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Biomedical subjects

Thomas N Tulenko

Publications and source records attributed to Thomas N Tulenko.

12 recordsLinked to original sources
PubMed2006

Sphingomyelinase-to-LDL molar ratio determines low density lipoprotein aggregation size: biological significance.

The subendothelial retention of low density lipoproteins (LDL) is believed to be the central pathogenic event in atherosclerosis, as stated by the response-to-retention hypothesis. Sphingomyelinase, an enzyme present in the arteries, has been proven to promote LDL aggregation. This study investigates the hypothesis that the extent of LDL aggregation is determined by the molar ratio of sphingomyelinase (SMase)-to-LDL, rather than the absolute concentrations. A mass action model is used to describe the aggregation process, and binding and dissociation rate constants are determined by fitting of dynamic light scattering data. The model predicts aggregate sizes that agree well with experimental observations. This study also tests the hypothesis that monocyte uptake of LDL correlates with aggregate size. LDL aggregates of three specific sizes (75, 100, and 150 nm) were incubated with J774A.1 cells and the net accumulation of LDL was monitored by measuring changes in the cellular cholesterol and protein content. Relative to a control sample, cholesterol accumulation was enhanced for aggregate sizes of 75 and 150 nm. The intermediate size aggregates, 100 nm, led to a very striking result demonstrating that cholesterol accumulation was markedly greater than the other samples, and was sufficient to cause cell death. These results underscore an important role of colloidal aggregation, and the influence of LDL aggregate size, in atherosclerosis.

Animals↗
PubMed2005

A membrane defect in the pathogenesis of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an often lethal birth defect resulting from mutations in the gene responsible for the synthesis of the enzyme 3beta-hydroxy-steroid-Delta7-reductase, which catalyzes the reduction of the double bond at carbon 7 on 7-dehydrocholesterol (7-DHC) to form unesterified cholesterol. We hypothesize that the deficiency in cholesterol biosynthesis and subsequent accumulation of 7-DHC in the cell membrane leads to defective composition, organization, dynamics, and function of the cell membrane. Using skin fibroblasts obtained from SLOS patients, we demonstrate that the SLOS membrane has increased 7-DHC and reduced cholesterol content and abnormal membrane fluidity. X-ray diffraction analyses of synthetic membranes prepared to mimic SLOS membranes revealed atypical membrane organization. In addition, calcium permeability is markedly augmented, whereas membrane-bound Na+/K+ATPase activity, folate uptake, inositol-1,4,5-trisphosphate signaling, and cell proliferation rates are markedly suppressed. These data indicate that the disturbance in membrane sterol content in SLOS, likely at the level of membrane caveolae, directly contributes to the widespread tissue abnormalities in this disease.

Calcium↗
PubMed2005

In vivo behavior of decellularized vein allograft.

BACKGROUND: We are investigating decellularized vein allograft as a scaffold to engineer a non-synthetic, small-diameter vascular graft. This study examines the in vivo behavior of this scaffolding after implantation into the arterial circulation. MATERIALS AND METHODS: Canine animals underwent bilateral carotid interposition grafting using jugular vein implanted as either: 1) fresh autograft, 2) fresh allograft, or 3) decellularized allograft. Decellularization was achieved using sodium dodecyl sulfate. Grafts were examined with duplex ultrasound biweekly to determine luminal diameter, thrombosis, stenosis, or anastomotic breakdown. After perfusion fixation at 2 or 8 weeks, grafts underwent histological, morphometric, and immunohistochemical examination. RESULTS: All animals survived without neurological or hemorrhagic complication. No deterioration of graft integrity (rupture, aneurysm) was observed in any group. Luminal narrowing was observed in both allograft groups, but secondary to different pathology. Fresh allografts had significant mononuclear cell infiltrate, intimal hyperplasia, and intramural hemorrhage consistent with rejection. Conversely, decellularized allografts had minimal evidence of rejection but instead had a compact fibrin layer formed along their lumen. This fibrin layer was absent in the peri-anastomotic regions where endothelium had migrated from the native artery. By 8 weeks, decellularized grafts had repopulated with cells staining positive for smooth muscle alpha-actin. CONCLUSIONS: After 8 weeks of arterial flow, decellularized vein allograft exhibits satisfactory strength, reduced antigenicity compared to fresh allograft, and supports cellular repopulation. These characteristics make it satisfactory for further tissue engineering; combined with luminal vascular cell seeding, it may prove useful as a small-diameter arterial bypass graft.

Animals↗
PubMed2005

Atherosclerotic plaque progression and vulnerability to rupture: angiogenesis as a source of intraplaque hemorrhage.

Observational studies of necrotic core progression identify intraplaque hemorrhage as a critical factor in atherosclerotic plaque growth and destabilization. The rapid accumulation of erythrocyte membranes causes an abrupt change in plaque substrate characterized by increased free cholesterol within the lipid core and excessive macrophage infiltration. Neoangiogenesis is associated closely with plaque progression, and microvascular incompetence is a likely source of intraplaque hemorrhage. Intimal neovascularization is predominantly thought to arise from the adventitia, where there are a plethora of pre-existing vasa vasorum. In lesions that have early necrotic cores, the majority of vessels invading from the adventitia occur at specific sites of medial wall disruption. A breech in the medial wall likely facilitates the rapid in-growth of microvessels from the adventitia, and exposure to an atherosclerotic environment stimulates abnormal vascular development characterized by disorganized branching and immature endothelial tubes with "leaky" imperfect linings. This network of immature blood vessels is a viable source of intraplaque hemorrhage providing erythrocyte-derived phospholipids and free cholesterol. The rapid change in plaque substrate caused by the excessive accumulation of erythrocytes may promote the transition from a stable to an unstable lesion. This review discusses the potential role of intraplaque vasa vasorum in lesion instability as it relates to plaque rupture.

Animals↗
PubMed2004

Aggregation kinetics of low density lipoproteins upon exposure to sphingomyelinase.

The response-to-retention hypothesis in atherosclerosis states that subendothelial retention of cholesterol-rich, atherogenic lipoproteins is the central pathogenic event that is both necessary and sufficient to provoke lesion initiation in an otherwise normal artery. Sphingomyelinase-induced aggregation of low density lipoproteins (LDL) is known to facilitate LDL retention, and the only available measurements of LDL aggregates suggest LDL aggregate size is approximately 100 nm. This study investigates the hypothesis that LDL aggregate size is determined by the relative rates of sphingomyelinase hydrolysis and LDL collisions. Using a combination of dynamic light scattering and UV-vis absorbance spectroscopy to measure aggregation kinetics and particle sizes, a mass action model was developed to describe the aggregation process. It is found that LDL aggregation is sensitive to the relative amounts of sphingomyelinase and LDL and to pH. Model rate parameters were fit to experimental data in vitro and used to predict LDL aggregate sizes in vivo. The value of 100 nm in vivo does not appear to be fixed; rather, it is the value expected for the prevailing enzyme-to-LDL molar ratio.

Kinetics↗
PubMed2004

Influence of serum cholesterol on atherogenesis and intimal hyperplasia after angioplasty: inhibition by amlodipine.

The objectives of the present study were to determine whether serum hypercholesterolemia (HC) promotes the development of spontaneous and angioplasty-induced lesions and whether amlodipine inhibits these lesions and cellular processes underlying their genesis. Rabbits were fed normal, 0.5%, or 2% cholesterol diets for 9 wk, which resulted in the development of increasing HC. After week one, balloon dilation of the abdominal aorta was performed while the thoracic aorta was not disturbed and monitored for the development of spontaneous lesions. Lesion size increased with the degree of HC and was accompanied by increased collagen synthesis and smooth muscle cell (SMC) proliferation at each site. Amlodipine (5 mg/kg p.o.) inhibited lesion size by 50% (P < 0.01) at both sites in cholesterol-fed animals but not at angioplasty sites in animals on a normal diet. Local collagen synthesis was inhibited at both sites by amlodipine in the diet animals. The increase in HC was accompanied by a 1.7-fold increase in basal Ca2+ uptake in SMCs in the thoracic aorta, which was not altered by amlodipine, nifedipine, Ni2+, or La3+, revealing an uninhibitable calcium leak during atherogenesis. In culture, cholesterol enrichment increased SMC proliferation, collagen synthesis, and the secretion of a soluble SMC mitogen, which were inhibited by amlodipine (10(-9) M). Finally, in SMC membranes, amlodipine uniquely restored the cholesterol-expanded membrane bilayer width without any effect on membrane fluidity. This study establishes a causal role between serum HC and the development of spontaneous and angioplasty-induced lesions and the ability of amlodipine to disrupt this action by a novel remodelling action on the SMC membrane.

Amlodipine↗
PubMed2004

Cholesterol crystal nucleation from enzymatically modified low-density lipoproteins: combined effect of sphingomyelinase and cholesterol esterase.

An assay detecting and quantifying cholesterol nucleation from low-density lipoproteins has been established. Förster resonance energy transfer between dehydroergosterol and dansylated lecithin becomes significantly alleviated as a consequence of conucleation of dehydroergosterol and cholesterol. The assay, in combination with dynamic light scattering, absorbance spectroscopy, and fluorescence microscopy, can be used to study aggregation and nucleation in model blood systems. Human plasma LDL was labeled with dehydroergosterol and dansylated lecithin by incubation with donor multilamellar liposomes and isolated by centrifugation. Exposure of labeled LDL (0.5 mg/mL of total lipids) to sphingomyelinase (0.0-0.2 unit/mL) led to modest particle aggregation but produced no changes in energy transfer and no crystallization. However, addition of sphingomyelinase produced significant particle aggregation, nucleation, and crystallization, in a dose-dependent fashion, in samples that were previously treated with the enzyme, cholesterol esterase (0.2 unit/mL). The combination of cholesterol esterase and sphingomyelinase led to a significant alleviation of energy transfer, which preceded by 24 h the appearance of fluorescent, microscopic sterol crystals. These results point to a synergistic effect between cholesterol esterase and sphingomyelinase, suggesting that mere aggregation of LDL is insufficient to promote nucleation, and crystal formation likely proceeds in the intracellular space after LDL uptake by macrophages.

Cholesterol↗
PubMed2004

Decellularized vein as a potential scaffold for vascular tissue engineering.

PURPOSE: Current strategies to create small-diameter vascular grafts involve seeding biocompatible, compliant scaffolds with autologous vascular cells. Our purpose was to study the composition and strength of decellularized vein to determine its potential as a vascular tissue-engineering scaffold. METHODS: Intact human greater saphenous vein specimens were decellularized by using sodium dodecyl sulfate (SDS). Residual cellular and extracellular matrix composition was studied with light and electron microscopy as well as immunohistochemistry. Burst and suture-holding strength was measured in vitro by insufflation and pull-through techniques. To assess initial handling and durability of decellularized vein in vivo, a canine model was developed wherein decellularized canine jugular veins were implanted as carotid interposition grafts in recipient animals. After two weeks of arterial perfusion, these grafts were studied with duplex imaging and histologic methods. RESULTS: Human saphenous vein decellularized by using SDS was devoid of endothelial cells and >94% of the cells resident within the vein wall. Collagen morphology appeared unchanged, and elastin staining decreased only slightly. Basement membrane collagen type IV remained intact. Compared with fresh vein, decellularized vein had similar in vitro burst (2480 +/- 460 mm Hg vs 2380 +/- 620 mm Hg; P >.05) and suture-holding (185 +/- 30 gm vs 178 +/- 66 gm; P >.05) strength. Decellularized canine vein functioned well in vivo without dilation, anastomotic complication, or rupture over 2 weeks of arterial perfusion. CONCLUSIONS: Vein rendered acellular with SDS has well-preserved extracellular matrix, basement membrane structure, and strength sufficient for vascular grafting. These properties suggest proof of concept for its use as a scaffold for further vascular tissue engineering. CLINICAL RELEVANCE: The following research examines the creation of a new small-diameter bypass graft. It is clinically relevant to patients who need distal arterial bypass, coronary artery bypass, or hemodialysis access, but who do not have adequate autologous vein for their surgeries. Future investigations will involve further tissue engineering of this vascular scaffold (eg, autologous endothelial seeding of its lumen) and testing the clinical usefulness of the completed graft.

Antigens, Surface↗
PubMed2003

Direct evidence for cholesterol crystalline domains in biological membranes: role in human pathobiology.

This review will discuss the use of small-angle X-ray diffraction approaches to study the organization of lipids in plasma membranes derived from two distinct mammalian cell types: arterial smooth muscle cells and ocular lens fiber cells. These studies indicate that cholesterol at an elevated concentration can self-associate and form immiscible domains in the plasma membrane, a phenomenon that contributes to both physiologic and pathologic cellular processes, depending on tissue source. In plasma membrane samples isolated from atherosclerotic smooth muscle cells, the formation of sterol-rich domains is associated with loss of normal cell function, including ion transport activity and control of cell replication. Analysis of meridional diffraction patterns from intact and reconstituted plasma membrane samples indicates the presence of an immiscible cholesterol domain with a unit cell periodicity of 34 A, consistent with a cholesterol monohydrate tail-to-tail bilayer, under disease conditions. These cholesterol domains were observed in smooth muscle cells enriched with cholesterol in vitro as well as from cells obtained ex vivo from an animal model of atherosclerosis. By contrast, well-defined cholesterol domains appear to be essential to the normal physiology of fiber cell plasma membranes of the human ocular lens. The organization of cholesterol into separate domains underlies the role of lens fiber cell plasma membranes in maintaining lens transparency. These domains may also interfere with cataractogenic aggregation of soluble lens proteins at the membrane surface. Taken together, these analyses provide examples of both physiologic and pathologic roles that sterol-rich domains may have in mammalian plasma membranes. These findings support a model of the membrane in which cholesterol aggregates into structurally distinct regions that regulate the function of the cell membrane.

Arteries↗
PubMedDate not supplied

The physiology of lipoproteins.

The seminal studies of Brown and Goldstein (Science 1986;232:34-47) coupled with the findings of the Framingham study revolutionized our understanding of the metabolic basis for vascular disease. These studies led to the widespread use of the coronary risk lipid profile, which uses the total cholesterol/high-density lipoprotein (HDL) ratio (or low-density lipoprotein [LDL]/HDL ratio) in predicting risk for vascular disease and as a tool for therapeutic management of patients at risk for vascular disease. However, although these methods are predictive of coronary artery disease (CAD) in general, it is also well known that the extent of occlusive disease and CAD varies greatly between individuals with similar cholesterol and HDL lipid profiles. For this reason, the National Cholesterol Education Program Expert Panel revised these guidelines and now recommends monitoring LDL and HDL cholesterol in the context of coronary heart disease risk factors and "risk equivalents." In addition, more recent findings indicate that specific alterations in individual lipoprotein subclasses may account for the variations in CAD in subjects with similar lipid profiles. For example, a preponderance of small, dense LDL particles correlates with a marked increase in risk for myocardial infarction independent of LDL levels. In particular, the association of small, dense LDL with elevated triglycerides (large, less dense VLDL) and reduced HDL has been defined as the atherogenic lipoprotein profile, and the key metabolic defect driving this profile may be elevated levels of triglycerides, specifically large, less dense VLDL. In an attempt to explain the physiologic basis for lipoprotein variations, this review describes the basic metabolic scheme underlying the traditional view of lipoprotein metabolism and physiology. It then examines the identity and role of the various lipoprotein subfractions in an attempt to distill a working model of how lipoprotein abnormalities might account for vascular disease in general and the metabolic syndrome in particular.

Anticholesteremic Agents↗