Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 1. Characterization of rabbit EPCs and comparison with HUVECs. A, B, Cobblestone morphology of EPCs and HUVEC 10 days after plating (40); C, D, immunohistochemical detec- tion of CD31; immunofluorescence detection of (E, F), VE-cadherin; (G, H), eNOS; (I, J), vWF. Preparations were viewed at 400 unless otherwise indicated. Preparations in G, H, I, and J were counterstained with Hoechst 33342.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Comparison, Immunohistochemical staining

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 2. Ex vivo retroviral transduction and transgene expression in EPCs. A, Confluent monolayer of EPCs trans- duced with MSCV retrovirus expressing GFP viewed under normal (white) light (100); B, view of same field under green fluorescent light; C, D, human eNOS transgene mRNA and protein expression in untransduced and trans- duced EPCs, respectively; E, F, HO-1 mRNA and protein expression in untransduced and transduced EPCs, respectively.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Ex Vivo, Retroviral, Transduction, Expressing

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 3. In vivo transgene expression after transplantation of EPCs onto injured carotid arteries. A, Immunohistochemical detection of eNOS (arrows) in frozen sections from denuded vessel trans- planted with eNOS-EPCs (250). B, eNOS immunoreactivity in sections from control vessel transplanted with GFP- EPCs (250); C, immunohistochemical detection of human HO-1 (arrows) in fro- zen sections from vessel transplanted with HO-1-EPCs (250); D, control ves- sels transplanted with GFP-EPCs (250); E, detection of human eNOS transgene mRNA expression by RT-PCR; F, human eNOS protein expression in homoge- nates from eNOS-EPC–transplanted ves- sels; G, human HO-1 transgene mRNA expression; H, HO-1 protein expression in homogenates from HO-1-EPC–trans- planted vessels. GFP-EPC–transplanted vessels served as controls.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: In Vivo, Expressing, Transplantation Assay, Immunohistochemical staining, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 4. Reendothelialization of injured arteries after transplan- tation of EPCs expressing GFP, eNOS, or HO-1 genes. Low- power (100) magnification of vessel profiles stained with anti- CD31 antibody in A, unseeded; B, GFP-EPCs; C, eNOS-EPCs; and D, HO-1-EPCs. Red CD31-positive staining indicates pres- ence of an endothelial monolayer. E, Quantification of endotheli- alization by planar morphometry. Endothelialization was enhanced in EPC-transplanted vessels compared with unseeded saline-treated controls (*P0.05, n4/group).

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Expressing, Staining, Saline

Journal: Circulation

Article Title: Enhanced Inhibition of Neointimal Hyperplasia by Genetically Engineered Endothelial Progenitor Cells

doi: 10.1161/01.cir.0000121732.85572.6f

Figure Lengend Snippet: Figure 5. Inhibition of neointimal proliferation by EPCs in injured carotid arteries. All sections were stained with Accustain elastic stain. Low (40, left) and high (200, right) magnification of vessel profiles. A, B, Saline-injured artery transplanted with eNOS-EPCs (n6); C, D, balloon-injured artery transplanted with EPC expressing GFP (n6); E, F, balloon-injured artery transplanted with EPC expressing eNOS (n6); G, H, balloon- injured artery transplanted with HO-1-EPCs (n6); I, neointima/ media ratios in unseeded and EPC-transplanted injured vessels. Arrows indicate neointima. *Saline vs GFP-EPCs, eNOS-EPCs, HO-1-EPCs; †eNOS-EPCs vs GFP-EPCs, P0.05.

Article Snippet: The cells were incubated with 1:50 dilution of monoclonal anti human CD31 (PECAM-1, Dako, Carpinteria, Calif), or polyclonal goat anti-mouse VE-cadherin or anti-human eNOS (R&D Systems Inc, Minneapolis, Minn), or anti-human von Willebrand factor (vWF) (Dako).

Techniques: Inhibition, Staining, Saline, Expressing

Journal: Journal of vascular surgery

Article Title: Neointimal hyperplasia on a cell-seeded polytetrafluoroethylene graft is promoted by transfer of tissue plasminogen activator gene and inhibited by transfer of nitric oxide synthase gene.

doi: 10.1016/j.jvs.2004.10.023

Figure Lengend Snippet: Fig. 2. Immunostaining of neointima. Paraffin sections of grafts seeded with SMC/tPA (a), SMC/tPANOS (c), or SMC (b, d) were stained with antibody against human tPA (a, b) or antibody against human eNOS (c, d). Black arrows indicate positively stained cells in brown. White arrows indicate negtively stained cells with nuclear stained in blue.

Article Snippet: Biotinylated anti-human eNOS goat immunoglobulin G (1:5 dilution) was from R & D Systems, Inc (Minneapolis, Minn).

Techniques: Immunostaining, Staining

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Mechanical stretch upregulated SGLT1/2 protein levels, promoted nuclear translocation of p‐ERK, and induced p‐eNOS expression. (a–d) Green fluorescence indicating SGLT1, SGLT2, p‐ERK, and p‐eNOS‐positive cells in cardiomyocytes after 24 h of 5%, 25% cyclic stretch. Cells were counterstained in 4,6‐diamidino‐2‐phenylindole to exhibit blue fluorescence. Scale bar = 50 μm. Bar graphs representing SGLT1, SGLT2, p‐ERK, and p‐eNOS fluorescence intensity. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test.

Article Snippet: Troponin I antibody (GeneTex; GTX113028), P‐ERK antibody (Cell Signaling; CS4370S), P‐eNOS S633 (BD 612665), SGLT1 (Abcam; ab14686) and SGLT2 (Abcam; ab85626) were used in this study.

Techniques: Translocation Assay, Expressing, Fluorescence, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Mechanical stretch induced SGLT1/2 expression, upregulated ERK and eNOS activation. (a) Fluorescence images showing cardiomyocytes positive for SGLT1/2, p‐ERK, and p‐eNOS. DAPA (SGLT2 inhibitor) or PD98059 (ERK inhibitor) were added to cells, and stretched at 5%, 25% for 24 h. Scale bar = 50 μm. (b–d) Quantifications of SGLT1/2 and p‐ERK. (e) p‐eNOS fluorescence intensity in the stretched cells. Data represented at least three independent experiments in median (interquartile range). Data were analyzed with the Mann–Whitney U test.

Article Snippet: Troponin I antibody (GeneTex; GTX113028), P‐ERK antibody (Cell Signaling; CS4370S), P‐eNOS S633 (BD 612665), SGLT1 (Abcam; ab14686) and SGLT2 (Abcam; ab85626) were used in this study.

Techniques: Expressing, Activation Assay, Fluorescence, MANN-WHITNEY

Journal: Physiological Reports

Article Title: Dapagliflozin prevents ERK activation and SGLT2‐dependent endoglin upregulation in a mechanically provoked cardiac injury model

doi: 10.14814/phy2.15990

Figure Lengend Snippet: Proposed mechanism for Dapagliflozin on troponin I, endoglin (CD105) and ERK activation in the mechanically injured cardiac cell model. Our model depicts the upregulation of troponin I and endoglin (CD105) after mechanically triggered cardiac injury. (a) Cardiomyocytes were seeded on stretchable PDMS membrane, subjected to cyclic stretch at 5% and 25% elongation. After cyclic stretch, troponin I and CD105 were increased, triggering downstream p‐ERK to enter cell nucleus, potentially altered gene transcription that promoted upregulation of p‐eNOS. The accumulated p‐ERK nuclear translocation and p‐eNOS activation led to an imbalanced regulation of sodium/calcium ions. Finally, the cardiomyocytes were overwhelmed by loss of sodium/calcium ions homeostasis, renting the cells susceptible to CVD‐related risk factor. (b) Dapagliflozin (DAPA), the inhibitor having higher affinity for SGLT2, is able to prevent the characteristics of the mechanically provoked biomarkers for cardiac injury. As a result, SGLT2 and/or SGLT1 is crucial in maintaining the normal function of cardiomyocytes.

Article Snippet: Troponin I antibody (GeneTex; GTX113028), P‐ERK antibody (Cell Signaling; CS4370S), P‐eNOS S633 (BD 612665), SGLT1 (Abcam; ab14686) and SGLT2 (Abcam; ab85626) were used in this study.

Techniques: Activation Assay, Membrane, Translocation Assay