An array of analytical methods including density gradient ultracentrifugation, capillary electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), were utilized to analyze serum high density lipoprotein (HDL) subfractions from two cohorts of normolipidemic individuals, which included subjects with diagnosed coronary artery disease (CAD), and angiographically proven non-CAD controls. These methods collectively provided characteristic information about the two populations of individuals including composition, electrophoretic mobilities, molecular weights, isoforms, and post-translational modifications of HDL apolipoproteins. This information proved useful in identifying potential biomarkers for CAD risk, and understanding the biological functions of a novel atherogenic HDL phenotype in individuals with CAD. Through the implementation of the aforementioned methodologies, new isoforms of apoC-I were identified. MALDI-MS, detected a shifting of approximately 90 Da in the mass to charge ratios corresponding to apoC-I peaks in the serum subfractions from all CAD cohort patients. This shifting was not observed in the non-CAD cohort, which displayed apoC-I peaks in accordance with the known mass of this protein. In addition to the shifting observed in the CAD cohort, some CAD patients showed further modifications of apoC-I that were indicative of oxidative processes. Interestingly, one patient, who has not been diagnosed with CAD, and has a family history of the disease, contained the apoC-I isoforms. This feature could underlie this subject’s known family history of CAD, and serve as an initial screening that could indicate the future development of CAD in this individual. Through collaborative work with Johns Hopkins University, it was initially observed that apoC-I enriched HDL induced apoptosis of aortic smooth muscle cells. Conversely, apoC-I depleted HDL induced minimal to no apoptosis, which led to the hypothesis that apoC-I is a contributor to atherogenic HDL and is a potential risk factor for CAD. Further collaborative work with Johns Hopkins assessed the apoptosis levels induced by HDL from both cohorts of patients. A distinct difference in apoptosis was identified between the two cohorts. High density lipoprotein subfractions from subjects in the CAD cohort, all of which contained the apoC-I isoforms, induced marked apoptosis compared to the non-CAD controls. These results further supported the hypothesis that apoC-I compromises the functionality of HDL and showed that through the induction of apoptosis, apoC-I can contribute to the destabilization of atherosclerotic plaque and the acceleration of CAD.

Background: Kawasaki disease (KD) affects mainly children younger than five years of age, leading to coronary artery lesions, and even to life-threatening myocardial infarctions. Since 1976, Kawasaki disease has occurred among thousands of children in Taiwan. Evidence suggests that inflammation plays a key role in the pathogenesis of atherosclerosis. Significant determinants of high sensitivity C-reactive protein (hs-CRP), which is a sensitive indicator of inflammation, as well as white blood cell (WBC) count, and high-density lipoprotein cholesterol (HDLc) and coronary artery lesion were identified. The relationships between these factors¡¦ concentration and arterial lesion were likewise investigated and had reported. The aim of this study was to determine the serum levels of the hs-CRP, WBC count, and plasma HDLc levels in patients with later phase of KD. Methods and Materials: From July 2005 to June 2006, 97 children with Kawasaki disease at least 1 year after diagnosis were recruited in this study. These participated children had been diagnosed as KD and collected at the interval of 2001 to 2004. Diagnosis was based on the 1984 revised by the KD Research Committee in Japan. The participants were grouped into 45 patients with KD and coronary aneurysms (Group I), 52 patients with KD and normal coronary arteries (Group II), and 50 healthy age-matched children (Control Group III). Their WBC count, systemic and diastolic blood pressures, body mass index, age, sex, fasting total cholesterol concentrations, triglyceride, high-density lipoprotein cholesterol (HDLc), low-density lipoprotein cholesterol, serum hs-CRP levels, and coronary artery lesion by cardioechography were recorded and compared. The analytical differences between hs-CRP, WBC count, and plasma HDLc levels and the coronary artery events in KD were examined. Results: Serum hs-CRP levels of Group I patients (mean 0.264 mg/dl) was significantly greater than that of Group II (mean 0.155 mg/dl, p=0.006) and Group III patients (mean 0.116 mg/dl, p =0.017). Similarly, the WBC count of Group I patients (mean 6,543.11/mm3) was significantly greater than that of Group II (mean 5,720.19/mm3, p=0.029), and Group III patients (mean 5,611.27/mm3, p =0.012). However, plasma HDLc levels of Group I patients (mean 41.42 mg/dl) was significantly lesser than that of Group II (mean 44.79 mg/dl, p=0.035), and Control Group III patients (mean 46.58 mg/dl, p=0.027). There was a positive association between hs-CRP and WBC count levels (r = 0.641, p < 0.05), but none between hs-CRP and plasma HDLc levels. Conclusions: There is the possibility of ongoing low-grade inflammation late after the convalescent phase of Kawasaki disease in children with coronary aneurysms, which may have a role in increasing coronary artery dysfunction. These results also suggest that hs-CRP, WBC count, and plasma HDLc levels are useful parameters for predicting formation of coronary artery lesion even in children after onset of KD.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成3年3月8日 葛谷雅文氏の博士論文として提出された

A method for the oxidation of lipoproteins in vitro was developed using the free radical initiator, 2,2?-azobis-(2-amidinopropane) dihydrochloride (AAPH). Following in vitro oxidation, the susceptibility to oxidation of the serum samples was studied using density gradient ultracentifugation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Shifts in mean buoyant density of the lipoprotein particles, specifically low density lipoprotein (LDL) and high density lipoprotein (HDL), were observed in the density profile following centrifugation. The degree of shift in the density proved to be proportional to the extent of oxidation. Changes in apolipoproteins were studied with MALDI-TOF-MS. Observed variations in the mass spectra include m/z shifts due to chemical modifications and change in isoform distributions. The oxidation procedure and analysis techniques were applied to a clinical application to study the effects of table grape consumption on lipoprotein susceptibility to oxidation. The main objective of the research, to show feasibility that these methods could be used in a clinical setting, was achieved.

The vesicle-mediated degradation of low-density lipoprotein (LDL) is an essential cellular function due to its role in cellular biosynthesis of membranes and steroids. Using multi-color single particle tracking fluorescence microscopy, the intracellular degradation of LDL was probed in live, intact cells. Unique to these experiments is the direct observation of LDL degradation using an LDL-based probe that increases fluorescence intensity upon degradation. Specifically, individual LDL particles were labeled with multiple fluorophores resulting in a quenched fluorescent signal. The characteristics of the vesicle responsible for degradation were determined and the vesicle dynamics involved in LDL degradation were quantified. Visualization of early endosomes, late endosomes and lysosomes was accomplished by fluorescently labeling vesicles with variants of GFP. Transient colocalization of LDL with specific vesicles and the intensity of the LDL particle were measured simultaneously. These studies, which are the first to directly observe the degradation of LDL within a cell, strive to completely describe the endo-lysosomal pathway and quantify the dynamics of LDL degradation in cells.

Traumatic brain injury (TBI) has become an increasingly unmet clinical need due to intense military conflicts worldwide. Directly impacted brain cells suffer massive death, with neighboring cells succumbing to progressive neurodegeneration accompanied by inflammatory and other secondary cell death events. Subsequent neurodegenerative events may extend to normal areas beyond the core of injury, thereby exacerbating the central nervous system’s inflammatory response to TBI. Recently CD-36 (cluster of differentiation 36/fatty acid translocase (FAT), a class B scavenger receptor of modified low-density lipoproteins (mLDLs) in macrophages, has been implicated in lipid metabolism, atherosclerosis, oxidative stress, and tissue injury in cerebral ischemia, and in certain neurodegenerative diseases. Accordingly, we proposed that CD-36 has a pivotal role in the neuroinflammatory cascade that further contributes to the pathology of TBI. First, we explored the neuroinflammatory role of CD-36 after acute and chronic stages of TBI. Second, we employed a neuroinflammatory model to test the therapeutic effect of the soluble receptor of advanced end-glycation product (sRAGE); previously shown to abrogate increased CD-36 expression in stroke. Third, we further examined ameliorating TBI related inflammation as a therapeutic pathway by combination of stem cell therapy and sRAGE. At acute stages of TBI, we observed brain co-localization of CD-36, monocyte chemoattractant protein 1 (MCP-1) and ionized calcium-binding adapter molecule 1 (Iba-1) on impacted cortical areas, significant increases of CD-36 and MCP-1 positive cells in the ipsilateral vs. contralateral hemispheres of TBI animals in acute, but no significant increases of Iba-1 expressing cells over time. In early acute stages of TBI immunoblotting showed overexpression of CD-36 in brain cortex when comparing ipsilateral and contralateral hemispheres vs. sham. Spleen CD-36 protein expression at acute post-TBI stages showed no significant difference between TBI and sham groups. In addition, immunohistochemistry revealed minimal CD-36 detection on the cortical area of impact on our chronic group. Spleen immunohistochemistry also showed co-localization of CD-36 and MCP-1 in the red pulp of spleen in acute stages of TBI animals when compared to sham. Ongoing ischemic and hyperlipidemic rodent models suggest that infiltrating monocytes/macrophages from the periphery are the major source of CD-36 in the post-ischemic brain. Likewise, CD-36 expressing monocytes in the spleen after TBI may suggest its role in peripheral immune response, which may exacerbates the inflammatory response after TBI. Therefore, CD-36 may play a key role as a pathological link between inflammation and TBI. Our results suggest an intimate involvement of CD-36 mediated inflammation in TBI, providing novel insights into the understanding of disease neuroinflammation and as a potent therapeutic target for TBI treatment. The critical timing (i.e., 24-48 hours) of CD-36 expression (from downregulation to upregulation) may signal the transition of functional effects of this immune response from pro-survival to cell death. This observed dynamic CD-36 expression also suggests the therapeutic window for TBI. The detection of CD-36 expression in brain areas proximal, as well as distal, to the site of impacted injury suggests its role in both acute and progressive evolution of TBI. CD-36 neuroinflammatory role has clinical relevance for treating patients who have suffered any TBI condition at acute and chronic stages.

No description available.

Apolipoprotein B (apoB) is the main protein of very-low-density lipoprotein (VLDL). As apoB is translated and moves through the secretory pathway, lipids from cytoplasmic lipid droplets (LDs) are added to form VLDL particles. Without adequate lipid availability, apoB is misfolded and undergoes proteasomal degradation; however, evidence now shows that apoB can be degraded through autophagy. Inhibiting autophagy decreased apoB localization to autophagosomes in HepG2 cells, but also decreased apoB recovered from cells and media. Inducing autophagy increased apoB localization to autophagosomes and decreased apoB recovery. LDs are also degraded through autophagy however LDs were not affected by autophagy modulation in HepG2 cells. In primary hamster hepatocytes, inhibiting autophagy reduced apoB-autophagosome co-localization and increased LD numbers. These data suggest that autophagy may play a complex role in VLDL assembly by regulating degradation of both apoB and LDs. This dual role is more evident in primary hepatocytes indicating a potential physiological role.

In recent years, studies have revealed a central role for the intestine in regulation of lipid homeostasis and development of insulin resistance and type-2 diabetes. The function of intestinal Scavenger Receptor Class-B type-I remains unknown, however it is believed to play a role in dietary lipid uptake. Recently, our laboratory demonstrated a correlation between intestinal SR-BI expression and chylomicron secretion. We hypothesized that intestinal SR-BI is involved in chylomicron secretion and contributes to chylomicron oversecretion in insulin resistance. I first characterized chylomicron production in healthy and insulin resistant Syrian golden hamsters. Inhibition of SR-BI resulted in reduced postprandial chylomicron accumulation in plasma, and resistance to diet-induced hyperlipidemia and weight-gain. Lower postprandial triglyceride levels were also observed in SR-BI-/- mice. In summary, these data demonstrate a key role for intestinal SR-BI in chylomicron secretion and control of lipid homeostasis, implicating intestinal SR-BI in chylomicron overproduction in insulin resistant states.

The primary objective of this research was to explore the impact of glucose ingestion on 2-back task performance (accuracy, discrimination, and reaction times (RT) to target), its relationship to neural activation, using functional magnetic resonance imaging, and potential modulation by insulin resistance (IR) and low density lipoprotein (LDL) in hypertensive but otherwise healthy older adults. While there was no effect of glucose ingestion on task performance or task-relevant neural activation patterns, this study uniquely observed that IR and LDL associated with all 3 measures of 2-back performance and task-relevant neural activation patterns. The left and right precuneus, left cingulate, and left insula were identified as task-associated regions according to our specific target minus nontarget contrast. Of particular importance was the task activation in the right precuneus as it both showed sensitivity to IR and predicted task RTs to targets, suggesting it plays a modulatory role linking IR to task performance.