Diego F. Alvarez

Transient Receptor Potential Vanilloid 4-Mediated Disruption of the Alveolar Septal Barrier. A Novel Mechanism of Acute Lung Injury

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.

Transient Receptor Potential Vanilloid 4–Mediated Disruption of the Alveolar Septal Barrier

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.

Human embryonic stem cell−derived vascular progenitor cells capable of endothelial and smooth muscle cell function

OBJECTIVE Previous studies have demonstrated development of endothelial cells (ECs) and smooth muscle cells (SMCs) as separate cell lineages derived from human embryonic stem cells (hESCs). We demonstrate CD34(+) cells isolated from differentiated hESCs function as vascular progenitor cells capable of producing both ECs and SMCs. These studies better define the developmental origin and reveal the relationship between these two cell types, as well as provide a more complete biological characterization. MATERIALS AND METHODS hESCs are cocultured on M2-10B4 stromal cells or Wnt1-expressing M2-10B4 for 13 to 15 days to generate a CD34(+) cell population. These cells are isolated using a magnetic antibody separation kit and cultured on fibronectin-coated dishes in EC medium. To induce SMC differentiation, culture medium is changed and a morphological and phenotypic change occurs within 24 to 48 hours. RESULTS CD34(+) vascular progenitor cells give rise to ECs and SMCs. The two populations express respective cell-specific transcripts and proteins, exhibit intracellular calcium in response to various agonists, and form robust tube-like structures when cocultured in Matrigel. Human umbilical vein endothelial cells cultured under SMC conditions do not exhibit a change in phenotype or genotype. Wnt1-overexpressing stromal cells produced an increased number of progenitor cells. CONCLUSIONS The ability to generate large numbers of ECs and SMCs from a single vascular progenitor cell population is promising for therapeutic use to treat a variety of diseased and ischemic conditions. The stepwise differentiation outlined here is an efficient, reproducible method with potential for large-scale cultures suitable for clinical applications.

Critical role for lactate dehydrogenase A in aerobic glycolysis that sustains pulmonary microvascular endothelial cell proliferation

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.

Lung Vascular Cell Heterogeneity: Endothelium, Smooth Muscle, and Fibroblasts

The pulmonary circulation represents a unique vascular bed, receiving 100% of the cardiac output while maintaining low blood pressure. Multiple different cell types, including endothelium, smooth muscle, and fibroblasts, contribute to normal vascular function, and to the vascular response to injury. Our understanding of the basic cell biology of these various cell types, and the roles they play in vascular homeostasis and disease, remains quite limited despite several decades of study. Recent advances in approaches that enable the mapping of cell origin and the study of the molecular basis of structure and function have resulted in a rapid accumulation of new information that is essential to vascular biology. A recent National Institutes of Health workshop was held to discuss emerging concepts in lung vascular biology. The findings of this workshop are summarized in this article.

Essential Role of a Ca2+-Selective, Store-Operated Current (ISOC) in Endothelial Cell Permeability. Determinants of the Vascular Leak Site

Store-operated calcium (SOC) entry is sufficient to disrupt the extra-alveolar, but not the alveolar, endothelial cell barrier. Mechanism(s) underlying such insensitivity to transitions in cytosolic calcium ([Ca2+]i) in microvascular endothelial cells are unknown. Depletion of stored Ca2+ activates a larger SOC entry response in extra-alveolar (pulmonary artery; PAECs) than alveolar (pulmonary microvascular; PMVECs) endothelial cells. In vivo permeation studies revealed that Ca2+ store depletion activates similar nonselective cationic conductances in PAECs and PMVECs, while only PAECs possess the calcium-selective, store-operated Ca2+ entry current, ISOC. Pretreatment with the type 4 phosphodiesterase inhibitor, rolipram, abolished thapsigargin-activated ISOC in PAECs, and revealed ISOC in PMVECs. Rolipram pretreatment shifted the thapsigargin-induced fluid leak site from extra-alveolar to alveolar vessels in the intact pulmonary circulation. Thus, our results indicate ISOC provides a [Ca2+]i source that is needed to disrupt the endothelial cell barrier, and demonstrate that intracellular events controlling ISOC activation coordinate the site-specific vascular response to inflammation.

Ca2+ Channels and Pulmonary Endothelial Permeability: Insights from Study of Intact Lung and Chronic Pulmonary Hypertension

Phenotypic heterogeneity in pulmonary vascular endothelial cells extends to regulation of endothelial permeability, a process which often depends upon Ca2 + entry from the extracellular space. Scanning electron microscopy of vascular corrosion casts has documented distinct patterns of barrier disruption. Store depletion and activation of Ca2 + entry via canonical transient potential channels (TRPC1 and TRPC4) disrupts the barrier in extraalveolar vessels. In contrast, numerous other models of acute lung injury, including high vascular pressure‐ or epoxyeicosatrienoic acid‐induced injury, specifically disrupt the alveolar septal barrier. This review discusses Ca2 + permeant channels which potentially could be involved in regulation of barrier integrity in the alveolar septal compartment: transient receptor potential channels, cyclic nucleotide gated channels, purinergic (P2X) channels, and T‐type voltage gated channels. The evidence for the vanilloid transient receptor potential channel TRPV4 in regulating septal barrier function is discussed. Adaptations in barrier function in chronic pulmonary hypertension are reviewed, notably the loss of a store depletion‐dependent permeability response in the intact lung. Finally, the authors propose that since specific disruption of the alveolar septal barrier will have deleterious functional consequences, such as alveolar flooding and impairment of gas exchange, identification of specific molecular targets for Ca2 + entry‐dependent regulation of barrier function in this compartment is needed.

Dysfunctional resident lung mesenchymal stem cells contribute to pulmonary microvascular remodeling

Pulmonary vascular remodeling and oxidative stress are common to many adult lung diseases. However, little is known about the relevance of lung mesenchymal stem cells (MSCs) in these processes. We tested the hypothesis that dysfunctional lung MSCs directly participate in remodeling of the microcirculation. We employed a genetic model to deplete extracellular superoxide dismutase (EC-SOD) in lung MSCs coupled with lineage tracing analysis. We crossed floxpsod3 and mT/mG reporter mice to a strain expressing Cre recombinase under the control of the ABCG2 promoter. We demonstrated In vivo that depletion of EC-SOD in lung MSCs resulted in their contribution to microvascular remodeling in the smooth muscle actin positive layer. We further characterized lung MSCs to be multipotent vascular precursors, capable of myofibroblast, endothelial and pericyte differentiation in vitro. EC-SOD deficiency in cultured lung MSCs accelerated proliferation and apoptosis, restricted colony-forming ability, multilineage differentiation potential and promoted the transition to a contractile phenotype. Further studies correlated cell dysfunction to alterations in canonical Wnt/β-catenin signaling, which were more evident under conditions of oxidative stress. Our data establish that lung MSCs are a multipotent vascular precursor population, a population which has the capacity to participate in vascular remodeling and their function is likely regulated in part by the Wnt/β-catenin signaling pathway. These studies highlight an important role for microenviromental regulation of multipotent MSC function as well as their potential to contribute to tissue remodeling.

The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY(+)), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoY(K81M); ExoY(K81M)), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY(+) induced interendothelial cell gaps, whereas ExoY(K81M) and ΔPcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intratracheal inoculation of ExoY(+) and ExoY(K81M) caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved 1 wk following ExoY(K81M) infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY(+) infection. In conclusion, ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury.

Perivascular fluid cuffs decrease lung compliance by increasing tissue resistance

Objective:Lung inflammation causes perivascular fluid cuffs to form around extra-alveolar blood vessels; however, the physiologic consequences of such cuffs remain poorly understood. Herein, we tested the hypothesis that perivascular fluid cuffs, without concomitant alveolar edema, are sufficient to decrease lung compliance. Design:Prospective, randomized, controlled study. Setting:Research laboratory. Subjects:One hundred twenty male CD40 rats. Interventions:To test this hypothesis, the plant alkaloid thapsigargin was used to activate store-operated calcium entry and increase cytosolic calcium in endothelium. Thapsigargin was infused into a central venous catheter of intact, sedated, and mechanically ventilated rats. Measurements:Static and dynamic lung mechanics and hemodynamics were measured continuously. Main Results:Thapsigargin produced perivascular fluid cuffs along extra-alveolar vessels but did not cause alveolar flooding or blood gas abnormalities. Lung compliance dose-dependently decreased after thapsigargin infusion, attributable to an increase in tissue resistance that was attributed to increased tissue damping and tissue elastance. Airway resistance was not changed. Neither central venous pressure nor left ventricular end diastolic pressure was altered by thapsigargin. Heart rate did not change, although thapsigargin decreased left ventricular systolic function sufficient to reduce cardiac output by 50%. Infusion of the type 4 phosphodiesterase inhibitor, rolipram, prevented thapsigargin from inducing perivascular cuffs and decreasing lung compliance. Rolipram also normalized pressure over time and corrected the deficit in cardiac output. Conclusions:Our findings resolve for the first time that perivascular cuff formation negatively impacts mechanical coupling between the bronchovascular bundle and the lung parenchyma, decreasing lung compliance without impacting central venous pressure.