Augmentation of Vasculogenesis by Injected Human Hematopoietic Stem Cells in Response to Ischemia Follows Early Activation of Innate Immune Response.

Abstract

The immunocompromised NOD.SCID murine model of hindlimb ischemia has been used extensively to test in vivo the impact of human hematopoietic stem cell (HSC) therapy on neovascularization. Although NOD.SCID lack T and B cell adaptive immune responses, innate responses are intact. We utilized this model to test the hypothesis whether injection of human HSC following femoral ligation impacts neovascularization within the ischemic bed and whether early murine innate cellular responses differ in animals treated with HSC. NOD.SCID underwent femoral ligation and were divided into 3 study groups: controls (media alone), human HSC (CD133+ HSC selected from umbilical cord blood-UCB), or human peripheral blood mononuclear cells. Laser Doppler studies day +28 after femoral ligation showed significantly improved blood flow ratio in animals treated with human HSC vs. controls, 0.55 ± 0.06 (n=9) and 0.37 ± 0.03 (n=12, p<0.05) respectfully. Histology day +42 confirmed increased capillary density in animals treated with human HSC (320±18 cells/mm2) when compared to control animals (131±.6.9 cells/mm2; p<0.0001). Gene expression microarrays (Affymetrix Murine GeneChip 430 2.0) of RNA extracted from tissues harvested from the ischemic limbs 3 days following femoral ligation and injection of cells (n=3 each experimental group) comparing control mice to those receiving human HSC vs. controls revealed 414 genes with 2-fold or greater change. Included in these were genes regulating innate immunity: monocyte chemoattractant protein-5 precursor (MCP-5), CCL11, CXCL5, CXCL9, IL4 receptor, CXCL10, CCL5 and toll-like receptor 3 (TLR3). Of note large expression differences were noted comparing animals treated with human HSC including kruppel-like factor 15 (4 fold increase), IL6 receptor (3.25 fold increase), cyclin A2 (4.25 fold decrease). No significant increases in genes regulating angiogenesis were noted comparing animals treated with human HSC vs controls, including VEGF, angiopoietin, or bFGF. In summary, NOD.SCID treated with human HSC after femoral ligation demonstate significantly improved capillary density and blood flow recovery at later time points (day 28–42). Proteins regulating innate immunity are significantly higher at early time points in animals treated with human HSC suggesting an important role for inflammatory responses leading to augmentation of vasculogenesis after acute vascular injury.