Y. Eugene Chen

Loss of Perivascular Adipose Tissue on Peroxisome Proliferator–Activated Receptor-γ Deletion in Smooth Muscle Cells Impairs Intravascular Thermoregulation and Enhances Atherosclerosis

Background— Perivascular adipose tissue (PVAT) surrounds most vessels and shares common features with brown adipose tissue (BAT). Although adaptive thermogenesis in BAT increases energy expenditure and is beneficial for metabolic diseases, little is known about the role of PVAT in vascular diseases such as atherosclerosis. We hypothesize that the thermogenic function of PVAT regulates intravascular temperature and reduces atherosclerosis. Methods and Results— PVAT shares similar structural and proteomics with BAT. We demonstrated that PVAT has thermogenic properties similar to BAT in response to cold stimuli in vivo. Proteomics analysis of the PVAT from mice housed in a cold environment identified differential expression in proteins highly related to cellular metabolic processes. In a mouse model deficient in peroxisome proliferator–activated receptor-&ggr; in smooth muscle cells (SMPG KO mice), we uncovered a complete absence of PVAT surrounding the vasculature, likely caused by peroxisome proliferator–activated receptor-&ggr; deletion in the perivascular adipocyte precursor cells as well. Lack of PVAT, which results in loss of its thermogenic activity, impaired vascular homeostasis, which caused temperature loss and endothelial dysfunction. We further showed that cold exposure inhibits atherosclerosis and improves endothelial function in mice with intact PVAT but not in SMPG KO mice as a result of impaired lipid clearance. Proinflammatory cytokine expression in PVAT is not altered on exposure to cold. Finally, prostacyclin released from PVAT contributes to the vascular protection against endothelial dysfunction. Conclusions— PVAT is a vasoactive organ with functional characteristics similar to BAT and is essential for intravascular thermoregulation of cold acclimation. This thermogenic capacity of PVAT plays an important protective role in the pathogenesis of atherosclerosis.

Systematic evaluation of coding variation identifies a candidate causal variant in TM6SF2 influencing total cholesterol and myocardial infarction risk.

Blood lipid levels are heritable, treatable risk factors for cardiovascular disease. We systematically assessed genome-wide coding variation to identify new genes influencing lipid traits, fine map known lipid loci and evaluate whether low-frequency variants with large effects exist for these traits. Using an exome array, we genotyped 80,137 coding variants in 5,643 Norwegians. We followed up 18 variants in 4,666 Norwegians and identified ten loci with coding variants associated with a lipid trait (P < 5 × 10−8). One variant in TM6SF2 (encoding p.Glu167Lys), residing in a known genome-wide association study locus for lipid traits, influences total cholesterol levels and is associated with myocardial infarction. Transient TM6SF2 overexpression or knockdown of Tm6sf2 in mice alters serum lipid profiles, consistent with the association observed in humans, identifying TM6SF2 as a functional gene within a locus previously known as NCAN-CILP2-PBX4 or 19p13. This study demonstrates that systematic assessment of coding variation can quickly point to a candidate causal gene.

Molecular recognition of nitrated fatty acids by PPAR[gamma]

Peroxisome proliferator activated receptor-γ (PPARγ) regulates metabolic homeostasis and adipocyte differentiation, and it is activated by oxidized and nitrated fatty acids. Here we report the crystal structure of the PPARγ ligand binding domain bound to nitrated linoleic acid, a potent endogenous ligand of PPARγ. Structural and functional studies of receptor-ligand interactions reveal the molecular basis of PPARγ discrimination of various naturally occurring fatty acid derivatives.

Covalent Peroxisome Proliferator-activated Receptor γ Adduction by Nitro-fatty Acids SELECTIVE LIGAND ACTIVITY AND ANTI-DIABETIC SIGNALING ACTIONS

The peroxisome proliferator-activated receptor-γ (PPARγ) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARγ include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARγ ligands are intermediates of lipid metabolism and oxidation that bind PPARγ with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO2-FA) are endogenous products of nitric oxide (•NO) and nitrite (NO2−)-mediated redox reactions that activate PPARγ at nanomolar concentrations. We report that NO2-FA act as partial agonists of PPARγ and covalently bind PPARγ at Cys-285 via Michael addition. NO2-FA show selective PPARγ modulator characteristics by inducing coregulator protein interactions, PPARγ-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO2-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency.

Zinc-finger nuclease, transcription activator-like effector nuclease and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) are becoming major tools for genome editing. Importantly, knock-in in several non-rodent species has been finally achieved thanks to these customizable nucleases; yet the rates remain to be further improved. We hypothesize that inhibiting non-homologous end joining (NHEJ) or enhancing homology-directed repair (HDR) will improve the nuclease-mediated knock-in efficiency. Here we show that the in vitro application of an HDR enhancer, RS-1, increases the knock-in efficiency by two- to five-fold at different loci, whereas NHEJ inhibitor SCR7 has minimal effects. We then apply RS-1 for animal production and have achieved multifold improvement on the knock-in rates as well. Our work presents tools to nuclease-mediated knock-in animal production, and sheds light on improving gene-targeting efficiencies on pluripotent stem cells.

Perivascular Adipose Tissue in Vascular Function and Disease A Review of Current Research and Animal Models

Perivascular adipose tissue (PVAT), long assumed to be nothing more than vessel-supporting connective tissue, is now understood to be an important, active component of the vasculature, with integral roles in vascular health and disease. PVAT is an adipose tissue with similarities to both brown and white adipose tissue, although recent evidence suggests that PVAT develops from its own precursors. Like other adipose tissue depots, PVAT secretes numerous biologically active substances that can act in both autocrine and paracrine fashion. PVAT has also proven to be involved in vascular inflammation. Although PVAT can support inflammation during atherosclerosis via macrophage accumulation, emerging evidence suggests that PVAT also has antiatherosclerotic properties related to its abilities to induce nonshivering thermogenesis and metabolize fatty acids. We here discuss the accumulated knowledge of PVAT biology and related research on models of hypertension and atherosclerosis.

miR-10a contributes to retinoid acid-induced smooth muscle cell differentiation.

MicroRNAs (miRs) have been reported to play a critical role in muscle differentiation and function. The purpose of this study is to determine the role of miRs during smooth muscle cell (SMC) differentiation from embryonic stem cells (ESCs). MicroRNA profiling showed that miR-10a expression is steadily increased during in vitro differentiation of mouse ESCs into SMCs. Loss-of-function approaches using miR-10a inhibitors uncovered that miR-10a is a critical mediator for SMC lineage determination in our retinoic acid-induced ESC/SMC differentiation system. In addition, we have documented for the first time that histone deacetylase 4 is a novel target of miR-10a and mediates miR-10a function during ESC/SMC differentiation. To determine the molecular mechanism through which retinoic acid induced miR-10a expression, a consensus NF-κB element was identified in the miR-10a gene promoter by bioinformatics analysis, and chromatin immunoprecipitation assay confirmed that NF-κB could bind to this element. Finally, inhibition of NF-κB nuclear translocation repressed miR-10a expression and decreased SMC differentiation from ESCs. Our data demonstrate for the first time that miR-10a is a novel regulator in SMC differentiation from ESCs. These studies suggest that miR-10a may play important roles in vascular biology and have implications for the diagnosis and treatment of vascular diseases.

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Generation of PPARγ mono-allelic knockout pigs via zinc-finger nucleases and nuclear transfer cloning

Nitro-Oleic Acid Inhibits Angiotensin II–Induced Hypertension

Rationale Nitro-oleic acid (OA-NO2) is a bioactive, nitric-oxide derived fatty acid with physiologically relevant vasculoprotective properties in vivo. OA-NO2 exerts cell signaling actions as a result of its strong electrophilic nature and mediates pleiotropic cell responses in the vasculature. Objective The present study sought to investigate the protective role of OA-NO2 in angiotensin (Ang) II–induced hypertension. Methods and Results We show that systemic administration of OA-NO2 results in a sustained reduction of Ang II–induced hypertension in mice and exerts a significant blood pressure lowering effect on preexisting hypertension established by Ang II infusion. OA-NO2 significantly inhibits Ang II contractile response as compared to oleic acid (OA) in mesenteric vessels. The improved vasoconstriction is specific for the Ang II type 1 receptor (AT1R)-mediated signaling because vascular contraction by other G-protein–coupled receptors is not altered in response to OA-NO2 treatment. From the mechanistic viewpoint, OA-NO2 lowers Ang II–induced hypertension independently of peroxisome proliferation-activated receptor (PPAR)&ggr; activation. Rather, OA-NO2, but not OA, specifically binds to the AT1R, reduces heterotrimeric G-protein coupling, and inhibits IP3 (inositol-1,4,5-trisphosphate) and calcium mobilization, without inhibiting Ang II binding to the receptor. Conclusions These results demonstrate that OA-NO2 diminishes the pressor response to Ang II and inhibits AT1R-dependent vasoconstriction, revealing OA-NO2 as a novel antagonist of Ang II–induced hypertension.

PPARγ and its ligands: therapeutic implications in cardiovascular disease

The relevance of PPARgamma (peroxisome-proliferator-activated receptor gamma) as an important therapeutic target for the treatment of diabetes arises from its hypoglycaemic effects in diabetic patients and also from the critical role in the regulation of cardiovascular functions. From a clinical perspective, differences between current FDA (Food and Drug Administration)-approved PPARgamma drugs have been observed in terms of atherosclerosis and cardiac and stroke events. The adverse effects of PPARgamma-specific treatments that hamper their cardiovascular protective roles, affirm the strong need to evaluate the efficacy of the current drugs. Therefore active research is directed towards high-throughput screening and pharmacological testing of a plethora of newly identified natural or synthetic compounds. In the present review we describe the rationale behind drug design strategies targeting PPARgamma, based on current knowledge regarding the effects of such drugs in experimental animal models, as well as in clinical practice. Regarding endogenous PPARgamma ligands, several fatty acid derivatives bind PPARgamma with different affinities, although the physiological relevance of these interactions is not always evident. Recently, NO-derived unsaturated fatty acids were found to be potent agonists of PPARs, with preferential affinity for PPARgamma, compared with oxidized fatty acid derivatives. Nitroalkenes exert important bioactivities of relevance for the cardiovascular system including anti-inflammatory and antiplatelet actions, and are important mediators of vascular tone. A new generation of insulin sensitizers with PPARgamma function for the treatment of diabetes may serve to limit patients from the increased cardiovascular burden of this disease.