Ameeth Vedre

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

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 degrees 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 degrees 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.

Effect of statins on cholesterol crystallization and atherosclerotic plaque stabilization.

Pleiotropic effects of statins have not been fully elucidated. Recently we demonstrated that cholesterol expands when crystallizing and may trigger plaque rupture. The present study evaluated the potential direct effects of statins in altering cholesterol crystallization as a possible mechanism for plaque stabilization independent of cholesterol lowering. Cholesterol powder was dissolved in oil with and without pravastatin, simvastatin, or atorvastatin (10 to 90 mg) and then allowed to crystallize to measure peak volume expansion (ΔVE) in graduated cylinders. Effect of ΔVE on fibrous membrane damage was also evaluated. Human coronary, carotid, and peripheral arterial plaques (65 plaques from 55 patients) were incubated with statin or saline solution using matched plaque segments to evaluate direct effects of statins on preformed crystals. Also, the effect of in vivo use of oral statins on crystal structure was examined by scanning electron microscopy and crystal content in plaques scored from 0 to +3. For all statins, ΔVE decreased significantly in a dose-dependent fashion (0.76 ± 0.1 vs 0 ml at 60 mg, p <0.001). By scanning electron microscopy crystal structure with statins had loss of pointed tip geometries, averting fibrous membrane damage. Cholesterol crystal density was markedly decreased and appeared dissolved in human plaques incubated with statins (+2.1 ± 1.1 vs +1.3 ± 1.0, p = 0.0001). Also, plaques from patients taking oral statins compared to controls had significantly more dissolving crystals (p = 0.03). In conclusion, statins decreased ΔVE by altering cholesterol crystallization and blunting sharp-tipped crystal structure and dissolving cholesterol crystals in human arteries in vivo and in vitro, providing plaque stabilization.

Physical factors that trigger cholesterol crystallization leading to plaque rupture

BACKGROUND Triggers of plaque rupture have been elusive. Recently it was demonstrated that cholesterol expands when transforming from a liquid to a crystal state, disrupting overlying plaque. This study examined the effect of physical conditions including saturation, temperature, hydration, pH on cholesterol crystallization. METHODS Graduated cylinders were filled with varying amount of cholesterol powder (1, 2 and 3g) and dissolved in corn oil at 37 degrees C. Change in volume expansion (DeltaVE) and time to crystallization were measured for each saturation. The same was repeated while varying temperature (22-44 degrees C); hydration (1-3ml H(2)O); pH (5-8.4) and combination of saturation and temperature. Scanning electron microscopy was performed to evaluate crystal morphology and X-ray diffractometry to assess molecular structure of cholesterol. RESULTS Increasing saturation raised both DeltaVE (3g: 0.53+/-0.1ml vs. 1g: 0.14+/-0.02ml and 2g: 0.3+/-0.1ml; p<0.0001; p<0.01) and rate of change over 3min (3g: 60% vs. 1g: 14%). Crystal morphology was the same seen with crystals perforating human plaques. Temperature drop increased DeltaVE (44 degrees C: 0.05+/-0.01ml vs. 22 degrees C: 0.5+/-0.07ml; p<0.0001) and initiated earlier crystallization. Hydration resulted in greater DeltaVE (3ml: 0.7+/-0.07 vs. 0ml: 0.1+/-0.05; p<0.001) with corresponding changes in cholesterol molecular structure. Rising pH was associated with increased DeltaVE (1.3+/-0.03ml vs. 0.1+/-0.02ml; p<0.001). Combined increase in saturation and temperature had greater DeltaVE than expected from the sum of each alone. CONCLUSIONS Physical factors influenced both volume and rate of cholesterol crystallization. This suggests that local factors may play an important role in triggering plaque rupture. Combination of several factors may even be a more powerful trigger for acute cardiovascular events.

Plaque Rupture and Thrombosis Are Reduced by Lowering Cholesterol Levels and Crystallization With Ezetimibe and Are Correlated With Fluorodeoxyglucose Positron Emission Tomography

Objective— This study evaluated effects of lipid lowering with ezetimibe on plaque burden and associated cholesterol crystallization and inflammation in a rabbit model of plaque disruption and thrombosis. Methods and Results— Atherosclerotic rabbits (Group I, n=10 without; Group II, n=12 with ezetimibe, 1 mg/kg per day) were pharmacologically triggered for plaque disruption. Fluorodeoxyglucose positron emission tomography, RAM 11 macrophage staining, and serum inflammatory markers detected arterial inflammation. Serum and aortic wall cholesterol levels were measured, and thrombus area was planimetered. Cholesterol crystal density on aortic surface was scored (0 to +3) by scanning electron microscopy. Serum and aortic wall cholesterol, plaque area, and thrombosis area were significantly lower in Group II versus Group I (83.4±106.4 versus 608±386 mg/dL, P=0.002; 3.12±1.40 versus 9.39±5.60 mg/g, P=0.003; 10.84±1.6 versus 17.48±1.8 mm2, P<0.001; and 0.05±0.15 versus 0.72±0.58 mm2, P=0.01, respectively). There were significant correlations between crystal density and plaque area (r=0.75, P<0.003) and between crystal density and RAM 11 (r=0.82, P<0.001). Scanning electron microscopy demonstrated that there were fewer crystals in Group II versus Group I (+1.2±0.61 versus +2.4±0.63, P<0.001) and less inflammation detected by fluorodeoxyglucose positron emission tomography and RAM 11 (P<0.004 and P<0.04, respectively). Conclusion— Lowering cholesterol levels with ezetimibe reduced plaque burden, crystallization, and inflammation, preventing plaque disruption and thrombosis.

Prophylaxis to Prevent Infective Endocarditis: To Use or Not to Use?

The American Heart Association (AHA) published their revised guidelines in 2007 in which they markedly limited the recommendations for the use of antimicrobial prophylaxis for the prevention of infective endocarditis (IE), except for patients who are at highest risk of adverse outcomes. A recent focused update on valvular heart diseases changed the recommendation for antibiotic use for patients with many underlying heart conditions including mitral valve prolapse (MVP) which were considered as “low risk” heart defects. In this article, we argue that antibiotic prophylaxis should be considered until concrete clinical evidence is provided to dispute against the use of this strategy, especially for patients with MVP. This approach is cost efficient, and provides a chance to prevent a dreadful disease. We have also enlisted 2 clinical cases to support our argument. These are not uncommon clinical scenarios, and emphasize that IE can be fatal in spite of optimum treatment. Patients have the right to make the final decision, and they should be allowed to participate in choosing for or against this approach until adequate clinical evidence is available. Copyright

The effect of ethanol on cholesterol crystals during tissue preparation for scanning electron microscopy.

Dr. Abela and colleagues correctly point out that the use of ethanol, a dehydrating agent, in the preparation of the coronary thrombi samples for scanning electron microscopy may have dissolved cholesterol crystals and led to an underestimation of the true content of cholesterol crystals in the coronary thrombi retrieved by thromboaspiration. The study by Abela et al. (1) demonstrated that the use of vacuum dehydration of tissues is likely the preferred technique to study the impact of cholesterol crystals on biological membranes in vitro. In our study (2), we focused on the 2 main pharmacological therapeutic targets in acute coronary syndromes that can be visualized by microscopy, platelets, and fibrin fibers. Therefore, we preferred to use the ethanol dehydration technique instead of vacuum dehydration, to avoid the shrinkage and distortion of fibrin fibers that can occur with the latter technique. It was important to understand how the coronary thrombi evolve in time to understand the usefulness of treatments, such as fast-acting P2Y12 inhibitors or glycoprotein IIb/IIIa inhibitors, and their optimal use in the time course of the event. However, the hypothesis of cholesterol crystals perforating the intima and being the underlying mechanism of plaque rupture is seductive and deserves more in vivo studies. Unfortunately, no currently available technology can detect cholesterols crystals in vivo in patients experiencing acute coronary syndromes, and even though angioscopy has been used to define thrombus colors, the lack of magnification and 3-dimensional analysis as provided by scanning electron microscopy does not allow the identification of such small structures (3). Finally, it must be considered that the thromboaspiration technique does not allow a transversal analysis from 1 side of the artery to the other, as done in histological analysis of postmortem coronary arteries, but is most likely to retrieve a piece of the thrombus in the inner lumen, distant from the site of plaque rupture and therefore with fewer cholesterol crystals. This difference, added to the possibility of cutting-induced translocation of cholesterol crystals from the plaque itself to the inner lumen during tissue preparation, could be an explanation for the discrepancies in the amount of cholesterol crystals found in our study.