Plaque Composition Such as Lipid Core and Fibrous Cap Determine Neointimal Formation Associated with In-Stent Restenosis: Results from a Clinicopathologic Study

Hatsue Ishibashi-Ueda
National Cardiovascular Center, Osaka, Japan

Atheromatous plaque composition, especially the presence of a large lipid core associated with a thin fibrous cap, is the most important element for excessive neointima formation in the development of in-stent restenosis (ISR), based on results from a study conducted by this speaker. Thus, stabilizing plaques and reducing the lipid core volume with statin therapy may inhibit neointima proliferation and thereby prevent ISR.

Post-stent restenosis remains a serious problem. Predicting the occurrence of ISR, associated with extensive neo-intima formation, is important. Several morphological determinants of neointima formation have been proposed, including pre-stent atheromatous plaque volume, plaque composition, positive remodeling due to vessel wall over-stretching/ vascular injury, thrombogenic factors and inflammation.

Study design

The present study by Ishibashi-Ueda and colleagues sought to 1) clarify histologically the structure of coronary arteries with in-stent restenosis compared to those without restenosis, and 2) evaluate the relation between native atheromatous plaque and neointimal formation in stented coronary arteries.

They examined 34 stented segments from 25 autopsied cadavers; the average age was 70 years, and 80% were male. Forty percent were hyperlipidemic and 24% treated with a statin. The stent implantation period was defined as 30 days for their histologic analysis, and the average was 198 days. The target vessel was LAD in 53% and right coronary artery in 32%. An acute myocardial infarction (AMI) was the indication for stenting in 56% and unstable angina in 28%. Figure 1 outlines the clinical characteristics in the study patients.

Study results

A representative comparison of coronary artery sections with and without ISR, on a micrograph, showed an atheromatous plaque protruding outward representing remodeling, with a large lipid core, occupying about 60% of the atheromatous plaque (Figure 2). In the stented area, proliferating neointima nearly occluded the lumen. In contrast, a concentric fibrous plaque with no obvious lipid-rich core was seen in the sections in non-ISR; the neointima was thin and compact, and the lumen was largely open, even 6 months post-stenting. 

Under higher magnification, plaque morphology revealed that in ISR the stent strut penetrated into the fibrous cap and a large lipid core was under the stent strut. Oil red O staining showed numerous lipid-laden macrophages. In the non-ISR case, a thick fibrous cap was seen beneath the stent and in a deeper layer a small lipid-rich core composed of CD68 positive macrophages.

A detailed histology of peri-stent strut in ISR showed that the stent implanted in a lipid-rich core, neovascularization, lymphocyte infiltration, and foreign body giant cells, and CD68 positive macrophage infiltration.

Figure 3 outlines the histological characteristics of the atheromatous plaque in coronary sections with and without ISR. In ISR, all the plaques were lipid-rich; no fibrous plaque was present. The plaques were primarily eccentric (86.5%), 13.5% concentric, and 59.5% of struts penetrated into plaque. Peri-stent strut inflammation was 21.6%s lymphocytes and 29.7% macrophages. In non-ISR, plaques were lipid rich (40.4%) or fibrous (59.6%), and were eccentric (42.3%) or concentric (57.7%); no struts penetrated into plaques. Peri-stent strut inflammation was limited.

Figure 4 outlines the morphometric results at the stented sites. Although remodeling was one of the determinants of ISR, their data showed no significant difference in the remodeling index between ISR and non-ISR (1.26 vs 1.19, respectively; Cox hazard ratio (HR) 1.08; p=0.39). However, the ratio of the lipid-rich core to plaque area was much larger in ISR compared to non-ISR (0.66 vs 0.34; Cox HR 2.48; p=0.011). The thickness of the fibrous cap was thinner in ISR (140.4m vs 317.8m in non-ISR; Cox HR 3.5; p<0.0001). A larger ratio for the neointima area to stent area was found in ISR versus non-ISR (0.81 vs 0.29, respectively; Cox HR 17.29; p=0.025). A positive correlation between the amount of neointima and the lipid rich core burden was found (r=0.79; p<0.001).

Pre-Procedural Small Platelet Aggregates Predict Restenosis After Percutaneous Coronary Intervention

Shinzo Miyamoto
Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

Previous studies have shown that platelet aggregation at follow-up is greater in patients with developing restenosis.  However, conventional assessment of pre-procedural platelet aggregation has not provided powerful predictors of restenosis after a percutaneous intervention (PCI). A novel platelet aggregometer using laser-light scattering has been shown to quantitatively evaluate aggregate size and number.Small platelet aggregates ultimately develop into medium and then large platelet aggregates as platelet aggregation proceeds. Because small platelet aggregates indicate the first step in the development of platelet aggregation, their measurement may be an important marker of initial thrombus formation.

Study design

To elucidate the relation between platelet aggregation and restenosis post-PCI, Miyamoto and colleagues evaluated 189 of the 196 enrolled patients (96% follow-up; 54 with acute coronary syndrome (ACS), 135 with stable angina).

All patients had characteristic symptoms or subjective clinical evidence of myocardial ischemia.The angioplasty procedure was performed by the femoral approach according to standard techniques.Angioplasty success was defined as a <50% residual luminal stenosis of the dilated segment using visual estimation, without major complications. Coronary artery stenosis was assessed in two or more projections before and after angioplasty and at follow-up. All patients received standard medical therapy during the follow-up period.

Follow-up coronary angiograms for asymptomatic patients were performed 4 to 12 months after PCI.Restenosis was defined as luminal reduction of ÿ50% at the previous PCI site. Stenosis was assessed using quantitative coronary angiography (QCA). All patients who had elective PCI were treated with a low dose (100mg) of aspirin daily and 200 mg of ticlopidine for 4 weeks when stents were implanted. Written informed consent was obtained from all patients.

In patients with stable angina, peripheral venous blood was drawn after an overnight fast before elective PCI.In patients with ACS, blood samplings were drawn before initiating intravenous heparin injection and before emergent PCI. Samples for platelet aggregation were left for 15 minutes at room temperature, and then centrifuged at 150 g for 10 minutes at room temperature to obtain platelet-rich plasma. The other samples were then centrifuged at 300 g for 10 minutes at room temperature to obtain platelet-poor plasma.

Platelet aggregation was measured with a PA-200-instrument. ADP 1.0 m was used as an aggregating agent and added to platelet-rich plasma 60 seconds after initiating the measurement.  Platelet aggregation was evaluated as the maximum value of light intensity induced by ADP.

Study results

The clinical characteristics of the study patients are outlined in Figure 1. Based on pre-procedural small platelet aggregation, patients were divided into 3 percentiles: <50 percentile, 50-75 percentile, and >75 percentile. A significantly higher triglyceride level was found in the >75 percentile (149 mg/dl vs 108 mg/dl in 50-75 percentile; p<0.05). A greater percentage of patients in the >75 percentile had ACS (51.7%) compared to 14.9% in the <50 percentile and 41.7% in the 50-75 percentile (p<0.01).

Restenosis rate was significantly higher in the ACS group compared to the stable angina group (43.5% vs 25.5%, p<0.05). In the 63 patients with restenosis, small platelet aggregation was significantly greater than in the 126 patients without restenosis (volume of 3.4x10⁴ vs 1.5x10⁴ ; p<0.001; Figure 2). Figure 3 shows the relation between restenosis and pre-procedural small platelet aggregates.

The type of lesion and pre-procedural small platelet aggregates were significant predictors of post-PCI restenosis on multivariate logistic regression analysis. However, only pre-procedural small platelet aggregates was an independent predictor of post-PCI restenosis (Figure 4). A preliminary study by this group shows that ticlopidine, compared to aspirin, significantly decreased the

Treatment recommendations

Based on their data, to prevent post-PCI restenosis they recommend: In AMI patients, reperfusion should be performed quickly in the acute phase, and in the chronic phase, pretreatment before PCI for residual stenosis. In patients with unstable angina, in the acute phase use drugs for stabilization if urgent coronary angiogram and PCI are not needed. In the chronic phase, pretreatment before PCI. In patients with stable angina, pretreatment before PCI.

Potent Inhibitory Effects of Sirolimus on Circulating Smooth Muscle Progenitor Cells

Daiju Fukuda
University of Tokyo, Tokyo, Japan

Sirolimus-eluting stents have emerged as a promising strategy to prevent in-stent restenosis (ISR). Sirolimus is a macrolide antibiotic with potent antifungal, immunosuppressive. and antimitotic properties. Sirolimus binds to a specific cytosolic protein, FK506 binding protein 12 (FKBP). The sirolimus FKBP complex binds to a specific cell cycle-regulatory protein, the mammalian target of rapamycin (mTOR), and inhibits its activation. The inhibition of mTOR ultimately induces cell-cycle arrest in the G1 phase and consequently arrests cell growth. Restenosis rates and clinical events in patients with complex lesions have been reduced in randomized clinical trials of sirolimus-eluting stents

The inhibitory effect of sirolimus on smooth muscle cell (SMC) proliferation and migration in vitro has been demonstrated. However, the exact mechanism by which locally delivered sirolimus prevents ISR is unknown. Although neointimal hyperplasia resulting from the excessive accumulation of SMC is the primary cause of ISR, the origin of neointimal SMC is not well understood.

This group recently demonstrated that bone marrow cells give rise to smooth muscle progenitor cells (SMPCs) that accumulate at the site injured arteries, differentiate, and contribute to neointimal hyperplasia. Neointimal SMCs are thought to derive from the media and from circulating SMPCs.

Study design

The effect of sirolimus on SMPCs was therefore studied by this group. In their assay system for SMPCs, peripheral mononuclear cells were isolated from the blood of healthy human volunteers by density gradient centrifugation. The isolated mononuclear cells were then cultured in fibronectin-coated wells with basic FGF and PDGF-BB. At day 14, the cells were differentiated into alpha-smooth muscle actin positive cells, which were then defined as smooth muscle-like cells. The assay was performed in the presence of sirolimus, and actin was expressed at both the mRNA and protein levels.

Study results

Mononuclear cells were cultured in the presence of 0 to 100 ng/mL sirolimus, which had little effect at day 4 on the number of adherent cells. However, at day 14, a dramatic decrease in the number of smooth muscle-like cells by 1.0 ng/mL sirolimus was found (2.4 vs 60.9 cells, p<0.001). Culturing human aortic smooth muscle cells with sirolimus inhibited their proliferation and the differentiation of SMPCs (Figure 1). However, SMPC differentiation was inhibited more effectively than the proliferation of the human aortic SMCs.

The expression of FKBP12 was more abundant in mononuclear cells than in SMCs. Sirolimus is thought to have other pathways, however this difference may be one explanation for the present results, that is, sirolimus may target cell types other than medial SMCs.

To investigate the effect of sirolimus on EPCs, mononuclear cells were cultured with hydrocortisone (1g/mL), bovine brain extract (3g/mL), and VEGF (10 ng/mL).  Endothelial-like cells were defined as cells double positive for Dil-acetylated LDL and FITC-lectin, as described in the literature. Differentiation of mononuclear cells to endothelial-like cells was inhibited, in the presence of sirolimus, as were SMPCs.

Culturing with sirolimus mildly inhibited the proliferation of human umbilical vein endothelial cells by 33.3% and inhibited the differentiation of EPCs (Figure 2). These results suggest that sirolimus also exerts an inhibitory effect on EPCs originating from mononuclear cells, thereby affecting re-endothelialization.

Summary

Sirolimus has been reported to inhibit medial SMC cell migration and proliferation.The present study suggests that sirolimus inhibits the differentiation of bone marrow-derived SMPCs to neointimal smooth muscle cells. Further, these results suggest that sirolimus also exerted an inhibitory effect on EPCs, thus affecting re-endothelialization.

Potent inhibitory effects of sirolimus on circulating SMPCs may, at least in part, mediate the clinical efficacy of sirolimus-eluting stent. Sirolimus potently affects re-endothelialization after stent-implantation.

Effects of Local Delivery of Evans Blue and Phenolsulfonphtaleine on Migration of Vascular Progenitor Cells into the Coronary Arterial Wall

Yasumi Uchida
Jikei University School of Medicine Tokyo, Japan

Previous work by this group found that vascular progenitor cells (VPCs) play an important role in restenosis by migrating into coronary wall through three routes. The aim of the present studies was to examine the roles of VPCs in coronary restenosis and to develop new treatment modalities by controlling migration of VPCs.

Study design

In anesthesized beagle dogs, coronary arteries were dilated by either balloon inflation (POBA) or by stent implantation and the role of VPCs examined. Mononuclear cells with beta-SM actin were considered ad VPCs because they also had CD34 and other vascular markers. Following mechanical intervention, VPCs migrated into the interstitial space. VPCs were found to migrate into the intima via 3 routes, from the adventitia, the lumen, and from new vessels in hyperplastic intima. Direct migration from the lumen into the intima was also observed. The migration continues for more than 4 weeks.

To prevent migration of VPCs into the intima, treatment approaches should include substances that prevent migration of VPCs and of smooth muscle cells (SMCs) pre-existing in the media. The substances should easily diffuse into the entire wall, including the adventitia, and remain there for more than 4 weeks. Stents should elute drugs for more than 4 weeks.

Further experiments by this group showed that Evans Blue (EB, 25 mg) or phenolsulfonphthaleine (PSP, 25 mg), delivered using a porous balloon into the dilated middle to distal coronary segments of anesthetized beagle dogs, prevented migration of VPCs, as found on coronary angiography. POBA-induced intimal hyperplasia was suppressed by EB and PSP. Also, POBA-induced stenosis was suppressed by EB and PSP. A significant reduction in the number of VPCs per unit area by the dyes was found. At 6 months, multi-layered dye-eluting stents (EB 20-25 mg) effectively prevented intimal hyperplasia and stenosis. VPC migration was also suppressed.

To clarify the mechanisms for the beneficial effects of these dyes, they compared the pharmacologic actions of EB and PSP in vitro and in vivo. EB has inhibitory actions on SMC proliferation, thrombosis, migration, and stenosis, and inhibits the cell cycle at G1 and inhibits von Willebrand factor action. In contrast, PSP inhibits only migration and stenosis.

Open Clinical Trials

Based on the results of the experimental studies, they conducted 2 open clinical trials using EB, which had been clinically used for the measurement of cardiac out put in patients. In Trial 1, EB (25 mg) was injected into the treated arteries through a guiding catheter immediately after PCI. In Trial 2, EB (20-25 mg) was delivered locally by a porous balloon after PCI in 25 patients with ACS.

In Trial 1, the intracoronary bolus injection of EB was associated with a reduction in restenosis in the setting of angina, AMI, stent, and cutting balloon (Figure 1). The restenosis rate was about 10% in the treated groups. EB also reduced the in-stent restenosis (ISR) rate, regardless of the angiographic coronary anatomy (Figure 2). The ISR rate was below 5% in the type AB1 group. In Trial 2, local delivery of EB to the stented segments also reduced the ISR rate below 5% in patients with AMI (Figure 3).

Conclusions

Following PCI, VPCs migrate into the intima via 3 routes, through adventitia through the media, directly from the lumen, and from neovasculature in the intima. VPCs play an important role in coronary stenosis in the canine model. The local delivery of EB and PSP and the novel dye-eluting stents prevented the migration of VPCs and inhibited PCI-induced coronary stenosis in dogs. EB prevented PCI-induced restenosis in patients with coronary artery disease. In addition to the inhibition of thrombosis and migration of SMCs pre-existing in the media, by preventing migration of VPCs into the intima, complete restenosis prevention may be attained.