Lung Endothelial Cells Research Articles

The alarmin, interleukin-33, increases vascular tone via extracellular signal regulated kinase-mediated Ca2+ sensitization and endothelial nitric oxide synthase inhibition.

Alarmins are classified by their release from damaged or ruptured cells. Many alarmins have been found to increase vascular tone and oppose endothelium-dependent dilatation (EDD). Interleukin (IL)-33 plays a prominent role in lung injury and can be released during vascular injury and in chronic studies found to be cardioprotective. Our recent work has implicated IL-33 in acute vascular dysfunction following inhalation of engineered nanomaterials (ENM). However, the mechanisms linking IL-33 to vascular tone have not been interrogated. We therefore aimed to determine whether IL-33 directly influenced microvascular tone and endothelial function. Isolated feed arteries and in vivo arterioles from male and female Sprague-Dawley rats were used to determine direct vascular actions of IL-33. Mesenteric feed arteries and arterioles demonstrated reduced intraluminal diameters when treated with increasing concentrations of recombinant IL-33. IL-33 activated extracellular signal regulated kinase (ERK)1/2 of rat aortic smooth muscle cells but not phosphorylation of myosin light chain kinase. This suggested IL-33may sensitize arterioles to Ca2+-mediated responses. Indeed, IL-33 augmented the myogenic- and phenylephrine-induced vasoconstriction. Additionally, incubation of arterioles with 1ng IL-33 attenuated ACh-mediated EDD. Mechanistically, in human aortic endothelial cells, we demonstrate that IL-33-mediated ERK1/2 activation leads to inhibitory phosphorylation of serine 602 on endothelial nitric oxide synthase. Finally, we demonstrate that IL-33-ERK1/2 contributes to vascular tone following two known inducers of IL-33; ENM inhalation and the rupture endothelial cells. The present study provides novel evidence that IL-33 increases vascular tone via canonical ERK1/2 activation in microvascular smooth muscle and endothelium. Altogether, it is suggested IL-33 plays a critical role in microvascular homeostasis following barrier cell injury. KEY POINTS: Interleukin (IL)-33 causes a concentration-dependent reduction in feed artery diameter. IL-33 acts on vascular smooth muscle cells to augment Ca2+-mediated processes. IL-33 causes inhibitory phosphorylation of endothelial nitric oxide synthase and opposes endothelium-dependent dilatation. Engineered nanomaterial-induced lung injury and endothelial cell rupture in part act through IL-33 to mediate increased vascular tone.

Abstract 4135700: Effects of Mitochondrial Dysfunction on Extracellular Vesicle Release and Subsequent Disruption of the Lung Vascular Endothelial Barrier

Background: Lung endothelial cell (EC) barrier disruption is a hallmark of acute lung injury (ALI), which is commonly caused by bacterial pneumonia. Alveolar epithelial cells (AEC) are the first line of defence against invading respiratory pathogens in the lungs and can mediate inflammatory signaling to the adjacent EC contributing to barrier disruption. We have previously demonstrated that pneumolysin (PLY), a major Streptococcus pneumoniae virulence factor, induces both mitochondrial dysfunction in lung AEC and release of large extracellular vesicles (EVs). These EVs, which are enriched with mitochondrial cargo, contribute to increased permeability of the lung EC barrier. Here, we investigate strategies to inhibit mitochondrial dysfunction in AEC and examine how these interventions impact EC barrier function. Methods: Human alveolar epithelial cells (A549) or immortalized AEC (Cell Biologics) were pre-treated with mitoTEMPO (mtROS scavenger), P110 (excessive fission inhibitor), or ensifentrine [dual phosphodiesterase (PDE) 3/4 inhibitor] prior to PLY treatment (100-300 ng/ml, 4 hrs). Mitochondrial function was assessed by the JC-1 assay. EVs were isolated from cell media and analyzed for their mitochondrial content (Tom20 and Tim23 expression). To assess their functional role, EVs were added to TNF-α treated human lung microvascular EC, and barrier function was then determined using the ECIS assay to measure transendothelial electrical resistance (TER). Results: PLY-induced mitochondrial dysfunction was reduced in the presence of mitoTempo or P110. EVs from PLY-treated AEC (EV PLY ; enriched mitochondrial content) exacerbated TNF-α-induced EC barrier permeability as indicated by the significant decrease in TER. EV PLY derived from MitoTEMPO or P110-pretreated AEC contained less mitochondrial cargo and resulted in less EC permeability after TNF-α. Compared to mitoTEMPO and P110, ensifentrine exhibited superior protection against mitochondrial dysfunction after PLY exposure. In addition, in the presence of ensifentrine, the quantity of EV release and their extracelllular mitochondrial content were both dramatically reduced. In contrast to control EV PLY , EV PLY from ensifentrine-pretreated AEC demonstrated no barrier disruptive properties. Conclusions: Mitochondrial dysfunction in AEC contributes to vascular endothelial injury in ALI through mechanisms involving the release of barrier-disruptive EVs, which can be effectively targeted by inhibiting PDE3/4.

MiR-144/451: A Regulatory Role in Inflammation.

Inflammation is the natural defense mechanism of the body in response to injury, infection, or other stimuli. Excessive or persistent inflammatory responses can lead to the development of inflammatory diseases. Therefore, elucidating the regulatory mechanisms of inflammatory cells is crucial for understanding the pathogenesis of such diseases and devising novel therapeutic approaches. Moreover, miR-144/451 plays an important role in erythroid maturity and tumour development. Herein, we have reviewed the regulatory role of miR-144/451 in inflammation. Papers on miR-144, miR-451, and inflammation were retrieved from PubMed and Web of Science to be analysed and summarised. miR-144/451 plays a significant role in modulating inflammatory responses. Pro- and anti-inflammatory gene transcription is regulated by miR-144/451 binding to the 3' untranslated regions. Studies have shown that miR-451 inhibits the activation of various inflammatory cells, including macrophages, neutrophils, and T lymphocytes, thereby reducing the release of inflammatory mediators. However, miR-144 expression varies in different inflammatory diseases. miR-144 expression is downregulated in macrophages after induction by lipopolysaccharide, cysteine, or Mycobacterium tuberculosis, which promotes the secretion of inflammatory mediators; nonetheless, miR-144-3p overexpression in macrophages can aggravate atherosclerosis. Meanwhile, miR-144 overexpression prevents disruption of the lung endothelial cell barrier, whereas it exacerbates endothelial cell injury in Crohn's disease. miR-144/451 may serve as a potential target for the treatment of inflammatory diseases.

Cannabidiol (CBD) Protects Lung Endothelial Cells from Irradiation-Induced Oxidative Stress and Inflammation In Vitro and In Vivo.

Objective: Radiotherapy, which is commonly used for the local control of thoracic cancers, also induces chronic inflammatory responses in the microvasculature of surrounding normal tissues such as the lung and heart that contribute to fatal radiation-induced lung diseases (RILDs) such as pneumonitis and fibrosis. In this study, we investigated the potential of cannabidiol (CBD) to attenuate the irradiation damage to the vasculature. Methods: We investigated the ability of CBD to protect a murine endothelial cell (EC) line (H5V) and primary lung ECs isolated from C57BL/6 mice from irradiation-induced damage in vitro and lung ECs (luECs) in vivo, by measuring the induction of oxidative stress, DNA damage, apoptosis (in vitro), and induction of inflammatory and pro-angiogenic markers (in vivo). Results: We demonstrated that a non-lethal dose of CBD reduces the irradiation-induced oxidative stress and early apoptosis of lung ECs by upregulating the expression of the cytoprotective mediator heme-oxygenase-1 (HO-1). The radiation-induced increased expression of inflammatory (ICAM-2, MCAM) and pro-angiogenic (VE-cadherin, Endoglin) markers was significantly reduced by a continuous daily treatment of C57BL/6 mice with CBD (i.p. 20 mg/kg body weight), 2 weeks before and 2 weeks after a partial irradiation of the lung (less than 20% of the lung volume) with 16 Gy. Conclusions: CBD has the potential to improve the clinical outcome of radiotherapy by reducing toxic side effects on the microvasculature of the lung.

Dual Inhibition of Phosphodiesterase 3 and 4 Enzymes by Ensifentrine Protects against MRSA-Induced Lung Endothelial and Epithelial Dysfunction.

Acute Respiratory Distress Syndrome (ARDS) is a severe lung condition with a high mortality rate for which there are no effective therapeutics. The failure of the alveolar-capillary barrier, composed of lung endothelial (EC) and alveolar epithelial (AEC) cells, is a critical factor leading to excessive inflammation and edema characteristic of acute lung injury (ALI) pathophysiology. Phosphodiesterases (PDE) are enzymes well-recognized for their roles in regulating endothelial permeability and inflammation. Although PDE inhibitors are used as therapeutics for inflammatory diseases like COPD (chronic obstructive pulmonary disease), their efficacy in treating ARDS has not yet been established. In this study, we investigated the effects of ensifentrine, an FDA-approved novel dual PDE 3/4 inhibitor, on lung endothelial and epithelial dysfunction caused by methicillin-resistant S. aureus (MRSA), a pathogen involved in bacterial ARDS. Human primary lung endothelial cells and alveolar epithelial cell lines (A549 and immortalized AEC) were treated with heat-killed MRSA, and their responses were assessed in the presence or absence of ensifentrine. Ensifentrine given either pre- or post-exposure attenuated MRSA-induced increased lung endothelial permeability. VE-cadherin junctions, which serve to stabilize the EC barrier, were disrupted by MRSA; however, ensifentrine effectively prevented this disruption. Pre-treatment with ensifentrine protected against MRSA-induced EC pro-inflammatory signaling by inhibiting the expression of VCAM-1, ICAM-1, and by reducing the IL-6 and IL-8 release. In AEC, MRSA caused the upregulation of ICAM-1, the activation of NF-kB, and the production of IL-8, all of which were inhibited by ensifentrine. These results indicate that the dual inhibition of phosphodiesterases 3 and 4 by ensifentrine is barrier protective and attenuates MRSA-induced inflammation in both lung endothelial and epithelial cells. The PDE3/4 inhibitor ensifentrine may represent a promising novel strategy for the treatment of MRSA-induced ARDS.

Decoding the multiple functions of ZBP1 in the mechanism of sepsis-induced acute lung injury.

Sepsis-induced acute lung injury (ALI), characterized by severe hypoxemia and pulmonary leakage, remains a leading cause of mortality in intensive care units. The exacerbation of ALI during sepsis is largely attributed to uncontrolled inflammatory responses and endothelial dysfunction. Emerging evidence suggests an important role of Z-DNA binding protein 1 (ZBP1) as a sensor in innate immune to drive inflammatory signaling and cell death during infections. However, the role of ZBP1 in sepsis-induced ALI has yet to be defined. We utilized ZBP1 knockout mice and combined single-cell RNA sequencing with experimental validation to investigate ZBP1's roles in the regulation of macrophages and lung endothelial cells during sepsis. We demonstrate that in sepsis, ZBP1 deficiency in macrophages reduces mitochondrial damage and inhibits glycolysis, thereby altering the metabolic status of macrophages. Consequently, this metabolic shift leads to a reduction in the differentiation of macrophages into pro-inflammatory states and decreases macrophage pyroptosis triggered by activation of the NLRP3 inflammasome. These changes significantly weaken the inflammatory signaling pathways between macrophages and endothelial cells and alleviate endothelial dysfunction and cellular damage. These findings reveal important roles for ZBP1 in mediating multiple pathological processes involved in sepsis-induced ALI by modulating the functional states of macrophages and endothelial cells, thereby highlighting its potential as a promising therapeutic target.

Control of inflammatory lung injury and repair by metabolic signaling in endothelial cells.

Sepsis-induced inflammatory lung injury includes acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). There are currently no effective treatments for ALI/ARDS, but clinical outcomes could be improved by inhibiting lung injury and/or promoting post-sepsis vascular repair. In this review, we describe studies of endothelial cell metabolic pathways in sepsis-induced ALI/ARDS and vascular repair and identify areas of research that deserve attention in future studies. We also describe studies of metabolic interventions that aim to inhibit ALI/ARDS and/or promote post-sepsis vascular repair, including those that target endothelial cell metabolites, endothelial cell metabolic signaling pathways, and endothelial cell metabolism. Endothelial cells are integral to both the injury and repair phases of ALI/ARDS. During the injury phase of ALI/ARDS, lung endothelial cell survival decreases, and lung endothelial cell-to-endothelial cell (EC-EC) junctions are weakened. During the repair phase after sepsis-induced lung injury, lung endothelial cell proliferation and lung EC-EC junction reannealing occur. These crucial aspects of ALI/ARDS and post-sepsis vascular repair, that is, endothelial cell viability, growth, and junction integrity, are controlled by a myriad of metabolites and metabolic signaling pathways in endothelial cells. Metabolic signaling pathways in endothelial cells represent a novel class of putative targets for the prevention and treatment of sepsis-induced inflammatory lung injury. Therapies that target metabolic signaling in endothelial cells are currently being explored as potential treatments for sepsis-induced inflammatory lung injury.

Heart-lung crosstalk in acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is initiated by a primary insult that triggers a cascade of pathological events, including damage to lung epithelial and endothelial cells, extracellular matrix disruption, activation of immune cells, and the release of pro-inflammatory mediators. These events lead to increased alveolar-capillary barrier permeability, resulting in interstitial/alveolar edema, collapse, and subsequent hypoxia and hypercapnia. ARDS not only affects the lungs but also significantly impacts the cardiovascular system. We conducted a comprehensive literature review on heart-lung crosstalk in ARDS, focusing on the pathophysiology, effects of mechanical ventilation, hypoxemia, and hypercapnia on cardiac function, as well as ARDS secondary to cardiac arrest and cardiac surgery. Mechanical ventilation, essential for ARDS management, can increase intrathoracic pressure, decrease venous return and right ventricle preload. Moreover, acidemia and elevations in transpulmonary pressures with mechanical ventilation both increase pulmonary vascular resistance and right ventricle afterload. Cardiac dysfunction can exacerbate pulmonary edema and impair gas exchange, creating a vicious cycle, which hinders both heart and lung therapy. In conclusion, understanding the heart-lung crosstalk in ARDS is important to optimize therapeutic strategies. Future research should focus on elucidating the precise mechanisms underlying this interplay and developing targeted interventions that address both organs simultaneously.

Inhibition of angiogenesis and regenerative lung growth in Lepob/ob mice through adiponectin-VEGF/VEGFR2 signaling.

Obesity is associated with impairment of wound healing and tissue regeneration. Angiogenesis, the formation of new blood capillaries, plays a key role in regenerative lung growth after unilateral pneumonectomy (PNX). We have reported that obesity inhibits angiogenesis. The effects of obesity on post-PNX lung vascular and alveolar regeneration remain unclear. Unilateral PNX is performed on Lep o b / o b obese mice to examine vascular and alveolar regeneration. Regenerative lung growth and expression of vascular endothelial growth factor (VEGF) and its receptor VEGFR2 induced after PNX are inhibited in Lep o b / o b obese mice. The levels of adiponectin that exhibits pro-angiogenic and vascular protective properties increase after unilateral PNX, while the effects are attenuated in Lep o b / o b obese mice. Post-PNX regenerative lung growth and increases in the levels of VEGF and VEGFR2 are inhibited in adiponectin knockout mice. Adiponectin stimulates angiogenic activities in human lung endothelial cells (ECs), which is inhibited by decreasing the levels of transcription factor Twist1. Adiponectin agonist, AdipoRon restores post-PNX lung growth and vascular and alveolar regeneration in Lep o b / o b obese mice. These findings suggest that obesity impairs lung vascular and alveolar regeneration and adiponectin is one of the key factors to improve lung regeneration in obese people.

Pathogenesis of Klebsiella Pneumoniae Induced Proteinopathy is Amyloid Strain Dependent

Abstract Introduction/Objective The World Health Organization has labeled Klebsiella pneumoniae (KP), a Gram-negative bacterium, as a ‘Critical Priority’ pathogen due to its increasing prevalence in serious conditions like pneumonia and sepsis, along with its growing resistance to multiple drugs. Infections with KP have been linked to the collapse of microtubules and the release of harmful proteins called amyloid-beta (Aβ) and tau from lung endothelial cells. These infection-induced lung endothelial amyloids can spread like prions and are stable under heat. Methods/Case Report However, the mechanisms behind these effects and whether the infection-generated proteinopathy depends on tau and/or Aβ were not well understood. This study hypothesized that KP infection triggers tau dysfunction, breakdown of microtubules, increased lung barrier permeability, and the production and release of toxic amyloids. To test this, researchers used CRISPR/Cas9 technology to create lung endothelial cells without tau and/or amyloid precursor protein (APP). They confirmed gene deletion through various methods and exposed these cells, along with regular cells, to a highly virulent KP strain. Results (if a Case Study enter NA) The results showed that the KP strain (Kp 1-008) caused disruption of cellular structures, increased permeability, and bacterial spread within hours of infection. Supernatants containing proteins from infected cells, when applied to healthy cells, led to barrier disruption, permeability, and cell death. Interestingly, different pathologies were induced by supernatants from cells lacking tau or APP, indicating that either tau or Aβ alone could propagate damage in a strain- specific manner. The most severe damage required both tau and Aβ together. Conclusion In conclusion, KP infection disturbs cellular structures, promoting barrier breakdown and bacterial dissemination. It also generates toxic tau and Aβ proteins. Either of these proteins can spread harm from cell to cell, but together, they enhance the severity and potency of the resulting lung endothelial protein damage caused by the infection. The study sheds light on the complex mechanisms of KP-related infections and their impact on cellular structures and protein pathways.

Endothelial ENaC-α Restrains Oxidative Stress in Lung Capillaries in Murine Pneumococcal Pneumonia-associated Acute Lung Injury.

Infection of lung endothelial cells with pneumococci activates the superoxide-generating enzyme NADPH oxidase 2 (NOX2), involving the pneumococcal virulence factor pneumolysin (PLY). Excessive NOX2 activity disturbs capillary barriers, but its global inhibition can impair bactericidal phagocyte activity during pneumococcal pneumonia. Depletion of the α subunit of the epithelial sodium channel (ENaC) in pulmonary endothelial cells increases expression and PMA-induced activity of NOX2. Direct ENaC activation by TIP peptide improves capillary barrier function -measured by electrical cell substrate impedance sensing in endothelial monolayers and by Evans Blue Dye incorporation in mouse lungs- following infection with pneumococci. PLY-induced hyperpermeability in HL-MVEC monolayers is abrogated by both NOX2 inhibitor gp91dstat and TIP peptide. Endothelial NOX2 expression is assessed by increased surface membrane presence of phosphorylated p47phox subunit (Western blotting) in vitro and by co-localization of CD31 and gp91phox in mouse lung slices using DuoLink, whereas NOX2-generated superoxide is measured by chemiluminescence. TIP peptide blunts PMA-induced NOX2 activity in cells expressing ENaC-α, but not in neutrophils, which lack ENaC. Conditional endothelial ENaC-α KO (enENaC-α KO) mice develop increased capillary leak upon i.t. instillation with PLY or pneumococci, compared to wild type (wt) animals. TIP peptide diminishes capillary leak in Sp-infected wt mice, without significantly increasing lung bacterial load. Lung slices from Sp-infected enENaC-α KO mice have a significantly increased endothelial NOX2 expression, as compared to infected CRE mice. In conclusion, endothelial ENaC may represent a novel therapeutic target to reduce NOX2-mediated oxidative stress and capillary leak in ARDS, without impairing host defense.

Mechanisms of Lung Endothelial Cell Injury and Survival in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a progressive, chronic, and incurable inflammatory pulmonary vascular disease characterized by significant sex bias and largely unexplored microbial-associated molecular mechanisms that may influence its development and sex prevalence across various subgroups. PAH can be subclassified as idiopathic, heritable, or associated with conditions such as connective tissue diseases, congenital heart defects, liver disease, infections, and chronic exposure to drugs or toxins. During PAH progression, lung vascular endothelial cells (ECs) undergo dramatic morphofunctional transformations in response to acute and chronic inflammation. These transformations include the appearance and expansion of abnormal vascular cell phenotypes such as those derived from apoptosis-resistant cell growth and endothelial-to-mesenchymal transition (EndoMT). Compelling evidence indicates that these endothelial phenotypes seem to be triggered by chronic lung vascular injury and dysfunction, often characterized by reduced secretion of vasoactive molecules like nitric oxide (NO) and exacerbated response to vasoconstrictors such as Endothelin-1 (ET-1); both long-term known contributors of PAH pathogenesis. This review sheds light on the mechanisms of EC dysfunction, apoptosis, and EndoMT in PAH, aiming to unravel the intricate interactions between ECs, pathogens, and other cell types that drive the onset and progression of this devastating disease. Ultimately, we hope to provide an overview of the complex functions of lung vascular ECs in PAH, inspiring novel therapeutic strategies that target these dysfunctional cells to improve the treatment landscape for PAH, particularly in the face of current and emerging global pathogenic threats.

Preferred Lung Accumulation of Polystyrene Nanoplastics with Negative Charges.

With the increasing presence of nanoplastics (NPs) in the human bloodstream, it is urgent to investigate their tissue accumulation and potential health risks. This study examines the effects of the size and surface charges of polystyrene (PS) NPs on lung accumulation. Using magnetic separation, we identified the protein corona composition on iron-core PS NPs, revealing the enrichment of vitronectin and fibrinogen. The corona promotes integrin αIIbβ3 receptor-mediated uptake by lung endothelial cells, explaining that both the corona composition and protein structure determine preferred localization of negatively charged PS NPs in the lung. This study uncovers the role of protein corona in NP uptake and the way NPs enter the lung, emphasizing the need to consider interactions between nanoplastics with varying surface characteristics and biological molecules in the nanotoxicological field.

Endothelial PHD2 deficiency induces apoptosis resistance and inflammation via AKT activation and AIP1 loss independent of HIF2α.

In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which was partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared with wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and patients with PAH all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds the potential for developing new treatments for endothelial dysfunction in PH.NEW & NOTEWORTHY HIF activation alone does not conclusively lead to human PH, suggesting that HIF-independent signaling may also contribute to hypoxia-induced PH. This study demonstrated that PHD2 silencing-induced pseudohypoxia in human lung endothelial cells suppresses apoptosis and activates STAT, effects that persist despite HIF2α inhibition or knockdown and are attributed to AKT and ERK activation, JNK inhibition, and AIP1 loss. These findings align with observations in lung endothelial cells and tissues from PAH rodent models and patients.

Rap1A Modulates Store-Operated Calcium Entry in the Lung Endothelium: A Novel Mechanism Controlling NFAT-Mediated Vascular Inflammation and Permeability.

Store-operated calcium entry mediated by STIM (stromal interaction molecule)-1-Orai1 is essential in endothelial cell (EC) functions, affecting signaling, NFAT (nuclear factor for activated T cells)-induced transcription, and metabolic programs. While the small GTPase Rap1 isoforms, including the predominant Rap1B, are known for their role in cadherin-mediated adhesion, EC deletion of Rap1A after birth uniquely disrupts lung endothelial barrier function. Here, we elucidate the specific mechanisms by which Rap1A modulates lung vascular integrity and inflammation. The role of EC Rap1A in lung inflammation and permeability was examined using in vitro and in vivo approaches. We explored Ca2+ signaling in human ECs following siRNA-mediated knockdown of Rap1A or Rap1B. Rap1A knockdown, unlike Rap1B, significantly increased store-operated calcium entry in response to a GPCR (G-protein-coupled receptor) agonist, ATP (500 µmol/L), or thapsigargin (250 nmol/L). This enhancement was attenuated by Orai1 channel blockers 10 μmol/L BTP2, 10 μmol/L GSK-7975A, and 5 μmol/L Gd3+. Whole-cell patch clamp measurements revealed enhanced Ca2+ release-activated Ca2+ current density in siRap1A ECs. Rap1A depletion in ECs led to increased NFAT1 nuclear translocation and activity and elevated levels of proinflammatory cytokines (CXCL1, CXCL11, CCL5 [chemokine (C-C motif) ligand 5], and IL-6 [interleukin-6]). Notably, reducing Orai1 expression in siRap1A ECs normalized store-operated calcium entry, NFAT activity, and endothelial hyperpermeability in vitro. EC-specific Rap1A knockout (Rap1AiΔEC) mice displayed an inflammatory lung phenotype with increased lung permeability and inflammation markers, along with higher Orai1 expression. Delivery of siRNA against Orai1 to lung endothelium using lipid nanoparticles effectively normalized Orai1 levels in lung ECs, consequently reducing hyperpermeability and inflammation in Rap1AiΔEC mice. Our findings uncover a novel role of Rap1A in regulating Orai1-mediated Ca2+ entry and expression, crucial for NFAT-mediated transcription and endothelial inflammation. This study distinguishes the unique function of Rap1A from that of the predominant Rap1B isoform and highlights the importance of normalizing Orai1 expression in maintaining lung vascular integrity and modulating endothelial functions.

GCN2 kinase activation mediates pulmonary vascular remodeling and pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is characterized by progressive increase of pulmonary vascular resistance and remodeling that result in right heart failure. Recessive mutations of EIF2AK4 gene (encoding general control nonderepressible 2 kinase, GCN2) are linked to heritable pulmonary veno-occlusive disease (PVOD) in patients but rarely in patients with PAH. The role of GCN2 kinase activation in the pathogenesis of PAH remains unclear. Here, we show that GCN2 was hyperphosphorylated and activated in pulmonary vascular endothelial cells (ECs) of hypoxic mice, monocrotaline-treated rats, and patients with idiopathic PAH. Unexpectedly, loss of GCN2 kinase activity in Eif2ak4-/- mice with genetic disruption of the kinase domain induced neither PVOD nor pulmonary hypertension (PH) but inhibited hypoxia-induced PH. RNA-sequencing analysis suggested endothelin-1 (Edn1) as a downstream target of GCN2. GCN2 mediated hypoxia-induced Edn1 expression in human lung ECs via HIF-2α. Restored Edn1 expression in ECs of Eif2ak4-/- mice partially reversed the reduced phenotype of hypoxia-induced PH. Furthermore, GCN2 kinase inhibitor A-92 treatment attenuated PAH in monocrotaline-treated rats. These studies demonstrate that GCN2 kinase activation mediates pulmonary vascular remodeling and PAH at least partially through Edn1. Thus, targeting GCN2 kinase activation is a promising therapeutic strategy for treatment of PAH in patients without EIF2AK4 loss-of-function mutations.

ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 μM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Inhibiting endothelial cell Mst1 attenuates acute lung injury in mice.

Lung endothelium plays a pivotal role in the orchestration of inflammatory responses to acute pulmonary insults. Mammalian sterile 20-like kinase 1 (Mst1) is a serine/threonine kinase that has been shown to play an important role in the regulation of apoptosis, stress responses, and organ growth. This study investigated the role of Mst1 in lung endothelial activation and acute lung injury (ALI). We found that Mst1 was significantly activated in inflamed lung endothelial cells (ECs) and mouse lung tissues. Overexpression of Mst1 promoted nuclear factor κ-B (NF-κB) activation through promoting JNK and p38 activation in lung ECs. Inhibition of Mst1 by either its dominant negative form (DN-Mst1) or its pharmacological inhibitor markedly attenuated cytokine-induced expression of cytokines, chemokines, and adhesion molecules in lung ECs. Importantly, in a mouse model of lipopolysaccharide-induced (LPS-induced) ALI, both deletion of Mst1 in lung endothelium and treatment of WT mice with a pharmacological Mst1 inhibitor significantly protected mice from LPS-induced ALI. Together, our findings identified Mst1 kinase as a key regulator in controlling lung EC activation and suggest that therapeutic strategies aimed at inhibiting Mst1 activation might be effective in the prevention and treatment of inflammatory lung diseases.

Endothelialized Bronchioalveolar Lung Organoids Model Endothelial Cell Responses to Injury.

Organoid 3D systems are powerful platforms to study development and disease. Recently, the complexity of lung organoid models derived from adult mouse and human stem cells has increased substantially in terms of cellular composition and structural complexity. However, a murine lung organoid system with a clear integrated endothelial compartment is still missing. Here, we describe a novel method that adds another level of intricacy to our published bronchioalveolar lung organoid (BALO) model by microinjection of FACS-sorted lung endothelial cells (ECs) into differentiated organoid cultures. Before microinjection, ECs obtained from the lung homogenate (LH) of young mice expressed typical ECs markers such as CD31 and vascular endothelial (VE)-Cadherin and showed tube formation capacity. Following microinjection, ECs surrounded BALO´s alveolar-like compartment aligning with both alveolar epithelial cells type I (AECI) and type II (AECII), as demonstrated by confocal and electron microscopy. Notably, expression of Car4 and Aplnr was as well detected, suggesting presence of EC microvascular phenotypes in the cultured ECs. Moreover, upon epithelial cell injury by lipopolysaccharides (LPS) and influenza A virus (IV), endothelialized BALO (eBALO) released proinflammatory cytokines leading to the upregulation of the intercellular adhesion molecule 1 (ICAM-1) in ECs. In summary, we characterized for the first time a organoid model that incorporates ECs into the alveolar structures of lung organoids, not only increasing our previous model ́s cellular and structural complexity but also providing a suitable niche to model lung endothelium responses to injury ex vivo.

Investigation of the cytotoxic and antimicrobial properties of new quinoline peptide conjugates

Background: Cancer is one of the most important health problems causing deaths in Turkey and the world. Nowadays, many treatment methods such as radiotherapy and chemotherapy are applied in cancer patients. Quinoline and its derivatives, which belong to heterocyclic compounds, have many biological activities for drug development. Quinoline compounds play a crucial role in the development of antitumor drugs due to their anticancer activity. Materials and Methods: In this study, the cytotoxic effects of synthesized seven different quinoline derivatives on lung cancer (A549) and healthy lung epithelial cell (BEAS2B), liver cancer (Hep 3B), and endothelial cell (HUVEC) were determined. Different concentrations were applied and IC50 values were calculated at different time courses. The antimicrobial activites of the compounds were also determined. Results: The IC50 values of compound 2 on the A549 cell line were determined to be 10.48 μg/mL, 9.738 μg/mL, and 10.14 μg/mL. IC50 values of compound 6 on the same cell line were determined to be 7.307 µg/mL, 9.888 µg/mL, 10.63 µg/mL. Conclusions: Compounds 2 and 6 had a cytotoxic effect on the BEAS2B cell line. The antimicrobial activity of the compounds was determined by the minimum inhibition concentration method on different strains of bacteria and yeast. The compounds showed no antimicrobial activity on bacteria and yeast.