Natriuretic peptide receptor-C mediates the inhibitory effect of atrial natriuretic peptide on neutrophil recruitment to the lung during acute lung injury.

Atrial natriuretic peptide (ANP) protects against acute lung injury (ALI), but the receptor that mediates this effect is not known. Transgenic mice with 0 (knockout), 1 (heterozygote), or 2 (wild-type) functional copies of Npr3, the gene that encodes for natriuretic peptide receptor-C (NPR-C), were treated with intravenous infusion of ANP or saline vehicle before oropharyngeal aspiration of Pseudomonas aeruginosa (PA103) or saline vehicle. Lung injury was assessed 4 h following aspiration by measurement of lung wet/dry (W/D) weight, whole lung leukocyte and cytokine levels, and protein, leukocyte, and cytokine concentration in bronchoalveolar lavage fluid (BALF). PA103 induced acute lung injury as evidenced by increases in lung W/D ratio and protein concentration in BALF. The severity of PA103-induced lung injury did not differ between NPR-C genotypes. Treatment with intravenous ANP infusion reduced PA103-induced increases in lung W/D and BALF protein concentration in all three NPRC genotypes. PA103 increased the percentage of leukocytes that were neutrophils and cytokine levels in whole lung and BALF in NPR-C wild-type and knockout mice. This effect was blunted by ANP in wild-type mice but not in the NPR-C knockout mice. NPR-C does not mediate the protective effect of ANP on endothelial cell permeability in settings of PA103-induced injury but may mediate the effect of ANP on inhibition of the recruitment of neutrophils to the lung and thereby attenuate the release of inflammatory cytokines.

Culture of pulmonary artery endothelial cells from pulmonary artery catheter balloon tips: considerations for use in pulmonary vascular disease.

Endothelial dysfunction is a hallmark of pulmonary arterial hypertension (PAH) but there are no established methods to study pulmonary artery endothelial cells (PAECs) from living patients. We sought to culture PAECs from pulmonary artery catheter (PAC) balloons used during right-heart catheterisation (RHC) to characterise successful culture attempts and to describe PAEC behaviour.PAECs were grown in primary culture to confluence and endothelial cell phenotype was confirmed. Standard assays for apoptosis, migration and tube formation were performed between passages three to eight. We collected 49 PAC tips from 45 subjects with successful PAEC culture from 19 balloons (39%).There were no differences in subject demographic details or RHC procedural details in successful versus unsuccessful attempts. However, for subjects who met haemodynamic criteria for PAH, there was a higher but nonsignificant (p=0.10) proportion amongst successful attempts (10 out of 19, 53%) versus unsuccessful attempts (nine out of 30, 30%). A successful culture was more likely in subjects with a lower cardiac index (p=0.03) and higher pulmonary vascular resistance (p=0.04). PAECs from a subject with idiopathic PAH were apoptosis resistant compared to commercial PAECs (p=0.04) and had reduced migration compared to PAECs from a subject with portopulmonary hypertension with high cardiac output (p=0.01). PAECs from a subject with HIV-associated PAH formed fewer (p=0.01) and shorter (p=0.02) vessel networks compared to commercial PAECs.Sustained culture and characterisation of PAECs from RHC balloons is feasible, especially in PAH with high haemodynamic burden. This technique may provide insight into endothelial dysfunction during PAH pathogenesis.

Activation of the sweet taste receptor, T1R3, by the artificial sweetener sucralose regulates the pulmonary endothelium.

A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin remodeling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor T1R3 is a G protein-coupled receptor, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified; however, no studies have focused on T1R3 within the vasculature. We hypothesize that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier-disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule sucralose attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signaling, through zinc sulfate, T1R3, or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of the key signaling molecules Src, p21-activated kinase (PAK), myosin light chain-2 (MLC2), heat shock protein 27 (HSP27), and p110α phosphatidylinositol 3-kinase (p110αPI3K). Activation of T1R3 by sucralose protects the pulmonary endothelium from edemagenic agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin remodeling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS.

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

p18, a novel adaptor protein, regulates pulmonary endothelial barrier function via enhanced endocytic recycling of VE-cadherin.

Vascular permeability is a hallmark of several disease states including acute lung injury (ALI). Endocytosis of VE-cadherin, away from the interendothelial junction (IEJ), causes acute endothelial barrier permeability. A novel protein, p18, anchors to the endosome membrane and plays a role in late endosomal signaling via MAPK and mammalian target of rapamycin. However, the fate of the VE-cadherin-positive endosome has yet to be elucidated. We sought to elucidate a role for p18 in VE-cadherin trafficking and thus endothelial barrier function, in settings of ALI. Endothelial cell (EC) resistance, whole-cell ELISA, and filtration coefficient were studied in mice or lung ECs overexpressing wild-type or nonendosomal-binding mutant p18, using green fluorescent protein as a control. We demonstrate a protective role for the endocytic protein p18 in endothelial barrier function in settings of ALI in vitro and in vivo, through enhanced recycling of VE-cadherin-positive early endosomes to the IEJ. In settings of LPS-induced ALI, we show that Src tethered to the endosome tyrosine phosphorylates p18 concomitantly with VE-cadherin internalization and pulmonary edema formation. We conclude that p18 regulates pulmonary endothelial barrier function in vitro and in vivo, by enhancing recycling of VE-cadherin-positive endosomes to the IEJ.

SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and ß-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients suffering from ALI.

Neovascularization in the pulmonary endothelium is regulated by the endosome: Rab4-mediated trafficking and p18-dependent signaling.

Neovascularization, the formation of new blood vessels, requires multiple processes including vascular leak, migration, and adhesion. Endosomal proteins, such as Rabs, regulate trafficking of key signaling proteins involved in neovascularization. The novel endosome protein, p18, enhances vascular endothelial (VE)-cadherin recycling from early endosome to cell junction to improve pulmonary endothelial barrier function. Since endothelial barrier integrity is vital in neovascularization, we sought to elucidate the role for endosome proteins p18 and Rab4, Rab7, and Rab9 in the process of vessel formation within the pulmonary vasculature. Overexpression of wild-type p18 (p18(wt)), but not the nonendosomal-binding mutant (p18(N39)), significantly increased lung microvascular endothelial cell migration, adhesion, and both in vitro and in vivo tube formation. Chemical inhibition of mTOR or p38 attenuated the proneovascularization role of p18(wt). Similar to the effect of p18(wt), overexpression of prorecycling wild-type (Rab4(WT)) and endosome-anchored (Rab4(Q67L)) Rab4 enhanced neovascularization processes, whereas molecular inhibition of Rab4, by using the nonendosomal-binding mutant (Rab4(S22N)) attenuated VEGF-induced neovascularization. Unlike p18, Rab4-induced neovascularization was independent of mTOR or p38 inhibition but was dependent on p18 expression. This study shows for the first time that neovascularization within the pulmonary vasculature is dependent on the prorecycling endocytic proteins Rab4 and p18.

PKC δ and ßII regulate angiotensin II-mediated fibrosis through p38: a mechanism of RV fibrosis in pulmonary hypertension.

Pulmonary hypertension (PH) eventually leads to right ventricular (RV) fibrosis and dysfunction that is associated with increased morbidity and mortality. Although angiotensin II plays an important role in RV remodeling associated with hypoxic PH, the molecular mechanisms underlying RV fibrosis in PH largely remain unresolved. We hypothesized that PKC-p38 signaling is involved in RV collagen accumulation in PH and in response to angiotensin II stimulation. Adult male Sprague-Dawley rats were exposed to 3 wk of normoxia or hypoxia (10% FiO2 ) as a model of PH. Hypoxic rats developed RV hypertrophy and fibrosis associated with an increase in PKC ßII and δ protein expression and p38 dephosphorylation in freshly isolated RV cardiac fibroblasts. Further mechanistic studies were performed in cultured primary cardiac fibroblasts stimulated with angiotensin II, a key activator of ventricular fibrosis in PH. Angiotensin II induced a reduction in p38 phosphorylation that was attenuated following chemical inhibition of PKC ßII and δ. Molecular and chemical inhibition of PKC ßII and δ abrogated angiotensin II-induced cardiac fibroblast proliferation and collagen deposition in vitro. The effects of PKC inhibition on proliferation and fibrosis were reversed by chemical inhibition of p38. Conversely, constitutive activation of p38 attenuated angiotensin II-induced increase of cardiac fibroblast proliferation and collagen accumulation. PKC ßII- and δ-dependent inactivation of p38 regulates cardiac fibroblast proliferation and collagen deposition in response to angiotensin II, which suggests that the PKC-p38 signaling in cardiac fibroblasts may be involved and important in the pathophysiology of RV fibrosis in PH.

Deletion of the Npr3 gene increases severity of acute lung injury in obese mice.

Previous studies have shown that atrial natriuretic peptide (ANP) attenuates agonist-induced pulmonary edema and that this effect may be mediated in part by the ANP clearance receptor, natriuretic peptide receptor-C (NPR-C). Obesity has been associated with lower plasma ANP levels due to increased expression of NPR-C, and with decreased severity of acute lung injury (ALI). Therefore, we hypothesized that increased expression of NPR-C may attenuate ALI severity in obese populations. To test this, we examined ALI in Npr3 wild-type (WT) and knockout (KO) mice fed normal chow (NC) or high-fat diets (HFD). After 12 weeks, ALI was induced with intra-tracheal administration of Pseudomonas aeruginosa strain 103 (PA103) or saline. ALI severity was determined by lung wet-to-dry ratio (W/D) along with measurement of cell count, protein levels from bronchoalveolar lavage fluid (BALF), and quantitative polymerase chain reaction was performed on whole lung to measure cytokine/chemokine and Npr3 mRNA expression. ANP levels were measured from plasma. PA103 caused ALI as determined by significant increases in W/D, BALF protein concentration, and whole lung cytokine/chemokine expression. PA103 increased Npr3 expression in the lungs of wild-type (WT) mice regardless of diet. There was a nonsignificant trend toward increased Npr3 expression in the lungs of WT mice fed HFD versus NC. No differences in ALI were seen between Npr3 knockout (KO) mice and WT-fed NC, but Npr3 KO mice fed HFD had a significantly greater W/D and BALF protein concentration than WT mice fed HFD. These findings support the hypothesis that Npr3 may help protect against ALI in obesity.

Acute protein kinase C beta inhibition preserves coronary endothelial function after cardioplegic hypoxia/reoxygenation.

Objective: Protein kinase C (PKC) influences myocardial contractility and susceptibility to long-term cardiac dysfunction after ischemia-reperfusion injury. In diabetes, PKC inhibition has a protective effect in terms of microvascular dysfunction. SK-channel dysfunction also influences endothelial dysfunction in cardioplegic hypoxia-reoxygenation (CP-H/R). Here, we examine whether acute inhibition of PKC beta protects against CP-H/R-induced coronary endothelial and SK channel dysfunction. Methods: Isolated mouse coronary arterioles, half pretreated with selective PKC inhibitor ruboxistaurin (RBX), were subjected to hyperkalemic, cardioplegic hypoxia (1 hour), and reoxygenation (1 hour) with Krebs buffer. Sham control vessels were continuously perfused with oxygenated Krebs buffer without CP-H/R. After 1 hour of reoxygenation, responses to the endothelium-dependent vasodilator adenosine-diphosphate (ADP) and the SK-channel activator NS309 were examined. Endothelial SK-specific potassium currents from mouse heart endothelial cells were examined using whole-cell path clamp configurations in response to NS309 and SK channel blockers apamin and TRAM34. Results: CP-H/R significantly decreased coronary relaxation responses to ADP (P = .006) and NS309 (P = .0001) compared with the sham control group. Treatment with selective PKC beta inhibitor RBX significantly increased recovery of coronary relaxation responses to ADP (P = .031) and NS309 (P = .004) after CP-H/R. Treatment with RBX significantly increased NS309-mediated potassium currents following CP-H/R (P = .0415). Apamin and TRAM34 sensitive currents were significantly greater in CP-H/R + RBX versus CP-H/R mouse heart endothelial cells (P = .0027). Conclusions: Acute inhibition of PKC beta significantly protected mouse coronary endothelial function after CP-H/R injury. This suggests that acute PKC beta inhibition may be a novel approach for preventing microvascular dysfunction during CP-H/R.

Impact of blood factors on endothelial cell metabolism and function in two diverse heart failure models.

Role of blood-based factors in development and progression of heart failure (HF) is poorly characterized. Blood contains factors released during pathophysiological states that may impact cellular function and provide mechanistic insights to HF management. We tested effects of blood from two distinct HF models on cardiac metabolism and identified possible cellular targets of the effects. Blood plasma was obtained from daunorubicin- and myocardial infarction-induced HF rabbits (Dauno-HF and MI-HF) and their controls (Dauno-Control and MI-Control). Effects of plasma on bioenergetics of myocardial tissue from healthy mice and cellular cardiac components were assessed using high-resolution respirometry and Seahorse flux analyzer. Since endothelial cell respiration was profoundly affected by HF plasma, effects of plasma on endothelial cell barrier function and death were further evaluated. Western-blotting and electron microscopy were performed to evaluate mitochondrial proteins and morphology. Brief exposure to HF plasma decreased cardiac tissue respiration. Endothelial cell respiration was most impacted by exposure to HF plasma. Endothelial cell monolayer integrity was decreased by incubation with Dauno-HF plasma. Apoptosis and necrosis were increased in cells incubated with Dauno-HF plasma for 24 h. Down-regulation of voltage-dependent anion-selective channel (VDAC)-1, translocase of outer membrane 20 (Tom20), and mitochondrial fission factor (MFF) in cells exposed to Dauno-HF plasma and mitochondrial signal transducer and activator of transcription 3 (Stat3) and MFF in cells exposed to MI-HF plasma were observed. Mitochondrial structure was disrupted in cells exposed to HF plasma. These findings indicate that endothelial cells and mitochondrial structure and function may be primary target where HF pathology manifests and accelerates. High-throughput blood-based screening of HF may provide innovative ways to advance disease diagnosis and management.

Transcriptional profiles of pulmonary artery endothelial cells in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by endothelial cell (EC) dysfunction. There are no data from living patients to inform whether differential gene expression of pulmonary artery ECs (PAECs) can discern disease subtypes, progression and pathogenesis. We aimed to further validate our previously described method to propagate ECs from right heart catheter (RHC) balloon tips and to perform additional PAEC phenotyping. We performed bulk RNA sequencing of PAECs from RHC balloons. Using unsupervised dimensionality reduction and clustering we compared transcriptional signatures from PAH to controls and other forms of pulmonary hypertension. Select PAEC samples underwent single cell and population growth characterization and anoikis quantification. Fifty-four specimens were analyzed from 49 subjects. The transcriptome appeared stable over limited passages. Six genes involved in sex steroid signaling, metabolism, and oncogenesis were significantly upregulated in PAH subjects as compared to controls. Genes regulating BMP and Wnt signaling, oxidative stress and cellular metabolism were differentially expressed in PAH subjects. Changes in gene expression tracked with clinical events in PAH subjects with serial samples over time. Functional assays demonstrated enhanced replication competency and anoikis resistance. Our findings recapitulate fundamental biological processes of PAH and provide new evidence of a cancer-like phenotype in ECs from the central vasculature of PAH patients. This "cell biopsy" method may provide insight into patient and lung EC heterogeneity to advance precision medicine approaches in PAH.

Protection against LPS-induced pulmonary edema through the attenuation of protein tyrosine phosphatase-1B oxidation.

One hallmark of acute lung injury is the disruption of the pulmonary endothelial barrier. Such disruption correlates with increased endothelial permeability, partly through the disruption of cell-cell contacts. Protein tyrosine phosphatases (PTPs) are known to affect the stability of both cell-extracellular matrix adhesions and intercellular adherens junctions (AJs). However, evidence for the role of select PTPs in regulating endothelial permeability is limited. Our investigations noted that the inhibition of PTP1B in cultured pulmonary endothelial cells (ECs), as well as in the vasculature of intact murine lungs via the transient overexpression of a catalytically inactive PTP1B, decreased the baseline resistance of cultured EC monolayers and increased the formation of edema in murine lungs, respectively. In addition, we observed that the overexpression of wild-type PTP1B enhanced basal barrier function in vitro. Immunohistochemical analyses of pulmonary ECs and the coimmunoprecipitation of murine lung homogenates demonstrated the association of PTP1B with the AJ proteins ß-catenin, p120-catenin, and VE-cadherin both in vitro and ex vivo. Using LPS in a model of sepsis-induced acute lung injury, we showed that reactive oxygen species were generated in response to LPS, which correlated with enhanced PTP1B oxidation, inhibited phosphatase activity, and attenuation of the interactions between PTP1B and ß-catenin, as well as enhanced ß-catenin tyrosine phosphorylation. Finally, the overexpression of a cytosolic PTP1B fragment, shown to be resistant to nicotinamide adenine dinucleotide phosphate-reduced oxidase-4 (Nox4)-mediated oxidation, significantly attenuated LPS-induced endothelial barrier dysfunction and the formation of lung edema, and preserved the associations of PTP1B with AJ protein components, independent of PTP1B phosphatase activity. We conclude that PTP1B plays an important role in maintaining the pulmonary endothelial barrier, and PTP1B oxidation appears to contribute to sepsis-induced pulmonary vascular dysfunction, possibly through the disruption of AJs.

Heterogeneity in apoptotic responses of microvascular endothelial cells to oxidative stress.

Oxidative stress contributes to disease and can alter endothelial cell (EC) function. EC from different vascular beds are heterogeneous in structure and function, thus we assessed the apoptotic responses of EC from lung and heart to oxidative stress. Since protein kinase Cδ (PKCδ) is activated by oxidative stress and is an important modulator of apoptosis, experiments assessed the level of apoptosis in fixed lung and heart sections of PKCδ wild-type (PKCδ(+/+)) and null (PKCδ(-/-)) mice housed under normoxia (21% O(2)) or hyperoxia (~95% O(2)). We noted a significantly greater number of TUNEL-positive cells in lungs of hyperoxic PKCδ(+/+) mice, compared to matched hearts or normoxic organs. We found that 33% of apoptotic cells identified in hyperoxic lungs of PKCδ(+/+) mice were EC, compared to 7% EC in hyperoxic hearts. We further noted that EC apoptosis was significantly reduced in lungs of PKCδ(-/-) hyperoxic mice, compared to lungs of PKCδ(+/+) hyperoxic mice. In vitro, both hyperoxia and H(2)O(2) promoted apoptosis in EC isolated from microvasculature of lung (LMVEC), but not from the heart (HMVEC). H(2)O(2) treatment significantly increased p38 activity in LMVEC, but not in HMVEC. Inhibition of p38 attenuated H(2)O(2)-induced LMVEC apoptosis. Baseline expression of total PKCδ protein, as well as the caspase-mediated, catalytically active PKCδ cleavage fragment, was higher in LMVEC, compared to HMVEC. PKCδ inhibition significantly attenuated H(2)O(2)-induced LMVEC p38 activation. Conversely, overexpression of wild-type PKCδ or the catalytically active PKCδ cleavage product greatly increased H(2)O(2)-induced HMVEC caspase and p38 activation. We propose that enhanced susceptibility of lung EC to oxidant-induced apoptosis is due to increased PKCδ→p38 signaling, and we describe a PKCδ-centric pathway which dictates the differential response of EC from distinct vascular beds to oxidative stress.

Genetic disruption of protein kinase Cδ reduces endotoxin-induced lung injury.

The pathogenesis of acute lung injury and acute respiratory distress syndrome is characterized by sequestration of leukocytes in lung tissue, disruption of capillary integrity, and pulmonary edema. PKCδ plays a critical role in RhoA-mediated endothelial barrier function and inflammatory responses. We used mice with genetic deletion of PKCδ (PKCδ(-/-)) to assess the role of PKCδ in susceptibility to LPS-induced lung injury and pulmonary edema. Under baseline conditions or in settings of increased capillary hydrostatic pressures, no differences were noted in the filtration coefficients (k(f)) or wet-to-dry weight ratios between PKCδ(+/+) and PKCδ(-/-) mice. However, at 24 h after exposure to LPS, the k(f) values were significantly higher in lungs isolated from PKCδ(+/+) than PKCδ(-/-) mice. In addition, bronchoalveolar lavage fluid obtained from LPS-exposed PKCδ(+/+) mice displayed increased protein and cell content compared with LPS-exposed PKCδ(-/-) mice, but similar changes in inflammatory cytokines were measured. Histology indicated elevated LPS-induced cellularity and inflammation within PKCδ(+/+) mouse lung parenchyma relative to PKCδ(-/-) mouse lungs. Transient overexpression of catalytically inactive PKCδ cDNA in the endothelium significantly attenuated LPS-induced endothelial barrier dysfunction in vitro and increased k(f) lung values in PKCδ(+/+) mice. However, transient overexpression of wild-type PKCδ cDNA in PKCδ(-/-) mouse lung vasculature did not alter the protective effects of PKCδ deficiency against LPS-induced acute lung injury. We conclude that PKCδ plays a role in the pathological progression of endotoxin-induced lung injury, likely mediated through modulation of inflammatory signaling and pulmonary vascular barrier function.

Interplay between FAK, PKCδ, and p190RhoGAP in the regulation of endothelial barrier function.

Disruption of either intercellular or extracellular junctions involved in maintaining endothelial barrier function can result in increased endothelial permeability. Increased endothelial permeability, in turn, allows for the unregulated movement of fluid and solutes out of the vasculature and into the surrounding connective tissue, contributing to a number of disease states, including stroke and pulmonary edema (Ermert et al., 1995; Lee and Slutsky, 2010; van Hinsbergh, 1997; Waller et al., 1996; Warboys et al., 2010). Thus, a better understanding of the molecular mechanisms by which endothelial cell junction integrity is controlled is necessary for development of therapies aimed at treating such conditions. In this review, we will discuss the functions of three signaling molecules known to be involved in regulation of endothelial permeability: focal adhesion kinase (FAK), protein kinase C delta (PKCδ), and p190RhoGAP (p190). We will discuss the independent functions of each protein, as well as the interplay that exists between them and the effects of such interactions on endothelial function.

Agonists for Bitter Taste Receptors T2R10 and T2R38 Attenuate LPS-Induced Permeability of the Pulmonary Endothelium in vitro.

One of the hallmarks of acute respiratory distress syndrome (ARDS) is an excessive increase in pulmonary vascular permeability. In settings of ARDS, the loss of barrier integrity is mediated by cell-cell contact disassembly and actin remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability seen in ARDS. Bitter taste receptors (T2Rs) belong to the superfamily of G-protein-coupled receptors found in several extraoral systems, including lung epithelial and smooth muscle cells. In the present study, we show for the first time that several T2Rs are expressed in human pulmonary arterial endothelial cells (HPAECs). Our results focus on those which are highly expressed as: T2R10, T2R14 and T2R38. Agonists for T2R10 (denatonium) and T2R38 (phenylthiourea), but not T2R14 (noscapine), significantly attenuated lipopolysaccharide (LPS)-induced permeability and VE-cadherin internalisation in HPAECs. In T2R10- or T2R38-siRNA knockdown cells, these endothelial-protective effects were abolished, indicating a direct effect of agonists in regulating barrier integrity. Our further findings indicate that T2R10 and T2R38 exert their barrier-protective function through cAMP but via Rac1-dependent and independent pathways, respectively. However, using an in vivo model of ARDS, the T2R38 agonist, phenylthiourea, was not able to protect against pulmonary edema formation. Taken together, these studies identify bitter taste sensing in the pulmonary endothelium to regulate barrier integrity in vitro through cAMP-Rac1 signalling.

Commercial human pulmonary artery endothelial cells have in-vitro behavior that varies by sex.

It is unknown whether biological sex influences phenotypes of commercially available human pulmonary artery endothelial cells (HPAECs). Ten lots of commercial HPAECs were used (Lonza Biologics; PromoCell). Five (50%) were confirmed to be genotypically male (SRY+) and five (50%) were confirmed to be female (SRY-). Experiments were conducted between passages five and eight. HPAEC phenotype was confirmed with a panel of cell expression markers. Standard assays for proliferation, migration and tube formation were performed in triplicate with technical replicates, under three treatment conditions (EndoGRO; Sigma-Aldrich). Apoptosis was assessed by exposing cells treated with complete media or low serum media to hypoxic (1% oxygen) or normoxic (20% oxygen) conditions. Laboratory staff was blinded. The median (range) age of male and female donors from whom the HPAECs were derived was 58 (48-60) and 56 (33-67), respectively. Our results suggest decreased proliferation in genotypically female cells compared with male cells (p = 0.09). With increasing donor age, female cells were less proliferative and male cells were more proliferative (p = 0.001). Female cells were significantly more apoptotic than male cells by condition (p = 0.001). Female cells were significantly more migratory than male cells in complete media but less migratory than male cells under vascular endothelial growth factor enriched conditions (p = 0.001). There are subtle sex-based differences in the behavior of HPAECs that depend on donor sex and, less so, age. These differences may undermine rigor and reproducibility. Future studies should define whether biological sex is an important regulator of HPAEC function in health and disease.

Inhibition of mitochondrial reactive oxygen species improves coronary endothelial function after cardioplegic hypoxia/reoxygenation.

OBJECTIVE: Cardioplegic ischemia-reperfusion and diabetes mellitus are correlated with coronary endothelial dysfunction and inactivation of small conductance calcium-activated potassium channels. Increased reactive oxidative species, such as mitochondrial reactive oxidative species, may contribute to oxidative injury. Thus, we hypothesized that inhibition of mitochondrial reactive oxidative species may protect coronary small conductance calcium-activated potassium channels and endothelial function against cardioplegic ischemia-reperfusion-induced injury. METHODS: Small coronary arteries and endothelial cells from the hearts of mice with and without diabetes mellitus were isolated and examined by using a cardioplegic hypoxia and reoxygenation model to determine whether the mitochondria-targeted antioxidant Mito-Tempo could protect against coronary endothelial and small conductance calcium-activated potassium channel dysfunction. The microvessels or mouse heart endothelial cells were treated with or without Mito-Tempo (0-10 µM) 5 minutes before and during cardioplegic hypoxia and reoxygenation. Microvascular function was assessed in vitro by vessel myography. K+ currents of mouse heart endothelial cells were measured by whole-cell patch clamp. The levels of intracellular cytosolic free calcium (Ca2+) concentration, mitochondrial reactive oxidative species, and small conductance calcium-activated potassium protein expression of mouse heart endothelial cells were measured by Rhod-2 fluorescence staining, MitoSox, and Western blotting, respectively. RESULTS: Cardioplegic hypoxia and reoxygenation significantly attenuated endothelial small conductance calcium-activated potassium channel activity, caused calcium overload, and increased mitochondrial reactive oxidative species of mouse heart endothelial cells in both the nondiabetic and diabetes mellitus groups. In addition, treating mouse heart endothelial cells with Mito-Tempo (10 µM) reduced cardioplegic hypoxia and reoxygenation-induced Ca2+ and mitochondrial reactive oxidative species overload in both the nondiabetic and diabetes mellitus groups, respectively (P < .05). Treatment with Mito-Tempo (10 µM) significantly enhanced coronary relaxation responses to adenosine 5'-diphosphate and NS309 (P < .05), and endothelial small conductance calcium-activated potassium channel currents in both the nondiabetic and diabetes mellitus groups (P < .05). CONCLUSIONS: Administration of Mito-Tempo improves endothelial function and small conductance calcium-activated potassium channel activity, which may contribute to its enhancement of endothelium-dependent vasorelaxation after cardioplegic hypoxia and reoxygenation.

Chitinase 3 like 1 contributes to the development of pulmonary vascular remodeling in pulmonary hypertension.

Chitinase 3 like 1 (CHI3L1) is the prototypic chitinase-like protein mediating inflammation, cell proliferation, and tissue remodeling. Limited data suggest CHI3L1 is elevated in human pulmonary arterial hypertension (PAH) and is associated with disease severity. Despite its importance as a regulator of injury/repair responses, the relationship between CHI3L1 and pulmonary vascular remodeling is not well understood. We hypothesize that CHI3L1 and its signaling pathways contribute to the vascular remodeling responses that occur in pulmonary hypertension (PH). We examined the relationship of plasma CHI3L1 levels and severity of PH in patients with various forms of PH, including group 1 PAH and group 3 PH, and found that circulating levels of serum CHI3L1 were associated with worse hemodynamics and correlated directly with mean pulmonary artery pressure and pulmonary vascular resistance. We also used transgenic mice with constitutive knockout and inducible overexpression of CHI3L1 to examine its role in hypoxia-, monocrotaline-, and bleomycin-induced models of pulmonary vascular disease. In all 3 mouse models of pulmonary vascular disease, pulmonary hypertensive responses were mitigated in CHI3L1-null mice and accentuated in transgenic mice that overexpress CHI3L1. Finally, CHI3L1 alone was sufficient to induce pulmonary arterial smooth muscle cell proliferation, inhibit pulmonary vascular endothelial cell apoptosis, induce the loss of endothelial barrier function, and induce endothelial-mesenchymal transition. These findings demonstrate that CHI3L1 and its receptors play an integral role in pulmonary vascular disease pathobiology and may offer a target for the treatment of PAH and PH associated with fibrotic lung disease.