Viviane Z. Rocha

Obesity, inflammation, and atherosclerosis

Understanding of the pathophysiology of atherogenesis has evolved substantially during the last few decades. Atherosclerosis was once identified as a lipid-storage disease, but is now recognized as a subacute inflammatory condition of the vessel wall, characterized by infiltration of macrophages and T cells, which interact with one another and with cells of the arterial wall. The pathological mechanisms of obesity recapitulate many features of the inflammatory processes at work in atherosclerosis. Our current appreciation of the similarities between obesity and atherosclerosis has already fostered innovations for the diagnosis, prognosis, and prevention of these two conditions.

Interferon-γ, a Th1 Cytokine, Regulates Fat Inflammation. A Role for Adaptive Immunity in Obesity

Adipose tissue (AT) can accumulate macrophages and secrete several inflammatory mediators. Despite its pivotal role in the progression of chronic inflammatory processes such as atherosclerosis, the adaptive role of immunity in obesity remains poorly explored. Visceral AT of diet-induced obese C57BL/6 mice had higher numbers of both CD4+ and CD8+ T cells than lean controls, monitored by flow cytometry. When stimulated in vitro, T cells from obese AT produced more interferon (IFN)&ggr; than those from controls. AT from obese animals also had more cells expressing I-Ab, a mouse class II histocompatibility marker implicated in antigen presentation, as determined by immunostaining. Differentiated 3T3-L1 cells stimulated with recombinant IFN&ggr; or T-helper 1–derived supernatant produced several chemokines and their mRNAs. Obese IFN&ggr;-deficient animals had significantly reduced AT expression of mRNA-encoding inflammatory genes such as tumor necrosis factor-&agr; and monocyte chemoattractant protein-1, decreased AT inflammatory cell accumulation, and better glucose tolerance than control animals consuming the same diet. Obese mice doubly deficient for IFN&ggr; receptor and apolipoprotein (Apo)E on a mixed 129SvEv/C57BL/6 (129/B6) genetic background, despite exhibiting similar AT mRNA levels of tumor necrosis factor-&agr; and monocyte chemoattractant protein-1 as 129/B6-ApoE−/− controls, had decreased expression of important T cell–related genes, such as IFN&ggr;-inducible protein-10 and I-Ab, and lower plasma triglycerides and glucose. These results indicate a role for T cells and IFN&ggr;, a prototypical T-helper 1 cytokine, in regulation of the inflammatory response that accompanies obesity.

Adiponectin Inhibits Pro-inflammatory Signaling in Human Macrophages Independent of Interleukin-10

Macrophages participate pivotally in the pathogenesis of many chronic inflammatory diseases including atherosclerosis. Adiponectin, a vasculoprotective molecule with insulin-sensitizing and anti-atherogenic properties, suppresses pro-inflammatory gene expression in macrophages by mechanisms that remain incompletely understood. This study investigated the effects of adiponectin on major pro-inflammatory signaling pathways in human macrophages. We demonstrate that pretreatment of these cells with adiponectin inhibits phosphorylation of nuclear factor κB inhibitor (IκB), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), induced by either lipopolysaccharide (LPS) or tumor necrosis factor (TNF) α, as well as STAT3 phosphorylation induced by interleukin-6 (IL6). Antagonism of IL10 by either neutralizing antibodies or siRNA-mediated silencing did not abrogate the anti-inflammatory actions of adiponectin, indicating that the ability of adiponectin to render human macrophages tolerant to various pro-inflammatory stimuli does not require this cytokine. A systematic search for adiponectin-inducible genes with established anti-inflammatory properties revealed that adiponectin augmented the expression of A20, suppressor of cytokine signaling (SOCS) 3, B-cell CLL/lymphoma (BCL) 3, TNF receptor-associated factor (TRAF) 1, and TNFAIP3-interacting protein (TNIP) 3. These results suggest that adiponectin triggers a multifaceted response in human macrophages by inducing the expression of various anti-inflammatory proteins that act at different levels in concert to suppress macrophage activation.

Inflammatory concepts of obesity.

Obesity, long considered a condition characterized by the deposition of inert fat, is now recognized as a chronic and systemic inflammatory disease, where adipose tissue plays a crucial endocrine role through the production of numerous bioactive molecules, collectively known as adipokines. These molecules regulate carbohydrate and lipid metabolism, immune function and blood coagulability, and may serve as blood markers of cardiometabolic risk. Local inflammatory loops operate in adipose tissue as a consequence of nutrient overload, and crosstalk among its cellular constituents-adipocytes, endothelial and immune cells-results in the elaboration of inflammatory mediators. These mediators promote important systemic effects that can result in insulin resistance, dysmetabolism and cardiovascular disease. The understanding that inflammation plays a critical role in the pathogenesis of obesity-derived disorders has led to therapeutic approaches that target different points of the inflammatory network induced by obesity.

Increased Glucose Uptake in Visceral Versus Subcutaneous Adipose Tissue Revealed by PET Imaging

OBJECTIVES The current study tested the hypothesis that glucose utilization differs between visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT), and investigated potential mechanisms for such a finding. BACKGROUND VAT burden correlates better with cardiovascular risk than does SAT burden. Beyond volumetric measurement, glucose uptake in adipose tissue (AT) might reflect metabolic activity and provide pathophysiologic insight and aid risk stratification. METHODS We retrospectively studied tissue-specific glucose uptake in vivo in clinically obtained whole-body fluorodeoxyglucose positron emission tomography (FDG-PET) scans in humans. We also assessed glucose uptake in vitro, using stromal vascular cells isolated from SAT and VAT of diet-induced obese C57BL/6 mice. Quantitative polymerase chain reaction (PCR) evaluated the expression of multiple genes involved in cellular glucose metabolism, including glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2) in SAT and VAT of obese C57BL/6 mice. RESULTS We analyzed whole-body FDG-PET scans from 31 obese and 26 lean patients. VAT exhibited higher FDG uptake compared with SAT (p < 0.0001) independent of age, sex, body mass index, comorbidities, and medications. To investigate mechanisms underlying this observation, we studied glucose uptake in the stromal vascular cell fraction of AT, which is rich in inflammatory cells. Stromal vascular cells from VAT of diet-induced obese C57BL/6 mice exhibited higher glucose uptake than those from SAT (p = 0.01). Evaluation of expression of glucose transporters (GLUT-1, -3, and -4) and hexokinases (HK-1 and -2), revealed increased expression of HK-1 in VAT-derived compared with SAT-derived stromal vascular cells, and also in visceral versus subcutaneous unfractionated AT. CONCLUSIONS In humans in vivo, VAT has increased glucose uptake compared with SAT, as determined noninvasively with FDG PET imaging. Differential stromal metabolic activity may be 1 mechanism underlying differences in metabolic activity of visceral and subcutaneous AT.

The Multiple Facets of the Fat Tissue

After a long history of relative neglect by the scientific community, white adipose tissue is finally emerging as a central component of body homeostasis. Indeed, the explosion of obesity statistics worldwide encouraged the study of adipose tissue and the complications of increased adiposity, such as insulin resistance, type 2 diabetes, and accelerated vascular disease. Far beyond merely furnishing free fatty acids from triglyceride depots, a growing list of fat tissue-derived mediators has increased the understanding of the regulatory role of adipose tissue in metabolic control. Recently, inflammation within the obese adipose tissue has surfaced as another important link of obesity to its undesirable metabolic consequences.

Deletion of EP4 on Bone Marrow–Derived Cells Enhances Inflammation and Angiotensin II–Induced Abdominal Aortic Aneurysm Formation

Objective—To examine whether a lack of prostaglandin E receptor 4 (EP4) on bone marrow–derived cells would increase local inflammation and enhance the formation of abdominal aortic aneurysm (AAA) in vivo. Methods and Results—Prostaglandin E2 (PGE2) through activation of EP4, can mute inflammation. Hypercholesterolemic low-density lipoprotein receptor knockout (LDLR−/−) mice transplanted with either EP4+/+ (EP4+/+/LDLR−/−) or EP4−/− (EP4−/−/LDLR−/−) bone marrow received infusions of angiotensin II to induce AAA. Deficiency of EP4 on bone marrow–derived cells increased the incidence (50% of male EP4+/+/LDLR−/− mice versus 88.9% of male EP4−/−/LDLR−/− mice developed AAA; and 22% of female EP4+/+/LDLR−/− mice versus 83.3% of female EP4−/−/LDLR−/− mice developed AAA) and severity of AAA, increased monocyte chemoattractant protein-1 (2.72-fold in males and 1.64-fold in females), and enhanced infiltration of macrophages (3.8-fold in males and 2.44-fold in females) and T cells (1.88-fold in males and 1.66-fold in females) into AAA lesions. Lack of EP4 on bone marrow–derived cells augmented elastin fragmentation, increased apoptotic markers, and decreased smooth muscle cell accumulation within AAA lesions. Conclusion—Deficiency of EP4 on bone marrow–derived cells boosted inflammation and AAA formation induced by angiotensin II in hyperlipidemic mice. This study affirms the pathophysiologic importance of PGE2 signaling through EP4 as an endogenous anti-inflammatory pathway involved in experimental aneurysm formation.

Familial hypercholesterolemia in Brazil: Cascade screening program, clinical and genetic aspects

BACKGROUND There is little knowledge about familial hypercholesterolemia in Brazil. This study presents the first results of genetic cascade screening performed in the city of Sao Paulo. MATERIAL AND METHODS Two-hundred and forty-eight suspected index cases were initially included. DNA was extracted from peripheral blood and the complete coding sequence of low-density lipoprotein receptor, exon 7 of proprotein convertase subtilisin/kexin type 9 gene and part of exon 26 of apolipoprotein B genes were sequenced. Multiplex Ligation-dependent Probe Amplification was performed on cases where a causal mutation was not identified through sequencing. After the identification of a causal mutation screening in first-degree relatives was pursued. RESULTS From 248 index cases, a mutation was found in 125 individuals (50.4%). 394 relatives were included in the cascade screening program and a mutation was identified in 59.4%. Seventy different causal mutations in the low-density lipoprotein receptor gene (97.2%) and 2 in the apolipoprotein B gene (2.8%) were found. No mutations were encountered in the proprotein convertase subtilisin/kexin type 9 gene. Mutations in exons 14 and 4 were the most prevalent and, 10 cases of true homozygotes (8 index cases and 2 relatives) and 1 compound heterozygote were identified. The most frequent mutation found was of Lebanese origin, the p.(Cys681*) mutation in exon 14 (8.5%). CONCLUSION Genetic familial hypercholesterolemia cascade screening is feasible in Brazil and leads to identification of a mutation in approximately half of the index cases with higher rates of success in their relatives.

Loss of Myeloid Related Protein-8/14 Exacerbates Cardiac Allograft Rejection

Background— The calcium-binding proteins myeloid-related protein (MRP)-8 (S100A8) and MRP-14 (S100A9) form MRP-8/14 heterodimers (S100A8/A9, calprotectin) that regulate myeloid cell function and inflammatory responses and serve as early serum markers for monitoring acute allograft rejection. Despite functioning as a proinflammatory mediator, the pathophysiological role of MRP-8/14 complexes in cardiovascular disease is incompletely defined. This study investigated the role of MRP-8/14 in cardiac allograft rejection using MRP-14−/− mice that lack MRP-8/14 complexes. Methods and Results— We examined parenchymal rejection after major histocompatibility complex class II allomismatched cardiac transplantation (bm12 donor heart and B6 recipients) in wild-type (WT) and MRP-14−/− recipients. Allograft survival averaged 5.9±2.9 weeks (n=10) in MRP-14−/− recipients compared with >12 weeks (n=15; P<0.0001) in WT recipients. Two weeks after transplantation, allografts in MRP-14−/− recipients had significantly higher parenchymal rejection scores (2.8±0.8; n=8) than did WT recipients (0.8±0.8; n=12; P<0.0001). Compared with WT recipients, allografts in MRP-14−/− recipients had significantly increased T-cell and macrophage infiltration and increased mRNA levels of interferon-&ggr; and interferon-&ggr;–associated chemokines (CXCL9, CXCL10, and CXCL11), interleukin-6, and interleukin-17 with significantly higher levels of Th17 cells. MRP-14−/− recipients also had significantly more lymphocytes in the adjacent para-aortic lymph nodes than did WT recipients (cells per lymph node: 23.7±0.7×105 for MRP-14−/− versus 6.0±0.2×105 for WT; P<0.0001). The dendritic cells (DCs) of the MRP-14−/− recipients of bm12 hearts expressed significantly higher levels of the costimulatory molecules CD80 and CD86 than did those of WT recipients 2 weeks after transplantation. Mixed leukocyte reactions with allo–endothelial cell–primed MRP-14−/− DCs resulted in significantly higher antigen-presenting function than reactions using WT DCs. Ovalbumin-primed MRP-14−/− DCs augmented proliferation of OT-II (ovalbumin-specific T cell receptor transgenic) CD4+ T cells with increased interleukin-2 and interferon-&ggr; production. Cardiac allografts of B6 major histocompatibility complex class II−/− hosts and of B6 WT hosts receiving MRP-14−/− DCs had significantly augmented inflammatory cell infiltration and accelerated allograft rejection compared with WT DCs from transferred recipient allografts. Bone marrow–derived MRP-14−/− DCs infected with MRP-8 and MRP-14 retroviral vectors showed significantly decreased CD80 and CD86 expression compared with controls, indicating that MRP-8/14 regulates B7-costimulatory molecule expression. Conclusions— Our results indicate that MRP-14 regulates B7 molecule expression and reduces antigen presentation by DCs and subsequent T-cell priming. The absence of MRP-14 markedly increased T-cell activation and exacerbated allograft rejection, indicating a previously unrecognized role for MRP-14 in immune cell biology.

Lack of EP4 receptors on bone marrow–derived cells enhances inflammation in atherosclerotic lesions

AIM prostaglandin E(2), by ligation of its receptor EP4, suppresses the production of inflammatory cytokines and chemokines in macrophages in vitro. Thus, activation of EP4 may constitute an endogenous anti-inflammatory pathway. This study investigated the role of EP4 in atherosclerosis in vivo, and particularly its impact on inflammation. METHODS AND RESULTS Ldlr(-/-) mice transplanted with EP4(+/+) or EP4(-/-) bone marrow consumed a high-fat diet for 5 or 10 weeks. Allogenic bone marrow transplantation promoted exacerbation of atherosclerosis irrespective of EP4 genotype, compatible with prior observations of exacerbated atherogenesis by allogenicity. EP4 deficiency had little effect on plaque size or morphology in early atherosclerosis, but at the later time point, mice deficient in EP4 displayed enhanced inflammation in their atherosclerotic plaques. Expression of monocyte chemoattractant protein-1 and interferon-γ inducible protein 10 increased, and there was a corresponding increase in macrophage and T-cell infiltration. These plaques also contained fewer smooth muscle cells. Despite these changes, mice deficient in EP4 in bone marrow-derived cells at an advanced stage had similar lesion size (in both aorta and aortic root) as mice with EP4. CONCLUSION this study shows that in advanced atherosclerosis, EP4 deficiency did not alter atherosclerotic lesion size, but yielded plaques with exacerbated inflammation and altered lesion composition.