Human Aldose Reductase Expression Prevents Atherosclerosis Regression in Diabetic Mice.

Guidelines to reduce cardiovascular risk in diabetes include aggressive LDL lowering, but benefits are attenuated compared with those in patients without diabetes. Consistent with this, we have reported in mice that hyperglycemia impaired atherosclerosis regression. Aldose reductase (AR) is thought to contribute to clinical complications of diabetes by directing glucose into pathways producing inflammatory metabolites. Mice have low levels of AR, thus raising them to human levels would be a more clinically relevant model to study changes in diabetes under atherosclerosis regression conditions. Donor aortae from Western diet-fed Ldlr-/- mice were transplanted into normolipidemic wild-type, Ins2Akita (Akita+/- , insulin deficient), human AR (hAR) transgenic, or Akita+/- /hAR mice. Akita+/- mice had impaired plaque regression as measured by changes in plaque size and the contents of CD68+ cells (macrophages), lipids, and collagen. Supporting synergy between hyperglycemia and hAR were the even more pronounced changes in these parameters in Akita+/- /hAR mice, which had atherosclerosis progression in spite of normolipidemia. Plaque CD68+ cells from the Akita+/- /hAR mice had increased oxidant stress and expression of inflammation-associated genes but decreased expression of anti-inflammatory genes. In summary, hAR expression amplifies impaired atherosclerosis regression in diabetic mice, likely by interfering with the expected reduction in plaque macrophage inflammation.

Hypoxia is present in murine atherosclerotic plaques and has multiple adverse effects on macrophage lipid metabolism.

RATIONALE: Human atherosclerotic plaques contain large numbers of cells deprived of O(2). In murine atherosclerosis, because the plaques are small, it is controversial whether hypoxia can occur. OBJECTIVE: To examine if murine plaques contain hypoxic cells, and whether hypoxia regulates changes in cellular lipid metabolism and gene expression in macrophages. METHODS AND RESULTS: Aortic plaques from apolipoprotein-E-deficient mice were immunopositive for hypoxia-inducible transcription factor (HIF-1α) and some of its downstream targets. Murine J774 macrophages rendered hypoxic demonstrated significant increases in cellular sterol and triglycerides. The increase in sterol content in hypoxic macrophages correlated with elevated 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase activity and mRNA levels. In addition, when macrophages were incubated with cholesterol complexes, hypoxic cells accumulated 120% more cholesterol, predominately in the free form. Cholesterol-efflux assays showed that hypoxia significantly decreased efflux mediated by ATP-binding cassette subfamily A member 1 (ABCA1), whose sub cellular localization was altered in both J774 and primary macrophages. Furthermore, in vivo expression patterns of selected genes from cells in hypoxic regions of murine plaques were similar to those from J774 and primary macrophages incubated in hypoxia. The hypoxia-induced accumulation of sterol and decreased cholesterol efflux was substantially reversed in vitro by reducing the expression of the hypoxia-inducible transcription factor, HIF-1α. CONCLUSION: Hypoxic regions are present in murine plaques. Hypoxic macrophages have increased sterol content due to the induction of sterol synthesis and the suppression of cholesterol efflux, effects that are in part mediated by HIF-1α.

Diabetes adversely affects macrophages during atherosclerotic plaque regression in mice.

OBJECTIVE: Patients with diabetes have increased cardiovascular risk. Atherosclerosis in these patients is often associated with increased plaque macrophages and dyslipidemia. We hypothesized that diabetic atherosclerosis involves processes that impair favorable effects of lipid reduction on plaque macrophages. RESEARCH DESIGN AND METHODS: Reversa mice are LDL receptor-deficient mice that develop atherosclerosis. Their elevated plasma LDL levels are lowered after conditional knockout of the gene encoding microsomal triglyceride transfer protein. We examined the morphologic and molecular changes in atherosclerotic plaques in control and streptozotocin-induced diabetic Reversa mice after LDL lowering. Bone marrow-derived macrophages were also used to study changes mediated by hyperglycemia. RESULTS: Reversa mice were fed a western diet for 16 weeks to develop plaques (baseline). Four weeks after lipid normalization, control (nondiabetic) mice had reduced plasma cholesterol (-77%), plaque cholesterol (-53%), and plaque cells positive for macrophage marker CD68+ (-73%), but increased plaque collagen (+116%) compared with baseline mice. Diabetic mice had similarly reduced plasma cholesterol, but collagen content increased by only 34% compared with baseline; compared with control mice, there were lower reductions in plaque cholesterol (-30%) and CD68+ cells (-41%). Diabetic (vs. control) plaque CD68+ cells also exhibited more oxidant stress and inflammatory gene expression and less polarization toward the anti-inflammatory M2 macrophage state. Many of the findings in vivo were recapitulated by hyperglycemia in mouse bone marrow-derived macrophages. CONCLUSIONS: Diabetes hindered plaque regression in atherosclerotic mice (based on CD68+ plaque content) and favorable changes in plaque macrophage characteristics after the reduction of elevated plasma LDL.

Reversal of hyperlipidemia with a genetic switch favorably affects the content and inflammatory state of macrophages in atherosclerotic plaques.

BACKGROUND: We previously showed that the progression of atherosclerosis in the Reversa mouse (Ldlr(-/-Apob100/100Mttpfl/fl) Mx1Cre(+/+)) was arrested when the hyperlipidemia was normalized by inactivating the gene for microsomal triglyceride transfer protein. Here, we tested whether atherosclerosis would regress if the lipid levels were reduced after advanced plaques formed. METHODS AND RESULTS: Reversa mice were fed an atherogenic diet for 16 weeks. Plasma lipid levels were then reduced. Within 2 weeks, this reduction led to decreased monocyte-derived (CD68(+)) cells in atherosclerotic plaques and was associated with emigration of these cells out of plaques. In addition, the fall in lipid levels was accompanied by lower plaque lipid content and by reduced expression in plaque CD68(+) cells of inflammatory genes and higher expression of genes for markers of antiinflammatory M2 macrophages. Plaque composition was affected more than plaque size, with the decreased content of lipid and CD68(+) cells balanced by a higher content of collagen. When the reduced lipid level was combined with the administration of pioglitazone to simulate the clinical aggressive lipid management and proliferator-activated receptor-γ agonist treatment, the rate of depletion of plaque CD68(+) cells was unaffected, but there was a further increase in their expression of antiinflammatory macrophage markers. CONCLUSION: The Reversa mouse is a new model of atherosclerosis regression. After lipid lowering, favorable changes in plaque composition were independent of changes in size. In addition, plaque CD68(+) cells became less inflammatory, an effect enhanced by treatment with pioglitazone.