Effects of gelsolin on macrophage inflammatory responses to orthopaedic implant wear debris.

The local effects of implant wear debris on surrounding tissue has been a major focus of many investigators. Although there have been improvements in implants, significant numbers of revision surgeries are performed to address these issues. Gelsolin (GSN) is a protein in the cytoplasm and circulating serum involved in actin breakdown as well as anti-inflammatory processes. In this study, we tested the hypothesis that GSN in the presence of wear debris in vitro decreases the inflammatory response of a human monocyte cell line. We utilized titanium-, polyethylene-, and cobalt-characterized wear particles in a 1:100 and a 1:500 cell-to-particle ratios in the presence of a low (0.2 µM) and normal (2.0 µM) concentrations of GSN and compared the inflammatory response to cells without GSN exposure. The results show that IL-6, IL-1, TNF-α, and PGE2 all increased with higher concentrations of GSN. Although the anti-inflammatory properties of GSN were not seen in this in vitro experiment, it has previously been shown that GSN does affect the inflammatory response of monocytes to orthopedic implant wear debris. The dose-response curve for GSN may have a bimodal profile, which should be further investigated.

Depot-specific overexpression of proinflammatory, redox, endothelial cell, and angiogenic genes in epicardial fat adjacent to severe stable coronary atherosclerosis.

BACKGROUND: Pro- and antiinflammatory genes are expressed in epicardial adipose tissue (EAT). Our objectives were to characterize genes in EAT that may contribute specifically to coronary atherogenesis and to measure circulating adipokines matched to their messenger RNAs (mRNAs) in EAT. We hypothesized that severe coronary atherosclerosis (CAD) would preferentially affect gene expression in EAT as compared to substernal fat or subcutaneous thoracic adipose tissue (SAT), as well as circulating levels of adipokines. METHODS: Fat mRNA was quantified using reverse transcription polymerase chain reaction (RT-PCR), and circulating adipokines were measured by enzyme-linked immunosorbent assays (ELISAs) in patients with severe stable CAD and controls without severe CAD undergoing open heart surgery. RESULTS: A total of 39 of 70 mRNAs in EAT were significantly increased in CAD. Only 4 and 3 of these mRNAs were increased in substernal fat and SAT, respectively. Of the mRNAs increased in EAT, 17 were either inflammatory adipokines or proteins known to be involved in inflammatory processes, 7 were involved in oxidative stress and or oxygen species regulation, whereas 15 were proteins involved in metabolism and regulation of gene transcription or proteins unique to fat cells. The largest increases, over three-fold, were seen in GPX3, gp91 phox, p47phox, heme oxygenase, and interleukin-8 (IL-8). Tpl2 mRNA was uniquely elevated in all three fat depots from CAD patients, and its expression in SAT, but not in EAT or substernal fat, was directly correlated with homeostasis model assessment of insulin resistance (HOMA-IR) values. Compared to controls, there were no associations between circulating levels of IL-8, lipocalin-2, nerve growth factor (NGF), RANTES, CD-163, GPX-3, monocyte chemotactic protein-1 (MCP-1)/CCL2, leptin, soluble vascular endothelial growth factor receptor-1 (sFLT1), fatty acid binding protein-4 (FABP-4), and plasminogen activator inhibitor-1 (PAI-1) and increases in their gene expression in EAT adjacent to CAD. CONCLUSIONS: Expression of proinflammatory, redox, endothelial cell, and angiogenic genes in EAT is depot specific and supports the hypothesis that pathophysiologically EAT contributes locally to CAD. CAD links with these fat depots might involve Tpl2 as a primary response indicator.

Inflammatory genes in epicardial fat contiguous with coronary atherosclerosis in the metabolic syndrome and type 2 diabetes: changes associated with pioglitazone.

OBJECTIVE: To determine changes in gene expression in epicardial adipose tissue (EAT) associated with coronary atherosclerosis (CAD) and effects of pioglitazone therapy. RESEARCH DESIGN AND METHODS: Genes were quantified by RT-PCR in EAT and thoracic subcutaneous adipose tissue (SAT) obtained during surgery in CAD patients with metabolic syndrome (MS) or type 2 diabetes and control subjects with minimal or no CAD and no MS or type 2 diabetes. RESULTS: Increased expression of interleukin-1 receptor antagonist (IL-1Ra) and IL-10, a trend for higher IL-1ß, and no change in peroxisome proliferator-activated receptor-γ (PPARγ) was found in EAT from MS or type 2 diabetes. Only PPARγ mRNA was reduced in SAT. Pioglitazone therapy in type 2 diabetes was associated with decreased expression of IL-1ß, IL-1Ra, and IL-10 in EAT; decreased IL-10 in SAT; and increased PPARγ in SAT. CONCLUSIONS: In MS and type 2 diabetes with CAD, proinflammatory and anti-inflammatory genes were differentially increased in EAT and selectively reduced in association with pioglitazone treatment.

Human epicardial adipokine messenger RNAs: comparisons of their expression in substernal, subcutaneous, and omental fat.

We compared the gene expression of inflammatory and other proteins by real-time quantitative polymerase chain reaction in epicardial, substernal (mediastinal) and subcutaneous sternal, upper abdominal, and leg fat from coronary bypass patients and omental (visceral) fat from extremely obese women undergoing bariatric surgery. We hypothesized that (1) epicardial fat would exhibit higher expression of inflammatory messenger RNAs (mRNAs) than substernal and subcutaneous fat and (2) epicardial mRNAs would be similar to those in omental fat. Epicardial fat was clearly different from substernal fat because there was a far higher expression of haptoglobin, prostaglandin D(2) synthase, nerve growth factor beta, the soluble vascular endothelial growth factor receptor (FLT1), and alpha1 glycoprotein but not of inflammatory adipokines such as monocyte chemoattractant protein-1, interleukin (IL)-8, IL-1beta, tumor necrosis factor alpha, serum amyloid A, plasminogen activator inhibitor-1, or adiponectin despite underlying coronary atherosclerosis. However, the latter inflammatory adipokines as well as most other mRNAs were overexpressed in epicardial fat as compared with the subcutaneous depots except for IL-8, fatty acid binding protein 4, the angiotensin II receptor 1, IL-6, and superoxide dismutase-2. Relative to omental fat, about one third of the genes were expressed at the same levels, whereas monocyte chemoattractant protein-1, cyclooxygenase-2, plasminogen activator inhibitor-1, IL-1beta, and IL-6 were expressed at far lower levels in epicardial fat. In conclusion, epicardial fat does not appear to be a potentially more important source of inflammatory adipokines than substernal mediastinal fat. Furthermore, the expression of inflammatory cytokines such as IL-6 and IL-1beta is actually higher in omental fat from obese women without coronary atherosclerosis. The data do not support the hypothesis that most of the inflammatory adipokines are expressed at high levels in epicardial fat of humans.

The inflammatory response seen when human omental adipose tissue explants are incubated in primary culture is not dependent upon albumin and is primarily in the nonfat cells.

BACKGROUND: The present studies were designed to investigate the changes in gene expression during in vitro incubation of human visceral omental adipose tissue explants as well as fat cells and nonfat cells derived from omental fat. METHODS: Adipose tissue was obtained from extremely obese women undergoing bariatric surgery. Explants of the tissue as well as fat cells and the nonfat cells derived by digestion with collagenase were incubated for 20 minutes to 48 h. The expression of interleukin 1beta [IL-1beta], tumor necrosis factor alpha [TNFalpha], interleukin 8 [IL-8], NFkappaB(1)p50 subunit, hypoxia-inducible factor 1alpha [HIF1alpha], omentin/intelectin, and 11beta-hydroxysteroid dehydrogenase 1 [11beta-HSD1] mRNA were measured by qPCR as well as the release of IL-8 and TNFalpha. RESULTS: There was an inflammatory response at 2 h in explants of omental adipose tissue that was reduced but not abolished in the absence of albumin from the incubation buffer for IL-8, IL-1beta and TNFalpha. There was also an inflammatory response with regard to upregulation of HIF1alpha and NFkappaB1 gene expression that was unaffected whether albumin was present or absent from the medium. In the nonfat cells derived by a 2 h collagenase digestion of omental fat there was an inflammatory response comparable but not greater than that seen in tissue. The exception was HIF1alpha where the marked increase in gene expression was primarily seen in intact tissue. The inflammatory response was not seen with respect to omentin/intelectin. Over a subsequent 48 h incubation there was a marked increase in IL-8 mRNA expression and IL-8 release in adipose tissue explants that was also seen to the same extent in the nonfat cells incubated in the absence of fat cells. CONCLUSION: The marked inflammatory response seen when human omental adipose tissue is incubated in vitro is reduced but not abolished in the presence of albumin with respect to IL-1beta, TNFalpha, IL-8, and is primarily in the nonfat cells of adipose tissue.

Release of 12 adipokines by adipose tissue, nonfat cells, and fat cells from obese women.

The relative release in vitro of endothelin-1, zinc-alpha2-glycoprotein (ZAG), lipocalin-2, CD14, RANTES (regulated on activation, normal T cell expressed and secreted protein), lipoprotein lipase (LPL), osteoprotegerin (OPG), fatty acid-binding protein 4 (FABP-4), visfatin/PBEF/Nampt, glutathione peroxidase-3 (GPX-3), intracellular cell adhesion molecule 1 (ICAM-1), and amyloid A was examined using explants of human adipose tissue as well as the nonfat cell fractions and adipocytes from obese women. Over a 48-h incubation the majority of the release of LPL was by fat cells whereas that of lipocalin-2, RANTES, and ICAM-1 was by the nonfat cells present in human adipose tissue. In contrast appreciable amounts of OPG, amyloid A, ZAG, FABP-4, GPX-3, CD14, and visfatin/PBEF/Nampt were released by both fat cells and nonfat cells. There was an excellent correlation (r = 0.75) between the ratios of adipokine release by fat cells to nonfat cells over 48 h and the ratio of their mRNAs in fat cells to nonfat cells at the start of the incubation. The total release of ZAG, OPG, RANTES, and amyloid A by incubated adipose tissue explants from women with a fat mass of 65 kg was not different from that by women with a fat mass of 29 kg. In contrast that of ICAM-1, FABP-4, GPX-3, visfatin/PBEF/Nampt, CD14, lipocalin-2, LP, and endothelin-1 was significantly greater in tissue from women with a total fat mass of 65 kg.

Dexamethasone and the inflammatory response in explants of human omental adipose tissue.

Dexamethasone is a synthetic glucocorticoid that is a potent anti-inflammatory agent. The present studies examined the changes in gene expression of 64 proteins in human omental adipose tissue explants incubated for 48h both in the absence and presence of dexamethasone as well as the release of 8 of these proteins that are putative adipokines. The proteins were chosen because they are inflammatory response proteins in other cells, are key regulatory proteins or are proteins with known functions. About 50% were significantly up-regulated while about 10% were unchanged and the remaining 40% were down-regulated. Dexamethasone significantly up-regulated the expression of about 33% of the proteins but down-regulated the expression of about 12% of the proteins. We conclude that dexamethasone is a selective anti-inflammatory agent since it inhibits only about one-fourth of the proteins up-regulated during in vitro incubation of human omental adipose tissue.

Uncoupling protein-1 and related messenger ribonucleic acids in human epicardial and other adipose tissues: epicardial fat functioning as brown fat.

CONTEXT: Uncoupling protein-1 (UCP-1) is the inner mitochondrial membrane protein that is a specific marker for and mediator of nonshivering thermogenesis in brown adipocytes. OBJECTIVE: This study was performed to better understand the putative thermogenic function of human epicardial fat. DESIGN: We measured the expression of UCP-1 and brown adipocyte differentiation transcription factors PR-domain-missing 16 (PRDM16) and peroxisome-proliferator-activated receptor gamma co-activator-1 alpha (PGC-1 alpha) in epicardial, substernal, and sc thoracic, abdominal, and leg fat. SETTING: The study was conducted at a tertiary care hospital cardiac center. PATIENTS: Forty-four patients had coronary artery bypass surgery, and six had heart valve replacement. INTERVENTIONS: Fat samples were taken at open heart surgery. RESULTS: UCP-1 expression was 5-fold higher in epicardial fat than substernal fat and barely detectable in sc fat. Epicardial fat UCP-1 expression decreased with age, increased with body mass index, was similar in women and men and patients on and not on statin therapy, and showed no relationship to epicardial fat volume or waist circumference. UCP-1 expression was similar in patients without and with severe coronary atherosclerosis and metabolic syndrome or type 2 diabetes. PRDM16 and PGC-1 alpha expression was 2-fold greater in epicardial than sc fat. Epicardial fat UCP-1, PRDM16, and PGC1-alpha mRNAs were similar in diabetics treated with thiazolidinediones compared to diabetics not treated with thiazolidinediones. CONCLUSION: Because UCP-1 is expressed at high levels in epicardial fat as compared to other fat depots, the possibility should be considered that epicardial fat functions like brown fat to defend the myocardium and coronary vessels against hypothermia. This process could be blunted in the elderly.

Stimulation of human omental adipose tissue lipolysis by growth hormone plus dexamethasone.

Growth hormone [GH] administration results in a reduction in adiposity of humans that is attributed to stimulation of lipolysis. We examined the effect of direct addition of human GH, in both the absence and presence of dexamethasone [Dex], as well as that of interferon beta on lipolysis by omental adipose tissue explants from obese women incubated for 48h in primary culture. There was a significant stimulation of lipolysis by GH in the presence of Dex but not by Dex or GH alone. There was also a significant further stimulation by GH in the presence of Dex of hormone-sensitive lipase, perilipin, lipoprotein lipase and beta1 adrenergic receptor mRNA. We conclude that the direct lipolytic effect of GH is accompanied by an increase in HSL mRNA in the presence of DEX, but GH also increased the mRNAs for other proteins that could explain all or part of its lipolytic action.

Release in vitro of adipsin, vascular cell adhesion molecule 1, angiotensin 1-converting enzyme, and soluble tumor necrosis factor receptor 2 by human omental adipose tissue as well as by the nonfat cells and adipocytes.

The relative release in vitro of adipsin, vascular cell adhesion molecule (VCAM) 1, angiotensin 1-converting enzyme (ACE), and soluble tumor necrosis factor alpha receptor 2 (sTNFR2) by explants of human omental and subcutaneous adipose tissue as well as the nonfat cell fractions and adipocytes from morbidly obese gastric bypass women was compared with that by tissue from obese abdominoplasty patients. Release of VCAM-1 and ACE by omental adipose tissue explants was 220% and 80% greater, respectively, over 48 hours of incubation than that by subcutaneous adipose tissue explants. However, this difference was not seen when release by adipocytes derived from omental fat was compared with that by adipocytes from subcutaneous fat. The release of adipsin and sTNFR2 by omental adipose tissue explants did not differ from that by subcutaneous tissue adipose tissue. The release of adipsin by tissue explants over 48 hours was 100-fold greater than that of VCAM-1, ACE, or sTNFR2. Most of the release of all 4 adipokines was due to the nonfat cells because adipsin release by adipocytes was only 13% of that by the nonfat cells derived from the same amount of adipose tissue, whereas ACE release by adipocytes was 7% and release of VCAM-1 as well as sTNFR2 by adipocytes was 4% or less of that by nonfat cells. The total release in vitro of ACE and sTNFR2, but not that of adipsin or VCAM-1, was enhanced in adipose tissue explants from morbidly obese women as compared with those by explants derived from obese women. We conclude that although human adipose tissue explants release appreciable amounts of adipsin and far smaller amounts of VCAM-1, ACE, and sTNFR2 in vitro, more than 87% of the release is due to the nonfat cells present in adipose tissue. Furthermore, the enhanced release of VCAM-1 and ACE seen in omental adipose tissue explants as compared with explants of subcutaneous adipose tissue is due to release by nonfat cells.

Comparison of the release of adipokines by adipose tissue, adipose tissue matrix, and adipocytes from visceral and subcutaneous abdominal adipose tissues of obese humans.

The purpose of this study was to examine the source of adipokines released by the visceral and sc adipose tissues of obese humans. Human adipose tissue incubated in primary culture for 48 h released more prostaglandin E(2), IL-8, and IL-6 than adiponectin, whereas the release of plasminogen activator inhibitor 1 and hepatocyte growth factor was less than that of adiponectin but greater than that of leptin. IL-10 and TNFalpha were released in amounts less than those of leptin, whereas vascular endothelial growth factor and IL1-beta were released in much lower amounts. The accumulation of adipokines was also examined in the three fractions (adipose tissue matrix, isolated stromovascular cells, and adipocytes) obtained by collagenase digestion of adipose tissue. Over 90% of the adipokine release by adipose tissue, except for adiponectin and leptin, could be attributed to nonfat cells. Visceral adipose tissue released greater amounts of vascular endothelial growth factor, IL-6, and plasminogen activator inhibitor 1 compared with abdominal sc tissue. The greatly enhanced total release of TNFalpha, IL-8, and IL-10 by adipose tissue from individuals with a body mass index of 45 compared with 32 was due to nonfat cells. Furthermore, most of the adipokine release by the nonfat cells of adipose tissue was due to cells retained in the tissue matrix after collagenase digestion.

Resistin release by human adipose tissue explants in primary culture.

Resistin, also known as Fizz3 or ADSF, is a protein found in murine adipose tissue and inflammatory lung exudates. The present studies found that resistin was released by explants of human adipose tissue but the release was quite variable ranging from 3 to 158 ng/g over 48 h. The release of resistin was 250% greater by explants of omental than by explants of human subcutaneous abdominal adipose tissue. Resistin release by adipocytes was negligible as compared to that by the non-fat cells of adipose tissue. Leptin formation by adipocytes was 8-fold greater than its formation by the non-fat cells, while the formation of PAI-1 by adipocytes was 38% of that by the non-fat cells. The conversion of glucose to lactate as well as the formation of PGE(2) and IL-8 was approximately 15% of that by the non-fat cells. In contrast the release of IL-6 and IL-1beta by adipocytes was 4-7% of that by the non-fat cells while the formation of resistin and IL-10 by adipocytes was 2% of that by non-fat cells. The release of adiponectin by explants ranged from 1000 to 5000 ng/g over 48 h but did not correlate with that of resistin. The present data suggest that resistin release by explants of human adipose tissue in primary culture is largely derived from the non-fat cells present in the explants.