Molecular sieving and mass spectroscopy reveal enhanced collagen degradation in rabbit atheroma.

BACKGROUND: Collagen degradation is the major mechanism of atherosclerotic plaque destabilization. It is unknown whether collagen breakdown is involved into formation of early atherosclerotic lesions. METHODS: Current paper describes a novel collagen degradation assay based on a combination of molecular sieving and mass spectroscopy. The first step of the assay consists of the extraction of total collagen from tissue. This extract includes both intact collagen and its breakdown products. Molecular sieving is used to isolate low molecular weight collagen fragments. Since the low molecular weight fraction of the extract may contain some non-collagenous molecular species, the collagen-specific amino acid hydroxyproline is quantified using mass spectroscopy. RESULTS: This assay was validated in various experimental systems with known/predictable level of collagen breakdown in vitro, ex vivo and in vivo. When applied to cholesterol-fed rabbit aorta, it revealed enhanced collagen degradation in rabbit atheromas compared to unaffected aortic regions. CONCLUSION: A novel assay has been developed to demonstrate enhanced collagen degradation in rabbit atherosclerotic plaques. Accurate quantification of collagen breakdown products should provide a new relevant end point in the analysis of plaque development and stability.

The combined effect of inhibiting both ACAT and HMG-CoA reductase may directly induce atherosclerotic lesion regression.

We hypothesized that coadministration of avasimibe and simvastatin would limit size, composition and extent of atherosclerotic lesions and potentially promote lesion regression, since bioavailable ACAT inhibitors decrease monocyte-macrophage enrichment and HMG-CoA reductase inhibitors limit smooth muscle cell migration and proliferation. Male New Zealand white rabbits were sequentially fed a 0.5% cholesterol, 3% peanut oil, 3% coconut oil diet for 9 weeks and a chow-fat diet for 6 weeks prior to drug administration. A time zero control group was necropsied prior to drug administration and the progression control was fed various diets but untreated. Avasimibe (10 mg/kg), simvastatin (2.5 mg/kg) or combination of avasimibe (10 mg/kg) with simvastatin (2.5 mg/kg) were administered in the chow-fat diet for 8 weeks. Plasma total cholesterol exposure was unchanged by avasimibe but was reduced 21% by both simvastatin alone and in combination with avasimibe. Combination of avasimibe and simvastatin decreased VLDL-cholesterol exposure by 56%. VLDL+IDL lipid composition was similar in the progression control and simvastatin-treated animals. Administration of avasimibe alone or in combination with simvastatin reduced the cholesteryl ester fraction and increased the triglyceride fraction to comparable extents. Relative to the progression control, avasimibe plus simvastatin markedly decreased thoracic aortic cholesteryl ester content and lesion coverage by 50% and aortic arch lesion size and macrophage area by 75 and 73%, respectively. With respect to lesion regression, avasimibe+simvastatin decreased aortic arch lesion size by 64% and monocyte-macrophage area by 73% when compared to time zero. Based on these data, we conclude that despite changes in plasma total and lipoprotein cholesterol exposure and lipoprotein composition comparable to monotherapy, inhibition of both ACAT and HMG-CoA reductase may not only directly blunt lesion progression but also promote regression of pre-established atherosclerotic lesions.

Hypercholesterolemia causes mechanical weakening of rabbit atheroma : local collagen loss as a prerequisite of plaque rupture.

Hypercholesterolemia may render atherosclerotic plaques prone to rupture. To test this hypothesis, catheters with matrix-covered balloons were implanted into the aorta of rabbits fed standard or 0. 5% cholesterol chow (n=70). In 1 month, fibrous plaques developed around the balloon. Time-dependent accumulation of cholesteryl esters and free cholesterol was detected in the plaques of the cholesterol-fed group only. The pressure needed to rupture the plaque by balloon inflation was used as an index of plaque strength. Three months after the catheter implantation, the breaking pressure was 2.1 times lower (P<0.05) in cholesterol-fed rabbits. It was accompanied by collagen loss, as measured by plaque hydroxyproline content, but not with deficiency of collagen cross-linking. Sirius red staining showed preservation of collagen originally covering the balloon and accumulation of nascent collagen in the lesions of standard chow-fed rabbits. In the cholesterol-fed group, both mature and new collagen underwent degradation predominantly in the plaque shoulders. Collagen breakdown was associated with local accumulation of foamy macrophages. Gel zymography demonstrated relative enhancement of gelatinolytic activity at 92 and 72 kDa, as well as caseinolytic activity at 57, 45, and 19 kDa in the lipid-laden plaques. Lipid accumulation in the plaque was also associated with a loss of smooth muscle cells, the cellular source of the collagen fibers. The remaining smooth muscle cells showed increased collagen synthesis, although it was insufficient to counterbalance collagen degradation and cell loss. Thus, we have obtained direct evidence that hypercholesterolemia is accompanied by enhanced local collagen degradation, which is potentially responsible for plaque weakening.

The ACAT inhibitor avasimibe reduces macrophages and matrix metalloproteinase expression in atherosclerotic lesions of hypercholesterolemic rabbits.

Given the significance of cholesteryl ester (CE) accumulation in macrophage foam cell formation, we hypothesized that inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT) would produce a histologically stable lesion by limiting macrophage enrichment and thereby a source of matrix metalloproteinases (MMPs). Male New Zealand White rabbits were sequentially fed a cholesterol/fat diet for 9 weeks, a fat-only diet for 6 weeks, and 25 mg/kg avasimibe for 7 to 8 weeks. Avasimibe had no effect on plasma total cholesterol exposure. Plasma avasimibe maximal concentration and 24-hour area-under-the-curve levels were 178 ng/mL and 2525 ng. h/mL, respectively, after 7 weeks of treatment with 25 mg/kg avasimibe. The median inhibitory concentration against human monocyte-macrophage ACAT was 12 ng/mL when determined in the absence of albumin, and aortic arch avasimibe levels were 25 ng/g of tissue wet weight. Avasimibe reduced thoracic aortic and iliac-femoral CE content by 39%, the extent of thoracic aortic lesions by 41%, aortic arch cross-sectional lesions area by 35%, and monocyte-macrophage area by 27%. The reduction in monocyte-macrophage area reflected a change in cell number and not cell size. In the iliac-femoral artery, avasimibe decreased monocyte-macrophage content by 77% and reduced the macrophage-to-lesion ratio from 0.16 to 0.05. Within the aortic arch, the catalytic activity of latent and active MMP-9 was reduced by 65% and 33%, respectively; latent and active MMP-1 and MMP-3 activity measured collectively was decreased by 52% and 60%, respectively, and MMP-2 was unchanged. Aortic arch MMP-9, tissue inhibitor of matrix metalloproteinase (TIMP)-1, and TIMP-2 mRNA levels were reduced 29% to 39%, and MMP-2 mRNA levels increased. We conclude that the bioavailable ACAT inhibitor avasimibe can directly limit macrophage accumulation, resulting in the histological appearance of mainly fibromuscular lesions, and can potentially stabilize preestablished atherosclerotic lesions by reducing MMP expression within the lesion.

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.

Inhibition of interleukin 1-induced biosynthesis of stromelysin by the calcium antagonist TMB-8 (8-(N, N-diethylamino)octyl-3,4,5-trimethoxybenzoate HCl).

This study was done to identify agents that can inhibit interleukin 1 (IL1)-induced stromelysin biosynthesis and to gain insight into the mechanism of IL1 action. For this purpose, various agents known to modulate calcium-dependent signal transduction pathway were evaluated in rabbit synovial fibroblast (RSF) cultures. Only the conditioned medium from RSF treated with the intracellular calcium antagonist TMB-8 (8-(N,N-diethylamino)-octyl 3,4,5-trimethoxybenzoate hydrochloride) had significantly lower proteoglycan-degrading metalloproteinase activity than controls. Biosynthetic labeling, immunoprecipitation and immunohistochemical studies, using a polyclonal antibody against rabbit stromelysin, demonstrated that TMB-8 inhibited synthesis stromelysin, the proteoglycan-degrading matrix metalloproteinase. Further evaluation of the TMB-8 effect revealed that the compound had no effect on secretion and that it was not acting by preventing activation of the proenzyme or by inhibiting the enzyme activity. These results suggest that TMB-8 may be inhibiting stromelysin synthesis by limiting intracellular calcium levels.

Insulin-secreting pancreatic (islet cell) carcinoma in a cat.

A functional, insulin-secreting pancreatic (islet cell) carcinoma was diagnosed in a 17-year-old male Siamese cat. Diagnosis was made on the basis of clinical signs (i.e., seizures and stupor) that resolved temporarily after correction of hypoglycemia with feeding or intravenous administration of glucose, the finding of an inappropriately increased serum insulin concentration in the face of hypoglycemia, and prolonged resolution of hypoglycemia after surgical removal of the tumor. Primary islet cell tumor of the pancreas was confirmed by biopsy. The cat died 18 months later, and necropsy revealed metastases to regional lymph nodes and liver. Specimens of the tumor and metastatic lesions both stained positively for insulin.