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