Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Increased expression of integrin β 5 in lung tissue from PAH patients and rats with cHx + EC clone PH. (a) Representative immunohistochemistry shows enhanced integrin‐β5 staining in pulmonary arteries from PAH patients, including elevated expression in PAECs (arrows), compared to the faint integrin‐β5 staining in pulmonary arteries from control subjects. Scale bar: 50 μm. (b) PAECs from PAH patients show increased ITGB5 mRNA expression compared to control PAECs. n = 4 (control) and n = 5 (PAH). (c) Representative integrin‐β5 immunohistochemistry images show increased endothelial integrin‐β5 staining in cHx + CD117 + EC clone rats compared to faint endothelial staining in normoxia + CD117 + EC clone rats. Scale bars: 25 μm. (d) cHx + CD117 + EC clone rats had increased lung tissue mRNA level of Itgb5 . (e) Electric cell impedance sensing (ECIS) measurement of transendothelial electrical resistance indicates accelerated EC barrier formation in EC clones compared to control ECs. (f) Gene array analysis shows integrin mRNA expression switch with reduced expression of Itgb1 and Itgb8 , and upregulation of Itgb5, Itgb3 , and Itgav in CD117 + EC clones compared to control ECs. Note that the most consistent upregulation in all four clonal EC lines was for Itgb5 . PAH, pulmonary arterial hypertension; PH, pulmonary hypertension. * p < 0.05, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Expressing, Immunohistochemistry, Staining, Control, Clone Assay

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin α ν β 5 affects TGF‐β1‐induced endothelial‐to‐mesenchymal transition gene expression in rat lung CD117 + EC clones versus control ECs. (a–g) Comparison of mRNA expression in rat lung control ECs and CD117 + EC clones following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml). Abbreviations: Vehicle for TGF‐β1 (veh), transforming growth‐factor‐β1 (TGF‐β1), isotype antibody (iso) and neutralizing anti‐integrin‐ανβ5 antibody (itgανβ5). (a) increased baseline levels of Itgb5 in EC clones, but significant induction of Itgb5 in control ECs by TGF‐β1, which was ameliorated by antibody inhibition. (b) induction of Vwf by anti‐integrin ανβ5 antibody in control ECs. (c) Cnn1 was induced by TGF‐β1 in control ECs, with a trend towards reduction by anti‐integrin ανβ5 antibody. In EC clones, Cnn1 was induced by TGF‐β1. (d) Tagln was induced by TGF‐β1 in control ECs and EC clones, and anti‐integrin ανβ5 antibody treatment reduced this increased Tagln expression in control ECs (trend) and EC clones. (e) Twist1 was induced in control ECs by TGF‐β1, and EC clones had higher baseline expression of Twist1 . (f) Snai1 was induced by TGF‐β1 in control ECs, and anti‐integrin ανβ5 antibody decreased Snai1 expression in control ECs and EC clones. (g) Serpine1 was induced by TGF‐β1 in control ECs and EC clones, and anti‐integrin ανβ5 antibody reduced Serpine1 expression in control ECs. n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Gene Expression, Clone Assay, Control, Comparison, Expressing

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 mainly affects TGF‐β1‐induced endothelial‐to‐mesenchymal transition on the gene expression level in human lung microvascular ECs. (a–e) Comparison of mRNA expression in human lung microvascular ECs following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml) for 72 h. Abbreviations: Vehicle (veh), isotype antibody (iso), transforming growth factor‐β1 (ΤGF‐β1) and neutralizing anti‐integrin‐ανβ5 antibody (itgανβ5). (a) Induction of ITGB5 expression in TGF‐β1 + anti‐integrin ανβ5 antibody treated ECs. (b, c) TGF‐β1 treatment reduced VCAM1 and PECAM1 mRNA expression and was not affected by anti‐integrin ανβ5 antibody. (d) TGF‐β1 induced ACTA2 expression, and anti‐integrin ανβ5 antibody reduced TGF‐β1‐induced ΑCTA2 expression. (e) While TGF‐β1 induced a trend towards elevated SNAI1 expression, the addition of anti‐integrin ανβ5 antibody elevated SNAI1 expression significantly. (f) Representative immunoblots for VCAM1 (short isoform), α‐SMA, SM22α, SNAIL1, and β‐actin (loading control) for HLMVECs following treatment with a neutralizing anti‐integrin ανβ5 antibody (10 μg/ml) and/or TGF‐β1 (10 ng/ml) for 72 h. (g–j) VCAM1 (short isoform, G), α‐SMA (h), SM22α (i) and SNAIL1 (j) protein expression. n = 3 per group. In (h) p Values are shown to demonstrate a trend. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Gene Expression, Comparison, Expressing, Western Blot, Control

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin ανβ5 reduces gap closure in human lung microvascular ECs. (a, b) Representative differential interference contrast (DIC) images (a) show reduced gap closure following 24 h treatment with anti‐integrin ανβ5 antibody and/or TGF‐β1. Scale bar: 200 μm. (b) Quantification of percent gap closure over time. n = 3 per group. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 promotes apoptosis in human lung microvascular ECs and rat lung CD117 + EC clones, but not in control rat lung ECs. (a, b) Representative dot plots of Annexin V (AV) and 7‐aminoactinomycin (7‐AAD) flow cytometry (a) and quantification of AV + 7‐AAD − (early apoptotic) human lung microvascular ECs (HLMVECs) following 24 h treatment with isotype antibody (10 μg/ml) or anti‐integrin‐ανβ5 (ITGανβ5) antibody (5 or 10 μg/ml). (c, d) Representative dot plots of AV and 7‐AAD flow cytometry (c) and quantification of AV + 7‐AAD − rat lung control ECs and CD117 + EC clones following 24 h treatment with isotype antibody or anti‐integrin‐aνβ5 antibody (10 μg/ml) and recombinant human TGF‐β1 (5 ng/ml) or vehicle. n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Clone Assay, Control, Flow Cytometry, Recombinant

Journal: Pulmonary Circulation

Article Title: Dichotomous role of integrin‐β5 in lung endothelial cells

doi: 10.1002/pul2.12156

Figure Lengend Snippet: Inhibition of integrin‐ανβ5 exaggerates pulmonary hypertension in the rat cHx + CD117 + EC clone model. Physiology and tissue harvest was performed on day 21. (a) Representative histology images show enhanced muscularization and occlusion in cHx + CD117 + EC clone rats treated with neutralizing anti‐integrin‐ανβ5 antibody (ανβ5) versus isotype control antibody (con) (100 μg/kg three times a week from day 1–21). Scale bar: 25 μm. (b) Right ventricular systolic pressure (RVSP). (c) Fulton index of right ventricular hypertrophy: (right ventricle weight (RV) versus left ventricle + septum weight [LV + S]). (d) Media wall thickness (MWT). (e) Fraction of fully and partially occluded pulmonary arteries and not occluded pulmonary arteries. (f) ratio of pulmonary artery acceleration time (PAAT) versus pulmonary ejection time (PET) as measured by echocardiography. (g) Tricuspid annular plane systolic excursion (TAPSE) as measured by echocardiography. (h) Cardiac output estimated by echocardiography. n = 4 per group. Data are shown as single values and mean ± SEM. n.s, not significant. * p < 0.05, *** p < 0.001.

Article Snippet: Slides for anti‐integrin‐ανβ5 antibody treated rats were sectioned, stained and scanned by Histowiz.

Techniques: Inhibition, Control