Joe L. Raya

Isolation, Characterization, and Functional Assessment of Oxidatively Modified Subfractions of Circulating Low-Density Lipoproteins

Objective—Current evidence suggests that oxidatively modified human plasma low-density lipoproteins (ox-LDLs) are proatherogenic and cytotoxic to endothelial and vascular smooth muscle cells. The present study describes a method using ion-exchange chromatography that is capable of large-scale subfractionation of LDL for adequate analyses of composition or bioactivities. Methods and Results—LDLs from normolipidemic (N-LDL) and homozygous familial hypercholesterolemic (FH-LDL) subjects were separated into 5 subfractions (L1 through L5) by high-capacity ion-exchange chromatography. The most strongly retained fraction from FH subjects, FH-L5, suppressed DNA synthesis in cultured bovine aortic endothelial cells and stimulated mononuclear cell adhesion to cultured endothelial cells under flow conditions in vitro. L5, which represented 1.1±0.2% and 3.7±1.7% of the LDL from N-LDL and FH-LDL, respectively, was more triglyceride-rich (17% versus 5%) and cholesteryl ester-poor (23% versus 33%) than were L1 through L4. Electrophoretic mobilities on agarose gels increased from L1 to L5. According to SDS-PAGE, apolipoprotein B-100 in N-LDL fractions L1 through L5 appeared as a single ≈500-kDa band. In contrast, the fractions isolated from FH-LDL showed substantial fragmentation of the apolipoprotein B-100, including bands between 200 and 116 kDa. Competitive ELISA analyses using a malondialdehyde-specific monoclonal antibody against Cu2+ ox-LDL suggest that FH-L5 is malondialdehyde-modified. Conclusions—Relative to N-LDL, FH-LDL contains higher concentrations of a fraction, L5, that exhibits distinctive physicochemical properties and biological activities that may contribute to initiation and progression of atherogenesis in vivo.

Dynamics of dense electronegative low density lipoproteins and their preferential association with lipoprotein phospholipase A2

Small, dense, electronegative low density lipoprotein [LDL(−)] is increased in patients with familial hypercholesterolemia and diabetes, populations at increased risk for coronary artery disease. It is present to a lesser extent in normolipidemic subjects. The mechanistic link between small, dense LDL(−) and atherogenesis is not known. To begin to address this, we studied the composition and dynamics of small, dense LDL(−) from normolipidemic subjects. NEFA levels, which correlate with triglyceride content, are quantitatively linked to LDL electronegativity. Oxidized LDL is not specific to small, dense LDL(−) or lipoprotein [a] (i.e., abnormal lipoprotein). Apolipoprotein C-III is excluded from the most abundant LDL (i.e., that of intermediate density: 1.034 < d < 1.050 g/ml) but associated with both small and large LDL(−). In contrast, lipoprotein-associated phospholipase A2 (LpPLA2) is highly enriched only in small, dense LDL(−). The association of LpPLA2 with LDL may occur through amphipathic helical domains that are displaced from the LDL surface by contraction of the neutral lipid core.

Increased Myocardial Susceptibility to Repetitive Ischemia With High-fat diet-induced Obesity

Obesity and diabetes are frequently associated with cardiovascular disease. When a normal heart is subjected to brief/sublethal repetitive ischemia and reperfusion (I/R), adaptive responses are activated to preserve cardiac structure and function. These responses include but are not limited to alterations in cardiac metabolism, reduced calcium responsiveness, and induction of antioxidant enzymes. In a model of ischemic cardiomyopathy inducible by brief repetitive I/R, we hypothesized that dysregulation of these adaptive responses in diet‐induced obese (DIO) mice would contribute to enhanced myocardial injury. DIO C57BL/6J mice were subjected to 15 min of daily repetitive I/R while under short‐acting anesthesia, a protocol that results in the development of fibrotic cardiomyopathy. Cardiac lipids and candidate gene expression were analyzed at 3 days, and histology at 5 days of repetitive I/R. Total free fatty acids (FFAs) in the cardiac extracts of DIO mice were significantly elevated, reflecting primarily the dietary fatty acid (FA) composition. Compared with lean controls, cardiac FA oxidation (FAO) capacity of DIO mice was significantly higher, concurrent with increased expression of FA metabolism gene transcripts. Following 15 min of daily repetitive I/R for 3 or 5 days, DIO mice exhibited increased susceptibility to I/R and, in contrast to lean mice, developed microinfarction, which was associated with an exaggerated inflammatory response. Repetitive I/R in DIO mice was associated with more profound significant downregulation of FA metabolism gene transcripts and elevated FFAs and triglycerides. Maladaptive metabolic changes of FA metabolism contribute to enhanced myocardial injury in diet‐induced obesity.

Differential effect of weight loss with low-fat diet or high-fat diet restriction on inflammation in the liver and adipose tissue of mice with diet-induced obesity

OBJECTIVE We studied the effects of weight loss induced by either a low-fat normal diet (ND) or restriction of high-fat diet (HFD) on hepatic steatosis, inflammation in the liver and adipose tissue (AT), and blood monocytes of obese mice. METHODS In mice with HFD-induced obesity, weight loss was achieved by switching from HFD to ND and maintaining on ND ad libitum or by restricting HFD intake to match body weight of mice with ND-induced weight loss. After diet interventions for 4 weeks, hepatic steatosis, hepatic and AT inflammation, and blood CD11c(+) monocytes were examined. RESULTS At 4 weeks after switching diets, body weight was reduced by 23% from baseline. To achieve the same reduced body weight required restricting calorie intake from HFD. Weight loss with either ND or HFD restriction decreased body fat mass and ameliorated liver steatosis; both effects were greater with ND-induced weight loss than HFD restriction-induced weight loss. Weight loss with ND but not HFD restriction normalized blood CD11c(+) monocytes and attenuated hepatic inflammation assessed by chemokine and CD11c expression. In contrast, weight loss with HFD restriction significantly reduced chemokine levels and CD11c(+) cells in AT compared to obese controls, and tended to reduce AT chemokines and CD11c(+) cells more than ND-induced weight loss. CONCLUSION In mice with diet-induced obesity, weight loss with ND was superior in alleviating hepatic inflammation and steatosis, whereas weight loss with HFD calorie restriction provided greater amelioration of AT inflammation.

Impaired Lipoprotein Processing in HIV Patients on Antiretroviral Therapy: Aberrant High-Density Lipoprotein Lipids, Stability, and Function

Objective—HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results—Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). Conclusions—Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.Objective— HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results— Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain ( P <0.001). Conclusions— Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness. # Significance {#article-title-40}

The Influence of an Obesogenic Diet on Oxysterol Metabolism in C57BL/6J Mice

Our current understanding of oxysterol metabolism during different disease states such as obesity and dyslipidemia is limited. Therefore, the aim of this study was to determine the effect of diet-induced obesity on the tissue distribution of various oxysterols and the mRNA expression of key enzymes involved in oxysterol metabolism. To induce obesity, male C57BL/6J mice were fed a high fat-cholesterol diet for 24 weeks. Following diet-induced obesity, plasma levels of 4β-hydroxycholesterol, 5,6α-epoxycholesterol, 5,6β-epoxycholesterol, 7α-hydroxycholesterol, 7β-hydroxycholesterol, and 27-hydroxycholesterol were significantly (P < 0.05) increased. In the liver and adipose tissue of the obese mice, 4β-hydroxycholesterol was significantly (P < 0.05) increased, whereas 27-hydroxycholesterol was increased only in the adipose tissue. No significant changes in either hepatic or adipose tissue mRNA expression were observed for oxysterol synthesizing enzymes 4β-hydroxylase, 27-hydroxylase, or 7α-hydroxylase. Hepatic mRNA expression of SULT2B1b, a key enzyme involved in oxysterol detoxification, was significantly (P < 0.05) elevated in the obese mice. Interestingly, the appearance of the large HDL1 lipoprotein was observed with increased oxysterol synthesis during obesity. In diet-induced obese mice, dietary intake and endogenous enzymatic synthesis of oxysterols could not account for the increased oxysterol levels, suggesting that nonenzymatic cholesterol oxidation pathways may be responsible for the changes in oxysterol metabolism.

Streptococcal serum opacity factor promotes cholesterol ester metabolism and bile acid secretion in vitro and in vivo

Plasma high density lipoprotein-cholesterol (HDL-C) concentrations negatively correlate with atherosclerotic cardiovascular disease. HDL is thought to have several atheroprotective functions, which are likely distinct from the epidemiological inverse relationship between HDL-C levels and risk. Specifically, strategies that reduce HDL-C while promoting reverse cholesterol transport (RCT) may have therapeutic value. The major product of the serum opacity factor (SOF) reaction versus HDL is a cholesteryl ester (CE)-rich microemulsion (CERM), which contains apo E and the CE of ~400,000 HDL particles. Huh7 hepatocytes take up CE faster when delivered as CERM than as HDL, in part via the LDL-receptor (LDLR). Here we compared the final RCT step, hepatic uptake and subsequent intracellular processing to cholesterol and bile salts for radiolabeled HDL-, CERM- and LDL-CE by Huh7 cells and in vivo in C57BL/6J mice. In Huh7 cells, uptake from LDL was greater than from CERM (2-4X) and HDL (5-10X). Halftimes for [(14)C]CE hydrolysis were 3.0±0.2, 4.4±0.6 and 5.4±0.7h respectively for HDL, CERM and LDL-CE. The fraction of sterols secreted as bile acids was ~50% by 8h for all three particles. HDL, CERM and LDL-CE metabolism in mice showed efficient plasma clearance of CERM-CE, liver uptake and metabolism, and secretion as bile acids into the gall bladder. This work supports the therapeutic potential of the SOF reaction, which diverts HDL-CE to the LDLR, thereby increasing hepatic CE uptake, and sterol disposal as bile acids.

Impaired Lipoprotein Processing in HIV Patients on Antiretroviral Therapy

Objective—HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results—Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). Conclusions—Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.Objective— HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results— Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain ( P <0.001). Conclusions— Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness. # Significance {#article-title-40}

Impaired Lipoprotein Processing in HIV Patients on Antiretroviral TherapySignificance

Objective—HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results—Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). Conclusions—Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.Objective— HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results— Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain ( P <0.001). Conclusions— Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness. # Significance {#article-title-40}

Impaired Lipoprotein Processing in HIV Patients on Antiretroviral Therapy: Aberrant HDL Lipids, Stability, and Function

Objective—HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results—Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain (P<0.001). Conclusions—Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness.Objective— HIV patients on antiretroviral therapy (HIV/ART) exhibit a unique atherogenic dyslipidemic profile with hypertriglyceridemia (HTG) and low plasma concentrations of high-density lipoprotein (HDL) cholesterol. In the Heart Positive Study of HIV/ART patients, a hypolipidemic therapy of fenofibrate, niacin, diet, and exercise reduced HTG and plasma non–HDL cholesterol concentrations and raised plasma HDL cholesterol and adiponectin concentrations. We tested the hypothesis that HIV/ART HDL have abnormal structures and properties and are dysfunctional. Approach and Results— Hypolipidemic therapy reduced the TG contents of low-density lipoprotein and HDL. At baseline, HIV/ART low-density lipoproteins were more triglyceride (TG)-rich and HDL were more TG- and cholesteryl ester-rich than the corresponding lipoproteins from normolipidemic (NL) subjects. Very-low-density lipoproteins, low-density lipoprotein, and HDL were larger than the corresponding lipoproteins from NL subjects; HIV/ART HDL were less stable than NL HDL. HDL-[3H]cholesteryl ester uptake by Huh7 hepatocytes was used to assess HDL functionality. HIV/ART plasma were found to contain significantly less competitive inhibition activity for hepatocyte HDL-cholesteryl ester uptake than NL plasma were found to contain ( P <0.001). Conclusions— Compared with NL subjects, lipoproteins from HIV/ART patients are larger and more neutral lipid-rich, and their HDL are less stable and less receptor-competent. On the basis of this work and previous studies of lipase activity in HIV, we present a model in which plasma lipolytic activities or hepatic cholesteryl ester uptake are impaired in HIV/ART patients. These findings provide a rationale to determine whether the distinctive lipoprotein structure, properties, and function of HIV/ART HDL predict atherosclerosis as assessed by carotid artery intimal medial thickness. # Significance {#article-title-40}