Novel HIF-1-target gene isthmin1 contributes to hypoxia-induced hyperpermeability of pulmonary microvascular endothelial cells monolayers

Hypoxia-induced pulmonary microvascular endothelial cell (PMVEC) monolayers hyperpermeability is vital for vascular leakage, which participates in vascular diseases, such as acute lung injury (ALI) and high altitude pulmonary edema (HAPE). We previously observed PMVEC permeability was markedly elevated in hypoxia when cocultured with primary type II alveolar epithelial cells (AECII) in which isthmin1(ISM1) was highly upregulated. However, whether the upregulation of ISM1 plays a role in hypoxia-induced PMVEC hyperpermeability is unclear. In this study, we assessed the role of AECII-derived ISM1 in hypoxia-induced PMVEC hyperpermeability with an AECII/PMVEC co-culture system and uncovered the underlying mechanism whereby hypoxia stimulates ISM1 gene expression. We found that ISM1 gene expression was upregulated in cultured AECII cells exposed to hypoxia (3% O2), and that AECII-derived ISM1 participated in hypoxia-induced hyperpermeability of PMVEC monolayers since siRNA-mediated knockdown of ISM1 in AECII markedly attenuated the increasement of PMVEC permeability in co-culture system under hypoxia. Additionally, we confirmed that ISM1 was regulated by hypoxia-inducible factor-1α (HIF1α) according to the evidence that silencing of HIF1α inhibited the hypoxia-mediated upregulation of ISM1. Mechanismly, overexpression of HIF1α transcriptionally activated ISM1 gene expression by directly binding to the conserved regulatory elements upstream of the ism1 locus. We identified a novel HIF-1-target gene ISM1, which involves in hyperpermeability of pulmonary microvascular endothelial cell monolayers under hypoxia. Our in vitro cell experiments implied that the upregulated ISM1 derived from alveolar epithelium might be a vital modulator in hypoxia-induced endothelial hyperpermeability and thereby implicates with hypoxic pulmonary-related diseases.

Polymer Optical Waveguide Grating-Based Biosensor to Detect Effective Drug Concentrations of Ginkgolide A for Inhibition of PMVEC Apoptosis

In this work, we successfully developed a fluorinated cross-linked polymer Bragg waveguide grating-based optical biosensor to detect effective drug concentrations of ginkgolide A for the inhibition of pulmonary microvascular endothelial cell (PMVEC) apoptosis. Fluorinated photosensitive polymer SU-8 (FSU-8) as the sensing core layer and polymethyl methacrylate (PMMA) as the sensing window cladding were synthesized. The effective drug concentration range (5–10 µg/mL) of ginkgolide A for inhibition of PMVEC apoptosis was analyzed and obtained by pharmacological studies. The structure of the device was optimized to be designed and fabricated by direct UV writing technology. The properties of the biosensor were simulated with various refractive indices of different drug concentrations. The actual sensitivity of the biosensor was measured as 1606.2 nm/RIU. The resolution and detection limit were characterized as 0.05 nm and 3 × 10−5 RIU, respectively. The technique is suitable for safe and accurate detection of effective organic drug dosages of Chinese herbal ingredients.

Targeting MALAT1 and miRNA-181a-5p for the intervention of acute lung injury/acute respiratory distress syndrome

Background ALI/ARDS is a severe lung injury leading to refractory respiratory failure, accounting for high morbidity and mortality. However, therapeutic approaches are rather limited. Targeting long non-coding RNA MALAT1 and microRNA miR-181a-5p might be potential option for ALI/ARDS intervention. Objective We aimed to investigate the role of MALAT and miR-181a-5p in the pathogenesis of ALI/ARDS, and test the therapeutic effects of targeting MALAT and miR-181a-5p for ALI/ARDS intervention in vitro. Methods MALAT1 and miR-181a-5p levels were measured in plasma from ALI/ARDS patients. In vitro human pulmonary microvascular endothelial cell (HPMEC) injury was induced by LPS treatment, and molecular targets of MALAT1 and miR-181a-5p were explored by molecular biology approaches, mainly focusing on cell apoptosis and vascular inflammation. Interaction between MALAT1 and miR-181a-5p was also detected. Finally, the effects of targeting MALAT1 and miR-181a-5p for ALI/ARDS intervention were validated in a rat ALI/ARDS model. Results MALAT1 upregulation and miR-181a-5p downregulation were observed in ALI/ARDS patients. Transfection of mimic miR-181a-5p into HPMECs revealed decreased Fas and apoptosis, along with reduced inflammatory factors. Fas was proved to be a direct target of miR-181a-5p. Similar effects were also present upon MALAT1 knockdown. As for the interaction between MALAT1 and miR-181a-5p, MALAT1 knockdown increased miR-181a-5p expression. Knocking down of MALAT1 and miR-181a-5p could both improve the outcome in ALI/ARDS rats. Conclusion MALAT1 antagonism or miR-181a-5p could both be potential therapeutic strategies for ALI/ARDS. Mechanistically, miR-181a-5p directly inhibits Fas and apoptosis, along with reduced inflammation. MALAT1 negatively regulates miR-181a-5p.

MiR-375-3p regulates rat pulmonary microvascular endothelial cell activity by targeting Notch1 during hypoxia

Objective Pulmonary microvascular endothelial cells (PMECs) exhibit specific responses in adaptation to hypoxia. However, the mechanisms regulating PMEC activities during hypoxia remain unclear. This study investigated the potential involvement of a microRNA, miR-375-3p, in the regulation of PMEC activities. Methods Primary PMECs were isolated from rats. The expression levels of miR-375-3p and Notch1 in the PMECs were detected by quantitative PCR and western blotting. Luciferase reporter assays were performed to explore the transcriptional regulation of Notch1 by miR-375-3p. The proliferation and chemotaxis of the PMECs were measured with the Cell Counting Kit-8 and Transwell invasion assays, respectively. Additionally, the capacity of hypoxia-treated PMECs for angiogenesis and inflammatory response was determined with tube formation assays and ELISA, respectively. Results The expression of miR-375-3p and Notch1 in the PMECs was significantly down-regulated and up-regulated during hypoxia, respectively. The results demonstrated that miR-375-3p directly targets Notch1 in PMECs, thereby suppressing the transcriptional expression of Notch1. It was further revealed that miR-375-3p regulates the proliferation, chemotaxis, angiogenesis, and inflammatory response of PMECs. Conclusions Our findings revealed the important role of miR-375-3p in the regulation of PMEC function and suggest the potential involvement of miR-375-3p in the development of lung diseases.