Loss of Neuronal Support to the Bone Marrow BM Promotes Increased Generation Of (C-C Motif) Receptor 2+ (CCR2+) Monocytes And Reduced Endothelial Progenitors Cells (EPC): Implications For Diabetic Retinopathy (DR) Pathogenesis

Abstract

Purpose: : The dysfunction of EPCs in diabetes contributes to the vasodegenerative phase of DR. Neurotransmitters in the BM are responsible for stimulating BM stromal cells to produce the growth factors needed for proper hematopoiesis and hematopoietic stem cells (HSC) differentiation into all blood lineages. We showed in type 1 diabetic (T1D) rats that reduced norepinephrine (NE) lead to abnormal release of EPC from BM into the circulation (Busik,2009). Using T1D rodents, we examined other key neurotransmitters/neuropeptides (NT/NP) to determine whether they were reduced; BM supernatants for growth factor and cytokine expression; differentiation fate of HSC and the degree of infiltration of BM derived cells into retina.

Methods: : Immunohistochemical analysis of NT/NP in T1D long bones was preformed. Using BM derived cells (BMDC) from 6, 9 and 12 mo T1D mice, quantifation of the side population (SP) cells was performed by colony forming assays and flow cytometry. Measurement of growth factors/cytokines was done in BM supernatant by ELISA. Chimeric mice (transplanted with gfp-expressing HSC) were generated and made T1D and their retinas isolated for either retinal BMDC infiltration by immunohistochemisty of flatmounts or by flow cytometry of enzymatically-dissociated retinal cells.

Results: : In T1D mice, immunodetection of calcitonin gene related peptide was reduced by 65% (p<0.05), neuropeptide Y by 67% (p<0.05), acetylcholine by 51% (p<0.05), and somatostatin by 50% (p<0.05). BM cells isolated from 9 mo. T1D mice showed depletion of SP-HSCs but increased numbers of monocyte/macrophage-colony forming units (M-CFU) were generated. BM supernatant of T1D mice contained increased levels (2.5-fold, p<0.002) of macrophage-colony stimulating factor (M-CSF/CSF-1, a stimulator of monocyte generation) and increased levels of the inflammatory cytokines IL-1β (2-fold, p<0.05) and IL-27 (3-fold, p<0.01). Retinas of T1D mice showed a 10-fold increase in CD11b⁺/CD45^hi/ (C-C motif) receptor 2⁺ (CCR2⁺) population, consistent with an inflammatory monocytic phenotype. In T1D retina, Iba⁺ microglia exhibited a less ramified morphology, suggesting their inflammatory activation.

Conclusions: : Our data suggests that loss of BM neurotransmitters is responsible for increased M-CSF production which shifts hematopoiesis towards generation of excessive numbers of monocytes in particular pro-inflammatory CCR2⁺ monocytes that can contribute to retinal inflammation. This combined with loss of key reparative cells (EPCs) could promote retinal pathology seen in DR.

Keywords: circadian rhythms • diabetic retinopathy • retinal neovascularization