Pulmonary Endothelium Research Articles

Mechanisms of pulmonary endothelial permeability and inflammation caused by extracellular histone subunits H3 and H4.

Extracellular DNA-binding proteins such as histones are danger-associated molecular pattern released by the injured tissues in trauma and sepsis settings, which trigger host immune response and vascular dysfunction. Molecular events leading to histone-induced endothelial cell (EC) dysfunction remain poorly understood. This study performed comparative analysis of H1, H2A, H2B, H3, and H4 histone subunits effects on human pulmonary EC permeability and inflammatory response. Analysis of transendothelial electrical resistance and EC monolayer permeability for macromolecues revealed that H3 and H4, but not H1, H2A, or H2B caused dose-dependent EC permeability accompanied by disassembly of adherens junctions. At higher doses, H3 and H4 activated nuclear factor kappa B inflammatory cascade leading to upregulation EC adhesion molecules ICAM1, VCAM1, E-selectin, and release of inflammatory cytokines. Inhibitory receptor analysis showed that toll-like receptor (TLR) 4 but not TLR1/2 or receptor for advanced glycation end inhibition significantly attenuated deleterious effects of H3 and H4 histones. Inhibitor of Rho-kinase was without effect, while inhibition of Src kinase caused partial preservation of cell-cell junctions, H3/H4-induced permeability and inflammation. Deleterious effects of H3/H4 were blocked by heparin. Activation of Epac-Rap1 signaling restored EC barrier properties after histone challenge. Intravenous injection of histones in mice caused elevation of inflammatory markers and increased vascular leak. Post-treatment with pharmacological Epac/Rap1 activator suppressed injurious effects of histones in vitro and in vivo. These results identify H3 and H4 as key histone subunits exhibiting deleterious effects on pulmonary vascular endothelium via TLR4-dependent mechanism. In conclusion, elevation of circulating histones may represent a serious risk of exacerbated acute lung injury (ALI) and multiple organ injury during severe trauma and infection.

Substrate stiffness modulates migration and local intercellular membrane motion in pulmonary endothelial cell monolayers.

The pulmonary artery endothelium forms a semipermeable barrier that limits macromolecular flux through intercellular junctions. This barrier is maintained by an intrinsic forward protrusion of the interacting membranes between adjacent cells. However, the dynamic interactions of these membranes have been incompletely quantified. Here, we present a novel technique to quantify the motion of the peripheral membrane of the cells, called paracellular morphological fluctuations (PMFs), and to assess the impact of substrate stiffness on PMFs. Substrate stiffness impacted large-length scale morphological changes such as cell size and motion. Cell size was larger on stiffer substrates, whereas the speed of cell movement was decreased on hydrogels with stiffness either larger or smaller than 1.25 kPa, consistent with cells approaching a jammed state. Pulmonary artery endothelial cells moved fastest on 1.25 kPa hydrogel, a stiffness consistent with a healthy pulmonary artery. Unlike these large-length scale morphological changes, the baseline of PMFs was largely insensitive to the substrate stiffness on which the cells were cultured. Activation of store-operated calcium channels using thapsigargin treatment triggered a transient increase in PMFs beyond the control treatment. However, in hypocalcemic conditions, such an increase in PMFs was absent on 1.25 kPa hydrogel but was present on 30 kPa hydrogel-a stiffness consistent with that of a hypertensive pulmonary artery. These findings indicate that 1) PMFs occur in cultured endothelial cell clusters, irrespective of the substrate stiffness; 2) PMFs increase in response to calcium influx through store-operated calcium entry channels; and 3) stiffer substrate promotes PMFs through a mechanism that does not require calcium influx.

Evolving Concepts in Endothelial Pathobiology of Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a deadly disease, characterized by increased vascular resistance, pulmonary arteriolar loss, and occlusive arterial remodeling, leading to eventual right heart failure. Evidence increasingly points to the pulmonary endothelium as a central actor in PAH. Endothelial cell apoptosis can result directly in distal lung arteriolar pruning and indirectly in the formation of complex and occlusive arterial lesions, reflecting an imbalance between endothelial injury and repair in the development and progression of PAH. Many of the mutations implicated in PAH are in genes, which are predominantly, or solely, expressed in endothelial cells, and the endothelium is a major target for therapeutic interventions to restore BMP signaling. We explore how arterial pruning can promote the emergence of occlusive arterial remodeling mediated by ongoing endothelial injury secondary to hemodynamic perturbation and pathological increases in luminal shear stress. The emerging role of endothelial cell senescence is discussed in the transition from reversible to irreversible arterial remodeling in advanced PAH, and we review the sometimes conflicting evidence that female sex hormones can both protect or promote vascular changes in disease. Finally, we explore the contribution of the endothelium to metabolic changes and the altered inflammatory and immune state in the PAH lung, focusing on the role of excessive TGFβ signaling. Given the complexity of the endothelial pathobiology of PAH, we anticipate that emerging technologies that allow the study of molecular events at a single cell level will provide answers to many of the questions raised in this review.

The Vasculature in Pulmonary Fibrosis.

The current paradigm of idiopathic pulmonary fibrosis (IPF) pathogenesis involves recurrent injury to a sensitive alveolar epithelium followed by impaired repair responses marked by fibroblast activation and deposition of extracellular matrix. Multiple cell types are involved in this response with potential roles suggested by advances in single-cell RNA sequencing and lung developmental biology. Notably, recent work has better characterized the cell types present in the pulmonary endothelium and identified vascular changes in patients with IPF. Lung tissue from patients with IPF has been examined at single-cell resolution, revealing reductions in lung capillary cells and expansion of a population of vascular cells expressing markers associated with bronchial endothelium. In addition, pre-clinical models have demonstrated a fundamental role for aging and vascular permeability in the development of pulmonary fibrosis. Mounting evidence suggests that the endothelium undergoes changes in the context of fibrosis, and these changes may contribute to the development and/or progression of pulmonary fibrosis. Additional studies will be needed to further define the functional role of these vascular changes.

The myosin II inhibitor, blebbistatin, ameliorates pulmonary endothelial barrier dysfunction in acute lung injury induced by LPS via NMMHC IIA/Wnt5a/β-catenin pathway

Acute lung injury (ALI) or its most advanced form, acute respiratory distress syndrome (ARDS), is a severe inflammatory pulmonary process triggered by varieties of pathophysiological factors, among which endothelial barrier disruption plays a critical role in the progression of ALI/ARDS. As an inhibitor of myosin II, blebbistatin inhibits endothelial barrier damage. This study aimed to investigate the effect of blebbistatin on lung endothelial barrier dysfunction in LPS induced acute lung injury and its potential mechanism. Mice were challenged with LPS (5 mg/kg) by intratracheal instillation for 6 h to disrupt the pulmonary endothelial barrier in the model group. Blebbistatin (5 mg/kg, ip) was administrated 1 h before LPS challenge. The results showed that blebbistatin could significantly attenuate LPS-induced lung injury and pulmonary endothelial barrier dysfunction. And we observed that blebbistatin inhibited the activation of NMMHC IIA/Wnt5a/β-catenin pathway in pulmonary endothelium after LPS treatment. In murine lung vascular endothelial cells (MLECs) and human umbilical vein endothelial cells (HUVECs), we further confirmed that Blebbistatin (1 μmol/L) markedly ameliorated endothelial barrier dysfunction in MLECs and HUVECs by modulating NMMHC IIA/Wnt5a/β-catenin pathway. Our data demonstrated that blebbistatin could inhibit the development of pulmonary endothelial barrier dysfunction and ALI via NMMHC IIA/Wnt5a/β-catenin signaling pathway.

Ultrastructure of the Alveolar Cell Complex of Human Lung and its Functional Correlation: A Review

The human lung is a dynamic organ of constant biotransformation. Out of 40 different cell types of lungs, Type‑1 and Type‑2 pneumocytes, pulmonary capillary endothelium, and cells of lung interstitium play a vital role in functioning of the lung. Exploring the structure of these cells would shed light on the pathogenesis of various diseases affecting the lungs. A systematic literature search was conducted using three research databases – Medline, Google Scholar, and Cochrane. Out of ninety articles published between the year 1981 and 2020, 41 articles were included in the study based on the inclusion and exclusion criteria. Among the cellular components of the alveolar cell complex of the lung, the Type‑2 pneumocytes show versatility in its function in the form of surfactant synthesis, immune reaction, and regenerative capacity. The pathogenesis of the lung injury is due to the loss of functions of the Type‑2 pneumocytes and the intercellular junction functional discrepancy. Irrespective of the cause of lung disease, there is an increase in permeability in the alveolocapillary membrane leading to interstitial edema. This, in turn, leads to hyperventilation, low oxygen saturation, and a decrease in lung capacity. The pericytes present in the interstitium have a preventive role to play in lung injury and potential stem cell function. Keywords: alveolar epithelial cells, capillary endothelium, lung interstitium, type-1 pneumocytes, type-2 pneumocytes

An Update on Advancements and Challenges in Inhalational Drug Delivery for Pulmonary Arterial Hypertension.

A lethal condition at the arterial–alveolar juncture caused the exhaustive remodeling of pulmonary arterioles and persistent vasoconstriction, followed by a cumulative augmentation of resistance at the pulmonary vascular and, consequently, right-heart collapse. The selective dilation of the pulmonary endothelium and remodeled vasculature can be achieved by using targeted drug delivery in PAH. Although 12 therapeutics were approved by the FDA for PAH, because of traditional non-specific targeting, they suffered from inconsistent drug release. Despite available inhalation delivery platforms, drug particle deposition into the microenvironment of the pulmonary vasculature and the consequent efficacy of molecules are influenced by pathophysiological conditions, the characteristics of aerosolized mist, and formulations. Uncertainty exists in peripheral hemodynamics outside the pulmonary vasculature and extra-pulmonary side effects, which may be further exacerbated by underlying disease states. The speedy improvement of arterial pressure is possible via the inhalation route because it has direct access to pulmonary arterioles. Additionally, closed particle deposition and accumulation in diseased tissues benefit the restoration of remolded arterioles by reducing fallacious drug deposition in other organs. This review is designed to decipher the pathological changes that should be taken into account when targeting the underlying pulmonary endothelial vasculature, especially with regard to inhaled particle deposition in the alveolar vasculature and characteristic formulations.

Pulmonary Endothelial Activation with COVID‐19: Possible Role of Reactive Oxygen Species

IntroductionRecent research suggests that endothelial activation plays a role in COVID‐19 pathogenesis by promoting a pro‐coagulative and pro‐inflammatory state. However, the mechanism by which the endothelium is activated in COVID‐19 is unclear.ObjectiveTo investigate the mechanism by which COVID‐19 activates the pulmonary endothelium.HypothesisThe pulmonary endothelium generates reactive oxygen species (ROS) upon exposure to the “inflammatory load” of the systemic circulation.MethodsCOVID‐19 was recreated in vitro and ex vivo, by exposing human lung endothelial cells (EC) or donor human lung slices (human precision‐cut lung slices or huPCLS) to medium supplemented with serum from COVID‐19 affected subjects. Sera were acquired from patients with COVID‐19 infection admitted to the Intensive Care Unit of the Hospital at the University of Pennsylvania. ROS (fluorescent dye, CellROX) and intercellular adhesion molecule (ICAM‐1) levels were assessed by fluorescence labeling and imaging.ResultsBoth EC activation (as monitored by ROS production) and pro‐inflammatory phenotype (as assessed by ICAM‐1), were significantly higher with COVID‐19 as compared to normal subjects.ConclusionsThe endothelium is activated with COVID‐19 via ROS production; thus, the ROS produced drive a pro‐inflammatory phenotype by inducing the expression of ICAM‐1, a pivotal marker of endothelium inflammation. As ROS mediates EC activation and inflammation during COVID‐19, ROS blockade could be a therapeutic target in maintaining vascular health.

Peroxiredoxin 6 suppresses erastin‐induced ferroptosis in lung endothelial cells

Ferroptosis is a newly described form of regulated cell death driven by an iron‐dependent accumulation of phospholipid hydroperoxides. Glutathione peroxidase 4 (GPx4) suppresses ferroptosis through its ability to reduce phospholipid hydroperoxides. Peroxiredoxin 6 (Prdx6) expresses phospholipid hydroperoxide glutathione peroxidase activity, but its role in the regulation of ferroptosis remains unexplored. We compared Prdx6 and GPx4 mRNA levels in murine lungs. Prdx6 expression was five times higher than GPx4 expression. We then visualized the spatial localization of lung Prdx6. Prdx6 was expressed in both pulmonary epithelial (epCAM+) and endothelial (Ve‐cadherin+) cells. We studied the role of Prdx6 on lung ferroptosis induced by erastin, an inhibitor of the cystine‐glutamate antiporter, which is critical to glutathione production. Erastin (1‐5μM, 24h) increased lipid peroxidation and cell death in mouse pulmonary microvascular endothelial cells (MPMVECs) in primary culture; this effect was higher in Prdx6‐KO MPMVECs than in WT‐MPMVECs. Co‐treatment with the ferroptosis suppressor ferrostatin‐1 prevented erastin‐induced lipid peroxidation and cell death in both WT and Prdx6‐KO MPMVECs. Similarly, knockdown (KD) of Prdx6 in human pulmonary microvascular endothelial cells (HPMVECs) increased erastin‐induced cell death, suggesting that Prdx6 limits ferroptosis in the pulmonary endothelium. We then conducted extracellular flux assays and RNA‐seq analyses in Prdx6‐deficient cells to further explore how the loss of Prdx6 sensitizes cells to ferroptosis. Cellular respiration and mitochondrial function were blunted in Prdx6‐deficient cells compared to corresponding controls. Top differentially expressed genes in Prdx6‐KD HPMVECs include known regulators of iron metabolism and ferroptosis such as hmox1, cybrd1, and sfxn1. Upregulated Reactome pathways enriched in Prdx6‐KD HPMVECs include mitochondrial and amino acid metabolism and p53‐mediated DNA damage response, while downregulated pathways include biological oxidations, transport of bile salts and organic acids, metal ions and amine compounds, cytochrome P450, and phase 1 metabolism. These results show that loss of Prdx6 sensitizes lung endothelial cells to ferroptosis by promoting lipid peroxidation and altering transcriptional signatures associated with cell cycle progression and mitochondrial metabolism.

Abstract 108: Mediator 1 Regulation Of The BMP / TGF-β Signaling In Endothelium: Implications For Pulmonary Hypertension

Rationale: Dysregulated bone morphogenetic protein (BMP) or transforming growth factor β (TGF-β) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. Mediator 1 (MED1) is a key transcriptional coactivator and Krüppel-like factor 4 (KLF4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-β axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive. Objectives: We investigate the MED1/KLF4 co-regulation of the BMP/TGF-β axes in endothelium by studying the epigenetic regulation of BMP receptor type II (BMPR2), ETS-related gene (ERG), and TGF-β receptor 2 (TGFBR2) and their involvement in the PH. Methods: High throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, KLF4 ATAC-seq, and high-throughput chromosome conformation capture (HiC) together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells (PAECs) and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic pulmonary arterial hypertension (IPAH), animals with experimental PH, and mice with endothelial ablation of MED1 (EC-MED1 -/- ) were used to study the PH-protective effect of MED1. Results: Levels of MED1 were decreased in lung tissue or PAECs from IPAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-MED1 -/- mice showed PH susceptibility. In contrast, MED1 overexpression mitigated the PH phenotype in rodents. Conclusions: A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-β signaling can lead to the development of PAH/PH.

Comparison Of Dynamic Calcium Signaling Profiles On Activation Of TRPV4 Channel And T‐Type Calcium Channel In Pulmonary Microvasculature

BackgroundAcute Lung Injury (ALI) is characterized by inflammation, barrier disruption and edema within lung microvasculature. A dysregulation in intercellular signaling of calcium (Ca2+) within pulmonary microvascular endothelium potentially damages lung endothelial barrier integrity. Recently, two plasma membrane ion channels, the vanilloid transient receptor potential IV (TRPV4) and the T‐type voltage gated calcium channels (TTCC) have been implicated as potential conduits for spatially and temporally targeted Ca2+ signals within lung endothelium. Ca2+ entry via TRPV4 causes barrier disruption and Ca2+ entry via TTCC regulates P‐selectin expression, but their underlying patterns of specific Ca2+ signaling events are unknown. Therefore, in this study we aim to determine whether these channels elicit distinct Ca2+ signaling events that might contribute to their different functions in lung endothelium.MethodPulmonary microvascular endothelial cells (PMVECs) grown in 35mm glass‐bottomed dishes were loaded with fluorescent Ca2+ indicator Cal‐520 AM and recorded for signals via spinning disk confocal microscopy. After baseline recording, cells were subjected to GSK1016790 (TRPV4 activator) and then to GSK2798754 (TRPV4 inhibitor). Another set of cells were treated with high K+ solution (TTCC activator) and then with mibefradil (TTCC inhibitor). Lung slices were mounted on Sylgard inserts, and loaded with Cal‐520 AM. Two different slices were recorded for baseline activities and were respectively subjected to same drug treatments as PMVECs. Time‐lapse image sequences recorded after each addition were analyzed for amplitude and frequency of events using ImageJ and customized signal detection software LC_Pro.ResultsIn PMVECs with TRPV4 channel activation, the amplitude and frequency of events increased 1.5‐fold when compared to baseline, whereas the TTCC activation showed an increase by 1.3‐fold. In lung slices, the TRPV4 channel activation exhibited an increase by 1.4‐fold compared to baseline, as opposed to an increase of only 1.1‐fold with TTCC activation. The addition of respective inhibitors brought the signals back to near baseline levels.ConclusionOur findings suggest both TRPV4 channel and TTCC activation caused an increase in Ca2+ dynamics, with the TRPV4 channel activation showing a greater increase in event amplitude and frequency than TTCCs, thereby indicating they both contribute to distinct signaling events in PMVECs and lung slices. Further studies will focus on how these different signatures contribute to the differential functional roles of the channels.

Interferon‐alpha or ‐beta Facilitates SARS‐CoV‐2 Pulmonary Vascular Infection by Inducing ACE2

Severe viral pneumonia caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is characterized by a hyperinflammatory state typified by elevated circulating pro‐inflammatory cytokines, frequently leading to potentially lethal vascular complications including thromboembolism, disseminated intracellular coagulopathy and vasculitis. Though endothelial infection and subsequent endothelial damage have been described in patients with fatal COVID‐19, the mechanism by which this occurs remains elusive, particularly given that, under naïve conditions, pulmonary endothelial cells demonstrate minimal cell surface expression of the SARS‐CoV‐2 binding receptor ACE2. Herein we describe SARS‐CoV‐2 infection of the pulmonary endothelium in postmortem lung samples from individuals who died of COVID‐19, demonstrating both heterogeneous ACE2 expression and endothelial damage (Figure). In primary endothelial cell cultures, we show that SARS‐CoV‐2 infection is dependent on the induction of ACE2 protein expression and that this process is facilitated by type 1 interferon‐alpha (IFNα) or ‐beta(β) ‐ two of the main anti‐viral cytokines induced in severe SARS‐CoV‐2 infection ‐ but not significantly by other cytokines (including interleukin 6 and interferon g /λ). Our findings suggest that the stereotypical anti‐viral interferon response may paradoxically facilitate the propagation of COVID‐19 from the respiratory epithelium to the vasculature, raising concerns regarding the use of exogenous IFNα/β in the treatment of patients with COVID‐19.

Cell‐Free Hemoglobin Increases Leukocyte Adhesion and Mitochondrial Oxidative Damage in the Pulmonary Microvascular Endothelium

Sepsis is a critical problem in intensive care units globally, and accounts for 20% of all annual deaths. During septic shock, the highly inflammatory state leads to the release of cell‐free hemoglobin (CFH) from lysed red blood cells. Once released into the vascular circulation, CFH can oxidize from the normal ferrous 2+ state to methemoglobin (3+, ferric). We have shown previously that CFH is a key driver of acute respiratory distress syndrome due to the breakdown of the alveolar capillary barrier. However, the underlying mechanism of how CFH disrupts the pulmonary endothelium remains unknown. Originally, it was believed hemoglobin acted only in an oxidative damage fashion. We hypothesized that oxidized CFH disrupts the pulmonary endothelium due to mitochondrial oxidative damage and leads to increased leukocyte adhesion. We utilized primary human lung microvascular endothelial cells (HLMVECs) to probe the mechanism underlying CFH‐induced barrier dysfunction. Endothelial barrier function was analyzed using electric cell‐substrate impedance sensing (ECIS) to quantify the transendothelial resistance changes in real‐time following treatment with CFH2+ and CFH3+ (1.0 and 0.5 mg/mL). Permeability was assessed using the SynVivo idealized network coculture system using HLMVECs and small airway epithelial cells and was quantified using fluorescent 60 kDa dextrans mixed with CFH2+,3+ (1.0 mg/mL) in media flowed through the channels while images were taken every 15 minutes for 4 h. Cytokines were measured via ELISA post CFH3+ treatment from conditioned culture media at 1, 3, 6, and 24 h. Adhesion of leukocytes was quantified using immunofluorescence by counting the number of fluorescent polymorphonuclear cells (PMNs) adhered to an endothelial monolayer following pretreatment with CFH3+ of the endothelial cells, the PMNs, or both. Mitochondrial superoxide production was quantified by MitoSOX staining followed by flow cytometry analysis of CFH‐treated HLMVECs. We saw a dose‐dependent decrease in transendothelial resistance following oxidized CFH3+ treatment but not with CFH2+ treatment (‐1270 vs 262 max TER drop, p<0.0001). Real‐time permeability assays using the SynVivo system showed an increase in barrier permeability from CFH3+ treatment compared to CFH2+ (47 vs 6 relative permeability). Surface expression of E‐selectin (p=0.037) and ICAM‐1 (p=0.0497) was increased following oxidized CFH3+ treatment versus vehicle. In addition, pretreatment of HLMVECs with CFH3+ increased adhesion of polymorphonuclear cells (PMNs) to the endothelium (1.69 x 108 vs 7.81 x 109 GFP intensity, p=0.0014), while pretreatment of the PMNs did not increase adhesion. Treatment of HLMVECs with CFH3+ for 24 h increased secretion of IL‐1b, IL‐6, and IL‐8 (p<0.05). HLMVECs exposed to CFH3+ for 6 h have significantly increased mitochondrial superoxide compared to vehicle treated cells (608 vs 283 adjusted MFI, p=0.0014). These data demonstrate that oxidized CFH decreases pulmonary endothelial function through both immune and oxidative damage pathways. We also show that CFH induces endothelial dysfunction in an oxidation‐state dependent manner. In summary, these studies provide new evidence to the potential mechanism in which CFH harms the endothelium during sepsis beyond the conventionally attributed oxidative cycling of the heme moiety.

Hemin-Induced Endothelial Dysfunction and Endothelial to Mesenchymal Transition in the Pathogenesis of Pulmonary Hypertension Due to Chronic Hemolysis.

Pulmonary hypertension in sickle cell disease is an independent predictor of mortality, yet the pathogenesis of pulmonary vascular disease in chronic hemolytic disorders remains incompletely understood and treatment options are limited primarily to supportive care. The release of extracellular hemoglobin has been implicated in the development of pulmonary hypertension, and in this study we explored the direct effects of hemin, the oxidized moiety of heme, on the pulmonary artery endothelium. We found that low dose hemin exposure leads to significantly increased endothelial cell proliferation, migration, and cytokine release as markers of endothelial dysfunction. Protein expression changes in our pulmonary artery endothelial cells showed upregulation of mesenchymal markers after hemin treatment in conjunction with a decrease in endothelial markers. Endothelial to mesenchymal transition (EndoMT) resulting from hemin exposure was further confirmed by showing upregulation of the transcription factors SNAI1 and SLUG, known to regulate EndoMT. Lastly, given the endothelial dysfunction and phenotypic transition observed, the endothelial cytoskeleton was considered a potential novel target. Inhibiting myosin light chain kinase, to prevent phosphorylation of myosin light chain and cytoskeletal contraction, attenuated hemin-induced endothelial hyper-proliferation, migration, and cytokine release. The findings in this study implicate hemin as a key inducer of endothelial dysfunction through EndoMT, which may play an important role in pulmonary vascular remodeling during the development of pulmonary hypertension in chronic hemolytic states.

Endothelial cells of pulmonary origin display unique sensitivity to the bacterial endotoxin lipopolysaccharide.

Acute respiratory distress syndrome (ARDS) is a major clinical problem without available therapies. Known risks for ARDS include severe sepsis, SARS‐CoV‐2, gram‐negative bacteria, trauma, pancreatitis, and blood transfusion. During ARDS, blood fluids and inflammatory cells enter the alveoli, preventing oxygen exchange from air into blood vessels. Reduced pulmonary endothelial barrier function, resulting in leakage of plasma from blood vessels, is one of the major determinants in ARDS. It is, however, unknown why systemic inflammation particularly targets the pulmonary endothelium, as endothelial cells (ECs) line all vessels in the vascular system of the body. In this study, we examined ECs of pulmonary, umbilical, renal, pancreatic, and cardiac origin for upregulation of adhesion molecules, ability to facilitate neutrophil (PMN) trans‐endothelial migration (TEM) and for endothelial barrier function, in response to the gram‐negative bacterial endotoxin LPS. Interestingly, we found that upon LPS stimulation, pulmonary ECs showed increased levels of adhesion molecules, facilitated more PMN‐TEM and significantly perturbed the endothelial barrier, compared to other types of ECs. These observations could partly be explained by a higher expression of the adhesion molecule ICAM‐1 on the pulmonary endothelial surface compared to other ECs. Moreover, we identified an increased expression of Cadherin‐13 in pulmonary ECs, for which we demonstrated that it aids PMN‐TEM in pulmonary ECs stimulated with LPS. We conclude that pulmonary ECs are uniquely sensitive to LPS, and intrinsically different, compared to ECs from other vascular beds. This may add to our understanding of the development of ARDS upon systemic inflammation.

A Novel Controlled Cold Storage System for Optimal Lung Preservation

Purpose Currently the on-ice method is the standard technique used to transport lungs for transplantation. This involves placing the organ in preservation solution and subsequently in a cooler filled with ice. This method is imprecise, difficult to monitor and results in temperatures below 2° C. These temperatures impair mitochondrial activity, damage pulmonary endothelium and can potentially lead to primary and/or long-term graft dysfunction. The objective of this study was to compare the standard on-ice technique to a controlled rate cooling system using the LUNGguard (Paragonix) device to maintain an organ temperature of 4° to 8° C. Methods Porcine lungs were procured and preserved for 1, 2, 4 and 6 hours (according to clinical protocol). For each time point, two sets of lungs were preserved using the standard on-ice technique and the LUNGguard device. Temperatures were measured with thermal surface imaging at the beginning and the end of each run. Results In the traditional on-ice preservation method the temperature dropped from 2.3° to -1.3° C after 6 hours. Controlled rate storage using the LUNGguard device was able to maintain organ temperatures between 4° and 8° C across all time periods. Conclusion The studied controlled rate cooling system offers a consistent hypothermic preservation method for the lungs between 4° to 8° C. This ideal and consistent storage temperature may reduce mitochondrial and endothelial cell damage of donor lungs and prevent primary and long-term graft failure. Currently the on-ice method is the standard technique used to transport lungs for transplantation. This involves placing the organ in preservation solution and subsequently in a cooler filled with ice. This method is imprecise, difficult to monitor and results in temperatures below 2° C. These temperatures impair mitochondrial activity, damage pulmonary endothelium and can potentially lead to primary and/or long-term graft dysfunction. The objective of this study was to compare the standard on-ice technique to a controlled rate cooling system using the LUNGguard (Paragonix) device to maintain an organ temperature of 4° to 8° C. Porcine lungs were procured and preserved for 1, 2, 4 and 6 hours (according to clinical protocol). For each time point, two sets of lungs were preserved using the standard on-ice technique and the LUNGguard device. Temperatures were measured with thermal surface imaging at the beginning and the end of each run. In the traditional on-ice preservation method the temperature dropped from 2.3° to -1.3° C after 6 hours. Controlled rate storage using the LUNGguard device was able to maintain organ temperatures between 4° and 8° C across all time periods. The studied controlled rate cooling system offers a consistent hypothermic preservation method for the lungs between 4° to 8° C. This ideal and consistent storage temperature may reduce mitochondrial and endothelial cell damage of donor lungs and prevent primary and long-term graft failure.

Paraquat Induces Lung Injury via miR-199-Mediated SET in a Mouse Model.

Objective: To explore the molecular mechanism of lung injury caused by paraquat (PQ) poisoning by investigating miR-199-mediated SET. Methods: A paraquat poisoning model was established in C57BL/6 male mice via intraperitoneal injection of paraquat. The mice were transfected with miR-199 siRNA and or mimic. After 14 days of treatment, pathophysiological changes of the lung were observed and lung tissue was analyzed via Hematoxylin-Eosin staining. The levels of miR-199, SETs, surfactant protein SP-A and SP-B, and inflammatory and oxidative factors were analyzed by qPCR, Western Blot, and ELISA kits. Results: A acute lung-injury (ALI) model was established using PQ treatment and confirmed with edema of pulmonary endothelium with low electronic density of endothelial cytoplasm, presence of protein-rich fluid, and numerous erythrocytes in alveolar space, concentric figures of damaged tubular myelin, alveolar destruction, and increase in inflammatory cell numbers. Compared with the control group, miR-199 and SET levels were reduced in the PQ-treated group. miR-199 siRNA increased the SET level, inflammatory and oxidative levels, and reduced the levels of SP-A and SP-B, and miR-199 mimic reduced the SET level, inflammatory and oxidative levels, and increased the levels of SP-A and SP-B. PQ treatment reduced miR-199 level. Conclusion: Paraquat induces ALI by affecting miR-199-mediated SET.

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.

Endothelial Senescence: A New Age in Pulmonary Hypertension.

Pulmonary hypertension is an enigmatic, deleterious disease driven by multiple heterogeneous causes with a burgeoning proportion of older patients with complex, chronic comorbidities without adequate treatment options. The underlying endothelial pathophenotypes that direct vasoconstriction and panvascular remodeling remain both controversial and incompletely defined. This review discusses emerging concepts centered on endothelial senescence in pulmonary vascular disease. This principle proposes a more heterogeneous, dynamic pulmonary endothelium in disease; it provides a potentially unifying feature of endothelial dysfunction in pulmonary hypertension irrespective of cause; and it supports a clinically relevant link between aging and pulmonary hypertension like other chronic illnesses. Thus, taking cues from studies on aging and age-related diseases, we present possible opportunities and barriers to diagnostic and therapeutic targeting of senescence in pulmonary hypertension.

Sodium tanshinone IIA sulfonate enhances the BMP9-BMPR2-Smad1/5/9 signaling pathway in rat pulmonary microvascular endothelial cells and human embryonic stem cell–derived endothelial cells

BackgroundRecent studies have demonstrated the beneficial effects of STS in treating pulmonary hypertension by inhibiting the pulmonary vascular remodeling and suppressing the abnormally elevated proliferation and migration of PASMCs. However, the roles of STS on pulmonary vascular endothelium remain largely known. MethodsIn this study, we investigated the effects and mechanisms of STS on pulmonary vascular endothelial dysfunction by using a chronic hypoxia-induced pulmonary hypertension (HPH) rat model, as well as in primarily cultured rat PMVECs and human ESC-ECs cell models. ResultsFirstly, a 21-day treatment of STS significantly prevents the disease development of HPH by normalizing the right ventricular systolic pressure and right ventricular hypertrophy, improving the cardiac output. Then, STS treatment markedly inhibits the hypoxia-induced medial wall thickening of the distal intrapulmonary arteries. Notably, STS significantly inhibits the hypoxia-induced apoptosis in both the pulmonary endothelium of HPH rats and primarily cultured PMVECs, through the stabilization of BMPR2 protein and protection of the diminished BMP9-BMPR2-Smad1/5/9 signaling pathway. In mechanism, STS treatment retrieves the hypoxic downregulation of BMPR2 by stabilizing the BMPR2 protein, inhibiting the BMPR2 protein degradation via lysosome system, and promoting the plasma membrane localization of BMPR2, all of which together reinforcing the BMP9-induced signaling transduction in both PMVECs and human ESC-ECs. However, these effects are absent in hESC-ECs expressing heterozygous dysfunctional BMPR2 protein (BMPR2+/R899X). ConclusionSTS may exert anti-apoptotic roles, at least partially, via induction of the BMP9-BMPR2-Smad1/5/9 signaling transduction in pulmonary endothelium and PMVECs.