Objective: T-cells are central to the immune response responsible for native atherosclerosis. The objective of this study is to investigate T-cell contribution to post-interventional accelerated atherosclerosis development, as well as the role of the CD28-CD80/86 co-stimulatory and Cytotoxic T-Lymphocyte Antigen (CTLA)-4 co-inhibitory pathways controlling T-cell activation status in this process. Methods and results: The role of T-cells and the CD28-CD80/86 co-stimulatory and CTLA-4 co-inhibitory pathways were investigated in a femoral artery cuff mouse model for post-interventional remodeling, with notable intravascular CTLA-4+ T-cell infiltration. Reduced intimal lesions developed in CD4 −/− and CD80 −/− CD86 −/− mice compared to normal C57Bl/6J controls. Systemic abatacept-treatment, a soluble CTLA-4Ig fusion protein that prevents CD28-CD80/86 co-stimulatory T-cell activation, prevented intimal thickening by 58.5% (p = 0.029). Next, hypercholesterolemic ApoE3*Leiden mice received abatacept-treatment which reduced accelerated atherosclerosis development by 78.1% (p = 0.040) and prevented CD4 T-cell activation, indicated by reduced splenic fractions of activated KLRG1 +, PD1 +, CD69+ and CTLA-4+ T-cells. This correlated with reduced plasma interferon-γ and elevated interleukin-10 levels. The role of CTLA-4 was confirmed using CTLA-4 blocking antibodies, which strongly increased vascular lesion size by 66.7% (p=0.008), compared to isotype-treated controls. Conclusions: T-cell CD28-CD80/86 co-stimulation is vital for post-interventional accelerated atherosclerosis development and is regulated by CTLA-4 co-inhibition, indicating promising clinical potential for prevention of post-interventional remodeling by abatacept.

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...

Objective-Annexin A5 (AnxA5) has antithrombotic, antiapoptotic, and antiinflammatory properties; we investigated its effectiveness against vascular inflammation, remodeling, and dysfunction in accelerated atherosclerosis. Methods and Results-AnxA5 (1 mg/kg per day or vehicle) was investigated in vascular injury models in hypercholesterolemic apolipoprotein E (ApoE)3*Leiden mice.

ObjectiveGenetic P300/CBP-associated factor (PCAF) variation affects restenosis-risk in patients. PCAF has lysine acetyltransferase activity and promotes nuclear factor kappa-beta (NFκB)-mediated inflammation, which drives post-interventional intimal hyperplasia development. We studied the contributing role of PCAF in post-interventional intimal hyperplasia.Methods and resultsPCAF contribution to inflammation and intimal hyperplasia was assessed in leukocytes, macrophages and vascular smooth muscle cells (vSMCs) in vitro and in a mouse model for intimal hyperplasia, in which a cuff is placed around the femoral artery. PCAF deficiency downregulate CCL2, IL-6 and TNF-alpha expression, as demonstrated on cultured vSMCs, leukocytes and macrophages. PCAF KO mice showed a 71.8% reduction of vSMC-rich intimal hyperplasia, a 73.4% reduction of intima/media ratio and a 63.7% reduction of luminal stenosis after femoral artery cuff placement compared to wild type (WT) mice. The association of PCAF and vascular inflammation was further investigated using the potent natural PCAF inhibitor garcinol. Garcinol treatment reduced CCL2 and TNF-alpha expression, as demonstrated on cultured vSMCs and leukocytes.To assess the effect of garcinol treatment on vascular inflammation we used hypercholesterolemic ApoE*3-Leiden mice. After cuff placement, garcinol treatment resulted in reduced arterial leukocyte and macrophage adherence and infiltration after three days compared to untreated animals.ConclusionsThese results identify a vital role for the lysine acetyltransferase PCAF in the regulation of local inflammation after arterial injury and likely the subsequent vSMC proliferation, responsible for intimal hyperplasia.

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...