Abed Janoudi

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.

Cholesterol crystal induced arterial inflammation and destabilization of atherosclerotic plaque

Evolution of plaque that is prone to rupture is characterized by inflammation and physical changes. Accumulation of low-density lipoprotein in the sub-intima provides esterified cholesterol (ESC) to macrophages and smooth muscle cells that convert it into free cholesterol (FRC) by cholesteryl ester hydrolases (CEHs). Membrane-bound cholesterol carriers transport FRC to high-density lipoprotein (HDL). Impaired HDL transport function and altered composition can lead to extracellular accumulation of FRC, whereas impaired membrane carrier activity can lead to intracellular FRC accumulation. Saturation of FRC can result in cholesterol crystallization with cell death and intimal injury. Disequilibrium between ESC and FRC can impact foam cell and cholesterol crystal (CC) formation. Cholesterol crystals initiate inflammation via NLRP3 inflammasome leading to interleukin-1β (IL-1β) production inducing C-reactive protein. Eventually, crystals growing from within the plaque and associated inflammation destabilize the plaque. Thus, inhibition of inflammation by antagonists to IL-1β or agents that dissolve or prevent CC formation may stabilize vulnerable plaques.

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.

Polycrystalline-Diamond MEMS Biosensors Including Neural Microelectrode-Arrays

Diamond is a material of interest due to its unique combination of properties, including its chemical inertness and biocompatibility. Polycrystalline diamond (poly-C) has been used in experimental biosensors that utilize electrochemical methods and antigen-antibody binding for the detection of biological molecules. Boron-doped poly-C electrodes have been found to be very advantageous for electrochemical applications due to their large potential window, low background current and noise, and low detection limits (as low as 500 fM). The biocompatibility of poly-C is found to be comparable, or superior to, other materials commonly used for implants, such as titanium and 316 stainless steel. We have developed a diamond-based, neural microelectrode-array (MEA), due to the desirability of poly-C as a biosensor. These diamond probes have been used for in vivo electrical recording and in vitro electrochemical detection. Poly-C electrodes have been used for electrical recording of neural activity. In vitro studies indicate that the diamond probe can detect norepinephrine at a 5 nM level. We propose a combination of diamond micro-machining and surface functionalization for manufacturing diamond pathogen-microsensors.

Building Young Engineers: TASEM for Third Graders in Woodcreek Magnet Elementary School

Following the success of summer-camp-based programs, a new program has been developed for in-school sessions focused around LEGO robotics to foster interest in STEM topics at a young age. The program has been implemented in a very diverse school, and preliminary results on the efficacy of the program are presented.

Effects of multiple applications of methyl jasmonate on fruit ripening, leaf gas exchange and vegetative growth in fruit trees

Summary Multiple applications of methyl jasmonate (Me-J), at 10.mM, enhanced the rate of fruit ripening in peach ‘Redhaven’. The effect of Me-J on red colour development and fruit firmness was most pronounced in fruit treated six times with Me-J. Lower concentrations of Me-J had no effect of fruit colour. Me-J, at 10.mM, significantly reduced stomatal conductance, photosynthetic rate and transpiration rate of crabapple leaves, but had no effect on these parameters in peach leaves. The short-term inhibitory effects of Me-J on leaf gas exchange in crabapple persisted for up to 24 h. Fourteen applications of Me-J over a period of 28 days resulted in a 60% decrease in the concentration of chlorophyll in leaves. These Me-J applications also reduced the length of new branches, leaf number and leaf fresh weight by 65%, 31% and 47%, respectively. In peach, Me-J induced some leaf chlorosis and early leaf senescence within two weeks of the treatments which resulted in significant reductions in shoot length.

Plaque Rupture and Thrombosis: the Value of the Atherosclerotic Rabbit Model in Defining the Mechanism

Persistent inflammation and mechanical injury associated with cholesterol crystal accretion within atherosclerotic plaques typically precedes plaque disruption (rupture and/or erosion) and thrombosis—often the terminal events of atherosclerotic cardiovascular disease. To elucidate the mechanisms of these events, the atherosclerotic rabbit model provides a unique and powerful tool that facilitates studies of atherogenesis starting with plaque buildup to eventual disruption. Examination of human coronary arteries obtained from patients who died with myocardial infarction demonstrates evidence of cholesterol crystals perforating the plaque cap and intimal surface of the arterial wall that can lead to rupture. These observations were made possible by omitting ethanol, an avid lipid solvent, from the tissue processing steps. Importantly, the atherosclerotic rabbit model exhibits a similar pathology of cholesterol crystals perforating the intimal surface as seen in ruptured human plaques. Local and systemic inflammatory responses in the model are also similar to those observed in humans. The strong parallel between the rabbit and human pathology validates the atherosclerotic rabbit model as a predictor of human pathophysiology of atherosclerosis. Thus, the atherosclerotic rabbit model can be used with confidence to evaluate diagnostic imaging and efficacy of novel anti-atherosclerotic therapy.

Role of cholesterol crystals in atherosclerosis is unmasked by altering tissue preparation methods.

Standard tissue preparation for light and scanning electron microscopy (SEM) uses ethanol as a dehydrating agent but that can also dissolve cholesterol crystals (CC) leaving behind empty tissue imprints or “clefts”. Cholesterol crystals may contribute to plaque rupture by their sharp tips that can tear membranes and trigger inflammation. Therefore, use of ethanol in tissue processing can mask the pathological role of CC. Here we evaluated the amount of cholesterol dissolved from CC with single and complete series of standard graded ethanol concentrations (25−100%) used in tissue preparation. Also, solubility of CC in ethanol at physiological levels was measured. Furthermore, we compared the effect of ethanol on CC in fresh human atherosclerotic plaques to matched segments dehydrated using vacuum (−1 atm, 12h). Tissue crystal density ranging from 0 to +3 was measured semi‐quantitatively by SEM. For CC exposed to 25% and 100% ethanol for 10 min each, 0.38% and 95% of CC were dissolved respectively. Also, increase in CC solubility was significant at physiological levels of ethanol (0.16%) compared to water (43.4 ± 18.0 ng/mL vs. 30.9 ± 13.9 ng/mL; p < 0.05). We speculate that this could represent a potential mechanism of cardio‐protective effects of alcohol consumption. In atherosclerotic plaques, CC density was lower in ethanol vs. saline treatment (+1.2 vs. +2.8; P < 0.01) with visible dissolving noted by SEM. Ethanol has been used for centuries in tissue preparation for microscopy. Here we demonstrate how current tissue preparation methods greatly alter histological findings with SEM by masking the potential mechanism of plaque rupture. Microsc. Res. Tech. 78:969–974, 2015.

Frequency of Cholesterol Crystals in Culprit Coronary Artery Aspirate During Acute Myocardial Infarction and Their Relation to Inflammation and Myocardial Injury

Cholesterol crystals (CCs) have been associated with plaque rupture through mechanical injury and inflammation. This study evaluated the presence of CCs during acute myocardial infarction (AMI) and associated myocardial injury, inflammation, and arterial blood flow before and after percutaneous coronary intervention. Patients presenting with AMI (n = 286) had aspiration of culprit coronary artery obstruction. Aspirates were evaluated for crystal content, size, composition, and morphology by scanning electron microscopy, crystallography, and infrared spectroscopy. These were correlated with inflammatory biomarkers, cardiac enzymes, % coronary stenosis, and Thrombolysis in Myocardial Infarction (TIMI) blush and flow grades. Crystals were detected in 254 patients (89%) and confirmed to be cholesterol by spectroscopy. Of 286 patients 240 (84%) had CCs compacted into clusters that were large enough to be measured and analyzed. Moderate to extensive CC content was present in 172 cases (60%). Totally occluded arteries had significantly larger CC clusters than partially occluded arteries (p <0.05). Patients with CC cluster area >12,000 µm2 had significantly elevated interleukin-1 beta (IL-1β) levels (p <0.01), were less likely to have TIMI blush grade of 3 (p <0.01), and more likely to have TIMI flow grade of 1 (p <0.01). Patients with recurrent AMI had smaller CC cluster area (p <0.04), lower troponin (p <0.02), and IL-1β levels (p <0.04). Women had smaller CC clusters (p <0.04). Macrophages in the aspirates were found to be attached to CCs. Coronary artery aspirates had extensive deposits of CCs during AMI. In conclusion, presence of large CC clusters was associated with increased inflammation (IL-1β), increased arterial narrowing, and diminished reflow following percutaneous coronary intervention.

CHOLESTEROL LOWERING WITH SIMVASTATIN AND EZETIMIBE INHIBITS CHOLESTEROL CRYSTAL FORMATION AND ASSOCIATED INFLAMMATION

Cholesterol crystals (CC) have been noted to be perforating plaques in patients with acute myocardial infarction. This study evaluated effects of simvastatin (SIMVA) and ezetimibe (EZET), alone and in combination (SIMVA/EZET) on CC formation and associated inflammation. Twenty four rabbits were