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MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia

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Abstract

Peripheral artery disease (PAD) is an occlusive disease of limb arteries. Critical limb ischemia (CLI) is an advanced form of PAD that is prognostically worse in subjects with diabetes and can result in limb loss, gangrene, and death, although the underlying signaling mechanisms that contribute to its development remain poorly understood. By comparing plasma samples from diabetic humans with PAD and mouse models of PAD, we identified miR-375 to be significantly downregulated in humans and mice during progression to CLI. Overexpression of miR-375 was pro-angiogenic in endothelial cells in vitro and induced endothelial migration, proliferation, sprouting, and vascular network formation, whereas miR-375 inhibition conferred anti-angiogenic effects. Intramuscular delivery of miR-375 improved blood flow recovery to diabetic mouse hindlimbs following femoral artery ligation (FAL) and improved neovessel growth and arteriogenesis in muscle tissues. Using RNA-sequencing and prediction algorithms, Kruppel-like factor 5 (KLF5) was identified as a direct target of miR-375 and siRNA knockdown of KLF5 phenocopied the effects of miR-375 overexpression in vitro and in vivo through regulatory changes in NF-kB signaling. Together, a miR-375-KLF5-NF-kB signaling axis figures prominently as a potential therapeutic pathway in the development CLI in diabetes.

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Data availability

All relevant data are available from the authors. The RNA-seq data are accessible at: GSE210778. Source data are provided with this paper.

Abbreviations

3′UTR:

3′ untranslated region

ALI:

Acute limb ischemia

CAECS:

Coronary artery endothelial cells

CASMCs:

Coronary artery smooth muscle cells

CD31:

Cluster of differentiation 31; PECAM

ChIP:

Chromatin immunoprecipitation

CLI:

Critical limb ischemia

Db/+:

Heterozygous leptin receptor deficient, non-diabetic

Db/Db:

Homozygous leptin receptor deficient, diabetic

EC:

Endothelial cell

FAL:

Femoral artery ligation

HAECs:

Human aortic endothelial cells

HIF1α:

Hypoxia-inducible factor alpha

HLI:

Hind limb ischemia

HUVECs:

Human umbilical vein endothelial cells

IKKB:

Inhibitor of Nuclear factor kappa-B kinase subunit

Beta IL-1β:

Interleukin-1 Beta

IL-8:

Interleukin-8

IPA:

Ingenuity pathway analysis

IkBα:

Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

KLF4:

Kruppel-like factor 4

KLF5:

Kruppel-like factor 5

LPS:

Lipopolysaccharide

miRNA:

microRNA

miR-375:

microRNA-375-3p

MOVAS:

Mouse vascular smooth muscle cells

NF-kB:

Nuclear factor kappa-light-chain-enhancer of activated B cells

Non-EC:

Non-endothelial cell

p65:

Nuclear factor NF-kappa-B p65 subunit

PAD:

Peripheral artery disease

RT-qPCR:

Real-time quantitative polymerase chain reaction

siRNA:

Short Interfering RNA

SLI:

Sub-acute limb ischemia

TNFα:

Tumor necrosis factor alpha

αSMA:

Smooth muscle actin alpha

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Acknowledgements

The authors would like to thank Ana Lay-Hong and Aniket P. Gad for their assistance with immunofluorescence imaging (Harvard Digestive Disease Center, NIH P30DK034854). This work was supported by the National Institutes of Health (Grant Nos. HL115141, HL134849, HL148207, HL148355, HL153356 to M.W.F.), and the American Heart Association (Grant Nos. 18SFRN33900144 and 20SFRN35200163 to M.W.F.; Grant No. 907663 to M.G.M.).

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Authors and Affiliations

  1. Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, 02115, USA

    Michael G. McCoy, Anurag Jamaiyar, Grasiele Sausen, Henry S. Cheng, Daniel Pérez-Cremades, Rulin Zhuang, Jingshu Chen, Marc S. Sabatine & Mark W. Feinberg

  2. Department of Physiology, INCLIVA Biomedical Research Institute, University of Valencia, 46010, Valencia, Spain

    Daniel Pérez-Cremades

  3. Department of Cardiovascular Surgery, Shanghai East Hospital, Tongji University School of Medicine, 200120, Shanghai, China

    Rulin Zhuang

  4. Heart and Vascular Center, Dartmouth-Hitchcock Medical Center and Geisel School of Medicine at Dartmouth, 03756, Lebanon, NH, USA

    Philip P. Goodney & Mark A. Creager

  5. CPC Clinical Research, University of Colorado, Denver, CO, 80045, USA

    Marc P. Bonaca

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  1. Michael G. McCoy
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Contributions

Conceived the hypothesis: MWF and MGM; performed the experiments: MGM, AJ, GS, HSC, DP-C, RZ, JC; designed or interpreted the results: MGM, AJ, GS, HSC, DP-C, RZ, JC, PPG, MAC, MSS, MPB, and MWF; wrote the manuscript: MGM and MWF.

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Correspondence to Mark W. Feinberg.

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McCoy, M.G., Jamaiyar, A., Sausen, G. et al. MicroRNA-375 repression of Kruppel-like factor 5 improves angiogenesis in diabetic critical limb ischemia. Angiogenesis 26, 107–127 (2023). https://doi.org/10.1007/s10456-022-09856-3

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