High-density lipoproteins (HDL) have many biological functions, including reducing endothelial activation and adhesion molecule expression. We recently reported that HDL transport and deliver functional microRNAs (miRNA). Here we show that HDL suppresses expression of intercellular adhesion molecule 1 (ICAM-1) through the transfer of miR-223 to endothelial cells. After incubation of endothelial cells with HDL, mature miR-223 levels are significantly increased in endothelial cells and decreased on HDL. However, miR-223 is not transcribed in endothelial cells and is not increased in cells treated with HDL from miR-223−/− mice. HDL inhibit ICAM-1 protein levels, but not in cells pretreated with miR-223 inhibitors. ICAM-1 is a direct target of HDL-transferred miR-223 and this is the first example of an extracellular miRNA regulating gene expression in cells where it is not transcribed. Collectively, we demonstrate that HDL’s anti-inflammatory properties are conferred, in part, through HDL-miR-223 delivery and translational repression of ICAM-1 in endothelial cells.

Key Points Blockmirs are designed against the miR-27 binding site in VE-cadherin and display restricted specificity. Blockmirs regulate VE-cadherin and endothelial cell junctions, inhibit edema, and promote angiogenesis associated with ischemia.

Rationale: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is well reported as an inducer of apoptosis in tumor models; however, its role and function in vivo in atherosclerosis and vascular injury has not been established. Objective: We sought to study the function of TRAIL in cardiovascular pathology and its regulation in vivo. Methods and Results: Here, we show that TRAIL was upregulated in medial vascular smooth muscle cells (VSMCs) 24 hours following perivascular cuff placement around femoral arteries of mice. We also show that TRAIL mRNA and promoter activity was induced in VSMCs following in vitro mechanical injury. Intimal thickening 15 days after cuff placement was reduced 2-to 3-fold in TRAIL ؊/؊ compared to wild-type mice and was reversible by administration of recombinant TRAIL. Additionally, reduced VSMC proliferation was observed in injured arteries of TRAIL ؊/؊ mice. Fibroblast growth factor (FGF)-2, a potent growth factor released following vascular injury, was also reduced in arteries of TRAIL ؊/؊ mice, and VSMCs isolated from these animals did not respond to FGF-2 in vitro. Injury and FGF-2 regulated TRAIL transcriptional activity via 2 specificity protein (Sp)1 elements in the proximal TRAIL promoter, a binding site also shared by nuclear factor (NF)B. Mutational studies confirmed a role for Sp1 in injury-and FGF-2-inducible TRAIL transcription. Furthermore, increased NFB expression after injury transactivated the TRAIL promoter. Interestingly, following mechanical injury, Sp1 phosphorylation (Thr453) and an increase in the physical interaction of p-Sp1(Thr453) with NFB was observed. Conclusions: We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NFB and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury. (Circ Res. 2010;106: 1061-1071.)Key Words: TRAIL Ⅲ VSMC proliferation Ⅲ injury Ⅲ transcriptional regulation T RAIL (tumor necrosis factor-related apoptosis inducing ligand) is expressed by a variety of cells, traditionally known to induce apoptosis via the death receptor pathway analogous to Fas ligand (FasL). Unlike FasL, however, TRAIL apoptotic signaling occurs following its engagement with 2 death domain-containing receptors, death receptors 4 and 5 (DR4 and DR5). Additional receptors are also present in humans, including decoy receptors 1 and 2 (DCR1 and DCR2) and osteoprotegerin, which may compete with DR4 and/or DR5 for TRAIL binding, thereby protecting cells from apoptosis. 1,2 TRAIL expression is regulated by insulin, interferons type I and II, and transcription factors Stat1, nuclear factor (NF)B, Egr2/3 (early growth response factor 2/3), and IRF-1/3/7 (interferon regulatory factor 1/3/7). 3 In the vessel wall, TRAIL is expressed in vascular smooth muscle cells (VSMCs), endothelial cells (ECs), macrophages, and T cells (reviewed 3 ); however, the role and regulation of TRAIL in cardiovascular disorders is currently controversial. Under certain conditions TRAIL can induce apoptosis of vascular cells which may...

Catalytic DNA molecules that target the transcription factor c- jun inhibit skin cancer growth in mice.

BackgroundTumor necrosis factor–related apoptosis‐inducing ligand (TRAIL) has the ability to inhibit angiogenesis by inducing endothelial cell death, as well as being able to promote pro‐angiogenic activity in vitro. These seemingly opposite effects make its role in ischemic disease unclear. Using Trail −/− and wildtype mice, we sought to determine the role of TRAIL in angiogenesis and neovascularization following hindlimb ischemia.Methods and ResultsReduced vascularization assessed by real‐time 3‐dimensional Vevo ultrasound imaging and CD31 staining was evident in Trail −/− mice after ischemia, and associated with reduced capillary formation and increased apoptosis. Notably, adenoviral TRAIL administration significantly improved limb perfusion, capillary density, and vascular smooth‐muscle cell content in both Trail −/− and wildtype mice. Fibroblast growth factor‐2, a potent angiogenic factor, increased TRAIL expression in human microvascular endothelial cell‐1, with fibroblast growth factor‐2‐mediated proliferation, migration, and tubule formation inhibited with TRAIL siRNA. Both fibroblast growth factor‐2 and TRAIL significantly increased NADPH oxidase 4 (NOX4) expression. TRAIL‐inducible angiogenic activity in vitro was inhibited with siRNAs targeting NOX4, and consistent with this, NOX4 mRNA was reduced in 3‐day ischemic hindlimbs of Trail −/− mice. Furthermore, TRAIL‐induced proliferation, migration, and tubule formation was blocked by scavenging H2O2, or by inhibiting nitric oxide synthase activity. Importantly, TRAIL‐inducible endothelial nitric oxide synthase phosphorylation at Ser‐1177 and intracellular human microvascular endothelial cell‐1 cell nitric oxide levels were NOX4 dependent.ConclusionsThis is the first report demonstrating that TRAIL can promote angiogenesis following hindlimb ischemia in vivo. The angiogenic effect of TRAIL on human microvascular endothelial cell‐1 cells is downstream of fibroblast growth factor‐2, involving NOX4 and nitric oxide signaling. These data have significant therapeutic implications, such that TRAIL may improve the angiogenic response to ischemia and increase perfusion recovery in patients with cardiovascular disease and diabetes.

Vascular endothelial growth factor-A (VEGF), a powerful factor involved in vasculogenesis and angiogenesis, is translationally regulated through 2 independent internal ribosome entry sites (IRESs A and B). IRESs enable an mRNA to be translated under conditions in which 5'-cap-dependent translation is inhibited, such as low oxygen stress. In the VEGF mRNA, IRES A influences translation at the canonical AUG codon, whereas the 5' IRES B element regulates initiation at an upstream, in frame CUG. In this study, we have developed transgenic mice expressing reporter genes under the control of these 2 IRESs. We reveal that although these IRESs display low activity in embryos and adult tissues, they permit efficient translation at early time points in ischemic muscle, a stress under which cap-dependent translation is inhibited. These results demonstrate the in vivo efficacy of the VEGF IRESs in response to a local environmental stress such as hypoxia.

Tumour suppressor p53 has been shown to inhibit ®broblast growth factor 2 expression post-transcriptionally in cultured cells. Here we have investigated the mechanism responsible for this post-transcriptional blockade. Deletion mutagenesis of the FGF-2 mRNA leader revealed the requirement of at least four RNA cisacting elements to mediate the inhibitory e ect of p53 in SK-Hep-1 transfected cells, suggesting the involvement of RNA secondary or tertiary structures. Recombinant wild-type, but not Ala 143 mutant p53, was able to speci®cally repress FGF-2 mRNA translation in rabbit reticulocyte lysate, in a dose dependent manner. Sucrose gradient experiments showed that p53 blocks translation initiation by preventing 80S ribosome formation on an mRNA bearing the FGF-2 mRNA leader sequence. Interaction of wild-type and mutant p53 with di erent RNAs showed no signi®cant correlation between p53 RNA binding activity and its translational inhibiting e ect. However, by checking the accessibility of the FGF-2 mRNA leader to complementary oligonucleotide probes, we showed that the binding to RNA of wild-type, but not mutant p53, induced RNA conformational changes that might be responsible for the translational blockade. This strongly suggests that p53 represses FGF-2 mRNA translation by a direct mechanism involving its nucleic acid unwinding ± annealing activity. Oncogene (2001) 20, 4613 ± 4620.

Due to the lack of an adequate conventional therapy against lower limb ischemia, gene transfer for therapeutic angiogenesis is seen as an attractive alternative. However, the possibility of side effects, due to the expression of large amounts of angiogenic factors, justifies the design of devices that express synergistic molecules in low controlled doses. We have developed an internal ribosome entry site (IRES)–based bicistronic vector expressing two angiogenic molecules, fibroblast growth factor 2 (FGF2), and Cyr61. Through electrotransfer into the ApoE−/− mice hindlimb ischemic muscle model, we show that the IRES-based vector gives more stable expression than either monocistronic plasmid. Furthermore, laser Doppler analysis, arteriography, and immunochemistry clearly show that the bicistronic vector promotes a more abundant and functional revascularization than the monocistronic vectors, despite the fact that the bicistronic system produces 5–10 times less of each angiogenic molecule. Furthermore, although the monocistronic Cyr61 vector accelerates B16 melanoma growth in mice, the bicistronic vector is devoid of such side effects. Our results show an active cooperation of FGF2 and Cyr61 in therapeutic angiogenesis of hindlimb ischemia, and validate the use of IRES-based bicistronic vectors for the coexpression of controlled low doses of therapeutic molecules, providing perspectives for a safer gene therapy of lower limb ischemia.