Abstract
Purpose We investigated the roles of stem cell factor(SCF)–c-kit and stromal derived factor-1(SDF-1)–chemokine receptor type 4(CXCR4) stem cell signaling axes in transmyocardial revascularization (TMR)-enhanced engraftment of transplanted mesenchymal stem cells (MSC) in infarcted hearts. Methods and Materials 3 weeks after LAD ligation, female Lewis rats underwent 10-channel needle TMR, followed by daily IV injections of 1 million male donor MSC for 5 days, either wild type (WT) or with knockdown (K/D) of c-kit or CXCR4, accomplished via a shRNA+plasmid in a lentiviral vector. Experimental groups included: WT MSC with or without TMR, c-kit K/D MSC with or without TMR, and CXCR4 K/D MSC with or without TMR (N=6/group). Results In vitro cell surface expression of c-kit (N=3) was reduced from 14±0.7% of WT MSC to 1.6±0.4% c-kit K/D MSC, and CXCR4 (N=3) was reduced from 39±10% WT MSCs to 3.7±0.7% CXCR4 K/D MSC after 1 week (p Conclusions Downregulation of either c-kit or CXCR4 in MSC decreased engraftment of circulating MSC and inhibited the reparative effects of TMR. Hence, both SCF–c-kit and SDF-1–CXCR4 signaling axes are required for TMR-augmented repair of the infarcted heart.
Highlights
Objectives5.3 Hypothesis 5.4 Experimental Protocol Chapter Six: Materials and Methods 6.1 Animal Characteristics 6.2 Left Anterior Descending (LAD) Coronary Artery Ligations 6.3 Transmyocardial Revascularization (TMR) 6.4 Mesenchymal Stem Cell (MSC) Isolation 6.5 Cell Transplantation 6.6 Procuring the Hearts 6.7 CXCR4 Receptor Knockdown 6.8 C-kit Receptor Knockdown 6.9 deoxyribonucleic acid (DNA) Isolation 6.10 Quantification of the Y Chromosomal DNA by Real-Time polymerase chain reaction (PCR) 6.11 RNA Isolation 6.12 Quantification of C-kit, stem cell factor (SCF), CXCR4, and stromal derived factor-1 (SDF-1) by Real-time PCR 6.13 Flow Cytometry 6.14 Immunohistochemistry 6.15 Vascular Densities 6.16 Left Ventricular Function 6.17 Statistical Analysis !
Transmyocardial revascularization (TMR) prior to mesenchymal stem cell (MSC) transplantation augments repair of infarcted hearts
We evaluated the importance of stem cell factor (SCF)—c-kit and stromal derived factor-1 (SDF-1)—CXCR4 signaling axes in transmyocardial revascularization (TMR) mediated enhanced engraftment of MSCs in infarcted tissue, as well as improvement in left ventricle (LV) function
Summary
5.3 Hypothesis 5.4 Experimental Protocol Chapter Six: Materials and Methods 6.1 Animal Characteristics 6.2 Left Anterior Descending (LAD) Coronary Artery Ligations 6.3 Transmyocardial Revascularization (TMR) 6.4 Mesenchymal Stem Cell (MSC) Isolation 6.5 Cell Transplantation 6.6 Procuring the Hearts 6.7 CXCR4 Receptor Knockdown 6.8 C-kit Receptor Knockdown 6.9 DNA Isolation 6.10 Quantification of the Y Chromosomal DNA by Real-Time PCR 6.11 RNA Isolation 6.12 Quantification of C-kit, SCF, CXCR4, and SDF-1 by Real-time PCR 6.13 Flow Cytometry 6.14 Immunohistochemistry 6.15 Vascular Densities 6.16 Left Ventricular Function 6.17 Statistical Analysis ! 8.2 Knockdown Construct Reduces In Vitro mRNA and Cell Surface Expression of C-kit and CXCR4 8.3 TMR Increases Donor MSC Engraftment 8.4 TMR Increases Expression of Signaling Cytokines 8.5 Effect of TMR on Vascular Densities 8.6 TMR Improves Left Ventricular Function Only When SCF—C-kit and SDF-1—CXCR4 Axes are Intact 8.7 Conclusions and Clinical Implications 8.8 Study Limitations 8.9 Future Directions Chapter Nine: References 9.1 References Appendix 1 Transmyocardial Revascularization Induces Mesenchymal Stem Cell Engraftment in Infarcted Hearts ! Knockdown of c-kit and CXCR4 was accomplished via a short hairpin RNA (shRNA)+plasmid, using a lentiviral vector delivery system
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