Endogenous reduction of miR‐185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K

Abstract Angiogenesis is critical for re‐establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR‐185‐5p were differently expressed in plasma from patients with ACS by high‐throughput RNA sequencing. The expressional levels of miR‐185‐5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT‐PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR‐185‐5p. In HUVECs, miR‐185‐5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR‐185‐5p inhibitors performed the opposites. Further, the inhibitory effects of miR‐185‐5p up‐regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus‐mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain‐function of miR‐185‐5p by agomir infusion down‐regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR‐185‐5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR‐185‐5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.


| INTRODUCTION
Acute coronary syndrome (ACS) remains a major cause of morbidity and mortality worldwide, in which ischaemic complication represents the leading cause in cardiovascular diseases, such as hypertension and atherosclerosis. 1 Angiogenesis is critical for re-establishing the blood supply to the surviving myocardium after myocardial infarction (MI) and, consequently, to the recovery of cardiac function. 2 In response to pro-angiogenic stimuli, vascular endothelial cells need to be activated rapidly to migrate to distant sites and proliferate to form new primary capillaries from existing ones. 3,4 Impaired angiogenic responses of endothelial cells have been linked to poor cardiac function and outcome after ACS clinically. Therefore, better understandings of the molecular mechanisms of angiogenesis provide a basis for therapeutic application in ACS patients.
MicroRNA is a kind of small, nonprotein-coding RNA that binds to specific 3′ untranslated regions (3′-UTR) of mRNA and represses target gene expression through inhibition of translation or transcript degradation. 5 In vascular cells, several microRNAs, such as miR-126, miR-217, miR-10a, miR-124, etc., have been identified to regulate multiple cellular functions including the glucose and lipid metabolisms, senescence and blood flow. [6][7][8][9] We have previously reported that miR-15b-5p regulates collateral artery formation by targeting protein kinase B in endothelial cells and miR-199 regulates normalise arachidonic acid metabolism in nitrate tolerance by prostaglandin synthase. 10,11 Interestingly, endogenous expressions of miR-133a and miR-130, are null or very low in quiescent endothelial cells, while their expressions are ectopically induced by risk factors, resulting in endothelial dysfunction. 12,13 These results indicate that expressional alterations of endogenous microRNAs are crucial to regulations of endothelial functions. However, the roles of microRNA in hypoxiainduced angiogenesis in endothelial cells remain poorly understood.
Based on the aforementioned studies, we tested the hypothesis that hypoxia affects endogenous microRNA expressions to promote angiogenesis in ischaemic heart. In this study, we firstly got 18 differentially expressed circulating microRNAs in plasma from ACS patients by using high-throughput sequencing and then identified the function of miR-185-5p as a key regulator of angiogenesis by targeting cathepsin K (CatK), which plays an important role in regulating vascular repair. 14 In vivo studies demonstrated that endogenous reduction of miR-185-5p was vital to the recovery of heart function after ischaemia in mice with MI. In perspectives, targeting miR-185-5p or CatK should be taken into consideration when treating ACS patients.

| Statistical analysis
All quantitative results are expressed as mean ± SEM. Comparisons between two groups were analysed by unpaired Student's t test.
Because miR-185-5p has been reported to inhibit angiogenesis and response to hypoxia, 17,18 we chose miR-185-5p as a candidate to regulate angiogenesis and the recovery of heart function after MI. Therefore, we confirmed the plasma levels of miR-185-5p by qPCR in 30 ACS patients (the demographic data were presented in Table S1).
As indicated in Figure 1B, plasma miR-185-5p levels were down-regulated in patients with ACS, compared to patients without ACS.

| MiR-185-5p is reduced in ischaemic heart in mice following MI
To determine whether miR-185-5p is vital to ischaemia-induced angiogenesis in heart, we performed FISH analysis to measure miR-185-5p expression in mice heart at 7 post-MI days. As depicted in Figure 1C, miR-185-5p expression was positively expressed in both cardio myocytes and endothelial cells under physiological condition. While, miR-185-5p expression in heart was remarkably reduced in ischaemic heart in mice with MI. The reduction of miR-185-5p expression in ischaemic heart was further confirmed by qPCR analysis ( Figure 1D). Further, decreased miR-185-5p expression was associated with increased capillary intensity ( Figure 1E), as indicated by quantitative analysis of CD31. These data suggest that miR-185-5p reduction may contribute to ischaemia-induced angiogenesis in heart.

| Hypoxia reduces miR-185-5p expression in endothelial cells
As the key cells of endothelial cells in angiogenesis, 19 Table S1. *P < 0.05 vs non-ACS. (C-E) FISH analysis of miR-185-5p in hearts were performed in mice following MI after 7 postoperative days in C. Red, miR-185-5p. Green, CD31. Blue, DAPI. Scar bar is 50 μm. Quantitative analyses of miR-185-5p levels in D and capillary intensities in E were conducted in data from C. N = 10 per group. *P < 0.05 vs Sham 3.4 | CatK is a target of miR-185-5p in endothelial cells In order to investigate how miR-185-5p regulates angiogenesis under hypoxia, we used TargetScan database to predict the potential target of miR-185-5p. Computational target scan analysis showed that a highly conserved putative target site of miR-185-5p located in 3′-UTR of CatK mRNA among human, rat and mouse ( Figure 2B). To examine whether miR-185-5p is able to repress CatK gene expression through direct 3′-UTR interaction, we cloned wild-type CatK 3′-UTR (WT-CatK-3′-UTR) or mutated CatK 3′-UTR (MT-CatK-3′-UTR) to luciferase reporter plasmid and performed reporter analysis in HEK293 cells. As shown in Figure 2C  3.10 | CatK regulates c-notch1, Hes1, Hey1 and

Hey2 mRNA expression in HUVECs
To uncover how CatK regulates angiogenesis, we measured notch signalling, which has been reported to play critical roles in vascular F I G U R E 3 MiR-185-5p negatively regulates HUVECs proliferations and migrations under hypoxia. Cultured HUVECs were transfected with control mimics, miR-185-5p mimics (50 nmol/L), control inhibitors and miR-185-5p inhibitors (100 nmol/L) for 48 h followed by 24-h hypoxia. a, Control. b, Hypoxia. c, Hypoxia plus control mimics. d, Hypoxia plus miR-185-5p mimics. e, Hypoxia plus control inhibitors. f, Hypoxia plus miR-185-5p inhibitors. (A and B) Cell proliferations were determined by CCK8 in A and BrdU in B. (C-E) Cell migration and tube formation were assayed in HUVECs after treatments and representative pictures were shown in C. Quantitative analysis of cell migration in D and tube formation in E were performed in data from C. N = 5 per group. *P < 0.05 vs control (a). # P < 0.05 vs hypoxia plus control mimics (c). & P < 0.05 vs hypoxia plus control inhibitors (e) development. 21,22 As shown in Figure S4A (Figure S4B), but activated by CatK gene overexpression ( Figure S4C).

| Gain-function of miR-185-5p inhibits ischaemia-induced angiogenesis in mice following MI
Knowing that miR-185-5p negatively regulates proliferations, migrations and tube formations in endothelial cells by targeting CatK, we next determined whether up-regulation of miR-185-5p inhibits angiogenesis in ischaemic heart in vivo. To this point, mice were infused with miR-185-5p agomir and were conducted with MI surgery ( Figure S5A). Infusion of miR-185-5p agomir obviously increased miR-185-5p levels in hearts, compared with control agomir ( Figure S5B). As indicated in Figure 6A, B, the capillary intensities in ischaemic area of hearts isolated from mice with MI after 28 postoperative days were significantly higher than hearts from mice with sham surgery, as determined by IHC analysis of CD31.
Treatment of miR-185-5p agomir but not control agomir completely reduced capillary numbers in mice with MI. Furthermore, administration of miR-185-5p agomir reduced fibrosis in ischaemic heart ( Figure 6A, C). These results suggest that miR-185-5p reduction contributes to ischaemia-induced angiogenesis in mice after MI.
3.12 | MiR-185-5p agomir delays the recovery of heart functions in mice following MI Angiogenesis is a key regenerative event to recover the blood supply and to repair cardiac function in ACS patients. 25 Next, we determined whether miR-185-5p agomir delays the recovery of heart functions in mice following MI by echocardiographic analysis ( Figure 6D). Echocardiographic parameters were shown in Table S2.
Both EF and FS were dramatically decreased in mice with MI, which were further reduced by miR-185-5p agomir, compared to mice with negative agomir treatment ( Figure 6E, F). 3.13 | Gain-function of miR-185-5p reduces CatK protein expression in mice following MI We next examined whether miR-185-5p represses CatK gene expression in vivo. As shown in Figure S6A, B, IFC analysis indicated that the numbers of both CatK + and CD31 + cells in ischaemic area were significantly enhanced in heart isolated from mice with MI, compared to mice with sham. Administration of miR-185-5p agomir but not negative agomir extensively repressed CatK protein expressions in CD31 + cells in mice after 28 postoperative days. These effects were further confirmed by qPCR analysis showing the mRNA expression of CatK in ischaemic area was significantly reduced by miR-185-5p agomir in mice with MI surgery ( Figure S6C).

| Knockdown of CatK impairs angiogenesis
and delays the recovery of heart functions in mice following MI Above, we have provided evidence to reveal that CatK mRNA is a target of miR-185-5p. As a result, we thought that silencing of CatK would mirror the effects of miR-185-5p agomir in mice with MI. To test this notion, in vivo loss-function of CatK was induced by infecting mice with adenovirus expressing CatK shRNA ( Figure S5C, D). We also detected the effects of CatK gene silencing on cardiac function in mice with MI by echocardiography and the echocardiographic parameters were shown in Table S3. As expected, knockdown of CatK gene expression reduced EF and FS in mice infected with adenovirus expressing CatK shRNA at 28 postoperative days after MI surgery ( Figure S7D-F). Taking these data together, it reveals that miR-185-5p/CatK axis plays a key role in angiogenesis and repair of heart function after MI.
3.15 | MiR-185-5p agonist and CatK inhibition suppress arteriogenesis in ischaemic heart isolated from mice with MI Growth of functional arteries from angiogenesis, which is driven by flow shear stress, is essential for the restoration of blood flow to ischaemic organs. 26,27 Finally, we examined the roles of miR-185-5p and CatK in arteriogenesis in mice heart by performing double staining of CD31 and ɑ-SMA shown in Figure S8A, B, either miR-185-5p Besides regulating angiogenesis, CatK, as a member of the lysosomal cysteine and aspartic proteinase family, has been shown to be one of the most potent mammalian collagenases. It plays a vital role in maintaining the homeostasis of extracellular matrix. 33 Pharmacological inhibition of CatK is a promising novel approach for postmenopausal osteoporosis therapy. 34 Overexpression of CatK in mice decreases collagen deposition and lung resistance in response to bleomycin-induced pulmonary fibrosis. 35 We further reported that CatK has some effects on the metabolism of collagen in the late stage of MI. This might be the reason why CatK is mediating a protective effect in cardiac function. Previous studies have reported that miR-185 plays important roles in regulation of cell proliferation and apoptosis in response to stress such as oscillating glucose. 36 As a tumour suppressive gene, miR-185 is a pivotal mediator in the cellular response to hypoxia, a state that directly affects angiogenesis. 37 In this study, we further uncovered the potential functions of miR-185 as a suppressor of angiogenesis in revascularisation in ischaemic heart by regulating CatK gene expression. Moreover, the miR-185-5p/CatK axis is a key mechanism contributing to the recovery of cardiac function after MI.
We demonstrated that miR-185-5p was a key regulation of revascularisation in heart after ischaemia, and may act as a potential therapeutic target in patients with ACS. As an endothelial rich microRNA, miR-185-5p is positive in blood and circulating miR-185 has been reported as a biomarker for clinical outcome in patients with dilated cardiomyopathy. 38 By using high-throughput RNA sequencing, we found 18 microRNAs including miR-185-5p in blood were differentially expressed in ACS patient. The reduction of circulating miR-185-5p in patients with ACS was further confirmed by quantitative PCR. In vivo experiments also indicate that infusion of miR-185-5p agomir delayed the recovery of heart function after MI. Combining the results, we thought that low level of miR-185-5p in blood may be associated with the good prognosis of ACS. Consistent with this notion, it has reported that circulating CatK, as a target of miR-185-5p identified by us, is a potential novel biomarker of coronary artery disease. 39 How miR-185-5p is down-regulated in endothelial cells by hypoxia and reduced in blood in ACS patient needs further investigation.
In summary, we identified that circulating miR-185-5p is substantially reduced in ACS patients. Endogenous inhibition of miR-185-5p in endothelial cells promotes angiogenesis and accelerates the repair of heart function after MI through targeting CatK gene expression ( Figure S6D). Therefore, modulation of CatK gene expression by miR-185-5p could be effective for an angiogenic therapy in treating ischaemic diseases, including MI, stroke, peripheral artery disease and wound healing in diabetes. 40