Brian E. Sansbury

Overexpression of Endothelial Nitric Oxide Synthase Prevents Diet-Induced Obesity and Regulates Adipocyte Phenotype

Rationale: Endothelial dysfunction is a characteristic feature of diabetes and obesity in animal models and humans. Deficits in nitric oxide production by endothelial nitric oxide synthase (eNOS) are associated with insulin resistance, which is exacerbated by high-fat diet. Nevertheless, the metabolic effects of increasing eNOS levels have not been studied. Objective: The current study was designed to test whether overexpression of eNOS would prevent diet-induced obesity and insulin resistance. Methods and Results: In db/db mice and in high-fat diet-fed wild-type C57BL/6J mice, the abundance of eNOS protein in adipose tissue was decreased without significant changes in eNOS levels in skeletal muscle or aorta. Mice overexpressing eNOS (eNOS transgenic mice) were resistant to diet-induced obesity and hyperinsulinemia, although systemic glucose intolerance remained largely unaffected. In comparison with wild-type mice, high-fat diet-fed eNOS transgenic mice displayed a higher metabolic rate and attenuated hypertrophy of white adipocytes. Overexpression of eNOS did not affect food consumption or diet-induced changes in plasma cholesterol or leptin levels, yet plasma triglycerides and fatty acids were decreased. Metabolomic analysis of adipose tissue indicated that eNOS overexpression primarily affected amino acid and lipid metabolism; subpathway analysis suggested changes in fatty acid oxidation. In agreement with these findings, adipose tissue from eNOS transgenic mice showed higher levels of PPAR-&agr; and PPAR-&ggr; gene expression, elevated abundance of mitochondrial proteins, and a higher rate of oxygen consumption. Conclusions: These findings demonstrate that increased eNOS activity prevents the obesogenic effects of high-fat diet without affecting systemic insulin resistance, in part, by stimulating metabolic activity in adipose tissue.

Bioenergetic function in cardiovascular cells: the importance of the reserve capacity and its biological regulation

The ability of the cell to generate sufficient energy through oxidative phosphorylation and to maintain healthy pools of mitochondria are critical for survival and maintenance of normal biological function, especially during periods of increased oxidative stress. Mitochondria in most cardiovascular cells function at a basal level that only draws upon a small fraction of the total bioenergetic capability of the organelle; the apparent respiratory state of mitochondria in these cells is often close to state 4. The difference between the basal and maximal activity, equivalent to state 3, of the respiratory chain is called the reserve capacity. We hypothesize that the reserve capacity serves the increased energy demands for maintenance of organ function and cellular repair. However, the factors that determine the volume of the reserve capacity and its relevance to biology are not well understood. In this study, we first examined whether responses to 4-hydroxynonenal (HNE), a lipid peroxidation product found in atherosclerotic lesions and the diseased heart, differ between vascular smooth muscle cells, adult mouse cardiomyocytes, and rat neonatal cardiomyocytes. In both types of cardiomyocytes, oxygen consumption increased after HNE treatment, while oxygen consumption in smooth muscle cells decreased. The increase in oxygen consumption in cardiomyocytes decreased the reserve capacity and shifted the apparent respiratory state closer to state 3. Neonatal rat cardiomyocytes respiring on pyruvate alone had a fourfold higher reserve capacity than cells with glucose as the sole substrate, and these cells were more resistant to mitochondrial dysfunction induced by 4-HNE. The integration of the concepts of reserve capacity and state-apparent are discussed along with the proposal of two potential models by which mitochondria respond to stress.

Metabolomic Analysis of Pressure-overloaded and Infarcted Mouse Hearts

Background—Cardiac hypertrophy and heart failure are associated with metabolic dysregulation and a state of chronic energy deficiency. Although several disparate changes in individual metabolic pathways have been described, there has been no global assessment of metabolomic changes in hypertrophic and failing hearts in vivo. Hence, we investigated the impact of pressure overload and infarction on myocardial metabolism. Methods and Results—Male C57BL/6J mice were subjected to transverse aortic constriction or permanent coronary occlusion (myocardial infarction [MI]). A combination of LC/MS/MS and GC/MS techniques was used to measure 288 metabolites in these hearts. Both transverse aortic constriction and MI were associated with profound changes in myocardial metabolism affecting up to 40% of all metabolites measured. Prominent changes in branched-chain amino acids were observed after 1 week of transverse aortic constriction and 5 days after MI. Changes in branched-chain amino acids after MI were associated with myocardial insulin resistance. Longer duration of transverse aortic constriction and MI led to a decrease in purines, acylcarnitines, fatty acids, and several lysolipid and sphingolipid species but a marked increase in pyrimidines as well as ascorbate, heme, and other indices of oxidative stress. Cardiac remodeling and contractile dysfunction in hypertrophied hearts were associated with large increases in myocardial, but not plasma, levels of the polyamines putrescine and spermidine as well as the collagen breakdown product prolylhydroxyproline. Conclusions—These findings reveal extensive metabolic remodeling common to both hypertrophic and failing hearts that are indicative of extracellular matrix remodeling, insulin resistance and perturbations in amino acid, and lipid and nucleotide metabolism.

Metabolic remodeling of white adipose tissue in obesity

Adipose tissue metabolism is a critical regulator of adiposity and whole body energy expenditure; however, metabolic changes that occur in white adipose tissue (WAT) with obesity remain unclear. The purpose of this study was to understand the metabolic and bioenergetic changes occurring in WAT with obesity. Wild-type (C57BL/6J) mice fed a high-fat diet (HFD) showed significant increases in whole body adiposity, had significantly lower V̇(O₂), V̇(CO₂), and respiratory exchange ratios, and demonstrated worsened glucose and insulin tolerance compared with low-fat-fed mice. Metabolomic analysis of WAT showed marked changes in lipid, amino acid, carbohydrate, nucleotide, and energy metabolism. Tissue levels of succinate and malate were elevated, and metabolites that could enter the Krebs cycle via anaplerosis were mostly diminished in high-fat-fed mice, suggesting altered mitochondrial metabolism. Despite no change in basal oxygen consumption or mitochondrial DNA abundance, citrate synthase activity was decreased by more than 50%, and responses to FCCP were increased in WAT from mice fed a high-fat diet. Moreover, Pgc1a was downregulated and Cox7a1 upregulated after 6 wk of HFD. After 12 wk of high-fat diet, the abundance of several proteins in the mitochondrial respiratory chain or matrix was diminished. These changes were accompanied by increased Parkin and Pink1, decreased p62 and LC3-I, and ultrastructural changes suggestive of autophagy and mitochondrial remodeling. These studies demonstrate coordinated restructuring of metabolism and autophagy that could contribute to the hypertrophy and whitening of adipose tissue in obesity.

MerTK cleavage limits proresolving mediator biosynthesis and exacerbates tissue inflammation

Significance Specialized proresolving mediators (SPMs) are lipids that temper inflammation, enhance efferocytosis, and repair tissue damage after inflammation. However, the upstream regulators of SPM production are not completely identified. We show here that Mer proto-oncogene tyrosine kinase (MerTK) signaling in macrophages promotes the production of SPMs both in vitro and in vivo, and thereby contributes to the resolution process. Moreover, inflammation-induced MerTK cleavage can limit this response, as shown by the improvement in resolution in a new MerTK-cleavage resistant mouse model. These findings increase our knowledge of the regulation of SPM biosynthesis and suggest new ideas for improving resolution in chronic inflammatory diseases. The acute inflammatory response requires a coordinated resolution program to prevent excessive inflammation, repair collateral damage, and restore tissue homeostasis, and failure of this response contributes to the pathology of numerous chronic inflammatory diseases. Resolution is mediated in part by long-chain fatty acid-derived lipid mediators called specialized proresolving mediators (SPMs). However, how SPMs are regulated during the inflammatory response, and how this process goes awry in inflammatory diseases, are poorly understood. We now show that signaling through the Mer proto-oncogene tyrosine kinase (MerTK) receptor in cultured macrophages and in sterile inflammation in vivo promotes SPM biosynthesis by a mechanism involving an increase in the cytoplasmic:nuclear ratio of a key SPM biosynthetic enzyme, 5-lipoxygenase. This action of MerTK is linked to the resolution of sterile peritonitis and, after ischemia–reperfusion (I/R) injury, to increased circulating SPMs and decreased remote organ inflammation. MerTK is susceptible to ADAM metallopeptidase domain 17 (ADAM17)-mediated cell-surface cleavage under inflammatory conditions, but the functional significance is not known. We show here that SPM biosynthesis is increased and inflammation resolution is improved in a new mouse model in which endogenous MerTK was replaced with a genetically engineered variant that is cleavage-resistant (MertkCR). MertkCR mice also have increased circulating levels of SPMs and less lung injury after I/R. Thus, MerTK cleavage during inflammation limits SPM biosynthesis and the resolution response. These findings contribute to our understanding of how SPM synthesis is regulated during the inflammatory response and suggest new therapeutic avenues to boost resolution in settings where defective resolution promotes disease progression.

MerTK receptor cleavage promotes plaque necrosis and defective resolution in atherosclerosis

Atherothrombotic vascular disease is often triggered by a distinct type of atherosclerotic lesion that displays features of impaired inflammation resolution, notably a necrotic core and thinning of a protective fibrous cap that overlies the core. A key cause of plaque necrosis is defective clearance of apoptotic cells, or efferocytosis, by lesional macrophages, but the mechanisms underlying defective efferocytosis and its possible links to impaired resolution in atherosclerosis are incompletely understood. Here, we provide evidence that proteolytic cleavage of the macrophage efferocytosis receptor c-Mer tyrosine kinase (MerTK) reduces efferocytosis and promotes plaque necrosis and defective resolution. In human carotid plaques, MerTK cleavage correlated with plaque necrosis and the presence of ischemic symptoms. Moreover, in fat-fed LDL receptor–deficient (Ldlr–/–) mice whose myeloid cells expressed a cleavage-resistant variant of MerTK, atherosclerotic lesions exhibited higher macrophage MerTK, lower levels of the cleavage product soluble Mer, improved efferocytosis, smaller necrotic cores, thicker fibrous caps, and increased ratio of proresolving versus proinflammatory lipid mediators. These findings provide a plausible molecular-cellular mechanism that contributes to defective efferocytosis, plaque necrosis, and impaired resolution during the progression of atherosclerosis.

Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

Resolvin D2 Enhances Postischemic Revascularization While Resolving Inflammation

Background: Resolvins are lipid mediators generated by leukocytes during the resolution phase of inflammation. They have been shown to regulate the transition from inflammation to tissue repair; however, it is unknown whether resolvins play a role in tissue revascularization following ischemia. Methods: We used a murine model of hind limb ischemia (HLI), coupled with laser Doppler perfusion imaging, microcomputed tomography, and targeted mass spectrometry, to assess the role of resolvins in revascularization and inflammation resolution. Results: In mice undergoing HLI, we identified resolvin D2 (RvD2) in bone marrow and skeletal muscle by mass spectrometry (n=4–7 per group). We also identified RvD2 in skeletal muscle biopsies from humans with peripheral artery disease. Monocytes were recruited to skeletal muscle during HLI and isolated monocytes produced RvD2 in a lipoxygenase-dependent manner. Exogenous RvD2 enhanced perfusion recovery in HLI and microcomputed tomography of limb vasculature revealed greater volume, with evidence of tortuous arterioles indicative of arteriogenesis (n=6–8 per group). Unlike other treatment strategies for therapeutic revascularization that exacerbate inflammation, RvD2 did not increase vascular permeability, but reduced neutrophil accumulation and the plasma levels of tumor necrosis factor-&agr; and granulocyte macrophage colony-stimulating factor. In mice treated with RvD2, histopathologic analysis of skeletal muscle of ischemic limbs showed more regenerating myocytes with centrally located nuclei. RvD2 enhanced endothelial cell migration in a Rac-dependent manner, via its receptor, GPR18, and Gpr18-deficient mice had an endogenous defect in perfusion recovery following HLI. Importantly, RvD2 rescued defective revascularization in diabetic mice. ConclusionS: RvD2 stimulates arteriogenic revascularization during HLI, suggesting that resolvins may be a novel class of mediators that both resolve inflammation and promote arteriogenesis.

Resolution of Acute Inflammation and the Role of Resolvins in Immunity, Thrombosis, and Vascular Biology

Acute inflammation is a host-protective response that is mounted in response to tissue injury and infection. Initiated and perpetuated by exogenous and endogenous mediators, acute inflammation must be resolved for tissue repair to proceed and for homeostasis to be restored. Resolution of inflammation is an actively regulated process governed by an array of mediators as diverse as those that initiate inflammation. Among these, resolvins have emerged as a genus of evolutionarily conserved proresolving mediators that act on specific cellular receptors to regulate leukocyte trafficking and blunt production of inflammatory mediators, while also promoting clearance of dead cells and tissue repair. Given that chronic unresolved inflammation is emerging as a central causative factor in the development of cardiovascular diseases, an understanding of the endogenous processes that govern normal resolution of acute inflammation is critical for determining why sterile maladaptive cardiovascular inflammation perpetuates. Here, we provide an overview of the process of resolution with a focus on the enzymatic biosynthesis and receptor-dependent actions of resolvins and related proresolving mediators in immunity, thrombosis, and vascular biology. We discuss how nutritional and current therapeutic approaches modulate resolution and propose that harnessing resolution concepts could potentially lead to the development of new approaches for treating chronic cardiovascular inflammation in a manner that is not host disruptive.

Unidirectional and sustained delivery of the proresolving lipid mediator resolvin D1 from a biodegradable thin film device.

Resolvin D1 (RvD1) belongs to a family of endogenously derived proresolving lipid mediators that have been shown to attenuate inflammation, activate proresolution signaling, and promote homeostasis and recovery from tissue injury. In this study we present a poly(lactic-co-glycolic acid) (PLGA) based thin-film device composed of layers of varying ratios of lactic and glycolic acid that elutes RvD1 unidirectionally to target tissues. The device demonstrated sustained release in vitro for 56 days with an initial burst of release over 14 days. The asymmetric design of the device released 98% of RvD1 through the layer with the lowest molar ratio of lactic acid to glycolic acid, and the remainder through the opposite side. We validated structural integrity of RvD1 released from the device by mass spectrometry and investigated its bioactivity on human vascular endothelial (EC) and smooth muscle cells (VSMC). RvD1 released from the device attenuated VSMC migration, proliferation, and TNF-α induced NF-κB activation, without evidence of cytotoxicity. Delivery of RvD1 to blood vessels was demonstrated ex vivo in a flow chamber system using perfused rabbit aortas and in vivo in a rat carotid artery model, with the devices applied as an adventitial wrap. Our results demonstrate a novel approach for sustained, local delivery of Resolvin D1 to vascular tissue at therapeutically relevant levels.