Peter F. Bodary

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.

Pioglitazone protects against thrombosis in a mouse model of obesity and insulin resistance.

Summary.  Background: As arterial thrombosis accounts for the vast majority of cardiovascular complications in obese, insulin resistant patients, we hypothesized that improving insulin sensitivity may be effective in reducing the thrombotic response following vascular injury. Objectives: We investigated the effect of the thiazolidinedione drug, pioglitazone, on the thrombotic response to injury in obese, insulin resistant mice. Methods: Insulin‐resistant, obesity‐prone mice (KK strain) were treated with pioglitazone, placebo, or the sulfonylurea compound, glipizide, for 2.5 weeks and then subjected to photochemical injury of the carotid artery. Results: KK mice have a significant increase in adiposity (7 weeks: 25.6%; 15 weeks: 34.4%; P < 0.0001) and thrombotic tendency (7 weeks: 21.2 ± 1.9 min; 15 weeks: 13.7 ± 1.7 min; P < 0.01) with age. Pioglitazone provided significant protection from thrombosis at both time points, prolonging the time to occlusive thrombosis by 40% and 68%, at 7 and 15 weeks of age, respectively (P < 0.05). Similarly, following a diet‐challenge to promote diabetes, pioglitazone provided protection from occlusive thrombus formation (Placebo: 11.3 ± 1.0 min; Pioglitazone: 22.3 ± 3.9 min; P < 0.05). However, despite a salient effect of glipizide on the hyperglycemia of the mice, there was no effect on the time to occlusive thrombus formation (13.2 ± 0.9 min, n = 4) compared with placebo‐treated mice. The pioglitazone protection was paralleled by significantly lower soluble P‐selectin and platelet P‐selectin expression providing evidence of an antiplatelet effect. Conclusions: We conclude that pioglitazone treatment provides protection against arterial thrombosis in an obese, insulin resistant, prothrombotic mouse model.

Obesity-Induced Endothelial Dysfunction Is Prevented by Deficiency of P-Selectin Glycoprotein Ligand-1

Endothelial dysfunction precedes atherosclerosis and represents an important link between obesity and cardiovascular events. Strategies designed to prevent endothelial dysfunction may therefore reduce the cardiovascular complications triggered by obesity. We tested the hypothesis that deficiency of P-selectin glycoprotein ligand-1 (Psgl-1) would improve the endothelial dysfunction associated with obesity. Psgl-1-deficient (Psgl-1−/−) and wild-type (Psgl-1+/+) mice were fed standard chow or a high-fat, high-sucrose diet (diet-induced obesity [DIO]) for 10 weeks. DIO increased mesenteric perivascular adipose tissue (mPVAT) macrophage content and vascular oxidative stress in Psgl-1+/+ mice but not in Psgl-1−/− mice. Pressure myography using mesenteric arteries demonstrated that relaxation responses to acetylcholine were significantly impaired in DIO Psgl-1+/+ mice, whereas DIO Psgl-1−/− mice were protected from endothelial dysfunction with similar relaxation responses to Psgl-1+/+ or Psgl-1−/− mice fed standard chow. The superoxide scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy (TEMPOL) partially recovered impaired endothelial function induced by DIO. A neutralizing Psgl-1 antibody was also effective in preventing endothelial dysfunction and reducing mPVAT macrophage content induced by DIO. These results indicate that obesity in mice leads to PVAT inflammation and endothelial dysfunction that is prevented by Psgl-1 deficiency. Psgl-1 inhibition may be a useful treatment strategy for targeting vascular disease associated with obesity.

Novel astaxanthin prodrug (CDX-085) attenuates thrombosis in a mouse model

BACKGROUND Cardiovascular disease remains the leading cause of morbidity and premature mortality in most industrialized countries as well as in developing nations. A pro-oxidative state appears to promote and/or exacerbate vascular disease complications. Furthermore, a state of low-grade chronic inflammation can promote increased oxidative stress and lead to endothelial cell and platelet dysfunction ultimately contributing to thrombogenesis. OBJECTIVES In this study, the effect of a proprietary astaxanthin prodrug (CDX-085) on thrombus formation was investigated using a mouse model of arterial thrombosis. The influence of free astaxanthin, the active drug of CDX-085, on human endothelial cells and rat platelets was evaluated to investigate potential mechanisms of action. METHODS AND RESULTS Oral administration of CDX-085 (0.4% in chow, approximately 500 mg/kg/day) to 6-8 week old C57BL/6 male mice for 14 days resulted in significant levels of free astaxanthin in the plasma, liver, heart and platelets. When compared to control mice, the CDX-085 fed group exhibited significant increases in basal arterial blood flow and significant delays in occlusive thrombus formation following the onset of vascular endothelial injury. Primary human umbilical vein endothelial cells (HUVECs) and platelets isolated from Wistar-Kyoto rats treated with free astaxanthin demonstrated significantly increased levels of released nitric oxide (NO) and significantly decreased peroxynitrite (ONOO-) levels. CONCLUSION Observations of increased NO and decreased ONOO- levels in endothelial cells and platelets support a potential mechanism of action for astaxanthin (CDX-085 active drug). These studies support the potential of CDX-085 and its metabolite astaxanthin in the treatment or prevention of thrombotic cardiovascular complications.

Blood Pressure and Vascular Effects of Leptin in Humans

BACKGROUND Leptin may play a role in mediating obesity-related hypertension. However, its effects on the vasculature and blood pressure (BP) remain poorly defined in humans. METHODS In the first study, we performed a short-term, placebo-controlled, randomized, double-blind, cross-over experiment investigating the actions of recombinant human leptin (r-metHuLeptin) in 15 nonobese adults. To compliment the acute study, we retrospectively analyzed available BP results from a previously performed 85-day, placebo-controlled, randomized, double-blind, parallel weight-loss study using r-metHuLeptin in 284 obese adults. RESULTS In the acute study, conduit artery endothelial function determined by brachial flow-mediated dilatation (FMD) increased 2 hours following 0.2 mg . Kg(1) subcutaneously (SC) of r-metHuLeptin versus placebo (+3.3% versus -2.8%, P = .02). BP remained unchanged 4 hours after injections. In the retrospective analysis of the weight loss study data, 10 mg every day before noon (QAM), 10 mg every day after noon (QPM), or 10 mg twice a day (BID) SC of r-metHuLeptin was found to not alter the degree of weight loss (-3.2 +/- 3.7 versus -2.9 +/- 3.2 Kg, P = .54), change in systolic (-1.6 + 12.9 versus -2.0 +/- 13.9 mmHg, P = .85) and diastolic BP (-0.2 +/- 8.7 versus -1.5 +/- 8.6, P = .30), as well as heart rate (-1.4 +/- 10.7 versus -1.4 +/- 10.4 beats/min, P = .98) compared to placebo. CONCLUSIONS In our acute study, marked hyperleptinemia rapidly enhanced endothelial function and did not alter BP. The available data from a longer-term study in healthy obese adults did not demonstrate a significant effect of hyperleptinemia upon BP. These combined findings do not support a direct role for leptin in linking obesity to hypertension, however more studies are required to corroborate these observations.

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.