Abstract 5596: Differential Paracrine Dysfunction and Hypoxic Desensitization in Diabetic CD34+ and CD14+ Bone Marrow Derived Cells (BMDC)
Purpose: To test the hypothesis that BMDC of diabetic (D) origin have impaired responsiveness to the hypoxic microenvironment and ineffective paracrine signaling.
Methods: FACS sorted CD34+ and CD14+ cells from D and non diabetic (n=10) (ND) individuals (n=10) were exposed to normoxia (pO2=40 mmHg) or hypoxia (pO2=5 mmHg). Levels of HIF1-α and surface expression of VEGFR1, VEGFR2 and CXCR4 were evaluated. CD34+ and CD14+ cells were placed in culture (24 hr) and conditioned-medium (CM) was concentrated and analyzed for cytokines and growth factors. The effects of CM were assessed on CD34+ cell and CD14+ cell migration, and proliferation. Streptozotocin-induced diabetic mice of 11 months duration or age matched controls received intravitreal injection of fluorescently labeled ND or D CD34+ cells or CD14+ cells. Cells were localized 48 hrs post-injection and the degree of vascular homing to acellular capillaries was evaluated immunohistologically and quantitated.
Results: With hypoxia (4hr), expression of VEGFR2 (not VEGFR1), CXCR4 and HIF1-α were significantly increased in ND CD34+ cells but this response was markedly blunted in D CD34+ cells. In contrast, both ND and D CD14+ cells showed decreases in surface expression for all 3 receptors following hypoxia. TNFα, MCP1 and TGFβ were 10-fold higher and SCF and HGF were 3-fold lower in CM from D CD34+ cells compared to ND CD34+ cells. CM from D CD34+ cells increased migration of ND CD14+ cells but not their proliferation (P<0.01). D CD14+ cells did not secrete SCF and Flt-3L and did not support proliferation. ND CD14+ secreted higher levels of IL6, IL1β, IL8 and TGFβ1 compared to D CD34+. ND CD34+ and both ND and D CD14+cells, but not DM CD34+ cells migrated through the vitreous and homed to areas of injured vasculature in diabetic mice, but not control mice (P<0.05).
Conclusions: D CD34+ are unresponsive to hypoxia and cannot home to areas of retinal injury, whereas D CD14+ remain hypoxia responsive, can home as efficiently as ND CD14+ and ND CD34+ and can support migration but not proliferation. Our studies show complex paracrine interactions between BMDC and distinct desensitization of CD34+ cells to hypoxia which supports their observed in vivo dysfunction.