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 Welten et al 14q32 MicroRNAs in Neovascularization 697Both arteriogenesis and angiogenesis are highly multifactorial processes, and yet clinical trials aiming to induce neovascularization in patients with occlusive arterial disease have so far only focused on single-factor therapeutics, such as growth factors (eg, vascular endothelial growth factor A [VEGFA] and basic fibroblast growth factor [bFGF]). Unfortunately, these trials were less successful than anticipated.1,3,4 Growth factors only target 1 of multiple processes required for efficient neovascularization. Therefore, there is a need for novel proarteriogenic and proangiogenic factors that can act as master switches in neovascularization.MicroRNAs are endogenous RNA molecules that downregulate expression of their target genes.5 MicroRNAs do not completely silence their target genes, but rather downtune their expression. However, because each microRNA has multiple, up to several hundred, target genes, changes in microR-NA expression can have a major impact. Inhibition of a single microRNA can thus lead to activation of entire multifactorial physiological processes.Several studies have been published on the effects of microRNA inhibition on neovascularization, but in general, the focus of these studies lies with angiogenesis alone, not arteriogenesis. [6][7][8][9][10][11][12][13][14] In the present study, we exploited the master switch character of microRNAs to identify microRNAs that play a regulat...
R NA-binding proteins are central regulators of gene expression in both health and disease. 1,2 The RNA-binding protein Quaking (QKI) is a member of the highly conserved signal transduction and activator of RNA (STAR) family of RNA-binding proteins. 3 Alternative splicing of the mammalian qkI transcript yields 3 protein isoforms, notably QKI-5, QKI-6, and QKI-7, 2 with dimerization of QKI isoforms being required for the regulation of pre-mRNA splicing, mRNA export, and stability. 2,4 QKI drives central and peripheral nervous system myelination by regulating oligodendrocyte and Schwann cell differentiation, respectively. 2,4,5 However, a role for QKI outside the neural network is poorly understood. In This
IntroductionAngiogenesis in the adult is associated with specific conditions such as tissue ischemia and wound repair, in which the formation of new blood vessels is temporally and spatially controlled. Tissue injury causes damage of blood vessels and the extravasation of plasma proteins, including fibrinogen, which results in the formation of a fibrin clot in the surrounding interstitium. This extracellular matrix of extravasated fibrin entangled with the existing collagen fibers is furthermore composed of a number of proteins such as vitronectin, fibronectin, laminin, hyaluronic acid, and proteoglycans. The fibrinous matrix formed serves as a provisional matrix, into which cells can infiltrate during the subsequent wound healing. [1][2][3] During the formation of a granulation tissue, a dynamic interaction between microvascular endothelial cells (MVECs) and the surrounding extracellular matrix occurs. The cells disrupt existing cell-matrix interactions and locally degrade the surrounding extracellular matrix; they migrate, proliferate, and form new capillarylike tubular structures, which become stabilized in the course of time. 4,5 The temporary fibrin matrix can be degraded by plasmin, which is activated from its zymogen by 2 types of plasminogen activators, tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA). 6 The activity of u-PA is directed to the cell surface by a cellular u-PA receptor (u-PAR). 7 In vitro 8,9 and in vivo 10,11 studies have shown that the u-PA/plasmin system plays a critical role in the process of angiogenesis. In addition to the u-PA/plasmin system, matrix metalloproteinases (MMPs) are also involved in the degradation of the extracellular matrix. [10][11][12][13][14][15] A role for both u-PA/plasmin and MMP systems in angiogenesis is supported by the fact that different components in the extracellular matrix are substrates for plasmin as well as for MMPs, including fibrin, 16 vitronectin, fibronectin, laminin, gelatins, and proteoglycans, whereas plasmin is unable to degrade collagens. 17 Furthermore, endothelial cells at the leading edge of a new blood vessel concomitantly express components of both protease systems, [18][19][20] and their expression is regulated by the same growth factors and cytokines. [21][22][23][24] MMPs are a family of zinc-dependent enzymes that can be divided into 2 structurally distinct groups, the secreted MMPs and the membrane-type MMPs (MT-MMPs). The MMPs are secreted as inactive zymogens, and in vitro studies have shown that the u-PA/plasmin system located at the cell membrane directly or indirectly activates a number of pro-MMPs, such as pro-MMP-1, pro-MMP-3, pro-MMP-9, pro-MMP-10, and pro-MMP-13. 25,26 In addition to zymogen activation, MMP activity is regulated by tissue inhibitors of metalloproteinases (TIMPs), of which to date 4 types have been characterized. 27 MT-MMPs, of which 6 types are known, are transmembrane proteins that are activated intracellularly in the secretory pathway by furinlike enzymes. 28-30 MT1...
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 inflammationdriven 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 Bastiaansen et al PCAF Regulates Arteriogenesis 1903growth are multifactorial and too complex to be modulated by therapeutics that target a single gene or pathway. In contrast, modulation of a factor that acts as a master switch for multiple relevant gene programs may be a more effective strategy to augment arteriogenesis.A protein with such master switch potential is p300-CBPassociated factor (PCAF), a transcriptional coactivator with intrinsic histone acetyltransferase activity. PCAF acetylates histones H3 and H4, but there is also increasing evidence that PCAF modulates nonhistone proteins, [13][14][15][16] including hypoxiainducible factor 1α 17 and Notch. 18 Furthermore, the histone acetylating activity of PCAF is essential for nuclear factor κB (NF-κB)-mediated gene transcription 19 and facilitates inflammatory gene regulation. 20 Because arteriogenesis is an inflammatory-like process, we hypothesized that PCAF acts as master switch that stimulates multiple inflammatory processes important for collateral remodeling.Recently, it was shown in a large patient population study (>3000 individuals)21 that a variation in the promoter region of PCAF is associated with coronary heart disease-related mortality. 22 In support of this observation, we recently demonstrated a role for PCAF in vascular remodeling in a mouse model for reactive stenosis. However, whether PCAF participates in arteriogenesis has not yet been investigated.In the present study, we investigated the contribution of PCAF to postischemic neovascularization in a hindlimb ischemia (HLI) model, 23 using PCAF-deficient (PCAF −/− ) mice. When studying arteriogenesis in a knockout model, it is possible that the gene deletion may affect vascular development in the embryo, including collaterogenesis, thus affec...
BackgroundIn 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 ResultsAlthough 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.ConclusionsThese 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.
AimsWe 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 ResultsRP105−/− 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.ConclusionsRP105 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.
Neural primordia of chick embryos were cultured for three days and the behaviour of migrating neural crest cells studied. Somite cells were used as a comparison. Crest cells were actively multipolar with narrow projections which extended and retracted rapidly, contrasting to the gradual extension of somite-cell lamelleae. On losing cell contact, somite cells were also more directionally persistent. The rate of displacement of isolated crest cells was particularly low when calculated over a long time base. Both crest and somite cells were monolayered; contact paralysis occurred in somite cell collisions but was not ascertained for crest cells. However, crest cells in a population were far more directionally persistent than isolated cells. Contact duration between crest cells increased with time and they formed an open network. Eventually, retraction clumping occurred, initially and chiefly at the periphery of the crest outgrowth. Crest cells did not invade cultured embryonic mesenchymal or epithelial populations but endoderm underlapped them. No effects were observed on crest cells prior to direct contact. Substrate previously occupied by endoderm of ectoderm caused crest cells to flatten while substrate previously occupied by the neural tube caused them to round up and clump prematurely.
e72P ostinterventional remodeling is a critical determinant of long-term efficacy of percutaneous coronary interventions. Restenosis is characterized by acute elastic recoil and intimal hyperplasia attributable to inflammation, smooth muscle cell (SMC) proliferation, and extracellular matrix turnover. 1 Under hypercholesterolemic conditions, this is accompanied by influx and accumulation of low-density lipoprotein (LDL) cholesterol in the vessel wall that becomes oxidized and taken up by macrophages. Thereby these macrophages become foam cells and initiate a process of accelerated atherosclerosis.2 Previously, we and others described an important causal role for extracellular toll-like receptors (TLRs) in postinterventional remodeling. It has been shown that TLR4 and the MyD88-dependent pathway play an important role in restenosis and postinterventional accelerated atherosclerosis. [3][4][5][6] Similarly, a crucial role for TLR2 has been described. TLRs, as part of the innate immune system, are pattern recognition receptors known to recognize exogenous ligands that originate from bacteria or viruses as well as endogenous ligands. These endogenous ligands may be released after tissue damage or cell stress, processes that may be initiated by percutaneous coronary interventions. MyD88-dependent signaling is the dominant activation pathway of TLR signaling leading to nuclear factor-kappaB activation and upregulation of several proinflammatory cytokines. Because TLR2 and TLR4 are known to be expressed on the cell surface of vascular cells and activated in vascular disease processes via damageassociated molecular patterns as endogenous ligands, such as heat shock proteins, fibronectin containing extradomain A, tenascin-C, and high-mobility group box 1 (HMGB1), [8][9][10][11] research in the cardiovascular field mainly focused on TLR2 and TLR4. Objective-The role of toll-like receptors (TLRs) in vascular remodeling is well established. However, the involvement of the endosomal TLRs is unknown. Here, we study the effect of combined blocking of TLR7 and TLR9 on postinterventional remodeling and accelerated atherosclerosis. Methods and Results-In hypercholesterolemic apolipoprotein E*3-Leiden mice, femoral artery cuff placement led to strong increase of TLR7 and TLR9 presence demonstrated by immunohistochemistry. Blocking TLR7/9 with a dual antagonist in vivo reduced neointimal thickening and foam cell accumulation 14 days after surgery by 65.6% (P=0.0079). Intima/media ratio was reduced by 64.5% and luminal stenosis by 62.8%. The TLR7/9 antagonist reduced the arterial wall inflammation, with reduced macrophage infiltration, decreased cytoplasmic high-mobility group box 1 expression, and altered serum interleukin-10 levels. Stimulation of cultured macrophages with TLR7 and TLR9 ligands enhanced tumor necrosis factor-α expression, which is decreased by TLR7/9 antagonist coadministration. Additionally, the antagonist abolished the TLR7/9-enhanced low-density lipoprotein uptake. The antagonist also reduced oxidized lowden...
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