Loss of Angiotensin-Converting Enzyme 2 Exacerbates Diabetic Retinopathy by Promoting Bone Marrow Dysfunction

Abstract

Angiotensin-converting enzyme 2 (ACE2) is the primary enzyme of the vasoprotective axis of the renin angiotensin system (RAS). We tested the hypothesis that loss of ACE2 would exacerbate diabetic retinopathy by promoting bone marrow dysfunction. ACE2^-/y were crossed with Akita mice, a model of type 1 diabetes. When comparing the bone marrow of the ACE2^-/y -Akita mice to that of Akita mice, we observed a reduction of both short-term and long-term repopulating hematopoietic stem cells, a shift of hematopoiesis toward myelopoiesis, and an impairment of lineage^- c-kit⁺ hematopoietic stem/progenitor cell (HS/PC) migration and proliferation. Migratory and proliferative dysfunction of these cells was corrected by exposure to angiotensin-1-7 (Ang-1-7), the protective peptide generated by ACE2. Over the duration of diabetes examined, ACE2 deficiency led to progressive reduction in electrical responses assessed by electroretinography and to increases in neural infarcts observed by fundus photography. Compared with Akita mice, ACE2^-/y -Akita at 9-months of diabetes showed an increased number of acellular capillaries indicative of more severe diabetic retinopathy. In diabetic and control human subjects, CD34⁺ cells, a key bone marrow HS/PC population, were assessed for changes in mRNA levels for MAS, the receptor for Ang-1-7. Levels were highest in CD34⁺ cells from diabetics without retinopathy. Higher serum Ang-1-7 levels predicted protection from development of retinopathy in diabetics. Treatment with Ang-1-7 or alamandine restored the impaired migration function of CD34⁺ cells from subjects with retinopathy. These data support that activation of the protective RAS within HS/PCs may represents a therapeutic strategy for prevention of diabetic retinopathy. Stem Cells 2018;36:1430-1440.

Keywords: Bone marrow; CD34+; Diabetes; Hematopoietic progenitors; Retina.

Figure 1.. Reduction of both short-term (ST-) and long-term repopulating (LT-) hematopoietic stem cells (HSCs) in bone marrow from ACE2^-/y-Akita mice at 9 months of diabetes.

(A): Random glucose levels were increased in both Akita and ACE2^-/y-Akita groups (n = 7–9 per group). (B): Diabetic groups showed consistent low body weight, while the nondiabetic mice had increased body weight over time (n = 9–18 per group). (C): Representative scheme of gating for flow cytometry studies enumerating HSCs in each of the four cohorts, WT, ACE2^-/y, Akita, ACE2^-/y-Akita. (D): The percentage of ST-HSCs was decreased in ACE2^-/y-Akita mice at 9-month time point (n = 12–22). (E): The percentage of LT-HSCs was reduced in the bone marrow of Akita mice compared to WT mice at 9-months. Loss of ACE2 further decreased the diabetes-induced reduction of LT-HSC enumeration at 9-month time point (n = 12–22). * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita; ** p<0.05, as compared to 3-month time point. Abbreviations: WT, wild type; ST-HSC, short-term repopulating hematopoietic stem cell; LT-HSC, long-term repopulating hematopoietic stem cell; 3 mon, 3 months of diabetes and age-matched control; 9 mon, 9 months of diabetes and age-matched control.

Figure 2.. Depletion of ACE2 worsens diabetes-mediated impairment of HS/PC proliferation and migration functions.

(A): Impaired proliferative function of HS/PCs was observed in Akita mice as early as 3 months of diabetes. Loss of ACE2 further reduced proliferation (n = 5–7). (B): Absence of ACE2 exacerbated diabetes-induced impairment of HS/PC proliferation, which was restored by the treatment of Ang-1–7 100nM or alamandine 100nM (n = 5–6). (C): Ang-1–7 or alamandine improved the migration of diabetic HS/PCs toward chemoattractant CXCL12 at 3 months of diabetes (n = 4–6). (D): HS/PCs from ACE2^-/y-Akita mice had no response to chemoattractant CXCL12. Ang-1–7 or alamandine partially restored the migration of the diabetic HS/PCs (n = 4–6). For A-B, * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, compared to Akita; ** p<0.05, as compared to no treatment. For C-D, * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita; ** p<0.05, Compared to no treatment; ***, Compared to CXCL12. Abbreviations: WT, wild type.

Figure 3.. Absence of ACE2 worsens diabetes-mediated imbalance in hematopoiesis.

(A): Colony forming unit (CFU) assay showed an increased number of CFU-G/M/GM when plating bone marrow cells and a decreased number of total CFUs when plating red blood cell-lysed blood cells. Loss of ACE2 exacerbated diabetes-mediated alterations in both bone marrow and blood CFU assay (n = 14–20). (B): Representative gating scheme for enumeration of common lymphoid progenitors (CLP) (Lineage^-CD127⁺Sca-1^medc-Kit^med). (C): The percentage of CLPs was reduced in Akita mice at 9-month time point. There was a further decrease in CLP percentage in ACE2^-/y-Akita (as compared to Akita alone) (n = 13–22). * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita. Abbreviations: CFU, colony forming units; BFU-E, burst-forming unit-erythroid; CFU-G/M/GM, CFU-granulocyte/monocyte/granulocyte-monocyte; CFU-GEMM, CFU-granulocyte/erythrocyte/monocyte/megakaryocyte; CLP, common lymphoid progenitors; WT, wild type.

Figure 4.. Reduced retinal thickness in Akita groups at 9 months of diabetes.

(A): Representative images of retina near the optic nerve by OCT. (B): Reduced retinal thickness was observed in both Akita and ACE2^-/y-Akita mice at the 9-month time point when compared to non-diabetic groups (n = 8 per group). (C): Color fundus photography showed increased white lesions in both ACE2^-/y and ACE2^-/y-Akita groups (n = 4–5 per group). (D): Quantification of the number of white lesions per retina. * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita. Abbreviations: OCT, optical coherence tomography; WT, wild type.

Figure 5.. Reduced electrical responses of retinal cells to scotopic light flash in ACE2^-/y-Akita mice.

(A): scotopic a-waves, which reflects the signal of rod photocurrents, did not change in Akita alone at 9 months of diabetes. Loss of ACE2 caused a decrease in scotopic a-wave amplitude in Akita mice at 9 months of diabetes (n = 5–7). (B): Both Akita mice and ACE2^-/y-Akita mice exhibited a reduction of scotopic b-wave, indicating low electrical responses of depolarizing bipolar cells and Müller cells. Only ACE2^-/y-Akita mice showed a reduction of both scotopic a- and b- wave over the duration of diabetes (n = 5–7). * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita; ** p<0.05, as compared to 3-month time point. Abbreviations: ERG, electroretinogram; WT, wild type.

Figure 6.. ACE2 ablation worsens diabetes-induced increase in acellular capillaries at 9 months of diabetes.

(A): representative images of retinal vasculature and acellular capillaries (arrow) from the different cohorts at both 3-month and 9-month time points. (B): Increased number of retinal acellular capillaries was observed in Akita mice. ACE2^-/y-Akita mice had a further increase in acellular capillary number (as compared to Akita mice), suggesting more advanced retinopathy (n = 7–12 per group). * p<0.05, as compared to WT; # p<0.05, as compared to ACE2^-/y; & p<0.05, as compared to Akita; ** p<0.05, as compared to 3-month time point. Abbreviations: WT, wild type.

Figure 7.. Loss of protective RAS axis and impaired migration function of HSPCs in diabetic subjects with non-proliferative diabetic retinopathy (NPDR).

(A): plasma Ang II and Ang-1–7 peptide levels in healthy control, diabetes with no complications (DM-NC), and diabetes with NPDR. Ang-1–7 level was increased in DM-NC followed by a reduction at NPDR stage (n = 5–10). (B): MAS, receptor for Ang-1–7 was also increased in CD34⁺ cells from DM-NC patients, but reduced in those from NPDR subjects (n = 5–17). (C): Impaired migration ability of CD34⁺ cells were observed at the NPDR stage, which can be restored by Ang-1–7 treatment (n = 6–14). (D): Alamandine treatment can also restore the migration function of CD34⁺ cells at both no complication and NPDR stages (n = 4–8). For A and B, * p<0.05 as compared to control; & P<0.05 as compared to DM-NC. For C and D, * p<0.05 as compared to control; & P<0.05 as compared to DM-NC; ** p<0.05 as compared to CXCL12+Ang-1–7 or CXCL12+Alamd group; # p<0.05 compared to no treatment group. Abbreviations: DM-NC, diabetes with no complications; NPDR, non-proliferative retinopathy; Alamd, alamandine.