Jim L. Kelley

Pathogenesis of the Atherosclerotic Lesion: Implications for diabetes mellitus

In this review, we have highlighted pivotal cellular and molecular events in the initiation and progression of atherosclerosis. Key components of lesion initiation are an enhanced focal intimal influx and accumulation of lipoproteins, including LDL in hemodynamically determined lesion-prone areas, focal monocyte-macrophage recruitment, intimal generation of ROS, and oxidative modification of lipoproteins (including LDL [Ox-LDL]). Modified lipoproteins are taken up by the non-downregulating macrophage scavenger receptor, with foam cell formation and the development of the so-called fatty streak. One transitional event in lesion progression is foam cell necrosis, likely attributable to the cytotoxicity of both intimal free radicals and Ox-LDL, with development of an extracellular metabolically inert lipid core. Another is the migration to and proliferation within the intima of medial SMCs, leading to the synthesis of plaque collagens, elastin, and proteoglycans. Mural thrombosis plays a significant role in the late-stage progression of lesions. Regression of lesions is considered a function of the dynamic balance among components of initiation, progression, plaque stabilization, and removal of plaque constituents—the so-called regression quartet. Here, we critically examine how components of diabetes mellitus might impact not only lesion development, but also lesion regression. It is concluded that some components of diabetes mellitus augment key mechanisms in lesion initiation and progression and will likely retard the processes of plaque regression. Specifically, we focus on the various influences of diabetes mellitus on lipoprotein influx and accumulation, free radical generation and Ox-LDL, monocyte-macrophage recruitment, thrombosis and impaired fibrinolysis, and the reverse cholesterol transport system. The importance of nonenzymatic protein glycosylation in modifying a number of these processes is emphasized.

Influence of infant and juvenile diets on serum cholesterol, lipoprotein cholesterol, and apolipoprotein concentrations in juvenile baboons (Papio sp.)☆

The long-term effects of infant diet (breast milk or formula containing 2, 30, or 60 mg/dl cholesterol) and subsequent dietary cholesterol (1 mg/kcal) and fat (saturated or unsaturated) on serum lipid and apolipoprotein concentrations were estimated using 82 juvenile baboons 4-6 years of age. A significant interaction of infant diet (breast vs formula) with type of fat (saturated vs unsaturated) at 4-6 years of age was observed on HDL cholesterol and apolipoprotein A-I (apoA-I) concentrations. That is, animals breast-fed as infants had higher HDL cholesterol and apoA-I concentrations when fed unsaturated fat from weaning to 4-6 years of age than those fed saturated fat (77 vs 68 mg/dl). In contrast, animals fed formulas in infancy followed by a diet containing unsaturated fat had lower HDL cholesterol and apoA-I concentrations at 4-6 years of age than did those fed saturated fat (67 vs 78 mg/dl). However, breast feeding or feeding formulas containing various levels of cholesterol for 3 months during infancy did not result in statistically significant differences in total serum cholesterol, VLDL + LDL cholesterol and apolipoprotein B (apoB) concentrations. Dietary cholesterol after infancy significantly increased serum total cholesterol, VLDL + LDL and HDL cholesterol, apoA-I and apoB concentrations. All of these response variables also were higher in animals fed saturated fat compared to those fed unsaturated fat on the same level of cholesterol. At 4-6 years of age, regardless of diet, females had significantly higher serum VLDL + LDL cholesterol (57 vs 43 mg/dl) and apoB concentrations (39 vs 30 mg/dl) than did males.

Toll-like receptors: new players in myocardial ischemia/reperfusion injury.

Innate immune and inflammatory responses have been implicated in myocardial ischemia/reperfusion (I/R) injury. However, the mechanisms by which innate immunity and inflammatory response are involved in myocardial I/R have not been elucidated completely. Recent studies highlight the role of Toll-like receptors (TLRs) in the induction of innate immune and inflammatory responses. Growing evidence has demonstrated that TLRs play a critical role in myocardial I/R injury. Specifically, deficiency of TLR4 protects the myocardium from ischemic injury, whereas modulation of TLR2 induces cardioprotection against ischemic insult. Importantly, cardioprotection induced by modulation of TLRs involves activation of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, suggesting that there is a crosstalk between TLRs and PI3K/Akt signaling pathways. In addition, TLRs also associate with other coreceptors, such as macrophage scavenger receptors in the recognition of their ligands. TLRs are also involved in the induction of angiogenesis, modulation of stem cell function, and expression of microRNA, which are currently important topic areas in myocardial I/R. Understanding how TLRs contribute to myocardial I/R injury could provide basic scientific knowledge for the development of new therapeutic approaches for the treatment and management of patients with heart attack.

Atherosclerosis as an inflammatory process. The roles of the monocyte-macrophage

In this brief review, we have addressed the roles of the monocyte-macrophage in atherogenesis, with emphasis on the recruitment to the arterial wall. We have presented summary data on the SMC-derived chemoattractant protein and also on the temporal evolution of monocyte-derived macrophages into cholesteryl ester-rich foam cells. The concept of atheroma as an inflammatory process has also been discussed.

Aortic intimal monocyte recruitment in the normo and hypercholesterolemic baboon (Papio cynocephalus) - An ultrastructural study: Implications in atherogenesis

The ultrastructural features of peripheral blood monocyte margination, migration, and aortic intimal accumulation have been described in the normo- and mildly hypercholesterolemic baboon. Intimal monocyte-macrophage recruitment over fatty streaks and fibro-fatty plaques was enhanced by dietary cholesterol-fat supplementation, resulting in an 8-fold increase in monocyte-macrophages and macrophage-derived foam cells in the subendothelial space. Margination or attachment observed over both plaques and normal areas was not associated with morphologic evidence of endothelial injury. Migration through continuous aortic endothelium was principally between endothelial cells via junctions. Transitional sequences from the typical morphology of the blood monocyte to the lipid-containing macrophage or foam cell were discerned. The intimal accumulation of monocytes and macrophages reinforces our view of atherosclerosis as an inflammatory process, in which monocyte attachment is likely to reflect changes in the endothelial surface-membrane complex and surface charge, while migration to and accumulation in the SES may result from one or more chemoattractants originating in the intima or media.

TLR3 plays a central role in cardiac dysfunction during polymicrobial sepsis

Objective: To determine the role of Toll-like receptor 3 in cardiac dysfunction during polymicrobial sepsis. Design: Controlled animal study. Setting: University research laboratory. Subjects: Male C57BL/6, wild-type, Toll-like receptor 3−/−. Intervention: Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. Toll-like receptors (TLRs) play a critical role in the pathophysiology of sepsis/septic shock. TLR3 is located in intracellular endosomes, and recognizes double-stranded RNA. This study examined the role of TLR3 in cardiac dysfunction following cecal ligation and puncture (CLP)-induced sepsis. TLR3 knockout (TLR3−/−, n = 12) and age-matched wild-type (n = 12) mice were subjected to CLP. Cardiac function was measured by echocardiography before and 6 hrs after CLP. Measurements and Main Results: CLP resulted in significant cardiac dysfunction as evidenced by decreased ejection fraction by 25.7% and fractional shortening by 29.8%, respectively. However, TLR3−/− mice showed a maintenance of cardiac function at pre-CLP levels. Wild-type mice showed 50% mortality at 58 hrs and 100% mortality at 154 hrs after CLP. In striking contrast, 70% of TLR3−/− mice survived indefinitely, that is, >200 hrs. TLR3 deficiency significantly decreased CLP-induced cardiac-myocyte apoptosis and attenuated CLP-induced Fas and Fas ligand expression in the myocardium. CLP-activation of TLR4-mediated nuclear factor-&kgr;B and Toll/IL-1 receptor-domain-containing adapter-inducing interferon-&bgr;-dependant interferon signaling pathways was prevented by TLR3 deficiency. In addition, CLP-increased vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression, and neutrophil and macrophage sequestration in the myocardium were also attenuated in septic TLR3−/− mice. More significantly, adoptive transfer of wild-type bone-marrow stromal cells to TLR3−/− mice abolished the cardioprotective effect in sepsis. Conclusions: These data indicate that TLR3 plays a deleterious role in mediating cardiac dysfunction in sepsis. Thus, modulation of the TLR3 activity may be useful in preventing cardiac dysfunction in sepsis.

Glucan phosphate attenuates myocardial HMGB1 translocation in severe sepsis through inhibiting NF-κB activation

Myocardial dysfunction is a major consequence of septic shock and contributes to the high mortality of sepsis. High-mobility group box 1 (HMGB1) serves as a late mediator of lethality in sepsis. We have reported that glucan phosphate (GP) attenuates cardiac dysfunction and increases survival in cecal ligation and puncture (CLP)-induced septic mice. In the present study, we examined the effect of GP on HMGB1 translocation from the nucleus to the cytoplasm in the myocardium of septic mice. GP was administered to mice 1 h before induction of CLP. Sham-operated mice served as control. The levels of HMGB1, Toll-like receptor 4 (TLR4), and NF-κB binding activity were examined. In an in vitro study, H9C2 cardiomyoblasts were treated with lipopolysaccharide (LPS) in the presence or absence of GP. H9C2 cells were also transfected with Ad5-IκBα mutant, a super repressor of NF-κB activity, before LPS stimulation. CLP significantly increased the levels of HMGB1, TLR4, and NF-κB binding activity in the myocardium. In contrast, GP administration attenuated CLP-induced HMGB1 translocation from the nucleus to the cytoplasm and reduced CLP-induced increases in TLR4 and NF-κB activity in the myocardium. In vitro studies showed that GP prevented LPS-induced HMGB1 translocation and NF-κB binding activity. Blocking NF-κB binding activity by Ad5-IκBα attenuated LPS-induced HMGB1 translocation. GP administration also reduced the LPS-stimulated interaction of HMGB1 with TLR4. These data suggest that attenuation of HMGB1 translocation by GP is mediated through inhibition of NF-κB activation in CLP-induced sepsis and that activation of NF-κB is required for HMGB1 translocation.

Growth, structure and function of baboon aortic smooth muscle cells in culture☆

Abstract Procedures for the successful culture of aortic medial smooth muscle cells (SMC) from the baboon ( Papio cynocephalus ) have been described. Growth rates, plating efficiency, and doubling times were determined. SMC showed increasing growth with increasing concentrations of heat-inactivated homologous serum. Growth on glass was accelerated relative to plastic surfaces, a difference reflected in the more numerous and extensive attachment sites on glass and more extensive cellular spreading. Attachment sites on both glass and plastic were associated with short parallel arrays of thin filaments (50 A). The fine structure of SMC grown on plastic or glass was examined at intervals from 7 to 74 days in culture. Cells exhibited many of the features traditionally associated with cultured SMC, including basement membrane material and extracellular matrix, predominantly thin cytoplasmic filaments (40–80 A) with dense bodies, coated pits and vesicles, caveolae and pinocytotic vesicles, and both cell-cell and internalized gap junctions. Changes with increasing time in culture included an increased extracellular matrix, more numerous lipid inclusions, residual bodies, myelin forms, multivesicular bodies, and crystalline lysosomal inclusions. Membranous multivesicular blebs and extruded autophagosomes were also seen with increasing frequency. An increasing culture age was also associated with changes in organelles, including less prominent Golgi profiles, more free ribosomes, dilatation of the cisternae of the RER, and less prominent shorter profiles of mitochondria with increasing electron density. Cultured SMC exhibited both a continuous and a homogeneous ruthenium red-staining glycocalyx around their perimeter, and the ability to respond to the polycationic ligand, CF, with a redistribution of surface-associated anionic binding sites. Additionally, they have been shown to possess high-affinity LDL receptors ( K d = 1 × 10 −7 M ) with binding showing half-maximal saturation at 20 μg/ml.

Detection of an abnormal lipoprotein in a large colony of pedigreed baboons using high-performance gel exclusion chromatography

High-performance gel exclusion chromatography was used to detect an abnormal lipoprotein in a large colony of baboons. Serum obtained from fasting baboons was adjusted to density 1.21 g/ml and ultracentrifuged to obtain lipoproteins. A small fraction (equivalent to 50 microliter serum) was separated using a gel filtration column (TSK 4000 SW) or a combination of TSK 4000 PW and TSK 3000 PW columns. The unusual lipoprotein was detected either as a distinct peak between low- and high-density lipoproteins or as a distinct shoulder to the high-density lipoprotein peak. In some baboons the unusual lipoprotein was present on both chow and atherogenic diet, but in most cases it was induced by feeding an atherogenic diet.

Influence of the acyl-CoA:cholesterol O-acyltransferase inhibitor, CL 277082, on cholesteryl ester accumulation in rabbit macrophage-rich granulomas and hepatic tissue

The influence of the acyl-CoA: cholesterol O-acyltransferase (ACAT) inhibitor, CL 277082, on macrophage cholesteryl ester accumulation in a rabbit carrageenan granuloma macrophage-foam cell model was studied. Diets were supplemented with 0.3% cholesterol and 6% peanut oil with or without the inhibitor (0.25%) for 4 weeks prior to granuloma induction, and macrophage-rich granuloma tissue was harvested 14 days after carrageenan injection. Serum cholesterol was monitored biweekly, and plasma lipoproteins were isolated terminally. Total, free and esterified cholesterol contents were measured in hepatic and granuloma tissue. In hepatic tissue, administration of CL 277082 resulted in an 80% reduction in the content of total cholesterol, a 37% decrease in free cholesterol, and a 90% decrease in esterified cholesterol. Similarly, in macrophage-rich granuloma tissue, total cholesterol content was decreased by 44%, and esterified cholesterol content by 61%, with no change in free cholesterol. Additionally, CL 277082 was shown to inhibit granuloma tissue ACAT activity by 45%, VLDL mass was decreased slightly, LDL mass increased 3.4-fold and HDL mass was similar in both the inhibitor-treated and control animals. CL 277082 resulted in a 57% decrease in VLDL cholesteryl ester content and a 4.5-fold increase in triacylglycerol. Cholesteryl ester content in LDL was decreased by 31% and LDL triacylglycerol was increased 5.2-fold, while the only change in HDL composition was a 3.5-fold increase in triacylglycerol. The reductions in both hepatic tissue and macrophage-rich granuloma tissue esterified cholesterol accumulation are considered to be due largely to cellular ACAT inhibition, and the altered distribution and composition of the plasma lipoproteins.