A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia
Henry S. Cheng, Rulin Zhuang, Daniel Pérez-Cremades, Jingshu Chen, Anurag Jamaiyar, Winona Wu, Grasiele Sausen, Aspasia Tzani, Jorge Plutzky, Jorge Henao-Mejia, Philip P. Goodney, Mark A. Creager, Marc S. Sabatine, Marc P. Bonaca, Mark W. Feinberg
Henry S. Cheng, Rulin Zhuang, Daniel Pérez-Cremades, Jingshu Chen, Anurag Jamaiyar, Winona Wu, Grasiele Sausen, Aspasia Tzani, Jorge Plutzky, Jorge Henao-Mejia, Philip P. Goodney, Mark A. Creager, Marc S. Sabatine, Marc P. Bonaca, Mark W. Feinberg
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Research Article Angiogenesis Vascular biology

A miRNA/CXCR4 signaling axis impairs monopoiesis and angiogenesis in diabetic critical limb ischemia

Abstract

Patients with peripheral artery disease (PAD) and diabetes have the highest risk of critical limb ischemia (CLI) and amputation, yet the underlying mechanisms remain incompletely understood. MicroRNA (miRNA) sequencing of plasma from diabetic patients with or without CLI was compared to diabetic mice with acute or subacute limb ischemia to identify conserved miRNAs. miRNA-KO mice on high-fat diet were generated to explore the impact on CLI. Comparison of dysregulated miRNAs from diabetic individuals with PAD and diabetic mice with limb ischemia revealed conserved miR-181 family members. High-fat–fed, diabetic Mir181a2b2-KO mice had impaired revascularization in limbs due to abrogation of circulating Ly6Chi monocytes, with reduced accumulation in ischemic skeletal muscles. M2-like KO macrophages under diabetic conditions failed to produce proangiogenic cytokines. Single-cell transcriptomics of the bone marrow niche revealed that the reduced monocytosis in diabetic KO mice was a result of impaired hematopoiesis, with increased CXCR4 signaling in bone marrow Lineage–Sca1+Kit+ (LSK) cells. Exogenous Ly6Chi monocytes from nondiabetic KO mice rescued the impaired revascularization in ischemic limbs of diabetic KO mice. Increased Cxcr4 expression was mediated by the miR-181 target, Plac8. Taken together, our results show that MiR-181a/b is a putative mediator of diabetic CLI and contributes to changes in hematopoiesis, monocytosis, and macrophage polarization.

Authors

Henry S. Cheng, Rulin Zhuang, Daniel Pérez-Cremades, Jingshu Chen, Anurag Jamaiyar, Winona Wu, Grasiele Sausen, Aspasia Tzani, Jorge Plutzky, Jorge Henao-Mejia, Philip P. Goodney, Mark A. Creager, Marc S. Sabatine, Marc P. Bonaca, Mark W. Feinberg

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Figure 2

Diabetes hinders revascularization in miR-181a2b2–KO mice.

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Diabetes hinders revascularization in miR-181a2b2–KO mice. (A) Left: Rep...
(A) Left: Representative LDI images of hind limbs of WT and KO mice on HFSC diet immediately after FAL surgeries and 3 weeks later. Right: Schema showing experimental setup and quantification of blood flow (surgical limb/contralateral limb) quantified by LDI, normalized to measurement immediate after surgery. Two-way ANOVA (n = 10–11). (B) Necrosis score of ischemic foot 3 weeks after FAL. (C) Left: Representative immunofluorescence images of ischemic gastrocnemius stained for SMA (green), CD31 (red), and with DAPI (blue). Scale bar: 100 μm. Right: Quantification of CD31+ areas per field of view (5 images per sample, n = 10–11), and quantification of SMA+ arterioles with different diameters (n = 10–11). Unpaired, 2-tailed Student’s t test. (D) Top: Scheme showing generation of bone marrow–transplanted (BMT) mice and experimental setup. Bottom: Blood flow (surgical limb/contralateral limb) quantified by LDI, normalized to measurement immediately after surgery. Two-way ANOVA (n = 7–8). (E and F) Flow cytometric analysis of mice 2 weeks after FAL. Unpaired, 2-tailed Student’s t test. (E) Time course (0 week [chow-fed diet, n =3], 2 weeks [HFSC diet, n = 9], and 6 weeks [HFSC diet, n = 14]) of total circulating neutrophils, CD3+ T cells, Ly6Clo and Ly6Chi monocytes. (F) Ischemic gastrocnemius neutrophils (neut.), CD3+ T cells, CD86+ and CD206+ macrophages. Unpaired, 2-tailed Student’s t test (n = 4–5). (G) Schema of experimental setup with tail-vein delivery of PBS, WT monocytes, or KO monocytes given 1 day after FAL surgeries. (H) Left: Representative LDI images of KO mouse hind limbs 2 weeks after FAL surgeries. Right: Quantification of blood flow percentage (surgical limb/contralateral limb) quantified by LDI. Two-way ANOVA (n = 6–7). (I) Left: Representative immunofluorescence images of ischemic gastrocnemius stained for SMA (green), CD31 (red), and with DAPI (blue). Scale bar: 100 μm. Right: Quantification of CD31+ areas per field of view. Unpaired, 2-tailed Student’s t test (4 images per sample, n = 6–7). *P < 0.05; **P < 0.01; ***P < 0.001.