Belinda A. Di Bartolo

TRAIL Promotes VSMC Proliferation and Neointima Formation in a FGF-2–, Sp1 Phosphorylation–, and NFκB-Dependent Manner

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)&kgr;B. Mutational studies confirmed a role for Sp1 in injury- and FGF-2–inducible TRAIL transcription. Furthermore, increased NF&kgr;B 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 NF&kgr;B was observed. Conclusions: We conclude that TRAIL induction involves FGF-2, Sp1-phosphorylation and NF&kgr;B and that TRAIL promotes VSMC proliferation and neointima formation after arterial injury.

Anti-inflammatory effects of apolipoprotein A-I in the rabbit

OBJECTIVE Infusions of apoA-I in the lipid-free form or as a constituent of discoidal reconstituted high-density lipoproteins, (A-I)rHDL, markedly inhibit acute vascular inflammation in normocholesterolemic New Zealand White (NZW) rabbits. This effect is apparent even when apoA-I is administered 24h prior to the inflammatory insult. The present study asks if this benefit is related to an improved anti-inflammatory capacity of the high-density lipoprotein (HDL) fraction, or to increased arterial expression of genes that inhibit inflammation. METHODS AND RESULTS The ability of apoA-I to increase the anti-inflammatory capacity of HDL was assessed by infusing normocholesterolemic NZW rabbits with saline, lipid-free apoA-I or (A-I)rHDL. The infused apoA-I incorporated rapidly into the rabbit HDL fraction. The animals were sacrificed at 5 or 360 min post-infusion and plasma was collected. HDL were isolated by ultracentrifugation and incubated with cytokine-activated cultured human coronary artery endothelial cells. HDL from animals sacrificed at 5 min post-apoA-I infusion had a slightly enhanced anti-inflammatory capacity relative to HDL from the saline-infused animals. The anti-inflammatory capacity of HDL from the animals sacrificed at 360 min post-apoA-I infusion was comparable to that of HDL from the saline-infused animals. The effect of (A-I)rHDL infusions on arterial 3β-hydroxysteroid-Δ24 reductase (DHCR24) and endothelial adhesion molecule expression was investigated in cholesterol-fed NZW rabbits. Relative to animals infused with saline, (A-I)rHDL infusions decreased aortic VCAM-1 and ICAM-1 protein expression by 73 and 54%, respectively (p<0.05), and increased DHCR24 mRNA levels by 56% (p<0.0001). CONCLUSION ApoA-I inhibits vascular inflammation in NZW rabbits, at least in part, by increasing DHCR24 expression.

The apolipoprotein A-I mimetic peptide ETC-642 exhibits anti-inflammatory properties that are comparable to high density lipoproteins

OBJECTIVES Mimetic peptides of apolipoprotein A-I (apoA-I) present a new strategy for promoting the biological activity of high density lipoproteins (HDL). This study aimed to compare the anti-inflammatory effects of ETC-642, a new apoA-I mimetic peptide, with discoidal reconstituted HDL (rHDL). METHODS New Zealand White rabbits (n=42) received daily infusions of saline, rHDL or discoidal complexes of an amphipathic peptide, ETC-642 (1-30 mg/kg), prior to insertion of non-occlusive carotid collars. Human coronary artery endothelial cells (HCAECs) were pre-incubated with ETC-642 or rHDL before TNF-α stimulation. Monocyte adhesion was investigated by pre-incubating HCAECs with rHDL or ETC-642, stimulating with TNF-α and incubating with THP-1 monocytes. RESULTS Infusion of ETC-642 resulted in dose-dependent reductions of collar-induced expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the artery wall (p<0.05). Pre-incubation of HCAECs with ETC-642 and rHDL reduced TNF-α-induced THP-1 monocyte adhesion (p<0.01). Furthermore, ETC-642 and rHDL treatment reduced TNF-α induced mRNA levels of inflammatory markers VCAM-1, fractalkine, MCP-1 and the p65 subunit of NF-κB (p<0.05). CONCLUSION These studies demonstrate that ETC-642 exhibits anti-inflammatory properties that are comparable to apoA-I both in vivo and in vitro and that these effects are mediated via the NF-κB signaling pathway.

Reconstituted high-density lipoprotein suppresses leukocyte NADPH oxidase activation by disrupting lipid rafts.

Reconstituted discoidal high-density lipoprotein (rHDL) has potent vascular protective actions. Native HDL suppresses cellular generation of reactive oxygen species, whereas this antioxidant effect of rHDL is less clear. This study examined the effects of rHDL on NADPH oxidase, a major source of cellular superoxide generation, in both leukocytes and human umbilical vein endothelial cells. Superoxide was measured with lucigenin-enhanced chemiluminescence. Expression of NADPH oxidase sub-units was determined by real-time PCR. Pre-treatment of HL-60 cells with rHDL (10 and 25 µM) for 1 h significantly reduced phorbol 12-myristate 13-acetate-stimulated superoxide production. Treatment with rHDL for up to 24 h did not change the mRNA expression of NADPH oxidase sub-units. In HL-60 cells, depletion of cholesterol from the plasma membrane by methyl-β-cyclodextrin mimicked the effect of rHDL, whereas cholesterol repletion blunted the effects of rHDL. Treatment with rHDL induced disruption of the lipid raft structures and blunted PMA-induced redistribution of p47phox into lipid rafts. In contrast, treatment of endothelial cells with rHDL for up to 18 h had no effect on either basal or tumour necrosis factor-α-stimulated NADPH oxidase activity, but markedly suppressed the cytokine-induced expression of proinflammatory adhesion molecules. The results suggest that rHDL inhibits NADPH oxidase activation in leukocytes, probably by interrupting the assembly of NADPH oxidase sub-units at the lipid rafts. This effect may contribute to the vascular protective actions of rHDL against inflammation-mediated oxidative damage.

Calcium and osteoprotegerin regulate IGF1R expression to inhibit vascular calcification

AIMS Osteoprotegerin (OPG) inhibits vascular calcification in vitro, and OPG(-/-) mice develop vascular calcification. Insulin-like growth factor-1 (IGF1) signalling has been implicated in vascular smooth muscle cell (VSMC) survival; however, the role of IGF1-receptor (IGF1R) expression in calcification is unclear. We sought to determine whether the protective effects of OPG in vascular calcification were mediated by IGF1R. METHODS AND RESULTS Calcium-induced mineralization of VSMCs was blocked in cells expressing the IGF1R and by treatment with OPG. OPG induced IGF1R mRNA, protein, and transcription optimally at 1 ng/mL. Calcium also positively regulated both OPG and IGF1R, and siRNA targeting of OPG inhibited calcium-inducible IGF1R mRNA. Addition of calcium to VSMCs reduced camptothecin-stimulated apoptosis and increased expression of survival genes Bcl2 and nuclear factor-kappa B without altering levels of proliferation. Calciums induction of IGF1R and OPG was dose and time dependent but was blunted at higher calcium doses. Calcium- and OPG-inducible IGF1R transcription occurred between -446 and -188 bp of the IGF1R promoter, and inducible-IGF1R expression was blocked by specificity protein-1 (Sp1) silencing studies. Furthermore, elevated IGF1R and OPG protein levels were present in calcified atherosclerotic tissue. CONCLUSION We have shown for the first time that IGF1R expression and activity via OPG can modulate VSMC calcification in vitro. We suggest a feedback mechanism: moderate calcium levels increase OPG, which then increases IGF1R to enhance VSMC survival and block calcification induced by calcium. In contrast, high calcium leads to inhibition of IGF1R expression and activity to stimulate VSMC calcification further.

Sp1, Acetylated Histone-3 and p300 Regulate TRAIL Transcription: Mechanisms of PDGF-BB-Mediated VSMC Proliferation and Migration

We recently reported that TNF‐related apoptosis‐inducing ligand (TRAIL) is important in atherogenesis, since it can induce vascular smooth muscle cell (VSMC) proliferation and arterial thickening following injury. Here we show the first demonstrate that TRAIL siRNA reduces platelet‐derived growth factor‐BB (PDGF‐BB)‐stimulated VSMC proliferation and migration. PDGF‐BB‐inducible VSMC proliferation was completely inhibited in VSMCs isolated from aortas of TRAIL−/− mice; whereas inducible migration was blocked compared to control VSMCs. TRAIL transcriptional control mediating this response is not established. TRAIL mRNA, protein and promoter activity was increased by PDGF‐BB and subsequently inhibited by dominant‐negative Sp1, suggesting that the transcription factor Sp1 plays a role. Sp1 bound multiple Sp1 sites on the TRAIL promoter, including two established (Sp1‐1 and ‐2) and two novel Sp1‐5/6 and ‐7 sites. PDGF‐BB‐inducible TRAIL promoter activity by Sp1 was mediated through these sites, since transverse mutations to each abolished inducible activity. PDGF‐BB stimulation increased acetylation of histone‐3 (ac‐H3) and expression of the transcriptional co‐activator p300, implicating chromatin remodelling. p300 overexpression increased TRAIL promoter activity, which was blocked by dominant‐negative Sp1. Furthermore, PDGF‐BB treatment increased the physical interaction of Sp1, p300 and ac‐H3, while chromatin immunoprecipitation studies revealed Sp1, p300 and ac‐H3 enrichment on the TRAIL promoter. Taken together, our studies demonstrate for the first time that PDGF‐BB‐induced TRAIL transcriptional activity requires the cooperation of Sp1, ac‐H3 and p300, mediating increased expression of TRAIL which is important for VSMC proliferation and migration. Our findings have the promising potential for targeting TRAIL as a new therapeutic for vascular proliferative disorders. J. Cell. Biochem. 113: 2597–2606, 2012.

The apolipoprotein A-I mimetic peptide, ETC-642, reduces chronic vascular inflammation in the rabbit

BackgroundHigh-density lipoproteins (HDL) and their main apolipoprotein, apoA-I, exhibit anti-inflammatory properties. The development of peptides that mimic HDL apolipoproteins offers a promising strategy to reduce inflammatory disease. This study aimed to compare the anti-inflammatory effects of ETC-642, an apoA-I mimetic peptide, with that of discoidal reconstituted HDL (rHDL), consisting of full-length apoA-I complexed with phosphatidylcholine, in rabbits with chronic vascular inflammation.ResultsNew Zealand White rabbits (n = 10/group) were placed on chow supplemented with 0.2% (w/w) cholesterol for 6-weeks. The animals received two infusions of saline, rHDL (8 mg/kg apoA-I) or ETC-642 (30 mg/kg peptide) on the third and fifth days of the final week. The infusions of rHDL and ETC-642 were able to significantly reduce cholesterol-induced expression of intracellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the thoracic aorta (p < 0.05). When isolated rabbit HDL was pre-incubated with human coronary artery endothelial cells (HCAECs), prior to stimulation with TNF-α, it was found that HDL from ETC-642 treated rabbits were more effective at inhibiting the TNF-α-induced increase in ICAM-1, VCAM-1 and p65 than HDL isolated from saline treated rabbits (p < 0.05). There were, however, no changes in HDL lipid composition between treatment groups.ConclusionsInfusion of ETC-642 causes anti-inflammatory effects that are comparable to rHDL in an animal model of chronic vascular inflammation and highlights that apoA-I mimetic peptides present a viable strategy for the treatment of inflammatory disease.

Tumor Necrosis Factor–Related Apoptosis‐Inducing Ligand (TRAIL) Promotes Angiogenesis and Ischemia‐Induced Neovascularization Via NADPH Oxidase 4 (NOX4) and Nitric Oxide–Dependent Mechanisms

Background Tumor 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 Results Reduced 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. Conclusions This 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.

CETP Inhibition in CVD Prevention: an Actual Appraisal

By virtue of their effects on low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, and cellular cholesterol efflux, there is considerable interest in the potential use of pharmacological inhibitors of cholesteryl ester transfer protein (CETP) as a novel approach for cardiovascular disease prevention. This is supported by observations from genetic and animal studies suggesting that less CETP activity has favorable cardiovascular effects. Despite the adverse effects of the first CETP inhibitor to move forward in clinical development, torcetrapib, there remains considerable interest in developing alternative CETP inhibitors without the off-target effects of torcetrapib. The clinical development programs leading to a number of promising CETP inhibitors will be reviewed.

Clinical trials with cholesteryl ester transfer protein inhibitors.

Purpose of review Inhibition of cholesteryl ester transfer protein (CETP) has received considerable interest by virtue of its favorable effects on atherogenic and protective lipid parameters. The impact of CETP inhibitors in large clinical outcome trials will be reviewed. Recent findings Population and genetic studies demonstrate that low CETP activity associates with lower rates of cardiovascular events. Inhibiting CETP activity in animal models has a favorable impact on experimental atherosclerosis. Although the first CETP inhibitor to advance to an outcome trial proved to have adverse clinical effects and the next agent, a more modest inhibitor, was clinically futile, there continues to be immense interest in the potential to develop nontoxic, potent CETP inhibitors to reduce cardiovascular risk. Summary The current status of CETP inhibitors in the context of large outcomes trials will be reviewed.