Rat Pulmonary Microvascular Endothelial Cells Research Articles

Loss of AC10 Impairs Neutrophil Recruitment and Alters Bacterial Colonization in the Lung During Bacterial Pneumonia

Rationale: Pseudomonas aeruginosa is a gram-negative bacterium and a common cause of nosocomial pneumonia that can progress to acute respiratory distress syndrome (ARDS), characterized by hypoxia, pulmonary edema, elevated bicarbonate, and widespread inflammation. Neutrophil recruitment is a critical line of defense against P. aeruginosa; however, excessive, or dysregulated neutrophil infiltration can exacerbate tissue damage and contribute to the severity of ARDS. Recently, AC10 has been implicated in neutrophil transendothelial migration via CD99. Despite the growing recognition and significance of AC10 in various physiological processes, its role in neutrophil recruitment during ARDS remains poorly understood. Thus, herein we hypothesize that loss of AC10 attenuates neutrophil recruitment during pneumonia and increase bacterial colonization in the lung. Methods: Wild-type and AC10 knockout mice were intratracheally inoculated with increasing doses of P. aeruginosa. Twenty fours after infection, bronchoalveolar lavage fluid (BALF) was collected and the percent of PMNs in the BALF determined. In separate experiments, mice were intratracheally inoculated with P. aeruginosa for 6 or 24 hours and organs (lungs, kidney, liver, spleen and brain) harvested, homogenized and the homogenate cultured overnight on Pseudomonas isolation agar for quantification of colony forming units (CFUs). Results: Our data confirmed CD99 expression in rat pulmonary microvascular endothelial cells and whole lung of both wild type and AC10 knockout mice. While there was no difference in the percentage of PMNs in the BALF between male wild type and knockout animals at any of the bacterial doses tested, in the female AC10 knockout mice, there was a decrease in the percentage of cells PMNs in the BALF compared to female wild type litter mates at several doses tested (1x103 up to 1x105, p<0.05). At a higher dose of P. aeruginosa (2.5x106), there was no difference in bacterial clearance between male WT and AC10 knockout mice, but a trend toward an increase in the CFU harvested from lung tissue 6 hours post infection in female KO mice. Conclusion: Thus, CD99 is expressed in the pulmonary endothelium. Further, in female mice, AC10 plays a role in neutrophil recruitment into the airspace. Further studies are required to resolve whether AC10 plays a role in bacterial clearance in female mice in a model of P. aeruginosa pneumonia. HL121513. 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.

Elevated circulating BMP9 aggravates pulmonary angiogenesis in hepatopulmonary syndrome rats through ALK1-Endoglin-Smad1/5/9 signalling.

Bone morphogenetic protein 9 (BMP9) is a hepatokine that plays a pivotal role in the progression of liver diseases. Moreover, an increasing number of studies have shown that BMP9 is associated with hepatopulmonary syndrome (HPS), but its role in HPS is unclear. Here, we evaluated the influence of CBDL on BMP9 expression and investigated potential mechanisms of BMP9 signalling in HPS. We profiled the circulating BMP9 levels in common bile duct ligation-induced HPS rat model, and then investigated the effects and mechanisms of HPS rat serum on pulmonary vascular endothelial dysfunction in rat model, as well as in primarily cultured rat pulmonary microvascular endothelial cells. Our data revealed that circulating BMP9 levels were significantly increased in the HPS rats compared to control group. Besides, the elevated BMP9 in HPS rat serum was not only crucial for promoting endothelial cell proliferation and tube formation through the activin receptor-like kinase1 (ALK1)-Endoglin-Smad1/5/9 pathway, but also important for accumulation of monocytes. Treatments with ALK1-Fc or silencing ALK1 expression to inhibit the BMP9 signalling pathway effectively eliminated these effects. In agreement with these observations, increased circulating BMP9 was associated with an increase in lung vessel density and accumulation of pro-angiogenic monocytes in the microvasculature in HPS rats. This study provided evidence that elevated circulating BMP9, secreted from the liver, promote pulmonary angiogenesis in HPS rats via ALK1-Endoglin-Smad1/5/9 pathway. In addition, BMP9-regulated pathways are also involved in accumulation of pro-angiogenic monocytes in the pulmonary microvasculature in HPS rats.

MicroRNA-141-3p reduces pulmonary hypoxia/reoxygenation injury through suppression of Beclin-1-dependent autophagy.

Alterations in autophagy are involved in pulmonary hypoxia/reoxygenation (H/R)-induced injury. Here, we intended to explain the function of microRNA-141-3p (miR-141-3p) in regulating autophagy under the H/R condition. Rat pulmonary microvascular endothelial cells (PMVECs) were applied for H/R cell model establishment, followed by tracing of autophagy formation. SIRT1 plays a critical role in controlling the lifespan of yeast, flies, and mice. Interaction between SIRT1 and Beclin-1, an indicator protein for autophagy, and between miR-141-3p and SIRT1 was assayed with their roles in PMVEC injury. Autophagy of PMVECs was activated after hypoxia treatment and further activated after H/R treatment. The binding of miR-141-3p and SIRT1 was verified. In H/R-treated PMVECs, the binding of miR-141-3p and SIRT1 was reduced. Furthermore, SIRT1 acted as a deacetylase to stabilize the Beclin-1 protein, promoting autophagy and PMVEC injury. H/R rat models were established, and in vivo, experiments further confirmed that miR-141-3p regulated autophagy and lung injury in H/R rats through SIRT1/Beclin-1 axis. The current study highlighted that reduced miR-141-3p in H/R-treated PMVECs promoted deacetylation of Beclin-1 by SIRT1, thus causing PMVEC injury.

Adenosine diphosphate ribosylation factor 6 inhibition protects burn sepsis induced lung injury through preserving vascular integrity and suppressing ASC inflammasome transmission

BackgroundSevere burns are devastating injuries with significant immune dysfunction and result in substantial mortality and morbidity due to sepsis induced organ failure. Acute lung injury is the most common type of organ injury in sepsis, however, the mechanisms of which are poorly understood and effective therapeutic measures are limited. This study is aimed to investigate the effect of a small Guanosine triphosphatase (GTPase), Adenosine diphosphate ribosylation factor 6 (ARF6), on burn sepsis induced lung injury, and discuss the possible mechanisms. MethodsBurn sepsis was established in male C57BL/6 mice. Mice were anesthetised by intramuscular injection of ketamine and xylazine hydrochloride, then 30% TBSA full thickness burn followed by sub-eschar injection of lipopolysaccharide. Animals were treated with intraperitoneal injection of a small molecule inhibitor of ARF6: NAV-2729, or vehicle, right after the burn and sepsis stimuli were inflicted. Lung tissues were harvested for histopathological observation and the acute lung injury scores were calculated. Organ permeability, Vascular Endothelial Cadherin (VE-cadherin) expression, inflammatory cytokine levels and myeloperoxidase activity in lung tissues were detected. Rat pulmonary microvascular endothelial cells (PMVECs) were stimulated by burn sepsis serum with or without 10 μM NAV-2729. The ARF6 activation, VE-cadherin expression, inflammasome activity, adapter protein apoptosis speck-like protein containing a caspase recruiting domain (ASC) specks and cytokines secretion were determined. Student’s t test was used for comparison between two groups. Multiple comparisons among groups were performed by using analysis of variance, with Tukey’s test for the post hoc test. ResultsNAV-2729 treatment attenuated burn sepsis induced lung injury and promoted survival of burn septic mice by preserving VE-cadherin expression in endothelial cell adherent junction and limited vascular hyperpermeability in lung tissues. Moreover, inflammatory cytokine expression and inflammatory injury in lung tissues were alleviated. Mechanistically, NAV-2729 enhanced vascular integrity by inhibiting ARF6 activation and restoring VE-cadherin expression in PMVECs. In addition, NAV-2729 inhibited ARF6-dependent phagocytosis of ASC specks, thus preventing inflammation propagation mediated by cell-to-cell transmission of ASC specks. ConclusionsARF6 inhibition preserved vascular integrity by restoring expression of VE-cadherin and suppressed the spread of inflammation by affecting phagocytosis of ASC specks, thus protected against sepsis induced lung injury and improve survival of burn septic animals. The findings of this study implied potential therapeutics by which ARF6 inhibition can protect lung function from septic induced lung injury and improve outcomes in burn sepsis.

ERRα protects against sepsis-induced acute lung injury in rats

BackgroundSepsis-induced acute lung injury (ALI) is associated with poor survival rates. The identification of potential therapeutic targets for preventing sepsis-induced ALI has clinical importance. This study aims to investigate the role of estrogen-related receptor alpha (ERRα) in sepsis-induced ALI.MethodsLipopolysaccharide (LPS) was used to simulate sepsis-induced ALI model in rat pulmonary microvascular endothelial cells (PMVECs). The effects of ERRα overexpression and knockdown on LPS-induced endothelial permeability, apoptosis and autophagy were determined by horseradish peroxidase permeability assay, TdT-mediated dUTP Nick End Labeling (TUNEL) assay, flow cytometry, immunofluorescence staining, RT-PCR and Western Blotting. The rat model with sepsis-induced ALI was established by cecal ligation and puncture in anesthetized rats to verify the results of in vitro experiments. Animals were randomly assigned to receive intraperitoneal injection of vehicle or ERRα agonist. Lung vascular permeability, pathological injury, apoptosis and autophagy were examined.ResultsOverexpression of ERRα ameliorated LPS-induced endothelial hyperpermeability, degradation of adherens junctional molecules, upregulation of bax, cleaved caspase 3 and cleaved caspase 9 levels, downregulation of anti-apoptotic protein Bcl-2 level, and promoted the formation of autophagic flux, while the knockdown of ERRα exacerbated LPS-induced apoptosis and inhibited the activation of autophagy. Administration of ERRα agonist alleviated the pathological damage of lung tissue, increased the levels of tight junction proteins and adherens junction proteins, and decreased the expression of apoptosis-related proteins. Promoting the expression of ERRα significantly enhanced the process of autophagy and reduced CLP-induced ALI. Mechanistically, ERRα is essential to regulate the balance between autophagy and apoptosis to maintain the adherens junctional integrity.ConclusionERRα protects against sepsis-induced ALI through ERRα-mediated apoptosis and autophagy. Activation of ERRα provides a new therapeutic opportunity to prevent sepsis-induced ALI.

Sema3A inactivates the ERK/JNK signalling pathways to alleviate inflammation and oxidative stress in lipopolysaccharide-stimulated rat endothelial cells and lung tissues

Semaphorin 3A (Sema3A) is a secretory member of the semaphorin family of immune response regulators. This research focuses on its effects on inflammation and oxidative stress in acute respiratory distress syndrome (ARDS). By analysing the GEO dataset GSE57011, we obtained Sema3A as the most downregulated gene in ARDS samples. Lipopolysaccharide (LPS) was used to stimulate rat pulmonary microvascular endothelial cells (PMVECs) and rats to induce ARDS-like symptoms in vitro and in vivo, respectively. LPS induced severe damage in rat lung tissues, in which reduced immunohistochemical staining of Sema3A was detected. Sema3A overexpression reduced apoptosis and angiogenesis of LPS-induced PMVECs and alleviated lung injury and pulmonary edoema of rats. Moreover, ELISA results showed that Sema3A overexpression downregulated the levels of inflammatory cytokines and oxidative stress markers both in PMVECs and the rat lung. Activation of ERK/JNK signalling aggravated LPS-induced damage on PMVECs; however, the aggravation was partly blocked by Sema3A, which suppressed phosphorylation of ERK/JNK. Overall, this study demonstrates that Sema3A inactivates the ERK/JNK signalling to ameliorate inflammation and oxidative stress in LPS-induced ARDS models. Sema3A might therefore represent a candidate option for ARDS treatment.

Abstract 15215: Autophagy Inhibition Attenuate the Development and Progression of Pulmonary Hypertension

Introduction: Autophagy plays an important role in the pathogenesis of pulmonary hypertension. However, the potential therapeutic benefit of autophagy inhibition remains undetermined. Hypothesis: ROC-325 was developed as a novel small molecule lysosomal autophagy inhibitor that has demonstrated significantly more potent anticancer activity than the antimalarial drug hydroxychloroquine (HCQ). Methods: Rats with monocrotaline-induced PH (MCT-PH) and hypoxia/Sugen-induced severe PH were treated with ROC-325 and the related effects on echocardiography, hemodynamics, right ventricle hypertrophy, and pulmonary vascular remodeling were assessed. Pulmonary arterial pressure (PAP) and isometric tension were measured in isolated perfused/ventilated mouse lungs and rat PA rings. The in vitro effects on ROC-325 on autophagy, proliferation, viability, apoptosis and endothelial nitric oxide (NO) synthase (eNOS)-NO signaling were tested in human and rat pulmonary artery smooth muscle cells (PASMCs) as well as human pulmonary artery endothelial cells (PAECs) and rat pulmonary microvascular endothelial cells (PMVECs). Results and Conclusions: The therapeutic benefits of a novel lysosomal autophagy inhibitor, ROC-325, in experimental pulmonary hypertension (PH) models. Specifically, ROC-325 treatment prevents the development of monocrotaline (MCT)-induced PH, right ventricular dysfunction, and pulmonary vascular remodeling, and reverses the progression of established MCT-PH. Meanwhile, ROC-325 treatment attenuates high K + or alveolar hypoxia-induced pulmonary vasoconstriction. Mechanistically, in PASMC and PAEC, ROC-325 inhibits autophagic pathways, reduces hypoxia induced-hypoxia inducible factor (HIF) accumulation and blocks endothelial nitric oxide (NO) synthase (eNOS)-NO signaling activation. These studies illustrate the discovery of ROC-325 as a novel promising agent for PH therapy.

Protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells.

To investigate the protective effects of areca nut polyphenols on hypoxic damage of rat pulmonary microvascular endothelial cells (PMVECs). Malondialdehyde and superoxide dismutase (SOD) were used to determine the optimal modeling of lung hypoxic injury cells. CCK-8 method was used to detect cell viability for determining the effective dose of areca nut polyphenols. Rat PMVECs were divided into control group, hypoxia model group and areca nut polyphenols group. BCA method was used to detect the protein concentration of each group, and the oxidative stress level in PMVECs was measured. Western blotting was used to detect the expression of inflammatory and apoptosis-related proteins. Immunofluorescence staining was used to detect the expression of occludin and zonula occludens (ZO) 1. Transwell chamber was used to detect transendothelial electrical resistance, and rhodamine fluorescent dye was used to detect PMVECs barrier permeability. The hypobaric hypoxia-induced cell injury model was established by culturing PMVECs for 48 h at 1% oxygen concentration. The 20 μg/mL areca nut polyphenols significantly reversed the survival rate and the oxidative stress of PMVECs in hypoxia model group (all P<0.05). Areca nut polyphenols had significant inhibitory effect on the up-regulation of inflammation-related proteins, including nuclear factor-κB (NF-κB) and nuclear factor-E2-related factor (Nrf) 2 in hypoxia model group (all P<0.05). And areca nut polyphenols could reduce hypoxia-induced PMVECs apoptosis by down-regulating the expressions of apoptosis-related proteins, including cysteine aspartic acid specific protease (caspase) 3, Bcl-2 associated X protein (Bax) in PMVECs (all P<0.05). In addition, areca nut polyphenols effectively improves the transendothelial electrical resistance and barrier permeability of PMVECs through elevating the expression of occludin and ZO-1 (all P<0.05). Areca nut polyphenols can inhibit the hypoxic damage of PMVECs by reducing oxidative stress and apoptosis down-regulating the expression of inflammatory proteins and reducing membrane permeability.

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.

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.

ROCK inhibitor fasudil reduces the expression of inflammatory factors in LPS-induced rat pulmonary microvascular endothelial cells via ROS/NF-\u03baB pathway

BackgroundInflammation plays a major role in the pulmonary artery hypertension (PAH) and the acute lung injury (ALI) diseases. The common feature of these complications is the dysfunction of pulmonary microvascular endothelial cells (PMVECs). Fasudil, the only Rho kinase (ROCK) inhibitor used in clinic, has been proved to be the most promising new drug for the treatment of PAH, with some anti-inflammatory activity. Therefore, in the present study, the effect of fasudil on lipopolysaccharide (LPS)-induced inflammatory injury in rat PMVECs was investigated.MethodsLPS was used to make inflammatory injury model of rat PMVECs. Thereafter, the mRNA and protein expression of pro-inflammatory factors was evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) assay respectively. Intracellular reactive oxygen species (ROS) levels were measured by the confocal laser scanning system. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malondialdehyde (MDA) were determined by using commercial kits according to the manufacturer’s instructions. Western blot assay was used to detect the protein expression of nuclear factor kappa B (NF-κB) p65.ResultsFasudil effectively prevented inflammatory injury induced by LPS, which is manifested by the decrease of pro-inflammatory cytokines interleukin-6 (IL-6) and monocyte chenotactic protein-1 (MCP-1). Meanwhile, fasudil dramatically reduced the levels of ROS and MDA, and also elevated the activities of SOD and GSH-Px. Furthermore, the nuclear translocation of NF-κB p65 induced by LPS was also suppressed by fasudil. Additionally, the ROS scavengers N-Acetylcysteine (N-Ace) was also found to inhibit the nuclear translocation of NF-κB and the mRNA expression of IL-6 and MCP-1 induced by LPS, which suggested that ROS was essential for the nuclear translocation of NF-κB.ConclusionsThe present study revealed that fasudil reduced the expression of inflammatory factors, alleviated the inflammatory and oxidative damage induced by LPS in rat PMVECs via ROS-NF-κB signaling pathway.

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

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

Ferulic acid regulates miR-17/PTEN axis to inhibit LPS-induced pulmonary microvascular endothelial cells apoptosis through activation of PI3K/Akt pathway.

Acute lung injury (ALI) is mainly mediated by the damage of pulmonary microvascular endothelial cells (PMVECs). LPS is one of the pathogenic factors leading to microcirculatory abnormalities of ALI. Ferulic acid (FA) exhibits therapeutic effects against various diseases. During lipopolysaccharide-induced acute respiratory distress syndrome, FA, when given beforehand, could depress inflammation and oxidative stress. However, the concrete role and underlying mechanism of FA in ALI have not been well characterized. Ten μg/mL Lipopolysaccharide (LPS) was used to treat rat PMVECs for 24 hr. qRT-PCR was used to detect the level of miR-17 and phosphatase and tensin homolog deleted on chromosome ten (PTEN). Western blot was used to analyze the associated proteins in the PI3K/Akt pathway, and the apoptosis-related proteins. Flow cytometric analysis was performed to detect the apoptosis of PMVECs. MTT assay was constructed to detect the cell viability. Luciferase assay was conducted to detect the target gene of miR-17 and PTEN. A cell model for in vitro studying the role of FA in ALI was established using PMVECs. Our data demonstrate that FA up-regulates miR-17 and declines apoptosis induced by LPS. FA inhibits apoptosis mediated by up-regulating miR-17. Furthermore, we found miR-17 targeted PTEN negatively. FA inhibits cleaved caspase-3 and Bax expression through the PI3K/Akt pathway mediated by up-regulating miR-17. Over-expression of PTEN could contribute to the similar expression trend of the PI3K/Akt signal pathway protein compared to miR-17 inhibitor transfected cells. FA inhibits PMVECs apoptosis induced by LPS via miR-17/PTEN to further regulate the activation of the PI3K/Akt pathway in ALI. We anticipate that our data will provoke additional studies for ALI clinical therapy.

Adiponectin ameliorates lung ischemia\u2013reperfusion injury through SIRT1-PINK1 signaling-mediated mitophagy in type 2 diabetic rats

BackgroundDiabetes mellitus (DM) is a key contributing factor to poor survival in lung transplantation recipients. Mitochondrial dysfunction is recognized as a critical mediator in the pathogenesis of diabetic lung ischemia–reperfusion (IR) injury. The protective effects of adiponectin have been demonstrated in our previous study, but the underlying mechanism remains unclear. Here we demonstrated an important role of mitophagy in the protective effect of adiponectin during diabetic lung IR injury.MethodsHigh-fat diet-fed streptozotocin-induced type 2 diabetic rats were exposed to adiponectin with or without administration of the SIRT1 inhibitor EX527 following lung transplantation. To determine the mechanisms underlying the action of adiponectin, rat pulmonary microvascular endothelial cells were transfected with SIRT1 small-interfering RNA or PINK1 small-interfering RNA and then subjected to in vitro diabetic lung IR injury.ResultsMitophagy was impaired in diabetic lungs subjected to IR injury, which was accompanied by increased oxidative stress, inflammation, apoptosis, and mitochondrial dysfunction. Adiponectin induced mitophagy and attenuated subsequent diabetic lung IR injury by improving lung functional recovery, suppressing oxidative damage, diminishing inflammation, decreasing cell apoptosis, and preserving mitochondrial function. However, either administration of 3-methyladenine (3-MA), an autophagy antagonist or knockdown of PINK1 reduced the protective action of adiponectin. Furthermore, we demonstrated that APN affected PINK1 stabilization via the SIRT1 signaling pathway, and knockdown of SIRT1 suppressed PINK1 expression and compromised the protective effect of adiponectin.ConclusionThese data demonstrated that adiponectin attenuated reperfusion-induced oxidative stress, inflammation, apoptosis and mitochondrial dysfunction via activation of SIRT1- PINK1 signaling-mediated mitophagy in diabetic lung IR injury.

NF‑κB/IκBα signaling pathways are essential for resistance to heat stress‑induced ROS production in pulmonary microvascular endothelial cells.

The results of a previous study demonstrated that heat stress (HS) triggered oxidative stress, which in turn induced the apoptosis of epithelial cells. These results uncovered a novel mechanism underlying the activation of NF-κB in primary human umbilical vein endothelial cells. The present study aimed to further investigate the role of NF-κB/IκBα signaling pathways in the inhibition of HS-induced reactive oxygen species (ROS) generation and cytotoxicity in endothelial cells. The results of the present study demonstrated that HS triggered a significant amount of NF-κB and IκBα nuclear translocation without IκBα degradation in a time-dependent manner. Mutant constructs of IκBα phosphorylation sites (Ser32, Ser36) were employed in rat pulmonary microvascular endothelial cells (PMVECs). Cell Counting Kit-8 assays demonstrated that both the small interfering (si)RNA-mediated knockdown of p65 and IκBα mutant constructs significantly decreased cell viability and aggravated ROS accumulation in HS-induced rat PMVECs compared with the control. Additionally, western blot analysis revealed that p65 siRNA attenuated the protein expression of IκBα. However, IκBα mutant constructs failed to attenuate NF-κB activation and nuclear translocation, indicating that IκBα-independent pathways contributed to NF-κB activity and nucleus translocation in a time-dependent manner following HS. Collectively, the results of the present study suggested that the NF-κB/IκBα pathway was essential for resistance to HS-induced ROS production and cytotoxicity in rat PMVECs, and that it could be a potential therapeutic target to reduce the mortality and morbidity of heat stroke.

Engineering Functional Vasculature in Decellularized Lungs Depends on Comprehensive Endothelial Cell Tropism.

Tissue engineering using decellularized whole lungs as matrix scaffolds began as a promise for creating autologous transplantable lungs for patients with end-stage lung disease and can also be used to study strategies for lung regeneration. Vascularization remains a critical component for all solid organ bioengineering, yet there has been limited success in generating functional re-endothelialization of most pulmonary vascular segments. We evaluated recellularization of the blood vessel conduits of acellular mouse scaffolds with highly proliferating, rat pulmonary microvascular endothelial progenitor cells (RMEPCs), pulmonary arterial endothelial cells (PAECs) or microvascular endothelial cells (MVECs). After 8 days of pulsatile perfusion, histological analysis showed that PAECs and MVECs possessed selective tropism for larger vessels or microvasculature, respectively. In contrast, RMEPCs lacked site preference and repopulated all vascular segments. RMEPC-derived endothelium exhibited thrombomodulin activity, expression of junctional genes, ability to synthesize endothelial signaling molecules, and formation of a restrictive barrier. The RMEPC phenotype described here could be useful for identifying endothelial progenitors suitable for efficient vascular organ and tissue engineering, regeneration and repair.

Heat shock protein 70 attenuates hypoxia‑induced apoptosis of pulmonary microvascular endothelialcells isolated from neonatal rats.

Pulmonary microvascular endothelial cell (PMVEC) apoptosis is the initial stage of adult pulmonary hypertension (PH), which involves high pulmonary arterial pressure and pulmonary vascular remodeling. However, the mechanism regulating PMVEC apoptosis and its involvement in the early stages of neonatal hypoxic PH (HPH) pathogenesis are currently unclear. The present study aimed to investigate the effects of heat shock protein 70 (HSP70) on hypoxia-induced apoptosis in PMVECs. PMVECs isolated from neonatal Sprague-Dawley rats were transfected with lentivirus with or without HSP70, or treated with the synthetic HSP70 inhibitor N-formyl-3,4-methylenedioxy-benzylidene-g-butyrolactam under hypoxic conditions (5% O2) for 24, 48 or 72 h. PMVEC apoptosis was evaluated by performing flow cytometry and mitochondrial membrane potential (MMP) assays. The expression levels of HSP70, hypoxia-inducible factor-1α (HIF-1α) and apoptosis-associated proteins were determined by conducting reverse transcription-quantitative PCR and western blotting. Following 24, 48 or 72 h of hypoxia, the apoptotic rates of PMVECs were significantly elevated compared with cells under normoxic conditions. The MMP was significantly reduced, whereas the mRNA and protein expression levels of HIF-1α, cytochrome c (cyt C), caspase-3 and HSP70 were enhanced by hypoxia compared with those under normoxic conditions. Additionally, the mRNA and protein expression levels of B-cell lymphoma 2 (Bcl-2) were significantly downregulated in the hypoxia group compared with those in the normoxia group. In hypoxic PMVECs, HSP70 overexpression decreased the apoptotic rate and the expression levels of cyt C, downregulated the expression levels of caspase-3 and HIF-1α, and increased the MMP and the expression levels of Bcl-2. HSP70 inhibition resulted in the opposite outcomes compared with those of HSP70 overexpression. Therefore, the results of the present study suggested that HSP70 may inhibit mitochondrial pathway-mediated apoptosis in isolated neonatal rat PMVECs in early-stage hypoxia, which may be associated with HSP70-mediated HIF-1α downregulation. Overall, HSP70 may be protective against neonatal HPH through the HSP70/HIF-1α pathway.

Effect of the Notch4/Dll4 signaling pathway in early gestational intrauterine infection on lung development.

Intrauterine infection is an important risk factor for bronchopulmonary dysplasia (BPD). BPD is characterized by arrested lung alveolarization and impaired pulmonary vascularization. The Notch4 signaling pathway is a key regulator of vascular remodeling and angiogenesis. Therefore, the presents study investigated the expression of Notch4, delta-like canonical Notch ligand 4 (Dll4) and related factors in an in vivo rat model and in rat pulmonary microvascular endothelial cells (PMVECs) in vitro, to study the mechanisms by which intrauterine infection affects rat lung development. A rat model of intrauterine infection was established by endocervical inoculation with Escherichia scoli on embryonic day 15. The date of birth was counted as postnatal day 0 (P0). Then, the lung tissues were collected from pups at days P3-P14. The expression of Notch4, Dll4 and related factors was measured by reverse transcription-quantitative PCR and western blotting. In addition, the γ-secretase inhibitor DAPT was used to examine the effect of Notch4 signaling on PMVECs. Intrauterine E. coli infection impaired normal lung development, as indicated by decreased microvessel density, fewer alveoli, fewer secondary septa, and larger alveoli compared with the control group. Furthermore, Notch4, Dll4 and NF-κB levels were significantly increased in the E. coli-infected group at P3 compared with the control group. Similarly, the mRNA expression levels of fetal liver kinase 1 (Flk-1, a VEGF receptor) were significantly increased in the E. coli-infected group at P3 and P7. In PMVECs, the inhibition of Notch4 signaling contributed to decreases in lipopolysaccharide (LPS)-induced expression of VEGF and its receptors. Furthermore, the inhibition of Notch4/Dll4 signaling accelerated cell proliferation and decreased the apoptosis rate of LPS-induced PMVECs. LPS-induced NF-κB expression in PMVECs was also attenuated by the Notch4/Dll4 inhibitor. In conclusion, intrauterine E. coli infection impaired normal lung development, possibly through Notch4/Dll4 signaling and effects on VEGF and its receptors.

Actin-binding protein, IQGAP1, regulates LPS-induced RPMVECs hyperpermeability and ICAM-1 upregulation via Rap1/Src signalling pathway

Pulmonary microvascular barrier dysfunction is a hallmark feature of acute lung injury (ALI). IQGAP1 is a ubiquitously expressed scaffolding protein known to regulate cancer metastasis, angiogenesis, and barrier stability. However, the function of IQGAP1 in lipopolysaccharide (LPS)-induced microvascular endothelial hyperpermeability remains poorly understood. In the present study, we demonstrated that IQGAP1 was markedly upregulated in LPS-induced ALI models and rat pulmonary microvascular endothelial cells (RPMVECs). Lentivirus-mediated knockdown of IQGAP1 significantly attenuated the formation of actin stress fibers, phosphorylation of myosin light chain (MLC), and disruption of VE-cadherin, thereby protecting the RPMVECs barrier failure from LPS damage. In addition, IQGAP1 depletion reduced the reactive oxygen species (ROS)-mediated increase in intracellular adhesion molecule-1 (ICAM-1) in RPMVECs stimulated with LPS. Mechanistically, we found that the upregulation of IQGAP1 affected the activity of Rap1 and the downstream phosphorylation of Src. In conclusion, these findings reveal an essential mechanism by which increased IQGAP1 in LPS-treated RPMVECs promotes barrier dysfunction and ICAM-1 upregulation, at least in part by regulating Rap1/Src signalling, indicating that IQGAP1 may be a potential therapeutic target to prevent endothelial hyperpermeability and inflammation in ALI.

Inflammatory Immune Cytokine TNF-α Modulates Ezrin Protein Activation via FAK/RhoA Signaling Pathway in PMVECs Hyperpermeability

Background: One of the important pathogenesis of acute respiratory distress syndrome (ARDS) is the dysfunction of pulmonary microvascular endothelial barrier induced by a hyperinflammatory immune response. However, the potential mechanisms of such an imbalance in pulmonary microvascular endothelial cells (PMVECs) are not yet understood. Purpose: Explore the molecular mechanism of endothelial barrier dysfunction induced by inflammatory immune cytokines in ARDS, and find a therapeutic target for this syndrome. Methods: Rat PMVECs were cultured to form a monolayer. Immunofluorescence, flow cytometry, and Western blotting were selected to detect the distribution and the expression level of phosphorylated Ezrin protein and Ezrin protein. Transendothelial electrical resistance (TER) and transendothelial fluxes of fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (BSA) were utilized to measure the permeability of the cell monolayer. Ezrin short hairpin RNA (shRNA) and Ezrin 567-site threonine mutant (EzrinT567A) were used to examine the role of Ezrin protein and phosphorylated Ezrin protein in endothelial response induced by tumor necrosis factor-alpha (TNF-α), respectively. The function of focal adhesion kinase (FAK) and Ras homolog gene family, member A (RhoA) signaling pathways were estimated by inhibitors and RhoA/FAK shRNA in TNF-α-stimulated rat PMVECs. The activation of FAK and RhoA was assessed by Western blotting or pull-down assay plus Western blotting. Results: The TER was decreased after TNF-α treatment, while the Ezrin protein phosphorylation was increased in a time- and dose-dependent manner. The phosphorylated Ezrin protein was localized primarily at the cell periphery, resulting in filamentous actin (F-actin) rearrangement, followed by a significant decrease in TER and increase in fluxes of FITC-BSA. Moreover, FAK and RhoA signaling pathways were required in the phosphorylation of Ezrin protein, and the former positively regulated the latter. Conclusion: The phosphorylated Ezrin protein was induced by TNF-α via the FAK/RhoA signaling pathway leading to endothelial hyperpermeability in PMVECs.