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J Am Coll Cardiol, 2006; 47:2118-2120, doi:10.1016/j.jacc.2006.02.025
(Published online 20 April 2006). © 2006 by the American College of Cardiology Foundation |
* Department of Geriatrics and Metabolic Diseases, Second University of Naples, Naples, Italy (Email: raffaele.marfella{at}unina2.it).
The study group consisted of 26 type 2 diabetic and 30 nondiabetic patients enlisted to undergo carotid endarterectomy for asymptomatic extracranial high-grade (>70%) internal carotid artery stenosis (6). Written informed consent was obtained from all patients. The local ethics review committee approved the study. After surgery, the specimens were cut perpendicular to the long axis into two halves. The first half was frozen in liquid nitrogen, lysed, and centrifuged for the following enzyme-linked immunosorbent assay analysis. Caspase-3, TNF-
, IL-1ß and nitrotyrosine levels were quantified in plaques using specific kits (R&D Systems, Minneapolis, Minnesota; Imgenex, Oxford, United Kingdom). A portion of the other half specimen was immediately immersion-fixed in 10% buffered formalin. Serial sections were incubated with specific antibodies -smooth muscle (SM) actin and anti-CD68; anti–TNF-, IL-1ß and active caspase-3 as well as terminal deoxynucleotidyl transferase end-labeling (TUNEL).
Clinical data for the study population are presented in Table 1. In nondiabetic subjects, the presence of type 2 diabetes mellitus or impaired glucose tolerance was tested by oral glucose tolerance test. Compared with nondiabetic patients, diabetic patients had significantly greater portion of plaque area occupied by macrophages (24.5 ± 5% vs. 5 ± 2% of the total area; p < 0.01) (Fig. 1) and lower content of interstitial collagen (p < 0.01) (Fig. 1). The nondiabetic plaques were composed primarily of longitudinally oriented SMCs that strongly expressed -SMC actin (18 ± 6% of the total area). However, -SMC immunoreactivity was much lower in the diabetic plaques (9 ± 7% of the total area) (Fig. 1). We detected IL-1ß and TNF- in 22 of 26 diabetic plaques, whereas the same cytokines were detected in only 12 of 30 nondiabetic plaques. Compared with nondiabetic lesions, diabetic plaques had higher levels of IL-1ß and TNF- (p < 0.01) (Fig. 1). Notably, IL-1ß and TNF- plaque levels were strongly dependent on glycemic control, as also reflected by the statistically significant correlation between plasma HbA1c and cytokine plaque levels (IL-1ß: r = 0.49, p < 0.001; TNF-: r = 0.51, p < 0.001). The TUNEL labeling disclosed evidence of apoptosis in 32 (57%) of the total 56 specimens studied. Among 26 diabetic plaques, 24 (92%) contained foci of apoptosis; in contrast, apoptosis was observed in only 8 (27%) of 30 nondiabetic plaques (p < 0.01). Among nondiabetic plaques, apoptosis was typically limited to <2% of cells. Among diabetic plaques, the frequency of apoptotic cells ranged from 0.10% to 18% (4.9 ± 6.5%). Compared with nondiabetic plaques, diabetic plaques revealed higher levels of caspase-3 (p < 0.01) (Fig. 1). Notably, caspase-3 levels in plaque specimens were strongly dependent on glycemic control (r = 0.533, p < 0.001). Diabetic plaques had significantly higher percentage of TUNEL-positive VSMCs compared with nondiabetic plaques (p < 0.01) (Fig. 1). Higher nitrotyrosine levels were found in diabetic as compared with nondiabetic plaques (p < 0.001) (Fig. 1). Most TUNEL-positive diabetic plaques (75%) showed higher levels of TNF- and nitrotyrosine levels. Conversely, most fields that were not TUNEL-positive (72%) showed moderate to low levels of TNF- and nitrotyrosine levels. Moreover, by multivariate analysis, HbA1c was independently related to VSMC apoptosis, as showed by TUNEL-positive VSMCs and caspase-3 levels (p < 0.030 and p < 0.026, respectively).
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and IL-1ß levels along with lower interstitial collagen and -SMC actin content. All this would make diabetic plaques more prone to inflammatory-dependent rupture and might increase the risk of cerebrovascular ischemic events (7,8). In our study, macrophages were more abundant in diabetic plaques and represented the major source of inflammatory cytokines, suggesting the presence of an active inflammatory reaction in diabetic plaques. In agreement with the difference in apoptosis staining pattern, the histologic milieu of the lesions appears different with regard to cellularity, but not in the degree of vessel stenosis, suggesting that diabetic and nondiabetic lesions are different only with regard to inflammatory burden. Hence, the differences in plaque behavior likely stem from differences in the presence of stimuli (i.e., persistent hyperglycemia and oxidative stress, as evidenced by high HbA1c and nitrotyrosine levels) for selective expression of TNF- or IL-1ß capable of disrupting plaque stability via VSMC apoptosis. Notably, the intriguing and novel proatherogenic mechanism of apoptosis in human diabetes is supported in this study not only by the observation that VSMC apoptosis is higher in diabetic plaques, but also by the information that it is strongly correlated with the intensity of glycemic control as reflected by HbA1c. The mechanism of the relationship of cytokines with the VSMC apoptotic process remains uncertain in diabetic plaques. However, TNF- and IL-1ß do not act in isolation, as macrophage-induced VSMC apoptosis through formation of peroxynitrite also requires nitric oxide and oxygen free radicals (9). This suggests that cooperative interactions might occur between these mediators, and the present study outlines such associations demonstrating a presence of overproduction of nitrotyrosine, a good marker of peroxynitrite formation (10), in diabetic plaques that was not present in nondiabetic lesions. Indeed, these stimuli of diabetic milieu can also induce apoptosis of endothelial cells, resulting in vascular leak and inflammation, which are implicated in the pathogenesis of vascular diseases (3). This study demonstrates enhanced VSMC apoptosis in diabetic carotid atherosclerotic lesions and provides evidence that the activation of this mechanism by inflammatory cells is associated with an increase in oxidative stress potentially promoting plaque rupture. These findings are potentially important from a fundamental standpoint, because they indicate a pathogenetic role for the VSMC apoptosis in the evolution of diabetic atherosclerotic lesions. From a practical standpoint, these findings provide further support for the role of glycemic control on plaque stabilization in diabetic patients with atherosclerotic disease.
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