Pulmonary Microvascular Endothelial Cells Research Articles

Knocking Out of CEACAM1 Can Reduce Oxidative Stress and Promote Cell Proliferation in the HPMVECs under Hypoxia.

Pulmonary hypertension (PH) induced by hypoxia is common in clinical practice and often suggests a poor prognosis. The oxidative stress and proliferation of pulmonary vascular endothelial cells caused by hypoxia are the major mechanisms involved in the pathophysiology of PH. It has been reported in recent years that the carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes angiogenesis. In this study, normal human pulmonary microvascular endothelial cells (HPMVECs) and HPMVECs with stable knockout of CEACAM1 by CRISPR-Cas9 were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) to induce hypoxic conditions. JC-1, ROS, and cell cycle profile were analyzed for each cell line and controls, using flow cytometry. A tube formation assay was used to detect angiogenesis, along with expression levels of CEACAM1, TNF-α, VEGF, VEGFR-2, p-P38/P38, and CyclinD1 proteins (to distinguish profiles of angiogenic growth and cell proliferation). We observed increased expression of CEACAM1 in HPMVECs after OGD/R, while ROS production was reduced and mitochondrial membrane potential was increased after OGD/R in CEACAM1−/− HPMVECs. Furthermore, we observed increased cell division in CEACAM−/− HPMVECs, accompanied by enhanced angiogenesis and reduced TNF-α protein expression and increased VEGF, VEGFR-2, and CyclinD1 expression. Together, these data suggest that upregulation of CEACAM1 in HPMVECs under hypoxic conditions may damage cells by increasing oxidative stress and inhibiting cell proliferation.

Yu-Ping-Feng Formula Ameliorates Alveolar-Capillary Barrier Injury Induced by Exhausted-Exercise via Regulation of Cytoskeleton.

Background: Yu-ping-feng powder (YPF) is a compound traditional Chinese medicine extensively used in China for respiratory diseases. However, the role of YPF in alveolar-capillary barrier dysfunction remains unknown. This study aimed to explore the effect and potential mechanism of YPF on alveolar-capillary barrier injury induced by exhausted exercise. Methods: Male Sprague–Dawley rats were used to establish an exhausted-exercise model by using a motorized rodent treadmill. YPF at doses of 2.18 g/kg was administrated by gavage before exercise training for 10 consecutive days. Food intake-weight/body weight, blood gas analysis, lung water percent content, BALF protein concentration, morphological observation, quantitative proteomics, real-time PCR, and Western blot were performed. A rat pulmonary microvascular endothelial cell line (PMVEC) subjected to hypoxia was applied for assessing the related mechanism. Results: YPF attenuated the decrease of food intake weight/body weight, improved lung swelling and hemorrhage, alleviated the increase of lung water percent content and BALF protein concentration, and inhibited the impairment of lung morphology. In addition, YPF increased the expression of claudin 3, claudin 18, occludin, VE-cadherin, and β-catenin, attenuated the epithelial and endothelial hyperpermeability in vivo and/or in vitro, and the stress fiber formation in PMVECs after hypoxia. Quantitative proteomics discovered that the effect of YPF implicated the Siah2-ubiquitin-proteasomal pathway, Gng12-PAK1-MLCK, and RhoA/ROCK, which was further confirmed by Western blot. Data are available via ProteomeXchange with identifier PXD032737. Conclusion: YPF ameliorated alveolar-capillary barrier injury induced by exhausted exercise, which is accounted for at least partly by the regulation of cytoskeleton.

Abstract 6313: Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia

Abstract Acute myeloid leukemia is a grievous illness. BiTE® (bispecific T cell engager) molecules redirect T cells by engaging CD3 and a tumor-associated antigen (TAA). These molecules have shown clinical efficacy but one mechanism of resistance is loss of a single TAA. We hypothesized that a BiTE® molecule targeting >1 TAA could reduce relapse frequency. FLT3 and CD123, were selected for a dual-targeting BiTE® (dBiTE࣪) molecule.A half-life extended (HLE) CD123-FLT3 dBiTE࣪ molecule was evaluated in vitro, in mouse xenografts, and in non-human primate (NHP) tolerability studies. The molecule had nM affinity for human and NHP FLT3, CD123, and CD3, and pM efficacy in cytotoxicity assays using human T cells or NHP PBMCs. The molecule achieved 100% killing against single-positive (sp) cells (CRISPR-generated isogenic cell lines) at potencies like those of double-positive (dp) cells (7.4 ±. 4.1 pM FLT3sp, 7.1 ±. 3.8 pM CD123sp and 3.4 ±. 1.5 pM dp, n=3). In a mouse xenograft model, the molecule induced significant activity and extended survival > 3 weeks (1.0, 0.1, and 0.01 mg/kg (p ≤ 0.001)). In mice with sp tumors, survival benefit could not be calculated due to high survival, demonstrating that both arms of the molecule are active. In NHP, the molecule had a half-life of 52 hours (0.3 or 3 µg/kg). FLT3 mRNA levels, a marker of FLT3-expressing cells, decreased in blood following dosing. Repeat dosing was not tolerated, and cytokine release was observed. Some cytokines were reduced while others increased. CD123 is reportedly expressed on endothelial cells (ECs), with increased expression in inflammatory conditions. An immunohistochemical survey found that CD123 is expressed on human and NHP monocyte/macrophages and ECs with limited distribution in lymphoid tissues and lamina propria of the gut. We hypothesized that cytokine release following administration may result in increased CD123 expression and in turn, further increased cytokine levels with repeat dosing. CD123 was detected on primary human umbilical vein ECs (HUVECs). In co-cultures of HUVECs and T cells, a CD123 BiTE® molecule induced expression of CD123 on HUVEC cells at concentrations that elicited redirected lysis (12.5 pM), T cell activation, and cytokine secretion. To better understand BiTE®-induced upregulation of CD123 on ECs, HUVECs were cultured with supernatants (SN) from a TDCC assay or recombinant IL-3, IL-6, TNFα, or IFNγ. Assay SN and TNFα induced >2-fold CD123 expression on HUVECs, but not on CD123-negative primary human pulmonary microvascular ECs. These data demonstrate that CD123 expression on ECs was increased upon exposure to a CD123 mono-targeting BiTE® molecule, potentially through BiTE®-induced secretion of TNFα. Additional studies are ongoing.In sum, a CD123-FLT3 HLE dBiTE࣪ molecule was active against both dp- and sp-positive target cells in vitro and in vivo. Careful selection of TAA for dBiTE࣪ molecules is necessary to increase efficacy and maintain safety. Citation Format: Rebecca Goldstein, Christine Karbowski, Anja Henn, Petra Deegen, Joachim Wahl, Katja Matthes, Christoph Dahlhoff, Brooke Rock, Sabrina Benchaar, Katie Hsu, Brandy Alexander, Matthias Friedrich, Joan Lane, Xiaoting Wang, Jonas Anlahr, Markus Muenz, Tobias Raum, Tara L. Arvedson. Evaluation of a dual CD123-FLT3 BiTE molecule for acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6313.

TLR7 Mediates Acute Respiratory Distress Syndrome in Sepsis by Sensing Extracellular miR-146a.

TLR7 (Toll-like receptor 7), the sensor for single-stranded RNA, contributes to systemic inflammation and mortality in murine polymicrobial sepsis. Recent studies show that extracellular miR-146a-5p serves as a TLR7 ligand and plays an important role in regulating host innate immunity. However, the role of miR-146a-5p and TLR7 signaling in pulmonary inflammation, endothelial activation, and sepsis-associated acute respiratory distress syndrome remains unclear. Here, we show that intratracheal administration of exogenous miR-146a-5p in mice evokes lung inflammation, activates endothelium, and increases endothelial permeability via TLR7-dependent mechanisms. TLR7 deficiency attenuates pulmonary barrier dysfunction and reduces lung inflammatory response in a murine sepsis model. Moreover, the impact of miR-146a-5p-TLR7 signaling on endothelial activation appears to be a secondary effect because TLR7 is undetectable in the human pulmonary artery and microvascular endothelial cells (ECs), which show no response to direct miR-146a-5p treatment invitro. Both conditioned media of miR-146a-5p-treated macrophages (Mϕ) and septic sera of wild-type mice induce a marked EC barrier disruption invitro, whereas Mϕ conditioned media or septic sera of TLR7-/- mice do not exhibit such effect. Cytokine array and pathway enrichment analysis of the Mϕ conditioned media and septic sera identify TNFα (tumor necrosis factor α) as the main downstream effector of miR-146a-5p-TLR7 signaling responsible for the EC barrier dysfunction, which is further supported by neutralizing anti-TNFα antibody intervention. Together, these data demonstrate that TLR7 activation elicits pulmonary inflammation and endothelial barrier disruption by sensing extracellular miR-146a-5p and contributes to sepsis-associated acute respiratory distress syndrome.

Autophagy regulates the effects of ADSC-derived small extracellular vesicles on acute lung injury

Small extracellular vesicles (sEVs) have been recognized to be more effective than direct stem cell differentiation into functional target cells in preventing tissue injury and promoting tissue repair. Our previous study demonstrated the protective effect of adipose-derived stem cells (ADSCs) on lipopolysaccharide (LPS)-induced acute lung injury and the effect of autophagy on ADSC functions, but the role of ADSC-derived sEVs (ADSC-sEVs) and autophagy-mediated regulation of ADSC-sEVs in LPS-induced pulmonary microvascular barrier damage remains unclear. After treatment with sEVs from ADSCs with or without autophagy inhibition, LPS-induced human pulmonary microvascular endothelial cell (HPMVECs) barrier damage was detected. LPS-induced acute lung injury in mice was assessed in vivo after intravenous administration of sEVs from ADSCs with or without autophagy inhibition. The effects of autophagy on the bioactive miRNA components of ADSC-sEVs were assessed after prior inhibition of cell autophagy. We found that ADSC-sEV effectively alleviated LPS-induced apoptosis, tight junction damage and high permeability of PMVECs. Moreover, in vivo administration of ADSC-sEV markedly inhibited LPS-triggered lung injury. However, autophagy inhibition, markedly weakened the therapeutic effect of ADSC-sEVs on LPS-induced PMVECs barrier damage and acute lung injury. In addition, autophagy inhibition, prohibited the expression of five specific miRNAs in ADSC-sEVs -under LPS-induced inflammatory conditions. Our results indicate that ADSC-sEVs protect against LPS-induced pulmonary microvascular barrier damage and acute lung injury. Autophagy is a positive mediator of sEVs function, at least in part through controlling the expression of bioactive miRNAs in sEVs.

Extracellular Signal-Regulated Kinase 1 Alone Is Dispensable for Hyperoxia-Mediated Alveolar and Pulmonary Vascular Simplification in Neonatal Mice.

Bronchopulmonary dysplasia (BPD) is a morbid lung disease distinguished by lung alveolar and vascular simplification. Hyperoxia, an important BPD causative factor, increases extracellular signal-regulated kinases (ERK)-1/2 expression, whereas decreased lung endothelial cell ERK2 expression reduces angiogenesis and potentiates hyperoxia-mediated BPD in mice. However, ERK1′s role in experimental BPD is unclear. Thus, we hypothesized that hyperoxia-induced experimental BPD would be more severe in global ERK1-knockout (ERK1-/-) mice than their wild-type (ERK1+/+ mice) littermates. We determined the extent of lung development, ERK1/2 expression, inflammation, and oxidative stress in ERK1-/- and ERK1+/+ mice exposed to normoxia (FiO2 21%) or hyperoxia (FiO2 70%). We also quantified the extent of angiogenesis and hydrogen peroxide (H2O2) production in hyperoxia-exposed neonatal human pulmonary microvascular endothelial cells (HPMECs) with normal and decreased ERK1 signaling. Compared with ERK1+/+ mice, ERK1-/- mice displayed increased pulmonary ERK2 activation upon hyperoxia exposure. However, the extent of hyperoxia-induced inflammation, oxidative stress, and interrupted lung development was similar in ERK1-/- and ERK1+/+ mice. ERK1 knockdown in HPMECs increased ERK2 activation at baseline, but did not affect in vitro angiogenesis and hyperoxia-induced H2O2 production. Thus, we conclude ERK1 is dispensable for hyperoxia-induced experimental BPD due to compensatory ERK2 activation.

Carbonic anhydrase IX proteoglycan-like and intracellular domains mediate pulmonary microvascular endothelial cell repair and angiogenesis.

The lungs of patients with acute respiratory distress syndrome (ARDS) have hyperpermeable capillaries that must undergo repair in an acidic microenvironment. Pulmonary microvascular endothelial cells (PMVECs) have an acid-resistant phenotype, in part due to carbonic anhydrase IX (CA IX). CA IX also facilitates PMVEC repair by promoting aerobic glycolysis, migration, and network formation. Molecular mechanisms of how CA IX performs such a wide range of functions are unknown. CA IX is composed of four domains known as the proteoglycan-like (PG), catalytic (CA), transmembrane (TM), and intracellular (IC) domains. We hypothesized that the PG and CA domains mediate PMVEC pH homeostasis and repair, and the IC domain regulates aerobic glycolysis and PI3k/Akt signaling. The functions of each CA IX domain were investigated using PMVEC cell lines that express either a full-length CA IX protein or a CA IX protein harboring a domain deletion. We found that the PG domain promotes intracellular pH homeostasis, migration, and network formation. The CA and IC domains mediate Akt activation but negatively regulate aerobic glycolysis. The IC domain also supports migration while inhibiting network formation. Finally, we show that exposure to acidosis suppresses aerobic glycolysis and migration, even though intracellular pH is maintained in PMVECs. Thus, we report that 1) the PG and IC domains mediate PMVEC migration and network formation, 2) the CA and IC domains support PI3K/Akt signaling, and 3) acidosis impairs PMVEC metabolism and migration independent of intracellular pH homeostasis.

CCL25 Inhibition Alleviates Sepsis-Induced Acute Lung Injury and Inflammation.

PurposeAcute lung injury (ALI) is a common clinical syndrome with high mortality. The chemokine ligand 25 (CCL25) is involved in inflammation, leukocyte trafficking and immunoregulation. However, the role and mechanism of CCL25 in ALI are not fully understood yet. The aim of this study was to explore the relationship between acute lung injury and CCL25.Patients and MethodsIn this study, we first examined chemokine expression in sepsis patients and found that serum CCL25 expression levels were relatively high in sepsis patients compared to healthy individuals. Based on this, we designed in vitro and in vivo experiments to verify the validity of the theory. In vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs). In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis.ResultsIn vitro, we used lipopolysaccharide-stimulated human pulmonary microvascular endothelial cells (HPMECs) and found significantly higher expression of CCL25 by enzyme-linked immunosorbent assay. Inhibition of CCL25 resulted in a significant decrease in the expression of inflammatory cytokines in HPMECs. In addition, we found that CCL25 promoted increased endothelial permeability by reducing the expression of tight junction proteins and was associated with activation of the P38 MAPK pathway by measuring the transepithelial electrical resistance and fluorescence intensity of fluorescein isothiocyanate. Results from luciferase assays and chromatin immunoprecipitation assays showed that inhibition of NF-κB activity in HPMECs decreased CCL25 expression, but addition of recombinant CCL25 increased cell permeability and inflammatory cytokine expression. In vivo, we established male C57BL/6 mice cecal ligation puncture (CLP) model of sepsis. We found that inhibition of CCL25 significantly reduced inflammatory cytokine expression in a CLP-induced sepsis model, thereby alleviating lung tissue damage in mice.ConclusionOur study suggests that CCL25 contributed to the development of ALI by modulating the functions of microvascular endothelial cells.

Hypoxic pulmonary endothelial cells release epidermal growth factor leading to vascular smooth muscle cell arginase-2 expression and proliferation.

The hallmark of pulmonary hypertension (PH) is vascular remodeling. We have previously shown that human pulmonary microvascular endothelial cells (hPMVEC) respond to hypoxia with epidermal growth factor (EGF) mediated activation of the receptor tyrosine kinase, EGF receptor (EGFR), resulting in arginase‐2 (Arg2)‐dependent proliferation. We hypothesized that the release of EGF by hPMVEC could result in the proliferation of human pulmonary arterial smooth muscle cells (hPASMC) via activation of EGFR on the hPASMC leading to Arg2 up‐regulation. To test this hypothesis, we used conditioned media (CM) from hPMVEC grown either in normoxia (NCM) or hypoxia (HCM). Human PASMC were incubated in normoxia with either HCM or NCM, and HCM caused significant induction of Arg2 and viable cell numbers. When HCM was generated with either an EGF‐neutralizing antibody or an EGFR blocking antibody the resulting HCM did not induce Arg2 or increase viable cell numbers in hPASMC. Adding an EGFR blocking antibody to HCM, prevented the HCM‐induced increase in Arg2 and viable cell numbers. HCM induced robust phosphorylation of hPASMC EGFR. When hPASMC were transfected with siRNA against EGFR the HCM‐induced increase in viable cell numbers was prevented. When hPASMC were treated with the arginase antagonist nor‐NOHA, the HCM‐induced increase in viable cell numbers was prevented. These data suggest that hypoxic hPMVEC releases EGF, which activates hPASMC EGFR leading to Arg2 protein expression and an increase in viable cell numbers. We speculate that EGF neutralizing antibodies or EGFR blocking antibodies represent potential therapeutics to prevent and/or attenuate vascular remodeling in PH associated with hypoxia.

Activation of the AMPK-ULK1 pathway mediated protective autophagy by sevoflurane anesthesia restrains LPS-induced acute lung injury (ALI)

BackgroundSevoflurane anesthesia is deemed as potential therapeutic drug for lipopolysaccharide (LPS)-induced acute lung injury (ALI), but the molecular mechanisms have not been fully delineated. AimThe present study explored the specific molecular mechanism of sevoflurane regulating autophagy to reduce LPS induced ALI. MethodsMale C57BL/6J mice and mouse pulmonary microvascular endothelial cells (MPVECs) were treated with LPS to construct ALI models, and the levels of inflammation, apoptosis and autophagy were detected after treatment with sevoflurane. Meanwhile, cells were treated with autophagy inhibitor or AMP-activated protein kinase (AMPK)/unc-51 like autophagy activating kinase 1 (ULK1) pathway inhibitor in vitro to detect their effects on cell survival. ResultsSevoflurane reduced inflammation, recovered cell division so as to suppress cell apoptosis and maintain cell survival, and activated autophagic flux in LPS-induced ALI models in vivo and in vitro. Of note, the suppressing effects of sevoflurane on LPS-induced cell death were abrogated by inhibiting autophagy. Moreover, we evidenced that sevoflurane promoted activation of the AMPK/ULK1 pathway in LPS-induced ALI models. Blockage of this pathway abrogated the promoting effects of sevoflurane on cell autophagy and cell viability in LPS-treated cells. ConclusionCollectively, sevoflurane suppresses apoptosis and inflammation via activating protective autophagy, thereby ameliorating LPS-induced ALI, and the AMPK/ULK1/ PIKFYVE pathway is responsible for the process.

KGF-2 Protects against Lung Ischemia-Reperfusion Injury by Inhibiting Inflammation-Induced Damage to Endothelial Barrier Function.

Lung ischemia-reperfusion injury (LIRI), which has a mortality rate of approximately 50%, is a popular topic in critical care research. Keratinocyte growth factor-2 (KGF-2) is secreted by mesenchymal cells, and it is effective in promoting the proliferation, migration, and differentiation of various epithelial cells. To date, however, only a few reports on KGF-2-related regulators in LIRI have been published. In the current study, an LIRI rat model is constructed, and the upregulation of the fibroblast growth factor receptor 2 (FGFR2) is observed in the LIRI rat model. In addition, LIRI induces NLRP1 inflammasome activation in vivo and in vitro, and KGF-2 inhibits LIRI-induced damage to pulmonary microvascular endothelial cells. Mechanistically, KGF-2 inhibits NLRP1 inflammasome and NF-κB activity. KGF-2 inhibition attenuates LIRI injury-induced damage to endothelial integrity. In conclusion, KGF-2 protects against LIRI by inhibiting inflammation-induced endothelial barrier damage.

Molecular mechanisms underlying the heterogeneous barrier responses of two primary endothelial cell types to sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) signals to enhance or destabilize the vascular endothelial barrier depending on the receptor engaged. Here, we investigated the differential barrier effects of S1P on two influential primary endothelial cell (EC) types, human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs). S1PR1 (barrier protective) and S1PR3 (barrier disruptive) surface and gene expression were quantified by flow cytometry and immunofluorescence, and RT-qPCR, respectively. Functional evaluation of EC monolayer permeability in response to S1P was quantified with transendothelial electrical resistance (TEER) and small molecule permeability. S1P significantly enhanced HUVEC barrier function, while promoting HPMEC barrier breakdown. Immunofluorescence and flow cytometry analysis showed select, S1PR3-high HPMECs, suggesting susceptibility to barrier destabilization following S1P exposure. Reevaluation of HPMEC barrier following S1P exposure under inflamed conditions demonstrated synergistic barrier disruptive effects of pro-inflammatory cytokine and S1P. The role of the Rho-ROCK signaling pathway under these conditions was confirmed through ROCK1/2 inhibition (Y-27632). Thus, the heterogeneous responses of ECs to S1P signaling are mediated through Rho-ROCK signaling, and potentially driven by differences in the surface expression of S1PR3.

Sulfated β-glucan from Agaricus subrufescens inhibits flavivirus infection and nonstructural protein 1-mediated pathogenesis

Despite substantial morbidity and mortality, no therapeutic agents exist for treatment of dengue or Zika, and the currently available dengue vaccine is only recommended for dengue virus (DENV)-immune individuals. Thus, development of therapeutic and/or preventive drugs is urgently needed. DENV and Zika virus (ZIKV) nonstructural protein 1 (NS1) can directly trigger endothelial barrier dysfunction and induce inflammatory responses, contributing to vascular leak in vivo. Here we evaluated the efficacy of the (1–6,1-3)-β-D-glucan isolated from Agaricus subrufescens fruiting bodies (FR) and its sulfated derivative (FR-S) against DENV-2 and ZIKV infection and NS1-mediated pathogenesis. FR-S, but not FR, significantly inhibited DENV-2 and ZIKV replication in human monocytic cells (EC50 = 36.5 and 188.7 μg/mL, respectively) when added simultaneously with viral infection. No inhibitory effect was observed when FR or FR-S were added post-infection, suggesting inhibition of viral entry as a mechanism of action. In an in vitro model of endothelial permeability using human pulmonary microvascular endothelial cells (HPMECs), FR and FR-S (0.12 μg/mL) inhibited DENV-2 NS1- and ZIKV NS1-induced hyperpermeability by 50% and 100%, respectively, as measured by Trans-Endothelial Electrical Resistance. Treatment with 0.25 μg/mL of FR and FR-S inhibited DENV-2 NS1 binding to HPMECs. Further, FR-S significantly reduced intradermal hyperpermeability induced by DENV-2 NS1 in C57BL/6 mice and protected against DENV-induced morbidity and mortality in a murine model of dengue vascular leak syndrome. Thus, we demonstrate efficacy of FR-S against DENV and ZIKV infection and NS1-induced endothelial permeability in vitro and in vivo. These findings encourage further exploration of FR-S and other glycan candidates for flavivirus treatment alone or in combination with compounds with different mechanisms of action.

Pulmonary endothelial cells from different vascular segments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression.

Sodium-hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined. Utilizing the ammonium-prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited unique recovery rates from the acid load dependent upon the concentration of the sodium transport inhibitor, amiloride; further, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium-dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two-gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular versus pulmonary artery endothelial cells, which was confirmed at the protein level. Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

PECAM EMPs regulate apoptosis in pulmonary microvascular endothelial cells in COPD by activating the Akt signaling pathway.

INTRODUCTIONEndothelial microparticles (EMPs) are partly associated with the progress of chronic obstructive pulmonary disease (COPD). We sought to measure the levels of EMPs in COPD patients and in human pulmonary microvascular endothelial cells (HPMECs) exposed to cigarette smoking extract (CSE) to elucidate the potential mechanisms of their action.METHODSWe obtained prospectively blood EMPs from 30 stable COPD patients and 20 non-COPD volunteers. EMP subpopulations were determined by flow cytometry in platelet-free plasma according to the expression of membrane specific antigens. Cell growth, proliferation, apoptosis and the expression of protein kinase B (Akt) in HPMECs after exposure to PECAM EMPs were assessed. After intervention with an antioxidant (Eukarion-134, EUK-134), apoptosis and the expression of Akt in HPMECs were also measured.RESULTSUnlike those of MCAM EMPs, VE-cadherin, PECAM and E-selectin EMP values were significantly higher in the stable COPD patients than in the non-COPD volunteers (p<0.05). Only PECAM EMPs were higher in HPMECs exposed to CSE (p<0.05). Further, in vitro studies showed that the apoptosis rate and expression of cleaved caspase 3/9 in HPMECs increased in a dose- and time-independent manner with PECAM EMPs. The expression of phospho-Akt (p-Akt) decreased in a time-independent manner with PECAM EMPs (p<0.05). Compared with the control group, the early apoptosis rate of HPMECs was higher, and the expression of p-Akt was lower in both the PECAM EMP group and EUK-134 + PECAM EMP group (p<0.05). The apoptosis rate declined markedly, and the expression of p-Akt was higher in the EUK-134 + PECAM EMP group, compared with the PECAM EMPs group (p<0.05).CONCLUSIONSThe present results suggest that PECAM EMPs positively regulate apoptosis in HPMECs in COPD, likely by decreasing Akt phosphorylation and can be protected by antioxidants.

Amyloid‐Beta Precursor Protein: Essential to Lung Capillary Barrier Defense During Acute Infection

RationalePneumonia is a leading cause of acute respiratory distress syndrome (ARDS) and sepsis. Despite resolution of active infection, pro‐inflammatory mediators and cytotoxins elicited during the acute phase promote secondary tissue injury, impede recovery, and contribute to poor patient prognoses. Lung infection with Pseudomonas aeruginosa, Klebsiella pneumoniae, and influenza elicit pathogenic amyloids that propagate injury and likely contribute to this inflammatory milieu. However, clinical trials targeting amyloid‐beta (Aβ) have consistently failed. Lung endothelial‐derived amyloids have been implicated as innate antimicrobial effectors yet the contribution of Aβ to host defense of the lung capillary barrier remains poorly resolved. We hypothesized that lung endothelial Aβ contributes to 1) the attenuation of active infection, and 2) endothelial barrier resilience to infection.MethodsTo test our hypotheses, CRISPR/Cas9 was targeted to Aβ precursor protein (APP) in rat pulmonary microvascular endothelial cells (PMVECs), and the deletion was confirmed via PCR, sequencing, and immunoblotting. The contribution of APP expression to cell proliferation, self‐replication, wound closure, angiogenesis, and barrier integrity (transwell permeability, bacterial dissemination, ECIS) both at baseline and during virulent infection was assessed in both WT and APP‐/‐ PMVECs. Amyloid content was monitored with immunoblotting and Thioflavin T staining. Antimicrobicity was measured with kinetic bactericidal assays, agglutination, plating, and SYTO 9/propidium iodide.ResultsAPP‐/‐ cells were ~49% less effective in reducing the bacterial load of an active infection as compared to APP expressing PMVECs. Immunodepletion of Aβ from WT endothelial cell supernatant reduced bactericidal efficacy in a comparable manner. APP‐/‐ cells also exhibited impaired proliferation (WT: 0.59 days, APP‐/‐ 0.91 days), a ~2.2‐fold reduction in replication capacity, poor wound closure and barrier formation, and little resilience to infection with loss of barrier integrity in roughly half the time of infected WT PMVECs. Moreover, conditioning of naïve cells with supernatant derived from WT but not APP‐/‐ PMVECs significantly increased proliferation and angiogenic capacity.ConclusionsLung endothelial APP is an important contributor to host defense. Both APP and its Aβ variant(s) appear to constrain infective bacterial burden whereas APP products appear to function as mitogens/morphogens within the microvascular milieu, likely contributing to tissue repair and recovery post‐insult.

Pulmonary endothelial cells from different vascular compartments exhibit unique recovery from acidification and Na+/H+ exchanger isoform expression

Sodium‐hydrogen exchangers (NHEs) tightly regulate intracellular pH (pHi), proliferation, migration and cell volume. Heterogeneity exists between pulmonary endothelial cells derived from different vascular segments, yet the activity and isoform expression of NHEs between these vascular segments has not been fully examined.Utilizing the ammonium‐prepulse and recovery from acidification technique in a buffer lacking bicarbonate, pulmonary microvascular and pulmonary artery endothelial cells exhibited a similar rate of recovery from the acid load regardless of the concentrations of the sodium transport inhibitor, amiloride; however, pulmonary artery endothelial cells required a higher dose of amiloride to inhibit sodium‐dependent acid recovery compared to pulmonary microvascular endothelial cells, suggesting a unique complement of NHEs between the different endothelial cell types. While NHE1 has been described in pulmonary endothelial cells, all NHE isoforms have not been accounted for. To address NHE expression in endothelial cells, qPCR was performed. Using a two‐gene normalization approach, Sdha and Ywhag were identified for qPCR normalization and analysis of NHE isoforms between pulmonary microvascular and pulmonary artery endothelial cells. NHE1 and NHE8 mRNA were equally expressed between the two cell types, but NHE5 expression was significantly higher in pulmonary microvascular and pulmonary artery endothelial cells.Thus, pulmonary microvascular and pulmonary artery endothelial cells exhibit unique NHE isoform expression and have a unique response to acid load revealed through recovery from cellular acidification.

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.

RNA‐Seq Analysis Revealed Sex Differences in Pulmonary Microvascular Endothelial Cells in the Response to Hypoxia

Pulmonary hypertension (PH) is characterized by the abnormal elevation of mean pulmonary arterial pressure which may lead to right ventricle (RV) failure and death. PH is classified into five groups, four of them have been reported to have sex dimorphism with females showing better RV functioning than males. The most marked sex dimorphism is observed in Group 1, pulmonary arterial hypertension (PAH), which is 2‐4 times more common in females than in males. It has only recently been discovered that sex chromosomes may play a role in the sex differences in PH that is independent of sex hormones, although the exact mechanisms are still unknown (1). One of the approaches to get insight into the role of the sex chromosomes in vitro is isolated cells, cultured in the absence of sex hormones. The aim of this study was to reveal sex differences in human pulmonary microvascular endothelial cells, using hypoxia as an in vitro model of pulmonary hypertension.Human pulmonary microvascular endothelial cells (HPMEC) from female (n=4) and male (n=3) healthy donors were exposed to hypoxia (1% O2) for 48 hours. RNA from these samples was sequenced on the Illumina NovaSeq 6000. Differentially expressed genes (DEGs) were identified using DESeq2 (Wald test, p(adj.)&lt;0.05) (2). Gene Set Enrichment Analysis (GSEA, Hallmark gene set collection, FDR&lt;0.05) was used to find enriched pathways (3,4).Hypoxia induced changes in the expression of 79 genes in male HPMEC and of 34 genes in female HPMEC compared to normoxic controls. Among them 19 genes were common between the two sexes, 69 genes changed in males only, 24 genes changed in females only in response to hypoxia. GSEA revealed that in both male and female HPMEC hypoxia induced the enrichment of gene sets “Hypoxia”, “Glycolysis”, “Epithelial Mesenchymal Transition”, “MTORC1 Signaling”, and “Myogenesis”. One gene set, “MYC Targets V1”, was changed in opposite directions in the two sexes: it was upregulated in hypoxia in male HPMEC and downregulated in hypoxia in female HPMEC. Comparisons between male and female HPMEC basally (in normoxia) and in the hypoxic conditions revealed that 11 Y‐encoded genes were expressed more highly in male HPMEC than in female HPMEC (DDX3Y, KDM5D, USP9Y, ZFY, EIF1AY, UTY, RPS4Y1, NLGN4Y, PRKY, TXLNGY, TTTY14). Two X‐encoded genes (XIST, RPS4X) were expressed more highly in female HPMEC in normoxia than in male HPMEC, and one X‐coded gene (XIST) was expressed more highly in female HPMEC in hypoxia than in male HPMEC in hypoxia.According to RNA‐seq analysis, sex differences exist in male and female HPMEC. Some of the identified DEGs and pathways changed in response to hypoxia in the opposite directions in male compared to female HPMEC. Sex differences were induced by sex hormone independent mechanisms (i.e. sex chromosomes), since HPMECs were cultured in the absence of sex hormones. Further research is needed to explore the contribution of the genes and pathways reported here in the development of sex differences in PH. Kostyunina, D. S., &amp; McLoughlin, P. (2021). Antioxidants, 10(5):779; Love, M. I., Huber, W., &amp; Anders, S. (2014). Genome Biology, 15(12), 1–21; Subramaniana et al.(2005). PNAS, 102(43), 15545–15550; Liberzon et al. (2015). Cell Syst, 1(6), 417–425.

Aquaporin 1 mediates microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary hypertension

Pulmonary arterial hypertension (PAH) is a devastating disease, with increased pulmonary arterial pressure leading to right heart failure. The mechanisms underlying PAH are unclear, but increased pulmonary arterial smooth muscle (PASMC) and microvascular endothelial cell (MVEC) migration and proliferation leads to vaso‐occlusive lesions and vascular remodeling observed in PAH. Aquaporins regulate water transport and are expressed in many cells. We previously identified that hypoxia upregulates the expression of aquaporin 1 (AQP1) in PASMC and elevated AQP1 was required for hypoxia‐induced migration and proliferation. In this study, we wanted to elucidate whether the same is true in MVECs, and used the SU5416 plus hypoxia (SuHx) rat model, a robust model of PAH, which mimics many of the features of human PAH, to determine whether AQP1 levels were altered in MVECs and contributed to a hyperproliferative/hypermigratory phenotype.Rats were injected with the vascular endothelial growth factor receptor inhibitor, SU5416, combined with hypoxia (3 wk at 10% O2) followed by 2 wk return to normoxia. Using MVECs isolated from the normoxia (N‐MVEC) or SuHx (SuHx‐MVEC) rats, we found that AQP1 protein levels were increased in SuHx‐MVECs and associated with upregulation of AQP1 mRNA levels, even in the absence of sustained hypoxia exposure. Silencing of AQP1 reduced migration and proliferation observed in SuHx‐MVEC, whereas augmenting AQP1 levels in N‐MVECs using forced expression with an adenoviral construct containing wild‐type AQP1 (AdAQP1) was sufficient to increase cell migration (transwell assay) and proliferation (BrdU incorporation) compared to control cells infected with green fluorescent protein (AdGFP). Since we formerly reported the C‐terminal tail of AQP1 contains putative calcium and protein biding sites and is required for enhanced PASMC migration and proliferation, we tested the role of the AQP1 C‐terminal tail in N‐MVEC. Cells were treated with AdGFP, AdAQP1 or adenovirus containing AQP1 lacking the entire cytoplasmic C‐terminal tail (AdAQP1CT). In contrast to the increase in migration and proliferation induced by AdAQP1, infection with AdAQP1CT did not alter N‐MVEC migration and proliferation.Our results suggest a key role for enhanced AQP1 expression in mediating abnormal migration and proliferation in microvascular endothelial cells from a rodent model that reflects many of the features of human PAH. Interestingly, the AQP1 C‐terminal tail is required for regulating MVEC function, similar to our previous findings in PASMC.