Posttranscriptional Regulation of 14q32 MicroRNAs by the CIRBP and HADHB during Vascular Regeneration after Ischemia.

After induction of ischemia in mice, 14q32 microRNAs are regulated in three distinct temporal patterns. These expression patterns, as well as basal expression levels, are independent of the microRNA genes' order in the 14q32 locus. This implies that posttranscriptional processing is a major determinant of 14q32 microRNA expression. Therefore, we hypothesized that RNA binding proteins (RBPs) regulate posttranscriptional processing of 14q32, and we aimed to identify these RBPs. To identify proteins responsible for this posttranscriptional regulation, we used RNA pull-down SILAC mass spectrometry (RP-SMS) on selected precursor microRNAs. We observed differential binding of cold-inducible RBP (CIRBP) and hydroxyacyl-CoA dehydrogenase trifunctional multienzyme complex subunit beta (HADHB) to the precursors of late-upregulated miR-329-3p and unaffected miR-495-3p. Immunohistochemical staining confirmed expression of both CIRBP and HADHB in the adductor muscle of mice. Expression of both CIRBP and HADHB was upregulated after hindlimb ischemia in mice. Using RBP immunoprecipitation experiments, we showed specific binding of CIRBP to pre-miR-329 but not to pri-miR-329. Finally, using CRISPR/Cas9, we generated HADHB-/- 3T3 cells, which display reduced expression of miR-329 and miR-495 but not their precursors. These data suggest a novel role for CIRBP and HADHB in posttranscriptional regulation of 14q32 microRNAs.

Inhibition of Mef2a Enhances Neovascularization via Post-transcriptional Regulation of 14q32 MicroRNAs miR-329 and miR-494.

Improving the efficacy of neovascularization is a promising strategy to restore perfusion of ischemic tissues in patients with peripheral arterial disease. The 14q32 microRNA cluster is highly involved in neovascularization. The Mef2a transcription factor has been shown to induce transcription of the microRNAs within this cluster. We inhibited expression of Mef2a using gene-silencing oligonucleotides (GSOs) in an in vivo hind limb ischemia model. Treatment with GSO-Mef2a clearly improved blood flow recovery within 3 days (44% recovery versus 25% recovery in control) and persisted until 14 days after ischemia induction (80% recovery versus 60% recovery in control). Animals treated with GSO-Mef2a showed increased arteriogenesis and angiogenesis in the relevant muscle tissues. Inhibition of Mef2a decreased expression of 14q32 microRNAs miR-329 (p = 0.026) and miR-494 (trend, p = 0.06), but not of other 14q32 microRNAs, nor of 14q32 microRNA precursors. Because Mef2a did not influence 14q32 microRNA transcription, we hypothesized it functions as an RNA-binding protein that influences processing of 14q32 microRNA miR-329 and miR-494. Mef2A immunoprecipitation followed by RNA isolation and rt/qPCR confirmed direct binding of MEF2A to pri-miR-494, supporting this hypothesis. Our study demonstrates a novel function for Mef2a in post-ischemic neovascularization via post-transcriptional regulation of 14q32 microRNAs miR-329 and miR-494.

Inhibition of 14q32 microRNA miR-495 reduces lesion formation, intimal hyperplasia and plasma cholesterol levels in experimental restenosis.

BACKGROUND AND AIMS: We aimed at investigating the role of 14q32 microRNAs in intimal hyperplasia and accelerated atherosclerosis; two major contributors to restenosis. Restenosis occurs regularly in patients treated for coronary artery disease and peripheral arterial disease. We have previously shown that inhibition of 14q32 microRNAs leads to increased post-ischemic neovascularization, and microRNA miR-494 also decreased atherosclerosis, while increasing plaque stability. We hypothesized that 14q32 microRNA inhibition has beneficial effects on intimal hyperplasia, as well as accelerated atherosclerosis. METHODS: Non-constrictive cuffs were placed around both femoral arteries of C57BL/6J mice to induce intimal hyperplasia. Accelerated atherosclerotic plaque formation was induced in hypercholesterolemic ApoE-/- mice by placing semi-constrictive collars around both carotid arteries. 14q32 microRNAs miR-329, miR-494 and miR-495 were inhibited in vivo using Gene Silencing Oligonucleotides (GSOs). RESULTS: GSO-495 administration led to a 32% reduction of intimal hyperplasia. Moreover, the number of macrophages in the arterial wall of mice treated with GSO-495 was reduced by 55%. Inhibition of miR-329 and miR-494 had less profound effects on intimal hyperplasia. GSO-495 administration also decreased atherosclerotic plaque formation by 52% and plaques of GSO-495 treated animals showed a more stable phenotype. Finally, cholesterol levels were also decreased in GSO-495 treated animals, via reduction of the VLDL-fraction. CONCLUSIONS: GSO-495 administration decreased our primary outcomes, namely intimal hyperplasia, and accelerated atherosclerosis. GSO-495 administration also favourably affected multiple secondary outcomes, including macrophage influx, plaque stability and total plasma cholesterol levels. We conclude that 14q32 microRNA miR-495 is a promising target for prevention of restenosis.

CCR7-CCL19/CCL21 Axis is Essential for Effective Arteriogenesis in a Murine Model of Hindlimb Ischemia.

BACKGROUND: In order to identify factors that stimulate arteriogenesis after ischemia, we followed gene expression profiles in two extreme models for collateral artery formation over 28 days after hindlimb ischemia, namely "good-responding" C57BL/6 mice and "poor-responding" BALB/c mice. METHODS AND RESULTS: Although BALB/c mice show very poor blood flow recovery after ischemia, most known proarteriogenic genes were upregulated more excessively and for a longer period than in C57BL/6 mice. In clear contrast, chemokine genes Ccl19, Ccl21a, and Ccl21c and the chemokine receptor CCR7 were upregulated in C57BL/6 mice 1 day after hindlimb ischemia, but not in BALB/C mice. CCL19 and CCL21 regulate migration and homing of T lymphocytes via CCR7. When subjecting CCR7-/-/LDLR-/- mice to hindlimb ischemia, we observed a 20% reduction in blood flow recovery compared with that in LDLR-/- mice. Equal numbers of α-smooth muscle actin-positive collateral arteries were found in the adductor muscles of both mouse strains, but collateral diameters were smaller in the CCR7-/-/LDLR-/-. Fluorescence-activated cell sorter analyses showed that numbers of CCR7+ T lymphocytes (both CD4+ and CD8+) were decreased in the spleen and increased in the blood at day 1 after hindlimb ischemia in LDLR-/- mice. At day 1 after hindlimb ischemia, however, numbers of activated CD4+ T lymphocytes were decreased in the draining lymph nodes of LDLR-/- mice compared with CCR7-/-/LDLR-/- mice. CONCLUSIONS: These data show that CCR7-CCL19/CCL21 axis facilitates retention CD4+ T lymphocytes at the site of collateral artery remodeling, which is essential for effective arteriogenesis.

Inhibition of MicroRNA-494 Reduces Carotid Artery Atherosclerotic Lesion Development and Increases Plaque Stability.

OBJECTIVES: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery. BACKGROUND: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions. METHODS: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature. RESULTS: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions. CONCLUSIONS: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.

Inhibition of 14q32 MicroRNAs miR-329, miR-487b, miR-494, and miR-495 increases neovascularization and blood flow recovery after ischemia.

RATIONALE: Effective neovascularization is crucial for recovery after cardiovascular events. OBJECTIVE: Because microRNAs regulate expression of up to several hundred target genes, we set out to identify microRNAs that target genes in all pathways of the multifactorial neovascularization process. Using www.targetscan.org, we performed a reverse target prediction analysis on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster. Microarray analyses showed upregulation of 14q32 microRNAs during neovascularization in mice after single femoral artery ligation. METHODS AND RESULTS: Gene silencing oligonucleotides (GSOs) were used to inhibit 4 14q32 microRNAs, miR-329, miR-487b, miR-494, and miR-495, 1 day before double femoral artery ligation. Blood flow recovery was followed by laser Doppler perfusion imaging. All 4 GSOs clearly improved blood flow recovery after ischemia. Mice treated with GSO-495 or GSO-329 showed increased perfusion already after 3 days (30% perfusion versus 15% in control), and those treated with GSO-329 showed a full recovery of perfusion after 7 days (versus 60% in control). Increased collateral artery diameters (arteriogenesis) were observed in adductor muscles of GSO-treated mice, as well as increased capillary densities (angiogenesis) in the ischemic soleus muscle. In vitro, treatment with GSOs led to increased sprout formation and increased arterial endothelial cell proliferation, as well as to increased arterial myofibroblast proliferation. CONCLUSIONS: The 14q32 microRNA gene cluster is highly involved in neovascularization. Inhibition of 14q32 microRNAs miR-329, miR-487b, miR-494, and miR-495 provides a promising tool for future therapeutic neovascularization.

TLR4 accessory molecule RP105 (CD180) regulates monocyte-driven arteriogenesis in a murine hind limb ischemia model.

AIMS: We investigated the role of the TLR4-accessory molecule RP105 (CD180) in post-ischemic neovascularization, i.e. arteriogenesis and angiogenesis. TLR4-mediated activation of pro-inflammatory Ly6Chi monocytes is crucial for effective neovascularization. Immunohistochemical analyses revealed that RP105+ monocytes are present in the perivascular space of remodeling collateral arterioles. As RP105 inhibits TLR4 signaling, we hypothesized that RP105 deficiency would lead to an unrestrained TLR4-mediated inflammatory response and hence to enhanced blood flow recovery after ischemia. METHODS AND RESULTS: RP105-/- and wild type (WT) mice were subjected to hind limb ischemia and blood flow recovery was followed by Laser Doppler Perfusion Imaging. Surprisingly, we found that blood flow recovery was severely impaired in RP105-/- mice. Immunohistochemistry showed that arteriogenesis was reduced in these mice compared to the WT. However, both in vivo and ex vivo analyses showed that circulatory pro-arteriogenic Ly6Chi monocytes were more readily activated in RP105-/- mice. FACS analyses showed that Ly6Chi monocytes became activated and migrated to the affected muscle tissues in WT mice following induction of hind limb ischemia. Although Ly6Chi monocytes were readily activated in RP105-/- mice, migration into the ischemic tissues was hampered and instead, Ly6Chi monocytes accumulated in their storage compartments, bone marrow and spleen, in RP105-/- mice. CONCLUSIONS: RP105 deficiency results in an unrestrained inflammatory response and monocyte over-activation, most likely due to the lack of TLR4 regulation. Inappropriate, premature systemic activation of pro-inflammatory Ly6Chi monocytes results in reduced infiltration of Ly6Chi monocytes in ischemic tissues and in impaired blood flow recovery.

The 14q32 microRNA-487b targets the antiapoptotic insulin receptor substrate 1 in hypertension-induced remodeling of the aorta.

OBJECTIVES: To study the role of microRNAs in hypertension-induced vascular pathology before the onset of symptoms of severe cardiovascular disease. BACKGROUND: MicroRNAs play a crucial role in cardiovascular disease. However, microRNAs are often studied in full-blown cardiovascular disease models, not during development of cardiovascular pathology. METHODS: Angiotensin II was infused into healthy adult rats, inducing chronic hypertension, and microRNA expression profiles were obtained. The most prominently regulated microRNA, miR-487b, was further investigated, using primary cultures of rat aortic and human umbilical cord arterial cells. RESULTS: MiR-487b is predicted to target insulin receptor substrate 1 (IRS1). IRS1 plays an important role in both insulin signaling and cell proliferation and survival. IRS1 mRNA and protein levels were downregulated in aortae of hypertensive rats. MiR-487b binds directly to both rat and human IRS1 3'UTR and inhibits reporter gene expression in vitro. In primary rat and human arterial adventitial fibroblasts, inhibition of miR-487b leads to upregulation of IRS1 expression. Upregulation of miR-487b had the opposite effect, confirming direct targeting of IRS1 by miR-487b.Immunohistochemistry of aortic cross sections and rt/qPCR analyses of the separate aortic wall layers showed that both IRS1 and miR-487b were present mainly in the adventitia and less or not at all in the intima and tunica media. IRS1 expression in adventitial fibroblasts was predominantly nuclear and nuclear IRS1 is known to have antiapoptotic effects. Indeed, inhibition of miR-487b protected adventitial fibroblasts, and also medial smooth muscle cells, against serum starvation-induced apoptosis and increased cell survival. CONCLUSIONS: Angiotensin II-induced hypertension leads to upregulation of miR-487b, which targets IRS1. Via downregulation of IRS1, miR-487b can contribute to cell death and loss of adventitial and medial integrity during hypertension-induced vascular pathology.

Lysine acetyltransferase PCAF is a key regulator of arteriogenesis.

OBJECTIVE: Therapeutic arteriogenesis, that is, expansive remodeling of preexisting collaterals, using single-action factor therapies has not been as successful as anticipated. Modulation of factors that act as a master switch for relevant gene programs may prove more effective. Transcriptional coactivator p300-CBP-associated factor (PCAF) has histone acetylating activity and promotes transcription of multiple inflammatory genes. Because arteriogenesis is an inflammation-driven process, we hypothesized that PCAF acts as multifactorial regulator of arteriogenesis. APPROACH AND RESULTS: After induction of hindlimb ischemia, blood flow recovery was impaired in both PCAF(-/-) mice and healthy wild-type mice treated with the pharmacological PCAF inhibitor Garcinol, demonstrating an important role for PCAF in arteriogenesis. PCAF deficiency reduced the in vitro inflammatory response in leukocytes and vascular cells involved in arteriogenesis. In vivo gene expression profiling revealed that PCAF deficiency results in differential expression of 3505 genes during arteriogenesis and, more specifically, in impaired induction of multiple proinflammatory genes. Additionally, recruitment from the bone marrow of inflammatory cells, in particular proinflammatory Ly6C(hi) monocytes, was severely impaired in PCAF(-/-) mice. CONCLUSIONS: These findings indicate that PCAF acts as master switch in the inflammatory processes required for effective arteriogenesis.