Enhancer of zeste homolog 2 (EZH2) regulates adipocyte lipid metabolism independent of adipogenic differentiation: Role of apolipoprotein E.

Enhancer of zeste homolog 2 (EZH2), an epigenetic regulator that plays a key role in cell differentiation and oncogenesis, was reported to promote adipogenic differentiation in vitro by catalyzing trimethylation of histone 3 lysine 27. However, inhibition of EZH2 induced lipid accumulation in certain cancer and hepatocyte cell lines. To address this discrepancy, we investigated the role of EZH2 in adipogenic differentiation and lipid metabolism using primary human and mouse preadipocytes and adipose-specific EZH2 knockout (KO) mice. We found that the EZH2-selective inhibitor GSK126 induced lipid accumulation in human adipocytes, without altering adipocyte differentiation marker gene expression. Moreover, adipocyte-specific EZH2 KO mice, generated by crossing EZH2 floxed mice with adiponectin-Cre mice, displayed significantly increased body weight, adipose tissue mass, and adipocyte cell size and reduced very low-density lipoprotein (VLDL) levels, as compared with littermate controls. These phenotypic alterations could not be explained by differences in feeding behavior, locomotor activity, metabolic energy expenditure, or adipose lipolysis. In addition, human adipocytes treated with either GSK126 or vehicle exhibited comparable rates of glucose-stimulated triglyceride accumulation and fatty acid uptake. Mechanistically, lipid accumulation induced by GSK126 in adipocytes was lipoprotein-dependent, and EZH2 inhibition or gene deletion promoted lipoprotein-dependent lipid uptake in vitro concomitant with up-regulated apolipoprotein E (ApoE) gene expression. Deletion of ApoE blocked the effects of GSK126 to promote lipoprotein-dependent lipid uptake in murine adipocytes. Collectively, these results indicate that EZH2 inhibition promotes lipoprotein-dependent lipid accumulation via inducing ApoE expression in adipocytes, suggesting a novel mechanism of lipid regulation by EZH2.

Epigenetic Regulation of Vascular Diseases.

Epigenetic regulatory mechanisms, encompassing diverse molecular processes including DNA methylation, histone post-translational modifications, and noncoding RNAs, are essential to numerous processes such as cell differentiation, growth and development, environmental adaptation, aging, and disease states. In many cases, epigenetic changes occur in response to environmental cues and lifestyle factors, resulting in persistent changes in gene expression that affect vascular disease risk during the lifetime of the individual. Biological aging-a powerful cardiovascular risk factor-is partly genetically determined yet strongly influenced by traditional risk factors, reflecting epigenetic modulation. Quantification of specific DNA methylation patterns may serve as an accurate predictor of biological age-a concept known as the epigenetic clock, which could help to refine cardiovascular risk assessment. Epigenetic reprogramming of monocytes rewires cellular immune signaling and induces a metabolic shift toward aerobic glycolysis, thereby increasing innate immune responses. This form of trained epigenetic memory can be maladaptive, thus augmenting vascular inflammation. Somatic mutations in epigenetic regulatory enzymes lead to clonal hematopoiesis of indeterminate potential, a precursor of hematologic malignancies and a recently recognized cardiovascular risk factor; moreover, epigenetic regulators are increasingly being targeted in cancer therapeutics. Thus, understanding epigenetic regulatory mechanisms lies at the intersection between cancer and cardiovascular disease and is of paramount importance to the burgeoning field of cardio-oncology (Graphic Abstract).

Remote Effects of Transplanted Perivascular Adipose Tissue on Endothelial Function and Atherosclerosis.

PURPOSE: Perivascular adipose tissue (PVAT) surrounds the arterial adventitia and plays an important role in vascular homeostasis. PVAT expands in obesity, and inflamed PVAT can locally promote endothelial dysfunction and atherosclerosis. Here, using adipose tissue transplantation, we tested the hypothesis that expansion of PVAT can also remotely exacerbate vascular disease. METHODS: Fifty milligrams of abdominal aortic PVAT was isolated from high-fat diet (HFD)-fed wild-type mice and transplanted onto the abdominal aorta of lean LDL receptor knockout mice. Subcutaneous and visceral adipose tissues were used as controls. After HFD feeding for 10 weeks, body weight, glucose/insulin sensitivity, and lipid levels were measured. Adipocytokine gene expression was assessed in the transplanted adipose tissues, and the thoracic aorta was harvested to quantify atherosclerotic lesions by Oil-Red O staining and to assess vasorelaxation by wire myography. RESULTS: PVAT transplantation did not influence body weight, fat composition, lipid levels, or glucose/insulin sensitivity. However, as compared with controls, transplantation of PVAT onto the abdominal aorta increased thoracic aortic atherosclerosis. Furthermore, PVAT transplantation onto the abdominal aorta inhibited endothelium-dependent relaxation in the thoracic aorta. MCP-1 and TNF-α expression was elevated, while adiponectin expression was reduced, in the transplanted PVAT tissue, suggesting augmented inflammation as a potential mechanism for the remote vascular effects of transplanted PVAT. CONCLUSIONS: These data suggest that PVAT expansion and inflammation in obesity can remotely induce endothelial dysfunction and augment atherosclerosis. Identifying the underlying mechanisms may lead to novel approaches for risk assessment and treatment of obesity-related vascular disease.

High-density lipoprotein reduces inflammation from cholesterol crystals by inhibiting inflammasome activation.

Interleukin-1ß (IL-1ß), a potent pro-inflammatory cytokine, has been implicated in many diseases, including atherosclerosis. Activation of IL-1ß is controlled by a multi-protein complex, the inflammasome. The exact initiating event in atherosclerosis is unknown, but recent work has demonstrated that cholesterol crystals (CC) may promote atherosclerosis development by activation of the inflammasome. High-density lipoprotein (HDL) has consistently been shown to be anti-atherogenic and to have anti-inflammatory effects, but its mechanism of action is unclear. We demonstrate here that HDL is able to suppress IL-1ß secretion in response to cholesterol crystals in THP-1 cells and in human-monocyte-derived macrophages. HDL is able to blunt inflammatory monocyte cell recruitment in vivo following intraperitoneal CC injection in mice. HDL appears to modulate inflammasome activation in several ways. It reduces the loss of lysosomal membrane integrity following the phagocytosis of CC, but the major mechanism for the suppression of inflammasome activation by HDL is decreased expression of pro-IL-1ß and NLRP3, and reducing caspase-1 activation. In summary, we have described a novel anti-inflammatory effect of HDL, namely its ability to suppress inflammasome activation by CC by modulating the expression of several key components of the inflammasome.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice.

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

Impact of housing temperature on adipose tissue HDAC9 expression and adipogenic differentiation in high fat-fed mice.

OBJECTIVE: Impaired adipogenic differentiation exacerbates metabolic disease in obesity. This study reported that high-fat diet (HFD)-fed mice housed at thermoneutrality exhibited impaired adipogenic differentiation, attributed to increased expression of histone deacetylase 9 (HDAC9). However, the impact of HFD on adipogenic differentiation is reportedly variable, possibly reflecting divergent environmental conditions such as housing temperature. METHODS: C57BL/6J (wild-type [WT]) mice were housed at either thermoneutral (28-30°C) or ambient (20-22°C) temperature and fed HFD or chow diet (CD) for 12 weeks. For acute exposure experiments, WT or transient receptor potential cation channel subfamily M member 8 (TRPM8) knockout mice housed under thermoneutrality were acutely exposed to ambient temperature for 6 to 24 h. RESULTS: WT mice fed HFD and housed at thermoneutrality, compared with ambient temperature, gained more weight despite reduced food intake. They likewise exhibited increased inguinal adipose tissue HDAC9 expression and reduced adipogenic differentiation in vitro and in vivo compared with CD-fed mice. Conversely, HFD-fed mice housed at ambient temperature exhibited minimal change in adipose HDAC9 expression or adipogenic differentiation. Acute exposure of WT mice to ambient temperature reduced adipose HDAC9 expression independent of sympathetic ß-adrenergic signaling via a TRPM8-dependent mechanism. CONCLUSIONS: Adipose HDAC9 expression is temperature sensitive, regulating adipogenic differentiation in HFD-fed mice housed under thermoneutrality.

Sex-Dependent Role of Adipose Tissue HDAC9 in Diet-Induced Obesity and Metabolic Dysfunction.

Obesity is a major risk factor for both metabolic and cardiovascular disease. We reported that, in obese male mice, histone deacetylase 9 (HDAC9) is upregulated in adipose tissues, and global deletion of HDAC9 protected against high fat diet (HFD)-induced obesity and metabolic disease. Here, we investigated the impact of adipocyte-specific HDAC9 gene deletion on diet-induced obesity in male and female mice. The HDAC9 gene expression was increased in adipose tissues of obese male and female mice and HDAC9 expression correlated positively with body mass index in humans. Interestingly, female, but not male, adipocyte-specific HDAC9 KO mice on HFD exhibited reduced body weight and visceral adipose tissue mass, adipocyte hypertrophy, and improved insulin sensitivity, glucose tolerance and adipogenic differentiation gene expression. Furthermore, adipocyte-specific HDAC9 gene deletion in female mice improved metabolic health as assessed by whole body energy expenditure, oxygen consumption, and adaptive thermogenesis. Mechanistically, compared to female mice, HFD-fed male mice exhibited preferential HDAC9 expression in the stromovascular fraction, which may have offset the impact of adipocyte-specific HDAC9 gene deletion in male mice. These results suggest that HDAC9 expressed in adipocytes is detrimental to obesity in female mice and provides novel evidence of sex-related differences in HDAC9 cellular expression and contribution to obesity-related metabolic disease.

Long-chain monounsaturated fatty acid-rich fish oil attenuates the development of atherosclerosis in mouse models.

SCOPE: Fish oil-derived long-chain monounsaturated fatty acids (LCMUFA) containing chain lengths longer than 18 were previously shown to improve cardiovascular disease risk factors in mice. However, it is not known if LCMUFA also exerts anti-atherogenic effects. The main objective of the present study was to investigate the effect of LCMUFA on the development of atherosclerosis in mouse models. METHODS AND RESULTS: LDLR-KO mice were fed Western diet supplemented with 2% (w/w) of either LCMUFA concentrate, olive oil, or not (control) for 12 wk. LCMUFA, but not olive oil, significantly suppressed the development of atherosclerotic lesions and several plasma inflammatory cytokine levels, although there were no major differences in plasma lipids between the three groups. At higher doses 5% (w/w) LCMUFA supplementation was observed to reduce pro-atherogenic plasma lipoproteins and to also reduce atherosclerosis in ApoE-KO mice fed a Western diet. RNA sequencing and subsequent qPCR analyses revealed that LCMUFA upregulated PPAR signaling pathways in liver. In cell culture studies, apoB-depleted plasma from LDLR-K mice fed LCMUFA showed greater cholesterol efflux from macrophage-like THP-1 cells and ABCA1-overexpressing BHK cells. CONCLUSION: Our research showed for the first time that LCMUFA consumption protects against diet-induced atherosclerosis, possibly by upregulating the PPAR signaling pathway.

Elevated interleukin-10: a new cause of dyslipidemia leading to severe HDL deficiency.

BACKGROUND: Low high-density lipoprotein cholesterol (HDL-C) is a risk factor for coronary artery disease. Investigating mechanisms underlying acquired severe HDL deficiency in noncritically ill patients ("disappearing HDL syndrome") could provide new insights into HDL metabolism. OBJECTIVE: To determine the cause of low HDL-C in patients with severe acquired HDL deficiency. METHODS AND RESULTS: Patients with intravascular large B-cell lymphoma (n = 2), diffuse large B-cell lymphoma (n = 1), and autoimmune lymphoproliferative syndrome (n = 1) presenting with markedly decreased HDL-C, low low-density lipoprotein cholesterol (LDL-C), and elevated triglycerides were identified. The abnormal lipoprotein profile returned to normal after therapy in all 4 patients. All patients were found to have markedly elevated serum interleukin-10 (IL-10) levels that also normalized after therapy. In a cohort of autoimmune lymphoproliferative syndrome patients (n = 93), IL-10 showed a strong inverse correlation with HDL-C (R(2) = 0.3720, P < .0001). A direct causal role for increased serum IL-10 in inducing the observed changes in lipoproteins was established in a randomized, placebo-controlled clinical trial of recombinant human IL-10 in psoriatic arthritis patients (n = 18). Within a week of initiating subcutaneous recombinant human IL-10 injections, HDL-C precipitously decreased to near-undetectable levels. LDL-C also decreased by more than 50% (P < .0001) and triglycerides increased by approximately 2-fold (P < .005). All values returned to baseline after discontinuing IL-10 therapy. CONCLUSION: Increased IL-10 causes severe HDL-C deficiency, low LDL-C, and elevated triglycerides. IL-10 is thus a potent modulator of lipoprotein levels, a potential new biomarker for B-cell disorders, and a novel cause of disappearing HDL syndrome.