Regulatory variants in TCF7L2 are associated with thoracic aortic aneurysm.

Thoracic aortic aneurysm (TAA) is characterized by dilation of the aortic root or ascending/descending aorta. TAA is a heritable disease that can be potentially life threatening. While 10%-20% of TAA cases are caused by rare, pathogenic variants in single genes, the origin of the majority of TAA cases remains unknown. A previous study implicated common variants in FBN1 with TAA disease risk. Here, we report a genome-wide scan of 1,351 TAA-affected individuals and 18,295 control individuals from the Cardiovascular Health Improvement Project and Michigan Genomics Initiative at the University of Michigan. We identified a genome-wide significant association with TAA for variants within the third intron of TCF7L2 following replication with meta-analysis of four additional independent cohorts. Common variants in this locus are the strongest known genetic risk factor for type 2 diabetes. Although evidence indicates the presence of different causal variants for TAA and type 2 diabetes at this locus, we observed an opposite direction of effect. The genetic association for TAA colocalizes with an aortic eQTL of TCF7L2, suggesting a functional relationship. These analyses predict an association of higher expression of TCF7L2 with TAA disease risk. In vitro, we show that upregulation of TCF7L2 is associated with BCL2 repression promoting vascular smooth muscle cell apoptosis, a key driver of TAA disease.

RNA sequencing reveals perivascular adipose tissue plasticity in response to angiotensin II.

Most blood vessels are surrounded by perivascular adipose tissue (PVAT), which is a unique adipose tissue that plays critical roles in vascular physiology and pathophysiology. PVAT displays regional differences that impact vascular homeostasis. Angiotensin II (Ang II) is the main biologically active component of the renin-angiotensin-aldosterone system (RAAS), which has been extensively studied in vascular biology. However, the effects of Ang II on PVAT are less explored and remain to be elucidated. In this study, we systematically investigated the regional heterogeneity of three portions of aortic PVAT, i.e., ascending thoracic aortic PVAT (ATA-PVAT), descending thoracic aortic PVAT (DTA-PVAT) and abdominal aortic PVAT (AA-PVAT), and their responses to 7-day Ang II infusion using RNA sequencing. We found that AA-PVAT is clearly distinguished from both ATA-PVAT and DTA-PVAT, with significantly down-regulated oxidative phosphorylation and up-regulated inflammatory response pathways. Furthermore, AA-PVAT expresses lower levels of brown adipocyte marker genes, such as Ucp1, Cidea, Cox8b, Dio2 and Pgc1α, but expresses higher levels of proinflammatory genes, such as Ccl2, Il1ß and Tnfα, and components of the RAAS, including Agt, Ace and Agtr1a. Ang II infusion significantly down-regulated oxidative phosphorylation in all regions of aortic PVAT and significantly up-regulated inflammatory response specifically in ATA-PVAT and DTA-PVAT. Moreover, ATA-PVAT was most responsive to Ang II induced inflammation. We further used CDGSH iron-sulfur domain-containing protein 1 (a.k.a. mitoNEET) transgenic mice that exhibit enhanced brown adipose tissue (BAT)-like phenotype in aortic PVAT, as indicated by elevated expression levels of brown adipocyte marker genes, and found that the enhanced BAT-like phenotype of aortic PVAT could counterbalance Ang II induced inflammatory and oxidative effects.

Endothelial TFEB (Transcription Factor EB) Improves Glucose Tolerance via Upregulation of IRS (Insulin Receptor Substrate) 1 and IRS2.

OBJECTIVE: Vascular endothelial cells (ECs) play a critical role in maintaining vascular homeostasis. Aberrant EC metabolism leads to vascular dysfunction and metabolic diseases. TFEB (transcription factor EB), a master regulator of lysosome biogenesis and autophagy, has protective effects on vascular inflammation and atherosclerosis. However, the role of endothelial TFEB in metabolism remains to be explored. In this study, we sought to investigate the role of endothelial TFEB in glucose metabolism and underlying molecular mechanisms. Approach and Results: To determine whether endothelial TFEB is critical for glucose metabolism in vivo, we utilized EC-selective TFEB knockout and EC-selective TFEB transgenic mice fed a high-fat diet. EC-selective TFEB knockout mice exhibited significantly impaired glucose tolerance compared with control mice. Consistently, EC-selective TFEB transgenic mice showed improved glucose tolerance. In primary human ECs, small interfering RNA-mediated TFEB knockdown blunts Akt (AKT serine/threonine kinase) signaling. Adenovirus-mediated overexpression of TFEB consistently activates Akt and significantly increases glucose uptake in ECs. Mechanistically, TFEB upregulates IRS1 and IRS2 (insulin receptor substrate 1 and 2). TFEB increases IRS2 transcription measured by reporter gene and chromatin immunoprecipitation assays. Furthermore, we found that TFEB increases IRS1 protein via downregulation of microRNAs (miR-335, miR-495, and miR-548o). In vivo, Akt signaling in the skeletal muscle and adipose tissue was significantly impaired in EC-selective TFEB knockout mice and consistently improved in EC-selective TFEB transgenic mice on high-fat diet. CONCLUSIONS: Our data revealed a critical role of TFEB in endothelial metabolism and suggest that TFEB constitutes a potential molecular target for the treatment of vascular and metabolic diseases.

Cyclodextrin Prevents Abdominal Aortic Aneurysm via Activation of Vascular Smooth Muscle Cell Transcription Factor EB.

BACKGROUND: Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate in the event of rupture. Pharmacological therapy is needed to inhibit AAA expansion and prevent aneurysm rupture. Transcription factor EB (TFEB), a master regulator of autophagy and lysosome biogenesis, is critical to maintain cell homeostasis. In this study, we aim to investigate the role of vascular smooth muscle cell (VSMC) TFEB in the development of AAA and establish TFEB as a novel target to treat AAA. METHODS: The expression of TFEB was measured in human and mouse aortic aneurysm samples. We used loss/gain-of-function approaches to understand the role of TFEB in VSMC survival and explored the underlying mechanisms through transcriptome and functional studies. Using VSMC-selective Tfeb knockout mice and different mouse AAA models, we determined the role of VSMC TFEB and a TFEB activator in AAA in vivo. RESULTS: We found that TFEB is downregulated in both human and mouse aortic aneurysm lesions. TFEB potently inhibits apoptosis in VSMCs, and transcriptome analysis revealed that TFEB regulates apoptotic signaling pathways, especially apoptosis inhibitor B-cell lymphoma 2. B-cell lymphoma 2 is significantly upregulated by TFEB and is required for TFEB to inhibit VSMC apoptosis. We consistently observed that TFEB deficiency increases VSMC apoptosis and promotes AAA formation in different mouse AAA models. Furthermore, we demonstrated that 2-hydroxypropyl-ß-cyclodextrin, a clinical agent used to enhance the solubility of drugs, activates TFEB and inhibits AAA formation and progression in mice. Last, we found that 2-hydroxypropyl-ß-cyclodextrin inhibits AAA in a VSMC TFEB-dependent manner in mouse models. CONCLUSIONS: Our study demonstrated that TFEB protects against VSMC apoptosis and AAA. TFEB activation by 2-hydroxypropyl-ß-cyclodextrin may be a promising therapeutic strategy for the prevention and treatment of AAA.

TMAVA, a Metabolite of Intestinal Microbes, Is Increased in Plasma From Patients With Liver Steatosis, Inhibits γ-Butyrobetaine Hydroxylase, and Exacerbates Fatty Liver in Mice.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is characterized by excessive hepatic accumulation of triglycerides. We aimed to identify metabolites that differ in plasma of patients with liver steatosis vs healthy individuals (controls) and investigate the mechanisms by which these might contribute to fatty liver in mice. METHODS: We obtained blood samples from 15 patients with liver steatosis and 15 controls from a single center in China (discovery cohort). We performed untargeted liquid chromatography with mass spectrometry analysis of plasma to identify analytes associated with liver steatosis. We then performed targeted metabolomic analysis of blood samples from 2 independent cohorts of individuals who underwent annual health examinations in China (1157 subjects with or without diabetes and 767 subjects with or without liver steatosis; replication cohorts). We performed mass spectrometry analysis of plasma from C57BL/6J mice, germ-free, and mice given antibiotics. C57BL/6J mice were given 0.325% (m/v) N,N,N-trimethyl-5-aminovaleric acid (TMAVA) in their drinking water and placed on a 45% high-fat diet (HFD) for 2 months. Plasma, liver tissues, and fecal samples were collected; fecal samples were analyzed by 16S ribosomal RNA gene sequencing. C57BL/6J mice with CRISPR-mediated disruption of the gene encoding γ-butyrobetaine hydroxylase (BBOX-knockout mice) were also placed on a 45% HFD for 2 months. Hepatic fatty acid oxidation (FAO) in liver tissues was determined by measuring liberation of 3H2O from [3H] palmitic acid. Liver tissues were analyzed by electron microscopy, to view mitochondria, and proteomic analyses. We used surface plasmon resonance analysis to quantify the affinity of TMAVA for BBOX. RESULTS: Levels of TMAVA, believed to be a metabolite of intestinal microbes, were increased in plasma from subjects with liver steatosis compared with controls, in the discovery and replication cohorts. In 1 replication cohort, the odds ratio for fatty liver in subjects with increased liver plasma levels of TMAVA was 1.82 (95% confidence interval [CI], 1.14-2.90; P = .012). Plasma from mice given antibiotics or germ-free mice had significant reductions in TMAVA compared with control mice. We found the intestinal bacteria Enterococcus faecalis and Pseudomonas aeruginosa to metabolize trimethyllysine to TMAVA; levels of trimethyllysine were significantly higher in plasma from patients with steatosis than controls. We found TMAVA to bind and inhibit BBOX, reducing synthesis of carnitine. Mice given TMAVA had alterations in their fecal microbiomes and reduced cold tolerance; their plasma and liver tissue had significant reductions in levels of carnitine and acyl-carnitine and their hepatocytes had reduced mitochondrial FAO compared with mice given only an HFD. Mice given TMAVA on an HFD developed liver steatosis, which was reduced by carnitine supplementation. BBOX-knockout mice had carnitine deficiency and decreased FAO, increasing uptake and liver accumulation of free fatty acids and exacerbating HFD-induced fatty liver. CONCLUSIONS: Levels of TMAVA are increased in plasma from subjects with liver steatosis. In mice, intestinal microbes metabolize trimethyllysine to TMAVA, which reduces carnitine synthesis and FAO to promote steatosis.

Perivascular Adipose Tissue Regulates Vascular Function by Targeting Vascular Smooth Muscle Cells.

Adipose tissues are present at multiple locations in the body. Most blood vessels are surrounded with adipose tissue which is referred to as perivascular adipose tissue (PVAT). Similarly to adipose tissues at other locations, PVAT harbors many types of cells which produce and secrete adipokines and other undetermined factors which locally modulate PVAT metabolism and vascular function. Uncoupling protein-1, which is considered as a brown fat marker, is also expressed in PVAT of rodents and humans. Thus, compared with other adipose tissues in the visceral area, PVAT displays brown-like characteristics. PVAT shows a distinct function in the cardiovascular system compared with adipose tissues in other depots which are not adjacent to the vascular tree. Growing and extensive studies have demonstrated that presence of normal PVAT is required to maintain the vasculature in a functional status. However, excessive accumulation of dysfunctional PVAT leads to vascular disorders, partially through alteration of its secretome which, in turn, affects vascular smooth muscle cells and endothelial cells. In this review, we highlight the cross talk between PVAT and vascular smooth muscle cells and its roles in vascular remodeling and blood pressure regulation.

BAF60a Deficiency in Vascular Smooth Muscle Cells Prevents Abdominal Aortic Aneurysm by Reducing Inflammation and Extracellular Matrix Degradation.

OBJECTIVE: Currently, there are no approved drugs for abdominal aortic aneurysm (AAA) treatment, likely due to limited understanding of the primary molecular mechanisms underlying AAA development and progression. BAF60a-a unique subunit of the SWI/SNF (switch/sucrose nonfermentable) chromatin remodeling complex-is a novel regulator of metabolic homeostasis, yet little is known about its function in the vasculature and pathogenesis of AAA. In this study, we sought to investigate the role and underlying mechanisms of vascular smooth muscle cell (VSMC)-specific BAF60a in AAA formation. Approach and Results: BAF60a is upregulated in human and experimental murine AAA lesions. In vivo studies revealed that VSMC-specific knockout of BAF60a protected mice from both Ang II (angiotensin II)-induced and elastase-induced AAA formation with significant suppression of vascular inflammation, monocyte infiltration, and elastin fragmentation. Through RNA sequencing and pathway analysis, we found that the expression of inflammatory response genes in cultured human aortic smooth muscle cells was significantly downregulated by small interfering RNA-mediated BAF60a knockdown while upregulated upon adenovirus-mediated BAF60a overexpression. BAF60a regulates VSMC inflammation by recruiting BRG1 (Brahma-related gene-1)-a catalytic subunit of the SWI/SNF complex-to the promoter region of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) target genes. Furthermore, loss of BAF60a in VSMCs prevented the upregulation of the proteolytic enzyme cysteine protease CTSS (cathepsin S), thus ameliorating ECM (extracellular matrix) degradation within the vascular wall in AAA. CONCLUSIONS: Our study demonstrated that BAF60a is required to recruit the SWI/SNF complex to facilitate the epigenetic regulation of VSMC inflammation, which may serve as a potential therapeutic target in preventing and treating AAA.

Integration of Transformative Platforms for the Discovery of Causative Genes in Cardiovascular Diseases.

Cardiovascular diseases are the leading cause of morbidity and mortality worldwide. Genome-wide association studies (GWAS) are powerful epidemiological tools to find genes and variants associated with cardiovascular diseases while follow-up biological studies allow to better understand the etiology and mechanisms of disease and assign causality. Improved methodologies and reduced costs have allowed wider use of bulk and single-cell RNA sequencing, human-induced pluripotent stem cells, organoids, metabolomics, epigenomics, and novel animal models in conjunction with GWAS. In this review, we feature recent advancements relevant to cardiovascular diseases arising from the integration of genetic findings with multiple enabling technologies within multidisciplinary teams to highlight the solidifying transformative potential of this approach. Well-designed workflows integrating different platforms are greatly improving and accelerating the unraveling and understanding of complex disease processes while promoting an effective way to find better drug targets, improve drug design and repurposing, and provide insight towards a more personalized clinical practice.

Suppression of Vascular Macrophage Activation by Nitro-Oleic Acid and its Implication for Abdominal Aortic Aneurysm Therapy.

PURPOSE: Abdominal aortic aneurysm (AAA) is one of the leading causes of death in the developed world and is currently undertreated due to the complicated nature of the disease. Herein, we aimed to address the therapeutic potential of a novel class of pleiotropic mediators, specifically a new drug candidate, nitro-oleic acid (NO2-OA), on AAA, in a well-characterized murine AAA model. METHODS: We generated AAA using a mouse model combining AAV.PCSK9-D377Y induced hypercholesterolemia with angiotensin II given by chronic infusion. Vehicle control (PEG-400), oleic acid (OA), or NO2-OA were subcutaneously delivered to mice using an osmotic minipump. We characterized the effects of NO2-OA on pathophysiological responses and dissected the underlying molecular mechanisms through various in vitro and ex vivo strategies. RESULTS: Subcutaneous administration of NO2-OA significantly decreased the AAA incidence (8/28 mice) and supra-renal aorta diameters compared to mice infused with either PEG-400 (13/19, p = 0.0117) or OA (16/23, p = 0.0078). In parallel, the infusion of NO2-OA in the AAA model drastically decreased extracellular matrix degradation, inflammatory cytokine levels, and leucocyte/macrophage infiltration in the vasculature. Administration of NO2-OA reduced inflammation, cytokine secretion, and cell migration triggered by various biological stimuli in primary and macrophage cell lines partially through activation of the peroxisome proliferator-activated receptor-gamma (PPARγ). Moreover, the protective effect of NO2-OA relies on the inhibition of macrophage prostaglandin E2 (PGE2)-induced PGE2 receptor 4 (EP4) cAMP signaling, known to participate in the development of AAA. CONCLUSION: Administration of NO2-OA protects against AAA formation and multifactorial macrophage activation. With NO2-OA currently undergoing FDA approved phase II clinical trials, these findings may expedite the use of this nitro-fatty acid for AAA therapy.

Endothelial TFEB (Transcription Factor EB) Positively Regulates Postischemic Angiogenesis.

RATIONALE: Postischemic angiogenesis is critical to limit the ischemic tissue damage and improve the blood flow recovery. The regulation and the underlying molecular mechanisms of postischemic angiogenesis are not fully unraveled. TFEB (transcription factor EB) is emerging as a master gene for autophagy and lysosome biogenesis. However, the role of TFEB in vascular disease is less understood. OBJECTIVE: We aimed to determine the role of endothelial TFEB in postischemic angiogenesis and its underlying molecular mechanism. METHODS AND RESULTS: In primary human endothelial cells (ECs), serum starvation induced TFEB nuclear translocation. VEGF (vascular endothelial growth factor) increased TFEB expression level and nuclear translocation. Utilizing genetically engineered EC-specific TFEB transgenic and KO (knockout) mice, we investigated the role of TFEB in postischemic angiogenesis in the mouse hindlimb ischemia model. We observed improved blood perfusion and increased capillary density in the EC-specific TFEB transgenic mice compared with the wild-type littermates. Furthermore, blood flow recovery was attenuated in EC-TFEB KO mice compared with control mice. In aortic ring cultures, the TFEB transgene significantly increased vessel sprouting, whereas TFEB deficiency impaired the vessel sprouting. In vitro, adenovirus-mediated TFEB overexpression promoted EC tube formation, migration, and survival, whereas siRNA-mediated TFEB knockdown had the opposite effect. Mechanistically, TFEB activated AMPK (AMP-activated protein kinase)-α signaling and upregulated autophagy. Through inactivation of AMPKα or inhibition of autophagy, we demonstrated that the AMPKα and autophagy are necessary for TFEB to regulate angiogenesis in ECs. Finally, the positive effect of TFEB on AMPKα activation and EC tube formation was mediated by TFEB-dependent transcriptional upregulation of MCOLN1 (mucolipin-1). CONCLUSIONS: In summary, our data demonstrate that TFEB is a positive regulator of angiogenesis through activation of AMPKα and autophagy, suggesting that TFEB constitutes a novel molecular target for ischemic vascular disease.

KLF11 (Krüppel-Like Factor 11) Inhibits Arterial Thrombosis via Suppression of Tissue Factor in the Vascular Wall.

Objective- Mutations in Krüppel like factor-11 ( KLF11), a gene also known as maturity-onset diabetes mellitus of the young type 7, contribute to the development of diabetes mellitus. KLF11 has anti-inflammatory effects in endothelial cells and beneficial effects on stroke. However, the function of KLF11 in the cardiovascular system is not fully unraveled. In this study, we investigated the role of KLF11 in vascular smooth muscle cell biology and arterial thrombosis. Approach and Results- Using a ferric chloride-induced thrombosis model, we found that the occlusion time was significantly reduced in conventional Klf11 knockout mice, whereas bone marrow transplantation could not rescue this phenotype, suggesting that vascular KLF11 is critical for inhibition of arterial thrombosis. We further demonstrated that vascular smooth muscle cell-specific Klf11 knockout mice also exhibited significantly reduced occlusion time. The expression of tissue factor (encoded by the F3 gene), a main initiator of the coagulation cascade, was increased in the artery of Klf11 knockout mice, as determined by real-time quantitative polymerase chain reaction and immunofluorescence. Furthermore, vascular smooth muscle cells isolated from Klf11 knockout mouse aortas showed increased tissue factor expression, which was rescued by KLF11 overexpression. In human aortic smooth muscle cells, small interfering RNA-mediated knockdown of KLF11 increased tissue factor expression. Consistent results were observed on adenovirus-mediated overexpression of KLF11. Mechanistically, KLF11 downregulates F3 at the transcriptional level as determined by reporter and chromatin immunoprecipitation assays. Conclusions- Our data demonstrate that KLF11 is a novel transcriptional suppressor of F3 in vascular smooth muscle cells, constituting a potential molecular target for inhibition of arterial thrombosis.

Novel gene regulatory networks identified in response to nitro-conjugated linoleic acid in human endothelial cells.

Endothelial cell (EC) dysfunction is a crucial initiation event in the development of atherosclerosis and is associated with diabetes mellitus, hypertension, and heart failure. Both digestive and oxidative inflammatory conditions lead to the endogenous formation of nitrated derivatives of unsaturated fatty acids (FAs) upon generation of the proximal nitrating species nitrogen dioxide (·NO2) by nitric oxide (·NO) and nitrite-dependent reactions. Nitro-FAs (NO2-FAs) such as nitro-oleic acid (NO2-OA) and nitro-linoleic acid (NO2-LA) potently inhibit inflammation and oxidative stress, regulate cellular functions, and maintain cardiovascular homeostasis. Recently, conjugated linoleic acid (CLA) was identified as the preferential FA substrate of nitration in vivo. However, the functions of nitro-CLA (NO2-CLA) in ECs remain to be explored. In the present study, a distinct transcriptome regulated by NO2-CLA was revealed in primary human coronary artery endothelial cells (HCAECs) through RNA sequencing. Differential gene expression and pathway enrichment analysis identified numerous regulatory networks including those related to the modulation of inflammation, oxidative stress, cell cycle, and hypoxic responses by NO2-CLA, suggesting a diverse impact of NO2-CLA and other electrophilic nitrated FAs on cellular processes. These findings extend the understanding of the protective actions of NO2-CLA in cardiovascular diseases and provide new insight into the underlying mechanisms that mediate the pleiotropic cellular responses to NO2-CLA.

Bmal1 in Perivascular Adipose Tissue Regulates Resting-Phase Blood Pressure Through Transcriptional Regulation of Angiotensinogen.

BACKGROUND: The perivascular adipose tissue (PVAT) surrounding vessels constitutes a distinct functional integral layer of the vasculature required to preserve vascular tone under physiological conditions. However, there is little information on the relationship between PVAT and blood pressure regulation, including its potential contributions to circadian blood pressure variation. METHODS: Using unique brown adipocyte-specific aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1) and angiotensinogen knockout mice, we determined the vasoactivity of homogenized PVAT in aortic rings and how brown adipocyte peripheral expression of Bmal1 and angiotensinogen in PVAT regulates the amplitude of diurnal change in blood pressure in mice. RESULTS: We uncovered a peripheral clock in PVAT and demonstrated that loss of Bmal1 in PVAT reduces blood pressure in mice during the resting phase, leading to a superdipper phenotype. PVAT extracts from wild-type mice significantly induced contractility of isolated aortic rings in vitro in an endothelium-independent manner. This property was impaired in PVAT from brown adipocyte-selective Bmal1-deficient (BA-Bmal1-KO) mice. The PVAT contractile properties were mediated by local angiotensin II, operating through angiotensin II type 1 receptor-dependent signaling in the isolated vessels and linked to PVAT circadian regulation of angiotensinogen. Indeed, angiotensinogen mRNA and angiotensin II levels in PVAT of BA-Bmal1-KO mice were significantly reduced. Systemic infusion of angiotensin II, in turn, reduced Bmal1 expression in PVAT while eliminating the hypotensive phenotype during the resting phase in BA-Bmal1-KO mice. Angiotensinogen, highly expressed in PVAT, shows circadian expression in PVAT, and selective deletion of angiotensinogen in brown adipocytes recapitulates the phenotype of selective deletion of Bmal1 in brown adipocytes. Furthermore, angiotensinogen is a transcriptional target of Bmal1 in PVAT. CONCLUSIONS: These data indicate that local Bmal1 in PVAT regulates angiotensinogen expression and the ensuing increase in angiotensin II, which acts on smooth muscle cells in the vessel walls to regulate vasoactivity and blood pressure in a circadian fashion during the resting phase. These findings will contribute to a better understanding of the cardiovascular complications of circadian disorders, alterations in the circadian dipping phenotype, and cross-talk between systemic and peripheral regulation of blood pressure.

Direct Reprogramming of Fibroblasts Into Smooth Muscle-Like Cells With Defined Transcription Factors-Brief Report.

Objective- To identify the transcription factors that could contribute to direct reprogramming of fibroblasts toward smooth muscle cell fate. Approach and Results- We screened various combinations of transcription factors, including Myocd (myocardin), Mef2C (myocyte enhancer factor 2C), Mef2B (myocyte enhancer factor 2B), Mkl1 (MKL [megakaryoblastic leukemia]/Myocd-like 1), Gata4 (GATA-binding protein 4), Gata5 (GATA-binding protein 5), Gata6 (GATA-binding protein 6), Ets1 (E26 avian leukemia oncogene 1, 5' domain), and their corresponding carboxyterminal fusions to the transactivation domain of MyoD (myogenic differentiation 1)-indicated by *-for their effects on reprogramming mouse embryonic fibroblasts and human adult dermal fibroblasts to the smooth muscle cell fate as determined by the expression of specific markers. The combination of 3 transcription factors, Myocd (or Myocd*) with Mef2C (or Mef2C*) and Gata6, was the most efficient in enhancing the expression of smooth muscle marker genes and decreasing fibroblast gene expression. Additionally, the derived induced smooth muscle-like cells showed a contractile phenotype in response to carbachol. Conclusions- Combination of Myocd and Gata6 with Mef2C* (MG2*) could sufficiently and efficiently direct differentiation of mouse embryonic and human dermal fibroblasts into induced smooth muscle-like cells, thus opening new opportunities for disease modeling, tissue engineering, and personalized medicine.

Brown Adipocyte-Specific PPARγ (Peroxisome Proliferator-Activated Receptor γ) Deletion Impairs Perivascular Adipose Tissue Development and Enhances Atherosclerosis in Mice.

Objective- Perivascular adipose tissue (PVAT) contributes to vascular homeostasis by producing paracrine factors. Previously, we reported that selective deletion of PPARγ (peroxisome proliferator-activated receptor γ) in vascular smooth muscle cells resulted in concurrent loss of PVAT and enhanced atherosclerosis in mice. To address the causal relationship between loss of PVAT and atherosclerosis, we used BA-PPARγ-KO (brown adipocyte-specific PPARγ knockout) mice. Approach and Results- Deletion of PPARγ in brown adipocytes did not affect PPARγ in white adipocytes or vascular smooth muscle cells or PPARα and PPARδ expression in brown adipocytes. However, development of PVAT and interscapular brown adipose tissue was remarkably impaired, associated with reduced expression of genes encoding lipogenic enzymes in the BA-PPARγ-KO mice. Thermogenesis in brown adipose tissue was significantly impaired with reduced expression of thermogenesis genes in brown adipose tissue and compensatory increase in subcutaneous and gonadal white adipose tissues. Remarkably, basal expression of inflammatory genes and macrophage infiltration in PVAT and brown adipose tissue were significantly increased in the BA-PPARγ-KO mice. BA-PPARγ-KO mice were crossbred with ApoE KO (apolipoprotein E knockout) mice to investigate the development of atherosclerosis. Flow cytometry analysis confirmed increased systemic and PVAT inflammation. Consequently, atherosclerotic lesions were significantly increased in mice with impaired PVAT development, thus indicating that the lack of normal PVAT is sufficient to drive increased atherosclerosis. Conclusions- PPARγ is required for functional PVAT development. PPARγ deficiency in PVAT, while still expressed in vascular smooth muscle cell, enhances atherosclerosis and results in vascular and systemic inflammation, providing new insights on the specific roles of PVAT in atherosclerosis and cardiovascular disease at large.

Deficiency of Cholesteryl Ester Transfer Protein Protects Against Atherosclerosis in Rabbits.

OBJECTIVE: CETP (cholesteryl ester transfer protein) plays an important role in lipoprotein metabolism; however, whether inhibition of CETP activity can prevent cardiovascular disease remains controversial. APPROACH AND RESULTS: We generated CETP knockout (KO) rabbits by zinc finger nuclease gene editing and compared their susceptibility to cholesterol diet-induced atherosclerosis to that of wild-type (WT) rabbits. On a chow diet, KO rabbits showed higher plasma levels of high-density lipoprotein (HDL) cholesterol than WT controls, and HDL particles of KO rabbits were essentially rich in apolipoprotein AI and apolipoprotein E contents. When challenged with a cholesterol-rich diet for 18 weeks, KO rabbits not only had higher HDL cholesterol levels but also lower total cholesterol levels than WT rabbits. Analysis of plasma lipoproteins revealed that reduced plasma total cholesterol in KO rabbits was attributable to decreased apolipoprotein B-containing particles, while HDLs remained higher than that in WT rabbits. Both aortic and coronary atherosclerosis was significantly reduced in KO rabbits compared with WT rabbits. Apolipoprotein B-depleted plasma isolated from CETP KO rabbits showed significantly higher capacity for cholesterol efflux from macrophages than that from WT rabbits. Furthermore, HDLs isolated from CETP KO rabbits suppressed tumor necrosis factor-α-induced vascular cell adhesion molecule 1 and E-selectin expression in cultured endothelial cells. CONCLUSIONS: These results provide evidence that genetic ablation of CETP activity protects against cholesterol diet-induced atherosclerosis in rabbits.

MitoNEET in Perivascular Adipose Tissue Prevents Arterial Stiffness in Aging Mice.

PURPOSE: Arterial stiffness is an inevitable consequence of the aging process and is considered an early stage in the development of cardiovascular diseases. The perivascular adipose tissue (PVAT) is a distinct functional integral layer of the vasculature actively involved in blood pressure regulation and atherosclerosis development via PVAT-derived paracrine/autocrine factors. However, there is little knowledge regarding the relationship between PVAT and arterial stiffness. METHODS: Using unique mice lacking PVAT, high-fat diet-induced obesity, and in mice overexpressing brown adipocyte selective mitoNEET, we investigated the relationship between PVAT and arterial stiffness in mice. RESULTS: We found that lack of PVAT enhanced arterial stiffness in aging mice. High-fat diet feeding of aging C57BL/6J wild-type mice significantly induced hypertrophic PVAT and enhanced arterial stiffness. Furthermore, the expression of mitoNEET, a mitochondrial membrane protein related to energy expenditure, was significantly increased by pioglitazone treatment, while reduced in the hypertrophic PVAT induced by high-fat diet. Overexpression of mitoNEET in PVAT reduced the expression of inflammatory genes and was associated with lower pulse wave velocity in aging mice. CONCLUSIONS: These data indicate that local PVAT homeostasis especially inflammation in PVAT is associated with arterial stiffness development. Pioglitazone-induced mitoNEET in PVAT prevents PVAT inflammation and is negatively associated with arterial stiffness. These findings provide new experimental insight into the roles of pioglitazone on PVAT in arterial stiffness and indicate that PVAT might be a target to treat or prevent cardiovascular disease.

Hepatic Transmembrane 6 Superfamily Member 2 Regulates Cholesterol Metabolism in Mice.

BACKGROUND & AIMS: The rs58542926 C>T variant of the transmembrane 6 superfamily member 2 gene (TM6SF2), encoding an E167K amino acid substitution, has been correlated with reduced total cholesterol (TC) and cardiovascular disease. However, little is known about the role of TM6SF2 in metabolism. We investigated the long-term effects of altered TM6SF2 levels in cholesterol metabolism. METHODS: C57BL/6 mice (controls), mice that expressed TM6SF2 specifically in the liver, and mice with CRISPR/Cas9-mediated knockout of Tm6sf2 were fed chow or high-fat diets. Blood samples were collected from all mice and plasma levels of TC, low-density lipoprotein cholesterol (LDL-c), high-density lipoprotein cholesterol, and triglycerides were measured. Liver tissues were collected and analyzed by histology, real-time polymerase chain reaction, and immunoblot assays. Adenovirus vectors were used to express transgenes in cultured Hep3B hepatocytes. RESULTS: Liver-specific expression of TM6SF2 increased plasma levels of TC and LDL-c, compared with controls, and altered liver expression of genes that regulate cholesterol metabolism. Tm6sf2-knockout mice had decreased plasma levels of TC and LDL-c, compared with controls, and consistent changes in expression of genes that regulate cholesterol metabolism. Expression of TM6SF2 promoted cholesterol biosynthesis in hepatocytes. CONCLUSIONS: TM6SF2 regulates cholesterol metabolism in mice and might be a therapeutic target for cardiovascular disease.

Mitotic Protein CSPP1 Interacts with CENP-H Protein to Coordinate Accurate Chromosome Oscillation in Mitosis.

Mitotic chromosome segregation is orchestrated by the dynamic interaction of spindle microtubules with the kinetochores. During chromosome alignment, kinetochore-bound microtubules undergo dynamic cycles between growth and shrinkage, leading to an oscillatory movement of chromosomes along the spindle axis. Although kinetochore protein CENP-H serves as a molecular control of kinetochore-microtubule dynamics, the mechanistic link between CENP-H and kinetochore microtubules (kMT) has remained less characterized. Here, we show that CSPP1 is a kinetochore protein essential for accurate chromosome movements in mitosis. CSPP1 binds to CENP-H in vitro and in vivo. Suppression of CSPP1 perturbs proper mitotic progression and compromises the satisfaction of spindle assembly checkpoint. In addition, chromosome oscillation is greatly attenuated in CSPP1-depleted cells, similar to what was observed in the CENP-H-depleted cells. Importantly, CSPP1 depletion enhances velocity of kinetochore movement, and overexpression of CSPP1 decreases the speed, suggesting that CSPP1 promotes kMT stability during cell division. Specific perturbation of CENP-H/CSPP1 interaction using a membrane-permeable competing peptide resulted in a transient mitotic arrest and chromosome segregation defect. Based on these findings, we propose that CSPP1 cooperates with CENP-H on kinetochores to serve as a novel regulator of kMT dynamics for accurate chromosome segregation.

Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro.

Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson's theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.