Joseph A. Cornicelli

Attenuation of diet-induced atherosclerosis in rabbits with a highly selective 15-lipoxygenase inhibitor lacking significant antioxidant properties.

15‐Lipoxygenase (15‐LO) has been implicated in the pathogenesis of atherosclerosis because of its localization in lesions and the many biological activities exhibited by its products. To provide further evidence for a role of 15‐LO, the effects of PD 146176 on the development of atherosclerosis in cholesterol‐fed rabbits were assessed. This novel drug is a specific inhibitor of the enzyme in vitro and lacks significant non specific antioxidant properties. PD 146176 inhibited rabbit reticulocyte 15‐LO through a mixed noncompetitive mode with a Ki of 197 nm. The drug had minimal effects on either copper or 2,2′‐azobis(2‐amidinopropane)hydrochloride (ABAP) induced oxidation of LDL except at concentrations 2 orders higher than the Ki. Control New Zealand rabbits were fed a high‐fat diet containing 0.25% wt./wt. cholesterol; treated animals received inhibitor in this diet (175 mg kg−1, b.i.d.). Plasma concentrations of inhibitor were similar to the estimated Ki (197 nm). During the 12 week study, there were no significant differences in weight gain, haematocrit, plasma total cholesterol concentrations, or distribution of lipoprotein cholesterol. The drug plasma concentrations achieved in vivo did not inhibit low‐density lipoprotein (LDL) oxidation in vitro. Furthermore, LDL isolated from PD 146176‐treated animals was as susceptible as that from controls to oxidation ex vivo by either copper or ABAP. PD 146176 was very effective in suppressing atherogenesis, especially in the aortic arch where lesion coverage diminished from 15±4 to 0% (P<0.02); esterified cholesterol content was reduced from 2.1±0.7 to 0 μg mg−1 (P<0.02) in this region. Immunostainable lipid‐laden macrophages present in aortic intima of control animals were totally absent in the drug‐treated group. Results of these studies are consistent with a role for 15‐LO in atherogenesis.

A specific 15-lipoxygenase inhibitor limits the progression and monocyte–macrophage enrichment of hypercholesterolemia-induced atherosclerosis in the rabbit

Oxidant signalling and lipoprotein oxidation may play important roles in atherosclerotic lesion development. Given coincident localization of 15-lipoxygenase (15-LO), stereospecific products of 15-LO and epitopes of modified LDL in atherosclerotic lesions, we hypothesized that inhibition of 15-LO by PD146176, an inhibitor of 15-LO with an IC50 in cells or isolated enzyme of 0.5-0.8 microM, may limit atherosclerotic lesion development through regulation of monocyte-macrophage enrichment. Rabbits exposed to chronic endothelial denudation of the iliac-femoral artery were meal-fed a 0.25% cholesterol (C), 3% peanut oil (PNO), 3% coconut oil (CNO) diet twice daily with and without 175 mg/kg PD146176 for 12 weeks. In a second study, atherosclerotic lesions were pre-established in rabbits through chronic endothelial denudation and meal-fed a 0.5% C, 3% PNO, 3% CNO diet for 9 weeks and a 0% C/fat diet for 6 weeks prior to an 8 week administration of PD146176 at 175 mg/kg, q.d. Plasma total and lipoprotein cholesterol exposure were similar in control and PD146176-treated animals in both studies but PD146176 increased plasma triglyceride exposure 2- to 4-fold. Plasma PD146176 concentrations ranged from 99 to 214 ng/ml at 2 h post-dose. In the progression study, the iliac-femoral monocyte-macrophage area was reduced 71%, cross-sectional lesion area was unchanged and cholesteryl ester (CE) content was reduced 63%. In the regression study, size and macrophage content of iliac-femoral, fibrous plaque-like lesions were decreased 34%, CE content was reduced 19% and gross extent of thoracic aortic lesions were reduced 41%. We conclude that PD146176 can limit monocyte macrophage enrichment of atherosclerotic lesions and can attenuate development of fibrofoamy and fibrous plaque lesions in the absence of changes in plasma total or lipoprotein cholesterol concentrations.

A spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid.

An assay for the detection of hydroperoxy derivatives of linoleic acid formed by the action of 15-lipoxygenase is described. The assay developed is based on a method first reported by Ohishi et al. (1985) Biochem. Int. 10, 205-211) with some important modifications. The assay described herein takes advantage of the ability of (9Z,11E)-13-hydroperoxyoctadecadienoic acid (13-HPODE), the product of the action of 15-lipoxygenase on linoleic acid, to oxidize N-benzoyl leucomethylene blue to methylene blue in the presence of hemoglobin. The resultant blue color is stable to light and air and can be quantified spectrophometrically at 660 nm. The linear range of the assay is 1.6-32 nmol (0.5-10 micrograms) of 13-HPODE. The utility of the assay can be extended to detect other peroxides as well as inhibitors of 15-lipoxygenase. The assay is a rapid, reliable method for the detection of lipid hydroperoxide production.

Metabolic effects of troglitazone in the Goto-Kakizaki rat, a non-obese and normolipidemic rodent model of non-insulin-dependent diabetes mellitus

Troglitazone (TRG) is an orally active antidiabetic agent that increases insulin sensitivity in models of non-insulin-dependent diabetes mellitus (NIDDM), subsequently reducing hyperinsulinemia and hyperglycemia. We examined the effects of TRG on the development and severity of diabetes in the Goto-Kakizaki (GK) rat, a spontaneous, non-obese model of NIDDM. TRG was administered at a dose of 30 mg/kg/d beginning at 4 weeks of age. TRG-treated GK rats were evaluated against Wistar and untreated GK rats at 8, 12, and 16 weeks of age. Untreated GK rats were nonketotic, normolipidemic, hyperglycemic, and had normal fasting insulin levels compared with Wistar rats. TRG treatment decreased glycosylated hemoglobin levels in the GK rat independently of its effects on plasma insulin. In untreated GK rats, intravenous glucose tolerance tests (IVGTTs) showed a hyperglycemic response to glucose loading with severely impaired glucose disposal relative to Wistar controls. TRG treatment was successful in decreasing the glucose area under the curve (AUC) (P < .03) but did not improve glucose disposal, suggesting a direct hepatic effect. Ex vivo evaluation of hepatic glucose output (HGO) further supported a direct hepatic action, with 50% reduction in HGO in TRG-treated GK rats (P < .004). A euglycemic-hyperinsulinemic clamp performed at 16 weeks of age showed severe insulin resistance in the untreated GK rat, with a glucose infusion rate (GIR) 33% lower than in Wistar rats (P < .004). TRG treatment had no effect on this insulin resistance. These results indicate that TRG selectively decreases hepatic glucose production in this unique model of NIDDM independently of its action on peripheral insulin sensitivity or hyperlipidemia.

Is there a role for 15-lipoxygenase in atherogenesis?

15-Lipoxygenase has been suggested to play a role in atherogenesis. The proposed action of this enzyme is to oxidize low density lipoprotein (LDL) to the extent that LDL becomes a ligand for the macrophage scavenger receptor. 15-Lipoxygenase and oxidized LDL are co-localized in atherosclerotic lesions; antioxidant drugs that block the lipoxygenase also block oxidation of LDL and the progression of experimental atherosclerosis. Biochemical experiments have demonstrated that the lipoxygenase can be induced by cytokines and/or another factor(s) associated with hypercholesterolemia. However, molecular biological work has shown that induction of the enzyme alone is not sufficient to induce lesion formation. Furthermore, the mechanism of action of 15-lipoxygenase in atherogenesis remains unclear. Predictions of the stereochemistry of enzyme-oxidized linoleate products appear to conflict with the available data. In fact, most studies have discovered substantial levels of racemic 13-hydroxyoctadecadienoic acid (13-HODE) in arterial lesions rather than the stereochemically pure 13(S)-HODE expected from purified enzyme. The possibility that the generation of products of 15-lipoxygenase metabolism must occur in a specific cellular location and during a brief time window in the development of the disease has been discussed. It is also possible that the true function of the linoleate metabolites is to modulate gene expression and regulate mitogenesis, and that oxidation of LDL may play a secondary role. The advent of transgenic species that both develop atherosclerosis and either fail to express or overexpress the lipoxygenase presents an opportunity to clarify some of these issues in the near future.

Human and Monkey Corneal Endothelium Expression of Low-Density Lipoprotein Receptors

Receptors for low-density lipoprotein are necessary for high-affinity uptake of lipid and protein essential to cell structure and function. Distinct receptors for acetoacetylated low-density lipoprotein internalize oxidized or enzymatically modified low-density lipoprotein and extracellular matrix components. We identified low-density lipoprotein receptors on cultured human and monkey corneal endothelial cells by the avid incorporation of fluorescently labeled low-density lipoprotein that was competitively inhibited by excess unlabeled low-density lipoprotein but not by unlabeled acetoacetylated low-density lipoprotein. Specific uptake of labeled low-density lipoprotein was greatest in nonconfluent, growing cells and increased after low-density lipoprotein deprivation. Intact endothelial monolayers of whole human cornea also incorporated low-density lipoprotein but not acetoacetylated low-density lipoprotein. After scratch injury of human corneas, spreading endothelium adjacent to areas of cell loss internalized more fluorescent low-density lipoprotein than cells distant from the injury. Blood-aqueous barrier breakdown occurring in ocular diseases and after surgical and nonsurgical trauma may allow leakage of circulating low-density lipoprotein, which provides a rich supply of lipid and protein for endothelial use. Efficient, receptor-mediated, low-density lipoprotein uptake may facilitate repair of damaged corneal endothelial membranes and regeneration of intact, functional cell monolayers.

Effect of cadmium ingestion on cadmium and zinc profile in male and female rat liver cytosol.

Abstract A study of the changes in rat liver cytosol zinc and cadmium metalloprotein profiles obtained by G-75 Sephadex chromatography of male and female rats given oral cadmium chloride chronically at different low doses showed that the changes were dose related. Marked disturbances in the zinc-containing regions of the protein profile preceded any extensive formation of metallothionein, and the earliest signs of cadmium metalloprotein formation involved three equally prominent regions, a high molecular weight region (I), the metallothionein region (V) and a low molecular weight region (VI). The low molecular weight region (estimated at 3500 daltons) is of interest in that it is a newly recognized zinc-containing region even in the controls. Significant quantitative differences were found between males and females. Female livers consistently contained higher concentrations of cadmium and zinc. There also were important quantitative differences in the zinc and cadmium-containg cytosol fractions of the protein profiles of livers of males and females exposed to oral cadmium.

Catechol based inhibitors of 15-lipoxygenase

Abstract A potent 15-lipoxygenase (15-LO) inhibitor, compound 6 , was identified by mass screening the Parke-Davis compound portfolio. The active moiety of compound 6 was determined to be the catechol functionality. Additional analogs were prepared and analyzed for inhibitory activity against 5-, 12-, and 15- lipoxygenase.