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Objective— To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)–lipoprotein levels. Approach and Results— Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression ( Apoe h/h Ldlr –/– Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non–high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C high monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions— Restoring apoE expression in Apoe h/h Ldlr –/– Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non–high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C high monocytes levels and genetic reprogramming of lesional macrophages.

Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5β1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)–PINCH–parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.

FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-β and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-β and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

Objectives— Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. Methods and Results— We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE ( Apoe T h/h or Apoe R h/h mice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, Apoe R h/h mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with Apoe T h/h mice. In addition, evidence of cellular dysfunction was identified in peritoneal Apoe R h/h macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. Conclusions— These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

Background Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis. Methodology/Principal Findings Hypomorphic apoE (Apoe h/h) mice expressing wildtype mouse apoE at ∼2–5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoe h/h allele in Apoe h/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoe h/hLysM-Cre and Apoe h/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoe h/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoe h/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7). On HCD, Apoe h/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoe h/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoe h/h mice (167×103±16×103 µm2 versus 259×103±56×103 µm2, n = 7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol. Conclusions/Significance Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.

Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE ( Apoe h/h Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.