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.
Apolipoprotein (Apo) C-III (ApoCIII) resides on the surface of plasma chylomicron (CM), very low density lipoprotein (VLDL) and high density lipoproteins (HDL). It has been recognized that high levels of plasma ApoCIII constitutea risk factor for cardiovascular diseases (CVD). Elevated plasma ApoCIII level often correlates with insulin resistance, obesity, and hypertriglyceridemia. Invaluable knowledge on the roles of ApoCIIIin lipid metabolisms and CVD has been obtained from transgenic mouse models including ApoCIII knockout (KO) mice; however, it is noted that the metabolism of lipoprotein in mice is different from that of humans in many aspects. It is not known until now whether elevated plasma ApoCIII is directly atherogenic. We worked to develop ApoCIII KO rabbits in the present study based on the hypothesis that rabbits can serve as a reasonablemodelfor studying human lipid metabolism and atherosclerosis. Zinc finger nuclease (ZFN) sets targeting rabbit ApoCIIIgene were subjected to in vitro validation prior to embryo microinjection. The mRNA was injected to the cytoplasm of 35 rabbit pronuclear stage embryos, and evaluated the mutation rates at the blastocyst state. Of sixteen blastocysts that were assayed, a satisfactory 50% mutation rate (8/16) at the targeting site was achieved, supporting the use of Set 1 for in vivo experiments. Next, we microinjected 145 embryos with Set 1 mRNA, and transferred these embryos to 7 recipient rabbits. After 30 days gestation, 21 kits were born, out of which five were confirmed as ApoCIII KO rabbits after PCR sequencing assays. The KO animal rate (#KO kits/total born) was 23.8%. The overall production efficiency is 3.4% (5 kits/145 embryos transferred). The present work demonstrated that ZFN is a highly efficient method to produce KO rabbits. These ApoCIII KO rabbits are novel resources to study the roles of ApoCIII in lipid metabolisms.
Transcription factors play an important role in the pathophysiology of many neurological disorders, including stroke. In the past three decades, an increasing number of transcription factors and their related gene signaling networks have been identified, and have become a research focus in the stroke field. Krüppel-like factors (KLFs) are members of the zinc finger family of transcription factors with diverse regulatory functions in cell growth, differentiation, proliferation, migration, apoptosis, metabolism, and inflammation. KLFs are also abundantly expressed in the brain where they serve as critical regulators of neuronal development and regeneration to maintain normal brain function. Dysregulation of KLFs has been linked to various neurological disorders. Recently, there is emerging evidence that suggests KLFs have an important role in the pathogenesis of stroke and provide endogenous vaso-or neuro-protection in the brains response to ischemic stimuli. In this review, we summarize the basic knowledge and advancement of these transcriptional mediators in the central nervous system, highlighting the novel roles of KLFs in stroke.
Peroxisome proliferator-activated receptor gamma (PPAR?) is emerging as a major regulator in neurological diseases. However, the role of (PPAR?) and its co-regulators in cerebrovascular endothelial dysfunction after stroke is unclear. Here, we have demonstrated that (PPAR?) activation by pioglitazone significantly inhibited both oxygen-glucose deprivation-induced cerebral vascular endothelial cell death and middle cerebral artery occlusion-triggered cerebrovascular damage. Consistent with this finding, selective (PPAR?) genetic deletion in vascular endothelial cells resulted in increased cerebrovascular permeability and brain infarction in mice after focal ischaemia. Moreover, we screened for (PPAR?) co-regulators using a genome-wide and high-throughput co-activation system and revealed KLF11 as a novel (PPAR?) co-regulator, which interacted with (PPAR?) and regulated its function in mouse cerebral vascular endothelial cell cultures. Interestingly, KLF11 was also found as a direct transcriptional target of (PPAR?). Furthermore, KLF11 genetic deficiency effectively abolished pioglitazone cytoprotection in mouse cerebral vascular endothelial cell cultures after oxygen-glucose deprivation, as well as pioglitazone-mediated cerebrovascular protection in a mouse middle cerebral artery occlusion model. Mechanistically, we demonstrated that KLF11 enhanced (PPAR?) transcriptional suppression of the pro-apoptotic microRNA-15a (miR-15a) gene, resulting in endothelial protection in cerebral vascular endothelial cell cultures and cerebral microvasculature after ischaemic stimuli. Taken together, our data demonstrate that recruitment of KLF11 as a novel (PPAR?) co-regulator plays a critical role in the cerebrovascular protection after ischaemic insults. It is anticipated that elucidating the coordinated actions of KLF11 and (PPAR?) will provide new insights into understanding the molecular mechanisms underlying (PPAR?) function in the cerebral vasculature and help to develop a novel therapeutic strategy for the treatment of stroke.
Endothelial activation characterized by the expression of multiple chemokines and adhesive molecules is a critical initial step of vascular inflammation, which results in recruitment of leucocytes into the sub-endothelial layer of the vascular wall and triggers vascular inflammatory diseases such as atherosclerosis. Although inhibiting endothelial inflammation has already been well recognized as a therapeutic strategy in vascular inflammatory diseases, the therapeutic targets are still elusive. In the present study we found that Zc3h12c (zinc finger CCCH-type-containing 12C), a recently discovered CCCH zinc finger-containing protein, significantly inhibited the endothelial cell inflammatory response in vitro. Overexpression of Zc3h12c significantly attenuated TNF? (tumour necrosis factor ?)-induced expression of chemokines and adhesive molecules, and thus reduced monocyte adherence to HUVECs (human umbilical vein endothelial cells). Conversely, siRNA (small interfering RNA)-mediated knockdown of Zc3h12c increased the TNF?-induced expression of chemokines and adhesive molecules in HUVECs. Furthermore, forced expression of Zc3h12c decreased TNF?-induced IKK?/? [I?B (inhibitor of nuclear factor ?B) kinase ?/?], I?B? phosphorylation and p65 nuclear translocation, suggesting that Zc3h12c exerted its anti-inflammatory function probably by suppressing the NF-?B (nuclear factor ?B) pathway. Thus Zc3h12c is an endogenous inhibitor of TNF?-induced inflammatory signalling in HUVECs and might be a therapeutic target in vascular inflammatory diseases.
Gluconeogenesis contributes to insulin resistance in type 1 and type 2 diabetes, but its regulation and the underlying molecular mechanisms remain unclear. Recently, calcium-regulated heat-stable protein 1 (CARHSP1) was identified as a biomarker for diabetic complications. In this study, we investigated the role of CARHSP1 in hepatic gluconeogenesis. We assessed the regulation of hepatic CARHSP1 expression under conditions of fasting and refeeding. Adenovirus-mediated CARHSP1 overexpression and siRNA-mediated knockdown experiments were performed to characterize the role of CARHSP1 in the regulation of gluconeogenic gene expression. Here, we document for the first time that CARHSP1 is regulated by nutrient status in the liver and functions at the transcriptional level to negatively regulate gluconeogenic genes, including the glucose-6-phosphatase catalytic subunit (G6Pc) and phosphoenolpyruvate carboxykinase 1 (PEPCK1). In addition, we found that CARHSP1 can physically interact with peroxisome proliferator-activated receptor-? (PPAR?) and inhibit its transcriptional activity. Both pharmacological and genetic ablations of PPAR? attenuate the inhibitory effect of CARHSP1 on gluconeogenic gene expression in hepatocytes. Our data suggest that CARHSP1 inhibits hepatic gluconeogenic gene expression via repression of PPAR? and that CARHSP1 may be a molecular target for the treatment of diabetes.
Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone-receptor superfamily. Originally cloned in 1990, PPARs were found to be mediators of pharmacologic agents that induce hepatocyte peroxisome proliferation. PPARs also are expressed in cells of the cardiovascular system. PPAR gamma appears to be highly expressed during atherosclerotic lesion formation, suggesting that increased PPAR gamma expression may be a vascular compensatory response. Also, ligand-activated PPAR gamma decreases the inflammatory response in cardiovascular cells, particularly in endothelial cells. PPAR alpha, similar to PPAR gamma, also has pleiotropic effects in the cardiovascular system, including antiinflammatory and antiatherosclerotic properties. PPAR alpha activation inhibits vascular smooth muscle proinflammatory responses, attenuating the development of atherosclerosis. However, PPAR delta overexpression may lead to elevated macrophage inflammation and atherosclerosis. Conversely, PPAR delta ligands are shown to attenuate the pathogenesis of atherosclerosis by improving endothelial cell proliferation and survival while decreasing endothelial cell inflammation and vascular smooth muscle cell proliferation. Furthermore, the administration of PPAR ligands in the form of TZDs and fibrates has been disappointing in terms of markedly reducing cardiovascular events in the clinical setting. Therefore, a better understanding of PPAR-dependent and -independent signaling will provide the foundation for future research on the role of PPARs in human cardiovascular biology.
A decreased plasma level of adiponectin is associated with obesity and metabolic syndrome and correlated with endothelial dysfunction. This study aimed to investigate the regulated expression of the newly identified adiponectin receptors (AdipoR1 and 2) and their roles in the endothelial expression of intercellular adhesion molecule-1 (ICAM-1) in response to tumor necrosis factor (TNF)-alpha.
Proliferation of vascular smooth muscle cells (VSMCs) in response to vascular injury plays a critical role in vascular lesion formation. Emerging data suggest that peroxisome proliferator-activated receptor ? coactivator 1 (PGC-1) is a key regulator of energy metabolism and other biological processes. However, the physiological role of PGC-1? in VSMCs remains unknown. A decrease in PGC-1? expression was observed in balloon-injured rat carotid arteries. PGC-1? overexpression substantially inhibited neointima formation in vivo and markedly inhibited VSMC proliferation and induced cell cycle arrest at the G(1)/S transition phase in vitro. Accordingly, overexpression of PGC-1? decreased the expression of minichromosome maintenance 4 (MCM4), which leads to a decreased loading of the MCM complex onto chromatin at the replication origins and decreased cyclin D1 levels, whereas PGC-1? loss of function by adenovirus containing PGC-1? shRNA resulted in the opposite effect. The transcription factor AP-1 was involved in the down-regulation of MCM4 expression. Furthermore, PGC-1? is up-regulated by metformin, and metformin-associated anti-proliferative activity in VSMCs is at least partially dependent on PGC-1?. Our data show that PGC-1? is a critical component in regulating DNA replication, VSMC proliferation, and vascular lesion formation, suggesting that PGC-1? may emerge as a novel therapeutic target for control of proliferative vascular diseases.
Endothelial cell (EC) inflammatory status is critical to many vascular diseases. Emerging data demonstrate that mutations of Krüppel-like factor-11 (KLF11), a gene coding maturity-onset diabetes mellitus of the young type 7 (MODY7), contribute to the development of neonatal diabetes mellitus. However, the function of KLF11 in the cardiovascular system still remains to be uncovered. In this study, we aimed to investigate the role of KLF11 in vascular endothelial inflammation.
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