Journal: International Journal of Molecular Sciences

Article Title: High-Content Imaging and Machine Learning Classify Phenotypical Change in Coronary Artery Endothelial Cells Caused by BPS

doi: 10.3390/ijms27073259

Figure Lengend Snippet: Representative high-content microscopy images of human coronary artery endothelial cells (HCAEC) exposed to vehicle control (CTRL) or 0.1 µM Bisphenol S (BPS) for 96 h and stained using the PhenoVue Cell Painting assay. For each condition, a representative field acquired at 40× magnification and a higher-magnification inset are shown. Rows correspond to the individual fluorescence channels: Hoechst 33342 (nuclei), PhenoVue Fluor 488 Concanavalin A (endoplasmic reticulum and intracellular membranes), PhenoVue 512 nucleic acid stain (RNA/nucleoli), PhenoVue Fluor 555 wheat germ agglutinin (plasma membrane), PhenoVue 641 mitochondrial stain (mitochondria), and the merged image. White boxes represent the part of the image used for the related inset. Scale bar: 50 µm, 40× objective.

Article Snippet: Primary human coronary artery endothelial cells (HCAEC; ATCC ® PCS-100-020TM, Innovation, VA, USA) were cultured according to the supplier’s recommendations.

Techniques: Microscopy, Control, Staining, Fluorescence, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in the kidney as assessed by western blot performed on total kidney extracts. ( b ) Gata5 is essentially expressed in the glomeruli as assessed by qPCR on isolated glomeruli (Glo) and microdissected tubules (Tub) from Wt mice kidneys. ( n =3–5 per group). The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice (Mann–Whitney test). ( c , d ) Specific deletion of Gata5 in endothelial cells (e Gata5-null mice) virtually abolished renal and glomerular expression of Gata5 ( n =3 per group). The results are reported as mean±s.e.m. * P <0.05 versus eGata5 +/+ mice (Mann–Whitney test). ( e ) The expression of the glomerular genes Nphs1 (nephrin) and Nphs2 (podocin) as measured by qPCR ( n =6 per group) was increased in Gata5 -null mice. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice; *** P <0.005 versus Gata5 +/+ mice ( t -test for nephrin; Mann–Whitney test for podocin). ( f – i ) Absence of Gata5 induces glomerular lesions (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5 per group) and renal inflammation as assessed by leucocytes CD45 immunostaining (scale bar, 200 μm; n =4 per group). The results are reported as mean±s.e.m. *** P <0.005 versus Gata5 +/+ mice (Mann–Whitney test). ( j – n ) Deletion of Gata5 from endothelial cells reproduces the renal phenotype of global Gata5 deletion: both Nephs1 and Nphs2 transcript levels were increased ( n =4–6 per group) as well as glomerular lesion score (scale bar, 30 μm; n =4–6 per group) and renal leucocytes infiltration (scale bar, 200 μm; n =4–5 per group) in e Gata5 -null mice in comparison with their controls. The results are reported as mean±s.e.m. * P <0.05 versus e Gata5 +/+ mice; *** P <0.005 versus e Gata5 +/+ mice (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Western Blot, Isolation, MANN-WHITNEY, Expressing, Staining, Immunostaining, Comparison

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 is expressed in human cardiac microvascular (CM), coronary artery (CA), dermal microvascular (DM) and pulmonary microvascular (PM) endothelial cells. ( b , c ) The vasoconstrictor response of Gata5 -null mice mesenteric arteries to norepinephrine is unaltered ( n =7 per group), while the vasodilatory response to acetylcholine is decreased ( n =10–11 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—effector concentration for half-maximum response (EC 50 ) and Hill slope—using an F-test). ( d – f ) Deletion of Gata5 in endothelial (e Gata5 -null mice; n =4–5 per group) but not smooth muscle cells (sm Gata5 -null mice; n =5 per group) decreases mesenteric arteries sensitivity to acetylcholine and increases BP (e Gata5 -null mice n =6–10 per group; sm Gata5 -null mice n =6–9 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (two-factor ANOVA). ( g ) The vasodilatory response of Gata5 -null mice to diethylamine NONOate, an NO donor, is unaltered ( n =7 per group). The results are reported as mean±s.e.m. (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( h , i ) NOS3 and Akt phosphorylation are decreased in Gata5 -null mice mesenteric arteries. PTEN and PDK1 phosphorylation and expression are unaltered ( n =5–7 per group). Phosphorylated proteins are normalized to total proteins. Total proteins are normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Gata5 +/+ mice ( t -test). ( j ) Quantification of protein nitrotyrosination in mesenteric arteries of Gata5 -null mice and their controls as measured by ELISA. 3-Nitrotyrosine content is expressed as picomole of nitrotyrosine per milligram of protein ( n =5–7 per group). The results are reported as mean±s.e.m. ( t -test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Concentration Assay, Phospho-proteomics, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) GATA5 expression is significantly decreased in human dermal microvascular endothelial cells infected with a lentiviral vector containing an anti- GATA5 shRNA (HDMEC-GATA5-KD). Control cells were infected with a vector containing a control shRNA (targets no known mammalian gene) (HDMEC-pLKO-Ctrl, referred here as Ctrl). ( b ) Heatmap representation of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls as identified by transcriptomic analysis. Colour is function of Log2 RMA (Affymetrix microarray, n =3 per group). ( c ) Functional analysis of the differentially regulated genes between HDMEC-GATA5-KD cells and their controls. Protein kinase A pathway is the most significantly enriched pathway. Fisher's exact test P value. ( d ) Validation by qPCR (upper panel) of genes predicted by microarray (lower panel) to be up- and downregulated in HDMEC-GATA5-KD endothelial cells. ( n =5 wells per condition). Downregulated genes: PRKACB codes for the PKA catalytic subunit β, PRKAR2B for the PKA regulator subunit 2β and PRKAA2 for the AMPK catalytic subunit α2. Upregulated genes: ICAM1 codes for the intercellular adhesion molecule 1, BMP4 for the bone morphogenetic protein 4 and IL6 for the interleukin 6. The results are reported as mean±s.e.m. ** P <0.01 versus Ctrl ( t -test). ( e ) Western blot representation of phospho-NOS3, NOS3 (Ser1177) and phospho-(Ser/Thr) PKA substrate motif in HDMEC-GATA5-KD cells and their controls. ( f ) Phosphorylation of NOS3 on Ser1177 is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-NOS3 is normalized to total NOS3. NOS3 is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( g ) Phosphorylation of (Ser/Thr) PKA substrate motif is decreased in HDMEC-GATA5-KD cells (performed twice, 2–3 wells per condition). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. * P <0.05 versus Ctrl (Mann–Whitney test). ( h ) Western blot representation of phospho-(Ser/Thr) PKA substrate motif in mesenteric arteries of Gata5 -null mice and their controls. ( i ) In mesenteric arteries of Gata5 -null mice, there is a trend to decrease in the (Ser/Thr) PKA substrate motif phosphorylation ( n =4–5 per group). Phospho-(Ser/Thr) PKA substrate motif (between 25 and 250 kDa) is normalized to actin. The results are reported as mean±s.e.m. (Mann–Whitney test).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Expressing, Infection, Plasmid Preparation, shRNA, Control, Microarray, Functional Assay, Biomarker Discovery, Western Blot, Phospho-proteomics, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Gata5 transcription factor regulates blood pressure

doi: 10.1038/ncomms9835

Figure Lengend Snippet: ( a ) Administration of hydralazine (a smooth muscle cell relaxant) for 4 weeks decreased blood pressure similarly in both Gata5 -null mice and their controls ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( b ) Hydralazine had no effect on Gata5 -null mice endothelial dysfunction ( n =5–7 per group). The results are reported as mean±s.e.m. * P <0.05 versus controls (comparison of best-fit values—EC 50 and Hill slope—using an F-test). ( c ) Hydralazine decreased partially glomerular injuries in Gata5 -null mice (sections are stained with periodic acid Schiff; scale bar, 30 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons). ( d ) Hydralazine decreased completely renal CD45+ cells infiltration in Gata5 -null mice and also their controls (scale bar, 200 μm; n =5–7 per group). * P <0.05 versus controls; # P <0.05 versus corresponding untreated mice (two-factor ANOVA followed by Bonferonni correction for multiple comparisons).

Article Snippet: Human primary endothelial cells were purchased from Promocell: Human Coronary artery endothelial cells (ref C-14022, Lot: 91001010.7P); Human Cardiac Microvascular Endothelial Cells (ref C-14029, Lot: 20224401P); Human Pulmonary Microvascular Endothelial Cells (ref C-14027, Lot: 9090301P) and HDMECs (ref C-14016, Lot: 4060603P).

Techniques: Comparison, Staining

Journal: bioRxiv

Article Title: Temporal AI model predicts drivers of cell state trajectories across human aging

doi: 10.64898/2026.03.30.715396

Figure Lengend Snippet: ( A ) Predicted impact of in silico inhibition vs. slope of expression change across human aging for genes in cardiac fibroblasts. ( B ) Concordant genes whose inhibition was predicted to be rejuvenating were upregulated across aging, and vice versa. ( C ) Predicted impact of in silico inhibition of genes across the indicated cardiac cell types. ( D ) Beta-gal staining and cell counts quantifying senescence of primary human cardiac capillary endothelial cells in response to predicted pro-aging perturbation of ZBTB16 inhibition. *p<0.05, Wilcoxon rank sums, n=4. ( E ) Predicted impact of in silico perturbation and expression change across human aging and in telomere-shortened mice in each cardiac cell type for top predicted cardiomyocyte age-modulating targets. ( F ) PCA plot and ( G ) differential gene expression heatmap of transcriptional response (measured by bulk RNA sequencing) to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes. p<0.05, Wald test with BH correction, n=4. ( H ) Gene set enrichment of genes differentially expressed in response to all predicted pro-aging perturbations in human iPSC-derived cardiomyocytes (hypergeometric test with g:Set Counts and Sizes (g:SCS) correction). ( I ) Slowed calcium cycle kinetics and ( J ) rhythm irregularities in response to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes. *p<0.05, Wilcoxon rank sums with BH correction. Rhythm n=10, time to peak n=180, 111, 139, 37, 237, 150, 191 (bars left to right), decay n=169, 30, 86, 11, 213, 105, 172 (bars left to right). ( K ) Differential gene expression heatmap of transcriptional response (measured by bulk RNA sequencing) to predicted pro-aging perturbations (AAV overexpression of indicated genes vs. GFP) in human iPSC-derived cardiomyocytes or human primary cardiac fibroblasts where dysregulation of the given gene was statistically significant across all four conditions. Statistical significance of differential expression: p<0.05, Wald test with BH correction, n=4. Statistical significance of overlap of shared dysregulation between the four conditions: p<0.05, one-sided binomial test. RASG.=RASGEF1B. ( L ) Beta-gal staining quantifying senescence of primary human cardiac fibroblasts in response to predicted pro-aging perturbations (AAV overexpression of indicated genes). *p<0.05, Wilcoxon rank sums with BH correction, empty n=6, P4HA1 and RASGEF1B n=4. ( M ) Schematic of in vivo validation experiment. o/e=overexpression. ( N ) In vivo systolic function echocardiographic measurements at 6 weeks post-AAV9 injection. EF: GFP n=8, P4ha1 n=10, Rasgef1b n=10; GLS: GFP n=8, P4ha1 n=9, Rasgef1b n=8. *p<0.05 one-way ANOVA with multiple hypothesis correction.

Article Snippet: Human cardiac capillary endothelial cells were purchased from PromoCell (C-12285) and cultured in endothelial basal medium (EBM, CC-3121, Lonza) supplemented with 10% fetal bovine serum (10100147, Gibco).

Techniques: In Silico, Inhibition, Expressing, Staining, Gene Expression, RNA Sequencing, Over Expression, Derivative Assay, Quantitative Proteomics, In Vivo, Biomarker Discovery, Injection

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMP9 and BMP10 selectively activate SMAD1/5/8 signaling and induce proliferation in pulmonary artery endothelial cells but not pulmonary artery smooth muscle cells. ( A ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PAECs treated with the indicated TGF-β superfamily ligands (0.8 nM) or untreated control (UT); β-actin serves as a loading control. ( B ) PAEC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. ( C ) Western blot analysis of phosphorylated SMAD1/5/8 and SMAD2/3 in PASMCs treated with the indicated ligands (0.8 nM); β-actin serves as a loading control. ( D ) PASMC proliferation measured by BrdU incorporation following ligand treatment (0.8 nM), normalized to UT. Data are shown as mean ± SD ( n = 3 replicate wells). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). Statistical significance was assessed by one-way ANOVA with Dunnett’s multiple-comparisons test (each ligand vs. UT). ** p < 0.01, *** p < 0.001; ns, not significant.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Western Blot, Control, BrdU Incorporation Assay

Journal: Cells

Article Title: BMPR2 Dosage Gates BMP9/10 Signaling Output in Pulmonary Artery Endothelium

doi: 10.3390/cells15060492

Figure Lengend Snippet: BMPR2 dosage-dependent model for BMP9/10 signaling output in pulmonary artery endothelial cells. Schematic illustrating how BMPR2 abundance constrains BMP9/10 (ALK1-dependent) canonical signaling output and downstream cellular programs in PAECs. ( A ) BMPR2-sufficient (~100%) state: BMP9/10 predominantly signal through ALK1–BMPR2 complexes, generating pSMAD1/5/8 output consistent with a threshold-like requirement for proliferation; bimagrumab (BiMab) produces no effect detected under BMPR2-replete conditions. ( B ) BMPR2-limiting (~50%) state: Reduced BMPR2 attenuates BMP9/10-induced canonical output and is associated with reduced proliferation and increased caspase-3/7 activity consistent with stress/injury. Under BMPR2-limiting conditions, residual canonical output becomes bimagrumab-sensitive, consistent with context-dependent contribution of Activin type II receptors (predominantly ACVR2A in PAECs; see for BMP10 affinity comparisons) to the remaining pSMAD1/5/8 signal. A putative non-canonical stress-signaling arm is shown as a proposed intermediate. Solid arrows denote observed relationships; dashed arrows and dashed-outline boxes denote proposed steps. Node shading and output gauges depict relative canonical signaling output.

Article Snippet: Cell Lines and Culture: Human primary pulmonary artery endothelial cells (PAECs; ATCC PCS-100-022), pulmonary artery smooth muscle cells (PASMCs; ATCC PCS-100-023), and HEK293 cells (ATCC CRL-1573) were obtained from the American Type Culture Collection (Manassas, VA, USA).

Techniques: Activity Assay

Journal: Theranostics

Article Title: Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors

doi: 10.7150/thno.45973

Figure Lengend Snippet: Antibody binding and metabolism in living cells in vitro . Representative immunofluorescence images show the binding of selected anti-mouse (A) and anti-human (B) mAb clones to living endothelial cells. (C, D) EC 50 values of anti-mouse (C) and anti-human (D) mAb clones after binding to living cells for 15 min, n=3. (E, F) Quantitative assessment of the disappearance (due to capture, uptake, and elimination) of anti-mouse (E) and anti-human (F) mAb clones in cell cultures, n=3. (G-I) Cytotoxic effects on living cells in vitro , after different incubation times for anti-mouse mAbs at concentrations of 1-1000 ng/mL (G) or 1-2000 ng/mL (H), or (I) anti-human mAbs at 1-1000 ng/mL, n=3. n.s. no significant difference. mAb: monoclonal antibody; HUVEC: human umbilical vein endothelial cells; HDMEC: human dermal microvascular cells; # P <0.01.

Article Snippet: Human endothelial cell lines (passage 7-10), human umbilical vein endothelial cells (HUVEC,) and human dermal microvascular endothelial cells (HDMEC) (PromoCell, Heidelberg, Germany), were cultured in endothelial cell growth medium and endothelial cell growth medium MV2 (PromoCell) respectively.

Techniques: Binding Assay, In Vitro, Immunofluorescence, Clone Assay, Incubation

Journal: Theranostics

Article Title: Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors

doi: 10.7150/thno.45973

Figure Lengend Snippet: Endothelial antibody capture in isolated perfused mouse livers, ex vivo . (A, B) Representative macroscopic (A) and microscopic (B) images of fluorescence microscopy after segment perfusion. Segments (S) are labeled RA, LA: right and left anterior; RP, LP: right and left posterior; RM: right middle; O: omental. (C) Antibody capture efficacy and tissue concentrations (D) after perfusion either the whole liver or a liver segment with 200 ng RPE-labeled clone 390 mAb, n=3. (E) EC 50 values for clone 390 at different degrees of labeling (DOL: fluorophore/protein ratio) in mouse pancreas. (F) Antibody capture efficacy in segments perfused with 200 ng of antibody at different DOLs; n=3. (G) Antibody capture efficacy after segments were perfused with 200 ng of four different fluorophore-labeled antibodies: AF488, RPE, high degree of FITC (DOL: 8.5), and high degree of AF647 (DOL: 9.3); n=3-4. (H) Mean fluorescence intensity (MFI) ratios indicate local antibody enrichment and imaging contrast for different fluorophore-labeled antibodies. n.s. no significant difference; * P <0.05. # P <0.01.

Article Snippet: Human endothelial cell lines (passage 7-10), human umbilical vein endothelial cells (HUVEC,) and human dermal microvascular endothelial cells (HDMEC) (PromoCell, Heidelberg, Germany), were cultured in endothelial cell growth medium and endothelial cell growth medium MV2 (PromoCell) respectively.

Techniques: Isolation, Ex Vivo, Fluorescence, Microscopy, Labeling, Imaging

Journal: Theranostics

Article Title: Fluorescence-guided fiber-optic micronavigation using microscopic identification of vascular boundary of liver segment and tumors

doi: 10.7150/thno.45973

Figure Lengend Snippet: Visualization and analysis of a perfused liver segment with confocal laser endomicroscopy (CLE). (A) Dose-response analysis of endothelial antibody capture efficacy for AF488-labeled clone 390 mAb; n=3. (B) Representative images compare resolutions of conventional fluorescence microscopy and CLE. (C) Antibody capture efficacy after segment perfusion with 1200 ng of different mAbs, n=3. (D) Tissue antibody concentration and (E) local AF488 dye enrichment after perfusion segments with 1200 ng of different antibodies. (F) Antibody capture efficacy after perfusion with different doses of clone YN1/1.7.4 antibody, n=3-4. (G) Representative images of segments after perfusion 1200 ng of clone YN1/1.7.4 antibody (CD54) or the combination of clones YN1/1.7.4 and 390 (1200 ng each). (H) Quantitative analysis of the change in mean fluorescence intensity (MFI) for two antibodies at different doses, compared to 800 ng of clone 390. n.s. no significant difference. * P <0.05. # P <0.01.

Article Snippet: Human endothelial cell lines (passage 7-10), human umbilical vein endothelial cells (HUVEC,) and human dermal microvascular endothelial cells (HDMEC) (PromoCell, Heidelberg, Germany), were cultured in endothelial cell growth medium and endothelial cell growth medium MV2 (PromoCell) respectively.

Techniques: Labeling, Fluorescence, Microscopy, Concentration Assay, Clone Assay