Effect of Cholesterol Crystals on Plaques and Intima in Arteries of Patients With Acute Coronary and Cerebrovascular Syndromes

doi:10.1016/j.amjcard.2008.12.019

The American Journal of Cardiology

Volume 103, Issue 7, 1 April 2009, Pages 959-968

https://doi.org/10.1016/j.amjcard.2008.12.019 Get rights and content

Plaque disruption (PD) causes most acute cardiovascular events. Although cholesterol crystals (CCs) have been observed in plaques, their role in PD was unknown. However, cholesterol expands with crystallization tearing and perforating fibrous tissues. This study tested the hypothesis that CCs can damage plaques and intima, triggering PD, as observed in tissues prepared without ethanol solvents that dissolve CCs. Coronary arteries of patients who died of acute coronary syndrome (n = 19) and non–acute coronary syndrome causes (n = 12) and carotid plaques from patients with (n = 51) and without (n = 19) neurologic symptoms were studied. Samples were examined for CCs perforating the intima using light and scanning electron microscopy (SEM) with ethanol or vacuum dehydration. In addition, fresh unfixed carotid plaques were examined at 37°C using confocal microscopy. Crystal content using SEM was scored from 0 to +3. SEM using vacuum dehydration had significantly higher crystal content compared with SEM using ethanol dehydration (+2.5 ± 0.53 vs +0.25 ± 0.46; p <0.0003), with enhanced detection of CC perforations. The presence of CCs using SEM and confocal microscopy was similar, suggesting that CC perforation can occur in vivo at 37°C. All patients with acute coronary syndrome had perforating CCs, but none was present in patients without acute coronary syndrome (p = 0.0001). For all plaques, there were strong associations of CCs with PD, thrombus, symptoms (p <0.0001), and plaque size (p <0.02). Crystal content was an independent predictor of thrombus and symptoms. In conclusion, by avoiding ethanol in tissue preparation, CCs perforating the intima were shown to be associated with PD. Crystal content was significantly associated with clinical events, suggesting that cholesterol crystallization may have a role in PD.

Section snippets

Methods

A case-control study design was used by selecting autopsies of patients who died of acute coronary syndrome or non–acute coronary syndrome causes (December 2005 to August 2008). Acute coronary syndrome was considered present in patients who died with symptoms of cardiac ischemia and those found dead with no evidence other than PD and/or ventricular rupture. Patients without acute coronary syndrome died of no cardiovascular cause. Medical history and demographics were obtained from the

Results

Coronary arteries from the hearts of 31 patients (23 men, 8 women; age 50 ± 15 years) who had died of acute coronary syndrome and non–acute coronary syndrome causes were examined within 24 hours after death. Coronary artery disease as the primary cause of death was reported in 19 patients. By history, 8 patients had symptoms of myocardial ischemia before death, 2 had left ventricular rupture, and 9 were found with PD as the only cause of death. Patients without acute coronary syndrome (n = 12)

Discussion

The ability to detect the extensive CC distribution and elucidate their potential role in PD was possible by processing tissues without an ethanol solvent. This study showed that arterial tissues and plaque prepared using vacuum dehydration for SEM markedly improved the detection of CCs disrupting plaque and penetrating the intimal surface. In addition, these observations using SEM were shown to reflect in vivo events by being concurrent with the presence of CCs in unprocessed plaque at body

Acknowledgment

The authors thank Dr. Ara Pridjian, MD, and Nadyne Abfalter, Cardiovascular Surgery, Sparrow Hospital, for carotid plaques; Ruiping Huang, PhD, for technical support; Geral Diggs, BS, RBP, for artwork; Ewa Danielewicz, MSc, and Melinda Frame, PhD, Center for Advanced Microscopy; Ralph Common, BS, Electron Microscopy Facility at the Division of Human Pathology, Department of Physiology; Amy Porter, HT, QIHC, Investigative Histopathology Laboratory, Michigan State University; Dr. Gregory Fink,

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Non-culprit plaque progression is associated with recurrent cardiac ischemic events and worse clinical outcomes. Given that atherosclerosis is a systemic disease, the pancoronary characteristics of patients with rapid plaque progression are unknown. This study aims to identify pancoronary plaque features in patients with ST-segment elevation myocardial infarction (STEMI) with and without rapid plaque progression, focused on the patient level.
From January 2017 to July 2019, 291 patients underwent 3-vessel optical coherence tomography imaging at the time of the primary procedure and a follow-up angiography interval of 12 months. The final analysis included 237 patients. Overall, 308 non-culprit lesions were found in 78 STEMI patients with rapid plaque progression, and 465 non-culprit plaques were found in 159 STEMI patients without rapid plaque progression. These patients had a higher pancoronary vulnerability (CLIMA-defined high-risk plaque: 47.4% vs. 33.3%; non-culprit plaque rupture: 25.6% vs. 14.5%) and a significantly higher prevalence of other vulnerable plaque characteristics (i.e., lipid-rich plaque, cholesterol crystal, microchannels, calcification, spotty calcification, and thrombus) at baseline versus those without rapid plaque progression. Lesions with rapid progression were highly distributed at the LAD, tending to be near the bifurcation. In multivariate analysis, age ≥ 65 years was an independent predictor of subsequent rapid lesion progression at the patient level, whereas microchannel, spotty calcification, and cholesterol crystal were independent predictors for STEMI patients ≥65 years old.
STEMI patients with subsequent rapid plaque progression had higher pancoronary vulnerability and commonly presented vulnerable plaque morphology. Aging was the only predictor of subsequent rapid plaque progression.
Cholesterol crystals induce mechanical trauma, inflammation, and neo-vascularization in solid cancers as in atherosclerosis
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Cancer and atherosclerosis share common risk factors and inflammatory pathways that promote their proliferation via vascular endothelial growth factor (VEGF). Because CCs cause mechanical injury and inflammation in atherosclerosis, we investigated their presence in solid cancers and their activation of IL-1β, VEGF, CD44, and Ubiquityl-Histone H2B (Ub-H2B), that promote cancer growth.
Tumor specimens from eleven different types of human cancers and atherosclerotic plaques were assessed for CCs, free cholesterol content and IL1-β by microscopy, immunohistochemistry, and biochemical analysis. Breast and colon cancer cell lines were cultured with and without CCs to select for expression of VEGF, CD44, and Ub-H2B. Western blot and immunofluorescence were performed on cells to assess the effect of CCs on signaling pathways.
Cancers displayed higher CC content (+2.29 ± 0.74 vs +1.46 ± 0.84, p < 0.0001), distribution (5.06 ± 3.13 vs 2.86 ± 2.18, p < 0.001) and free cholesterol (3.63 ± 4.02 vs 1.52 ± 0.56 μg/mg, p < 0.01) than cancer free marginal tissues and similarly for atherosclerotic plaques and margins (+2.31 ± 0.51 vs +1.44 ± 0.79, p < 0.02; 14.0 ± 5.74 vs 8.14 ± 5.52, p < 0.03; 0.19 ± 0.14 vs 0.09 ± 0.04 μg/mg, p < 0.02) respectively. Cancers displayed significantly increased expression of IL1-β compared to marginal tissues. CCs treated cancer cells had increased expression of VEGF, CD44, and Ub-H2B compared to control. By microscopy, CCs were found perforating cancer tumors similar to plaque rupture.
These findings suggest that CCs can induce trauma and activate cytokines that enhance cancer growth as in atherosclerosis.
An Update on Inflammation in Atherosclerosis: How to Effectively Treat Residual Risk
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This study reviewed the contribution of inflammation to atherosclerotic cardiovascular disease (ASCVD), which has gained widespread recognition in recent years.
This critical review evaluated how recent publications and ongoing clinical trials in atherosclerotic inflammation will affect clinical care.
Key trials, including CANTOS (Canakinumab Anti-Inflammatory Thrombosis Outcomes Study) with canakinumab (interleukin-1β inhibition), and COLCOT (Colchicine Cardiovascular Outcomes Trial) and LoDoCo2 (Low Dose Colchicine 2) with colchicine, have shown that suppressing inflammation can improve outcomes in ASCVD. Cholesterol crystals play an important role in activating the NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome and subsequent cytokine cascade. Inflammation contributes to significant residual risk after optimal lipid-lowering therapy. High-sensitivity C-reactive protein is a recognized biomarker of residual risk, and newer biomarkers such as the neutrophil to lymphocyte ratio may add additional information. The role of lipoprotein(a) as a proinflammatory agent or possible inflammatory biomarker is under investigation. The contribution of clonal hematopoiesis of indeterminate potential and trained immunity are in the early stages of investigation. Ongoing clinical trials of suppressing inflammation with NOD-, LRR-, and pyrin domain-containing protein 3 inflammasome inhibition (colchicine) and alternative approaches with downstream interleukin-6 ligand inhibition (ziltivekimab) will expand the evidence base for the use of anti-inflammatory agents in ASCVD.
Based on current evidence and ongoing clinical trials, targeting inflammation alongside optimal lipid lowering is likely to be central to the future treatment of ASCVD. (Clin Ther. 2023;45:XXX–XXX) © 2023 Elsevier HS Journals, Inc.
Cholesterol crystals in atherosclerotic plaques: A future target to reduce the risk of plaque rupture?
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Birefringent crystals deposition and inflammasome expression in human atheroma plaques by levels of uricemia
2022, Joint Bone Spine
To verify the monosodium urate (MSU) crystal deposition in artery walls following a structure assessment and to assess NLRP3 inflammasome expression in human atheroma plaques by levels of uricemia.
Patients with peripheral arterial disease who were candidates for amputation were recruited and classified as normouricemic or hyperuricemic. During surgery, an artery segment from the amputated limb was sampled, divided and fixed separately by cryo-embedding, 100% ethanol or Glyo-fixx. Samples were assessed by compensated polarized-light microscopy to identify MSU crystals on the artery walls. Afterwards, macrophages, neutrophils and NLRP3 inflammasome components at the plaque were categorized by immunostaining and compared between normouricemics and hyperuricemics.
Thirty artery samples from 27 patients were studied; 10 (37.0%) participants were hyperuricemic. Birefringent needle-shaped crystals were found in three samples (10.0%), all processed by frozen sectioning. Other methods showed no crystals. No accompanying inflammatory process was noted, and the presence of crystals was equally distributed across ranges of uricemia, making it unlikely they were MSU crystals. Regarding immunostaining, 28 artery samples were available for analysis, with similar infiltration of macrophages and neutrophils. NLRP3 and gasdermin-D expression were significantly greater in hyperuricemics compared to normouricemics (P = 0.044 and P = 0.017, respectively). ASC content was numerically larger in hyperuricemics as well, while caspase-1 and IL-1beta expression were similar between groups.
The presence of MSU crystals on artery walls was not confirmed. Hyperuricemia was associated with greater NLRP3 and gasdermin-D expression on human atheroma plaques in patients with peripheral artery disease.
Cholesterol crystals in non-culprit plaques of STEMI patients: A 3-vessel OCT study
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Cholesterol crystals (CCs) are regular microstructures found within the necrotic core of atherosclerotic plaques and have been hypothesized to be related to plaque destabilization. We attempted to investigate the potential association between CCs and non-culprit plaque vulnerability in patients with ST-segment elevated myocardial infarction (STEMI) and study morphological features of CCs in ruptured non-culprit plaques.
A total of 261 patients with ST-segment elevation myocardial infarction who underwent 3-vessel optical coherence tomography (OCT) imaging were included. Non-culprit plaques were divided into two groups according to the presence or absence of CCs in the plaque to compare the morphological characteristics of the plaques. The differences in parameters of the non-culprit plaque CCs were explored between ruptured plaques and unruptured plaques.
Totally, 530 non-culprit plaques (29 ruptured plaques and 501 unruptured plaques) were identified by OCT. The incidence of CCs was 21.1%. Compared with non-culprit plaques without CCs, those with CCs had a larger lipid burden. Macrophages (p < 0.001) and spotty calcification (p = 0.002) were more frequently observed in non-culprit plaques with CCs. The frequency of CCs was significantly higher (p = 0.001) and the CCs were larger (p = 0.046) and more superficial (p = 0.005) in ruptured non-culprit plaques than in unruptured non-culprit plaques. The maximum lipid arc and fibrous cap thickness were independent predictors of plaque rupture, but the presence of CCs was not.
Non-culprit plaques with CCs have more vulnerable features. CCs are more frequently found in ruptured non-culprit plaques and larger and more superficial CCs are associated with plaque rupture.

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: This work was supported by Grant No. IRGP147 from Michigan State University, East Lansing, Michigan, and Sparrow Hospital, Lansing, Michigan.

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The American Journal of Cardiology

Section snippets

Methods

Results

Discussion

Acknowledgment

References (20)

Uptake and compartmentalization of fluorescent lipid analogs in larval Schistosoma mansoni

J Lipid Res

Frequency and distribution of thin-cap fibroatheroma and ruptured plaques in human coronary arteries

J Am Coll Cardiol

The severity of coronary atherosclerosis at sites of plaque rupture with occlusive thrombosis

J Am Coll Cardiol

Effect of histologic preparation on the cross-sectional area of arterial rings

J Surg Res

Plaque fissures in human coronary thrombosis

J Atheroscler Res

Plaque fissuring: the cause of acute myocardial infarction causing sudden ischaemic death, and crecendo angina

Br Heart J

Terminology for high-risk and vulnerable coronary artery plaques

Eur Heart J

Colour Atlas of Histopathology

Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events: a novel insight into plaque rupture by scanning electron microscopy

Scanning

Cholesterol crystals cause mechanical damage to biological membranes: a proposed mechanism of plaque rupture and erosion leading to arterial thrombosis

Clin Cardiol

Cited by (150)

Pancoronary plaque characteristics in STEMI patients with rapid plaque progression: An optical coherence tomography study

Cholesterol crystals induce mechanical trauma, inflammation, and neo-vascularization in solid cancers as in atherosclerosis

An Update on Inflammation in Atherosclerosis: How to Effectively Treat Residual Risk

Cholesterol crystals in atherosclerotic plaques: A future target to reduce the risk of plaque rupture?

Birefringent crystals deposition and inflammasome expression in human atheroma plaques by levels of uricemia

Cholesterol crystals in non-culprit plaques of STEMI patients: A 3-vessel OCT study

Coronary artery diseaseEffect of Cholesterol Crystals on Plaques and Intima in Arteries of Patients With Acute Coronary and Cerebrovascular Syndromes

Section snippets

Methods

Results

Discussion

Acknowledgment

J Lipid Res

J Am Coll Cardiol

J Am Coll Cardiol

J Surg Res

Plaque fissures in human coronary thrombosis

J Atheroscler Res

Plaque fissuring: the cause of acute myocardial infarction causing sudden ischaemic death, and crecendo angina

Br Heart J

Terminology for high-risk and vulnerable coronary artery plaques

Eur Heart J

Colour Atlas of Histopathology

Cholesterol crystals rupture biological membranes and human plaques during acute cardiovascular events: a novel insight into plaque rupture by scanning electron microscopy

Scanning

Cholesterol crystals cause mechanical damage to biological membranes: a proposed mechanism of plaque rupture and erosion leading to arterial thrombosis

Clin Cardiol

Coronary artery disease
Effect of Cholesterol Crystals on Plaques and Intima in Arteries of Patients With Acute Coronary and Cerebrovascular Syndromes