Yuechun Shen

Visceral Adipose Tissue Inflammation Accelerates Atherosclerosis in Apolipoprotein E–Deficient Mice

Background— Fat inflammation may play an important role in comorbidities associated with obesity such as atherosclerosis. Methods and Results— To first establish feasibility of fat transplantation, epididymal fat pads were harvested from wild-type C57BL/6J mice and transplanted into leptin-deficient (Lepob/ob) mice. Fat transplantation produced physiological leptin levels and prevented obesity and infertility in Lepob/ob mice. However, the transplanted fat depots were associated with chronically increased macrophage infiltration with characteristics identical to those observed in fat harvested from obese animals. The inflammation in transplanted adipose depots was regulated by the same factors that have been implicated in endogenous fat inflammation such as monocyte chemoattractant protein-1. To determine whether this inflamed adipose depot could affect vascular disease in mice, epididymal fat depots were transplanted into atherosclerosis-prone apolipoprotein E-deficient ApoE−/− mice. Plasma from ApoE−/− mice receiving fat transplants contained increased leptin, resistin, and monocyte chemoattractant protein-1 compared with plasma from sham-operated ApoE−/− mice. Furthermore, mice transplanted with visceral fat developed significantly more atherosclerosis compared with sham-operated animals, whereas transplants with subcutaneous fat did not affect atherosclerosis despite a similar degree of fat inflammation. Treatment of transplanted ApoE−/− mice with pioglitazone decreased macrophage content of the transplanted visceral fat pad and reduced plasma monocyte chemoattractant protein-1. Importantly, pioglitazone also reduced atherosclerosis triggered by inflammatory visceral fat but had no protective effect on atherosclerosis in the absence of the visceral fat transplantation. Conclusions— Our results indicate that visceral adipose-related inflammation accelerates atherosclerosis in mice. Drugs such as thiazolidinediones might be a useful strategy to specifically attenuate the vascular disease induced by visceral inflammatory fat.

Recombinant Leptin Promotes Atherosclerosis and Thrombosis in Apolipoprotein E–Deficient Mice

Objective—The direct role of leptin in vascular disease remains controversial. The objective of this study was to examine the effects of leptin treatment on atherosclerosis and thrombosis in atherosclerotic-prone mice. Methods and Results—Sixteen-week-old, male apolipoprotein E–deficient mice were treated with injections of recombinant leptin (125 &mgr;g per day IP; n=10) or vehicle (n=10) for 4 weeks. Leptin treatment resulted in reduced epididymal fat (352±30.7 versus 621±61.5 mg; P=0.005) and fasting insulin (0.57±0.25 versus 1.7±0.22 ng/mL; P=0.014). Despite these metabolic benefits, leptin treatment resulted in an increase in atherosclerosis (8.0±0.95% versus 5.4±0.59% lesion surface coverage; P<0.05). Leptin treatment also resulted in a shortened time to occlusive thrombosis after vascular injury (21±2.1 versus 34.6±5.4 minutes; P=0.045). Conclusions—These studies indicate that exogenous leptin promotes atherosclerosis and thrombosis and support the concept that elevations of leptin may increase the risk for cardiovascular disease.

α-Galactosidase A Deficiency Accelerates Atherosclerosis in Mice With Apolipoprotein E Deficiency

Background—&agr;-Galactosidase A (Gla) deficiency leads to widespread tissue accumulation of neutral glycosphingolipids and is associated with premature vascular complications such as myocardial infarction and stroke. Glycosphingolipids have been shown to accumulate in human atherosclerotic lesions, although their role in atherogenesis is unclear. Methods and Results—To determine whether Gla affects the progression of atherosclerosis, mice were generated with combined deficiencies of apolipoprotein E and Gla. At 45 weeks of age, Gla-deficient mice had developed more atherosclerosis than mice with normal Gla expression (25.1±14.0 versus 12.3±9.3 mm2 of total lesion area, P<0.02). This increase in atherosclerosis was associated with the presence of increased Gb3, enhanced inducible nitric oxide synthase expression, and increased nitrotyrosine staining. Conclusions—These findings suggest that deficiency of Gla leads to increased inducible nitric oxide synthase expression and accelerated atherosclerosis.

Leptin Regulates Neointima Formation After Arterial Injury Through Mechanisms Independent of Blood Pressure and the Leptin Receptor/STAT3 Signaling Pathways Involved in Energy Balance

Background—Leptin is an adipocyte-derived hormone critical for energy homeostasis and implicated in vascular disease processes. The relevant cellular leptin receptor pools and signaling pathways involved in leptin-related vascular phenotypes in vivo are unclear. Methods and Results—Arterial injury was induced in wild-type (wt), leptin-deficient (lepob/ob), and leptin receptor–deficient (leprdb/db) mice. Compared with wt mice, lepob/ob and leprdb/db mice were protected from the development of neointima. Bone marrow transplantation experiments between wt and leprdb/db mice indicated that the vascular protection in leprdb/db mice was not attributable to lack of leptin receptor expression on bone marrow–derived elements. To investigate the role of the lepr-mediated signal transducer and activator of transcription 3 (STAT3) signaling pathway in the response to vascular injury, leprs/s mice homozygous for a leptin receptor defective in STAT3 signaling underwent femoral arterial injury. Despite similar obesity and blood pressure levels, the neointimal area in leprs/s mice was significantly increased compared with leprdb/db mice. Conclusions—The molecular mechanism by which the leptin receptor mediates neointima formation and vascular smooth muscle cell proliferation is largely independent of the STAT3-dependent signaling pathways involved in energy balance.

Fabry Disease in Mice Is Associated With Age-Dependent Susceptibility to Vascular Thrombosis

Fabry disease is an X-linked lysosomal storage disorder due to deficiency of alpha-galactosidase A (GLA) activity that results in the widespread accumulation of neutral glycosphingolipids. Renal failure, neuropathy, premature myocardial infarction, and stroke occur in patients with this condition primarily due to deposition of glycosphingolipids in vascular endothelial cells. The clinical consequences of Fabry disease suggest that vascular thrombosis may play a prominent role in the pathogenesis of this disease; however, the vasculopathy associated with Fabry disease has not been extensively studied. To determine if mice genetically deficient in Gla are susceptible to vascular thrombosis, a photochemical carotid injury model was used to induce occlusive thrombosis. In this model, Gla-/0 mice displayed a progressive age-dependent shortening of the time to occlusive thrombosis after vascular injury that correlated with progressive accumulation of globotriasylceramide (Gb3) in the arterial wall. Bone marrow transplantation from Gla-/0 to Gla+/0 mice and from Gla+/0 to Gla-/0 mice did not change the thrombotic phenotype of the host. These studies reveal a potent vascular prothrombotic phenotype in Gla-deficient mice and suggest that antithrombotic therapies as well as therapies designed to reduce the vascular accumulation of Gb3 may have beneficial effects on thrombotic complications in patients with Fabry disease.

Homozygosity for Factor V Leiden Leads to Enhanced Thrombosis and Atherosclerosis in Mice

Background—Activated protein C resistance due to factor V Leiden (FVL) is a common genetic risk factor for venous thrombosis in humans. Although the impact of FVL on the development of venous thrombosis is well established, its effect on arterial thrombosis and atherosclerosis is controversial. Methods and Results—To determine the effect of the FVL mutation on arterial thrombosis in the mouse, wild-type (Fv+/+), heterozygous FVL (FvQ/+), and homozygous FVL (FvQ/Q) mice underwent photochemical carotid arterial injury to induce occlusive thrombosis. FvQ/Q mice formed occlusive thromboses 27±3 minutes (n=7) after the onset of injury, which was significantly shorter than that observed for Fv+/+ mice (56±7 minutes, n=9, P<0.01), whereas FvQ/+ mice (41±7 minutes, n=5) were intermediate (P=0.5, compared with Fv+/+). To determine the source of FVL relevant to the enhanced vascular thrombosis, bone marrow transplantation experiments were performed between Fv+/+ and FvQ/Q mice. FvQ/Q mice transplanted with Fv+/+ bone marrow formed occlusive thromboses at 35±5 minutes (n=7, P<0.05 compared with Fv+/+ mice), whereas Fv+/+ mice transplanted with FvQ/Q bone marrow occluded at 59±7 minutes (n=6, P<0.001 compared with FvQ/Q mice). To assess the effect of the FVL mutation on the development of atherosclerosis, FvQ/Q mice were crossed with the atherosclerosis-prone apolipoprotein E (ApoE)–deficient strain (ApoE−/−) to generate FvQ/Q,ApoE−/− mice. By 52 weeks of age, FvQ/Q,ApoE−/− mice (n=8) had developed more aortic atherosclerosis (40±6% lesion area) compared with Fv+/+,ApoE−/− mice (15±3% lesion area; n=12, P<0.02). Conclusions—In conclusion, homozygosity for the FVL mutation in mice leads to enhanced arterial thrombosis and atherosclerosis. The source of the FVL leading to accelerated thrombosis appears to be circulating, non–platelet-derived plasma FVL.

Atherosclerosis in Mice Is Not Affected by a Reduction in Tissue Factor Expression

Objective—To determine whether tissue factor (TF) contributes to the progression of atherosclerotic lesions in mice. Methods and Results—We determined the effect of a 50% reduction of TF levels in all cells on atherosclerosis in apolipoprotein E-deficient (apoE−/−) mice. No differences were observed in the extent of atherosclerosis in apoE−/−/TF+/+ and apoE−/−/TF+/− mice fed regular chow for 34 weeks. Atherosclerosis could not be analyzed in apoE−/− mice expressing low levels of TF because of premature death of these mice. Macrophages are a major source of TF in atherosclerotic plaques. Therefore, in a second series of experiments, we investigated the effect on atherosclerosis of selectively reducing hematopoietic cell-derived TF by transplanting bone marrow from mice expressing low levels of TF into low-density lipoprotein receptor deficient (LDLR−/−) mice. Atherosclerosis within the arterial tree and aortic root were similar in LDLR−/− mice with low-TF bone marrow compared with control bone marrow (TF+/+ or TF+/−) after 4 and 16 weeks on an atherogenic diet. Furthermore, the cellular composition of the aortic root lesions was similar between the 2 groups. Conclusions—Our data indicate that either a 50% reduction of TF in all cells or a selective reduction in hematopoietic cell-derived TF does not affect the development of atherosclerotic lesions in mice.

α-Galactosidase A Deficiency Leads to Increased Tissue Fibrin Deposition and Thrombosis in Mice Homozygous for the Factor V Leiden Mutation

Background— Factor V Leiden (FVL) is a common genetic risk factor for vascular thrombosis in humans. Fabry disease, an X-linked lysosomal storage disorder attributable to α-galactosidase A (GLA) deficiency, is associated with premature vascular events that may be thrombotic in nature. Methods and Results— To examine a potential interaction between FvL and Gla deficiency in vivo, we analyzed tissue fibrin deposition in mice carrying combined mutations in FvL and Gla. Gla deficiency markedly increased tissue fibrin deposition in mice carrying the FvL mutation (0.33±0.03%; n=7) compared with FvL mutation (0.14±0.02%; n=10; P<0.0005). Conclusions— These observations demonstrate a synergistic interaction between Gla deficiency and FvL toward tissue fibrin deposition in mice. Concomitant mutations in these genes may increase the penetrance of vascular thrombotic events in humans.

Leptin receptor-induced STAT3-independent signaling pathways are protective against atherosclerosis in a murine model of obesity and hyperlipidemia

AIMS Leptin is an adipocyte-derived hormone that has been shown to exert both beneficial metabolic effects and potentially adverse vascular effects in preclinical studies. The primary aim of this study was to determine the effects of leptin receptor signaling pathways on atherosclerosis in the setting of obesity and hyperlipidemia. METHODS AND RESULTS Mice were generated with deficiency of apolipoprotein E (ApoE(-/-)) and either wild-type leptin receptor expression (Lepr(+/+), ApoE(-/-)), mutant leptin receptor expression defective in all leptin receptor signaling pathways (Lepr(db/db), ApoE(-/-)), or mutant leptin receptor expression with selective deficiency of leptin receptor-STAT3 signaling (Lepr(s/s), ApoE(-/-)). At 27 weeks of age (including 7 weeks on a Western diet), Lepr(db/db), ApoE(-/-) developed severe obesity, hypercholesterolemia, and increased atherosclerosis compared to Lepr(+/+), ApoE(-/-) mice. Despite similar obesity and hyperlipidemia to Lepr(db/db), ApoE(-/-) mice, Lepr(s/s), ApoE(-/-) developed less atherosclerosis than Lepr(db/db), ApoE(-/-) mice. Adipose tissue macrophage content, monocyte chemoattractant protein-1 and fatty-acid-binding protein 4 levels were also reduced in Lepr(s/s), ApoE(-/-) mice compared to Lepr(db/db), ApoE(-/-) mice. CONCLUSIONS In a mouse model of obesity and hyperlipidemia, leptin receptor-mediated STAT3-independent signaling pathways confer protection against atherosclerosis. These differences occur independently of leptin effects on energy balance.