The types of lesion instability responsible for the majority of acute coronary events frequently include plaque disruption and plaque erosion with superimposed thrombosis. The term ‘vulnerable plaque is used to describe atherosclerotic (ATS) plaques that are particularly prone to rupture and susceptible to thrombus formation, such as the thin-cap fibroatheroma (TCFA). The aim of the present study was to assess the morphological and histological differences between plaques that are unstable and those that are vulnerable to instability. Carotid artery endarterectomy specimens were obtained from 26 patients with carotid artery stenosis, consisting of 20 men and 6 women (age range, 35–80 years). Histological and morphometric methods were used to visualize and characterize the ATS plaques. Among the 26 carotid ATS plaques, 23% were stable, 23% were unstable and 54% were vulnerable. With regard to morphometric characteristics, the following mean values were obtained for the TCFA and unstable plaques, respectively: Fibrous cap thickness, 21.91 and 11.66 µM; proportion of necrotic core area in the total plaque area, 25.90 and 22.03%; and the proportion of inflammatory area in the total plaque area, 8.41 and 3.04%. No plaque calcification was observed in any of them. Since ATS coronary artery disease is considerably widespread and fatal, it is crucial to further study ATS lesions to obtain an improved understanding of the nature of vulnerable and unstable plaques. The methods used to detect plaque size, necrotic core area and fibrous cap thickness are considered to be particularly useful for identifying vulnerable and unstable plaques.
Atherosclerotic (ATS) plaques consists of two main components: (i) Soft, lipid-rich atheromatous mass and (ii) hard, collagen-rich sclerotic tissue (
The majority of disrupted plaques have been shown to have a large necrotic core with a thin overlying fibrous cap, with substantial inflammation and limited calcification (
The aim of the present study was to characterize a number of carotid ATS plaques, which were obtained by endarterectomy, for the purpose of determining the importance of various pathological characteristics, including fibrous cap thickness and plaque, necrotic core, macrophage and calcification areas. The study included stable plaques [SP; including fibroatheromas (FAs) and fibrocalcified plaques (FCs)], vulnerable plaques [VP; including thin-cap fibroatheromas (TCFAs)], and unstable plaques [USP; featuring plaque rupture (PR), plaque erosion (PE) and calcified nodules (CNs)], as previously defined by pathologists (
The present study elucidated the associations between plaque characteristics and the risk factors for the development of CVD including age, smoking, arterial hypertension, hyperlipidemia and diabetes mellitus. We hypothesized that the present study may contribute to the improved understanding of acute coronary events and underlying plaque composition and its association with coronary risk factors, in order to improve the timely identification of vulnerable plaques.
A total of 26 patients that underwent carotid artery endarterectomy to treat high-grade internal carotid artery stenosis at the ‘Prof. Dr. George I.M. Georgescu’ Institute of Cardiovascular Diseases (Iasi, Romania) between January and December 2013 participated in the present study. Excised carotid ATS plaques were obtained from 20 male and 6 female patients, aged 35–80 years, who presented with transient ischemic attack symptoms upon diagnostic consultation. Ethical approval for the present study was granted by the Institutional Board at Prof. Dr. George I.M. Georgescu’ Institute of Cardiovascular Diseases. Written informed consent was obtained from the patients.
Carotid endarterectomy was performed under anesthesia using fentanyl (5–15 mcg/kg; Gedeon Richter Plc., Budapest, Hungary), midazolam (0.05–0.1 mg/kg; Hofman-La Roche Ltd., Basel, Switzerland), and rocuronium (0.6 mg/kg; Fresenius Kabi Austria GmbH, Graz, Austria) for induction and maintained with isoflurane (Rompharm Company, Otopeni, Romania). The entire intimal carotid plaques (length, ~1 cm) were removed from the carotid arteries. Excessively fragmented plaques (>2 pieces) were not subjected to further analysis. The carotid artery atherosclerotic plaques were subsequently sectioned at 3–4-mm intervals and processed for histological examination. All sections were stained with hematoxylin and eosin, elastic Van Gieson, Masson's trichrome and movat pentachrome (all Bio Optica Milano SpA, Milan, Italy). Hematoxylin stains the nuclei and calcified material in cells blue, whereas eosin stains eosinophilic structures in various shades of red. Elastic Van Gieson is a histological stain for elastic and collagen fibers and Masson's trichrome and movat pentachrome stain muscle fibers, collagen and nuclei. Masson's trichome stain is used to detect connective tissue (green) and muscle tissue (red) characterized by fibrotic and degenerative changes; whereas movat pentachrome stain differentiates between the various ages and types of collagen and connective tissue matrix in the ATS plaques. Microscopic sections were analyzed with the observer blinded to the clinical status of the patients whose plaques were being examined.
ATS plaques were classified as either VP, SP or USP, as previously described (
Histological assessment was performed by an experienced pathologist using an optical microscope (CX41; Olympus Corporation, Tokyo, Japan). The measurements were visualized using color image analysis software (QuickPHOTO MICRO 3.0, PROMICRA, S.r.o., Prague, Czech Republic). Quantitative morphometry included measurement of the plaque, lipid necrotic core, inflammatory and calcified areas and fibrous cap thickness.
The following potential risk factors (RFs) for atherosclerosis were also assessed: Age, gender, diabetes mellitus (DM), arterial hypertension, history of cigarette smoking, cerebrovascular diseases and hyperlipidemia. All RF data was obtained from the patients' files.
The association among the pathological characteristics of the three defined plaque types, as well as among plaque types and ATS RFs were evaluated. Data are expressed as mean values and percentages, calculated using Excel software (Microsoft Corporation, Redmond, WA, USA).
A total of 26 carotid ATS plaques were histologically analyzed and classified as follows (
Morphometric analysis was performed to evaluate the fibrous cap thickness and necrotic core size, in addition to the inflammatory and calcified plaque areas of the ATS plaques (
USPs exhibited the smallest fibrous cap thickness and a large lipid core area in the total plaque area, and exhibited inflammatory infiltrate. VPs were found to have a fibrous cap thickness of <164 µm and lipid core areas of varying sizes, and showed considerable inflammatory infiltrate. Plaque instability was found to be highly associated with fibrous cap thickness, lipid core area and plaque size. The incidence of the various RFs is summarized in
The age of the patients, as well as the prevalence of the RFs for ATS differed between the SP and USP groups (data not shown). Arterial hypertension was found to be coexistent with the development of ATS plaques in 96.15% of all cases. Furthermore, age appeared to be an associated factor in ATS development. Hyperlipidemia was detected in 50% of all cases. DM was present in 34.61% of cases and was one of the investigated RFs involved in ATS progression in the vessels. Smoking was involved in 26.92% of all cases, suggesting that it is an important risk factor in early and late thrombosis. Involvement of other peripheral vessels in ATS process was identified in 57.69% of all cases.
The age of the patients and the prevalence of the RFs for ATS in the SP group differed from the USP group: In order of decreasing frequency, the RFs were higher in VPs compared with the USPs and SPs (data not shown).
Taking into consideration the number of the RFs involved in the pathogenesis of ATS in each patient, the following frequencies were observed (
Atherosclerosis is a major cause of morbidity and mortality worldwide, and despite the advances in the understanding of its pathogenesis, the factors that determine atheromatous plaque instability remain largely unknown. The prediction of plaque vulnerability to rupture and subsequent thrombosis would be useful in the development of diagnostic and therapeutic approaches (
Cap thickness has been identified as a crucial characteristic for the distinction between TCFAs and FAs, as TCFAs are known to have a thinner cap (
Further separation of plaque ruptures and TCFAs from FAs is indicated by the simultaneous presence of plaque inflammation and necrotic core area (
Excluding fibrous cap thickness from the analysis, plaque inflammation allowed for the separation of plaque rupture and TCFAs from FAs with a proportion of ≥3 times greater in VPs and RPs compared with FAs (
In a previous study, the mean area of macrophages per plaque area, infiltrating the region of the fibrous cap and the shoulder was significantly greater in the non-calcified plaque sections compared with the calcified sections (
Calcification in ATS lesions is relatively common and has been implicated as a risk factor for increased cardiovascular morbidity and mortality (
In the present study, 14 plaques were defined as TCFAs. These plaques were not ruptured but were considered of high risk; thus, detection of such plaques is the key aim of diagnosis.
PRs and fissures usually occur in the fibrous cap and shoulder regions. Rupture of the ATS plaques is responsible for the majority of acute coronary events, and such lesions have been shown to exhibit distinct histopathological features (
Intimal thickness and lipid core area have been associated with RFs for coronary ATS disease (
Currently, among the most promising fields in the study of atherosclerosis is the development of imaging techniques that facilitate gross, microscopic and molecular characterization of
Atheromatous plaques may become unstable due to increases in size, increased intra- and extracellular lipid accumulation, as well as intraplaque hemorrhage (
The most common RFs that were found to be associated with stable, vulnerable and unstable ATS plaques were age and hypertension. Hypertension in atherosclerosis indicates disease progression and may become life-threatening (
(A) Fibroatheromatous plaque (stain, Masson's trichrome; magnification, ×10); (B) Fibro-calcified plaque (stain, elastic Van Gieson; magnification, ×10); (C) Thin-cap fibroatheroma (stain, elastic Van Gieson; magnification, ×10).
(A) Erosion plaque (stain, Masson's trichrome; magnification, ×10); (B) Ruptured plaque (stain, movat pentachrome; magnification, ×10); (C) Calcified nodule (stain, hematoxylin and eosin; magnification, ×10).
Carotid atherosclerotic plaques.
Patients (n=26) | ||
---|---|---|
Plaque type | n | % |
Stable | 6 | 23 |
Fibroatheroma | 4 | |
Fibro-calcified | 2 | |
Vulnerable | 14 | 54 |
Thin-cap fibroatheroma | 14 | |
Unstable | 6 | 23 |
Plaque erosion | 2 | |
Plaque rupture | 2 | |
Calcified nodules | 2 |
Carotid atherosclerotic plaque measurements.
Plaque type | Fibrous cap, µm | Necrotic core, % | Inflammatory infiltrate, % | Calcification, % |
---|---|---|---|---|
FA | 351 (173–654) | 56.99 (32.24–87.19) | Minimum | – |
FC | 270 (170–371) | 46.86 (37–56.72) | – | 6.23 (8.11–4.36) |
TCFA | 21.91 (5–44) | 25.90 (3.04–60.22) | 8.41 (0–25.87) | – |
PR | 11.66 (6–20) | 22.03 (2.64–32.50) | 3.04 (2.64–32.50) | – |
PE | 13.13 (10–16) | 70.29 (51.97–87.74) | 6.69 (5–8.35) | 17.85 (14.11–21.46) |
CN | 20.33 (11–35) | – | Minimum | 136.599 |
FA, fibroatheroma; FC, fibro-calcified plaque; TCFA, thin-cap fibroatheroma; PR, plaque rupture; PE, plaque erosion; CN, calcified nodule.
Incidence rates of risk factors in relation to ATS lesions.
Risk factors (n) | ||||||
---|---|---|---|---|---|---|
ATS lesion | Age >50 years | Smoking | AHT | HL | DM | CVD |
SP | 2 | 1 | 5 | 2 | 1 | 0 |
VP | 10 | 4 | 14 | 8 | 4 | 10 |
USP | 5 | 2 | 6 | 3 | 4 | 5 |
Total patients (%) | 17 (65.38) | 7 (26.92) | 25 (96.15) | 13 (50.00) | 9 (34.61) | 15 (57.69) |
ATS, atherosclerotic; AHT, arterial hypertension; HL, hyperlipidemia; DM, diabetes mellitus; CVD, cardiovascular diseases; SP, stable plaques; VP, vulnerable plaques; USP, unstable plaques.
Incidence of the cumulative cardiovascular risk factors in each patient.
Cardiovascular risk factors (n) | ||||||
---|---|---|---|---|---|---|
ATS lesion | 6 | 5 | 4 | 3 | 2 | 1 |
SP | 0 | 0 | 2 | 0 | 0 | 0 |
VP | 3 | 4 | 10 | 1 | 0 | 0 |
USP | 2 | 1 | 3 | 0 | 0 | 0 |
Total patients (%) | 5 (19.23) | 5 (19.23) | 15 (57.69) | 1 (3.84) | 0 (0.00) | 0 (0.00) |
ATS, atherosclerotic; SP, stable plaques; VP, vulnerable plaques; USP, unstable plaques.