Human Lung Microvascular Endothelial Cells Research Articles

Hemoglobin-Mediated Oxidation of Low-Density Lipoprotein (oxLDL) Contributes to Lung Microvascular Endothelial Dysfunction Via Lectin-Like Oxidized LDL Receptor 1 (LOX-1)

Introduction: Lung microvascular endothelial barrier disruption is a critical feature of acute lung injury during several pathologies, including sepsis. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. However, the precise signaling mechanisms involved in CFH-mediated lung microvascular barrier dysfunction are incompletely understood. One potential mechanism by which CFH might disrupt the microvascular endothelial barrier is through its known ability to oxidize lipids and lipoproteins, including low-density lipoprotein (LDL). Oxidized LDL (oxLDL) is a known endothelial barrier disruptor, primarily signaling through scavenger receptors such as lectin-like oxidized LDL receptor 1 (LOX-1). We hypothesized that CFH-mediated oxidation of LDL exacerbates lung microvascular endothelial barrier dysfunction via LOX-1. Methods: For experimental studies, LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxidation of LDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC), barrier dysfunction was assessed with expert permeability testing (XPerT) assay. HLMVEC were stimulated with vehicle control (PBS), LDL (0.05 mg/mL), CFH (0.5 mg/mL), or LDL oxidized by CFH (LDL+CFH) with or without one-hour pre-treatment of hemoglobin scavenger haptoglobin (1:1) or LOX-1 inhibitor BI-0115 (Boehringer Ingelheim, 10 μM). Cleaved caspase-3 (CST 9661, 1:500) was assessed with immunofluorescence microscopy. To test whether CFH and oxLDL are associated in clinical sepsis, circulating levels of CFH and oxLDL were measured in 60 sepsis patients via ELISA. Results: Oxidation of LDL by CFH (p=0.0006 by TBARS) exacerbated HLMVEC barrier dysfunction (p<0.0001 by XPerT) but was partially prevented by scavenging CFH with haptoglobin (p<0.0001 by XPerT) or blocking LOX-1 receptor (p<0.0001 by XPerT). Oxidation of LDL by CFH stimulation of HLMVEC also increased caspase-3 activation (p<0.0001 by immunofluorescence of cleaved caspase-3). In sepsis patients, circulating oxLDL levels correlated with CFH (r=0.4, p=0.002). Conclusions: Oxidation of LDL by CFH contributes to lung microvascular endothelial injury via LOX-1 receptor and activation of caspase-3; targeting this pathway may hold therapeutic potential to treat pathologies in which CFH is elevated, including sepsis-induced acute lung injury/ARDS. Funding: Parker B. Francis Fellowship (JEM); NIH K99HL166865 (JEM), R01HL158906 (LBW), R01HL164937 (LBW), R21GM144915 (LBW, JAB), R35HL150783 (JAB), RF1AG075341 (JAB). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vascular endothelial growth factor receptor 2 contributes to shear stress-induced endothelial nitric oxide synthase expression in human lung microvascular endothelial cells

Introduction: Physiological shear stress (pSS) helps to maintain endothelial homeostasis via numerous intracellular signaling pathways. We previously showed that pSS upregulates mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in primary human lung microvascular endothelial cells (hLMVECs), and that phosphorylation of Akt (p-Akt), a phosphatidylinositol 3-kinase (PI3K) target, is increased. Upstream regulatory mechanisms for these responses have not been fully studied. Since vascular endothelial growth factor receptor 2 (VEGFR2) is an integral component of the mechanosensory complex that transduces shear stress-mediated signaling pathways in endothelial cells, we hypothesized that VEGFR2 is required for shear stress-induced eNOS expression and PI3K activation in hLMVECs. Methods: We cultured early passage (p≤7) primary hLMVECs from 4 unique donor lots in standard 6-well culture plates using a fixed volume (depth) and viscosity of tissue culture media. We exposed cells to shear stress for 24 hours using an orbital shaker and calculated the magnitude of shear stress exerted on hLMVECs using the extended solution to Stokes’ second problem. We defined the magnitude of pSS as 12 dyn/cm2. In some experiments, we exposed cells to pSS in the presence of chemical inhibitors of VEGFR2 (SU5416; 5 or 10 μM) or PI3K (LY294002; 10 μM). We collected whole cell lysates and performed immunoblotting to probe for phosphorylated eNOS (p-eNOS; Serine 1177), total eNOS, p-Akt (Serine 473), total Akt and housekeeping protein β-tubulin. Potential interactions between chemical inhibitors and pSS on protein expression and phosphorylation were analyzed by two-way ANOVA. Results: After 24 hours of pSS exposure, total eNOS levels were significantly (p<0.05) increased in hLMVECs, although the p-eNOS/eNOS ratio was not changed (p=0.85). Conversely, 24 hours of pSS exposure increased the p-Akt/Akt ratio (p<0.05) but did not increase total Akt levels (p=0.88). Inhibition of VEGFR2 activity with SU5416 attenuated pSS-induced changes in total eNOS levels (p<0.05 for interaction) but did not attenuate pSS-induced increases in the p-Akt/Akt ratio (p=0.91 for interaction). Inhibition of PI3K activity using LY294002 did not modify the effects of pSS on total eNOS levels (p=0.25 for interaction) or p-Akt/Akt ratio (p=0.47 for interaction). Conclusions: We conclude that VEGFR2 activity is necessary for pSS-mediated increases in eNOS protein levels, but not PI3K activity, in hLMVECs. Furthermore, neither VEGFR2 nor PI3K activity appear to be required for pSS-mediated eNOS phosphorylation in these cells. Collectively, these data suggest that shear stress regulates hLMVEC eNOS signaling via multiple signaling pathways. This complex regulation may have important implications for local nitric oxide bioavailability under pathological conditions that modify shear stress (e.g., pulmonary embolism, pulmonary hypertension). This work is supported by NIH grants HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Liraglutide protects against microvascular dysfunction and sepsis-mediated inflammation and organ injury

Introduction: Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection characterized by increased systemic inflammation and microvascular injury. Recently, commonly used diabetes and obesity medications, glucagon-like peptide-1 (GLP-1) receptor agonists, have demonstrated unexpected anti-inflammatory effects. We tested GLP-1 receptor agonist liraglutide in a clinically relevant two-hit murine model of polymicrobial abdominal sepsis and hyperoxia and microvascular endothelial injury in vitro, hypothesizing that GLP-1 receptor agonists exert protective effects during sepsis, limiting microvascular permeability, inflammation, organ injury and death. Methods: Sepsis was induced in male and female mice by intraperitoneal (IP) injection of cecal contents (CS;1.8 mg/g body weight) collected from euthanized donor mice. Mice were also exposed to hyperoxia (FiO2 90-96%) for 24 hours. Six and 18 hours after CS injection, mice were administered saline or liraglutide (0.1 mg/kg). At 12 hours, mice were given fluids and antibiotics. At 24 hours, mice were euthanized for plasma and bronchoalveolar lavage (BAL; right lung) collection. Plasma cytokines, organ injury markers, and BAL immune cells and protein were measured. The left lung was tied off and excised to measure wet-to-dry lung weight ratios. Additionally, for in vitro studies, primary human lung microvascular endothelial cells (HLMVECs) were treated with saline or liraglutide for 24 hours prior to saline (control) or LPS (100 ng/mL). HLMVEC barrier dysfunction was quantified using express permeability testing (XPerT) assay or Electric Cell-Substrate Impedance Sensing (ECIS) to measure transendothelial electrical resistance (TER). Results: In murine sepsis, illness severity scores and lung injury were improved in mice pretreated with liraglutide (N=12). Plasma blood urea nitrogen (BUN; P=0.0581), alanine transaminase (ALT; P=0.0311) and aspartate aminotransferase (AST; P=0.0263), and cytokines IL-1β (P=0.0078), IL-6 (P=0.0038), and TNF-α (P=0.0183) in plasma were reduced in mice treated with liraglutide. In HLMVECs, liraglutide (1.5 nM) significantly reduced LPS-induced barrier dysfunction measured by XPerT assay (P=0.0030) and ECIS (P=0.0075). Conclusion: Liraglutide reduces illness severity, inflammation, and organ injury in a two-hit model of sepsis-induced ALI. Liraglutide has direct effects in the microvascular endothelium, limiting LPS-mediated barrier dysfunction. These findings support a protective role for GLP-1 receptor agonism in sepsis, mediated through the microvasculature. HL150783, AG075341, HL158906. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Shear Stress Induces Morphology Changes in Human Lung Microvascular Endothelial Cells via Calcium Influx

Introduction: The pulmonary vascular endothelium is in contact with laminar blood flow, resulting in a physiologic degree of shear stress exerted on endothelial cells (ECs). In vitro cell culture of ECs is typically performed under static conditions, leading to underappreciation of the impact of shear stress. Calcium signaling is among the most ubiquitous cell signaling mechanisms, and changes in intracellular calcium concentration ([Ca2+]i) have been linked to shear stress. The impact of shear stress on human lung microvascular endothelial cell (hLMVEC) morphology and cell signaling is not well described. We hypothesized that hLMVECs exposed to shear stress would undergo Ca2+-dependent morphologic changes. Methods: The effect of shear stress on [Ca2+]i was first tested using ratiometric measurement. Early passage (p≤8) primary hLMVECs from three unique donor lots were plated in gelatin-coated Ibidi 0.2 uM depth plates for 16 h, then incubated with the calcium indicator Fura-2 AM for 1 h. Measurement of [Ca2+]i was obtained over 15 minutes under static and physiologic shear stress (0 and 12 dyn/cm2, respectively), using validated flow rates from the Ibidi system. Measured [Ca2+]i was compared between cells treated vehicle or an inhibitor of the mechanosensitive Ca2+-permeable channel TRPV4 (HC-067047). HC treatment was for 1 h prior to and throughout the duration of shear exposure. Morphology changes were then assessed in hLMVECs from the same three cell lots grown in standard 6-well plates; one plate kept in static culture, while another was exposed to 24 h of 12 dyn/cm2 shear stress using an orbital shaker. Treatments in each plate were control, vehicle, and HC. Representative photos of each well were taken at 0 h and at 24 h. Average ratios of shortest to longest dimensions from 30 cells/field were measured using ImageJ and compared between treatments and shear conditions. A 2-way ANOVA with post-test was used to test for statistically significant changes in morphology. Results: Shear stress increased [Ca2+]i within 15 minutes, and both baseline [Ca2+]i and observed change in response to shear stress were decreased by TRPV4 inhibition with HC. Morphologic changes were observed after exposure to shear stress on microscopic examination, with cells grown in static culture having typical cobblestone appearance and shear-adapted cells having an elongated, spindle like appearance aligned in the direction of flow. The calculated ratio (shortest to longest dimension) of all wells at 0 h was ~0.70 with no significant differences between groups. 24 h of shear stress caused a nearly 80% decrease in ratio compared to static culture in untreated cells (p<0.05), with a decrease of only 30% in HC-treated cells after 24 h of shear (p<0.05). Conclusions: Exposure to shear stress at physiological levels results in a rapid increase [Ca2+]i through mechanosensitive, Ca2+-permeable TRPV4 channels as well as notable morphologic changes to hLMVECs with prolonged exposure to shear stress that appear to be mediated in large part by TRPV4. Further investigation into the impact of shear stress on hLMVECs is warranted to elucidate biochemical pathways and other downstream functional consequences of increased [Ca2+]i. NIH-T32HL007534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Unveiling the role of PD-L1 in vascular endothelial dysfunction: Insights into the mtros/NLRP3/caspase-1 mediated pyroptotic pathway

BackgroundProgrammed death ligand-1(PD-L1) has been postulated to play a crucial role in the regulation of barrier functions of the vascular endothelium, yet how this novel molecule mediates dysfunction in endothelial cells (ECs) during acute lung injury (ALI) remains largely unknown. MethodsPD-L1 siRNA and plasmids were synthesized and applied respectively to down- or up-regulate PD-L1 expression in human lung microvascular endothelial cells (HMVECs). RNA sequencing was used to explore the differentially expressed genes following PD-L1 overexpression. The expression levels of tight junction proteins (ZO-1 and occludin) and the signaling pathways of NLRP-3/caspase-1/pyroptosis were analyzed. A mouse model of indirect ALI was established through hemorrhagic shock (HEM) followed by cecal ligation and puncture (CLP), enabling further investigation into the effects of intravenous delivery of PD-L1 siRNA. ResultsA total of 1502 differentially expressed genes were identified, comprising 532 down-regulated and 970 up-regulated genes in ECs exhibiting PD-L1overexpression. Enrichment of PD-L1-correlated genes were observed in the NOD-like receptor signaling pathway and the TNF signaling pathway. Western blot assays confirmed that PD-L1 overexpression elevated the expression of NLRP3, cleaved-caspase-1, ASC and GSDMD, and concurrently diminished the expression of ZO-1 and occludin. This overexpression also enhanced mitochondrial oxidative phosphorylation and mitochondrial reactive oxygen species (mtROS) production. Interestingly, mitigating mitochondrial dysfunction with mitoQ partially countered the adverse effects of PD-L1 on the functionality of ECs. Furthermore, intravenous administration of PD-L1 siRNA effectively inhibited the activation of the NLRP3 inflammasome and pyroptosis in pulmonary ECs, subsequently ameliorating lung injury in HEM/CLP mice. ConclusionPD-L1-mediated activation of the inflammasome contributes significantly to the disruption of tight junction and induction of pyroptosis in ECs, where oxidative stress associated with mitochondrial dysfunction serves as a pivotal mechanism underpinning these effects.

Species-specific responses during Seoul orthohantavirus infection in human and rat lung microvascular endothelial cells.

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.

Distinct functional neutrophil phenotypes in sepsis patients correlate with disease severity.

Sepsis is a clinical syndrome defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Sepsis is a highly heterogeneous syndrome with distinct phenotypes that impact immune function and response to infection. To develop targeted therapeutics, immunophenotyping is needed to identify distinct functional phenotypes of immune cells. In this study, we utilized our Organ-on-Chip assay to categorize sepsis patients into distinct phenotypes using patient data, neutrophil functional analysis, and proteomics. Following informed consent, neutrophils and plasma were isolated from sepsis patients in the Temple University Hospital ICU (n=45) and healthy control donors (n=7). Human lung microvascular endothelial cells (HLMVEC) were cultured in the Organ-on-Chip and treated with buffer or cytomix ((TNF/IL-1β/IFNγ). Neutrophil adhesion and migration across HLMVEC in the Organ-on-Chip were used to categorize functional neutrophil phenotypes. Quantitative label-free global proteomics was performed on neutrophils to identify differentially expressed proteins. Plasma levels of sepsis biomarkers and neutrophil extracellular traps (NETs) were determined by ELISA. We identified three functional phenotypes in critically ill ICU sepsis patients based on ex vivo neutrophil adhesion and migration patterns. The phenotypes were classified as: Hyperimmune characterized by enhanced neutrophil adhesion and migration, Hypoimmune that was unresponsive to stimulation, and Hybrid with increased adhesion but blunted migration. These functional phenotypes were associated with distinct proteomic signatures and differentiated sepsis patients by important clinical parameters related to disease severity. The Hyperimmune group demonstrated higher oxygen requirements, increased mechanical ventilation, and longer ICU length of stay compared to the Hypoimmune and Hybrid groups. Patients with the Hyperimmune neutrophil phenotype had significantly increased circulating neutrophils and elevated plasma levels NETs. Neutrophils and NETs play a critical role in vascular barrier dysfunction in sepsis and elevated NETs may be a key biomarker identifying the Hyperimmune group. Our results establish significant associations between specific neutrophil functional phenotypes and disease severity and identify important functional parameters in sepsis pathophysiology that may provide a new approach to classify sepsis patients for specific therapeutic interventions.

Lung lymphatic endothelial cells undergo inflammatory and prothrombotic changes in a model of chronic obstructive pulmonary disease.

The lymphatic vasculature regulates lung homeostasis through drainage of fluid and trafficking of immune cells and plays a key role in the response to lung injury in several disease states. We have previously shown that lymphatic dysfunction occurs early in the pathogenesis of chronic obstructive pulmonary disease (COPD) caused by cigarette smoke (CS) and that this is associated with increased thrombin and fibrin clots in lung lymph. However, the direct effects of CS and thrombin on lymphatic endothelial cells (LECs) in COPD are not entirely clear. Studies of the blood vasculature have shown that COPD is associated with increased thrombin after CS exposure that causes endothelial dysfunction characterized by changes in the expression of coagulation factors and leukocyte adhesion proteins. Here, we determined whether similar changes occur in LECs. We used an in vitro cell culture system and treated human lung microvascular lymphatic endothelial cells with cigarette smoke extract (CSE) and/or thrombin. We found that CSE treatment led to decreased fibrinolytic activity in LECs, which was associated with increased expression of plasminogen activator inhibitor 1 (PAI-1). LECs treated with both CSE and thrombin together had a decreased expression of tissue factor pathway inhibitor (TFPI) and increased expression of adhesion molecules. RNA sequencing of lung LECs isolated from mice exposed to CS also showed upregulation of prothrombotic and inflammatory pathways at both acute and chronic exposure time points. Analysis of publicly available single-cell RNA sequencing of LECs as well as immunohistochemical staining of lung tissue from COPD patients supported these data and showed increased expression of inflammatory markers in LECs from COPD patients compared to those from controls. These studies suggest that in parallel with blood vessels, the lymphatic endothelium undergoes inflammatory changes associated with CS exposure and increased thrombin in COPD. Further research is needed to unravel the mechanisms by which these changes affect lymphatic function and drive tissue injury in COPD.

HSP70 Is a Critical Regulator of HSP90 Inhibitor's Effectiveness in Preventing HCl-Induced Chronic Lung Injury and Pulmonary Fibrosis.

Exposure to hydrochloric acid (HCl) can provoke acute and chronic lung injury. Because of its extensive production for industrial use, frequent accidental exposures occur, making HCl one of the top five chemicals causing inhalation injuries. There are no Food and Drug Administration (FDA)-approved treatments for HCl exposure. Heat shock protein 90 (HSP90) inhibitors modulate transforming growth factor-β (TGF-β) signaling and the development of chemical-induced pulmonary fibrosis. However, little is known on the role of Heat Shock Protein 70 (HSP70) during injury and treatment with HSP90 inhibitors. We hypothesized that administration of geranylgeranyl-acetone (GGA), an HSP70 inducer, or gefitinib (GFT), an HSP70 suppressant, alone or in combination with the HSP90 inhibitor, TAS-116, would improve or worsen, respectively, HCl-induced chronic lung injury in vivo and endothelial barrier dysfunction in vitro. GGA, alone, improved HCl-induced human lung microvascular endothelial cells (HLMVEC) barrier dysfunction and, in combination with TAS-116, improved the protective effect of TAS-116. In mice, GGA reduced HCl toxicity and while TAS-116 alone blocked HCl-induced chronic lung injury, co-administration with GGA, resulted in further improvement. Conversely, GFT potentiated HCl-induced barrier dysfunction and impaired the antidotal effects of TAS-116. We conclude that combined treatments with HSP90 inhibitors and HSP70 inducers may represent a novel therapeutic approach to manage HCl-induced chronic lung injury and pulmonary fibrosis.

Pseudomonas Aeruginosa Affects the Function of Pulmonary Vascular Endothelial Cells

Objective To investigate the impact of Pseudomonas aeruginosa(PA) infection on the function of pulmonary vascular endothelial cells,and explore the mechanism of this bacterium in exacerbating lung inflammation in mice. Methods Two hours after human lung microvascular endothelial cell(HULEC-5a) were infected with the PA strain PAO1,the mRNA levels of autophagy-related gene 5(ATG5),6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3),and calcium adhesion protein 5(CDH5) were determined by reverse transcription real-time fluorescent quantitative PCR(RT-qPCR).The protein levels of ATG5,PFKFB3,and vascular endothelial calcium adhesion protein(VE-cadherin) were detected by immunofluorescence.After the expression of ATG5 and PFKFB3 was respectively knocked down by small interfering RNA(siRNA),RT-qPCR was employed to measure the mRNA levels of ATG5,PFKFB3,and CDH5,and immunofluorescence to detect the protein levels of PFKFB3 and VE-cadherin.In addition,the lactate assay kit was used to determine the level of lactate in the cells.After mice were infected with PAO1,lung inflammation was assessed through histopathological section staining.Confocal microscopy was employed to capture and analyze fluorescence-labeled PFKFB3 and VE-cadherin in endothelial cells. Results Compared with the control group,the HULEC-5a cells infected with PAO1 showed up-regulated mRNA and protein levels of PFKFB3(all P<0.05),down-regulated mRNA level of CDH5(P=0.023),disrupted continuity and down-regulated protein level of VE-cadherin(P<0.001),and elevated lactate level(P=0.017).Compared with PAO1-infected HULEC-5a cells,knocking down PFKFB3 led to the up-regulated mRNA level of CDH5(P=0.043),lowered lactate level(P=0.047),and restored continuity of VE-cadherin;knocking down ATG5 led to up-regulated mRNA and protein levels of PFKFB3(P=0.013 and P=0.003),elevated lactate level(P=0.015),and down-regulated mRNA level of CDH5(P=0.020) and protein level of VE-cadherin(P=0.001).The HE staining results showed obvious red blood cell leakage,inflammatory cell infiltration,alveolar septal widening,and partial detachment of vascular endothelial cells in the alveoli of PA-infected mice.Immunofluorescence staining showed up-regulated expression of PFKFB3 and decreased fluorescence signal of VE-cadherin in endothelial cells of infected mice compared with normal mice. Conclusion PA may regulate the PFKFB3 pathway via AGT5 to disrupt the function of pulmonary vascular endothelial cells,thereby exacerbating the inflammation in the lungs of mice.

A Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 Regulates Pulmonary Vascular Hyperpermeability through Destruction of Glycocalyx in Acute Respiratory Distress Syndrome.

Acute respiratory distress syndrome (ARDS) has no specific and effective treatment, and there is an urgent need to understand its pathogenesis. Therefore, based on the hypothesis that molecules whose expression is upregulated in injured pulmonary vascular endothelial cells (VECs) are involved in the pathogenesis of ARDS, we conducted a study to elucidate the molecular mechanisms and identify target factors for treatment. Primary human lung microvascular endothelial cells (HMVEC-Ls) were stimulated with lipopolysaccharide (LPS) or poly (I:C) and analyzed via a microarray to identify target genes for ARDS. We found that a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) was induced in murine lung VECs in an LPS-mediated ARDS model. Elevated ADAMTS4 was also observed by the immunostaining of lung samples from ARDS patients. The suppression of ADAMTS4 by siRNA in VECs ameliorated LPS-stimulated vascular permeability. The impairment of the cell surface expression of syndecan-1, a marker of the glycocalyx that is an extracellular matrix involved in vascular permeability, was dramatically inhibited by ADAMTS4 suppression. In addition, the suppression of ADAMTS4 protected against LPS-induced reductions in syndecan-1 and the adherens junction protein vascular endothelial cadherin. These results suggest that ADAMTS4 regulates VEC permeability in ARDS and may be a predictive marker and therapeutic target for ARDS.

Abstract 14589: Impairment of Endothelial Function by Chronic Cannabis Use: An Interim Analysis of the CANDIDE Study

Background: Increasing cannabis legalization has led to a surge in cannabis use. To understand the consequences for cardiovascular health, we have initiated the CANnabis: Does It Damage Endothelium (CANDIDE) study. Here, we report results of an interim analysis. Aims: To investigate the effects of chronic cannabis product use on endothelial function. Approach: We recruited 44 healthy non-tobacco smokers/vapers, 21-50 yrs, in 3 groups based on chronic cannabis use: cannabis smokers, THC edible users, and nonusers. Participants underwent assessment of brachial artery flow-mediated dilation (FMD) and carotid-femoral pulse wave velocity (PWV). Human umbilical vein endothelial cells (ECs) were exposed to sera from each individual with and without VEGF to determine serum effects on endothelial NO production. Human lung microvascular ECs were also exposed to the individual sera and cell permeability was measured by electric cell-substrate impedance sensing. Results: FMD and VEGF-stimulated NO levels were both significantly lower in cannabis smokers than nonusers (5.7±3.3% vs 12.0±8.0%, p=.037, and 1.1±0.3 nM vs 1.5±0.3 nM, p=.018, respectively; figure), comparable effects to what we have reported for tobacco smokers. In contrast, FMD, but not VEGF-stimulated NO levels, was lower in the THC edible users than nonusers (5.0±4.1% vs 12.0±8.0%, p=.031, and 1.6±0.3 nM vs 1.5±0.3 nM, p=.54). There were no significant differences in other functional properties (p&gt;.48). CD31 and IL-1β, which are circulating inflammatory biomarkers that we showed are elevated in tobacco smokers, were not elevated in cannabis smokers. The inflammatory biomarker PTX3 was lower in cannabis smokers than nonusers (p=.038). Conclusion: Chronic cannabis use impairs endothelial function but may reduce inflammation. Our findings suggest that the impairment caused by smoking cannabis vs edible THC use occurs via distinct mechanisms differing in the involvement of endothelial NO production.

Abstract 18413: Therapeutic Genome Editing of Endothelial FABP4 by Endothelium-Targeted Nanoparticle Delivery of the CRISPR/Cas9 System Inhibits Pulmonary Arterial Hypertension in Mice and Rats

Introduction: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive vasoconstriction and obliterative vascular remodeling. The molecular mechanisms of obliterative pulmonary vascular remodeling remain elusive. Hypothesis: Therapeutic genome editing of endothelial Fabp4 will knockout FABP4 expression selectively in endothelial cells and inhibit PAH in mice and rats. Methods: Employing the newly developed endothelial cell (EC)-targeted nanoparticle delivery technology, plasmid DNA expressing CRISPR/Cas9 controlled by Cdh5 promoter and Fabp4 -specific guide RNAs driven by U6 promoter was administered i.v. to adult SD rats and Egln1 Tie2Cre mice which exhibit severe PAH and obliterative pulmonary vascular remodeling. One week after nanoparticle administration, the rats were injected with a single dose of monocrotaline (MCT) to induce PAH. At 4 weeks post-MCT, right ventricular systolic pressure (RVSP), ratio of right ventricular versus left ventricular plus septum (RV/LV+S) and pulmonary vascular remodeling were determined. Pharmacological FABP4 inhibitor was also employed to treat MCT rats as a comparison. Similarly, the pulmonary hypertensive phenotype in genome-edited Egln1 Tie2Cre mice was also characterized. Results: Nanoparticle delivery of the all-in-one CRISPR rCdh5 plasmid DNA knockout Fabp4 selectively in endothelial cells in adult rats and Egln1 Tie2Cre mice leading to reduced RVSP, RV hypertrophy and pulmonary vascular remodeling in MCT rats and also in Egln1 Tie2Cre mice. Obliterative pulmonary vascular remodeling seen in Egln1 Tie2Cre mice was inhibited and the mortality rate in endothelial Fabp4-deficient Egln1 Tie2Cre mice was reduced. Treatment of FABP4 inhibitor also inhibits PAH in MCT rats. We also observed a marked increase of FABP4 expression in endothelial cells of lung samples of IPAH patients. Overexpression of FABP4 in primary cultures of human lung microvascular endothelial cells resulted in a 70% increase of cell proliferation. Conclusions: These findings demonstrate endothelial FABP4 is a promising target for PAH therapy. EC-targeted nanoparticle delivery of the genome editing system may represent an effective and safer gene therapy approach for PAH treatment.

Abstract 17370: Activation of Endothelial GCN2 Kinase Contributes to Pulmonary Vascular Remodeling and Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and pulmonary vascular remodeling that result in right heart failure. Published studies show that recessive mutations in the EIF2AK4 gene (encoding GCN2, General control nonderepressibe 2 kinase) resulting in reduction of GCN2 are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients and EIF2AK4 mutation was also found in PAH patients although very rare. Objective: To determine the role of endothelial GCN2 kinase in the pathogenesis of PAH. Results: Employing Eif2ak4 -/- mice, we found that loss of GCN2 induced neither spontaneous PVOD nor pulmonary hypertension (PH). In response to chronic hypoxia, loss of GCN2 inhibited hypoxia-induced PH and pulmonary vascular remodeling. RNA-sequencing analysis of mouse lung samples indicated Endothelin-1 as the downstream target of GCN2 in regulating pulmonary vascular remodeling and PH. In cultured human lung microvascular endothelial cells, GCN2 was phosphorylated and activated in response to hypoxia, which mediated hypoxia-induced Endothelin-1 expression through the GCN2/ATF4/HIF-2a/Endothelin-1 pathway. Endothelial cell-targeted nanoparticle delivery of plasmid DNA expressing Endothelin-1 selectively in endothelial cells in Eif2ak4 -/- mice markedly reversed the phenotype in response to hypoxia. Furthermore, nanoparticle delivery of the CRIPSR/Cas9 system-mediated knockout of Eif2ak4 in adult C57BL/6 mice also attenuated hypoxia-induced PH. Additionally, monocrotaline-induced vascular remodeling and PH in rats were inhibited by GCN2 inhibitor A-92 treatment. In lung samples of idiopathic PAH patients, we observed endothelial GCN2 hyperphosphorylation of pulmonary vascular lesions, without marked differences in GCN2 mRNA and protein levels compared to normal donor lungs. Conclusion: These studies demonstrate that endothelial GCN2 activation mediates pulmonary vascular remodeling and PAH development through the GCN2/ATF4/HIF-2a/Endothelin-1 pathway. Targeting GCN2 signaling is a promising therapeutic strategy for treatment of PAH in patients without GCN2 loss of function mutations.

Role of interleukin 6 and its soluble receptor on the diffusion barrier dysfunction of alveolar tissue.

During respiratory infection, barrier dysfunction in alveolar tissue can result from "cytokine storm" caused by overly reactive immune response. Particularly, interleukin 6 (IL-6) is implicated as a key biomarker of cytokine storm responsible for and further progression to pulmonary edema. In this study, alveolar-like tissue was reconstructed in a microfluidic device with: (1) human microvascular lung endothelial cells (HULEC-5a) cultured under flow-induced shear stress and (2) human epithelial cells (Calu-3) cultured at air-liquid interface. The effects of IL-6 and the soluble form of its receptor (sIL-6R) on the permeability, electrical resistance, and morphology of the endothelial and epithelial layers were evaluated. The diffusion barrier properties of both the endothelial and epithelial layers were significantly degraded only when IL-6 treatment was combined with sIL-6R. As suggested by recent review and clinical studies, our results provide unequivocal evidence that the barrier dysfunction occurs through trans-signaling in which IL-6 and sIL-6R form a complex and then bind to the surface of endothelial and epithelial cells, but not by classical signaling in which IL-6 binds to membrane-expressed IL-6 receptor. This finding suggests that the role of both IL-6 and sIL-6R should be considered as important biomarkers in developing strategies for treating cytokine storm.

Hantavirus: an overview and advancements in therapeutic approaches for infection.

Hantaviruses are a significant and emerging global public health threat, impacting more than 200,000 individuals worldwide each year. The single-stranded RNA viruses belong to the Hantaviridae family and are responsible for causing two acute febrile diseases in humans: Hantavirus pulmonary syndrome (HPS) and hemorrhagic fever with renal syndrome (HFRS). Currently, there are no licensed treatments or vaccines available globally for HTNV infection. Various candidate drugs have shown efficacy in increasing survival rates during the early stages of HTNV infection. Some of these drugs include lactoferrin, ribavirin, ETAR, favipiravir and vandetanib. Immunotherapy utilizing neutralizing antibodies (NAbs) generated from Hantavirus convalescent patients show efficacy against HTNV. Monoclonal antibodies such as MIB22 and JL16 have demonstrated effectiveness in protecting against HTNV infection. The development of vaccines and antivirals, used independently and/or in combination, is critical for elucidating hantaviral infections and the impact on public health. RNA interference (RNAi) arised as an emerging antiviral therapy, is a highly specific degrades RNA, with post-transcriptional mechanism using eukaryotic cells platform. That has demonstrated efficacy against a wide range of viruses, both in vitro and in vivo. Recent antiviral methods involve using small interfering RNA (siRNA) and other, immune-based therapies to target specific gene segments (S, M, or L) of the Hantavirus. This therapeutic approach enhances viral RNA clearance through the RNA interference process in Vero E6 cells or human lung microvascular endothelial cells. However, the use of siRNAs faces challenges due to their low biological stability and limited in vivo targeting ability. Despite their successful inhibition of Hantavirus replication in host cells, their antiviral efficacy may be hindered. In the current review, we focus on advances in therapeutic strategies, as antiviral medications, immune-based therapies and vaccine candidates aimed at enhancing the body's ability to control the progression of Hantavirus infections, with the potential to reduce the risk of severe disease.

SIRT6 Protects Against Lipopolysaccharide-Induced Inflammation in Human Pulmonary Lung Microvascular Endothelial Cells.

Inflammatory response in the pulmonary endothelium drives the pathogenesis of acute lung injury and sepsis. Sirtuin 6 (SIRT6), a member of class III NAD+-dependent deacetylases belonging to the sirtuin family, regulates senescence, metabolism, and inflammation and extends lifespan in mice and model organisms. However, the role of SIRT6 in pulmonary endothelial inflammation is unknown. Thus, we hypothesized that SIRT6 suppresses inflammatory response in human lung microvascular cells (HLMEC) and ensues monocyte adhesion to endothelial cells. Primary HLMECs were treated with control or SIRT6 adenovirus or SIRT6 agonist, with or without lipopolysaccharide (LPS) treatment. We observed that treatment with LPS did not affect the protein expression of SIRT6 in HLMECs. However, adenovirus-mediated SIRT6 overexpression attenuated LPS-induced VCAM1 gene and protein expression, followed by decreased monocyte adhesion to endothelial cells. Similarly, activation of SIRT6 by a recently reported SIRT6 activator UBCS039, but not the regioisomer negative control compound UBCS060, ameliorated LPS-induced VCAM1 mRNA and protein expression as well as monocyte adhesion. Moreover, luciferase assay revealed that SIRT6 adenovirus decreased the activity of NF-κB, the master regulator of vascular inflammation. Taken together, these results indicate that molecular and pharmacological activation of SIRT6 protects against lung microvascular inflammation via suppressing NF-κB activation, implicating the therapeutic potential of the SIRT6 activators for lung disorders associated with microvascular inflammation.

Reduction of double-strand DNA break repair exacerbates vascular aging.

Advanced age is the greatest risk factor for cardiovascular disease (CVD), the leading cause of death. Arterial function is impaired in advanced age which contributes to the development of CVD. One underexplored hypothesis is that DNA damage within arteries leads to this dysfunction, yet evidence demonstrating the incidence and physiological consequences of DNA damage in arteries, and in particular, in the microvasculature, in advanced age is limited. In the present study, we began by assessing the abundance of DNA damage in human and mouse lung microvascular endothelial cells and found that aging increases the percentage of cells with DNA damage. To explore the physiological consequences of increases in arterial DNA damage, we evaluated measures of endothelial function, microvascular and glycocalyx properties, and arterial stiffness in mice that were lacking or heterozygous for the double-strand DNA break repair protein ATM kinase. Surprisingly, in young mice, vascular function remained unchanged which led us to rationalize that perhaps aging is required to accumulate DNA damage. Indeed, in comparison to wild type littermate controls, mice heterozygous for ATM that were aged to ~18 mo (Old ATM +/-) displayed an accelerated vascular aging phenotype characterized by increases in arterial DNA damage, senescence signaling, and impairments in endothelium-dependent dilation due to elevated oxidative stress. Furthermore, old ATM +/- mice had reduced microvascular density and glycocalyx thickness as well as increased arterial stiffness. Collectively, these data demonstrate that DNA damage that accumulates in arteries in advanced age contributes to arterial dysfunction that is known to drive CVD.

Pulmonary arterial hypertension drugs can partially restore altered angiogenic capacities in bmpr2-silenced human lung microvascular endothelial cells.

Mutations in the bone morphogenetic protein receptor type 2 (bmpr2) gene and signaling pathway impairment are observed in heritable and idiopathic pulmonary arterial hypertension (PAH). In PAH, endothelial dysfunction is currently handled by drugs targeting the endothelin-1 (ET-1), nitric oxide (NO), and prostacyclin (PGI2) pathways. The role of angiogenesis in the disease process and the effect of PAH therapies on dysregulated angiogenesis remain inconclusive. We aim to investigate in vitro whether (i) bmpr2 silencing can impair angiogenic capacity of human lung microvascular endothelial cells (HLMVECs) and (ii) PAH therapies can restore them. The effects of macitentan (ET-1), tadalafil (NO), and selexipag (PGI2), on BMPRII pathway activation, endothelial barrier function, and angiogenesis were investigated in bmpr2-silenced HLMVECs. Stable bmpr2 silencing resulted in impaired migration and tube formation in vitro capacity. Inhibition of ET-1 pathway was able to partially restore tube formation in bmpr2-silenced HLMVECs, whereas none of the therapies was able to restore endothelial barrier function, no deleterious effects were observed. Our findings highlight the potential role of BMPRII signaling pathway in driving pulmonary endothelial cell angiogenesis. In addition, PAH drugs display limited effects on endothelial function when BMPRII is impaired, suggesting that innovative therapeutic strategies targeting BMPRII signaling are needed to better rescue endothelial dysfunction in PAH.

Simultaneous Targeting of IL-1-Signaling and IL-6-Trans-Signaling Preserves Human Pulmonary Endothelial Barrier Function During a Cytokine Storm.

Systemic inflammatory diseases, such as sepsis and severe COVID-19, provoke acute respiratory distress syndrome in which the pathological hyperpermeability of the microvasculature, induced by uncontrolled inflammatory stimulation, causes pulmonary edema. Identifying the inflammatory mediators that induce human lung microvascular endothelial cell barrier dysfunction is essential to find the best anti-inflammatory treatments for critically ill acute respiratory distress syndrome patients. We have compared the responses of primary human lung microvascular endothelial cells to the main inflammatory mediators involved in cytokine storms induced by sepsis and SARS-CoV2 pulmonary infection and to sera from healthy donors and severely ill patients with sepsis. Endothelial barrier function was measured by electric cell-substrate impedance sensing, quantitative confocal microscopy, and Western blot. The human lung microvascular endothelial cell barrier was completely disrupted by IL (interleukin)-6 conjugated with soluble IL-6R (IL-6 receptor) and by IL-1β, moderately affected by TNF (tumor necrosis factor)-α and IFN (interferon)-γ and unaffected by other cytokines and chemokines, such as IL-6, IL-8, MCP (monocyte chemoattractant protein)-1 and MCP-3. The inhibition of IL-1 and IL-6R simultaneously, but not separately, significantly reduced endothelial hyperpermeability on exposing human lung microvascular endothelial cells to a cytokine storm consisting of 8 inflammatory mediators or to sera from patients with sepsis. Simultaneous inhibition of IL-1 and JAK (Janus kinase)-STAT (signal transducer and activator of transcription protein), a signaling node downstream IL-6 and IFN-γ, also prevented septic serum-induced endothelial barrier disruption. These findings strongly suggest a major role for both IL-6 trans-signaling and IL-1β signaling in the pathological increase in permeability of the human lung microvasculature and reveal combinatorial strategies that enable the gradual control of pulmonary endothelial barrier function in response to a cytokine storm.