We investigated the role of microRNAs (miRNA) in endothelial dysfunction in the setting of cardiometabolic disorders represented by type 2 diabetes mellitus (T2DM). miR‐29 was dysregulated in resistance arterioles obtained by biopsy in T2DM patients. Intraluminal delivery of miR‐29a‐3p or miR‐29b‐3p mimics restored normal endothelium‐dependent vasodilation (EDVD) in T2DM arterioles that otherwise exhibited impaired EDVD. Intraluminal delivery of anti‐miR‐29b‐3p in arterioles from non‐DM human subjects or rats or targeted mutation of Mir29b‐1/a gene in rats led to impaired EDVD and exacerbation of hypertension in the rats. miR‐29b‐3p mimic increased, while anti‐miR‐29b‐3p or Mir29b‐1/a gene mutation decreased, nitric oxide levels in arterioles. The mutation of Mir29b‐1/a gene led to preferential differential expression of genes related to nitric oxide including Lypla1. Lypla1 was a direct target of miR‐29 and could abrogate the effect of miR‐29 in promoting nitric oxide production. Treatment with Lypla1 siRNA improved EDVD in arterioles obtained from T2DM patients or Mir29b‐1/a mutant rats or treated with anti‐miR‐29b‐3p. These findings indicate miR‐29 is required for normal endothelial function in humans and animal models and has therapeutic potential for cardiometabolic disorders.

Background miR-29 is a master regulator of extracellular matrix genes, but conflicting data on its anti-fibrotic effect have been reported. miR-29 improves nitric oxide (NO) production in arterioles by targeting Lypla1. Mir29b1 targeted mutation exacerbates hypertension in a model derived from the Dahl salt-sensitive rat. We examined the effect of Mir29b1 mutation on tissue fibrosis and NO levels with a focus on kidney regions. Methods Mir29b1 targeted mutant rats on the genetic background of SS-Chr13 BN rats were studied. Masson trichrome staining, molecular and biochemical assays, metabolic cage studies, and bioinformatic analysis of human genomic data were performed. Findings The abundance of miR-29b and the co-transcribed miR-29a was substantially lower in mutant rats. Tissue fibrosis was significantly increased in the renal outer medulla, but not in the renal cortex, heart or liver in mutant rats on a 0.4% NaCl diet. Lypla1 protein abundance was significantly higher and NO levels lower in the renal outer medulla, but not in the renal cortex. After 14 days of a 4% NaCl diet, 24 h urine volume and urinary sodium excretion was significantly lower in mutant rats, and tissue fibrosis became higher in the heart. NO levels were lower in the renal outer medulla and heart, but not in the renal cortex. Human miR-29 genes are located in proximity with blood pressure-associated single nucleotide polymorphisms. Interpretation The renal outer medulla might be particularly susceptible to the injurious effects of a miR-29 insufficiency, which might contribute to the development of hypertension in Mir29b1 mutant rats.

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MicroRNA miR-29 promotes endothelial function in human arterioles in part by targeting LYPLA1 and increasing nitric oxide production. Endothelial dysfunction is common in cardiovascular diseases such as hypertension and diabetic microvascular complications. In addition, miR-29 is a master inhibitor of extracellular matrix gene expression, which may attenuate fibrosis but could also weaken tissue structure. The goal of this study was to develop an effective miR-29 therapeutic for multiple cardiovascular diseases using mouse models. Substantial accumulation of miR-29b and effective knockdown of Lypla1 in mouse tissues were achieved using a chitosan-packaged, chemically modified miR-29b mimic (miR-29b-CH) injected systemically at 200 μg/kg body weight. miR-29b-CH, injected once every three days, significantly attenuated angiotensin II-induced hypertension over two weeks (mean arterial pressure of 128 ± 5 vs 149 ± 5 mmHg, N = 7 Scr-CH, 8 miR-29b-CH p<0.05). In db/db mice, miR-29b-CH treatment for 12 weeks decreased cardiac (0.98 ± 0.13 vs 1.55 ± 0.17 % fibrosis) and renal (2.85 ± 0.28 vs 4.85 ± 0.47 % fibrosis) fibrosis and urinary albuminuria 0.0051 ± 0.0004 vs 0.0069 ± 0.0005 albumin/creatinine, N = 12 Scr-CH, 9 miR-29b-CH, p<0.05). In uninephrectomized db/db mice, the miR-29b-CH treatment for 20 weeks significantly improved myocardial performance index (0.36 ± 0.02 vs 0.57 ± 0.05) in addition to attenuating proteinuria (0.015 ± 0.004 vs 0.033 ± 0.005 protein/creatinine) (N = 8 Scr-CH, 8 miR-29b-CH, p<0.05). miR-29b-CH did not worsen abdominal aortic aneurysm in ApoE knockout mice treated with angiotensin II (33.7 ± 8.2 vs 29.8 ± 6.7% increase in suprarenal abdominal aorta diameter from baseline, N = 8 Scr-CH and 7 miR-29b-CH). miR-29b-CH caused aortic root fibrotic cap thinning (13.8 ± 2.5 vs 20.8 ± 1.8 % of total plaque area) in ApoE knockout mice fed a high cholesterol and high fat diet but did not worsen the necrotic zone (15.2 ± 1.4 vs 12.5 ± 1.0 % of total plaque area) or the mortality (11/12 control mice survived until end of study vs 10/12 miR-29b-CH treated, N = 8 Scr-CH, 7 miR-29b-CH). In conclusion, systemic delivery of low dose miR-29b-CH is an effective therapeutic for several forms of hypertension and diabetic complications in mice.