Miina K. Öhman

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.

Perivascular visceral adipose tissue induces atherosclerosis in apolipoprotein E deficient mice

OBJECTIVE Epicardial adipose tissue is associated with coronary artery disease, however the causal relationship between perivascular adipose tissue and local atherogenesis is unclear. METHODS AND RESULTS Apolipoprotein E deficient (ApoE(-/-)) mice underwent transplantation of visceral or subcutaneous adipose tissue immediately adjacent to the right common carotid artery. Carotid arteries with fat transplants were analyzed for atherosclerosis by surface oil-red-O staining and cross-sectional analysis. Vascular function of the carotid arteries was assessed using pressure myography. Visceral fat transplants were also performed to ApoE(-/-) mice with neutralization of P-selectin glycoprotein ligand-1 (Psgl-1). Atherosclerosis surface area and lesion thickness were greater in mice receiving the perivascular visceral fat compared to the subcutaneous fat. Mice with visceral fat transplants also displayed more complicated atherosclerotic lesions with evidence of atherothrombosis. Serum Mcp-1 was higher in mice receiving visceral fat transplants compared to subcutaneous transplants. Visceral fat transplantation also caused impaired endothelial-dependent relaxation of the carotid artery. Psgl-1 deficiency or neutralization of Psgl-1 with an anti-Psgl-1 antibody was protective against perivascular visceral adipose tissue-induced atherosclerosis and was associated with reduced Mcp-1 levels. CONCLUSIONS Perivascular visceral fat leads to endothelial dysfunction and accelerated atherosclerosis. This proatherogenic effect of perivascular adipose tissue is blocked by neutralization of Psgl-1.

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.

Visceral Adipose Tissue and Atherosclerosis

Obesity is a risk factor for complications of atherosclerotic vascular disease such as myocardial infarction and stroke. Recent studies have demonstrated that the vascular risk associated with obesity is correlated particularly with visceral adiposity. These clinical observations indicate that various adipose tissue depots may have differential effects on vascular risk. Cellular constituents of adipose tissue secrete cytokines and chemokines that may affect vascular disease. Visceral fat has been demonstrated to express more inflammatory cytokines than subcutaneous fat in obese states. The adipose tissue secretory profile may reflect the influx of macrophages that has been shown to occur with expansion of fat stores. This macrophage infiltration may lead to a chronic low grade, systemic, inflammatory state. Since circulating markers of inflammation are associated with cardiovascular events, the inflammation triggered by adipose tissue may contribute to increased vascular disease. While the vasculopathic effects of visceral obesity may be best treated by weight loss, long term weight loss is difficult to achieve, even with currently available pharmacotherapies. Therapies that target macrophage accumulation in fat or the adipocyte expression profile may be potentially beneficial in reducing the vascular risk associated with obesity. Further characterization of the factors responsible for promoting atherosclerosis in the setting of visceral obesity may lead to new targets for the prevention of atherosclerosis.

Lymphatic Vessel Insufficiency in Hypercholesterolemic Mice Alters Lipoprotein Levels and Promotes Atherogenesis

Objective—Lymphatic vessels collect extravasated fluid and proteins from tissues to blood circulation as well as play an essential role in lipid metabolism by taking up intestinal chylomicrons. Previous studies have shown that impairment of lymphatic vessel function causes lymphedema and fat accumulation, but clear connections between arterial pathologies and lymphatic vessels have not been described. Approach and Results—Two transgenic mouse strains with lymphatic insufficiency (soluble vascular endothelial growth factor 3 [sVEGFR3] and Chy) were crossed with atherosclerotic mice deficient of low-density lipoprotein receptor and apolipoprotein B48 (LDLR−/−/ApoB100/100) to study the effects of insufficient lymphatic vessel transport on lipoprotein metabolism and atherosclerosis. Both sVEGFR3×LDLR−/−/ApoB100/100 mice and Chy×LDLR−/−/ApoB100/100 mice had higher plasma cholesterol levels compared with LDLR−/−/ApoB100/100 control mice during both normal chow diet (16.3 and 13.7 versus 8.2 mmol/L, respectively) and Western-type high-fat diet (eg, after 2 weeks of fat diet, 45.9 and 42.6 versus 30.2 mmol/L, respectively). Cholesterol and triglyceride levels in very-low-density lipoprotein and low-density lipoprotein fractions were increased. Atherosclerotic lesions in young and intermediate cohorts of sVEGFR3×LDLR−/−/ApoB100/100 mice progressed faster than in control mice (eg, intermediate cohort mice at 6 weeks, 18.3% versus 7.7% of the whole aorta, respectively). In addition, lesions in sVEGFR3×LDLR−/−/ApoB100/100 mice and Chy×LDLR−/−/ApoB100/100 mice had much less lymphatic vessels than lesions in control mice (0.33% and 1.07% versus 7.45% of podoplanin-positive vessels, respectively). Conclusions—We show a novel finding linking impaired lymphatic vessels to lipoprotein metabolism, increased plasma cholesterol levels, and enhanced atherogenesis.

P-Selectin Glycoprotein Ligand-1 Regulates Adhesive Properties of the Endothelium and Leukocyte Trafficking Into Adipose Tissue

Rationale Adhesive interactions between endothelial cells and leukocytes affect leukocyte trafficking in adipose tissue. The role of P-selectin glycoprotein ligand-1 (Psgl-1) in this process is unclear. Objective The goal of this study was to determine the effect of Psgl-1 deficiency on adhesive properties of the endothelium and on leukocyte recruitment into obese adipose depots. Methods and Results A genetic model of obesity was generated to study the effects of Psgl-1 deficiency on leukocyte trafficking. Leukocyte-endothelial interactions were increased in obese leptin receptor mutant mice (Leprdb/db,Psgl-1+/+) but not obese Psgl-1–deficient mice (Leprdb/db,Psgl-1−/−), when compared with lean mice (Lepr+/+,Psgl-1+/+). This effect of Psgl-1 deficiency was due to indirect effects of Psgl-1, because Psgl-1+/+ adoptively transferred leukocytes did not exhibit enhanced rolling in Leprdb/db,Psgl-1−/− mice. Additionally, circulating levels of P-selectin, E-selectin, monocyte chemoattractant protein-1, and macrophage content of visceral adipose tissue were reduced in Leprdb/db,Psgl-1−/− compared with Leprdb/db,Psgl-1+/+ mice. Reduced leukocyte-endothelial interactions and macrophage content of visceral adipose tissue due to Psgl-1 deficiency was also observed in a diet-induced obese mouse model. Psgl-1−/− mice were resistant to the endothelial effects of exogenous IL-1&bgr;, suggesting that defective cytokine signaling contributes to the effect of Psgl-1 deficiency on leukocyte-endothelial interactions. Mice deficient in the IL-1 receptor also had reduced levels of circulating P-selectin, similar to those observed in Psgl-1−/− mice. Conclusions Deficiency of Psgl-1 is associated with reduced IL-1 receptor-mediated adhesive properties of the endothelium and is protective against visceral fat inflammation in obese mice.

Monocyte Chemoattractant Protein-1 Deficiency Protects Against Visceral Fat–Induced Atherosclerosis

Objective—To determine the role of monocyte chemoattractant protein-1 (Mcp-1) on the progression of visceral fat–induced atherosclerosis. Methods and Results—Visceral fat inflammation was induced by transplantation of perigonadal fat. To determine whether recipient Mcp-1 status affected atherosclerosis induced by inflammatory fat, apolipoprotein E–deficient (ApoE−/−) and ApoE−/− and Mcp-1–deficient (Mcp-1−/−) mice underwent visceral fat transplantation. Intravital microscopy was used to study leukocyte-endothelial interactions. To study the primary tissue source of circulating Mcp-1, both fat and bone marrow transplantation experiments were used. Transplantation of visceral fat increased atherosclerosis in ApoE−/− mice but had no effect on atherosclerosis in ApoE−/−, Mcp-1−/− mice. Intravital microscopy revealed increased leukocyte attachment to the endothelium in ApoE−/− mice compared with ApoE−/−, Mcp-1−/− mice after receiving visceral fat transplants. Transplantation of visceral fat increased plasma Mcp-1, although donor adipocytes were not the source of circulating Mcp-1 because no Mcp-1 was detected in plasma from ApoE−/−, Mcp-1−/− mice transplanted with Wt fat, indicating that recipient Mcp-1-producing cells were affecting the atherogenic response to the fat transplantation. Consistently, transplantation of Mcp-1−/− fat to ApoE−/− mice did not lead to atheroprotection in recipient mice. Bone marrow transplantation between Wt and Mcp-1−/− mice indicated that the primary tissue source of circulating Mcp-1 was the endothelium. Conclusion—Recipient Mcp-1 deficiency protects against atherosclerosis induced by transplanted visceral adipose tissue.

P-selectin glycoprotein ligand-1 deficiency leads to cytokine resistance and protection against atherosclerosis in apolipoprotein E deficient mice.

Adhesive interactions between endothelial cells and leukocytes contribute to atherosclerotic plaque growth. However, mechanism(s) responsible for endothelial priming and deactivation in inflammatory diseases such as atherosclerosis are not clear. Apolipoprotein E deficient mice were generated with deficiency of P-selectin glycoprotein ligand-1 (Psgl-1(-/-), ApoE(-/-)). On both standard chow and Western diet, Psgl-1(-/-), ApoE(-/-) mice were protected against atherosclerosis compared to Psgl-1(+/+), ApoE(-/-) controls. Psgl-1(-/-), ApoE(-/-) mice also showed reduced leukocyte rolling and firm attachment on endothelial cells, however, adoptively transferred Psgl-1(+/+), ApoE(-/-) leukocytes into Psgl-1(-/-), ApoE(-/-) hosts displayed similar reduced rolling as Psgl-1(-/-), ApoE(-/-) leukocytes. Hematopoietic deficiency of Psgl-1 conferred resistance to the effects of interleukin-1β (IL-1β) on leukocyte rolling along with reduced circulating levels of sP-sel and sE-sel. Antibody blockade of Psgl-1 also reduced endothelial activation in response to IL-1β, eliminated leukocyte rolling, and was protective against atherosclerosis in ApoE(-/-) mice. Monocyte depletion with clodronate restored the endothelial response to IL-1β in Psgl-1(-/-) mice. This study suggests that Psgl-1 deficiency leads to reduced atherosclerosis and adhesive interactions between endothelial cells and leukocytes by indirectly regulating endothelial responses to cytokine stimulation.

Targeting MCP-1 to Reduce Vascular Complications of Obesity

Obesity is a risk factor for complications of atherosclerotic vascular disease such as myocardial infarction. Recent studies and several patents have demonstrated that the cardiovascular risk associated with obesity is correlated particularly with visceral adiposity. Excess visceral adiposity may increase vascular risk due to secretion of cytokines and chemokines by cellular constituents of the adipose tissue. The secretory profile of various adipose depots may be regulated by the influx of macrophages that has been shown to occur with expansion of fat stores. This macrophage infiltration may lead to a chronic low grade, systemic, inflammatory state. Since circulating markers of inflammation are associated with cardiovascular events, the inflammation triggered by visceral fat may contribute to an increased risk for vascular complications. While the vasculopathic effects of central obesity may be best treated by weight loss, long term weight loss is difficult to achieve, even with currently available pharmacotherapies. Therapies that target macrophage accumulation in fat or secretory products of adipose tissue may be potentially beneficial in reducing the vascular risk associated with obesity. A potential therapeutic target is monocyte chemoattractant 1 (MCP-1), which is a potent chemokine that is elevated in obesity. Since MCP-1 promotes atherosclerosis, inhibition of MCP-1 may be effective in reducing the vascular risk associated with obesity.

Atherosclerosis and leukocyte–endothelial adhesive interactions are increased following acute myocardial infarction in apolipoprotein E deficient mice

OBJECTIVE To determine the effect of myocardial infarction (MI) on progression of atherosclerosis in apolipoprotein E deficient (ApoE-/-) mice. METHODS AND RESULTS MI was induced following left anterior descending coronary artery (LAD) ligation in wild-type (WT) (n=9) and ApoE-/- (n=25) mice. Compared to sham-operated animals, MI mice demonstrated increased intravascular leukocyte rolling and firm adhesion by intravital microscopy, reflecting enhanced systemic leukocyte-endothelial interactions. To determine if MI was associated with accelerated atherogenesis, LAD ligation was performed in ApoE-/- mice. Six weeks following surgery, atherosclerosis was quantitated throughout the arterial tree by microdissection and Oil-Red-O staining. There was 1.6-fold greater atherosclerotic burden present in ApoE-/- MI mice compared to sham-operated mice. CONCLUSIONS Acute MI accelerates atherogenesis in mice. These results may be related to the increased risk of recurrent ischemic coronary events following MI in humans.