Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: AMs aggravate LPS-induced injury in PMVECs via M1 polarization-mediated inflammatory response. (A) PMVECs were cultured in the presence of AMs or alone, followed by 100 ng/mL LPS treatment. Equivalent PBS was used as the control. After 24 h of culture, (B) PMVEC permeability was assessed using HRP leakage assays. (C) Western blot analysis revealed the relative expressions of both Claudin-5 and ZO-1 proteins, with GAPDH as the internal reference. (D) Annexin V-FITC/PI staining and (E) TUNEL assays demonstrated the apoptosis rate of PMVECs. (E) ELISA was used for inflammatory cytokines (F) IL-6 and (G) TNF-α concentration determination. (H) The western blot analysis for iNOS and Arg-1 represented the level of M1 polarization. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, *** p < 0.001. PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Mouse pulmonary microvascular endothelial cells (PMVECs; CP-M001) were purchased from Procell Life Science (Wuhan, China) and maintained in endothelial cell growth medium (Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (p/s).

Techniques: Cell Culture, Control, Permeability, Western Blot, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Biology Direct

Article Title: Enhanced itaconic acid secretion from macrophages mediates the protection of mesenchymal stem cell-derived exosomes on lipopolysaccharide-induced acute lung injury mice

doi: 10.1186/s13062-024-00586-8

Figure Lengend Snippet: MSC-exos attenuate LPS-induced PMVEC injury by promoting M2 polarization of AMs. MSC-exos were added to the LPS-treated co-culture system to evaluate their effects on PMVECs. (A) PMVEC permeability was assessed using HRP leakage assays. (B) Western blot analysis was performed to detect the expression levels of tight junction proteins Claudin-5 and ZO-1. (C) Annexin V-FITC/PI staining and (D) TUNEL assays demonstrated the apoptosis rate of PMVECs to determine the anti-apoptosis effect of MSC-exos. (E-F) ELISA for inflammatory cytokines IL-6 and TNF-α in cell supernatant. (G) Western blot analysis for M1 polarization markers assessed the relative levels of iNOS and Arg-1 proteins. (H) M2 polarization was determined via flow cytometry by calculate the percentage of CD206-positive cells. (I) Gas chromatography-mass spectrometry was used to measure ITA concentration. Data are presented as mean ± SEM. Statistical significance was determined using appropriate methods, with * p < 0.05, ** p < 0.01, and *** p < 0.001. MSC-exos: mesenchymal stem cell-derived exosome; PMVECs: pulmonary microvascular endothelial cells; AMs: alveolar macrophages; HRP: horseradish peroxidase

Article Snippet: Mouse pulmonary microvascular endothelial cells (PMVECs; CP-M001) were purchased from Procell Life Science (Wuhan, China) and maintained in endothelial cell growth medium (Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (p/s).

Techniques: Co-Culture Assay, Permeability, Western Blot, Expressing, Staining, TUNEL Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Gas Chromatography, Mass Spectrometry, Concentration Assay, Derivative Assay

Journal: Biomolecules

Article Title: Iron Replacement Attenuates Hypoxic Pulmonary Hypertension by Remodeling Energy Metabolism via Regulating the HIF2α/Mitochondrial Complex I, III/ROS Axis

doi: 10.3390/biom15050742

Figure Lengend Snippet: HIF2α inhibitors improve the activity and expression of mitochondrial complexes I and III, reduce mitochondrial ROS production, and attenuate mitochondrial respiratory depression of PAECs under hypoxia. ( A ) Representative transmission electron microscopy images of endothelial cells from mouse pulmonary arteries with PT2385 intervention (Red arrows indicate swollen mitochondria; scale bars: 500 nm (upper panel), 200 nm (lower panel). ( B ) Summary of data on the activity of mitochondrial complexes I and III in RPAECs. ( C , D ) The representative images of Western blot from mice lung tissues and RPAECs. ( E ) Representative immunofluorescence images and data of MitoSOX in RPAECs (scale bars: 50 μm). ( F ) Summary of data on the intracellular ROS. NC: Normoxia control group; NP: Normoxia combined with PT2385 group; HC: Hypoxia control group; HP: Hypoxia combined with PT2385 group; HCP: Hypoxia combined with PT2385 group (animal model); NIP: PT2385 intervention for 1 week after 4-week hypoxia exposure (animal model); LDH: Lactic dehydrogenase; GLUT1: Glucose transporter 1; ISCU: Iron/sulfur cluster assembly enzyme; MitoSOX: Mitochondrial superoxide; ROS: Reactive oxygen species. The original images of Western Blot can be found in the . In the statistical graph, each point represents the number of experimental animals or repetitions. The data are presented as mean ± SD; ns: No statistical significance; * p < 0.05; ** p < 0.01; *** p < 0.0001.

Article Snippet: Rat pulmonary artery endothelial cells (RPAECs, CD31 fluorescence identification is reported in ) and human pulmonary artery endothelial cells (HPAECs, the short tandem repeat (STR) assay report is shown in ) were supplied by Wuhan Procell Life Technology Co., Ltd., (Wuhan, China) and Bena Biological Technology Co., Ltd. (Xinyang, China)., respectively, and cultured with endothelial cell medium (ECM, ScienCell Research Laboratories, CA, USA) containing vascular endothelial growth factor at 37 °C with 5% CO 2 .

Techniques: Activity Assay, Expressing, Transmission Assay, Electron Microscopy, Western Blot, Immunofluorescence, Control, Animal Model

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3‐activated lncRNA XIST accelerates the inflammatory response and apoptosis of LPS‐induced acute lung injury

doi: 10.1111/jcmm.16653

Figure Lengend Snippet: LncRNA XIST was up‐regulated in LPS‐induced ALI. A, Heatmap demonstrated the differently expressed multiple alternative candidate lncRNAs in LPS‐induced or control human PMVECs. B, RT‐PCR demonstrated the expression of lncRNA XIST in LPS‐induced PMVECs as compared to the control. C, The LPS‐induced mice models were constructed to mimic the ALI. LncRNA XIST was detected using RT‐PCR in LPS‐induced mice ALI models and controls. D, ELISA was performed in bronchoalveolar lavage fluid (BALF) to detect the enrichment of inflammatory cytokines (IL‐6, IL‐1β, TNF‐α). ** P < .01

Article Snippet: Then, human PMVECs were purchased from ScienCell Research Laboratories and cultured in Dulbecco's modified Eagle medium (DMEM) added with 10% FCS (foetal calf serum, HyClone).

Techniques: Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca 2+ entry

doi: 10.1152/ajpheart.00540.2017

Figure Lengend Snippet: Epicholesterol (Epichol) reduces the endogenous membrane cholesterol (Chol) content of cultured pulmonary microvascular endothelial cells (PMVECs). A: representative images of filipin fluorescence from PMVECs treated with vehicle, methyl-β-cyclodextrin (MβCD), Chol, or Epichol. B: mean filipin fluorescence [in arbitrary units (AU)] in PMVECs from each group. Data were compared by the Kruskal-Wallis H-test and Dunn’s multiple comparison test. n = 5 (filipin fluorescence of 4–16 cells were measured per field of interest and averaged for an n = 1). *P < 0.05 vs. vehicle; #P < 0.05 vs. MβCD and Epichol. C: Amplex Red Chol assay to detect Chol concentration in PMVECs. Data were compared by one-way ANOVA followed by the Student-Newman-Keuls test. n = 3. *P < 0.05 vs. Vehicle; #P < 0.05 vs. MβCD and Epichol.

Article Snippet: To complement experiments using freshly isolated PAEC sheets, cultured PMVECs (VEC Technologies) were also used in this study.

Techniques: Membrane, Cell Culture, Fluorescence, Comparison, Cholesterol Activity Assay, Concentration Assay

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca 2+ entry

doi: 10.1152/ajpheart.00540.2017

Figure Lengend Snippet: Orai1 siRNA knockdown inhibits membrane cholesterol (Chol)-dependent store-operated Ca2+ entry (SOCE) in pulmonary microvascular endothelial cells (PMVECs). A: representative Western blot of Orai1 and β-actin protein bands in PMVECs transfected with Orai1 siRNA or nontargeting (NT) siRNA. F, fluorescence intensity at 360-nm excitation; F0, fluorescence intensity at time 0. B: mean Western blot data of Orai1 expression from NT and Orai1 siRNA-treated PMVECs. Orai1 levels are normalized to those of β-actin. n = 4/group. *P < 0.05 vs. NT. C: SOCE in cultured PMVECs. n = 3–4/group. *P < 0.05 vs. NT + Chol over the range of 60–120 s.

Article Snippet: To complement experiments using freshly isolated PAEC sheets, cultured PMVECs (VEC Technologies) were also used in this study.

Techniques: Knockdown, Membrane, Western Blot, Transfection, Fluorescence, Expressing, Cell Culture

Journal: American Journal of Physiology - Heart and Circulatory Physiology

Article Title: Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca 2+ entry

doi: 10.1152/ajpheart.00540.2017

Figure Lengend Snippet: Epicholesterol (Epichol) substitution reduces the stromal interaction molecule 1 (STIM1)-Orai1 interaction in pulmonary microvascular endothelial cells (PMVECs) as assessed by an in situ proximity ligation assay. A: representative images of STIM1-Orai1 interactions (red puncta) in response to cyclopiazonic acid (CPA) in cultured PMVECs with manipulated membrane cholesterol (Chol). Nuclei area labeled with SYTOX (green). B: summarized data of the STIM1-Orai1 interaction expressed as average number of puncta/cell in cultured PMVECs. Chol supplementation increased the CPA-induced STIM1-Orai1 interaction, whereas Epichol substitution significantly inhibited this response. Groups were compared by two-way ANOVA followed by multiple comparisons testing using the Student-Newman-Keuls test. n = 5/group. *P < 0.05 vs. Chol Veh; #P < 0.05 vs. Chol CPA.

Article Snippet: To complement experiments using freshly isolated PAEC sheets, cultured PMVECs (VEC Technologies) were also used in this study.

Techniques: In Situ, Proximity Ligation Assay, Cell Culture, Membrane, Labeling

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: Analysis of hypoxia-induced changes in VEGF/Notch signaling effector molecules and related miRNAs in PMVECs. ( A ) PMVECs were cultured under hypoxia for 0, 24, 48, or 72 h. With prolongation of hypoxic exposure the cells lost their original morphology and cell population density decreased (200X). ( B ) Relative expression of miR-30b-5p, miR-532-5p, miR-203a-3p, miR-101a-5p, and miR-16-3p assessed by qRT-PCR. ( C ) Relative expression of Hes-1, Notch1, VEGF-A, and VEGFR2 mRNA measured by qRT-PCR. ( D ) Hes-1, Notch1, VEGF-A, and VEGFR2 expression assessed by western blotting. Data are mean ± SEM. * P < 0.05, ** P < 0.01 compared with the normoxic control group; # P < 0.05, ## P < 0.01 compared with the 24-h hypoxia group; & P < 0.05, && P < 0.01 compared with the 48-h hypoxia group.

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: miR-203a-3p mimics expression inhibits the expression of VEGF-A and downstream genes. ( A ) Strong GFP fluorescence in PMVECs transduced with miR-203a-3p mimics indicated significantly increased expression of miR-203a-3p, compared with the corresponding control (no transduction) (P < 0.01) (100X). ( B ) Morphological changes in PMVECs transduced with lentiviral vectors encoding miR-203a-3p-NC (negative miRNA-203a-3p mimics control) or miR-203a-mimics (100X). ( C ) Results of qRT-PCR analysis showing decreased expression of Hes-1, VEGF-A, and VEGFR2 in miR-203a-mimics-transduced PMVECs (P < 0.01). ( D ) Western blotting results showing reduction on Hes-1, VEGF-A and VEGFR2 expression in PMVECs transduced with miR-203a-3p-mimics. Data are mean ± SEM. ** P < 0.01 compared with the control or normoxic control groups; ## P < 0.01 compared with miR-203a-3p-NC or 24-h hypoxia group; & P < 0.05, && P < 0.01 compared with the 48-h hypoxia group.

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Expressing, Fluorescence, Transduction, Control, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: miR-203a-3p mimics expression decreases survival and angiogenic activity and induces a pro-inflammatory response in PMVECs. ( A ) Apoptosis assay results showing increased apoptosis rate in PMVECs expressing miR-203a-3p mimics. ( B ) In vitro angiogenesis assay results. PMVECs transduced with miR-203a-3p mimics showed weak angiogenic ability, which improved however with prolonged hypoxic incubation time. ( C ) ELISA assay results showing increased IL-6, IL-10, and TNF-α secretion in PMVECs transfected with miR-203a-3p. ( D , E ) CCK8 assay results indicating decreased survival rate in PMVECs transfected with miR-203a-3p mimics. Data are mean ± SEM. * P < 0.05, ** P < 0.01 compared with the control (no transduction) or normoxic control groups; # P < 0.05, ## P < 0.01 compared with the miR-203a-3p-NC (negative miRNA-203a-3p mimics control) or 24-h hypoxia groups; & P < 0.05, && P < 0.01 compared with the 48-h hypoxia group.

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Expressing, Activity Assay, Apoptosis Assay, In Vitro, Angiogenesis Assay, Transduction, Incubation, Enzyme-linked Immunosorbent Assay, Transfection, CCK-8 Assay, Control

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: VEGF-A is a direct target of miR-203a-3p. ( A ) Sequence information of miR-203 binding sites in the 3′-UTR region of the VEGF-A mRNA. ( B ) Dual luciferase assay indicating decreased fluorescence in PMVECs co-expressing pmirGLO/VEGF-UTR and miR-203-3p mimics. ( C ) Western blotting detection of target genes of miR-203a-3p. Compared with mimics-NC (negative miR-203a-3p mimics control), VEGF expression decreased after transfection with miR-203-mimics, and increased after transfection with miR-203-ASO. ( D ) qRT-PCR detection of target genes of miR-203a-3p. VEGF mRNA levels decreased after transfection with miR-203-mimics, and increased after transfection with miR-203-ASO. Data are mean ± SEM. * P < 0.05, ** P < 0.01 compared with mimics-NC; ## P < 0.01 compared with miR-203a-3p-mimics; && P < 0.01 compared with miR-203a-3p-ASO-NC (negative miR-203a-3p-ASO control).

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Sequencing, Binding Assay, Luciferase, Fluorescence, Expressing, Western Blot, Control, Transfection, Quantitative RT-PCR

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: VEGF-A expression is rescued by miR-203a-3p knockdown. ( A ) The validation of VEGF-A expression reduction in VEGF-shRNA-transduced cells by qRT-PCR. ( B ) Western blotting data showing reduced expression of VEGF-A in PMVECs transduced with VEGF-shRNA. ( C ) Morphological evaluation in PMVECs transduced with VEGF-shRNA+ASO-NC (negative VEGF-shRNA+ASO control) or VEGF-shRNA+ASO (miR-203a-3p knockdown). Note that inhibition of miR-203a-3p activity reversed morphological impairment caused by VEGF silencing. ( D ) Flow cytometry apoptosis assay indicates increased apoptosis in PMVECs expressing VEGF-shRNA, and reversal of the effect by ASO-mediated miR-203a-3p silencing. ( E – F ) CCK-8 assay showing improved viability in PMVECs expressing VEGF-shRNA+ASO, compared with the VEGF-shRNA+ASO-NC group. ( G ) qRT-PCR results showing reduced expression of VEGF-A and its downstream mediators, VEGFR2 and Hes-1, in cells transfected with VEGF-shRNA. The expression of these mRNAs increased instead in cells co-expressing miR-203a-3p-ASO. ( H ) Western blotting results showing protein expression changes consistent with the mRNA data showed in ( G ). Data are mean ± SEM. * P < 0.05, ** P < 0.01 compared with VEGF-shRNA negative control (VEGF-shRNA-NC) and normoxia groups; # P < 0.05, ## P < 0.01 compared with the VEGF-shRNA or 24-h hypoxia group; & P < 0.05, && P < 0.01 compared with the VEGF-shRNA+ASO-NC or 48-h hypoxia group.

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Expressing, Knockdown, Biomarker Discovery, shRNA, Quantitative RT-PCR, Western Blot, Transduction, Control, Inhibition, Activity Assay, Flow Cytometry, Apoptosis Assay, CCK-8 Assay, Transfection, Negative Control

Journal: Aging (Albany NY)

Article Title: Downregulation of lung miR-203a-3p expression by high-altitude hypoxia enhances VEGF/Notch signaling

doi: 10.18632/aging.102878

Figure Lengend Snippet: Hypoxia stimulates VEGF/Notch signaling by downregulating miR-203a-3p expression. ( A ) Hypoxia upregulates the expression of VEGF-A and downregulates the expression of its negative regulator miR-203a-3p (red box). ( B ) VEGF-A binds and activates VEGFR2 in tip cells, leading to the activation of the Dll4 promoter (purple box). ( C ) Dll4 activates Notch1 receptor in neighboring stem cells. Gamma secretase (GS) cleaves the Notch1 receptor intracellular domain (NICD), which is transferred to the nucleus to enhance the expression of transcription factors (TFs) such as Hes-1. These TFs inhibit the expression of VEGFR2 in stem cells and promote the expression of downstream genes that regulate budding, proliferation, and differentiation of PMVECs (blue box).

Article Snippet: Pulmonary microvascular endothelial cells (PMVECs) were purchased from Cyagen Biosciences (Guangzhou, China) and grown in M199 medium (Cyagen Biosciences) supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Cyagen) and endothelial growth factor (EGFS) at 37°C under 5% CO 2 and 2% O 2 .

Techniques: Expressing, Activation Assay