Hematopoietic stem/progenitor involvement in retinal microvascular repair during diabetes: Implications for bone marrow rejuvenation

Abstract

The widespread nature of diabetes affects all organ systems of an individual including the bone marrow. Long-term damage to the cellular and extracellular components of the bone marrow leads to a rapid decline in the bone marrow-hematopoietic stem/progenitor cells (HS/PCs) compartment. This review will highlight the importance of bone marrow microenvironment in maintaining bone marrow HS/PC populations and the contribution of these key populations in microvascular repair during the natural history of diabetes. The autonomic nervous system can initiate and propagate bone marrow dysfunction in diabetes. Systemic pharmacological strategies designed to protect the bone marrow-HS/PC population from diabetes induced-oxidative stress and advanced glycation end product accumulation represent a new approach to target diabetic retinopathy progression. Protecting HS/PCs ensures their participation in vascular repair and reduces the risk of vasogdegeneration occurring in the retina.

Keywords: Bone marrow microenvironment; Diabetic retinopathy; Hematopoietic stem cells.

Figure 1:. Localization of c-Kit⁺ cells in mouse bone marrow.

Upper panel: immunofluorescence staining of N-cadherin (red) and c-Kit (green) in demineralized mouse femurs. Some c-Kit⁺ cells (white arrow) localized to endosteal niche (blue arrow) are defined as long-term repopulating (LTR)-hematopoietic stem cells (HSCs); Lower panel: mouse femurs stained for VE-cadherin (red) and c-Kit (green). c-Kit⁺ cells (white arrow) located at vascular niche are defined as short-term repopulating (STR)-HSCs. Representative images showing a reduced number of LTR-HSCs and increased STR-HSCs/LTR-HSCs in the STZ-induced diabetic bone marrow. ATM^−/− intensified diabetes-mediated defects of LTR- and STR-HSCs imbalance in the bone marrow. Abbreviations: WT, wild type; STZ, streptozotocin; ATM, ataxia telangiectasia mutated.

Figure 2:. Inhibition of acid sphingomyelinase in diabetic CACs corrects their dysfunction.

Diabetic animals received intravitreal injections of control (A), diabetic (B), or diabetic CACs treated with siRNA for ASM (C). CACs (green) were isolated from gfp⁺ mice, retinal vasculature was stained with anti-collagen IV antibody (red), and localization (yellow) indicates vascular association. Diabetic CACs show reduced localization with vasculature (middle), while ASM inhibition improved vascular association of diabetic CACs (right). Abbreviations: ASM, acid sphingomyelinase; BM, bone marrow; CAC, circulating angiogenic cell.

Figure 3:. BBZ/Wor T2D rats exhibit reduced blood flow to the bone marrow.

A significant reduction in blood flux is observed in T2D rats compared to controls in both the BM and sciatic nerve. (A) Background showing exposed femur under retraction. Target area was imaged using a laser Doppler (LDPI) for relative blood flux and color charted (arrow and inset). Examples of LDPI of bone marrow (B & C) and sciatic nerve (D & E) in a control and T2D rat respectively. Warm (red) colors represent regions of relative high flux whereas cool colors (blue) represent areas of low flux. F: Quantification of LDPI imaging is shown in control (n = 4) and diabetic (n = 4) rats.

Figure 4:. Increased density of lba-1⁺ microglia and a decreased density of NeuN⁺ neurons in the hypothalamus of type 2 diabetic (T2D) rats.

Brain sections of hypothalamic region of control (A), T2D (B) rats showing lba-1 staining in red and NeuN in green. (C) Bar chart showing relative quantitation. Scale bar =100 μm.

Figure 5:. BM-ASM inhibition prevents activation of microglia in diabetic retinas.

Confocal images of retinal flat mounts from bone marrow chimeras with GFP (green), or GFP-ASM^−/− bone marrow donors and wild type recipient mice. The retinas are stained with collagen IV for the retinal vasculature (red). (A) Control retina showing resting microglia with the typical ramified and branching shape. (B) Diabetic animals show activated microglia with more compact cells with fewer ramifications. (C) Diabetic animal showing that BM-ASM inhibition prevents activation of microglia. (D) Bar chart showing the quantification of the percentage of activated microglia in the three conditions. Scale bar is 50 μM, n=8

Figure 6:. Decrease in SST⁺ cells in the hypothalamus in type 2 diabetic rats.

Brain sections from age-matched nondiabetic lean BBZ rats (A) show robust cellular SST staining compared to that observed in diabetic BBZ/Wor rats with 4 months of diabetes (B). 3V = third ventricle. Scale bar = 50μm. (C) Quantitation shows a statistically significant decrease in the number of SST⁺ cells with diabetes (n=4, *P<0.05).

Figure 7:. Somatostatin+ nerves project from the hypothalamus to the bone marrow.

Confocal (60X) z-projection of hypothalamic periventricular region from a rat that received femoral bone marrow injection of PRV-152 (green) also immune-labeled for SST (red). 3V = third ventricle. Scale bar = 24μm.

Figure 8:. The bone marrow is a critical target organ of diabetes and its dysfunction can adversely affect diabetic retinopathy.

The release of bone marrow cells and hematopoiesis is under the regulation of the autonomic nervous system (ANS) and in late stage diabetes denervation of the bone marrow results in a shift in hematopoiesis with loss of generation of reparative cells but an increased production of pro-inflammatory cells. Therapeutic targets and novel strategies may include modulation of bone marrow ATM, ASM, and FOXO, systemic administration of Ang 1-7 or exogenous replacement of SST using a somatostatin analogue that crosses the BBB to influence the brain. Preservation of bone marrow function may represent a new opportunity for treatment and management of diabetic retinopathy.