Journal: American Journal of Translational Research

Article Title: microRNA-1246 mediates lipopolysaccharide-induced pulmonary endothelial cell apoptosis and acute lung injury by targeting angiotensin-converting enzyme 2

doi:

Figure Lengend Snippet: LPS induces miR-1246 to target ACE2 expression in PMVECs. A: qRT-PCR analysis of miR-1246 expression in PMVECs treated with LPS or vehicle control. *P < 0.05 vs. vehicle. B: Western blot analysis of ACE2 protein in PMVECs transfected with miR-1246 mimic or negative control miRNA. Bar graphs (bottom) show densitometric analysis of ACE2 protein. *P < 0.05 vs. vehicle. C: Luciferase reporter assay showed that miR-1246 repressed the expression of the luciferase reporter containing the wild-type (wt) but not mutated (mut) ACE2 3’-UTR. The predicted target site for miR-1246 in the ACE2 3’-UTR is shown in top panels. *P < 0.05 vs. vehicle. D: Western blot analysis of ACE2 protein in PMVECs pretransfected with anti-miR-1246 or control inhibitors before exposure to LPS. *P < 0.05.

Article Snippet: Cell culture and LPS treatment Human PMVECs were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS; GE Healthcare HyClone, Logan, UT, USA).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Transfection, Negative Control, Luciferase, Reporter Assay

Journal: American Journal of Translational Research

Article Title: microRNA-1246 mediates lipopolysaccharide-induced pulmonary endothelial cell apoptosis and acute lung injury by targeting angiotensin-converting enzyme 2

doi:

Figure Lengend Snippet: miR-1246 is involved in LPS-triggered apoptosis and inflammatory response in PMVECs. A: TUNEL staining of PMVECs pretransfected with anti-miR-1246 or control inhibitors (C-inhibitor) before exposure to LPS. Scale bar, 50 μm. *P < 0.05. B: ELISA analysis of IL-1β and TNF-α levels in conditioned medium from PMVECs with indicated treatments. *P < 0.05. C: TUNEL staining of PMVECs transfected with miR-1246 mimic or negative control miRNA. Scale bar, 50 μm. D: Measurement of IL-1β and TNF-α levels in conditioned medium from PMVECs transfected with miR-1246 mimic or negative control miRNA. *P < 0.05 vs. control.

Article Snippet: Cell culture and LPS treatment Human PMVECs were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS; GE Healthcare HyClone, Logan, UT, USA).

Techniques: TUNEL Assay, Staining, Control, Enzyme-linked Immunosorbent Assay, Transfection, Negative Control

Journal: American Journal of Translational Research

Article Title: microRNA-1246 mediates lipopolysaccharide-induced pulmonary endothelial cell apoptosis and acute lung injury by targeting angiotensin-converting enzyme 2

doi:

Figure Lengend Snippet: miR-1246 promotes PMVEC apoptosis by repressing ACE2 expression. A: Western blot analysis of ACE2 protein in PMVECs transfected with indicated constructs. B: Detection of apoptosis by TUNEL staining in PMVECs transfected with indicated constructs. *P < 0.05. C: Western blot analysis of Bcl-2, Bcl-xL, and Bax proteins in PMVECs transfected with indicated constructs.

Article Snippet: Cell culture and LPS treatment Human PMVECs were purchased from ScienCell Research Laboratories, Inc. (Carlsbad, CA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS; GE Healthcare HyClone, Logan, UT, USA).

Techniques: Expressing, Western Blot, Transfection, Construct, TUNEL Assay, Staining

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

Journal: PLoS ONE

Article Title: Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

doi: 10.1371/journal.pone.0018399

Figure Lengend Snippet: Hierarchical cluster analysis of differentially expressed genes in primary lung endothelial cells, PMVECs (A) and PAECs (B) successfully segregates cultures treated with vehicle from those treated with 5 µM hemin. The unpaired student's t-test was used on a gene-by-gene basis to test for differential expression between hemin and vehicle-treated cultures. (C) Venn diagram showing the number of genes differentially regulated by hemin in PMVECs and PAECs.

Article Snippet: The following primary human endothelial cells: pulmonary artery (PAECs), pulmonary microvascular (PMVECs) and dermal microvascular (DMVECs) were purchased from Lonza Inc (Allendale, NJ), while brain microvascular endothelial cells (BMVECs) were purchased from Cell System Corp. (Kirkland, WA).

Techniques: Quantitative Proteomics

Journal: PLoS ONE

Article Title: Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

doi: 10.1371/journal.pone.0018399

Figure Lengend Snippet: Genes differentially regulated by hemin in PAECs.

Article Snippet: The following primary human endothelial cells: pulmonary artery (PAECs), pulmonary microvascular (PMVECs) and dermal microvascular (DMVECs) were purchased from Lonza Inc (Allendale, NJ), while brain microvascular endothelial cells (BMVECs) were purchased from Cell System Corp. (Kirkland, WA).

Techniques:

Journal: PLoS ONE

Article Title: Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

doi: 10.1371/journal.pone.0018399

Figure Lengend Snippet: (A) Central role of Nrf2 in the response of the endothelium to heme. Genes whose expression was altered by hemin in both PAECs and PMVECs are shown. ROS = reactive oxygen species. (B) Microarray data of differentially expressed genes regulated by Nrf2 in PAECs and PMVECs treated with hemin. Data shown is mean fold change in gene expression as arbitrary units relative to the level of expression by control cells (n = 5).

Article Snippet: The following primary human endothelial cells: pulmonary artery (PAECs), pulmonary microvascular (PMVECs) and dermal microvascular (DMVECs) were purchased from Lonza Inc (Allendale, NJ), while brain microvascular endothelial cells (BMVECs) were purchased from Cell System Corp. (Kirkland, WA).

Techniques: Expressing, Microarray, Gene Expression, Control

Journal: PLoS ONE

Article Title: Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

doi: 10.1371/journal.pone.0018399

Figure Lengend Snippet: (A) Total RNA from PAECs and PMVECs treated with hemin (0–25 µM) was analyzed for the expression of HO-1 and NQO1. Data shown is mean fold change relative to the vehicle (0 µM hemin) +/− SD for three independent experiments each in triplicate (n = 9). (B) Low-density array data showing changes in expression of fourteen genes in PMVECs treated with a concentration range (0–25 µM) of hemin for 7 days. Note the concentration-dependent increase in the number of genes altered by hemin (n = 12).

Article Snippet: The following primary human endothelial cells: pulmonary artery (PAECs), pulmonary microvascular (PMVECs) and dermal microvascular (DMVECs) were purchased from Lonza Inc (Allendale, NJ), while brain microvascular endothelial cells (BMVECs) were purchased from Cell System Corp. (Kirkland, WA).

Techniques: Expressing, Concentration Assay

Journal: PLoS ONE

Article Title: Global Gene Expression Profiling of Endothelium Exposed to Heme Reveals an Organ-Specific Induction of Cytoprotective Enzymes in Sickle Cell Disease

doi: 10.1371/journal.pone.0018399

Figure Lengend Snippet: (A) Western blot analysis confirming concentration dependent induction of HO-1 and NQO1 in PMVECs and PAECs treated with hemin for 7 days. Blots were probed for EF-1αto control protein loading. (B, C) Quantification of HO-1 and NQO1 protein expression in PAECs, PMVECs, BMVECs and DMVECs treated with hemin and vehicle, assayed by western blot analysis. Data shown is mean fold change in protein level as arbitrary units relative to the EF-1α-normalized expression in vehicle treated cells (n = 9). (D) Quantification of western blot showing variable timing of HO-1 and NQO1 induction by hemin (5 µM) in PAECs (n = 9).

Article Snippet: The following primary human endothelial cells: pulmonary artery (PAECs), pulmonary microvascular (PMVECs) and dermal microvascular (DMVECs) were purchased from Lonza Inc (Allendale, NJ), while brain microvascular endothelial cells (BMVECs) were purchased from Cell System Corp. (Kirkland, WA).

Techniques: Western Blot, Concentration Assay, Control, Expressing

Journal: Advanced Science

Article Title: Pulmonary‐Targeted Nanoparticles Interrupt the Malignant Mechanical and Biochemical Signaling Crosstalk for Idiopathic Pulmonary Fibrosis Therapy

doi: 10.1002/advs.202512658

Figure Lengend Snippet: Characterization and multifunctional properties of the VB‐RT NPs. A) Particle size of different formulations. B) Zeta potential of different formulations. C‐D) Average size distribution and TEM images of VB NPs and VB‐RT NPs. E) UV–vis spectra of different formulations across the full wavelength range. F) Seven‐day stability of VB NPs, VB‐R NPs, and VB‐RT NPs. G) Comparison of particle size before and after nebulization. H) Heatmap of PDI before and after nebulization. I) Fluorescence imaging of ROS levels in 16HBE cells under different formulations using DCFH DA. J) The NO detection in 16HBE cells under different formulations using DAF‐FM DA. K) Quantification of mean fluorescence intensity (MFI) of NO in 16HBE cells under different formulations (n = 3). L) Uptake of different formulations in PMVECs measured by flow cytometry. M) Quantification of MFI of NO in A549 cells under different formulations using DAF‐FM DA (n = 3). N) Illustration of the in vitro collagen barrier model, showing DiI‐labeled NPs crossing the epithelial cell‐collagen layer to reach myofibroblasts. O) CLSM images of myofibroblasts treated with different formulations in Transwell chambers with collagen barrier for 4 h at 37 °C. P) Quantification of MFI in myofibroblasts analyzed using ImageJ (n = 3). All data are presented as the Mean ± SD (n = 3). *** p < 0.001.

Article Snippet: PMVECs were purchased from ProCell, and 16HBE cells were obtained from ATCC (Shanghai, China).

Techniques: Zeta Potential Analyzer, Comparison, Fluorescence, Imaging, Flow Cytometry, In Vitro, Labeling