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: 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: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Western Blot, Infection, BrdU Incorporation Assay

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Pulmonary TSP1 and CD47 are upregulated in human subjects with PAH and in experimental PAH. (A) Lung tissue from a historical cohort of non-PAH, IPAH or SCD was probed by western blot for TSP1 and β-actin. (western blot is the only representative). Densitometry is the result of 10 non-PAH patients, five SCD, 5 IPAH patients and is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (B) Western blot analysis of lung samples from a prospective cohort of non-PAH and PAH patients probed for CD47 and β-actin. Densitometry is the result of three non-PAH and five PAH patients. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with non-PAH patients. (C) TSP1 and CD47 mRNA expression levels in lung samples from the prospective cohort. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over non-PAH and normalized with 18S ribosomal subunit levels as endogenous non-PAH. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with control. Hash (#) indicates statistically significant difference (P < 0.05) compared with non-PAH. (D) Representative western blot of lung tissue from normoxic and chronically hypoxic (21 days normobaric 10% O2) C57BL/6 wild-type and TSP1 null mice probed against TSP1 and β-actin. Densitometry is presented as mean ratio of TSP1 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxic mice and is based on an analysis of eight mice per group. (E) Representative western blot probed against TSP1 and α-tubulin from lung samples from normoxic and acute hypoxia (normobaric 7.5% O2) challenged wild-type mice. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of TSP1 to α-tubulin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared to normoxia and is based on four mice at each time point. (F) TSP1 mRNA expression levels in lung samples from wild-type and TSP1 null mice exposed to normoxia or hypoxia (normobaric 7.5% O2) for the indicated time points. Representative data from three independent experiments are presented. mRNA levels are expressed as fold change over normoxic conditions and normalized with HPRT levels as endogenous control. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Representative western blot of wild-type and TSP1 null pulmonary microvascular endothelial cells exposed to normoxia or hypoxia (normobaric 1% O2) for 12 h probed against CD47 and α-tubulin. Representative data from three independent experiments are presented. Densitometry is presented as mean ratio of CD47 to α-tubulin (± SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (H) Representative western blot from wild-type mice exposed to acute hypoxia for the indicated time points probed against CD47 and β-actin. Densitometry from analysis of western results from four mice in each treatment group is presented as mean ratio of CD47 to β-actin (±SD). Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Western Blot, Expressing, Control

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Activated CD47 regulates hypoxic eNOS by altering Cav-1CD47 co-association. (A) Western blot of lung tissue from wild-type and TSP1 null mice exposed to chronic hypoxia or room air probed against total eNOS protein, eNOS phosphorylation at serine-1176 (murine residue), Cav-1, and β-actin. Quantification of densitometric analysis of the ratio of (B) total eNOS to β-actin, (C) p-eNOS to total eNOS, (D) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. Hash (#) indicates statistically significant difference (P < 0.05) compared with wild-type normoxic. All ratios represent mean ± SD and are the result of the total cohort (n = 8 animals per group). (E) Co-immunoprecipitation in human pulmonary microvascular endothelial cells of Cav-1 and CD47. Immunoprecipitation was with monoclonal antibodies to Cav-1, CD47, or an isotype-matched control IgG antibody. Results presented are representative of four separate experiments. (F) Human pulmonary microvascular endothelial cells were serum starved and then treated with TSP1 (2.2 nmol/L) ± hypoxia (1% O2 for 4 h), lysate prepared and immunoprecipitation for CD47 performed with protein blotted for Cav-1. Results are presented as the quantification of densitometric analysis from three separate experiments of the ratio of Cav-1 to CD47 following immunoprecipitation. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with normoxia. (G) Western blot of pulmonary Cav-1 from lung lysate from chronically hypoxic (21 days normobaric 10% O2) wild-type (n = 3) and CD47 null (n = 4) mice. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with wild-type.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Western Blot, Phospho-proteomics, Residue, Immunoprecipitation, Bioprocessing, Control

Journal: Cardiovascular Research

Article Title: Activated CD47 promotes pulmonary arterial hypertension through targeting caveolin-1

doi: 10.1093/cvr/cvr356

Figure Lengend Snippet: Blocking CD47 activation upregulates Cav-1 to inhibit eNOS-derived superoxide production in hypoxic pulmonary arterial endothelial cells. (A) Representative western blot of hPAEC exposed to normoxia, hypoxia (1% O2 for 12 h), or hypoxia plus a CD47-blocking antibody (αCD47, clone B6H12, 1 µg/mL). Quantification of densitometric analysis of the ratio of (B) TSP1 to β-actin and (C) Cav-1 to β-actin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (D) Superoxide production in hPAEC exposed to normoxia, hypoxia (1% O2 for 12 h), hypoxia plus L-NAME (100 mmol/L), or hypoxia plus a CD47-blocking antibody (clone B6H12, 1 µg/mL). Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic control. (E) Western blot analysis for Cav-1 expression in hPAEC transfected with control or Cav-1 siRNA. (F) Superoxide production in hPAEC transfected with control or Cav-1 siRNA then exposed to 24 h hypoxia (1% O2) with or without a CD47-blocking antibody. Data represent three independent experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) compared with hypoxia control alone. Hash (#) indicates statistically significant difference (P < 0.05) compared with hypoxia alone. (G) Wild-type and TSP1 null pulmonary microvascular endothelial cells harvested from 10-week-old male animals were grown to 80% confluence, treated with normoxia or hypoxia (1% O2) ± TSP1 (2.2 or 22 nmol/L) as indicated for 24 h, cell lysate prepared and western blot analysis for Cav-1 expression performed. A representative blot and accompanying densitometry from three separate experiments are presented. Quantification of densitometric analysis of the mean ratio of Cav-1 to α-tubulin. Asterisk (*) indicates statistically significant difference (P < 0.05) from normoxic untreated. Hash (#) indicates statistically significant difference (P < 0.05) from normoxic wild-type + 22 nmol/L TSP1 and hypoxic wild-type. (H) Western blot analysis for phospho-Cav-1Tyr14 expression in normoxic hPAEC treated with the indicated amounts of TSP1 (2.2 nmol/L) ± a CD47 antibody (B6H12, 1 µg/mL) for 30 m. Quantification of densitometric analysis of mean ratio p-Cav-1 to total Cav-1 of three separate experiments. Asterisk (*) indicates statistically significant difference (P < 0.05) from untreated and antibody alone. Hash (#) indicates statistically significant difference (P < 0.05) from TSP1 treated.

Article Snippet: 26 Human pulmonary arterial and microvascular endothelial cells were purchased from Lonza (Switzerland).

Techniques: Blocking Assay, Activation Assay, Derivative Assay, Western Blot, Control, Expressing, Transfection