P Coutinho

Interactions Between Vascular Wall and Perivascular Adipose Tissue Reveal Novel Roles for Adiponectin in the Regulation of Endothelial Nitric Oxide Synthase Function in Human Vessels

Background— Adiponectin is an adipokine with potentially important roles in human cardiovascular disease states. We studied the role of adiponectin in the cross-talk between adipose tissue and vascular redox state in patients with atherosclerosis. Methods and Results— The study included 677 patients undergoing coronary artery bypass graft surgery. Endothelial function was evaluated by flow-mediated dilation of the brachial artery in vivo and by vasomotor studies in saphenous vein segments ex vivo. Vascular superoxide (O2−) and endothelial nitric oxide synthase (eNOS) uncoupling were quantified in saphenous vein and internal mammary artery segments. Local adiponectin gene expression and ex vivo release were quantified in perivascular (saphenous vein and internal mammary artery) subcutaneous and mesothoracic adipose tissue from 248 patients. Circulating adiponectin was independently associated with nitric oxide bioavailability and O2− production/eNOS uncoupling in both arteries and veins. These findings were supported by a similar association between functional polymorphisms in the adiponectin gene and vascular redox state. In contrast, local adiponectin gene expression/release in perivascular adipose tissue was positively correlated with O2− and eNOS uncoupling in the underlying vessels. In ex vivo experiments with human saphenous veins and internal mammary arteries, adiponectin induced Akt-mediated eNOS phosphorylation and increased tetrahydrobiopterin bioavailability, improving eNOS coupling. In ex vivo experiments with human saphenous veins/internal mammary arteries and adipose tissue, we demonstrated that peroxidation products produced in the vascular wall (ie, 4-hydroxynonenal) upregulate adiponectin gene expression in perivascular adipose tissue via a peroxisome proliferator-activated receptor-&ggr;–dependent mechanism. Conclusions— We demonstrate for the first time that adiponectin improves the redox state in human vessels by restoring eNOS coupling, and we identify a novel role of vascular oxidative stress in the regulation of adiponectin expression in human perivascular adipose tissue.

Adiponectin as a Link Between Type 2 Diabetes and Vascular NADPH Oxidase Activity in the Human Arterial Wall: The Regulatory Role of Perivascular Adipose Tissue

Oxidative stress plays a critical role in the vascular complications of type 2 diabetes. We examined the effect of type 2 diabetes on NADPH oxidase in human vessels and explored the mechanisms of this interaction. Segments of internal mammary arteries (IMAs) with their perivascular adipose tissue (PVAT) and thoracic adipose tissue were obtained from 386 patients undergoing coronary bypass surgery (127 with type 2 diabetes). Type 2 diabetes was strongly correlated with hypoadiponectinemia and increased vascular NADPH oxidase–derived superoxide anions (O2˙−). The genetic variability of the ADIPOQ gene and circulating adiponectin (but not interleukin-6) were independent predictors of NADPH oxidase–derived O2˙−. However, adiponectin expression in PVAT was positively correlated with vascular NADPH oxidase–derived O2˙−. Recombinant adiponectin directly inhibited NADPH oxidase in human arteries ex vivo by preventing the activation/membrane translocation of Rac1 and downregulating p22phox through a phosphoinositide 3-kinase/Akt-mediated mechanism. In ex vivo coincubation models of IMA/PVAT, the activation of arterial NADPH oxidase triggered a peroxisome proliferator–activated receptor-γ–mediated upregulation of the adiponectin gene in the neighboring PVAT via the release of vascular oxidation products. We demonstrate for the first time in humans that reduced adiponectin levels in individuals with type 2 diabetes stimulates vascular NADPH oxidase, while PVAT “senses” the increased NADPH oxidase activity in the underlying vessel and responds by upregulating adiponectin gene expression. This PVAT-vessel interaction is identified as a novel therapeutic target for the prevention of vascular complications of type 2 diabetes.

Mutual Regulation of Epicardial Adipose Tissue and Myocardial Redox State by PPAR-γ/Adiponectin Signalling.

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Tracking Monocyte Recruitment and Macrophage Accumulation in Atherosclerotic Plaque Progression Using a Novel hCD68GFP/ApoE-/- Reporter Mouse-Brief Report.

Objective— To create a model of atherosclerosis using green fluorescent protein (GFP)–targeted monocytes/macrophages, allowing analysis of both endogenous GFP+ and adoptively transferred GFP+ myeloid cells in arterial inflammation. Approach and Results— hCD68GFP reporter mice were crossed with ApoE−/− mice. Expression of GFP was localized to macrophages in atherosclerotic plaques and in angiotensin II–induced aortic aneurysms and correlated with galectin 3 and mCD68 expression. Flow cytometry confirmed GFP+ expression in CD11b+/CD64+, CD11c+/MHC-IIHI, and CD11b+/F4/80+ myeloid cells. Adoptive transfer of GFP+ monocytes demonstrated monocyte recruitment to both adventitia and atherosclerotic plaque, throughout the aortic root, within 72 hours. We demonstrated the biological utility of hCD68GFP monocytes by comparing the recruitment of wild-type and CCR2−/− monocytes to sites of inflammation. Conclusions— hCD68GFP/ApoE−/− mice provide a new approach to study macrophage accumulation in atherosclerotic plaque progression and to identify cells recruited from adoptively transferred monocytes.

YIA2: A NOVEL CROSS-TALK BETWEEN PERIVASCULAR ADIPOSE TISSUE AND THE ARTERIAL WALL CONTROLS REDOX STATE IN HUMAN ATHEROSCLEROSIS

Background Endothelial nitric oxide synthase (eNOS) plays a crucial role in maintenance of vascular homeostasis. However, loss of eNOS co-factor tetrahydrobiopterin (BH4) due to oxidative degradation leads to uncoupling of the enzyme, that is turned into a source of superoxide radicals (O2−) instead of nitric oxide (NO). Adipose tissue has been identified as the source of hormone-like molecules termed adipokines, which can exert endocrine and paracrine effects on the vascular wall. However, little is known about the role of adipokines such as adiponectin (AdN) in the regulation of vascular redox signalling in the human arterial wall or the mechanisms regulating their synthesis in perivascular adipose tissue (PVAT). Methods In Study 1, 677 patients undergoing coronary bypass surgery (CABG) were recruited. Blood samples were obtained preoperatively and internal mammary artery (IMA) segments as well as PVAT surrounding them were harvested during surgery. Serum AdN was quantified ELISA, vascular O2− was determined by lucigenin chemiluminescence (+/−LNAME to estimate eNOS coupling) and qRTPCR was performed to determine AdN gene expression in PVAT. In Study 2, IMA segments from 17 patients undergoing CABG were exposed to AdN (10u2005µg/ml, 6h)±wortmannin (W, a PI3K/Akt inhibitor) ex vivo to determine the effects of AdN on vascular redox state, eNOS phosphorylation status and BH4 content. In addition, PVAT was exposed to 4-hydroxynonenal (4HNE, a peroxidation product released by the vascular wall in the presence of high vascular oxidative stress) ex vivo to determine its effects on AdN gene expression. Results In Study 1, serum AdN was inversely related with resting O2− (p<0.01) and positively with the degree of eNOS coupling (p<0.05) in the IMA. However, the expression of AdN gene in PVAT was positively related with resting O2- (A) and eNOS uncoupling (B) in the same vessels. In Study 2, AdN reduced O2− (p<0.05) by restoring eNOS coupling (p<0.05) in human arteries ex vivo. This was due to Akt-dependent eNOS phosphorylation at Ser1177 (C&D) and an increase in vascular BH4 (p<0.05). Vascular O2− triggered the release of 4HNE, while ex vivo exposure of PVAT to 4HNE up-regulated the expression of PPARγ (P<0.05) and subsequently the expression of AdN gene (P<0.05). Conclusions We describe a novel cross-talk between adipose tissue and the vascular wall in humans. Oxidative stress triggers the release of 4HNE from the vascular wall, which in turn up-regulates the expression of AdN in PVAT; then AdN exerts a paracrine effect on the vascular wall by activating eNOS and improving its BH4-mediated coupling. Figure 1 Figure 2