Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondria are efficiently internalized by HPMEC. (A) Representative live imaging of mitochondrial transplantation. Mitochondria isolated from MSC (MSC-mt) were prestained with MitoTracker Red and co-cultured with HPMEC prestained with MitoTracker Green for 24 h. Top panels: control. Middle panels: MSC-mt. Confocal imaging of internalized MSC-mt mitochondria (Mitotracker Red) co-localized to endogenous mitochondria network of non-stimulated HPMEC (MitoTracker Green). Lower panel: LPS + MSC-mt. MSC-mt internalized and co-localized to the endogenous mitochondrial network of HPMEC stimulated with LPS. The images were taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of orthogonal views (Scale bar = 20 μm) and 3D reconstruction (Scale bar = 5 μm) on the LPS + MSC-mt group. (C) Analysis of region of interest (ROI) colocalization of control, positive control, and MSC-mt group. Data presented as mean ± SD.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Imaging, Transplantation Assay, Isolation, Cell Culture, Control, Microscopy, Positive Control

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: MSC-mt transplantation effects on mtDNA copies, viability, inflammatory activation, and mitochondrial respiration of recipient cells. (A) Quantification of mtDNA copy numbers in LPS-stimulated HPMEC after mitochondrial transplantation (24 h). (B) Effects on HPMEC viability measured by levels of LDH release. ( n = 3–4). (C) Levels of interleukin (IL)-8 secretion by HPMEC measured by ELISA ( n = 3–4). (D) Levels of TNF-α secreted by THP1 macrophages measured by ELISA ( n = 3–4). (E) Representation of Seahorse Mito Stress assay curve showing OCR in HPMEC. (F–H) Values for respiratory parameters: basal respiration (F), maximal respiration (G), and ATP production (H) ( n = 4–5). Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Activation Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation alleviates mitochondrial dysfunction in HPMEC. (A) Representative images of JC-1 fluorescence in HPMEC. Green areas indicate depolarized mitochondrial membranes (JC-1 monomers), and red areas indicate polarized mitochondrial membranes (JC-1 aggregates). FCCP was used to induce mitochondria depolarization. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (B) Representative images of HPMEC mitochondrial superoxide production detected with MitoSOX. Mitotempo (MT) was used as positive control for ROS quenching. Images taken using Leica SP8 confocal microscope (Scale bar = 20 μm). (C) Quantification of red to green JC-1 fluorescence intensity ratio in HPMEC analyzed by ImageJ software. (D) Quantitative MitoSOX fluorescence intensity (MFI) analyzed by ImageJ software. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. Differences were assessed using one-way ANOVA analysis with post hoc Bonferroni’s test.

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Fluorescence, Microscopy, Positive Control, Software

Journal: Stem Cells Translational Medicine

Article Title: Transplantation of mesenchymal stromal cell-derived mitochondria alleviates endothelial dysfunction in pre-clinical models of acute respiratory distress syndrome

doi: 10.1093/stcltm/szaf053

Figure Lengend Snippet: Mitochondrial transplantation restores HPMEC barrier integrity in the presence of LPS or ARDS plasma. (A) Real-time impedance analysis of HPMEC exposed to LPS and treated with MSC-mt and their respective cell impedance analysis of XCelligence RTCA measurements at a 24-h timepoint. Data are illustrated as boxplots. The band indicates the median, the box indicates the interquartile range (IQR) of 25%–75%, and the whiskers denote the rest of the data distribution. (B) Representative real-time impedance analysis of HPMEC exposed to hypoinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. (C) Representative real-time impedance analysis of HPMEC exposed to hyperinflammatory ARDS plasma and their respective cell impedance analysis of XCelligence RTCA measurements at a 24 and 48-h timepoint. Data presented as mean ± SD. Differences were assessed using Kruskal–Wallis with post-hoc Dunn’s test (A) and one-way ANOVA analysis with post hoc Bonferroni’s test (B, C).

Article Snippet: Primary human pulmonary microvascular endothelial cells (HPMEC) were obtained from commercial vendor Innoprot, (Spain).

Techniques: Transplantation Assay, Clinical Proteomics

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: 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: 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