Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

doi: 10.1073/pnas.2022091118

Figure Lengend Snippet: Fig. 1. Differentiation of B6 and B6HIP iPSCs into iCMs and iECs. (A) B6 iPSCs were differentiated into iECs and iCMs. (B) During the differentiation, the cells lost their typical pluripotent features of iPSCs and B6 iECs adopted genetic markers of endothelial cells and B6 iCMs of cardiomyocytes (representative gels of two independent experiments). (C and D) B6 iECs expressed Cd31 and VE-cadherin (C, representative pictures of five independent experiments) and formed tubular structures in vitro (D, representative pictures of five independent experiments). (E) B6 iCMs expressed alpha-sarcomeric actinin and troponin I (representative pictures of five independent experiments). (F) B6HIP iPSCs were differentiated into iECs and iCMs. (G) During the differentiation, the cells lost their typical pluripotent features of iPSCs and B6HIP iECs adopted genetic markers of endothelial cells and B6HIP iCMs of cardiomyocytes (representative gels of two independent experiments). (H and I) B6HIP iECs expressed Cd31 and VE-cadherin (H, representative pictures of five independent experiments) and formed tubular structures in vitro (I, representative pictures of five independent experiments). (J) B6HIP iCMs expressed alpha-sarcomeric actinin and troponin I (representative pictures of five independent experiments).

Article Snippet: The iEC phenotype was confirmed by immunofluorescence (IF) for expression of Cd31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Biotechnology) with secondary antibodies conjugated with AF488 or AF555 (Invitrogen).

Techniques: In Vitro

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Hypoimmune induced pluripotent stem cell-derived cell therapeutics treat cardiovascular and pulmonary diseases in immunocompetent allogeneic mice.

doi: 10.1073/pnas.2022091118

Figure Lengend Snippet: Fig. 2. Allogeneic HIP iECs facilitate ischemic limb preservation. (A) In BALB/c mice, the superficial femoral artery was ligated and partially resected to induce left lower limb ischemia. (B) Mice were left untreated or received fan-shaped injections of allo or alloHIP iECs into the surrounding tissue. (C) The study protocol included assessment of iEC survival and laser Doppler perfusion imaging and histology after 28 d. (D and E) The survival of FLuc+ iEC grafts was longitudinally followed by BLI. All allo iEC grafts were rejected over 15 d (D, 5 animals), while all alloHIP iEC grafts survived and some grafts even showed proliferation (E, 15 animals). BLI signals of individual animals are plotted, representative pictures are shown. (F) Left lower extremity perfusion was serially assessed by laser Doppler imaging and showed an improvement over time only after transplantation of alloHIP iECs (mean ± SD, 15 animals with no cell injection, 5 animals in the allo group, and 15 animals in the alloHIP group; ANOVA with Bonferroni post hoc test). (G) The sequelae of CLI after 28 d were graded according to a standardized scoring system and showed improved limb preservation with alloHIP iEC treatment (parts of whole graphs, 15 animals with no cell injection, 5 animals in the WT group, 15 animals in the HIP group; Kruskal–Wallis test). (H) Immunofluorescence staining showed no engrafted FLuc+ allo iECs in allogeneic BALB/c recipients, but engraftment of FLuc+ alloHIP iECs located along the endothelial layer of larger and smaller intramuscular vessels. Costaining showed that transplanted cells retained their VE-cadherin expression (representative pictures of five independent experiments).

Article Snippet: The iEC phenotype was confirmed by immunofluorescence (IF) for expression of Cd31 (ab28364, Abcam), and VE-cadherin (sc-6458, Santa Cruz Biotechnology) with secondary antibodies conjugated with AF488 or AF555 (Invitrogen).

Techniques: Preserving, Imaging, Transplantation Assay, Injection, Immunofluorescence, Staining, Expressing

Journal: International journal of molecular medicine

Article Title: Cathepsin L stimulates autophagy and inhibits apoptosis of ox-LDL-induced endothelial cells: potential role in atherosclerosis.

doi: 10.3892/ijmm.2012.1201

Figure Lengend Snippet: Figure 6. CATL inhibitor reverses the decrease of VE-cadherin induced by ox-LDL. (A) ox-LDL decreased the VE-cadherin protein content of ECs in a dose- dependent manner. (B) CATL inhibitor pretreatment increased the VE-cadherin protein content of ECs treated by ox-LDL. *P<0.05, n=3.

Article Snippet: Antibodies for CATL, beclin 1, LC3, caspase-3, Bcl-2, VE-cadherin, and HRP-labeled and alkaline phosphatase-labeled goat anti-rabbit IgG were obtained from Proteintech Biotechnology (Chicago, IL, USA).

Techniques:

Journal: Computational and Structural Biotechnology Journal

Article Title: Structure-based discovery and in vitro validation of inhibitors of chloride intracellular channel 4 protein

doi: 10.1016/j.csbj.2022.12.040

Figure Lengend Snippet: AMPhB and RAPA inhibits CLIC4 membrane translocation and endothelial cell migration. Top Panel: (A) HUVECs (Control) immunostained for CLIC4 (red), VE-cadherin (cell junctions, green) and DAPI (nucleus, blue) under confluent conditions show the presence of CLIC4 within cytosol and nucleus with the maintenance of endothelial cell barrier indicated by the tight cell-cell junctions. (B) HUVECs treated with 0.003% hydrogen peroxide (H 2 O 2 ) significantly increased the expression levels of CLIC4, including at the plasma membrane with disruption to the barrier functions. These effects were partially reversed when treated with (C)10 μM AMPhB or (D) 10 μM RAPA as indicated by decreased CLIC4 staining in the cells, especially at the plasma membrane. Scale bar = 200 µM. Bottom Panel Representative images of HUVECs “scratch-wounded” using a universal 10 μl pipette tip (scratched area indicated by the broken lines) that are treated with (E) control adenovirus (AdControl) or (F) adenovirus to overexpress CLIC4 (AdCLIC4) alone or with (G) 10 μM AMPhB or (F) 10 μM RAPA. Migration pattern of cells into the ‘scratch-wounded’ area indicate that both drugs attenuated AdCLIC4- induced cell migration. Scale bar = 100 μM.

Article Snippet: Non-specific protein-protein interactions were blocked with incubating the cells in 1% BSA for an hour followed by incubation with anti-CLIC4 antibody (Santa Cruz Biotechnology, clone 356.1, 1/100 dilution) and anti-VE cadherin antibody (Bio-Techne, AF938-SP, 1/500 dilution) overnight at 4 °C.

Techniques: Membrane, Translocation Assay, Migration, Expressing, Disruption, Staining, Transferring