MicroRNA-615-5p Regulates Angiogenesis and Tissue Repair by Targeting AKT/eNOS (Protein Kinase B/Endothelial Nitric Oxide Synthase) Signaling in Endothelial Cells

Abstract

Objective- In response to tissue injury, the appropriate progression of events in angiogenesis is controlled by a careful balance between pro and antiangiogenic factors. We aimed to identify and characterize microRNAs that regulate angiogenesis in response to tissue injury. Approach and Results- We show that in response to tissue injury, microRNA-615-5p (miR-615-5p) is rapidly induced and serves as an antiangiogenic microRNA by targeting endothelial cell VEGF (vascular endothelial growth factor)-AKT (protein kinase B)/eNOS (endothelial nitric oxide synthase) signaling in vitro and in vivo. MiR-615-5p expression is increased in wounds of diabetic db/db mice, in plasma of human subjects with acute coronary syndromes, and in plasma and skin of human subjects with diabetes mellitus. Ectopic expression of miR-615-5p markedly inhibited endothelial cell proliferation, migration, network tube formation in Matrigel, and the release of nitric oxide, whereas miR-615-5p neutralization had the opposite effects. Mechanistic studies using transcriptomic profiling, bioinformatics, 3' untranslated region reporter and microribonucleoprotein immunoprecipitation assays, and small interfering RNA dependency studies demonstrate that miR-615-5p inhibits the VEGF-AKT/eNOS signaling pathway in endothelial cells by targeting IGF2 (insulin-like growth factor 2) and RASSF2 (Ras-associating domain family member 2). Local delivery of miR-615-5p inhibitors, markedly increased angiogenesis, granulation tissue thickness, and wound closure rates in db/db mice, whereas miR-615-5p mimics impaired these effects. Systemic miR-615-5p neutralization improved skeletal muscle perfusion and angiogenesis after hindlimb ischemia in db/db mice. Finally, modulation of miR-615-5p expression dynamically regulated VEGF-induced AKT signaling and angiogenesis in human skin organoids as a model of tissue injury. Conclusions- These findings establish miR-615-5p as an inhibitor of VEGF-AKT/eNOS-mediated endothelial cell angiogenic responses and that manipulating miR-615-5p expression could provide a new target for angiogenic therapy in response to tissue injury. Visual Overview- An online visual overview is available for this article.

Keywords: AKT/eNOS signaling; angiogenesis; endothelial cells; microRNAs; tissue repair.

Figure 1.

MiR-615–5p is regulated by pro-angiogenic stimuli and inhibits endothelial cell growth. (A) Real-time qPCR analysis of miR-615–5p expression in response to VEGF and bFGF in HUVECs. * P < 0.005 compared to Ctrl. Data is representative of n=4 experiments. (B) Circulating miR-615–5p levels are increased in plasma from human subjects with acute coronary syndrome ((ACS) (n = 40; 28 male, 12 female)) compared to subjects with normal coronary angiograms ((NCA) (n = 20; 9 male, 11 female)). * P < 0.05 compared to normal coronary angiogram. (C-D) Expression of miR-615–5p is increased in skin and plasma of patients with diabetes (n = 13; 9 male, 4 female) compared to controls (n=10; 8 male, 2 female). * P < 0.05 compared to controls. (E) WT and db/db mice (n=4 – 6/group) underwent punch-biopsy wounding of the dorsal skin, and wounds were collected for qPCR analyses for miR-615–5p on the indicated days post-wounding. (F) HUVECs transfected with miR negative control (NS_m), miR-615–5p mimics (miR-615–5p_m), miR inhibitor negative control (NS_i), or miR-615–5p inhibitor (miR-615–5p_i) and stimulated with VEGF or bFGF as indicated were subjected to BrdU cell proliferation assay. * P < 0.05 compared to controls. All data represent mean ± s.e.m.

Figure 2.. MiR-615–5p inhibits pro-angiogenic functions in ECs in vitro.

HUVECs transfected with miR negative control (NS_m), miR-615–5p mimics (miR-615–5p_m), miR inhibitor negative control (NS_i), or miR-615–5p inhibitor (miR-615–5p_i) were subjected to (A) tube-like network formation in matrigel; (B) EC migration in transwell Boyden chambers; (C) scratch assay. Data is representative of n = 6 per group and 3 independent experiments. * P < 0.05 compared to NS_m or NS_i ** P < 0.001 compared to NSi. Scale bars, 150μm (A) and 100μm (D). All data represent mean ± s.e.m.

Figure 3.. Bioinformatics and miR-615–5p gene profiling predicts AKT as a targeted signaling pathway

(A) Gene ontology analysis of 337 genes repressed by miR-615–5p overexpression in endothelial cells (ECs) identified from transcriptomic profiling. (B-C) AKT signaling pathway is predicted to be the top regulated signaling network regulated by miR-615–5p. GO, gene ontology.

Figure 4.. MiR-615–5p regulates the expression of downstream AKT/eNOS signaling in ECs.

HUVECs transfected with (A) miR negative control (NS_m) or miR-615–5p mimics (miR-615–5p_m) or (B) miR inhibitor negative control (NS_i) or miR-615–5p inhibitor (miR-615–5p_i) were subjected to Western analysis using antibodies to p-AKT, p-eNOS, AKT, eNOS, p-p38, p38, p-ERK1/2, ERK1/2 and β-actin (n = 3 to 5 experiments). (C) NO release was measured by Griess assay. All data represent mean ± s.e.m. * P < 0.05 compared to controls.

Figure 5.. IGF2 and RASSF2 are bona fide targets of miR-615–5p in ECs.

(A) Discovery and validation of MiR-615–5p target genes. HUVECs transfected with miR negative control (NS_m) and miR-615–5p mimics (miR-615–5p_m) were subjected to microarray gene profiling. Potential gene targets were further narrowed down by sequential use of bioinformatics and prediction algorithms, RT-qPCR, Western blot analyses, 3’-UTR reporter studies, and microribonucleoprotein immunoprecipitation (miRNP-IP) analysis. (B-C) HUVECs transfected with NSm or miR-615–5p_m were subjected RT-qPCR for IGF2 and RASSF2 expression (B) or Western blot analyses using antibodies to IGF2, RASSF2, and GAPDH (n = 3 experiments) (C). (D) Luciferase activity of IGF2 3’-untranslated region (UTR) and RASSF2 3’-UTR normalized to total protein was quantified in HUVECs transfected with NS_m, miR-615–5p_m, NS_i, or miR-615–5p_i (n = 3 experiments). (E) Luciferase activity of IGF2 or RASSF2 3’-UTRs bearing a deletion of the miR-615 binding site (miR-615 DEL) normalized to total protein was quantified in HUVECs transfected with NS_m or miR-615_m (n=3 experiments). (F) miRNP-IP analysis of enrichment of IGF2 and RASSF2 mRNA in HUVECs transfected with NS_m or miR-615–5p_m. *P < 0.01. RT-qPCR was performed to detect IGF2, RASSF2 or SMAD1. Results are representative of n = 3 replicates per group and 2 independent experiments. *P < 0.01. All data represent means ± s.e.m.

Figure 6.. SiRNA-mediated knockdown of IGF2 and RASSF2 recapitulates miR-615–5p functional effects in ECs.

(A) HUVECs were transfected with siRNA to RASSF2 (A-C), IGF2 (D-F), IGF2 and RASSF2 (G) or scrambled control (ctrl) siRNA. Protein expression was determined by Western analysis under baseline conditions (A, D) or in response to VEGF treatment (B,E) using antibodies to RASSF2, IGF2, p-Akt, Akt and GAPDH (n = 2 experiments). *P < 0.01. (C,F,G) migration of ECs were quantified by scratch assay. *P < 0.01. Results are representative of n = 3 replicates per group. All data represent means ± s.e.m.

Figure 7.. Local delivery of LNA-anti-miR-615–5p promotes wound healing in db/db mice.

(A) After two local injections in mice of LNA-anti-miR-615–5p (MiR-615–5p_i) or scrambled non-specific control LNA-anti-miRs (NS_i) (n =11–12 per group), mice underwent dorsal skin wounding. (B-D) Wound analyses included: (B) wound closure areas (C) granulation tissue thickness (GTT) and (D) confocal immunofluorescence staining for CD31. (E-G) After two local injections in mice of miR-615–5p mimics (MiR-615–5p_m) or scrambled non-specific control miRs (NS_m) (n =10 per group), mice underwent dorsal skin wounding. (E-G) Wound analyses included: (E) wound closure areas (F) granulation tissue thickness (GTT) and (G) confocal immunofluorescence staining for CD31. Scale bars, 5mm (B,E), 500 μm (C,F) and 100 μm (D,G) All data represent means ± s.e.m. * P <0.05, ** P<0.001.

Figure 8.. Inhibition of miR-615–5p promotes angiogenesis in human skin organoids.

(A) Punch biopsies of human skin were embedded into a collagen matrix, transfected with miR inhibitor negative control (NS_i), miR-615–5p inhibitor (miR-615–5p_i), miR negative control (NS_m) or miR-615–5p mimics (miR-615–5p_m), and cultured for indicated number of days. (B-C) Human skin organoids were transduced with the indicated miRNAs and cultured for 9 days followed by confocal immunofluorescence staining for CD31. (D-E) Human skin organoids (n=3–6) were transduced with the indicated miRNAs and cultured for 3 days with or without VEGF and followed by Western blot analyses for p-AKT, AKT, and β-actin at the indicated times (D-E). Scale bars, 50 μm (B-C). All data represent means ± s.e.m. * P <0.05.