Journal: bioRxiv

Article Title: Tissue fibroblasts are a critical source of prostacyclin and anti-thrombotic protection

doi: 10.1101/2022.01.11.475814

Figure Lengend Snippet: (A) FACS gating strategy (representative plots) and (B) prostacyclin release (measured as 6kPGF 1α after arachidonic acid 30μM stimulation) (n=4) of endothelial cells from matched mouse aorta and lung parenchyma. (C) FACS gating strategy (representative plots) and (D) prostacyclin release (n=4 independent isolations from n=2 donors) of endothelial cells from matched human pulmonary artery and lung. In each, endothelial cells were defined as DAPI-, CD41-, CD45-, CD31+ events. (E) FACS gating strategy (representative plots) and (F) prostacyclin release from endothelial cells (EC), leucocytes (Leuco), type 1 (T1 Epi) and other epithelial cells (Other Epi), smooth muscle cells (SMC) and adventitial (Fibro Adv), alveolar (Fibro Alv) and peribronchial fibroblasts (Fibro PeriB) from mouse lung (n=6-12). (G) FACS gating strategy (representative plots) and (H) prostacyclin release from EC, Leuco, T1 Epi, Other Epi, SMC and negatively selected mesenchymal cells (Neg Mesenc) from human lung (n=7-11 donors). (I) Representative brightfield images and (J) prostacyclin release (after arachidonic acid 30μM stimulation) (n=3 donors) from cultured primary human lung microvascular endothelial cells (Lung EC) and human lung fibroblasts (Lung Fibro). (K) EGFP fluorescence (green) in lung segments of ROSA mT/mG mice with/without a Fsp1-Cre transgene (representative of n=3/genotype). Prostacyclin release (after A23187 Ca 2+ ionophore 30μM stimulation) from (L) lung parenchyma segments (n=12-16), (M) aorta (n=12-16), (N) pulmonary artery (n=6-7) and (O) bronchi (n=6) from fibroblast-specific cyclo-oxygenase-1 knockout (Fibro COX1 KO) and floxed littermate control mice (Flox Ctrl). Plots show 5% density contours. Data are mean ± SEM with p values by repeated measures one-way ANOVA with Holm-Sidak post-test (F,H) or unpaired (B, D, J, L-O) t-test indicated where p<0.05.

Article Snippet: Human primary lung microvascular endothelial cells and human primary lung fibroblasts (Promocell, Germany) from 3 individual donors were cultured according to suppliers instructions in full endothelial growth factor-2 media (Promocell, Germany) supplemented with 10% fetal calf serum (Biosera, UK) and penicillin/streptomycin (Sigma, UK).

Techniques: Cell Culture, Fluorescence, Knock-Out

Journal: bioRxiv

Article Title: Aging-regulated TUG1 is dispensable for endothelial cell function

doi: 10.1101/2022.02.27.482212

Figure Lengend Snippet: (A) Top 10 expressed lncRNAs based on transcript counts from HUVEC bulk RNA sequencing data (n = 4). TUG1 is highlighted in green. Glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ) and Kinase Insert Domain Receptor ( KDR ) were used as controls. (B) RNA expression levels of TUG1 in different human cell types of the cardiovascular system (n=3). Vascular ECs are highlighted by grey bars. AoEC: Aortic ECs, PAEC: Pulmonary Artery ECs, CAEC: Coronary Artery ECs, CMEC: Cardiac Microvascular ECs, DMEC: Dermal Microvascular ECs, PMVEC: Pulmonary Microvascular ECs, SaVEC: Saphenous Vein ECs, HUVEC: Human Umbilical Vein ECs, DLEC: Dermal Lymphatic ECs, MSC: Mesenchymal Stem Cells, AoAF: Aortic Arterial Fibroblasts, AoSMC: Aortic Smooth Muscle Cells, CM: Cardiomyocytes (C) TUG1 expression levels in low (P3) vs. high (P16) passage HUVECs as determined by RT-qPCR. Expression is relative to GAPDH (n = 5-6; SEM; Mann-Whitney-test). (D) Tug1 expression from bulk RNA-sequencing data of the intima of the carotid arteries of young (10 weeks) vs. aged mice (18 months) (n = 3; SEM; Mann-Whitney-test).. (E) Quantification of the expression levels of the lncRNAs Differentiation Antagonizing Non-Protein Coding RNA ( DANCR ), TUG1 and Metastasis Associated Lung Adenocarcinoma Transcript 1 ( MALAT1 ) in subcellular fractions of wild type HUVECs using RT-qPCR (n=3). Results are expressed as percentages of the subcellular fractions associated to cytoplasm, nucleoplasm and chromatin. Expression is normalized to GAPDH as determined by RT-qPCR.

Article Snippet: Alternatively, total RNA was isolated from cell pellets from cardiomyocytes, aortic fibroblasts, pericytes, aortic smooth muscle cells, mesenchymal stem cells, dermal lymphatic endothelial cells, umbilical vein endothelial cells, saphenous vein endothelial cells, pulmonary microvascular endothelial cells, dermal microvascular endothelial cells, cardiac microvascular endothelial cells, coronary artery endothelial cells, pulmonary artery endothelial cells and aortic endothelial cells (all human; Promocell) with the miRNeasy Micro Kit (Qiagen) according to the manufacturer’s instructions including DNase digest.

Techniques: RNA Sequencing Assay, RNA Expression, Expressing, Quantitative RT-PCR, MANN-WHITNEY

Journal: Shock (Augusta, Ga.)

Article Title: Fibrinogen protects against barrier dysfunction through maintaining cell surface syndecan-1 in-vitro

doi: 10.1097/SHK.0000000000001207

Figure Lengend Snippet: A. Human lung microvascular endothelial cells (HLMECs) were cultured in EBM-2 containing 2% FBS for 2 hours and then incubated with the media containing 10% LR, 10% FFP, and 10 mg/ml fibrinogen for 6 hours. Cell lysates were analyzed with Western blot for syndecan-1 protein levels (top panel). Cells were also immunostained with anti-syndecan-1 antibody and images were captured with Nikon E800 fluorescence microscope with an original magnification of 600 (middle panel, same below). The fluorescence intensity was quantified using Quantity One and reported as relative fluorescence units (RFU). Results are presented as means±SEM, n=4/group (bottom panel). B. Fibrinogen increases cell surface syndecan-1 at a dose comparable to FFP. HLMECs were cultured in EBM-2 containing 2% FBS for 2 h and then incubated with the media containing 10% LR, 2.5, 5.0 and 10.0 mg/ml fibrinogen for 6 h. Cells were immunostained with anti-syndecan-1 antibody and images were captured (top panel). The fluorescence intensity results are presented as means±SEM, n=4/group (bottom panel). C. Fibrinogen increases cell surface fibrinogen. HLMECs were treated as described in A. Cells were immunostained with anti-fibrinogen antibody and the images were captured (top panel). Fibrinogen fluorescence intensity was reported as relative fluorescence units (RFU). Results are presented as means±SEM, n=4/group (bottom panel).

Article Snippet: Primary cell culture Human lung microvascular endothelial cells (HLMVEC; PromoCell) were grown to confluence in endothelial basic medium-2 (EBM-2; Lonza) supplemented with 10% fetal bovine serum (FBS), human recombinant epidermal growth factor, human recombinant insulin-like growth factor-1, human basic fibroblast growth factor, vascular endothelial growth factor, hydrocortisone, ascorbic acid, heparin, gentamicin, and amphotericin B. Endothelial cells (passages 6–8) were incubated with EBM-2 containing 2% FBS prior to experiments.

Techniques: Cell Culture, Incubation, Western Blot, Fluorescence, Microscopy

Journal: The American Journal of Pathology

Article Title: Treatment with Anti–Gremlin 1 Antibody Ameliorates Chronic Hypoxia/SU5416–Induced Pulmonary Arterial Hypertension in Mice

doi: 10.1016/j.ajpath.2013.07.017

Figure Lengend Snippet: Gremlin 1 expression in pulmonary arterial hypertension (PAH) clinical samples. A: Representative of high magnification images of colocalized expression of vWF and Grem1 in human pulmonary vessel endothelium. Arrows indicate positive staining. B: Lung sections from biopsies of controls and PAH patients were stained with an anti–Gremlin 1 antibody. Gremlin 1 staining in PAH patient is associated with plexiform lesions. Arrows indicate positive Gremlin 1 staining. C: Bone morphogenetic protein receptor type 2 (BMPR2) and Gremlin 1 mRNA analysis in primary microvascular endothelial cells (HMVEC) and pulmonary arterial smooth muscle cells (PASMCs). Quantitative changes in gene expression were analyzed by RT-PCR normalized to GAPDH (ΔΔCt method). Data shown are representative from one donor of PASMCs and HMVECs. The experiments were performed in triplicate, with similar results observed in at least one other donor. D: Gremlin 1 mRNA expression in lungs from PAH patients. Secondary PAH lung samples were obtained from NDRI (n = 3). Quantitative changes in gene expression were analyzed by RT-PCR normalized to GAPDH (ΔΔCt method). Original magnification, ×20 (A and B).

Article Snippet: Cell Culture of HMVECs and PASMCs HMVECs and PASMCs were obtained from Promocell; additional pulmonary arterial smooth muscle cells were purchased from Lonza.

Techniques: Expressing, Staining, Reverse Transcription Polymerase Chain Reaction