Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress activates PI3K/Akt in human PMVECs. Left: Representative immunoblots showing protein levels of phosphorylated Akt (p‐Akt), total Akt and β‐tubulin in whole cell lysates of human PMVECs exposed to different shear stress protocols. Right: Densitometry analysis showing corresponding ratio of p‐Akt to Akt. (a) Cells were pretreated with vehicle (0.1% DMSO) or PI3K inhibitor (LY294002; 10 μM) for 30 min and then exposed to physiological shear stress (12 dyn/cm 2 ) for 1 h, with static cells as controls. (b) After adapting to 0 or 12 dyn/cm 2 of shear stress for 24 h, human PMVECs were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) while maintaining at respective static or shear stress conditions for another 24 h. (c) Human PMVECs were first adapted to shear stress (12 dyn/cm 2 ) for 24 h and then exposed to 30 min of reduced (3 or 0 dyn/cm 2 ) shear stress, with cells maintained at 12 dyn/cm 2 as control. For all graphs, symbols represent different results from different donors ( n = 4; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values. Data were analyzed by one‐way ANOVA with Dunnett's multiple comparisons test (panel (a) interaction p = 0.0003 and panel (c) interaction p = 0.0005) or two‐way ANOVA with Tukey's multiple comparisons test (panel (b) interaction p = 0.1159).

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Western Blot, Control

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress reduces apoptosis in human PMVECs. After adapting PMVECs to 0 or 12 dyn/cm 2 of shear stress for 24 h, cells were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) while maintaining at respective static conditions or shear stress for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm, red arrows denote condensed chromatin; Right: Summary data); (b) caspase 3/7 activity and (c) DNA fragmentation (image representative of 3 separate experiments). Bar graphs show mean ± SD values; symbols represent results in cells from different donors ( n = 4–5; open circles represent cells from male and closed circles represent cells from female). Data were analyzed by three‐way ANOVA (Factors are shear stress, STS and LY294002; panel (a) interaction p = 0.0451; panel (b) interaction p = 0.0393) followed by Sidak's multiple comparisons test. Shear stress groups are the same as respective panels in Figure .

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress attenuates STS‐induced apoptosis in human PMVECs. After culture under static conditions or adaptation to 12 dyn/cm 2 for 24 h, human PMVECs were treated with either vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) and maintained at their respective static or shear stress conditions for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm; Right: Summary data); (b) caspase 3/7 activity and (c) DNA fragmentation (image representative of 3 separate experiments). Bar graphs show mean ± SD values; symbols represent results in cells from different donors ( n = 5; open circles represent cells from male and closed circles represent cells from female). Data were analyzed by two‐way ANOVA (panel (a) interaction p = 0.0830; panel (b) interaction p = 0.6835) followed by Tukey's multiple comparisons test.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Shear stress‐induced increases in PI3K activity are not inhibited by STS. After adaptation to 0 or 12 dyn/cm 2 for 24 h, human PMVECs were treated with either vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) while maintaining at their respective shear conditions (0 or 12 dyn/cm 2 ) for another 24 h. Immunoblot was performed in whole cell lysates to probe for p‐Akt, Akt and β‐tubulin. (a) Representative blot and (b) Densitometry analysis for p‐Akt to Akt ratio. Symbols represent different results from different donors ( n = 5; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values; Data were analyzed by two‐way ANOVA (Interaction p = 0.0005) followed by Tukey's multiple comparisons test.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Activity Assay, Western Blot

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: PI3K/Akt contributes to shear stress‐dependent apoptotic resistance to STS. After adaption to 0 or 12 dyn/cm of shear stress for 24 h, human PMVECs were treated with vehicle (0.1% DMSO) or LY294002 (10 μM) in combination with vehicle (0.01% DMSO) or staurosporine (STS; 20 nM) while maintaining at respective static or shear conditions for another 24 h. Apoptosis was evaluated by (a) Hoechst staining (Left: Representative images, white scale bar is 100 μm; Right: Summary data), (b) caspase 3/7 activity, and (c) DNA fragmentation (representative of three experiments). Symbols represent different results from different donors ( n = 4–5; open circles represent cells from male and closed circles represent cells from female). Bar graphs show mean ± SD values; Data were analyzed by three‐way ANOVA (Factors are shear stress, STS and LY294002; panel (a) interaction p = 0.0451; panel (b) interaction p = 0.0393) with Sidak's multiple comparisons test. Vehicle and vehicle LY294002 groups are the same as respective panels in Figure .

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Staining, Activity Assay

Journal: Physiological Reports

Article Title: Physiological shear stress suppresses apoptosis in human pulmonary microvascular endothelial cells

doi: 10.14814/phy2.70269

Figure Lengend Snippet: Schematic summary of major findings. In human PMVECs, physiological shear stress rapidly and sustainably activates PI3K/Akt signaling, which contributes to apoptotic resistance upon staurosporine challenge. Apoptotic resistance conveyed by shear stress is not disrupted by PI3K inhibition, suggesting the existence of additional pro‐survival pathways.

Article Snippet: Primary human PMVECs (Lonza; CC‐2527) were cultured using microvascular endothelial cell complete media (Lonza; CC‐3202) on flasks (for cell expansion) or 6‐well plates (for experiments) coated with attachment factor (Gibco; S006‐100).

Techniques: Shear, Inhibition

Journal: Protein & Cell

Article Title: Single-cell profiling reveals a potent role of quercetin in promoting hair regeneration

doi: 10.1093/procel/pwac062

Figure Lengend Snippet: Que treatment promotes the migration and proliferation of endothelial cells via the activation of HIF-1α . (A) Western blot analysis of HIF-1α in pMVECs treated with vehicle or Que. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 50 μm. (B) Immunostaining of HIF-1α and Lamin A/C in pMVECs treated with vehicle or Que. Right, the quantitative data of mean fluorescence intensity of HIF-1α in the nucleus. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 50 μm. (C) Heatmap showing the relative expression level (row-scaling) of DEGs in pMVECs treated with Que versus vehicle. (D) Representative GO terms and pathways of upregulated and downregulated DEGs in pMVECs treated with Que versus vehicle. (E) GSEA showing HIF-1 signaling pathway (top) and HIF-1α target genes (bottom) enriched in pMVEC treated with vehicle or Que. NES, normalized enrichment score; FDR, false discovery rate. (F) Immunostaining of Ki67 in vehicle or Que-treated pMVECs. Right, the quantification of Ki67-positive cells in vehicle or Que-treated pMVECs. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 20 μm. Arrows indicate Ki67-positive cells. (G) Immunofluorescence staining of Phospho-H3 (Ser10) in pMVECs treated with vehicle or Que. Right, the quantification of Phospho-H3 (Ser10)-positive cells in vehicle or Que-treated pMVECs. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 25 μm. Arrows indicate Phospho-H3 (Ser10)-positive cells. (H) Cell cycle analysis of pMVECs treated with vehicle or Que. n = 3 biological replicates. Two-way ANOVA with Sidak’s test was used and data are represented as mean ± SEMs. (I) Clonal expansion analysis of pMVECs treated with vehicle or Que. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. (J) Wound scratch assay for detecting the migration of pMVECs treated with vehicle or Que. n = 3 biological replicates. Two-tailed unpaired Student’s t -test is used and data are presented as the mean ± SEMs. Scale bars, 100 μm. (K) Angiogenesis was assessed by the formation of capillary-like tubes of pMVECs treated with vehicle or Que in vitro . n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 100 μm. (L) NO production ability of pMVECs treated with vehicle or Que by FACS analysis. n = 3 biological replicates. Two-tailed unpaired Student’s t -test was used and data are presented as the mean ± SEMs. Scale bars, 50 μm.

Article Snippet: Human pMVECs were purchased form Lonza and were cultured in endothelial cell growth medium (LONZA) in 5% CO 2 at 37°C.

Techniques: Migration, Activation Assay, Western Blot, Two Tailed Test, Immunostaining, Fluorescence, Expressing, Immunofluorescence, Staining, Cell Cycle Assay, Wound Healing Assay, In Vitro

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A) Cartoon of BMP9 signaling in HHT. B) Western blot verification of BMP9 in PMVECs. C-E) siRNA-mediated knockdown verification of ALK1, ENG, and SMAD4 by qPCR. F and G) Validation of BMP9 signaling in ALK1, ENG, and SMAD4 knockdown conditions in PMVECs by qPCR (read out ID1).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Western Blot, Knockdown, Biomarker Discovery

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Functional consequence of ALK1, ENG, and SMAD4 knockdown in PMVECs (proliferation, apoptosis and tube formation).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Functional Assay, Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A and B) Venn diagram of common upregulated or downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 (20ng/ml) for 2 or 24hrs (RNAseq). C) Heatmap of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. D) SinyGo biological processes analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with BMP9 for 2 and 24hrs. E) Panther pathway analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated BMP9 for 2 and 24hrs.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: STRING network analysis of the common upregulated and downregulated genes following knockdown of ALK1, ENG and SMAD4 in PMVECs stimulated with 2h and 24h of BMP9.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Validation of common upregulated or downregulated genes signatures with BMP9 stimulation at 2 (A and B) or 24 hrs (C and D) and functional consequences of the knockdown of LYVE1, GPNMB and MC5R in PMVECs. After 72 hrs knockdown of LYVE1, MC5R, and GPNMB PMVECs proliferation (E), apoptosis (F), tube formation (G) were assessed.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Biomarker Discovery, Functional Assay, Knockdown

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Drug prediction based on the common upregulated downstream targets after ALK1, ENG and SMAD4 knockdown and experimental validation in PMVECs. A) Experimental strategy: the common upregulated genes 117 (2h) and 112 (24h) after knockdown of the three HHT genes and stimulation with BMP9 for 2 and 24hrs (HHT disease signature) were uploaded separately on the Clue query app. The criteria of the drugs rank include samples >=3, normalized connectivity score, tas value >=0.20, and FDR value. B) top scoring 5 HHT (positive values) and anti-HHT drugs (negative values, indicated by brown color) are represented. (C-K) Effect of Brivanib on the expression of the common persistent downstream targets (LYVE1, GPNMB, RRAGD, ANKRD33, HS3ST2, MC5R, SLC25A47, FGF19, SHISA9, FRG2C, HIST1H2BE, and CPA4) after ALK1, ENG and SMAD4 knockdown was assessed by qRT-PCR in PMVECs. MOA, mode of action; cs, connectivity score; tas, transcriptional activity score. Data are represented as mean ± standard error mean (n=3). Two-way repeated measures ANOVA with a Bonferroni post-hoc test, * P= <0.5, ** P= <0.01, *** P= <0.001, **** P= <0.0001.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Knockdown, Biomarker Discovery, Expressing, Quantitative RT-PCR, Activity Assay

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: Effect of Brivanib on the expression of the common persistent downstream targets after ALK1, ENG and SMAD4 knockdown were assessed by qRT-PCR in PMVECs (A-C).

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Expressing, Knockdown, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Identifying transcriptomic downstream targets of genes commonly mutated in Hereditary Hemorrhagic Telangiectasia

doi: 10.1101/2022.11.25.517570

Figure Lengend Snippet: A) PMVECs were treated with 10uM Brivanib or DMSO for 24 hrs followed by 20ng/mL VEGF or PBS for 10 mins. Protein was harvested and the effect of Brivanib on VEGF-induced phosphorylation of ERK1/2 was assessed in PMVECs by western blotting. B) The effect of Brivanib on VEGF-induced cell proliferation was assessed by MTT assay in PMVECs. C) Angiogenesis was assessed in a Matrigel tube formation assay using PMVECs treated with either Brivanib 1, 10, 50 uM or DMSO. D) Proposed model for the mechanism by which Brivanib might influence AVM and HHT pathogenesis.

Article Snippet: Human healthy control pulmonary microvascular endothelial cells (PMVECs) (Cat # C12281, PromoCell GmbH, Heidelberg, Germany) were cultured in microvascular endothelial cell basal media (Cat # C-22120; PromoCell GmbH) supplemented with growth factors (Growth Medium MV SupplementPack, Cat # C-39220, PromoCell GmbH) and 100 U/mL Penicillin-Streptomycin Solution (Gibco) and used between passages 4 to 8.

Techniques: Phospho-proteomics, Western Blot, MTT Assay, Tube Formation Assay