Effect of aspirin on cholesterol crystallization: A potential mechanism for plaque stabilization

BackgroundThe interaction between pathogenic bacteria and cholesterol crystals (CCs) has not been investigated. However, CCs are found extensively in atherosclerotic plaques and sclerotic cardiac valves. Interactions between pathogenic bacteria and CCs could provide insights into destabilization of atherosclerotic plaques and bacterial adhesion to cardiac valves.MethodsStaphylococcus aureus and Pseudomonas aeruginosa were used to assess in vitro bacterial adhesion to CCs and proliferation in the presence of CCs compared to plastic microspheres and glass shards as controls. Ex vivo studies evaluated bacterial adhesion to atherosclerotic rabbit arteries compared to normal arteries and human atherosclerotic carotid plaques compared to normal carotid arteries. Scanning electron microscopy (SEM) was used to visualize bacterial adhesion to CCs and confocal microscopy was used to detect cholesterol binding to bacteria grown in the presence or absence of CCs.ResultsIn vitro, S. aureus and P. aeruginosa displayed significantly greater adhesion, 36% (p<0.0001) and 89% (p<0.0001), respectively, and growth upon exposure to CCs compared to microspheres or glass shards. Rabbit and human atherosclerotic arteries contained significantly greater bacterial burdens compared to controls (4× (p<0.0004); 3× (p<0.019), respectively. SEM demonstrated that bacteria adhered and appeared to degrade CCs. Consistent with this, confocal microscopy indicated increased cholesterol bound to the bacterial cells.ConclusionsThis study is the first to demonstrate an interaction between bacteria and CCs showing that bacteria dissolve and bind to CCs. This interaction helps to elucidate adhesion of bacteria to sclerotic valves and atherosclerotic plaques that may contribute to endocarditis and plaque destabilization.

Phospholipid nanoparticles: Therapeutic potentials against atherosclerosis via reducing cholesterol crystals and inhibiting inflammation

BackgroundThe involvement of cholesterol crystals (CCs) in plaque progression and destabilization of atherosclerotic plaques has been recently recognized. This study aimed to evaluate the association between the intraplaque localization of CCs and plaque vulnerability.MethodsWe investigated 55 acute coronary syndrome (ACS) and 80 stable angina pectoris (stable AP) lesions using optical frequency domain imaging (OFDI) prior to percutaneous coronary intervention. The distance between CCs and the luminal surface of coronary plaques was defined as CC depth.ResultsAlthough the incidence of CCs had similar frequencies in the ACS and stable AP groups (95% vs. 89%, p = 0.25), CC depth was significantly less in patients with ACS than in those with stable AP (median [25th to 75th percentile]: 68 μm [58 to 92 μm] vs. 152 μm [115 to 218 μm]; p < 0.001). The incidences of plaque rupture, thrombus, lipid-rich plaques, and thin-cap fibroatheroma were significantly greater in patients with ACS than in those with stable AP (62% vs. 18%, p < 0.001; 67% vs. 16%, p < 0.001; 84% vs. 57%, p < 0.01; and 56% vs. 19%, p < 0.001, respectively).ConclusionOFDI analysis revealed that CCs were found in the more superficial layers within the coronary atherosclerotic plaques in patients with ACS than in those with stable AP, suggesting that CC depth is associated with plaque vulnerability. CC depth, a novel OFDI-derived parameter, could be potentially used as an alternative means of evaluating plaque vulnerability in coronary arteries.

Plaque rupture and/or erosion is the leading cause of cardiovascular events; however, the process is not well understood. Although certain morphologic characteristics have been associated with ruptured plaques, these observations are of static histological images and not of the dynamics of plaque rupture. To elucidate the process of plaque rupture, we investigated the transformation of cholesterol from liquid to solid crystal to determine whether growing crystals are capable of injuring the plaque cap. We hypothesized that during cholesterol crystallization the spatial configuration rapidly changes, causing forceful expansion of sharp-edged crystals that can damage the plaque cap. Two experiments were performed in vitro: first, cholesterol powder was melted in graduated cylinders and allowed to crystallize at room temperature. Volume changes from liquid to solid state were measured and timed. Second, thin biological membranes (20-40 microm) were put in the path of growing crystals to determine damage during crystallization. As cholesterol crystallized, the peak volume increased rapidly by up to 45% over 3 min and sharp-tipped crystals cut through and tore membranes. The amount of cholesterol and peak level of crystal growth correlated directly (r = 0.98; p < 0.01), as did the amount of cholesterol and rate of crystal growth (r = 0.99; p < 0.01). These observations suggest that crystallization of supersaturated cholesterol in atherosclerotic plaques can induce cap rupture and/or erosion. This novel insight may help in the development of therapeutic strategies that can alter cholesterol crystallization and prevent acute cardiovascular events.

Objective To investigate the localization coexpression in situ of matrix metalloproteinase(MMP)-2,-8 and vascular endothelial growth factor(VEGF)in human atherosclerotic unstable plaques with monocytes,smooth muscle cells(SMCs)and endothelial cells(ECs).Methods The histopathologic changes of unstable human atherosclerotic plaques were observed by hematoxylin and eosin(HE)staining,and the localization coexpression of MMP-2,MMP-8 and VEGF in the unstable human atherosclerotic plaques were observed by double fluorescent immunochemistry technology and confocal microscopy.Results The human atherosclerotic plaques in 6 cases had typical histopathologic instability,which was classified as super-Ⅳ type unstable plaques.The MMP-2 coexpression was the most obvious in the smooth muscle cells of fibrous cap infiltrated by monocyts,and in the monocytes of shoulder of plaques,and more expression of MMP-2 in the microvascular endothelial cells at the edge of shoulder and lipid necrosis;MMP-8 coexpressed obviously with the monocytes in the fibrous cap and lipid cores of plaques,and next to coexpressing in the smooth muscle cells of fibrous cap,while coexpression in endothelial cells was very little;VEGF coexpression was significant in the proliferative microvascular endothelial cells of plaques;The fibrous cap,which consisted of the smooth muscle cells mainly,and the edge of lipid necrosis infiltrated by more monocyts,were over-expression areas of VEGF.Conclnsions MMP-2,-8 and VEGF can coexpress with monocytes,SMCs and ECs in unstable plaques,and the major expression areas are in the fibrous cap,shoulder and micro-vessel at the edge of lipid necrosis,which are infiltrated by monocytes.Moreover,the outer membrane of vessel is involved in the pathogenesis of atherosclerosis. Key words: Carotid artery diseases; Gelatinase A; Neutrophil collagenase; Vascular endothelial growth factor A; Muscle,smooth,vascular; Monocytes

The probability of atherosclerotic plaque rupture and its thrombotic sequelae are thought to be increased at sites of macrophage accumulation. Folate receptor β (FR-β) is present on activated macrophages but not on quiescent macrophages or other immune cells. By conjugating the ligand folate with a fluorescent contrast agent, fluorescein isothiocyanate (FITC), we aimed to explore the potential role of FR-β fluorescence imaging in the distinction of vulnerable sites from more stable regions. Carotid specimens were taken from 20 patients and incubated with folate-FITC for 30 min. Ex vivo fluorescence imaging was performed to determine the exact location of folate-FITC uptake. Sections displaying regions of high uptake (determined as hot spots) were compared with sections showing low uptake (cold spots) through immunohistochemistry and real-time quantitative reverse-transcription polymerase chain reaction for FR-β. Hot spots showed significantly higher folate-FITC uptake than cold spots (P < 0.001). Hot spots tended to contain more macrophages and areas of hypoxia than cold spots. A positive correlation between messenger RNA levels of CD68 (marker for macrophages), FR-β (r = 0.53, P = 0.045), and hypoxia-inducible factor-1α expression (marker for intraplaque hypoxia; r = 0.55, P = 0.034) was found. Compared with areas with low folate-FITC uptake, areas of high folate-FITC uptake within human atherosclerotic plaques had an increased number of activated macrophages and higher areas of hypoxia. These characteristics of vulnerability imply that molecular imaging of FR-β through folate conjugates might be a good indicator for plaque vulnerability in future noninvasive imaging studies.

The Effect of Estrogen and Testosterone on Cholesterol Crystallization

Atherosclerosis is caused by prolonged dyslipidemia and chronic inflammation. Although much is known about advanced stages of atherosclerosis, events leading to its initiation are poorly understood. Cholesterol crystals (CC) have been associated with advanced plaque, but their origin and potential role in the initiation of atherosclerosis and early plaque development are unknown. Using polarized light, scanning and transmission electron microscopy we show for the first time that CC are present in the very early plaque induced after only 1 week of high fat diet in ldlr-null mice. We determined that the source of CC is the endothelial monolayer by treatment of human aortic endothelial cells with LDL for 5 days. Other cell types like monocytes, fibroblasts and human umbilical vein endothelial cells failed to form CC upon LDL, AcLDL or oxLDL treatment. To identify in vitro formed CC we used polarized light, transmission electron and confocal microscopy. Furthermore, we identified that CC are generated within autophagolysosomes of the endothelium. The formation of CC by endothelial cells is accompanied with changes in intracellular cAMP levels. The consequences of early subendothelial CC formation are incomplete junction formation with subsequent impaired endothelial barrier integrity, processes known to be involved in atherosclerosis initiation and progression. Interestingly, increase of intracellular cAMP inhibited endothelial CC production in vitro and in vivo. By performing western Blot, flow cytometry and various microscopy techniques we could show that the inhibition of CC-formation as the results of cAMP increase is accompanied by a decrease autophagolysosomes as the CC producing organelles. We could furthermore demonstrate that this protective effect of cAMP could be inhibited by additional application of an eNOS inhibitor. In summary, we could demonstrate that CC are present in very early stages of atherosclerosis. These early subendothelial CC depositions are produced by autophagolysosomes within the endothelial monolayer in a cAMP/eNOS dependent manner. With these findings we might have found the so far missing link to initiation of plaque development in atherosclerosis.

MicroRNAs (miRNA, miR) are noncoding RNAs that regulate gene expression by hindering translation. miRNA expression profiles have been shown to differ in vivo and in vitro in many cellular processes associated with cardiovascular diseases (CVDs). The progression of CVDs has also been shown to alter the blood miRNA profile in humans. We summarize the results of animal and cell experiments concerning the miRNA profile in the atherosclerotic process and the changes which occur in the blood miRNA profile of individuals with CVD. We also survey the relationship of these CVD-related miRNAs and their expression in the human advanced atherosclerotic plaque, thereby providing more insight into miRNA function in human atherosclerotic lesions. The miRNAs miR-126, -134, -145, -146a, -198, -210, -340*, and -92a were found to be expressed differently in the blood of individuals affected and unaffected by CVD. These differences paralleled those seen in tissue comparisons of miRNA expression in advanced atherosclerotic plaques and healthy arteries. Furthermore, several miRNAs associated with atherosclerosis in in vitro studies (such as miR-10a, -126, -145, -146a/b, -185, -210, and -326) were expressed in plaques in a similar pattern as was predicted by the in vitro experiments. The clinical implications of miRNAs in atherosclerosis as biomarkers and as possible drug targets are also reviewed. miRNA profiles in in vitro and in vivo studies as well as in human peripheral blood are quite representative of the miRNA expression in human atherosclerotic plaques. miRNAs appear promising in terms of future clinical applications.

Funding Acknowledgements Type of funding sources: Other. Main funding source(s): Health Holland Visual Sonics Background Checkpoint inhibitor therapy (ICI), a potent oncologic treatment, has been associated with an enhanced risk for atherosclerotic cardiovascular events and increased plaque progression. Among the key players affected by ICI are tissue resident memory T cells (Trm), a subset residing in tissues and characterized by CD44, CD69, PD-1, and CD103. Although Trms have been associated with chronic inflammatory conditions, their role in atherosclerosis and accelerated atherosclerosis, as observed in postinterventional diseases such as vein graft disease, remains elusive Here, we present an investigation into the role of PD-1 positive cells and Trms in accelerated atherosclerosis as found in vein graft disease. Methods Human carotid atherosclerotic plaques were stained with a MOVAT stain and scored to determine plaque stability. 25 Stable and 25 unstable plaques were stained for CD3+ T cells and PD-1. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery and were sacrificed at t7, t14, t28 (n=6 per time point). The vein grafts were processed for flow cytometry to quantify the Trms population, using markers including CD4+, CD8+, CD44+, CD25-, CD69+, CD103+ and PD-1+. Hypercholesterolemic ApoE3*Leiden mice underwent vein bypass surgery. Biweekly, mice received ICI (anti-PD-L1) or control antibody (n=7). After 28 days vein graft morphology was assed using MOVAT staining and vein graft inflammation by staining MAC3 and CXCL10. Results Within human atherosclerotic plaques, PD-1-expressing T cells were predominantly located in the shoulder regions of the necrotic core. Unstable lesions exhibited significantly higher levels of PD-1 expressing CD3+ T cells compared to stable lesions (p=0.001). Furthermore, the percentage of PD-1 expression positively correlated with immune cell infiltration scores (p=0.019). In murine vein grafts, Trms were identified through flow cytometry. A trend was observed indicating an increase in the number of CD4 and CD8 Trms at t14 compared to t7 Trms (CD8+, CD44+, CD69+/CD103+). At t28, the number of CD4 and CD8 Trms decreased compared to t14, with PD1+ expressing Trms showing a similar trend, peaking at t14. Vein grafts in mice receiving anti-PD-L1 mAb showed no morphological differences compared to the control group. However, a significant difference in inflammation was noted in the anti-PD-L1 treated group, characterized by a higher number of macrophages (p=0.02) and increased expression of CXCL10 (p=0.008). Conclusion Here we demonstrate the association of PD-1+ T cells with unstable lesions and reveals that Trms increase during the inflammatory phase and decrease during the stabilizing phase of accelerated atherosclerosis. Trms emerge as potential detrimental targets of ICI therapy, which is characterized by increased inflammation.

Viewing atherosclerosis through a crystal lens: How the evolving structure of cholesterol crystals in atherosclerotic plaque alters its stability

Atherosclerosis is a chronic inflammatory disease of the arteries that can lead to cardiovascular diseases. Cholesterol crystals are one of the elements found in atherosclerotic plaques, and they play a significant role in the development and progression of the disease. In this study, we investigated the effects of cholesterol crystals on mRNA modification in macrophages, which are involved in plaque formation and inflammation. We used RAW 264.7 cells, a murine macrophage cell line, and exposed them to cholesterol crystals. We found that cholesterol crystals stimulated the production of pro-inflammatory cytokines, including IL-1β, IL-6, and TNFα. Furthermore, cholesterol crystals were taken up by the macrophages and were detected within the cells. Interestingly, cholesterol crystals upregulated demethylating enzymes FTO and ALKBH5, which are involved in RNA modification. However, the expression of methyltransferases, including METTL3, METTL14, NSUN2, and WTAP remained unchanged. These results suggest that cholesterol crystals can modulate mRNA modification by affecting demethylating enzymes and pro-inflammatory cytokines, potentially contributing to the development of atherosclerosis. Understanding the molecular mechanisms underlying cholesterol crystalinduced atherosclerosis may provide new insights into preventive and therapeutic strategies for cardiovascular diseases.

Ursodeoxycholic acid impairs atherogenesis and promotes plaque regression by cholesterol crystal dissolution in mice

Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by platelet-derived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease.

Vascular smooth muscle cells (VSMCs) were induced to mineralise with β-glycerophosphate (β-GP). Controls were cultured without β-GP. FGF-2 mRNA (~40-fold increase, P<0.001) and protein (~2-fold increase, P<0.05) expression are significantly...