Loss of Antigen Presentation in Adipose Tissue Macrophages or in Adipocytes, but Not Both, Improves Glucose Metabolism.

Macrophages, B cells, and adipocytes are among the adipose tissue (AT) APCs that differentiate and activate naive CD4+ T cells. Mice with adipocyte loss of MHC class II (MHC II) are more insulin sensitive. Because macrophages are professional APCs, mice with genetic myeloid MHC II depletion (myeloid MHC II knockout [mMHCII-/-]) were created and metabolically characterized. FITC+ glucan-coated particles (glucan-encapsulated small interfering RNA [siRNA] particles [GeRPs]) were also used to target MHC II knockout specifically in AT macrophages (ATMs). Mice with total body mMHCII-/- were generated by crossing LyzMCre with H2Ab1 floxed mice. For specific ATM depletion of H2Ab1, GeRPs containing H2Ab1 siRNA were administered to high-fat diet-fed C57BL/6 mice. Unexpectedly, mMHCII-/- mice had loss of both macrophage and adipocyte H2Ab1, one of only two Ag-presenting arms; thus, neither cell could present Ag and activate CD4+ T cells. This inability led to a reduction in AT immunosuppressive regulatory T cells, increased AT CD8+ T cells, and no improvement in systemic metabolism. Thus, with combined systemic myeloid and adipocyte MHC II loss, the impact of ATM-specific alterations in APC activity could not be delineated. Therefore, GeRPs containing H2Ab1 siRNA were administered to specifically reduce ATM H2Ab1 which, in contrast, revealed improved glucose tolerance. In conclusion, loss of either ATM or adipocyte APC function, but not both, improves systemic glucose metabolism because of maintenance of AT regulatory T cells.

Low visceral adipose tissue regulatory T cells are associated with higher comorbidity severity in patients undergoing bariatric surgery.

BACKGROUND: Adipose tissue (AT) inflammation is linked to the development of type 2 diabetes (T2DM) and atherosclerosis in murine models of obesity. Reduced AT regulatory T cells (Tregs), which are anti-inflammatory immune cells, play an important part in this pathogenesis, and we have shown that AT-Tregs are inversely correlated to increasing body-mass-index. The purpose of this study was to evaluate the association between AT-Treg abundance and comorbidity status in patients undergoing bariatric surgery (BS). METHODS: Visceral (intra-abdominal) AT was harvested at that time of primary BS (n = 80) and collagenase digested. AT-Treg abundance (CD4+/CD25+/FOXP3+) was characterized using flow cytometry from the AT stromal vascular fraction. The median AT-Treg abundance (3.03%) was utilized to define high (High-Tregs, n = 39) and low AT-Treg (low-Tregs, n = 38) abundance within this cohort. These two groups were compared in terms of baseline demographic data, preoperative obesity-related comorbidities, glycemic parameters, including insulin resistance (HOMA-IR). RESULTS: Age, excess body weight, and sex were not different between groups. Prevalence of hypertension, hyperlipidemia, or T2DM preoperatively were not different between groups. Compared to High-Tregs, patients with low-Tregs were more likely to have insulin-dependent type 2 diabetes (12.5% vs 2.9%, p = 0.04). Within patients with T2DM, low-Treg patients had higher plasma insulin levels compared to high-Tregs (31.8 (28.4-56.5) vs 15.5 (10.1-23.1), p = 0.04) and trended towards higher insulin resistance (HOMA-IR) (9.0 (5.3-18.3) vs 3.5 (2.2-7.7), p = 0.08). Within those diagnosed with hyperlipidemia, preoperative statin use was higher in Low-Treg patients compared to the control cohort (91% vs 50%, p = 0.056). Low-Treg patients with hypertension were more likely to need 2 + anti-hypertensive agents preoperatively compared to their counterparts (71% vs 44%, p = 0.058). CONCLUSION: Within bariatric candidates, lower visceral AT-Treg abundance was associated with increased baseline medication requirements for type 2 diabetes, hypertension, and hyperlipidemia. This suggests that reduced AT-Tregs may be associated with higher obesity-related comorbidity severity.

Obesity-associated microbiomes instigate visceral adipose tissue inflammation by recruitment of distinct neutrophils.

Neutrophils are increasingly implicated in chronic inflammation and metabolic disorders. Here, we show that visceral adipose tissue (VAT) from individuals with obesity contains more neutrophils than in those without obesity and is associated with a distinct bacterial community. Exploring the mechanism, we gavaged microbiome-depleted mice with stool from patients with and without obesity during high-fat or normal diet administration. Only mice receiving high-fat diet and stool from subjects with obesity show enrichment of VAT neutrophils, suggesting donor microbiome and recipient diet determine VAT neutrophilia. A rise in pro-inflammatory CD4+ Th1 cells and a drop in immunoregulatory T cells in VAT only follows if there is a transient spike in neutrophils. Human VAT neutrophils exhibit a distinct gene expression pattern that is found in different human tissues, including tumors. VAT neutrophils and bacteria may be a novel therapeutic target for treating inflammatory-driven complications of obesity, including insulin resistance and colon cancer.

Myeloid deletion of nuclear factor erythroid 2-related factor 2 increases atherosclerosis and liver injury.

OBJECTIVE: To determine the impact of hematopoietic deletion of nuclear factor- (erythroid-derived 2) like 2 factor (Nrf2) on the development of atherosclerosis and liver injury in an obese, hypercholesterolemic mouse model. METHODS AND RESULTS: Two-month-old male low-density lipoprotein receptor-deficient mice were lethally irradiated and transplanted with either wild type or Nrf2-deficient (Nrf2(-/-)) bone marrow cells. At 3 months of age, mice were placed on an obesogenic high-fat diet (HFD), high-cholesterol diet for 7 months. Despite no differences in body weight, body fat percentage, liver fat, plasma glucose, lipids, or insulin, the HFD-fed Nrf2(-/-) bone marrow recipients had increased proinflammatory vascular gene expression, a significant increase in atherosclerosis area (18% versus 28%; P=0.018) and lesion complexity, and a marked increase in liver fibrosis. The acceleration of vascular and liver injury may arise from enhanced macrophage migration, inflammation, and oxidative stress resulting from myeloid Nrf2 deficiency. CONCLUSIONS: Myeloid-derived Nrf2 activity attenuates atherosclerosis development and liver inflammation and fibrosis associated with obesity. Prevention of oxidative stress in macrophage and other myeloid lineage cells may be an important therapeutic target to reduce inflammation-driven complications of obesity.

Interferon gamma mediates the reduction of adipose tissue regulatory T cells in human obesity.

Decreased adipose tissue regulatory T cells contribute to insulin resistance in obese mice, however, little is known about the mechanisms regulating adipose tissue regulatory T cells numbers in humans. Here we obtain adipose tissue from obese and lean volunteers. Regulatory T cell abundance is lower in obese vs. lean visceral and subcutaneous adipose tissue and associates with reduced insulin sensitivity and altered adipocyte metabolic gene expression. Regulatory T cells numbers decline following high-fat diet induction in lean volunteers. We see alteration in major histocompatibility complex II pathway in adipocytes from obese patients and after high fat ingestion, which increases T helper 1 cell numbers and decreases regulatory T cell differentiation. We also observe increased expression of inhibitory co-receptors including programmed cell death protein 1 and OX40 in visceral adipose tissue regulatory T cells from patients with obesity. In human obesity, these global effects of interferon gamma to reduce regulatory T cells and diminish their function appear to instigate adipose inflammation and suppress adipocyte metabolism, leading to insulin resistance.

Rosiglitazone attenuates age- and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein receptor knockout mice.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) is a common complication of obesity that can progress to nonalcoholic steatohepatitis (NASH), a serious liver pathology that can advance to cirrhosis. The mechanisms responsible for NAFLD progression to NASH remain unclear. Lack of a suitable animal model that faithfully recapitulates the pathophysiology of human NASH is a major obstacle in delineating mechanisms responsible for progression of NAFLD to NASH and, thus, development of better treatment strategies. We identified and characterized a novel mouse model, middle-aged male low-density lipoprotein receptor (LDLR)(-/-) mice fed a high-fat diet (HFD), which developed NASH associated with four of five metabolic syndrome (MS) components. In these mice, as observed in humans, liver steatosis and oxidative stress promoted NASH development. Aging exacerbated the HFD-induced NASH such that liver steatosis, inflammation, fibrosis, oxidative stress, and liver injury markers were greatly enhanced in middle-aged versus young LDLR(-/-) mice. Although expression of genes mediating fatty acid oxidation and antioxidant responses were up-regulated in young LDLR(-/-) mice fed HFD, they were drastically reduced in MS mice. However, similar to recent human trials, NASH was partially attenuated by an insulin-sensitizing peroxisome proliferator-activated receptor-gamma (PPARγ) ligand, rosiglitazone. In addition to expected improvements in MS, newly identified mechanisms of PPARγ ligand effects included stimulation of antioxidant gene expression and mitochondrial ß-oxidation, and suppression of inflammation and fibrosis. LDLR-deficiency promoted NASH, because middle-aged C57BL/6 mice fed HFD did not develop severe inflammation and fibrosis, despite increased steatosis. CONCLUSION: MS mice represent an ideal model to investigate NASH in the context of MS, as commonly occurs in human disease, and NASH development can be substantially attenuated by PPARγ activation, which enhances ß-oxidation.

Age-accelerated atherosclerosis correlates with failure to upregulate antioxidant genes.

Excess food intake leads to obesity and diabetes, both of which are well-known independent risk factors for atherosclerosis, and both of which are growing epidemics in an aging population. We hypothesized that aging enhances the metabolic and vascular effects of high fat diet (HFD) and therefore examined the effect of age on atherosclerosis and insulin resistance in lipoprotein receptor knockout (LDLR(-/-)) mice. We found that 12-month-old (middle-aged) LDLR(-/-) mice developed substantially worse metabolic syndrome, diabetes, and atherosclerosis than 3-month-old (young) LDLR(-/-) mice when both were fed HFD for 3 months, despite similar elevations in total cholesterol levels. Microarray analyses were performed to analyze the mechanism responsible for the marked acceleration of atherosclerosis in middle-aged mice. Chow-fed middle-aged mice had greater aortic expression of multiple antioxidant genes than chow-fed young mice, including glutathione peroxidase-1 and -4, catalase, superoxide dismutase-2, and uncoupling protein-2. Aortic expression of these enzymes markedly increased in young mice fed HFD but decreased or only modestly increased in middle-aged mice fed HFD, despite the fact that systemic oxidative stress and vascular reactive oxygen species generation, measured by plasma F2alpha isoprostane concentration (systemic) and dihydroethidium conversion and p47phox expression (vascular), were greater in middle-aged mice fed HFD. Thus, the mechanism for the accelerated vascular injury in older LDLR(-/-) mice was likely the profound inability to mount an antioxidant response. This effect was related to a decrease in vascular expression of 2 key transcriptional pathways regulating the antioxidant response, DJ-1 and forkhead box, subgroup O family (FOXOs). Treatment of middle-aged mice fed HFD with the antioxidant apocynin attenuated atherosclerosis, whereas treatment with the insulin sensitizer rosiglitazone attenuated both metabolic syndrome and atherosclerosis. Both treatments decreased oxidative stress. A novel effect of rosiglitazone was to increase expression of Nrf2 (nuclear factor [erythroid-derived 2]-like 2), a downstream target of DJ-1 contributing to enhanced expression of vascular antioxidant enzymes. This investigation underscores the role of oxidative stress when multiple atherosclerotic risk factors, particularly aging, converge on the vessel wall and emphasizes the need to develop effective strategies to inhibit oxidative stress to protect aging vasculature.

PPARdelta-mediated antiinflammatory mechanisms inhibit angiotensin II-accelerated atherosclerosis.

Activation of the nuclear hormone receptor peroxisome proliferator-activated receptor delta (PPARdelta) has been shown to improve insulin resistance, adiposity, and plasma HDL levels. However, its antiatherogenic role remains controversial. Here we report atheroprotective effects of PPARdelta activation in a model of angiotensin II (AngII)-accelerated atherosclerosis, characterized by increased vascular inflammation related to repression of an antiinflammatory corepressor, B cell lymphoma-6 (Bcl-6), and the regulators of G protein-coupled signaling (RGS) proteins RGS4 and RGS5. In this model, administration of the PPARdelta agonist GW0742 (1 or 10 mg/kg) substantially attenuated AngII-accelerated atherosclerosis without altering blood pressure and increased vascular expression of Bcl-6, RGS4, and RGS5, which was associated with suppression of inflammatory and atherogenic gene expression in the artery. In vitro studies demonstrated similar changes in AngII-treated macrophages: PPARdelta activation increased both total and free Bcl-6 levels and inhibited AngII activation of MAP kinases, p38, and ERK1/2. These studies uncover crucial proinflammatory mechanisms of AngII and highlight actions of PPARdelta activation to inhibit AngII signaling, which is atheroprotective.

Characterization of inflammatory changes in the breast cancer associated adipose tissue and comparison to the unaffected contralateral breast.

BACKGROUND: Adipose tissue has emerged as an important window into cancer pathophysiology, revealing potential targets for novel therapeutic interventions. The goal of this study was to compare the breast adipose tissue (BrAT) immune milieu surrounding breast carcinoma and contralateral unaffected breast tissue obtained from the same patient. MATERIALS AND METHODS: Patients undergoing bilateral mastectomy for unilateral breast cancer were enrolled for bilateral BrAT collection at the time of operation. After BrAT was processed, adipocyte and stromal vascular fraction (SVF) gene expression was quantified by PCR. SVF cells were also processed for flow cytometric immune cell characterization. RESULTS: Twelve patients underwent bilateral mastectomy for unilateral ductal carcinoma. BrAT adipocyte CXCL2 gene expression trended higher in the tumor-affected breast as compared to the unaffected breast. Macrophage MCP-1 and PPARγ gene expression also tended to be higher in the tumor-affected breasts. T cell gene expression of FOXP3 (p = 0.0370) were significantly greater in tumor-affected breasts than unaffected breasts. Affected BrAT contained higher numbers of Th2 CD4+ cells (p = 0.0165) and eosinophils (p = 0.0095) while trending towards increased macrophage and lower Th1 CD4+ cells infiltration than tumor-affected BrAT. CONCLUSION: This preliminary study aimed to identify the immunologic environment present within BrAT and is the first to directly compare this in individual patients' tumor-associated and unaffected BrAT. These findings suggest that cancer-affected BrAT had increased levels of T cell specific FOXP3 and higher levels of anti-inflammatory/regulatory cells compared to the contralateral BrAT.

Critical role for osteopontin in diabetic nephropathy.

The profibrotic adhesion molecule, osteopontin (OPN), is upregulated in kidneys of humans and mice with diabetes. The thiazolidinedione (TZD) insulin sensitizers decrease albuminuria in diabetic nephropathy (DN) and reduce OPN expression in vascular and cardiac tissue. To examine whether OPN is a critical mediator of DN we treated db/db mice with insulin, rosiglitazone, or pioglitazone to achieve similar fasting plasma glucose levels. The urine albumin-to-creatinine ratio and glomerular OPN expression were increased in diabetic mice, but both were reduced by the TZDs more than by insulin. We administered streptozotocin to OPN-null and OPN-wild-type mice, and OPN-null mice were bred into both type 1 (Ins2(akita/+)) and 2 (db/db) diabetic mice. In each case, OPN deletion decreased albuminuria, mesangial area, and glomerular collagen IV, fibronectin and transforming growth factor (TGF)-beta in the diabetic mice compared with their respective controls. In cultured mouse mesangial cells, TZDs but not insulin decreased angiotensin II-induced OPN expression, while recombinant OPN upregulated TGF-beta, ERK/MAPK, and JNK/MAPK signaling. These studies show that OPN expression in DN mouse models enhances glomerular damage, likely through the expression of TGF-beta, while its deletion protects against disease progression, suggesting that OPN might serve as a therapeutic target.

Obesogenic Memory Maintains Adipose Tissue Inflammation and Insulin Resistance.

BACKGROUND: Obesity is characterized by visceral adipose tissue (AT) inflammation. Immunosuppressive regulatory T cells (Tregs), phagocytic M2-like macrophages, and innate lymphoid cells type 2 (ILC2) control lean AT inflammation to maintain systemic insulin sensitivity, while the loss of these cells in obesity leads to AT inflammation and insulin resistance (IR). OBJECTIVE: The objective of this study was to determine if weight loss following obesity would correct AT inflammation and systemic metabolism. RESULTS: After six months of high fat diet (HFD) in male C57/Bl6 mice, flow analyses of epidydimal AT stromal vascular fraction (SVF) revealed depleted Tregs by 50%, doubling of CD8+ T cells, tripling of pro-inflammatory M1-like macrophages, and an 80% drop in ILC2 cells associated with changes in pro-inflammatory adipocyte and macrophage gene expression. Despite normalization of body weight, fat, and adipocyte size, mice ingesting 3 months of high-fat diet (HFD) followed by 3 months of chow-diet remained more insulin resistant and glucose intolerant than chow-fed animals. Adipocytes, AT Tregs, CD8+ T cells, ILC2 cells, and M1-like macrophages all failed to normalize with weight loss. CONCLUSIONS: Persistent AT inflammation contributes to the maintenance of IR despite body weight and fat normalization in previously obese mice. These findings highlight the importance of obesity prevention to avoid the consequences of "obesogenic memory."

Human Visceral Adipose Tissue Macrophages Are Not Adequately Defined by Standard Methods of Characterization.

Obesity is associated with a state of chronic low-grade inflammation both systemically and within specific tissues, including adipose tissue (AT). In murine models of obesity, there is a shift in the inflammatory profile of the AT immune cells, with an accumulation of proinflammatory M1 macrophages that surround the expanding adipocyte. However, much less is known about the immune cell composition and how to best define AT macrophages in humans. Objective. The goals of the current study were to determine the contribution of macrophages to the stromal vascular fraction (SVF) in lean versus obese human visceral AT (VAT); examine the expression of common M1, M2, and pan macrophage markers; and determine the association of specific macrophage types with known biomarkers of obesity-related cardiometabolic disease. Research Design and Methods. VAT biopsies were obtained from obese (n = 50) and lean (n = 8) patients during elective surgery. Adipocytes and SVF were isolated, and the SVF was subjected to flow cytometry analyses. Results. Our results indicate that VAT macrophages are increased in obesity and associate with biomarkers of CVD but that many macrophages do not fall into currently defined M1/M2 classification system based on CD206 receptor expression levels. Conclusions. VAT macrophages are increased in obese subjects, but the current markers used to define macrophage populations are inadequate to distinguish differences in human obesity. Further studies are needed to delineate the function of AT macrophages in the maintenance and progression of human AT inflammation in obesity.

Clusterin Impairs Hepatic Insulin Sensitivity and Adipocyte Clusterin Associates With Cardiometabolic Risk.

OBJECTIVE: Components of the adipose tissue (AT) extracellular matrix (ECM) are recently discovered contributors to obesity-related cardiometabolic disease. We identified increased adipocyte expression of ECM-related clusterin (apolipoprotein J) in obese versus lean women by microarray. Our objective was to determine 1) whether subcutaneous AT adipocyte (SAd) clusterin and serum clusterin are associated with insulin resistance (IR) and known markers of cardiometabolic risk and 2) how clusterin may contribute to increased risk. RESEARCH DESIGN AND METHODS: We validated increased clusterin expression in adipocytes from a separate group of 18 lean and 54 obese individuals. The relationship of clusterin gene expression and plasma clusterin with IR, cardiovascular biomarkers, and risk of cardiovascular disease (CVD) was then determined. Further investigations in human cultured cells and in aged LDLR-/- mice prone to development of obesity-associated complications were performed. RESULTS: SAd clusterin correlated with IR, multiple CVD biomarkers, and CVD risk, independent of traditional risk factors. Circulating human clusterin exhibited similar associations. In human adipocytes, palmitate enhanced clusterin secretion, and in human hepatocytes, clusterin attenuated insulin signaling and APOA1 expression and stimulated hepatic gluconeogenesis. LRP2 (megalin), a clusterin receptor, highly expressed in liver, mediated these effects, which were inhibited by LRP2 siRNA. In response to Western diet feeding, an increase in adipocyte clusterin expression was associated with a progressive increase in liver fat, steatohepatitis, and fibrosis in aged LDLR-/- mice. CONCLUSIONS: Adipocyte-derived clusterin is a novel ECM-related protein linking cardiometabolic disease and obesity through its actions in the liver.

Rosiglitazone inhibits acyl-CoA synthetase activity and fatty acid partitioning to diacylglycerol and triacylglycerol via a peroxisome proliferator-activated receptor-gamma-independent mechanism in human arterial smooth muscle cells and macrophages.

Rosiglitazone is an insulin-sensitizing agent that has recently been shown to exert beneficial effects on atherosclerosis. In addition to peroxisome proliferator-activated receptor (PPAR)-gamma, rosiglitazone can affect other targets, such as directly inhibiting recombinant long-chain acyl-CoA synthetase (ACSL)-4 activity. Because it is unknown if ACSL4 is expressed in vascular cells involved in atherosclerosis, we investigated the ability of rosiglitazone to inhibit ACSL activity and fatty acid partitioning in human and murine arterial smooth muscle cells (SMCs) and macrophages. Human and murine SMCs and human macrophages expressed Acsl4, and rosiglitazone inhibited Acsl activity in these cells. Furthermore, rosiglitazone acutely inhibited partitioning of fatty acids into phospholipids in human SMCs and inhibited fatty acid partitioning into diacylglycerol and triacylglycerol in human SMCs and macrophages through a PPAR-gamma-independent mechanism. Conversely, murine macrophages did not express ACSL4, and rosiglitazone did not inhibit ACSL activity in these cells, nor did it affect acute fatty acid partitioning into cellular lipids. Thus, rosiglitazone inhibits ACSL activity and fatty acid partitioning in human and murine SMCs and in human macrophages through a PPAR-gamma-independent mechanism likely to be mediated by ACSL4 inhibition. Therefore, rosiglitazone might alter the biological effects of fatty acids in these cells and in atherosclerosis.

Adipocyte DIO2 Expression Increases in Human Obesity but Is Not Related to Systemic Insulin Sensitivity.

Deiodinase type II (D2), encoded by DIO2, catalyzes the conversion of T4 to bioactive T3. T3 not only stimulates adaptive thermogenesis but also affects adipose tissue (AT) lipid accumulation, mitochondrial function, inflammation, and potentially systemic metabolism. Although better defined in brown AT, the precise role of DIO2 expression in white AT remains largely unknown, with data derived only from whole fat. Therefore, the purpose of this study was to determine whether subcutaneous (SAT) and visceral (VAT) adipocyte-specific gene expression of DIO2 differs between obese and lean patients and whether these differences relate to alterations in mitochondrial function, fatty acid flux, inflammatory cytokines/adipokines, and ultimately insulin sensitivity. Accordingly, adipocytes of 73 obese and 21 lean subjects were isolated and subjected to gene expression analyses. Our results demonstrate that obese compared to lean human individuals have increased adipocyte-specific DIO2 expression in both SAT and VAT. Although higher DIO2 was strongly related to reduced fatty acid synthesis/oxidation and mitochondrial function, we found no relationship to proinflammatory cytokines or insulin resistance and no difference based on diabetic status. Our results suggest that adipocyte-derived DIO2 may play a role in weight maintenance but is likely not a major contributor to obesity-related insulin resistance.

Activation of peroxisome proliferator-activated receptor-gamma decreases pancreatic cancer cell invasion through modulation of the plasminogen activator system.

Cancer cell invasion and metastasis require the concerted action of several proteases that degrade extracellular matrix proteins and basement membranes. Recent reports suggest the plasminogen activator system plays a critical role in pancreatic cancer biology. In the present study, we determined the contribution of the plasminogen activator system to pancreatic cancer cell invasion in vitro. Moreover, the effect of peroxisome proliferator-activated receptor (PPAR)-gamma ligands, which are currently in clinical use as antidiabetic drugs and interestingly seem to display antitumor activities, on pancreatic cancer cell invasion and the plasminogen activator system was assessed. Expression of components of the plasminogen activator system [i.e., urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1, and uPA receptor] was detected in six human pancreatic cancer cell lines. Inhibition of urokinase activity by specific synthetic compounds reduced baseline pancreatic cancer cell invasion. The PPAR-gamma ligands 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone also attenuated pancreatic cancer cell invasion. This effect was abrogated by dominant-negative PPAR-gamma receptors and pharmacologic PPAR-gamma inhibitors. Moreover, activation of PPAR-gamma by ligands increased plasminogen activator inhibitor-1 and decreased uPA levels in pancreatic cancer cells, and this was accompanied by a reduction in total urokinase activity. The present study shows that the plasminogen activator system plays an integral role in pancreatic cancer cell invasion in vitro. Activation of the nuclear receptor PPAR-gamma by ligands reduced pancreatic cancer cell invasion, which was largely mediated by modulation of the plasminogen activator system. These findings further underscore the potential role of PPAR-gamma ligands as therapeutic agents in pancreatic cancer.

Growth stimulation of COX-2-negative pancreatic cancer by a selective COX-2 inhibitor.

Cyclooxygenase 2 (COX-2) inhibitors are promising antiangiogenic agents in several preclinical models. The aim of the present study was to evaluate the effect of selective COX-2 inhibitors on vascular endothelial growth factor (VEGF) production in vitro and angiogenesis and growth of pancreatic cancer in vivo, focusing on putative differences between COX-2-negative and COX-2-positive tumors. VEGF production and angiogenesis in vitro were determined by ELISA and endothelial cell migration assay. To determine whether the effect of COX-2 inhibitors was mediated by peroxisome proliferator-activated receptor gamma (PPAR-gamma), we used a dominant-negative PPAR-gamma and a pharmacologic inhibitor. In vitro findings were validated in a pancreatic cancer animal model. Microvessel density was assessed by CD31 immunostaining. Intratumoral prostaglandin and VEGF levels were measured by mass spectroscopy and ELISA. Selective COX-2 inhibitors had a concentration-dependent effect on VEGF production in vitro. Higher concentrations increased VEGF levels and stimulated angiogenesis by activating PPAR-gamma. In vivo, nimesulide increased VEGF production by cancer cells in COX-2-positive and COX-2-negative pancreatic tumors. In COX-2-negative pancreatic cancer, this effect was associated with an increase in angiogenesis and growth. In COX-2-positive pancreatic cancer, the nimesulide-induced increase of VEGF production by the cancer cells was offset by a decrease in VEGF production by the nonmalignant cell types leading to reduced tumor angiogenesis and growth. Selective COX-2 inhibitors had opposite effects on growth and angiogenesis in pancreatic cancer depending on COX-2 expression. These findings imply that assessing the COX-2 profile of the pancreatic tumor is mandatory before initiating therapy with a selective COX-2 inhibitor.

Regulation of the growth arrest and DNA damage-inducible gene 45 (GADD45) by peroxisome proliferator-activated receptor gamma in vascular smooth muscle cells.

Peroxisome proliferator-activated receptor (PPAR) gamma is activated by thiazolidinediones (TZDs), widely used as insulin-sensitizing agents for the treatment of type 2 diabetes. TZDs have been shown to induce apoptosis in a variety of mammalian cells. In vascular smooth muscle cells (VSMCs), proliferation and apoptosis may be competing processes during the formation of restenotic and atherosclerotic lesions. The precise molecular mechanisms by which TZDs induce apoptosis in VSMCs, however, remain unclear. In the present study, we demonstrate that the TZDs rosiglitazone (RSG), troglitazone (TRO), and a novel non-TZD partial PPARgamma agonist (nTZDpa) induce caspase-mediated apoptosis of human coronary VSMCs. Induction of VSMC apoptosis correlated closely with an upregulation of growth arrest and DNA damage-inducible gene 45 (GADD45) mRNA expression and transcription, a well-recognized modulator of cell cycle arrest and apoptosis. Using adenoviral-mediated overexpression of a constitutively active PPARgamma mutant and the irreversible PPARgamma antagonist GW9662, we provide evidence that PPARgamma ligands induce caspase-mediated apoptosis and GADD45 expression through a receptor-dependent pathway. Deletion analysis of the GADD45 promoter revealed that a 153-bp region between -234 and -81 bp proximal to the transcription start site, containing an Oct-1 element, was crucial for the PPARgamma ligand-mediated induction of the GADD45 promoter. PPARgamma activation induced Oct-1 protein expression and DNA binding and stimulated activity of a reporter plasmid driven by multiple Oct-1 elements. These findings suggest that activation of PPARgamma can lead to apoptosis and growth arrest in VSMCs, at least in part, by inducing Oct-1-mediated transcription of GADD45. The full text of this article is available online at http://www.circresaha.org.

Liver X receptor agonists suppress vascular smooth muscle cell proliferation and inhibit neointima formation in balloon-injured rat carotid arteries.

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1-->S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1-->S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase-associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1-->S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand-mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org.

Peroxisome proliferator-activated receptor gamma inhibits expression of minichromosome maintenance proteins in vascular smooth muscle cells.

Using a cDNA array consisting only of cell cycle genes, we found that a novel nonthiazolidinedione partial peroxisome proliferator-activated receptor gamma (PPARgamma) agonist (nTZDpa) inhibited expression of minichromosome maintenance (MCM) proteins 6 and 7 in vascular smooth muscle cells. MCM proteins are required for the initiation and elongation stages of DNA replication and are regulated by the transcription factor E2F. Mitogen-induced MCM6 and MCM7 mRNA expression was potently inhibited by nTZDpa and to a lesser degree by the full PPARgamma agonist, rosiglitazone. Inhibition of MCM6 and MCM7 expression by nTZDpa and rosiglitazone paralleled their effect to inhibit phosphorylation of the retinoblastoma protein and cell proliferation. Transient transfection experiments revealed that the nTZDpa inhibited mitogen-induced MCM6 and MCM7 promoter activity, implicating a transcriptional mechanism. Adenoviral-mediated E2F overexpression reversed the suppressive effect of nTZDpa on MCM6 and MCM7 expression. Furthermore, activity of a luciferase reporter plasmid driven by multiple E2F elements was inhibited by nTZDpa, indicating that their down-regulation by nTZDpa involves an E2F-dependent mechanism. Overexpression of dominant-negative PPARgamma or addition of a PPARgamma antagonist, GW 9662, blocked nTZDpa inhibition of MCM7 transcription. Adenovirus-mediated overexpression of constitutively active PPARgamma inhibited MCM7 expression in a similar manner as the nTZDpa. These findings provide strong evidence that activation of PPARgamma attenuates MCM7 transcription and support the important role of this nuclear receptor in regulating vascular smooth muscle cell proliferation.