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Mitochondrial dysfunction is associated with various forms of lung injury and disease that also involve alterations in pulmonary endothelial permeability, but the relationship, if any, between the two is not well understood. This question was addressed by perfusing the isolated intact rat lung with a buffered physiological saline solution in the absence or presence of the mitochondrial complex I inhibitor rotenone (20 uM). As compared to control, rotenone depressed whole lung tissue ATP from 5.66 ± 0.46 (SEM) to 2.34 ± 0.15 (SEM) μmol·gram−1 dry lung, with concomitant increases in the ADP:ATP and AMP:ATP ratios. Rotenone also increased lung perfusate lactate (from 12.36 ± 1.64 (SEM) to 38.62 ± 3.14 μmol·15 min−1 perfusion·gm−1 dry lung) and the lactate:pyruvate ratio, but had no detectable impact on lung tissue GSH:GSSG redox status. The amphipathic quinone, coenzyme Q1 (CoQ1; 50 μM) mitigated the impact of rotenone on the adenine nucleotide balance, wherein mitigation was blocked by NAD(P)H:quinone oxidoreductase 1 (NQO1) or mitochondrial complex III inhibitors. In separate studies, rotenone increased the pulmonary vascular endothelial filtration coefficient (Kf) from 0.043 ± 0.010 (SEM) to 0.156 ± 0.037 (SEM) ml·min−1·cm H2O−1·gm−1 dry lung weight, and CoQ1 protected against the effect of rotenone on Kf. A second complex I inhibitor, piericidin A, qualitatively reproduced the impact of rotenone on Kf and the lactate/pyruvate ratio. Taken together, the observations imply that pulmonary endothelial barrier integrity depends on mitochondrial bioenergetics as reflected in lung tissue ATP levels and that compensatory activation of whole lung glycolysis cannot protect against pulmonary endothelial hyperpermeability in response to mitochondrial blockade. The study further suggests that low molecular weight amphipathic quinones may have therapeutic utility in protecting lung barrier function in mitochondrial insufficiency.

Activation of vascular endothelial small- (KCa2.3, SK3) or intermediate- (KCa3.1, IK1) conductance Ca2+-activated potassium channels induces vasorelaxation via an endothelium-derived hyperpolarization (EDH) pathway. Although the activation of SK3 and IK1 channels converges on EDH, their subcellular effects on signal transduction are different and not completely clear. In this study, a novel endothelium-specific SK3 knockout (SK3−/−) mouse model was utilized to specifically examine the contribution of SK3 channels to mesenteric artery vasorelaxation, endothelial Ca2+ dynamics, and blood pressure. The absence of SK3 expression was confirmed using real-time quantitative PCR and Western blot analysis. Functional studies showed impaired EDH-mediated vasorelaxation in SK3−/− small mesenteric arteries. Immunostaining results from SK3−/− vessels confirmed the absence of SK3 and further showed altered distribution of transient receptor potential channels, type 4 (TRPV4). Electrophysiological recordings showed a lack of SK3 channel activity, while TRPV4-IK1 channel coupling remained intact in SK3−/− endothelial cells. Moreover, Ca2+ imaging studies in SK3−/− endothelium showed increased Ca2+ transients with reduced amplitude and duration under basal conditions. Importantly, SK3−/− endothelium lacked a distinct type of Ca2+ dynamic that is sensitive to TRPV4 activation. Blood pressure measurements showed that the SK3−/− mice were hypertensive, and the blood pressure increase was further enhanced during the 12-h dark cycle when animals are most active. Taken together, our results reveal a previously unappreciated SK3 signaling microdomain that modulates endothelial Ca2+ dynamics, vascular tone, and blood pressure.

Here, we tested the hypothesis that animals with severe pulmonary arterial hypertension (PAH) display increased sensitivity to vascular permeability induced by activation of store-operated calcium entry. To test this hypothesis, wild-type and transient receptor potential channel 4 (TRPC4) knockout Fischer 344 rats were given a single injection of Semaxanib (SU5416; 20 mg/kg) followed by 3 wk of exposure to hypoxia (10% oxygen) and a return to normoxia (21% oxygen) for an additional 2–3 wk. This Semaxanib/hypoxia/normoxia (i.e., SU5416/hypoxia/normoxia) treatment caused PAH, as evidenced by development of right ventricular hypertrophy, pulmonary artery medial hypertrophy, and occlusive lesions within precapillary arterioles. Pulmonary artery pressure was increased fivefold in Semaxanib/hypoxia/normoxia-treated animals compared with untreated, Semaxanib-treated, and hypoxia-treated controls, determined by isolated perfused lung studies. Thapsigargin induced a dose-dependent increase in permeability that was dependent on TRPC4 in the normotensive perfused lung. This increase in permeability was accentuated in PAH lungs but not in Semaxanib- or hypoxia-treated lungs. Fluid accumulated in large perivascular cuffs, and although alveolar fluid accumulation was not seen in histological sections, Evans blue dye conjugated to albumin was present in bronchoalveolar lavage fluid of hypertensive but not normotensive lungs. Thus PAH is accompanied by a TRPC4-dependent increase in the sensitivity to edemagenic agents that activate store-operated calcium entry.

The pulmonary vasculature comprises three anatomic compartments connected in series: the arterial tree, an extensive capillary bed, and the venular tree. Although, in general, this vasculature is thin-walled, structure is nonetheless complex. Contributions to structure (and thus potentially to function) from cells other than endothelial and smooth muscle cells as well as those from the extracellular matrix should be considered. This review is multifaceted, bringing together information regarding (i) classification of pulmonary vessels, (ii) branching geometry in the pulmonary vascular tree, (iii) a quantitative view of structure based on morphometry of the vascular wall, (iv) the relationship of nerves, a variety of interstitial cells, matrix proteins, and striated myocytes to smooth muscle and endothelium in the vascular wall, (v) heterogeneity within cell populations and between vascular compartments, (vi) homo- and heterotypic cell-cell junctional complexes, and (vii) the relation of the pulmonary vasculature to that of airways. These issues for pulmonary vascular structure are compared, when data is available, across species from human to mouse and shrew. Data from studies utilizing vascular casting, light and electron microscopy, as well as models developed from those data, are discussed. Finally, the need for rigorous quantitative approaches to study of vascular structure in lung is highlighted.

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca2+ influx in response to diverse environmental stimuli. However, it is not clear whether Ca2+ influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the α1G T-type Ca2+ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca2+, immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K+ perfusate induces Ca2+ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca2+ entry in septal endothelium evoked by the selective TRPV4 activator 4α-phorbol-12,13-didecanoate (4α-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of α1G abolishes depolarization-induced Ca2+ entry and surface expression of P-selectin but does not prevent 4α-PDD-activated Ca2+ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4α-PDD-activated Ca2+ entry and the increase in permeability, while not impacting depolarization-induced Ca2+ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca2+ entry through α1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (Kf) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH2O PIP at both 35°C and 40°C. Ventilation with 35 cmH2O PIP increased Kf by 2.2-fold at 35°C and 3.3-fold at 40°C compared with baseline, but Kf increased significantly with time at 40°C with 9 cmH2O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in Kf. In TRPV4−/− knockout mice, the high PIP ventilation protocol did not increase Kf at either temperature. We have also found that lung distention caused Ca2+ entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4−/− and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4−/− lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation.

Our previous work has shown that the increased lung endothelial permeability response to 14,15-epoxyeicosatrienoic acid (14,15-EET) in rat lung requires Ca(2+) entry via vanilloid type-4 transient receptor potential (TRPV4) channels. Recent studies suggest that activation of TRPV4 channels in systemic vascular endothelium prolongs agonist-induced hyperpolarization and amplifies Ca(2+) entry by activating Ca(2+)-activated K(+) (KCa) channels, resulting in vessel relaxation. Activation of endothelial KCa channels thus has potential to increase the electrochemical driving force for Ca(2+) influx via TRPV4 channels and to amplify permeability responses to TRPV4 activation in lung. To examine this hypothesis, we used Western blot analysis, electrophysiological recordings, and isolated-lung permeability measurements to document expression of TRPV4 and KCa channels and the potential for functional coupling. The results show that rat pulmonary microvascular endothelial cells express TRPV4 and 3 KCa channels of different conductances: large (BK), intermediate (IK), and small (SK3). However, TRPV4 channel activity modulates the IK and SK3, but not the BK, channel current density. Furthermore, the TRPV4-mediated permeability response to 14,15-EET in mouse lung is significantly attenuated by pharmacologic blockade of IK and SK3, but not BK, channels. Collectively, this functional coupling suggests that endothelial TRPV4 channels in rodent lung likely form signaling microdomains with IK and SK3 channels and that the integrated response dictates the extent of lung endothelial injury caused by 14,15-EET.

Transient receptor potential channel vanilloid 4 (TRPV4) is expressed in numerous cell types in lung, including vascular endothelium and smooth muscle, alveolar macrophages, airway epithelium, and airway smooth muscle. To date, our understanding of the role of TRPV4 in lung disease has been ­largely limited to its involvement in direct acute lung injury and lung injury induced by mechanical stress, specifically ventilator-induced lung injury and that due to high vascular pressure and edema formation in heart failure. We will discuss these areas in some depth. We will also introduce the evidence, albeit limited, for other lung diseases where TRPV4 may potentially play a role, including pulmonary hypertension, airway disease and cough, and thus therapeutics may prove beneficial. Although literature on the translation of bench research related to TRPV4 in lungs to clinical therapeutics is limited, we will discuss the available information.

Disruption of the alveolar septal barrier leads to acute lung injury, patchy alveolar flooding, and hypoxemia. Although calcium entry into endothelial cells is critical for loss of barrier integrity, the cation channels involved in this process have not been identified. We hypothesized that activation of the vanilloid transient receptor potential channel TRPV4 disrupts the alveolar septal barrier. Expression of TRPV4 was confirmed via immunohistochemistry in the alveolar septal wall in human, rat, and mouse lung. In isolated rat lung, the TRPV4 activators 4α-phorbol-12,13-didecanoate and 5,6- or 14,15-epoxyeicosatrienoic acid, as well as thapsigargin, a known activator of calcium entry via store-operated channels, all increased lung endothelial permeability as assessed by measurement of the filtration coefficient, in a dose- and calcium-entry dependent manner. The TRPV antagonist ruthenium red blocked the permeability response to the TRPV4 agonists, but not to thapsigargin. Light and electron microscopy of rat and mouse lung revealed that TRPV4 agonists preferentially produced blebs or breaks in the endothelial and epithelial layers of the alveolar septal wall, whereas thapsigargin disrupted interendothelial junctions in extraalveolar vessels. The permeability response to 4α-phorbol-12,13-didecanoate was absent in TRPV4 −/− mice, whereas the response to thapsigargin remained unchanged. Collectively, these findings implicate TRPV4 in disruption of the alveolar septal barrier and suggest its participation in the pathogenesis of acute lung injury.

This study tested the hypothesis that epoxyeicosatrienoic acids (EETs) derived from arachidonic acid via P-450 epoxygenases are soluble factors linking depletion of endoplasmic reticulum Ca2+ stores and store-dependent regulation of endothelial cell (EC) permeability in rat lung. EC permeability was measured via the capillary filtration coefficient ( Kf,c) in isolated, perfused rat lungs. 14,15-EET and 5,6-EET increased EC permeability, a response that was significantly different from that of 8,9-EET, 11,12-EET, and vehicle control. The permeability response to 14,15-EET was not significantly attenuated by the nonspecific Ca2+ channel blocker Gd3+ ( P = 0.068). In lungs perfused with low [Ca2+], 14,15-EET tended to increase EC permeability, although a significant increase in Kf,c was observed only following Ca2+ add-back. As positive control, we showed that the 3.7-fold increase in Kf,c evoked by thapsigargin (TG), a known activator of store depletion-induced Ca2+ entry, was blocked by both Gd3+ and low [Ca2+] buffer. Nonetheless, the permeability response to TG could not be blocked by the phospholipase A2 inhibitors mepacrine or methyl arachidonyl fluorophosphonate or the P-450 epoxygenase inhibitors 17-octadecynoic acid or propargyloxyphenyl hexanoic acid. Similarly, combined pretreatment with ibuprofen and dicyclohexylurea to block EET metabolism had no effect on the permeability response to TG. We conclude that EETs have a heterogeneous impact on EC permeability. Despite a requirement for Ca2+ entry with both TG and 14,15-EET, our data suggest that distinct signaling pathways or heterogeneity in EC responsiveness is responsible for the observed EC injury evoked by EETs and store depletion in the isolated rat lung.

The controversy over the use of nonpulsatile versus pulsatile pumps for maintenance of normal organ function during ex vivo perfusion has continued for many years, but resolution has been limited by lack of a congruent mathematical definition of pulsatility. We hypothesized that the waveform frequency and amplitude, as well as the underlying mean distending pressure are all key parameters controlling vascular function. Using discrete Fourier Analysis, our data demonstrate the complexity of the pulmonary arterial pressure waveform in vivo and the failure of commonly available perfusion pumps to mimic in vivo dynamics. In addition, our data show that the key harmonic signatures are intrinsic to the perfusion pumps, are similar for flow and pressure waveforms, and are unchanged by characteristics of the downstream perfusion circuit or perfusate viscosity.

Ca2+entry through transient receptor potential vanilloid 4 (TRPV4) results in swelling, blebbing, and detachment of the epithelium and capillary endothelium in the intact lung. Subsequently, increased permeability of the septal barrier and alveolar flooding ensue. In this study, we tested the hypothesis that TRPV4 activation provides a Ca2+source necessary for proteolytic disruption of cell-cell or cell-matrix adhesion by matrix metalloproteinases (MMPs) 2 and 9, thus increasing septal barrier permeability. In our study, C57BL/6 or TRPV4−/−mouse lungs were perfused with varying doses of the TRPV4 agonist GSK-1016790A (Sigma) and then prepared for Western blot. Lung injury, assessed by increases in lung wet-to-dry weight ratios and total protein levels in the bronchoalveolar lavage fluid, was increased in a dose-dependent fashion in TRPV4+/+but not TRPV4−/−lungs. In concert with lung injury, we detected increased active MMP2 and MMP9 isoforms, suggesting that TRPV4 can provide the Ca2+source necessary for increased MMP2/9 activation. Furthermore, tissue inhibitor of metalloproteinases (TIMP) 2 levels in the TRPV4-injured lungs were decreased, suggesting that TRPV4 activation increases the availability of these active MMPs. We then determined whether MMP2 and MMP9 mediate TRPV4-induced lung injury. Pharmacological blockade (SB-3CT, 1 μM; Sigma) of MMP2 and MMP9 resulted in protection against TRPV4-induced lung injury. We conclude that TRPV4 activation and the subsequent Ca2+transient initiates a rapid cascade of events leading to release and activation of the gelatinase MMPs, which then contribute to lung injury.

In isolated rat lung perfused with a physiological saline solution (5.5 mM glucose), complex I inhibitors decrease lung tissue ATP and increase endothelial permeability (Kf), effects that are overcome using an amphipathic quinone (CoQ1) [Free Radic. Biol. Med. 65:1455–1463; 2013]. To address the microvascular endothelial contribution to these intact lung responses, rat pulmonary microvascular endothelial cells in culture (PMVEC) were treated with the complex I inhibitor rotenone and ATP levels and cell monolayer permeability (PS) were measured. There were no detectable effects on ATP or permeability in experimental medium that, like the lung perfusate, contained 5.5 mM glucose. To unmask a potential mitochondrial contribution, the glucose concentration was lowered to 0.2 mM. Under these conditions, rotenone decreased ATP from 18.4±1.6 (mean±SEM) to 4.6±0.8 nmol/mg protein, depolarized the mitochondrial membrane potential (Δψm) from −129.0±3.7 (mean±SEM) to −92.8±5.5 mV, and decreased O2 consumption from 2.0±0.1 (mean±SEM) to 0.3±0.1 nmol/min/mg protein. Rotenone also increased PMVEC monolayer permeability (reported as PS in nl/min) to FITC–dextran (~40 kDa) continually over a 6 h time course. When CoQ1 was present with rotenone, normal ATP (17.4±1.4 nmol/mg protein), O2 consumption (1.5±0.1 nmol/min/mg protein), Δψm (−125.2±3.3 mV), and permeability (PS) were maintained. Protective effects of CoQ1 on rotenone-induced changes in ATP, O2 consumption rate, Δψm, and permeability were blocked by dicumarol or antimycin A, inhibitors of the quinone-mediated cytosol–mitochondria electron shuttle [Free Radic. Biol. Med. 65:1455–1463; 2013]. Key rotenone effects without and with CoQ1 were qualitatively reproduced using the alternative complex I inhibitor, piericidin A. We conclude that, as in the intact lung, PMVEC ATP supply is linked to the permeability response to complex I inhibitors. In contrast to the intact lung, the association in PMVEC was revealed only after decreasing the glucose concentration in the experimental medium from 5.5 to 0.2 mM.

Objective:After congestive heart failure (CHF), lung endothelial permeability responses to a number of perturbations, including acute barotrauma, angiotensin II, and thapsigargin are blunted. Our hypothesis was that similar attenuation of permeability responses occurs in peripheral vascular beds after CHF. We compared peripheral microvascular permeability responses to the autacoid histamine in control dogs and in dogs paced to heart failure (245 bpm for ∼36 days). Since catecholamines attenuate autacoid-induced increases in microvascular permeability in skin and muscle in normal animals, we also tested whether the known elevation in catecholamines in CHF was involved in any downregulation of permeability responses in this group. Methods:Control and paced dogs were anesthetized, intubated, and ventilated, and a hindpaw lymphatic cannulated. The reflection coefficient for total proteins (σ) was measured at baseline and during one-hour, local intra-arterial histamine infusion. Results:In controls, σ fell fro...

Current emphasis on translational application of genetic models of lung disease has renewed interest in the measurement of the gravimetric filtration coefficient ( Kf) as a means to assess vascular permeability changes in isolated perfused lungs. The Kfis the product of the hydraulic conductivity and the filtration surface area, and is a sensitive measure of vascular fluid permeability when the pulmonary vessels are fully recruited and perfused. We have observed a remarkable consistency of the normalized baseline Kfvalues between species with widely varying body weights from mice to sheep. Uniformity of Kfvalues can be attributed to the thin alveolar capillary barrier required for gas exchange and the conserved matching of lung vascular surface area to the oxygen requirements of the body mass. An allometric correlation between the total lung filtration coefficient ( Kf,t) and body weight in several species ( r2= 1.00) had a slope that was similar to those reported for alveolar and pulmonary capillary surface areas and pulmonary diffusion coefficients determined by morphometric methods in these species. A consistent Kfis dependent on accurately separating the filtration and vascular volume components of lung weight gain, then Kfis a consistent and repeatable index of lung vascular permeability.

Unique features of the pulmonary circulation impact its function in health and disease, not the least of which is the existence of developmentally distinct, functionally heterogeneous extra-alveolar and septal capillary networks. The impact of ion channel expression and regulation in lung vascular smooth muscle or endothelium in these vascular compartments provides a focus for this special topics issue. Reviews and original contributions from experts in the field discuss two broad groups of ion channels, drawing on studies utilizing biophysical and molecular approaches in heterologous expression systems, in vitro approaches in pulmonary vascular smooth muscle and endothelial cells, and physiologic studies in animal models of chronic pulmonary hypertension. First, channels involved in membrane depolarization and related alterations in vascular tone, and shear sensing or exocytosis by endothelium are discussed: voltage-gated potassium channels, ATP-regulated potassium channels and L- and T- type voltage-gated calcium channels. The second series of reviews discusses the role of calcium influx pathways provided by transient receptor potential channels in regulation of pulmonary vascular tone or vascular remodeling, and endothelial barrier function. Understanding the role of ion channels in pulmonary vascular pathophysiology may be critical to development of new therapeutic strategies.

In previous studies, blockade or gene deletion of either myosin light chain kinase (MLCK) or the mechanogated transient receptor potential vanilloid 4 (TRPV4) channel attenuated mechanical lung injury. To determine their effects on calcium entry, rat pulmonary microvascular endothelial cells (RPMVEC) were labeled with fluo-4 and calcium entry initiated with the TRPV4 agonist, 4α-phorbol 12, 13-didecanoate (4αPDD). Mean calcium transients peaked at ∼25 sec and persisted ∼500 sec. The 4αPDD response was essentially abolished in calcium-free media, or after pretreatment with the MLCK inhibitor, ML-7. ML-7 also attenuated the 4αPDD-induced inward calcium current measured directly using whole-cell patch clamp. Pretreatment with dynasore, an inhibitor of dynamin produced an initial calcium transient followed by a 4αPDD transient of unchanged peak intensity. Automated averaging of areas under the curve (AUC) of calcium transients in individual cells indicated total calcium activity with a relationship between treatment groups of ML-7 + 4αPDD < 4αPDD only < dynasore + 4αPDD. Measurement of biotinylated surface TRPV4 protein indicated a significant reduction after ML-7 pretreatment, but no significant change with dynasore treatment. RPMVEC monolayer electrical resistances were decreased by only 3% with 10 μmol/L 4αPDD and the response was dose-related. Dynasore alone produced a 29% decrease in resistance, but neither ML-7 nor dynasore affected the subsequent 4αPDD resistance response. These studies suggest that MLCK may inhibit mechanogated calcium responses through reduced surface expression of stretch activated TRPV4 channels in the plasma membrane.

This review highlights our current knowledge regarding expression of transient receptor potential (TRP) cation channels in lung endothelium and evidence for their involvement in regulation of lung endothelial permeability. Six mammalian TRP families have been identified and organized on the basis of sequence homology: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPML (mucolipin), TRPP (polycystin), and TRPA (ankyrin). To date, only TRPC1/4, TRPC6, TRPV4, and TRPM2 have been extensively studied in lung endothelium. Calcium influx through each of these channels has been documented to increase lung endothelial permeability, although their channel-gating mechanisms, downstream signaling mechanisms, and impact on endothelial structure and barrier integrity differ. While other members of the TRPC, TRPV, and TRPM families may be expressed in lung endothelium, we have little or no evidence linking these to regulation of lung endothelial permeability. Further, neither the expression nor functional role(s) of any TRPML, TRPP, and TRPA family members has been studied in lung endothelium. In addition to this assessment organized by TRP channel family, we also discuss TRP channels and lung endothelial permeability from the perspective of lung endothelial heterogeneity, using outcomes of studies focused on TRPC1/4 and TRPV4 channels. The diversity within the TRP channel family and the relative paucity of information regarding roles of a number of these channels in lung endothelium make this field ripe for continued investigation.

The effects of angiotensin II(ANG II) on pulmonary vascular resistance and microvascular permeability were studied in isolated, blood-perfused, ventilated canine lung lobes from control animals (n = 40) and animals with pacing-induced heart failure (n = 15). Conditioned dogs were paced (245 beats/min) for 30.6 +/- 0.9 (SE) days until left ventricular shortening fraction decreased by 56% (P < 0.05). Baseline pulmonary arterial resistance (Ra) (19.1 +/- 1.6 vs. 8.0 +/- 1.1 cmH2O.1(-1).min.100g) and venous resistance (Rv) (17.1 +/- 2.3 vs. 7.8 +/- 1.0 cmH2O.1(-1).min.100 g) were greater (P < 0.05) in the paced group compared with controls, respectively. Increments in Ra (delta Ra) and Rv(delta Rv) were measured after intra-arterial boluses of ANG II (1-10 micrograms). ANG II produced a dose-dependent response in delta Ra that was enhanced after pacing (P < 0.05). There was no effect on delta Rv in either group. At increased venous pressure (Pv = 20 cmH2O), the increments in delta Ra were significantly attenuated in both groups. In control lobes at low Pv, delta Ra and delta Rv both tended to decrease with increased lobar blood flow, suggesting that blood flow affects the pulmonary vascular response of ANG II. The baseline capillary filtration coefficient (Kf,c) was not different in the paced group compared with control, nor was there any effect of ANG II on Kf,c in the paced group. However, Kf,c did increase after ANG II in the control groups evaluated at either low or high Pv (P < 0.05). This difference in Kf,c was not seen if the experiment was done at increased Pv but without ANG II administration. We conclude that the pulmonary vasoconstrictor activity of ANG II is modestly enhanced in canine pacing-induced heart failure. Nonetheless, ANG II does not likely contribute to increased pulmonary vascular resistance in vivo in heart failure, since this effect was abolished at increased Pv. Finally, the absence of any effect of ANG II on pulmonary microvascular permeability in the paced group is suggestive of some adaptive remodeling of the capillary endothelial barrier.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

The effects of platelet-activating factor (PAF) on vascular resistance and capillary permeability were studied in the isolated rat hindquarter. Six groups were studied (n = 30): control; PAF alone (1.4 microM); and PAF (1.4 microM) pretreated with ibuprofen (30 mg/kg), thromboxane A2 (TxA2)-receptor antagonist (BM-13505, 2 mg/kg), PAF-receptor antagonist (WEB-2086, 5 mg/kg), or dexamethasone (5 mg/kg). The vascular resistance was calculated, and the reflection coefficient (sigma) was determined as an index of capillary permeability. Exogenous PAF caused a threefold increase in vascular resistance peaking at 5 min and a 2.5-fold increase in capillary permeability. The increased vascular resistance caused by PAF alone was significantly attenuated by ibuprofen, BM-13505, and dexamethasone. The PAF-induced permeability was neither attenuated by ibuprofen nor BM-13505. However, both the increased vascular resistance and permeability were blocked and attenuated by WEB-2086 and dexamethasone, respectively. We conclude that TxA2 mediates the PAF-induced increased vascular resistance; however, the increased vascular permeability is independent of the formation of TxA2 in the isolated hindquarter.

Although lymphatic washout of hyaluronan during lung hydration has been postulated to deplete lung interstitial hyaluronan content and thereby contribute to the decreased interstitial exclusion of albumin observed under these conditions, this hypothesis has not been directly tested. In anesthetized, ventilated mongrel dogs, a prenodal lung lymphatic was cannulated for measurement of lymph flow and hyaluronan concentration. Following baseline measurements, Pla was increased in four steps of 5 cm H2O in Group 1 or set to one pressure ranging between 6 and 32 cm H2O in Group 2. In Group 3, saline (15% body weight) was infused over 30 min and then Pla increased as in Group 2. Invariably, as lymph flow increased in Groups 1 through 3, lymph hyaluronan concentration and hyaluronan flux increased significantly (p0.05). In a separate control group, there were no changes in lymph flow, hyaluronan concentration, or hyaluronan flux. In Group 3, lung hyaluronan content at 5 h (0.76 +/- 0.08 mg/g dry weight) was not significantly less than that during baseline (0.88 +/- 0.05 mg/g dry weight), although total uronic acid content actually increased by 38% over the same time course. In contrast, in the control group, both lung hyaluronan and uronic acid content remained stable over the experimental period. From these data, approximately 2 to 3% of lung hyaluronan is predicted to leave the interstitium via lymphatic flux per day under baseline conditions. The daily turnover of interstitial hyaluronan by this route increased to 15 to 18% of total content when Pla was elevated and to 54% following saline infusion. Thus, lung hyaluronan can be rapidly mobilized with increased lymph flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary hypertension in congestive heart failure causes medial hypertrophy in pulmonary vessels and thickening of the endothelial basement membrane. In this study, the functional consequences of such pulmonary vascular adaptations were evaluated. Heart failure was induced in dogs by rapid ventricular pacing (240 beats per minute) for 28 days, at which time left ventricular shortening fraction was decreased by 57% compared with that at baseline. Lung lobes from paced (n = 56) and control dogs (n = 68) were isolated and perfused with autologous blood. Total, arterial (Ra), and venous (Rv) vascular resistances were significantly increased and vascular capacitance decreased in lobes from paced animals compared with controls. Increments in Ra and Rv after intra-arterial boluses of norepinephrine were measured before and after sequential addition of the alpha 1- and alpha 2-receptor antagonists prazosin (16 mumol/L) and yohimbine (0.1 mumol/L) in the presence or absence of propranolol (5 mumol/L). Norepinephrine (1 to 40 micrograms) had little effect on Ra in the absence of propranolol, a pattern that persisted in control lobes after propranolol. However, when lobes from paced animals were pretreated with propranolol, norepinephrine increased Ra, Rv was increased after norepinephrine in control lobes, an effect that was enhanced in the paced group. In both groups, the increment in Rv was greater after propranolol. Irrespective of propranolol pretreatment, prazosin significantly attenuated, if not abolished, the response to norepinephrine. The enhancement in venous vascular reactivity in lobes from paced animals remained when venous pressure was elevated to 20 cm H2O. In control lobes under conditions of elevated tone or when endothelium-dependent relaxing factor was blocked, responses to norepinephrine did not mimic those observed in the paced group. Microvascular permeability, as measured by the capillary filtration coefficient, was not altered in the paced group. We conclude that the pulmonary adaptations to 4 weeks of rapid ventricular pacing include functional changes in pulmonary hemodynamics and vascular reactivity but not in microvascular permeability.

The permselectivity of the parietal pleura was determined in spontaneously breathing anesthetized rabbits and dogs. In rabbits, we injected intrapleurally 5 ml of 1-g/dl albumin solution containing 100 microCi of 131I-labeled albumin plus 100 microCi of either lactate dehydrogenase (LDH) or alpha 2–125I-macroglobulin. Dogs received 100 ml of 1-g/dl albumin solution containing 100 microCi of 131I-albumin plus 100 microCi of alpha 2–125I-macroglobulin. A transpleural pressure gradient was set, lowering the intracapsular pressure to -30 cmH2O. The solvent drag reflection coefficients (sigma f) were calculated as the ratio between tracer concentrations in capsular and pleural liquid collected at 60–180 min. In rabbits sigma f was 0.44 +/- 0.2 (SD) for albumin, 0.84 +/- 0.1 for LDH, and 0.93 +/- 0.05 for alpha 2-macroglobulin. In dogs sigma f was 0.30 +/- 0.19 for albumin and 0.53 +/- 0.15 for alpha 2-macroglobulin. The hydraulic conductivity of the parietal pleura was 2.18 +/- 1.54 microliters.h-1.cmH2O-1.cm-2 in rabbits and 1.22 +/- 1.13 microliters.h-1.cmH2O-1.cm-2 in dogs. The parietal pleura could be modeled by two pore populations with radii of 83–89 and 156-222 A. The permeability coefficient averaged 0.08–0.21 x 10(-6) cm/s for albumin, 0.06–0.09 x 10(-6) cm/s for LDH, and 0.01-0.03 x 10(-6) cm/s for alpha 2-macroglobulin.

In a canine hind leg model, lymph flow (QL), lymph (CL), and plasma (CP) total protein concentrations, the reflection coefficient for total proteins (sigma d), and the filtration coefficient (Kf) were determined before and for 6 hours after a 5-second 100 degrees C hind paw scald (3% total body surface area, TBSA). Before injury, hind leg venous pressure was elevated and maintained by outflow restriction until a minimal, steady-state CL/CP ratio was achieved. Albumin (5%) was infused 30 minutes after the scald at low (0.4 mL/kg/% TBSA) or high (2 mL/kg/% TBSA) doses. Scald uniformly increased QL, CL/CP, Kf, and paw weight gain (PWG). Whereas postburn infusion of low-dose albumin mildly attenuated increases in CL/CP and PWG noted in scald-alone animals, no differences were noted between the scald and scald/high-dose albumin groups.

Hyaluronan is a structural component of the interstitial matrix in skin and is catabolized locally in skin as well as by lymphatic removal and subsequent degradation in lymph nodes and liver. The present experiments were designed to evaluate the role of interstitial fluid flux in turnover of hyaluronan in skin by measuring the maximal lymphatic flux of hyaluronan. Lymph flow, total protein concentration, hyaluronan concentration, and flux were measured every 15 min in prenodal lymph from the hind paw in 12 pentobarbital-anesthetized mongrel dogs at normal and increased interstitial fluid flux. An intravenous saline load (15% of body weight during the course of 30 min) was followed by a one-step increase in local venous pressure 30 min later (to a maximum of 50 mm Hg) and maintained at this level for the next 240 min. Lymph flow and hyaluronan concentration during the control period averaged 25.6 +/- (SD) 23.2 (range 4.7-61.9) microliters/min and 8.6 +/- 2.8 (range 2.0-11.6) micrograms/ml, respectively. The hyaluronan concentration fell by 30% during the experimental period, while the lymph flow increased up to ten times above control. Total tissue water increased from 1.73 +/- 0.11 ml/g dry weight during the control period to 1.91 +/- 0.12 ml/g dry weight at the end of the experiment (p0.01). The corresponding tissue contents of hyaluronan averaged 3.5 +/- 0.9 and 3.6 +/- 0.6 mg/g dry weight, respectively (p0.05). The average lymphatic hyaluronan flux was 8.4 +/- 5.4 micrograms/h during the control period, peaked at about 60 micrograms/h, and averaged 33.6 +/- 13.9 micrograms/h during the last hour of the experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)

We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4−/−and TRPV4+/+mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients ( Kf) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH2O PIP did not significantly increase in lungs from TRPV4−/−mice but increased >2.2-fold in TRPV4+/+lungs, TRPV4+/+lungs instilled with TRPV4−/−macrophages, and TRPV4−/−lungs instilled with TRPV4+/+macrophages after ventilation with 35 cmH2O PIP. Activation of TRPV4 with 4-α-phorbol didecanoate (4αPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4+/+macrophages but not TRPV4−/−macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4+/+macrophages compared with TRPV4−/−macrophages after 4αPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4+/+lungs compared with low PIP ventilated TRPV4+/+or high PIP ventilated TRPV4−/−lungs. Thus TRPV4+/+macrophages restored susceptibility of TRPV4−/−lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4+/+macrophages but not TRPV4−/−macrophages. Kfincreases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.

Microcirculation (2010) 17, 1–10. doi: 10.1111/j.1549-8719.2009.00013.x Abstract Objective: Epoxyeicosatrienoic acids (EETs) are protective in both myocardial and brain ischemia, variously attributed to activation of KATP channels or blockade of adhesion molecule upregulation. In this study, we tested whether EETs would be protective in lung ischemia–reperfusion injury. Methods: The filtration coefficient (Kf), a measure of endothelial permeability, and expression of the adhesion molecules vascular cell adhesion molecule (VCAM) and intercellular adhesion molecule (ICAM) were measured after 45 minutes ischemia and 30 minutes reperfusion in isolated rat lungs. Results: Kf increased significantly after ischemia–reperfusion alone vs time controls, an effect dependent upon extracellular Ca2+ although not on the EET-regulated channel TRPV4. Inhibition of endogenous EET degradation or administration of exogenous 11,12- or 14,-15-EET at reperfusion significantly limited the permeability response to ischemia–reperfusion. The beneficial effect of 11,12-EET was not prevented by blockade of KATP channels nor by blockade of TRPV4. Finally, 11,12-EET-dependent alteration in adhesion molecules expression is unlikely to explain its beneficial effect, since the expression of the adhesion molecules VCAM and ICAM in lung after ischemia–reperfusion was similar to that in controls. Conclusion: EETs are beneficial in the setting of lung ischemia–reperfusion, when administered at reperfusion. However, further study will be needed to elucidate the mechanism of action.

The transient receptor potential vanilloid 4 (TRPV4) non-selective cation channel has emerged as a critical channel for initiating the increased vascular permeability induced by high airway or vascular pressures in the lung. TRPV4 gating is regulated by multiple factors: mechanical stress, heat, epoxyeicosatrienoic acids (EETs) – the arachidonic acid metabolites of P450 epoxygenases, and phorbol esters. Increased pulmonary venous pressure and ventilation with high peak inflation pressures increase endothelial calcium influx, nitric oxide production, and vascular permeability in a TRPV4 dependent fashion in intact lungs. The permeability response to excess mechanical stress is attenuated by inhibition of cytosolic phospholipase A2 or P450 epoxygenases, and permeability increases in response to infusion of EETs. Various molecular mechanisms have been implicated for regulating TRPV4 gating, including channel translocation, direct ligand binding and phosphorylation. However, the mechanisms for EET dependent regulation of TRPV4 or amplification of TRPV4 by phosphorylation in intact lungs subjected to mechanical stress have not been clarified.

Administration of hyperosmolar fluids to burn patients has been proposed as a means of decreasing resuscitative fluid volume and, subsequently, wound edema accumulation. To test this hypothesis, canine hindpaw lymph flow (QL) and lymph (CL) and plasma (CP) total protein concentrations were measured, and capillary filtration coefficient (Kf) was calculated before and for 6 h after a 5-s 100 degrees C footpaw scald. Scald was followed 30 min later by bolus infusion (4 ml/kg) of 7% saline or 6% Dextran 70 or the two in combination. Before scald, venous pressure was elevated until a minimal CL/CP was reached. The reflection coefficient (sigma d) was calculated by the formula: 1-minimal CL/CP. Scald increased QL, CL/CP, and Kf, but sigma d was decreased (P less than 0.05). Compared with burn alone, 7% saline exacerbated burn-induced increases in QL and Kf. In contrast, infusion of Dextran 70 did not exacerbate these changes in QL or Kf. Perhaps more importantly, Dextran 70 may have attenuated the increase in CL/CP and reduced edema formation in the burned hindpaw. However, the addition of 7% saline to the dextran eliminated these beneficial effects.