EXpansion of stents after intravascular lithoTripsy versus conventional predilatation in CALCified coronary arteries.

BACKGROUND: Coronary artery calcification is a strong predictor for procedural failure and is independently associated with adverse events after percutaneous coronary intervention (PCI). An important contributor to the impaired outcome is the inability to achieve optimal results due to stent underexpansion or stent deformation/fracture. Intravascular lithotripsy (IVL) has emerged as an alternative technique to change the integrity of calcified plaques. AIMS: Our aim was to investigate if pre-treatment with IVL in severely calcified lesions increases stent expansion, assessed by optical coherence tomography (OCT), when compared to predilatation with conventional and/or specialty balloon strategy. METHODS: EXIT-CALC was a prospective, single-centre, randomised controlled study. Patients with an indication for PCI and severe calcification of the target lesion were allocated to predilatation with conventional angioplasty balloons or pre-treatment with IVL, followed by drug-eluting stenting and mandatory postdilatation. Primary endpoint was stent expansion assessed by OCT. Secondary endpoints were the occurrence of peri-procedural events and major adverse cardiac events (MACE) in hospital and during follow-up. RESULTS: A total of 40 patients were included. The minimal stent expansion in the IVL-group (n = 19) was 83.9 ± 10.3% and 82.2 ± 11.5% in the conventional group (n = 21) (p = 0.630). Minimal stent area was 6.6 ± 1.5 mm2 and 6.2 ± 1.8 mm2, respectively (p = 0.406). No peri-procedural, in-hospital and 30-day follow-up MACE were reported. CONCLUSIONS: In severely calcified coronary lesions we found no significant difference in stent expansion measured by OCT when comparing IVL, as plaque modification, with conventional and/or specialty angioplasty balloons.

The epigenetic factor PCAF regulates vascular inflammation and is essential for intimal hyperplasia development.

OBJECTIVE: Genetic 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 RESULTS: PCAF 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. CONCLUSIONS: These 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.

TLR accessory molecule RP105 (CD180) is involved in post-interventional vascular remodeling and soluble RP105 modulates neointima formation.

BACKGROUND: RP105 (CD180) is TLR4 homologue lacking the intracellular TLR4 signaling domain and acts a TLR accessory molecule and physiological inhibitor of TLR4-signaling. The role of RP105 in vascular remodeling, in particular post-interventional remodeling is unknown. METHODS AND RESULTS: TLR4 and RP105 are expressed on vascular smooth muscle cells (VSMC) as well as in the media of murine femoral artery segments as detected by qPCR and immunohistochemistry. Furthermore, the response to the TLR4 ligand LPS was stronger in VSMC from RP105(-/-) mice resulting in a higher proliferation rate. In RP105(-/-) mice femoral artery cuff placement resulted in an increase in neointima formation as compared to WT mice (4982 ± 974 µm(2) vs.1947 ± 278 µm(2),p = 0.0014). Local LPS application augmented neointima formation in both groups, but in RP105(-/-) mice this effect was more pronounced (10316±1243 µm(2) vs.4208 ± 555 µm(2),p = 0.0002), suggesting a functional role for RP105. For additional functional studies, the extracellular domain of murine RP105 was expressed with or without its adaptor protein MD1 and purified. SEC-MALSanalysis showed a functional 2∶2 homodimer formation of the RP105-MD1 complex. This protein complex was able to block the TLR4 response in whole blood ex-vivo. In vivo gene transfer of plasmid vectors encoding the extracellular part of RP105 and its adaptor protein MD1 were performed to initiate a stable endogenous soluble protein production. Expression of soluble RP105-MD1 resulted in a significant reduction in neointima formation in hypercholesterolemic mice (2500 ± 573 vs.6581 ± 1894 µm(2),p<0.05), whereas expression of the single factors RP105 or MD1 had no effect. CONCLUSION: RP105 is a potent inhibitor of post-interventional neointima formation.

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 Ly6C(hi) 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.

Blocking toll-like receptors 7 and 9 reduces postinterventional remodeling via reduced macrophage activation, foam cell formation, and migration.

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 low-density lipoprotein-induced foam cell formation, most likely not via decreased influx but via increased efflux, because CD36 expression was unchanged whereas interleukin-10 levels were higher (36.1 ± 22.3 pg/mL versus 128.9 ± 6.6 pg/mL; P=0.008). CONCLUSIONS: Blocking TLR7 and TLR9 reduced postinterventional vascular remodeling and foam cell accumulation indicating TLR7 and TLR9 as novel therapeutic targets.

Future potential biomarkers for postinterventional restenosis and accelerated atherosclerosis.

New circulating and local arterial biomarkers may help the clinician with risk stratification or diagnostic assessment of patients and selecting the proper therapy for a patient. In addition, they may be used for follow-up and testing efficacy of therapy, which is not possible with current biomarkers. Processes leading to postinterventional restenosis and accelerated atherosclerosis are complex due to the many biological variables mediating the specific inflammatory and immunogenic responses involved. Adequate assessment of these processes requires different and more specific biomarkers. Postinterventional remodeling is associated with cell stress and tissue damage causing apoptosis, release of damage-associated molecular patterns and upregulation of specific cytokines/chemokines that could serve as suitable clinical biomarkers. Furthermore, plasma titers of pathophysiological process-related (auto)antibodies could aid in the identification of restenosis risk or lesion severity. This review provides an overview of a number of potential biomarkers selected on the basis of their role in the remodeling process.

Annexin A5 therapy attenuates vascular inflammation and remodeling and improves endothelial function in mice.

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. AnxA5 treatment reduced adhesion and infiltration of leukocytes by 71% to 69% (P=0.015, P=0.031) and macrophages by 51% to 87% (P=0.014, P=0.018), as well as monocyte chemotactic protein-1 and tumor necrosis factor-α expression in a femoral artery inflammation model (perivascular cuff for 3 days), indicating reduced vascular inflammation. In a vein graft model, 28 days of AnxA5 treatment reduced vein graft thickening (48%; P=0.006) and leukocyte infiltration (46%; P=0.003). In these mice, reduced plasma concentrations of IFN-γ (-72%; P=0.040), granulocyte colony-stimulating factor (-41%; P=0.010), and macrophage inflammatory protein-1ß (MIP-1ß) (-66%; P=0.020) were measured, indicating reduced systemic inflammation. An in vitro endothelial cell model shows the importance of AnxA5's anticoagulant properties in reducing vascular inflammation. Endothelium-mediated dilatation in hypercholesterolemic ApoE((-/-)) mice was improved by 3 days of AnxA5 treatment, shown by improved systolic and diastolic blood pressure reductions in response to metacholine, which could be abolished by l-Nitro-Arginine-Methyl Ester (l-NAME), indicating nitric oxide involvement. CONCLUSIONS: AnxA5 reduced local vascular and systemic inflammation and vascular remodeling and improved vascular function, indicating that it has a therapeutic potential against atherosclerotic cardiovascular diseases.

Genetic variation in PCAF, a key mediator in epigenetics, is associated with reduced vascular morbidity and mortality: evidence for a new concept from three independent prospective studies.

AIMS: This study was designed to investigate the counterbalancing influence of genetic variation in the promoter of the gene encoding P300/CBP associated factor (PCAF), a lysine acetyltransferase (KAT), on coronary heart disease (CHD) and mortality. METHODS AND RESULTS: The association of genetic variation in the PCAF-gene with CHD, restenosis and mortality was investigated in three large cohorts. The results were combined to examine overall effects on CHD mortality and on restenosis risk. Compared with the homozygous -2481G allele in the PCAF promoter, a significant reduction in CHD mortality risk with the homozygous -2481C PCAF promoter allele was observed. A combined risk reduction for CHD death for the three studies was 21% (15-26%; p=8.1×10(-4)). In elderly patients (>58 years) the effects were stronger. Furthermore, this PCAF allele was significantly associated with all-cause mortality (p=0.001). Functional analysis showed that nuclear factors interact in vitro with the oligonucleotides encompassing the -2481G/C polymorphism and that this interaction might be influenced by this polymorphism in the PCAF promoter. Moreover, modulation of PCAF gene expression was detectable upon cuff-placement in an animal model of reactive stenosis. CONCLUSION: We showed in three large prospective studies that the -2481C allele in the PCAF promoter is associated with a significant survival advantage in elderly patients. Our observations promote the concept that epigenetic processes are under genetic control and that, other than environment, variation in genes encoding KATs may also determine susceptibility to CHD outcomes and mortality.