MicroRNA-132/212 family enhances arteriogenesis after hindlimb ischaemia through modulation of the Ras-MAPK pathway

Arteriogenesis is a complicated process induced by increased local shear-and radial wall-stress, leading to an increase in arterial diameter. This process is enhanced by growth factors secreted by both inflammatory and endothelial cells in response to physical stress. Although therapeutic promotion of arteriogenesis is of great interest for ischaemic diseases, little is known about the modulation of the signalling cascades via microRNAs. We observed that miR-132/212 expression was significantly upregulated after occlusion of the femoral artery. miR-132/212 knockout (KO) mice display a slower perfusion recovery after hind-limb ischaemia compared to wildtype (WT) mice. Immunohistochemical analysis demonstrates a clear trend towards smaller collateral arteries in KO mice. Although Ex vivo aortic ring assays score similar number of branches in miR-132/212 KO mice compared to WT, it can be stimulated with exogenous miR-132, a dominant member of the miR-132/212 family. Moreover, in in vitro pericyte-endothelial co-culture cell assays, overexpression of miR-132 and mir-212 in endothelial cells results in enhanced vascularization, as shown by an increase in tubular structures and junctions. Our results suggested that miR-132/212 may exert their effects by enhancing the Ras-Mitogen-activated protein kinases MAPK signalling pathway through direct inhibition of Rasa1, and Spred1. The miR-132/212 cluster promotes arteriogenesis by modulating Ras-MAPK signalling via direct targeting of its inhibitors Rasa1 and Spred1.


Introduction
Under physiological circumstances, normal adult blood vessels stay quiescent by using various inhibitors to counteract pro-angiogenic signal fluctuation [1]. Dysregulation of this balance may cause diseases such as capillary and arterio-venous malformations [2]. However, under ischaemic conditions, the compensatory growth of blood vessels is an appreciated response, which can be achieved in two ways: by branching from existing vessels (called angiogenesis), or by enlargement of pre-existing collaterals (termed arteriogenesis) [3]. The increase in diameter via arteriogenesis weights much more than the number of newly formed capillaries via angiogenesis and has therefore the potential to become a future therapeutic approach [4] in chronic and acute ischaemic diseases. Many attempts have been made to modulate the pro-and anti-arteriogenic balance [5][6][7]. However, effective therapeutic approaches to promote arteriogenesis are still lacking.
Initial studies have shown an important role for microRNAs (miRNAs) in neovascularization [8][9][10][11][12][13][14], but a clear understanding of all players involved is still lacking. It has previously been shown that miR-132 is upregulated in endothelial cells by various proangiogenic stimuli such as hypoxia [15], VEGF [10,15], and angiotensin II [16]. Overexpression of miR-132 in human umbilical venous endothelial cells (HUVECs) promoted proliferation and migration in vitro, and transplanting these cells promoted vascularization in vivo [17]. In cancer, miR-132 promoted angiogenesis by suppressing one of the GTPase-activating proteins, called RASA1 [10]. Very low density lipoprotein receptor knockout (KO) mice, displayed an aberrant neovascularization in the retina, associated with increased expression of miR-132. Moreover, inhibition of miR-132 in the retina could reduce aberrant neovascularization [18] or corneal neovascularization [19]. Although, the role of this microRNA family was explored for angiogenesis, its influence on hind-limb ischaemia induced arteriogenesis has not been explored. In this study, we combine in vitro assays and in vivo animal models to explore the role of miR-132/212 in vascular growth during arteriogenesis and to unravel the underlying mechanism.

Materials and methods Generation and genotyping of miR-132/212 KO mice
The generation of miR-132/212 KO mice has been described as previously [20]. For genotyping, DNA samples were obtained by ear clipping and used in a GC-Rich PCR kit (Cat. 12140306001; Roche, Switzerland) with the MiR-132/212 primers as shown in the Table S1. PCR products were revealed on a 1% agarose gel: wildtype (WT) genotype shows a predicted band at 1076 bp and the KO genotype at 392 bp.

Hind-limb ischaemia
This study was approved by the Animal Ethical Experimentation Committee (Utrecht University) and was carried out in accordance with the Guide for the care and use of Laboratory Animals.
Hind-limb ischaemia was applied on 10-12 week old mice [10 WT (C57B6) and 13 miR-132/212 KO] as described previously [21]. In brief, mice were anaesthetized with fentanyl (0.05 mg/kg), midazolam (5 mg/ kg) and medetomidine (0.5 mg/kg) by intraperitoneal injection and surgical procedures were performed under sterile conditions. A vertical longitudinal incision was made in the right hind-limb and the femoral artery was dissected. To achieve slower recovery, ligation was performed using an electricoagulator at the most proximal position and thereby separating them into two parts. After closure, mice received atipamezole (2.5 mg/kg) and flumazenil (0.5 mg/kg) to recover. Temgesic (0.1 mg/kg) was given every 8 hrs after surgery for 6 times. Measurement of blood flow was performed by scanning both rear paws with an LDI analyzer (Moor Infrared Laser Doppler Imager Instrument, Wilmington, DE, USA), before and after the surgical procedure (days 0, 4, 7, and 14). During the procedure, the animal was kept under 2% isoflurane anaesthesia and its body temperature was strictly maintained between 36.5 and 37.5°C. The images obtained were quantitatively converted into histograms with Moor LDI processing software as described before [22]. Data were reported as the ratio of blood flow in the right over left (R/L) hindlimb.

RNA isolation and RT-PCR
DNA-free RNA was extracted with Tripure (Roche Applied Science, Switzerland). To perform quantitative PCR (qPCR) for gene expression RNA is transcribed to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, USA) according to manufacturer's instructions, and quantitative real-time polymerase chain reaction was performed on a MyIQ single-color qRT-PCR system (Bio-Rad) as described previously [24]. All the primers used for qPCR analysis are listed in the Table S1. Mature miR-132 and miR-212 expression levels were measurement by TaqMan â MicroRNA Assay following manufactory's instruction, using U6 as control.

Aortic ring assay
Aortas from both WT and miR-132/212 KO mice were surgically isolated, cleaned, dissected into 0.5 mm segments and embedded into fibrin as described before [25]. For rescue, aortic ring segments were transfected overnight either with 50 nmol/l microRNA mimics as indicated by siPORT NeoFX prior to embedding. 25 ng/ml recombinant mouse vascular endothelial cell growth factor (VEGF164; 493-MV-005; R&D, USA) was added and replaced on day 4. Pictures were taken on day 7 and the number of branches were counted under an inverted microscope.
3 0 -untranslated region reporter generation and luciferase assay A 1 kb fragment, which flanks conserved miR-132-binding sequences of the spred1 untranslated region (UTR), and the full-length Spry1 3 0 UTR were cloned into the pMIR-REPORT Luciferase vector (Ambion, USA), as described previously [23]. Mutations in the seed-region were generated by Q5 Site-Directed Mutagenesis kit (New England Biolabs, USA). All the primers used for cloning and mutagenesis are listed in the Table S1. To determine suppression efficiency of miR-132 and 212 on these targets, HEK293 cells were co-transfected with 200 ng of pMIR-REPORT-3 0 UTR Luciferase vectors, or one of the mutated vectors, and a pMIR-REPORTbgal control plasmid to normalize for transfection efficiency. In addition, 25 nmol/l miR mimic controls, miR-132 mimics or miR-212 mimics were introduced by using Lipofectamine 2000 (Life Technologies). Luciferase and b-galactosidase activity was assessed after 48 hrs with the Luciferase Assay System and b-galactosidase Enzyme Assay System (both from Promega, USA), respectively, as previously described [24].

In vitro angiogenesis assay
Human umbilical vein endothelial cells (Lonza) and human brain vascular pericytes (#1200; Sciencell, San Diego, USA) were cultured on gelatincoated plates in EGM2 medium (EBM2 medium supplemented with EGM2 bullet kit and 2% FCS; Lonza) and DMEM (10% FCS; Lonza), respectively, in 5% CO 2 at 37°C. Lentiviral transfected HUVECs expressing green fluorescent protein (GFP) and pericytes were used at passage 6-8. miR-132 and miR-212 were inhibited or enhanced in HUVECs only, either by using anti-miR-132 and anti-miR-212, or by supplementing miR-132 mimics and miR-212 mimics, respectively. Control cells were transfected with non-targeting miR and anti-miR controls. To monitor the effects of miR-132 and miR-212 in angiogenesis, transfected HUVEC-GFP and PKH26 stained pericytes were suspended in a 2.5 mg/ml collagen type I (BD Biosciences, USA) as described by Stratman et al. [26]. Co-cultures were imaged after 96 hrs incubation in 5% CO 2 at 37°C by fluorescence microscopy, thereby acquiring four planes of images, followed by 3Danalysis using a commercial analysis system (Angiosys, Buckingham, UK).

Phospho-ERK1/2 Bio-PlexPro TM assay
In brief, 48 hrs after transfection of indicated siRNAs or microRNA mimics, EGM2 was removed and replaced with EBM2 for 3 hrs to starve the HUVECs. Subsequently, 25 ng/ml recombinant human VEGF165 (293-E-010; R&D) was added and cells were harvested at indicated time points and lysed with EDTA-free lysis buffer (Cat. 04719964001; Roche Applied Science) with 19 protease/phosphatase inhibitor cocktail (#5872; Cell Signaling). Protein concentrations were measured with the BCA protein assay kit (23227; Thermo Scientific) and diluted into 200 lg/ml. Phospho-ERK1/ 2 Bio-PlexPro TM assay was performed according to the manufacture's instruction. Of each sample, 100 ll was incubated with capture antibodies (171-v50006M, 171-v60003M; Bio-Rad), and after washing, streptavidin-PE was applied for visualization. Samples were processed with the Bio-plex 200 (Bio-Rad) and data were analysed with Bio-Plex Data Pro software (Bio-Rad) and Graphpad Prism 6.0.

Statistical analysis
Data were using Graphpad Prism 6 and comparisons were performed with t-test or paired t-test between two groups, and ANOVA for multiple comparisons. Data were presented as mean AE SEM. P-values are indicated as follows: *P < 0.05; **P < 0.01; ***P < 0.001, P < 0.05 is considered as significant.

MiRNA-132 and miR-212 is upregulated upon hind-limb ischaemia
To understand the function of miR-132 and miR-212 in arteriogenesis, we performed hind-limb ischaemia on WT mice and checked the expression of these two microRNAs in the thigh muscle at different time points after hind-limb ischaemia. By qRT-PCR, we found that miR-132 and miR-212 levels were significantly increased on day 4 and day 7 ( Fig. 1A and B) after hindlimb ischaemia in the adductor muscle.
The miR-132/212 locus is positioned in the first intron of the human 1700016p03 gene, whose function remains to be investigated. miR-132 and 212 are transcribed as a single transcript and further processed into two mature microRNAs, which are highly conserved among different species (Fig. S1A). Although these two miRNAs share the same seed sequences and hereby belong to the same miRNA family, the level of mature miR-132 expression is significantly higher than that of miR-212 in the thigh muscle, indicating that miR-132 might be more active in the arteriogenic response, as previously reported for miR-212 being a more dominant miRNA in angiogenesis (Fig. 1A).
To further understand its function, we analysed which cell types express miR-132 in hind-limb tissue by in situ hybridization. As expected, we found that miR-132 is expressed in endothelial cells (lectin BS-1 positive cells) of blood vessels and in cells surrounding the endothelial cell layer in WT mice only (Fig. S1B).

MicroRNA-132/212 is involved in arteriogenesis after hindlimb ischaemia
The increased expression upon hindlimb ischaemia and the vascular localization of miR-132 suggests that miR-132 may play a role in vascular growth, for example, in arteriogenesis. To test this, we compared the arteriogenic response between WT and KO mice. Blood flow perfusion ratio in the miR-132/212 KO mice was significantly lower compared to their WT litters at day 7 and 14 as measured by laser Doppler ( Fig. 2A and B), indicating a slower perfusion restoration. The total number of aSMA positive vessels in the adductor muscle was similar between WT and KO mice (Fig. 2C), but there is a clear trend towards smaller collateral arteries in KO mice, as determined by the cross-sectional diameter of aSMA-positive arteries (Fig. 2D and E).

miR-132/212 promotes endothelial cells neovascularization responses in vitro
To further investigate the effect of microRNA-132/212 on vascular growth, we modulated miR-132/212 activity with overexpression or inhibition approaches in different in vitro neovascularization assays.
Firstly, we performed WT and miR-132/212 KO mice-derived aortic ring assay [25]. In the growth factor rich environment, we observed a slight decrease in the number of branches in aortic rings from KO mice compared to WT control. Interestingly, transfection of miR-132 mimics rescued, and even significantly enhanced activation via increasing vascular branching. Although similar effect were observed with miR-212, the effects were less pronounced as compared with miR-132 ( Fig. S1C and D).
Secondly, we performed a HUVEC and pericyte co-culture assay, thereby better mimicking the in vivo situation by taking the interplay between endothelial cells and pericytes into account [26]. As shown in Figure 3A, supplementing miR-132 and miR-212 mimics to HUVECs enhanced the total number of junctions, tubules and tubule length compared to that of miR controls. Conversely, inhibiting miR-132 and miR-212 using anti-miRs resulted in some decline in the total number of junctions, tubules and tubule length (Fig. 3B). Spred-1, Spry1 and Rasa1 are direct targets of the microRNA-132/212 family Based on Ago-Hits-clip [27], PAR-CLIP [28] and CLASH studies [29] and our results above, we decided to focus on targets related to growth factor signalling. In combination with bioinformatics target site prediction algorithms (Targetscan), we selected Rasa1, Spred1 and Spry1 which have a high prediction context score and are conserved among species, as shown in Figure 4A and D. Since Rasa1 was already a confirmed miR-132 target [10], we only cloned the 3 0 UTR of Spred1 and Spry1 into a luciferase reporter vector and analysed whether miR-132 and miR-212 could suppress luciferase activity in HEK293 cells. We found that both miR-132 and miR-212 can significantly suppress the Spred1-3 0 UTR and Spry1-3 0 UTR luciferase activity at 25 nmol/l, compared with scramble control miRNAs ( Fig. 4B and E). Additionally, we performed a dose-response assay with Spred1-3 0 UTR, Spry1-3 0 UTR and 3 0 UTRs with three mutated nucleotides in indicated binding regions (Fig. 4A and D). Inhibitory effects were dosedependent and mutations within the seed region significantly affected the suppressing effects on luciferase activity, even completely abolishing the suppressive effect at a concentration of 1 nmol/l ( Fig. 4C and F).
We next investigated the direct regulation of these targets in HU-VEC cells. Following overexpression of miR-132 and miR-212 in HU-VECs in the co-culture assays, we detected reduced levels of Spred1, Spry1 and Rasa1 protein; while inhibition of the miR-132 and miR-212 led to elevated Spred1, Spry1 and Rasa1 expression. This indicates that these genes are also regulated by miR-132/212 in HUVECs ( Fig. 4G and H).

Spred1, Spry1 and Rasa1 knockdown promotes endothelial cells neovascularization responses in vitro
We have shown that overexpression of miR-132 or miR-212 promotes endothelial cells neovascularization. As microRNA functions by inhibiting its targets, we reason that knockdown of Spred1, Spry1 and Rasa1 should have similar effect as overexpression of miR132 and miR-212. As expected, subsequent knockdown of Spred1, Spry1, Rasa1 and a combination of these three via siRNA knockdown in HUVECs (Fig. 6C) showed similar neovascularization responses as overexpression of miR-132 or miR-212, total number of junctions, tubules and tubule length were increased compared to control conditions ( Fig. 6A and B).  We reasoned that if Spred1, Spry1 and Rasa1 are in vivo targets of miR-132 or 212, they should be expressed in the arteries of the thigh muscle. By immunofluorescent staining, we observed that Spred1, Spry1 and Rasa1 could all be detected in the vascular wall (Fig. S2). By comparing the expression of Spred1, Spry1 and Rasa1 in the adductor muscle from WT and KO after femoral artery ligation using Western blot, we observed that Spred1 and Rasa1 are significantly more present in the KO mice ( Fig. 7A and B). Surprisingly we found that there is no difference in the Spry1 protein between WT and KO mice ( Fig. 7A and B). Next we asked if higher level of Spred1 and Rasa1 expression could have an effect on the Ras-MAPK pathway. By Western blotting we detected lower phosphorylated ERK1/2 in the area of blood vessel growth in KO mice 14 days after hindlimb ischaemia ( Fig. 7C and D).

Discussion
Here, we show that upregulation of miR-132 and miR-212 upon hindlimb ischaemia is involved in the arteriogenic response: microRNA-132/212 KO animals display delayed perfusion restoration upon femoral artery occlusion. Furthermore, we demonstrate that this effect is attributable to miR-132/212 modulation of the Ras-MAPK signalling pathway through direct targeting of Spred1 and Rasa1. To the best of our knowledge, this is the first study showing a single microRNA family, and probably mediated via the more abundantly expressed miR-132, that can facilitate the arteriogenic responses by suppressing multiple targets within the Ras-MAPK pathway.
The Ras-MAPK pathway is very important in neovascularization during development and after ischaemic challenges. In the developing retina, for example, this physiological pathway only becomes apparent in active sprouting endothelial cells [18]. Reduced ERK1/2 activation leads to reduced lumen formation, whereas excessive activation of ERK1/2 results in larger arteries [34,35]. Upon ischaemic challenge, e.g. in hindlimb ischaemia models, shear stress-stimulated endothelial cells induce Monocyte Chemotactic Protein 1(MCP-1) expression, which in turn attracts neutrophil granulocytes and macrophages [36]. These circulating inflammatory cells start producing growth factors which eventually activate Ras-MAPK signalling pathway in the smooth muscle cells and endothelial cells further and promote their proliferation and extracellular matrix remodelling. Recently, attempts have been made to interfere in arteriogenesis through manipulation of Ras-MAPK signalling genetically and chemically, for example by suppression of Sproutys to promote blood flow recovery in the hindlimb ischaemia model [7]. Since therapeutic activation of Ras-MAPK signalling is still challenging, inhibition of their endogenous inhibitors using microRNA therapeutics holds a great promise as an alternative strategy.  We confirmed that Rasa1 is a direct target of miR-132 and miR-212, and further expanded their target spectrum thereby including Spred1 and Spry1. Using 3 0 UTR reporters of Spred1 and Spry1, we demonstrated a direct binding of miR-132 and 212, which was abolished by disrupting the corresponding binding sites. Knockdown of these three targets mimicked overexpression of miR-132 or miR-212 in the in vitro neovascularization assay and on the modulation of phosphorylated ERK1/2. Our data show that Rasa1 is the most potent regulator among the three targets in promoting neovascularization and prolonging pERK1/2 activation in HUVECs, probably as being the most upstream signalling molecule as compared to Spry1 and Spred1. Another possibility is genetic redundancy, in which the loss of Spred1 or Spry1 can be compromised partially by other family members; Spry1 has at least four homologues [37] and Spred1 has at least two homologues in mice [38]. Our in vitro observations were confirmed in vivo where levels of Rasa1 and Spred1 were significantly higher in the adductor muscle in the miR-132/212 KO mice upon hind-limb ischaemia. Although Spred1 and Rasa1 protein levels were higher in miR-132/ 212 KO mice, Spry1 expression levels were similar between WT and KO mice. Accordingly, we demonstrated lower levels of phosphorylated ERK1/2 as a downstream effect of lower active Ras-MAPK signalling.
The biological function of miR-132 and miR-212 may be different, althoughtheysharethesameseedsequence.Ithasbeenshownthatmicr-oRNA targets determination is beyond the seed sequence [39]. Consistent with this notion, we observe different effects in various assays. Since bothmiR-132andmiR-212areremovedintheKOmice,itisimpossible to determine which one should be responsible for the impaired arteriogenesis response. Given the fact that the expression of the mature miR-132 is 40-fold higher than miR-212 (Fig. S1A), we tend to believe that miR-132 plays a major role in the arteriogenic response after hindlimb ischaemia. However, it is still possible that a specific cell population, highly expressing miR-212 but not miR-132, is more important for the vascular growth after hindlimb ischaemia. In line with this hypothesis, a recent study showed that miR-212 is stronger in the regulation of vasodilatation than miR-132 [40]. To exclusively clarify the different roles and locations of thesetwomicroRNAs,improvedmicroRNAinsitutechniqueswithhigher sensitivity are needed that can detect low abundant expressed microRNA incombinationwithmicewithindividualmicroRNAKOs.Ourresultsdemonstrate a new role for miR-132 and miR-212 in the facilitation of the arteriogenic responses after hind-limb induced by targeting and enhancing Ras-MAPK signalling. This extends the role for miR-132 beyond the ischaemic challenges and promoting angiogenesis. It would be interesting to test if we can enhance arteriogenesis by specifically deliver these two microRNAs in the ischaemia vasculature or in combination with other Ras-MAPK activators such as growth factors to further boost their proarteriogenic capacity. However, local delivery strategies should still be furtherimproved [41].

Conflicts of interest
None.

Supporting information
Additional Supporting Information may be found in the online version of this article: Figure S1. The expression of the miR-132/212 and their roles in Ex vivo neovascularization assay. Figure S2. The expression of miR-132/212 targets Spred1, Spry1 and Rasa1 in wildtype thigh blood vessels characterized by immunofluorescent staining. Table S1. Primers and oligos nucleotides used in this study.