Ataxia Telangiectasia Mutated Dysregulation Results in Diabetic Retinopathy

Abstract

Ataxia telangiectasia mutated (ATM) acts as a defense against a variety of bone marrow (BM) stressors. We hypothesized that ATM loss in BM-hematopoietic stem cells (HSCs) would be detrimental to both HSC function and microvascular repair while sustained ATM would be beneficial in disease models of diabetes. Chronic diabetes represents a condition associated with HSC depletion and inadequate vascular repair. Gender mismatched chimeras of ATM(-/-) on wild type background were generated and a cohort were made diabetic using streptozotocin (STZ). HSCs from the STZ-ATM(-/-) chimeras showed (a) reduced self-renewal; (b) decreased long-term repopulation; (c) depletion from the primitive endosteal niche; (d) myeloid bias; and (e) accelerated diabetic retinopathy (DR). To further test the significance of ATM in hematopoiesis and diabetes, we performed microarrays on circulating angiogenic cells, CD34(+) cells, obtained from a unique cohort of human subjects with long-standing (>40 years duration) poorly controlled diabetes that were free of DR. Pathway analysis of microarrays in these individuals revealed DNA repair and cell-cycle regulation as the top networks with marked upregulation of ATM mRNA compared with CD34(+) cells from diabetics with DR. In conclusion, our study highlights using rodent models and human subjects, the critical role of ATM in microvascular repair in DR.

Keywords: Ataxia telangiectasia mutated; Diabetic retinopathy; Hematopoietic stem cells.

Figure 1

Hematopoietic stem cell (HSC) imbalance in diabetic ataxia telangiectasia mutated (ATM)^−/−→ wild type (WT) chimeras. (A): Representative scheme for flow cytomtery dot plot used to differentiate and quantify long-term repopulating (LTR) and short-term repopulating (STR)-HSCs. Lin⁻Scal1⁺ c-kit⁺ CD34⁻ and Lin⁻Scal1⁺ c-kit⁺ CD34⁺ were identified as LTR-HSCs and STR-HSCs, respectively. Bar chart showing a quantification of (B) LTR-HSCs and (C) STR-HSCs. WT (n = 12), WT+ STZ (n = 5), ATM^−/−→WT (n = 5), ATM^−/−→WT (n = 5). Statistical test one way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; FSCA, forward-scattered light area; HSC, hematopoietic stem cell; LTR, long-term repopulating; SSCA, side-scattered light area; STZ, streptozotocin; WT, wild type.

Figure 2

Decrease in quiescent long-term repopulating (LTR)-hematopoietic stem cells (HSCs) and short-term repopulating (STR)-HSCs in ataxia telangiectasia mutated (ATM)^−/−→ wild type (WT) mice. Cell cycle status for LTR and STR-HSCs was determined following staining with Pyronin Y and Hoechst blue. Top panel showing a pie chart for the percentage of LTR-HSCs in different stages of cell cycle; diabetes and ATM^−/−→WT caused a significant decrease in quiescent (G0) LTR-HSCs with an increase in the numbers of cells in active cell cycle (G1 and G2). Bottom panel showing a significant lack in quiescent cells with an increase in cell numbers in the phase of the active cycle in all three groups except wild type (WT) animals. WT (n = 12), WT+ STZ (n = 5), ATM^−/−→WT (n = 5), ATM^−/−→WT (n = 5) Statistical test-one way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; HSC, hematopoietic stem cell; LTR, long-term repopulating; STR, short-term repopulating; STZ, streptozotocin; WT, wild type.

Figure 3

mRNA expression of cell cycle check points and anti-oxidant enzymes in bone marrow cells. mRNA expression on bone marrow cell pellet was determined using q-RT-PCR. Bar chart showing expression of P53, P21, CDK2, CDC25a, SOD1 and SOD2. Wild type (WT) (n = 4), WT+ STZ (n = 4), ataxia telangiectasia mutated (ATM)^−/−→WT (n = 5), ATM^−/−→WT (n = 5). Statistical Test- One way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; SOD, Superoxide dismutase; STZ, streptozotocin; WT, wild type.

Figure 4

Reduced bone marrow engraftment in diabetic ataxia telangiectasia mutated (ATM)^−/−→wild type (WT) chimeras. (A): Demineralized mouse femurs were stained for n-cadherin (red) and c-kit (green) antibodies to identify long-term repopulating (LTR)-hematopoietic stem cells (HSCs). Representative photomicrographs from respective groups showing c-kit positive cells (white arrows) in an endosteal niche (open arrow) while bar chart showing a quantification of LTR-HSCs. (B): Mouse femurs stained with ve-cadherin (red) to define the vascular niche. c-kit⁺ cells (white arrows) in ve-cadherin positive regions identified as short-term repopulating (STR)-HSC. Bar chart showing ratio of STR-HSCs to LTR-HSCs. Scale bar = 20 μm. WT (n = 6), WT→WT (n = 4), WT+ STZ (n = 5), WT→WT + STZ (n = 7), ATM^−/−→WT (n = 5), ATM^−/−→WT (n = 5). Statistical Test- One way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; HSC, hematopoietic stem cell; LTR, long-term repopulating; STZ, streptozotocin; WT, wild type.

Figure 5

Accelerated diabetic retinopathy (DR) in ataxia telangiectasia mutated (ATM)^−/−→ wild type (WT) chimeras. (A): Isolated mouse retinas were trypsin digested and stained with PAS-hematoxylin stain, top panel showing representative pictures of retinal trypsin digests from respective groups. Red arrows showing acellular capillaries as a marker of DR. Scale bar = 50 μm (B): Bar chart showing quantification of numbers of acellular capillaries. WT (n = 6), WT→WT (n = 8) WT+ STZ (n = 5), ATM^−/−→WT (n = 5) WT→WT + STZ (n = 9), ATM^−/−→WT (n = 5) Statistical Test- One way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; STZ, streptozotocin; WT, wild type.

Figure 6

Increase in retinal inflammation of ataxia telangiectasia mutated (ATM)^−/−→ wild type (WT) chimeric animals. qRT-PCR was performed on isolated retinas, bar chart showing mRNA expression of a variety of retinal inflammatory markers (HIF-1α, PDGF-β, vascular endothelial growth factor [VEGF], Glial fibrillary acidic protein [GFAP], CD45, Lipocalin-2 [LCN-2], and CCL2). WT (n = 5), WT→WT (n = 8) WT+ STZ (n = 4), WT→WT + STZ (n = 13) ATM^−/−→WT (n = 5), ATM^−/−→WT (n = 5). Statistical test-one way analysis of variance followed by Student’s Newman Keul Test. Abbreviations: ATM, ataxia telangiectasia mutated; CCL2, The chemokine (C-C motif) ligand 2; GFAP, Glial fibrillary acidic protein; HIF-1α, Hypoxia-inducible factor 1-alpha; LCN-2, Lipocalin-2; PDGF-β, Platelet-Derived Growth Factor Beta; STZ, streptozotocin; VEGF, vascular endothelial growth factor; WT, wild type.

Figure 7

Diabetic patients protected from microvascular complications map unique targets involved in DNA repair and cell cycle in CD34⁺ cells. (A): Gene set enrichment cluster analysis showing a heat map exhibiting distinct signature in diabetic group as compared with control. (B): a similar comparison between protected patients from diabetic retinopathy (DR) and patients with DR. (C): Dot plot showing mRNA expression of ataxia telangiectasia mutated in CD34⁺ cells of study participants; Control (n = 5), Diabetes w/o DR (n = 5), Diabetes w/ DR (n = 4). Statistical test-one way analysis of variance followed by Student’s Newman Keul Test. Abbreviation: DR, diabetic retinopathy.