Kenneth B. Pomerantz

Human glial cell production of lipoxygenase-generated eicosanoids: a potential role in the pathophysiology of vascular changes following traumatic brain injury

Acute cerebrovascular changes which occur following traumatic brain injury represent a highly complex, multifactorial pathophysiologic process which is poorly understood. It is now recognized that, under normal conditions, the brain is a source of a variety of arachidonic acid metabolites which are synthesized by both cyclooxygenase and lipoxygenase. The specific cellular source of these highly vasoactive substances remains controversial. Recent work has demonstrated that lipoxygenase products were detected by immunosensitive assay in whole brain samples from a gerbil concussive injury model, yet the production of leukotrienes could not be accounted for by cerebral vessels and their contents alone. It has been theorized that the probable source for these metabolites is the cortical neuron. We sought to elucidate whether cultured human glial cells, obtained from specimens removed at the time of surgery, are a significant source of lipoxygenase products as measured by high performance liquid chromatography (HPLC). We observed that these cells consistently produced 5, 12, and 15-HETE class eicosanoids despite failure to produce significant cyclooxygenase products. These preliminary findings are of considerable interest because these lipoxygenase products are known to be highly vasoactive as well as potent mediators of increased vascular permeability. Since it is known that mechanical perturbation of cell membranes stimulates the release of arachidonic acid from membrane phospholipids, it is conceivable that the production of these eicosanoids following traumatic brain injury could account for local cerebrovascular changes including both vasospasm and interstitial edema formation.

Signal Transduction in Atherosclerosis: Second Messengers and Regulation of Cellular Cholesterol Trafficking

The data summarized in this review demonstrate that the regulation of intracellular cholesterol trafficking is mediated not only by extracellular lipoprotein concentrations and transcriptional responses to alterations in intracellular free cholesterol content. Rather, the modulation of cholesterol trafficking is also regulated by the products synthesized following activation of signal transduction pathways originating at the cell surface. Furthermore, we have identified those cell-derived factors which utilize these signal transduction pathways to elicit alterations in cholesterol trafficking, and demonstrated the importance of the generation of second messengers, most notably eicosanoids, and cyclic AMP in promoting a modulatory influence on specific pro-atherogenic effects of mitogens.

Role of Eicosanoids and the Cytokine Network in Transmembrane Signaling in Vascular Cells

The concept that atherosclerosis is an inflammatory response to injury is based on observations that cells of monocytic origin, including macrophages and T-cells populate the developing atherosclerotic lesion. This is in addition to the well-characterized smooth muscle cell infiltrate1. Since endothelial cell activation or injury is a prerequisite for monocyte adhesion and diapedesis, the histologic evidence described above support the hypothesis that endothelial cell injury is an important initial event in the development of the inflammatory lesion. Activation or injury may thus initiate a series of process that lead to intimai hyperplasia and choiesteryl ester deposition within the macrophage and smooth muscle cell.

Leukotriene Production by Human Glia

Elevated intracranial pressure and acute cerebrovascular changes following head injury remain the principle challenge in the management of traumatic brain injury. Recent work has demonstrated that leukotrienes can induce increases in blood brain barrier permeability and alter cerebrovascular dynamics. We investigated whether human astroglia in culture: 1. generate specific leukotrienes; 2. how they metabolize leukotrienes, and; 3. if astroglia generate leukotrienes in response to barotraumatic injury. Human astroglial cultures established from normal human brain obtained at surgery were exposed to either ionophore, exogenous 3H-LTC4, or barotraumatic injury. Supernatants were assayed for specific leukotrienes by one of three methods: HPLC, radioimmunoassay, or enzyme-immunoassay. Glial cells exposed to exogenous LTC4 metabolized nearly all of the LTC4 to LTD4 and LTE4 within 20 minutes. Glial cells stimulated with ionophore produced mostly LTC4 at five minutes after stimulation and LTD4 and LTE4 at fifteen minutes after stimulation. Glial cells subject to barotraumatic injury produced LTC4 in concentrations of 40-200 pg/ml 15 minutes after injury. These results demonstrate that human astroglial cells are capable of rapidly generating and degrading LTC4 and this capability of glial cells may play an important role in the pathophysiology of cerebrovascular changes following head injury.

Inhibition of cholesterol esterification in macrophages and vascular smooth muscle foam cells: Evaluation of E5324, an Acyl-CoA cholesterol acyltransferase inhibitor

Cholesteryl esters (CE) comprise the principal lipid class that accumulates within macrophages and smooth muscle cells of the atherosclerotic lesion. Acyl-CoA cholesterol acyl-transferase (ACAT) is the major enzyme responsible for esterification of intracellular cholesterol. We evaluated the ability of E5324 (n-butyl-N″-[-2-[3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxyl]-6-methyl-phenyllurea), a novel, orally absorbable ACAT inhibitor, to inhibit esterification of fatty acids to cholesterol and CE accumulation in macrophages and in smooth muscle cells. E5324 significantly inhibited cholesterol esterification in rat aortic smooth muscle cells and in macrophages. In addition, E5324 reduced the cellular mass of CE, the significant measure of the efficacy of drugs designed to modulate cholesterol metabolism. E5324 treatment of macrophages exposed to acetylated low-density lipoprotein reduced CE mass by 97%, and treatment of lipid-loaded smooth muscle cells reduced CE mass by 29%. Although free cholesterol increased approximately twofold, this free cholesterol would presumably be accessible to the membrane for effluxin vivo (reverse cholesterol transport). These results demonstrate that E5324 can inhibit cholesterol esterification and CE mass in atherosclerotic foam cells, derived from either macrophages or arterial smooth muscle cells.

Molecular motions and thermotropic phase behavior of triacylglycerols and cholesterol esters in herpesvirus-infected arterial smooth muscle cells: A deuterium nuclear magnetic resonance study

The physical state of lipids in arterial smooth muscle cells (SMC) may contribute to lipid accumulation following injury. We have previously demonstrated that herpes simplex virus (HSV) infection alters the physical state of the neutral lipid accumulating in arterial SMC, as determined by differential scanning calorimetry (Biochem. J. 268 (1990) 693-697). To more precisely determine the molecular packing of neutral lipids in HSV-infected cells, the influence of HSV-infection on the thermotropic and phase-behavior of the lipids in intact arterial smooth muscle cells and in cell-free lipid extracts was evaluated using [2H]-NMR, employing U-[2H]-oleic acid incorporated into cells. Inspection of the [2H]-line-widths indicate that the lipid of HSV-infected cells exhibited more restricted motion or a greater chemical shift dispersity than lipids from uninfected cells, as evidenced by significant broadening of the -CD = CD- signals at 25 degrees C and 45 degrees C. Fatty acid compositional analysis of the neutral lipids of control and HSV-infected cells following C18:1 supplementation (an amount added similar to the NMR experiments) reveals that: (1) there is approximately 55-fold more triacylglycerols (TG) than cholesteryl esters (CE) in control cells and 40-fold more TG than CE in the HSV-infected cells; (2) HSV infection significantly increases the C18:1 content of CE, and C18:3 and C20:4 in TG; and (3) HSV-infection does not alter the ratio of TG to CE. These data support the hypothesis that the greater restriction of the neutral lipids in HSV-infected cells may be due to the rigidifying effects of C18:1 on lipid mobility. Thus, alterations in the physical state of neutral lipids in HSV-infected cells may lead to reduced CE hydrolysis which, in turn, may contribute to or exacerbate lipid accumulation.

Biochemical Mechanisms Associated with the Lipolytic Effects of Calcium Channel Blockers

Calcium channel blockers are now widely used for the treatment of hypertension and angina. However, recent evidence suggests that calcium channel blockers may also be beneficial in controlling processes leading to atherosclerosis. In these studies, we evaluated the effects of two dihydropyridine calcium channel blockers, Nifedipine and Nicardipine on cholesterol metabolism in aortic smooth muscle cells. Nicardipine increased LDL receptor activity that was paralleled by an increase in the steady state level of LDL receptor mRNA. These calcium channel blockers also increased lysosomal and cytoplasmic cholesteryl ester hydrolase activities, but did not alter ACAT activity. Since we have demonstrated that these processes are modulated by prostacyclin (PGI2) and cyclic AMP, we evaluated the effects of calcium channel blockers on PGI2 release and cyclic AMP levels. We found that these agents increased both PGI2 release and cyclic AMP production. Finally, several calcium channel blockers reduced cholesterol content in cholesterol-enriched smooth muscle cells derived from atherosclerotic rabbits, and reduced cholesterol content in aortic biopsies taken from patients undergoing coronary bypass surgery. Taken together, our data demonstrate that calcium channel blockers reduce cholesterol content in vascular tissue by stimulating LDL catabolism through a process that is mediated by PGL2 and cyclic AMP. Our results engender support for the use of calcium channel blockers as anti-atherosclerotic agents.

Arterial Cell Interactions: Mechanistic Studies Related to Eicosanoid and Growth Factor-Induced Alterations in Cholesterol Metabolism

Intracellular cholesterol accumulation in the arterial wall and the proliferation of intimal smooth muscle cells are the hallmarks of atherogenesis. Humoral fluid-phase interactions between arterial endothelial and smooth muscle cells have been studied in vivo and in vitro in an attempt to delineate whether these cells communicate in the regulation of intimal hyperplasia and cholesterol and lipoprotein metabolism in arterial smooth muscle cells.