces Egr-1 via activation of ERK1/2 and contributes to pulmonary vascular remodeling
MicroRNA-143 activation regulates smooth muscle and endothelial cell crosstalk in pulmonary arterial hypertension
Rationale: The pathogenesis of PAH remains unclear. The four microRNAs representing the miR-143 and miR-145 stem loops are genomically clustered.
Objective: To elucidate the transcriptional regulation of the miR-143/145 cluster, and the role of miR-143 in PAH.
Methods and Results: We identified the promoter region that regulates miR-143/145 miRNA expression in pulmonary artery smooth muscle cells (PASMCs). We mapped PAH-related signalling pathways, including estrogens receptor (ER), liver X factor/retinoic X receptor (LXR/RXR), TGF-Ξ² (Smads), and hypoxia (HRE) that regulated levels of all pri-miR stem loop transcription and resulting miRNA expression. We observed that miR-143-3p is selectively upregulated compared to miR-143-5p during PASMC migration. Modulation of miR-143 in PASMCs significantly altered cell migration and apoptosis. In addition, we found high abundance of miR-143-3p in PASMCs-derived exosomes. Using assays with pulmonary arterial endothelial cells (PAECs) we demonstrated a paracrine pro-migratory and pro-angiogenic effect of miR-143-3p enriched exosomes from PASMC. Quantitative PCR and in situ hybridisation showed elevated expression of miR-143 in calf models of PAH as well as in samples from PAH patients. Moreover, in contrast to our previous findings that had not supported a therapeutic role in vivo, we now demonstrate a protective role for miR-143 in experimental PH in vivo in miR-143-/- and antimiR143-3p-treated mice exposed to chronic hypoxia in both preventative and reversal settings.
Conclusions: MiR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while inhibition of miR-143-3p blocked experimental PH. Taken together these findings confirm an important role for the miR-143/145 cluster in PAH pathobiology
HIF1A-Dependent Induction of Alveolar Epithelial PFKFB3 Dampens Acute Lung Injury
Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI
SBC2007-175993 MEASUREMENT OF IN-VIVO PULMONARY VASCULAR IMPEDANCE IN TWO ANIMAL MODELS OF PULMONARY HYPERTENSION
INTRODUCTION Pulmonary vascular input impedance has been increasingly promoted as an important diagnostic for pulmonary arterial hypertension (PAH) The human studies noted above have understandably not examined detailed associations between impedance and vascular behavior and structure, since the latter data are obtainable only through focused drug studies or ex-vivo measurements. Mechanical changes to a vascular network should be reflected in its input impedance; thus, such investigation should be useful in determining how impedance varies with changes in vascular condition, such as chronic stiffening due to vascular remodeling or acute stiffening due to smooth muscle cell response and/or pressure-induced strain-stiffening. Naturally, clinical identification of such stiffness changes on a routine basis could greatly impact diagnosis. Here, we demonstrate simple-toimplement impedance measurements in two animal models as part of a larger effort to establish said links between clinically-viable diagnostics, such as impedance, and physiological changes that occur to the vasculature as part of the progression of PAH. METHODS Animal Preparation: The two animal models examined here develop PAH due to chronic exposure to a hypoxic environment. The first model consisted of 10 male Sprague-Dawley rats (300-400g), half exposed to hypoxia via hypobaric chamber for 3-4 weeks (barometric pressure β 410 mmHg) and half retained at standard conditions in Denver, CO (barometric pressure β 630 mmHg). The second model utilized 4 male Holstein calves (70-110lb), again with half exposed to hypoxia for two weeks (barometric pressure β 460 mmHg) and the other half remaining normoxic. Both models were exposed to a 12:12-h light-dark cycle, and water and appropriate food were made available ad libitum. Animal care and use committees at both the University of Colorado Health Science Center (rat) and Colorado State University (calf) approved all protocols and procedures. Animal Data Collection and Analysis: The measurements obtained from each animal are identical; the main differences between collection methods are equipment size and type. For all measurements, rats are anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine (10 mg/kg) intraperitoneally, while cows remain conscious. Right jugular access is then obtained in each animal, and a fluid filled catheter, consisting of PV1 tubing for the rat or a commercial Swan-Ganz catheter for the calf, is inserted into the main pulmonary artery (MPA) for pressure measurements. During collection of MPA pressure, blood velocity at the midline of the MPA is obtained with pulse-wave Doppler echocardiography using an FPA probe on a commercial ultrasound scanner (Vivid 5, GE Medical Systems Inc). The imaging depth dictates the probe frequency
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