Differential response of delayed healing and persistent inflammation at sites of overlapping sirolimus- or paclitaxel-eluting stents.

BACKGROUND: Although effective coverage of challenging coronary lesions has warranted the use of overlapping drug-eluting stents, the histopathological response to stent overlap is unknown. METHODS AND RESULTS: The arterial reaction to overlapping Cypher or Taxus drug-eluting stents was examined in rabbits with bare metal stents, BxVelocity or Express, serving as controls. Single iliac artery balloon injury was followed by placement of 2 overlapping 3.0-mm-diameter drug-eluting stents or bare metal stents in 60 animals (mean length of overlap, 9.8+/-3.6 mm). Stented arteries were harvested at 28 and 90 days for histology. Overlapped segments exhibited delayed healing compared with proximal and distal nonoverlapping sites at 28 days. Overlapped segments in Taxus stents induced significantly more luminal heterophils/eosinophils and fibrin deposition than Cypher; peristrut giant cell infiltration, however, was more frequent in the latter. Overlapping bare metal stents also showed mild delayed healing compared with nonoverlapped segments, but not to the same extent as drug-eluting stents. Although neointimal thickness within the overlap was similar in 28- and 90-day Cypher stents, there was a significant increase with Taxus (P=0.03). CONCLUSIONS: Compared with bare metal stents, drug-eluting stents further delay arterial healing and promote inflammation at sites of overlap. Taxus stents induced greater fibrin deposition, medial cell loss, heterophils/eosinophils, and late neointimal hyperplasia. Patients receiving overlapping drug-eluting stents need more frequent follow-up than patients with nonoverlapping stents.

Extracellular matrix changes in stented human coronary arteries.

BACKGROUND: Restenosis after stenting occurs secondary to the accumulation of smooth muscle cells (SMCs) and extracellular matrix (ECM), with the ECM accounting for >50% of the neointimal volume. The composition of the in-stent ECM has not been well characterized in humans. METHODS AND RESULTS: Postmortem human coronary arteries (n=45) containing stents underwent histological assessment of neointimal proteoglycans, hyaluronan, collagen (types I and III), SMCs, and CD44 (a cell surface receptor for hyaluronan). The mean duration of stent implantation was 18.7 months; stents in place > or =3 to <9 months (n=17) were assigned to group 1, stents > or =9 to <18 months old (n=19) to group 2, and stents > or =18 months old (n=9) to group 3. In groups 1 and 2, neointimal versican and hyaluronan staining was strongly positive, colocalized with alpha-actin-positive SMCs, and was greater in intensity compared with group 3. Conversely, decorin staining was greatest in group 3. The neointima of both group 1 and 2 stents was rich in type III collagen, with reduced staining in group 3. Type I collagen staining was weakest in group 1 stents, with progressively stronger staining in groups 2 and 3. SMC density and stent stenosis were significantly reduced in group 3 stents compared with groups 1 and 2. CD44 staining colocalized with macrophages and was associated with increased neointimal thickness. CONCLUSIONS: The ECM within human coronary stents resembles a wound that is not fully healed until 18 months after deployment, followed by neointimal retraction. ECM contraction may be a target for therapies aimed at stent restenosis prevention.

Morphologic findings of coronary atherosclerotic plaques in diabetics: a postmortem study.

OBJECTIVE: Coronary atherosclerotic plaque composition of diabetic subjects and localization of receptor for advanced glycation end products (RAGE) and its ligands have not been extensively studied. METHODS AND RESULTS: Hearts from diabetic subjects and age, race, and sex-matched nondiabetic subjects dying suddenly were examined. Coronary arteries were dissected and lesions were evaluated for plaque burden, necrotic core size, and inflammatory infiltrate. The expression of RAGE, the RAGE-binding protein (S100-A12, EN-RAGE), and cell death (apoptosis) were also determined. Lesions from type II diabetic subjects had larger mean necrotic cores (P=0.01) and greater total and distal plaque load (P<0.001) than nondiabetic subjects. Necrotic core size correlated positively with diabetic status, independent of other risk factors. Intimal staining for macrophages, T-cells, and HLA-DR was also significantly greater in diabetic subjects (P=0.03, P=0.003, and P<0.0001), respectively. The association of increased macrophage infiltrate was independent of cholesterol levels and patient age. Expression of RAGE and EN-RAGE was significantly greater in diabetic subjects (P=0.004) and was associated with apoptotic smooth muscle cells and macrophages. CONCLUSIONS: In sudden coronary death, inflammation and necrotic core size play a greater role in the progression of atherosclerosis in diabetic subjects. The expression of RAGE and EN-RAGE may further compromise cell survival and promote plaque destabilization.

Uptake of 111In-Z2D3 on SPECT imaging in a swine model of coronary stent restenosis correlated with cell proliferation.

UNLABELLED: Small targets such as cell proliferation in the coronary arteries may potentially be detected with single-photon imaging using high-radiotracer-specific activity. We hypothesized that an antibody linked to polymers to increase specific radioactivity can be visualized on SPECT images and that counts in the target will correlate with the strength of the biologic signal. METHODS: Twenty-four stents were placed using the balloon overexpansion technique in the coronary arteries of 14 juvenile domestic swine. One week later, the animals received 74 MBq of (111)In-diethylenetriaminepentaacetic acid-polylysine Z2D3-F(ab')(2), and SPECT imaging was performed at 24 h. The coronary vessels were removed, and the stented vessels were processed with plastic embedding and sectioning. Medial and neointimal areas, percentage of vessel stenosis, and cell proliferation indices were quantified using a 5-bromo-2-deoxyuridine (BrdU) labeling index. Reconstructed SPECT images were interpreted for tracer uptake in coronary vessels. RESULTS: Sixteen of the vessels were positive on SPECT imaging and 10 were negative. The percentage injected dose was 0.85 +/- 0.28 x 10(-3) in scan-positive vessels and 0.34 +/- 0.11 x 10(-3) in scan-negative vessels (P < 0.001). The medial-plus-neointimal proliferative index was 42 +/- 11 in scan-positive vessels and 11 +/- 11 in scan-negative vessels (P < 0.0001). The percentage stenoses were 21% +/- 22% versus 19% +/- 15% (not statistically significant). When individual values for the stented-to-control vessel counts were plotted against BrdU labeling index, a significant relationship was found (r(2) = 0.441; P = 0.0014). CONCLUSION: These data indicate that small targets relevant to human coronary vascular disease may be detected using polymer-modified radiolabeled antibodies.

Targeting of apoptotic macrophages and experimental atheroma with radiolabeled annexin V: a technique with potential for noninvasive imaging of vulnerable plaque.

BACKGROUND: Apoptosis is common in advanced human atheroma and contributes to plaque instability. Because annexin V has a high affinity for exposed phosphatidylserine on apoptotic cells, radiolabeled annexin V may be used for noninvasive detection of apoptosis in atherosclerotic lesions. METHODS AND RESULTS: Atherosclerotic plaques were produced in 5 rabbits by deendothelialization of the infradiaphragmatic aorta followed by 12 weeks of cholesterol diet; 5 controls were studied without manipulation. Animals were injected with human recombinant annexin V labeled with technetium-99m before imaging. Aortas were explanted for ex vivo imaging, macroautoradiography, and histological characterization of plaque. Radiolabeled annexin V cleared rapidly from the circulation (T1/2, alpha 9 and beta 46 minutes). There was intense uptake of radiolabel within lesions by 2 hours; no uptake was seen in controls. The results were confirmed in the ex vivo imaging of the explanted aorta. Quantitative annexin uptake was 9.3-fold higher in lesion versus nonlesion areas; the lesion-to-blood ratio was 3.0+/-0.37. Annexin uptake paralleled lesion severity and macrophage burden; no correlation was observed with smooth muscle cells. DNA fragmentation staining of apoptotic nuclei was increased in advanced lesions with evolving necrotic cores, predominantly in macrophages; the uptake of radiolabel correlated with the apoptotic index. CONCLUSIONS: Because annexin V clears rapidly from blood and targets apoptotic macrophage population, it should constitute an attractive imaging agent for the noninvasive detection of unstable atherosclerotic plaques.

Intraplaque hemorrhage and progression of coronary atheroma.

BACKGROUND: Intraplaque hemorrhage is common in advanced coronary atherosclerotic lesions. The relation between hemorrhage and the vulnerability of plaque to disruption may involve the accumulation of free cholesterol from erythrocyte membranes. METHODS: We stained multiple coronary lesions from 24 randomly selected patients who had died suddenly of coronary causes with an antibody against glycophorin A (a protein specific to erythrocytes that facilitates anion exchange) and Mallory's stain for iron (hemosiderin), markers of previous intraplaque hemorrhage. Coronary lesions were classified as lesions with pathologic intimal thickening, fibrous-cap atheromas with cores in an early or late stage of necrosis, or thin-cap fibrous atheromas (vulnerable plaques). The arterial response to plaque hemorrhage was further defined in a rabbit model of atherosclerosis. RESULTS: Only traces of glycophorin A and iron were found in lesions with pathologic intimal thickening or fibrous-cap atheromas with cores in an early stage of necrosis. In contrast, fibroatheromas with cores in a late stage of necrosis or thin caps had a marked increase in glycophorin A in regions of cholesterol clefts surrounded by iron deposits. Larger amounts of both glycophorin A and iron were associated with larger necrotic cores and greater macrophage infiltration. Rabbit lesions with induced intramural hemorrhage consistently showed cholesterol crystals with erythrocyte fragments, foam cells, and iron deposits. In contrast, control lesions from the same animals had a marked reduction in macrophages and lipid content. CONCLUSIONS: By contributing to the deposition of free cholesterol, macrophage infiltration, and enlargement of the necrotic core, the accumulation of erythrocyte membranes within an atherosclerotic plaque may represent a potent atherogenic stimulus. These factors may increase the risk of plaque destabilization.

Differential accumulation of proteoglycans and hyaluronan in culprit lesions: insights into plaque erosion.

OBJECTIVE: The importance of the extracellular matrix molecules versican, biglycan, decorin, and hyaluronan in plaque instability has not been recognized. METHODS AND RESULTS: Coronary lesions with acute thrombi and stable plaques were examined for the accumulation and distribution of specific proteoglycans and hyaluronan at culprit sites. The cell surface receptor for hyaluronan, CD44, and smooth muscle (SM) cell maturation markers were also assessed. Proteoglycans and hyaluronan accumulated in distinct patterns depending on plaque type. The fibrous cap of stable lesions was enriched in versican and biglycan, with considerably less staining for decorin and hyaluronan, whereas picrosirius red revealed a heavy accumulation of collagen type I. In contrast, intense staining for hyaluronan and versican was found in erosions at the plaque/thrombus interface, with weak staining for biglycan and decorin; collagen content was predominantly type III. Rupture sites showed little immunoreactivity for proteoglycans or hyaluronan. CD44 was localized along the plaque/thrombus interface in erosions, whereas in ruptures and stable plaques, it was mostly confined to inflammatory cells. Positive immunostaining for immature SM cells (SM myosin heavy chain SM1 and SMemb) was present in stable and eroded plaques, whereas the presence of SM2 and smoothelin was weak or nonexistent. CONCLUSIONS: Specific accumulation of versican, hyaluronan, and CD44 at the sites of plaque erosion implicates an involvement of these molecules in events associated with acute coronary thrombosis.

A novel rat model of carotid artery stenting for the understanding of restenosis in metabolic diseases.

The effects of risk modifiers such as diabetes, obesity and hypertension on vascular healing after stent deployment are largely unknown, because of a lack of an appropriate animal model to study. Since many inbred strains of rats expressing these phenotypes are available, we validated a carotid artery model of in-stent restenosis in the rat. A detailed histomorphometric analysis was performed on 2-cell Multi-Link(TM) stents (1.5 x 5 mm) deployed in the common carotid artery of male Wistar rats. Early focal thrombus formation around stent struts with adherent leukocytes was evident by day 3. The number of ED-1-positive macrophages was maximal by day 7 and declined markedly thereafter. Neointimal cell proliferation peaked by day 7 (19.3 +/- 6.9) and progressively decreased to <2% by day 60. By day 14, neointimal area was significantly increased (0.39 +/- 0.03 vs. 0.18 +/- 0.05 mm(2) at day 7, p = 0.003) characterized by an enhanced number of alpha-actin-positive smooth muscle cells surrounded by extracellular matrix rich in versican and hyaluronan. At day 28, neointimal area was maximal accompanied by an appreciable decrease in the staining intensity for hyaluronan and versican. By day 60, neointimal area decreased significantly (0.28 +/- 0.04 vs. 0.45 +/- 0.07 mm(2) at day 28, p = 0.04) independent of a change in cell density. This regression phase was accompanied by a marked increase in elastin fibrils and collagen type I. In summary, vascular healing following carotid artery stenting in the rat parallels that of larger animals; however, it is accelerated relative to humans.

Morphological predictors of restenosis after coronary stenting in humans.

BACKGROUND: Experimental studies suggest that arterial injury and inflammation lead to increased neointimal growth after stenting. Despite the increased use of coronary stents in humans, there are only limited pathological data on the morphological features of in-stent restenosis. METHODS AND RESULTS: Detailed histology was performed on 116 stents, implanted > or =90 days in 87 coronary arteries, from 56 patients (mean age, 59+/-13 years). The mean duration of stent implant was 10 months. In-stent restenosis was defined as a stent area stenosis of >75%. Lumen area increased as stent area increased (r2=0.27, P=0.0001), but there was a much stronger correlation between stent area and neointimal area (r2=0.70, P<0.0001). Arterial medial fracture was associated with a 29% increase (P<0.01) in neointimal thickness compared with arteries with an intact media. Neointimal thickness (P=0.0001), inflammatory cell density (P<0.0001), and neointimal vascular channel density (P<0.0001) were greater when struts were in contact with a ruptured arterial media compared with fibrous plaque or an intact fibrous cap. Stent strut penetration into a lipid core was associated with increased neointimal thickness (P=0.04) and inflammatory cell density (P=0.03). Neointimal inflammatory cell content was 2.4-fold greater in stents with restenosis versus no restenosis, and inflammation was associated with increased neoangiogenesis. CONCLUSIONS: Coronary stenting that is accompanied by medial damage or penetration of the stent into a lipid core induces increased arterial inflammation, which is associated with increased neointimal growth. These data suggest the use of stenting strategies that reduce inflammation and neoangiogenesis to reduce the incidence of restenosis.