Sabine M.J. Welten

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.

The multifactorial nature of microRNAs in vascular remodelling

Vascular remodelling is a multifactorial process that involves both adaptive and maladaptive changes of the vessel wall through, among others, cell proliferation and migration, but also apoptosis and necrosis of the various cell types in the vessel wall. Vascular remodelling can be beneficial, e.g. during neovascularization after ischaemia, as well as pathological, e.g. during atherosclerosis and aneurysm formation. In recent years, it has become clear that microRNAs are able to target many genes that are involved in vascular remodelling processes and either can promote or inhibit structural changes of the vessel wall. Since many different processes of vascular remodelling are regulated by similar mechanisms and factors, both positive and negative vascular remodelling can be affected by the same microRNAs. A large number of microRNAs has been linked to various aspects of vascular remodelling and indeed, several of these microRNAs regulate multiple vascular remodelling processes, including both the adaptive processes angiogenesis and arteriogenesis as well as maladaptive processes of atherosclerosis, restenosis and aneurysm formation. Here, we discuss the multifactorial role of microRNAs and microRNA clusters that were reported to play a role in multiple forms of vascular remodelling and are clearly linked to cardiovascular disease (CVD). The microRNAs reviewed are miR-126, miR-155 and the microRNA gene clusters 17-92, 23/24/27, 143/145 and 14q32. Understanding the contribution of these microRNAs to the entire spectrum of vascular remodelling processes is important, especially as these microRNAs may have great potential as therapeutic targets for treatment of various CVDs.

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 Ly6Chi 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.

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.

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 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.

Letter Regarding Article, "MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization".

We read with interest the article by Pankratz and colleagues1 in Circulation in which the authors identified miR-155 as a dual player in postischemic neovascularization following hindlimb ischemia in mice. Although this study elegantly describes an antiangiogenic but proarteriogenic role for miR-155 in postischemic neovascularization, it remains unclear on several other aspects.1 Using microRNA expression profiling, miR-155 was identified as one of the strongest downregulated miRs at 7 days after induction of hindlimb ischemia.1 However, in a previous study from 2010 by the same research group, Grundmann et al already used microarrays to explore microRNA expression patterns during adaptive neovascularization in the same mouse model.2 MicroRNA analyses in both studies were performed on RNA …

Inhibition of Individual 14q32 MicroRNAs Drastically Increases Neovascularization and Blood Flow Recovery after Ischemia

Introduction and Hypothesis: Neovascularization, i.e. angiogenesis and arteriogenesis, is a multifactorial process. As microRNAs can regulate expression of up to several hundred target genes, we hypothesized that specific microRNAs may target not just single aspects of neovascularization, but neovascularization as a whole. We set out to identify microRNAs that target genes in all pathways of neovascularization. Using www.targetscan.org, we performed a reverse target prediction on a set of 197 genes involved in neovascularization. We found enrichment of binding sites for 27 microRNAs in a single microRNA gene cluster on the long arm of human chromosome 14. MicroArray analyses showed that 14q32 microRNAs were down-regulated during effective neovascularization in mice subjected to single femoral artery ligation. Methods and Results: Gene Silencing Oligonucleotides (GSOs), were injected (1mg/mouse) to inhibit four 14q32 microRNAs, miR-487b, miR-494, miR-329 and miR-495, one day prior to double ligation of the femoral artery. 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 vs. 15% in control animals) and those treated with GSO-329 showed a remarkable full recovery of perfusion after 7 days (vs. 60% perfusion in control animals). In vivo arteriogenesis was enhanced as 3-fold increased collateral artery diameters were observed in adductor muscles of GSO-treated mice. Simultaneously, in vivo angiogenesis was also enhanced as we observed up to 10-fold increased capillary densities in the ischemic soleus muscles of GSO-treated mice. Furthermore, in vitro treatment with GSO-329, GSO-495 and GSO-487b led to increased proliferation of primary human arterial endothelial cells whereas treatment with GSO-494 led to increased proliferation of primary human arterial fibroblasts. Conclusions: Inhibition of 14q32 microRNAs leads to drastic increases in post-ischemic blood flow recovery in vivo via stimulation of both arteriogenesis and angiogenesis. In conclusion, 14q32 microRNA inhibition may offer an alternative to growth factors in therapeutic neovascularization.