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 1

Identification of miR-181 family dysregulation in experimental PAD in diabetic mice and in PAD individuals with diabetes mellitus.

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Identification of miR-181 family dysregulation in experimental PAD in di...
(A) Left: Representative images from laser Doppler imaging (LDI) of db/+ and db/db mouse hind limbs 3 days after femoral artery ligation (FAL; acute ischemia) or ameroid constrictor implantation (subacute ischemia). Right: Quantification of blood flow recovery (surgical limb/contralateral limb). Significance was assessed within time points by unpaired, 2-tailed Student’s t test (n = 5–17). (B) Volcano plot highlighting downregulated miRNAs in plasma comparing acute versus subacute ischemia in db/db mice. (C) Venn diagram indicating number of miRNAs commonly dysregulated from human and mice miRNA-seq analysis. F, Fontaine. (D) miRNA-seq log2-normalized counts of miR-181 members in human and mouse platforms (n = 6–7 human, n = 3 mice). A, acute; SA, subacute. (E) Genomic location of miR-181 clusters in mice (Mir181a1b1, Mir181a2b2, and Mir181cd). (F) Mature miR-181a/b/c expression normalized to U6 in ischemic gastrocnemius of db/+ and db/db mice at different time points after FAL. Comparison between groups at specific time points by unpaired, 2-tailed Student’s t test (d0, n = 11–12; d3, n = 7–12; d11, n = 5–6; d31, n = 4–7). (G) Primary miR-181-a1/b1, -a2, -b2, and -c/d expression normalized to Gapdh in ischemic gastrocnemius of db/+ and db/db mice at different time points after FAL (comparison statistics similar to F). (H) HUVECs transfected with 50 nM nonspecific (NS-i) or miR-181 (181-i) inhibitors prior to scratch assay. Images captured every hour to determine area closure and area under curve (AUC) quantified and compared using unpaired, 2-tailed Student’s t test (n = 3). (I) Top: Representative images of 3D spheroids composed of HUVECs transfected with 50 nM NS-i or 181-i. Scale bar: 200 μm. Bottom: Additive sprout lengths and numbers quantified and compared using unpaired, 2-tailed Student’s t test (n = 10). (J) Top: Schema showing the generation of tamoxifen-induced (TAM-induced) miR-181a2b2ΔEC mice and experimental setup. Bottom: Blood flow (surgical limb/contralateral limb) quantified by LDI, normalized to measurement immediately after surgery. Two-way ANOVA (n = 6–7). *P < 0.05; **P < 0.01.