The Vascular Protective Effects of Over-expression of MicroRNA126 in Macrophages

Objectives: Construct a lentivirus vector expressing miR126 to test the hypothesis that genetically manipulated macrophage expression of miR126 will facilitate vascular protection
Methods: Primary miR126 DNA sequence was amplified by PCR and the product was cloned into Lenti-SMP-GFP (lentivirus vector with super macrophage promoter) by cold fusion to replace GFP reporter gene. Validation via sequencing, restriction enzyme digestion and luciferase activity was performed followed by cytotoxicity assays to determine the effect of miR126 on its endogenous targets in macrophages. Whole mouse genome microarray analysis of macrophages over-expressing miR126 was used to detect changes in genes/pathways relevant to macrophage function and atherosclerosis pathology. Lastly 8 week old apoE-/-mice were transplanted with HSC-enriched bone marrow cells transduced with lentivectors expressing either GFP (Lenti-SMP-GFP, control) or miR126 (Lenti-SMP-miR126) and after 4 weeks recovery, and 8weeks of of high fat diet, animals were sacrificed and various parameters relevant to atherosclerosis pathology measured.

Results: Over-expression of miR126 in macrophages displayed the ability of miR126 to effectively repress its endogenous targets without any cytotoxic effects as determined by proliferation, phagocytosis and cytokine profiles. Furthermore, miR126 over-expression in vivo displays a therapeutic reduction in atherosclerosis plaque size that may be attributed to miR126 regulation of progenitor cell mobilization and/or modulation of lipid biosynthesis via SCD1, an enzyme involved in lipid biosynthesis and regulation of cholesterol transporters
Conclusions: Over-expression of miR126 in macrophages represents a novel therapeutic in atherosclerosis pathology. NIDCR, Grant# DE023285-01A1