The Interplay between Aquaporin-1 and the Hypoxia-Inducible Factor 1α in a Lipopolysaccharide-Induced Lung Injury Model in Human Pulmonary Microvascular Endothelial Cells.

Aquaporin-1 (AQP1), a water channel, and the hypoxia-inducible factor 1α (HIF1A) are implicated in acute lung injury responses, modulating among others pulmonary vascular leakage. We hypothesized that the AQP1 and HIF1A systems interact, affecting mRNA, protein levels and function of AQP1 in human pulmonary microvascular endothelial cells (HPMECs) exposed to lipopolysaccharide (LPS). Moreover, the role of AQP1 in apoptosis and wound healing progression was examined. Both AQP1 mRNA and protein expression levels were higher in HPMECs exposed to LPS compared to untreated HPMECs. However, in the LPS-exposed HIF1A-silenced cells, the mRNA and protein expression levels of AQP1 remained unaltered. In the permeability experiments, a statistically significant volume increase was observed at the 360 s time-point in the LPS-exposed HPMECs, while LPS-exposed HIF1A-silenced HPMECs did not exhibit cell swelling, implying a dysfunctional AQP1. AQP1 did not seem to affect cell apoptosis yet could interfere with endothelial migration and/or proliferation. Based on our results, it seems that HIF1A silencing negatively affects AQP1 mRNA and protein expression, as well as AQP1 function, in the setting of lung injury.

PEAR1 suppresses the proliferation of pulmonary microvascular endothelial cells via PI3K/AKT pathway in ALI model.

BACKGROUND: Activation of the proliferation of pulmonary microvascular endothelial cells (PMVECs) is a key step in the recovery of the integrity of endothelial monolayer, which helps to alleviate acute lung injury (ALI). Platelet endothelial aggregation receptor-1 (PEAR1), expressed on endothelial cells, was reported to inhibit the proliferation of vascular endothelial cells and angiogenesis. However, little is known about its role and mechanism in vascular endothelial disorders in ALI. OBJECTIVE: The aim of this study was to investigate the impact of PEAR1 on the proliferation of pulmonary microvascular endothelial cells in ALI. METHODS: We tested the expression level of PEAR1 in the lungs of WT mice in ALI model induced by intestinal IR. Primary human pulmonary microvascular endothelial cells (HPMECs) were stimulated by 1 mg/L LPS in vitro. We synthesized siPEAR1 and Flag-PEAR1 plasmid to verify the role of PEAR1 on regulating the proliferation of HPMECs under LPS condition and to explore related signaling pathways. RESULTS: The expression level of PEAR1 significantly increased in ALI induced by intestinal IR. PEAR1 knockdown enhanced the proliferation level of HPMECs, which, however, was inhibited by PEAR1 overexpression. PEAR1 knockdown activated PI3K/AKT pathway both in steady state and under LPS condition. PI3K inhibitor, LY294002, reversed the increasing proliferation level and cell progression of HPMECs induced by PEAR1 knockdown after LPS challenge. CONCLUSIONS: PEAR1 acts as a negative regulator in the proliferation of HPMECs in ALI model via the PI3K/AKT pathway.

Knockdown of bone morphogenetic protein type II receptor leads to decreased aquaporin 1 expression and function in human pulmonary microvascular endothelial cells.

Bone morphogenetic proteins (BMPs) were once considered only to have a role in bone formation. It is now known that they have pivotal roles in other organ diseases, including heritable pulmonary arterial hypertension (PAH), where genetic mutations in the type II BMP receptor (BMPR2) are the commonest cause of receptor dysfunction. However, it has also recently been demonstrated that aquaporin 1 (Aqp1) dysfunction may contribute to PAH, highlighting that PAH development may involve more than one pathogenic pathway. Whether reduction in BMPR2 affects Aqp1 is unknown. We therefore studied Aqp1 in BMPR2-silenced human pulmonary microvascular endothelial cells (HPMECs). We demonstrated reduced Aqp1 mRNA, protein, and function in the BMPR2-silenced cells. Additionally, BMPR2-silenced cells exhibited lower expression of BMP-signaling molecules. In conclusion, decreased BMPR2 appears to affect Aqp1 at the mRNA, protein, and functional levels. This observation may identify a contributory mechanism for PAH.

Molecular Analysis of Fetal and Adult Primary Human Liver Sinusoidal Endothelial Cells: A Comparison to Other Endothelial Cells.

In humans, Factor VIII (F8) deficiency leads to hemophilia A and F8 is largely synthesized and secreted by the liver sinusoidal endothelial cells (LSECs). However, the specificity and characteristics of these cells in comparison to other endothelial cells is not well known. In this study, we performed genome wide expression and CpG methylation profiling of fetal and adult human primary LSECs together with other fetal primary endothelial cells from lung (micro-vascular and arterial), and heart (micro-vascular). Our results reveal expression and methylation markers distinguishing LSECs at both fetal and adult stages. Differential gene expression of fetal LSECs in comparison to other fetal endothelial cells pointed to several differentially regulated pathways and biofunctions in fetal LSECs. We used targeted bisulfite resequencing to confirm selected top differentially methylated regions. We further designed an assay where we used the selected methylation markers to test the degree of similarity of in-house iPS generated vascular endothelial cells to primary LSECs; a higher similarity was found to fetal than to adult LSECs. In this study, we provide a detailed molecular profile of LSECs and a guide to testing the effectiveness of production of in vitro differentiated LSECs.

The MK2/HuR signaling pathway regulates TNF-α-induced ICAM-1 expression by promoting the stabilization of ICAM-1 mRNA.

BACKGROUND: Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by acute lung inflammation. Intercellular adhesion molecule-1 (ICAM-1) and interleukin-8 (IL-8) play an important role in the development of these diseases. Mitogen-activated protein kinase (MAPK) p38/activated protein kinase 2 (MK2) regulates the expression of ICAM-1 and IL-8 in human lung microvascular endothelial cells (HPMECs) stimulated by tumor necrosis factor-α (TNF-α); however, the underlying molecular mechanism remains unclear. Here, we show that human antigen R (HuR), an RNA binding protein which binds preferentially to AU-rich elements (AREs) and stabilizes mRNAs, regulates TNF-α-induced ICAM-1 expression in the MK2/HuR signaling pathway. METHOD: MK2 and HuR were silenced respectively in HPMECs and then HPMECs were stimulatied with TNF-α. Nucleo-cytoplasmic shuttling of HuR was detected by subcellular fractionation and confocal microscopy in MK2 knockdown HPMECs. In HuR silencing cells, protein and mRNA levels of ICAM-1 and IL-8 were measured by western blot analysis, ELISA and real-time PCR; mRNA stabilization were measured by real-time PCR after actinomycin D (ActD) blocking transcription. Furthermore, we performed neutrophil adhesion assay to assess the adhering capacity after HuR silencing. RESULTS: MK2 were subjected to a knockdown by interfering RNA, the mRNA and protein levels of HuR in human pulmonary microvascular endothelial cells (HPMECs) were not affected. However, after the stimulation of TNF-α, silencing MK2 inhibited HuR accumulation to cytoplasm from nucleus in HPMECs. Consequently, knockdown of HuR by RNA interference in HPMECs, there was reduction in the stability of ICAM-1 mRNA and ICAM-1 protein level. This event was accompanied by a decrease in the adhesion of neutrophils towards HPMECs. Nevertheless, HuR silencing had no effect on the mRNA and protein levels of IL-8. CONCLUSION: These results indicate that MK2 post-transcriptionally regulates TNF-α-induced ICAM-1 expression by altering the cytoplasmic localization of HuR in HPMECs.

Adrenomedullin deficiency potentiates hyperoxic injury in fetal human pulmonary microvascular endothelial cells.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease of premature infants that is characterized by alveolar simplification and decreased lung angiogenesis. Hyperoxia-induced oxidative stress and inflammation contributes to the development of BPD in premature infants. Adrenomedullin (AM) is an endogenous peptide with potent angiogenic, anti-oxidant, and anti-inflammatory properties. Whether AM regulates hyperoxic injury in fetal primary human lung cells is unknown. Therefore, we tested the hypothesis that AM-deficient fetal primary human pulmonary microvascular endothelial cells (HPMEC) will have increased oxidative stress, inflammation, and cytotoxicity compared to AM-sufficient HPMEC upon exposure to hyperoxia. Adrenomedullin gene (Adm) was knocked down in HPMEC by siRNA-mediated transfection and the resultant AM-sufficient and -deficient cells were evaluated for hyperoxia-induced oxidative stress, inflammation, cytotoxicity, and Akt activation. AM-deficient HPMEC had significantly increased hyperoxia-induced reactive oxygen species (ROS) generation and cytotoxicity compared to AM-sufficient HPMEC. Additionally, AM-deficient cell culture supernatants had increased macrophage inflammatory protein 1α and 1ß, indicating a heightened inflammatory state. Interestingly, AM deficiency was associated with an abrogated Akt activation upon exposure to hyperoxia. These findings support the hypothesis that AM deficiency potentiates hyperoxic injury in primary human fetal HPMEC via mechanisms entailing Akt activation.

RU486 Metabolite Inhibits CCN1/Cyr61 Secretion by MDA-MB-231-Endothelial Adhesion.

Successful adhesion of circulating tumor cells (CTCs) to microvascular endothelium of distant metastatic tissue is the key starting step of metastatic cascade that could be effectively chemoprevented as we demonstrated previously. Here, we hypothesize that the hetero-adhesion may produce secretory biomarkers that may be important for both premetastatic diagnosis and chemoprevention. We show that co-incubation of triple-negative breast cancer (TNBC) cell line MDA-MB-231 with human pulmonary microvascular endothelial monolayers (HPMEC) secretes Cyr61 (CCN1), primarily from MDA-MB-231. However, addition of metapristone (RU486 metabolite) to the co-incubation system inhibits Cyr61 secretion probably via the Cyr61/integrin αvß1 signaling pathway without significant cytotoxicity on both MDA-MB-231 and HPMEC. Transfection of MDA-MB-231 with Cyr61-related recombinant plasmid or siRNA enhances or reduces Cyr61 expression, accordingly. The transfection significantly changes hetero-adhesion and migration of MDA-MB-231, and the changed bioactivities by overexpressed CYR61 could be antagonized by metapristone in vitro. Moreover, the circulating MDA-MB-231 develops lung metastasis in mice, which could be effectively prevented by oral metapristone without significant toxicity. The present study, for the first time, demonstrates that co-incubation of MDA-MB-231 with HPMEC secrets CYR61 probably via the CYR61/integrin αvß1 signaling pathway to promote adhesion-invasion of TNBC (early metastatic step). Metapristone, by interfering the adhesion-invasion process, prevents metastasis from happening.

Different involvement of the MAPK family in inflammatory regulation in human pulmonary microvascular endothelial cells stimulated with LPS and IFN-γ.

Pulmonary endothelial injury is central in the pathogenesis of acute lung injury (ALI). The MAPK signaling cascades are generally thought to be involved in the molecular mechanism underlying the ALI development, but their roles in pulmonary endothelial injury is poorly understood. We thus examined the involvement of the MAPK family member in inflammatory responses of human pulmonary microvascular endothelial cells (HPMVECs) stimulated with LPS and IFN-γ. HPMVECs were found to exhibit the upregulation of expression of Toll-like receptor 4 by IFN-γ, resulting in potentiation of inflammatory cytokine release by LPS stimulation. All MAPKs, ERK1/2, JNK, and p38, were activated by simultaneous stimulation with LPS/IFN-γ. JNK activation in cells stimulated with LPS/IFN-γ was significantly potentiated by the two different p38 inhibitors, SB203580 and RWJ67657, suggesting the negative regulation of JNK activation by p38 in HPMVECs. The mRNA and protein expression levels of ICAM-1 were eliminated by the JNK inhibitor, suggesting that ICAM-1 expression is positively regulated by JNK. The p38 inhibitor significantly enhanced ICAM-1 expression. ERK1/2 activation was not responsible for the LPS/IFN-γ-induced ICAM-1 upregulation in HPMVECs. THP-1 monocyte adhesion to HPMVECs under LPS/IFN-γ stimulation was inhibited by the JNK inhibitor and enhanced by the p38 inhibitor. We conclude that, in HPMVECs stimulated with LPS/IFN-γ, JNK mediates ICAM-1 expression that can facilitate leukocyte adherence and transmigration, while p38 MAPK negatively regulates the upregulation of ICAM-1 through inhibition of JNK activation.

The pivotal role of HIF-1α in lung inflammatory injury induced by septic mesenteric lymph.

Hypoxia inducible factor-1α (HIF-1α) plays an essential role in hypoxia and inflammatory response. Oxygen metabolic dysfunction and cascade amplification of inflammatory response are prominent pathophysiological characteristics in sepsis induced acute lung injury (ALI). In this study, we started with septic mesenteric lymph injection model to investigate whether HIF-1α played a role in the pathogenesis of ALI induced by septic lymph. The data demonstrated that rats injected with septic lymph showed a significant higher Lung Injury Scale and MPO(myeloperoxidase) levels than that of rats injected with normal saline/lymph. ALI was associated with a higher degree of HIF-1α expression in the lungs infused by septic lymph. Intratracheal delivery of YC-1(3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole) significantly attenuated lung inflammatory damages. Furthermore, in vitro studies, human alveolar type II epithelial cell (A549)/human pulmonary microvascular endothelial cell (HPMEC) incubated by septic lymph showed dramatically decreased cell viability, higher levels of inflammatory cytokines (TNF-α, IL-6 and IL-1ß) and excitation of HIF-1α expression (Immunofluorescence localization/RT-PCR test) simultaneously. Nevertheless, compared with the non-silencing cell lines, A549/HPMEC with HIF-1α gene silencing manifested increased viability and restrained cytokines' expression after incubation with septic lymph. These results indicate that HIF-1α expression can be induced and activated in rats during the acute lung inflammatory damages triggered by septic lymph injection and that lung inflammatory injuries occur via a HIF-1α-dependent pathway.

Role of GDF15 (growth and differentiation factor 15) in pulmonary oxygen toxicity.

GDF15 (growth and differentiation factor 15) is a secreted cytokine, a direct target of p53 and plays a role in cell proliferation and apoptosis. It is induced by oxidative stress and has anti-apoptotic effects. The role of GDF15 in hyperoxic lung injury is unknown. We tested the hypothesis that GDF15 will be induced in vitro, in a model of pulmonary oxygen toxicity, and will play a critical role in decreasing cell death and oxidative stress. BEAS-2B (human bronchial epithelial cells) and human pulmonary vascular endothelial cells (HPMEC) were exposed to hyperoxia, and expression of GDF15 and effect of GDF15 disruption on cell viability and oxidative stress was determined. Furthermore, we studied the effect of p53 knockdown on GDF15 expression. In vitro, both BEAS-2B and HPMEC cells showed a significant increase in GDF15 expression upon exposure to hyperoxia. After GDF15 knockdown, there was a significant decrease in cell viability and increase in oxidative stress compared to control cells transfected with siRNA with a scrambled sequence. Knockdown of p53 significantly decreased the induction of GDF15 by hyperoxia. In conclusion, we show that GDF15 has a pro-survival and anti-oxidant role in hyperoxia and that p53 plays a key role in its induction.

Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles.

As the lung is one of the main routes of exposure to manufactured nanoparticles, we developed an in vitro model resembling the alveolo-capillary barrier for the study of nanoparticle translocation. In order to provide a relevant and ethical in vitro model, cost effective and easy-to-implement human cell lines were used. Pulmonary epithelial cells (Calu-3 cell line) and macrophages (THP-1 differentiated cells) were cultivated on the apical side and pulmonary endothelial cells (HPMEC-ST1.6R cell line) on the basal side of a microporous polyester membrane (Transwell®). Translocation of non-functionalized (51 and 110 nm) and aminated (52 nm) fluorescent polystyrene (PS) nanobeads was studied in this system. The use of Calu-3 cells allowed high transepithelial electrical resistance (TEER) values (>1000 Ω cm2) in co-cultures with or without macrophages. After 24 h of exposure to non-cytotoxic concentrations of non-functionalized PS nanobeads, the relative TEER values (%/t0) were significantly decreased in co-cultures. Epithelial cells and macrophages were able to internalize PS nanobeads. Regarding translocation, Transwell® membranes per se limit the passage of nanoparticles between apical and basal side. However, small non-functionalized PS nanobeads (51 nm) were able to translocate as they were detected in the basal side of co-cultures. Altogether, these results show that this co-culture model present good barrier properties allowing the study of nanoparticle translocation but research effort need to be done to improve the neutrality of the porous membrane delimitating apical and basal sides of the model.