Aging is associated with impaired scavenging of reactive oxygen species (ROS). Here, we show that TAp73, a p53 family member, protects against aging by regulating mitochondrial activity and preventing ROS accumulation. TAp73-null mice show more pronounced aging with increased oxidative damage and senescence. TAp73 deletion reduces cellular ATP levels, oxygen consumption, and mitochondrial complex IV activity, with increased ROS production and oxidative stress sensitivity. We show that the mitochondrial complex IV subunit cytochrome C oxidase subunit 4 (Cox4i1) is a direct TAp73 target and that Cox4i1 knockdown phenocopies the cellular senescence of TAp73-null cells. Results indicate that TAp73 affects mitochondrial respiration and ROS homeostasis, thus regulating aging.

Endothelial dysfunction and impaired autophagic activity have a crucial role in aging-related diseases such as cardiovascular dysfunction and atherosclerosis. We have identified miR-216a as a microRNA that is induced during endothelial aging and, according to the computational analysis, among its targets includes two autophagy-related genes, Beclin1 (BECN1) and ATG5. Therefore, we have evaluated the role of miR-216a as a molecular component involved in the loss of autophagic function during endothelial aging. The inverse correlation between miR-216a and autophagic genes was conserved during human umbilical vein endothelial cells (HUVECs) aging and in vivo models of human atherosclerosis and heart failure. Luciferase experiments indicated BECN1, but not ATG5 as a direct target of miR-216a. HUVECs were transfected in order to modulate miR-216a expression and stimulated with 100 μg/ml oxidized low-density lipoprotein (ox-LDL) to induce a stress repairing autophagic process. We found that in young HUVECs, miR-216a overexpression repressed BECN1 and ATG5 expression and the ox-LDL induced autophagy, as evaluated by microtubule-associated protein 1 light chain 3 (LC3B) analysis and cytofluorimetric assay. Moreover, miR-216a stimulated ox-LDL accumulation and monocyte adhesion in HUVECs. Conversely, inhibition of miR-216a in old HUVECs rescued the ability to induce a protective autophagy in response to ox-LDL stimulus. In conclusion, mir-216a controls ox-LDL induced autophagy in HUVECs by regulating intracellular levels of BECN1 and may have a relevant role in the pathogenesis of cardiovascular disorders and atherosclerosis.

OBJECTIVEAtherosclerosis is accelerated in subjects with type 2 diabetes by unknown mechanisms. We identified tissue inhibitor of metalloproteinase 3 (TIMP3), the endogenous inhibitor of A disintegrin and metalloprotease domain 17 (ADAM17) and other matrix metalloproteinases (MMPs), as a gene modifier for insulin resistance and vascular inflammation in mice. We tested its association with atherosclerosis in subjects with type 2 diabetes and identified Sirtuin 1 (SirT1) as a major regulator of TIMP3 expression.RESEARCH DESIGN AND METHODSWe investigated ADAM10, ADAM17, MMP9, TIMP1, TIMP2, TIMP3, and TIMP4 expression levels in human carotid atherosclerotic plaques (n = 60) from subjects with and without diabetes. Human vascular smooth muscle cells exposed to several metabolic stimuli were used to identify regulators of TIMP3 expression. SirT1 small interference RNA, cDNA, and TIMP3 promoter gene reporter were used to study SirT1-dependent regulation of TIMP3.RESULTSHere, we show that in human carotid atherosclerotic plaques, TIMP3 was significantly reduced in subjects with type 2 diabetes, leading to ADAM17 and MMP9 overactivity. Reduced expression of TIMP3 was associated in vivo with SirT1 levels. In smooth muscle cells, inhibition of SirT1 activity and levels reduced TIMP3 expression, whereas SirT1 overexpression increased TIMP3 promoter activity.CONCLUSIONSIn atherosclerotic plaques from subjects with type 2 diabetes, the deregulation of ADAM17 and MMP9 activities is related to inadequate expression of TIMP3 via SirT1. Studies in vascular cells confirmed the role of SirT1 in tuning TIMP3 expression.

Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population of immature myeloid cells with major regulatory functions and rise during pathological conditions, including cancer, infections and autoimmune conditions. MDSC expansion is generally linked to inflammatory processes that emerge in response to stable immunological stress, which alter both magnitude and quality of the myelopoietic output. Inability to reinstate physiological myelopoiesis would fall in an “emergency state” that perpetually reprograms myeloid cells toward suppressive functions. While differentiation and reprogramming of myeloid cells toward an immunosuppressive phenotype can be considered the result of a multistep process that originates in the bone marrow and culminates in the tumor microenvironment, the identification of its driving events may offer potential therapeutic approaches in different pathologies. Indeed, whereas expansion of MDSCs, in both murine and human tumor bearers, results in reduced immune surveillance and antitumor cytotoxicity, placing an obstacle to the effectiveness of anticancer therapies, adoptive transfer of MDSCs has shown therapeutic benefits in autoimmune disorders. Here, we describe relevant mechanisms of myeloid cell reprogramming leading to generation of suppressive MDSCs and discuss their therapeutic ductility in disease.

BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Prevention and risk reduction are important and the identification of specific biomarkers for early diagnosis of HCC represents an active field of research. Increasing evidence indicates that fat accumulation in the liver, defined as hepatosteatosis, is an independent and strong risk factor for developing an HCC. MacroH2A1, a histone protein generally associated with the repressed regions of chromosomes, is involved in hepatic lipid metabolism and is present in two alternative spliced isoforms, macroH2A1.1 and macroH2A1.2. These isoforms have been shown to predict lung and colon cancer recurrence but to our knowledge, their role in fatty-liver associated HCC has not been investigated previously.MethodsWe examined macroH2A1.1 and macroH2A1.2 protein expression levels in the liver of two murine models of fat-associated HCC, the high fat diet/diethylnistrosamine (DEN) and the phosphatase and tensin homolog (PTEN) liver specific knock-out (KO) mouse, and in human liver samples of subjects with steatosis or HCC, using immunoblotting and immunohistochemistry.ResultsProtein levels for both macroH2A1 isoforms were massively upregulated in HCC, whereas macroH2A1.2 was specifically upregulated in steatosis. In addition, examination of human liver samples showed a significant difference (p<0.01) in number of positive nuclei in HCC (100% of tumor cells positive for either macroH2A1.1 or macroH2A1.2), when compared to steatosis (<2% of hepatocytes positive for either isoform). The steatotic areas flanking the tumors were highly immunopositive for macroH2A1.1 and macroH2A1.2.ConclusionsThese data obtained in mice and humans suggest that both macroH2A1 isoforms may play a role in HCC pathogenesis and moreover may be considered as novel diagnostic markers for human HCC.

Objective-Tissue inhibitor of metalloproteinase 3 (TIMP3) is a stromal protein that inhibits the activity of proteases and receptors. TIMP3 is downregulated in metabolic and inflammatory disorders, such as type 2 diabetes mellitus and atherosclerosis, particularly in regions enriched with monocyte/macrophage cells. To investigate the role of TIMP3 in atherosclerosis, we generated a new mouse model in which Timp3 was overexpressed in the atherosclerotic plaque via a macrophage-specific promoter (MacT3). We elucidated any potential antiatherosclerotic effects of TIMP3, including regulation of monocyte/macrophage recruitment within atherosclerotic plaques, in MacT3 mice crossbred with low-density lipoprotein receptor knockout (LDLR Ϫ/Ϫ ) mice. Methods and Results-MacT3/LDLRϪ/Ϫ mice had an improvement of atherosclerosis and metabolic parameters compared with LDLR Ϫ/Ϫ . En face aorta and aortic root examination of MacT3/LDLR Ϫ/Ϫ mice revealed smaller atherosclerotic plaques with features of stability, such as increased collagen content and decreased necrotic core formation. Atherosclerotic plaques in MacT3/LDLR Ϫ/Ϫ mice contained fewer T cells and macrophages. Furthermore, TIMP3 overexpression in macrophages resulted in reduced oxidative stress signals, as evidenced by lower lipid peroxidation, protein carbonylation, and nitration in atheromas. Conclusion-Our study confirmed that macrophage-specific overexpression of TIMP3 decreases the inflammatory content and the amplitude of atherosclerotic plaques in mice. Key Words: atherosclerosis Ⅲ macrophages Ⅲ metalloproteinases Ⅲ inflammation Ⅲ lipotoxicity I n patients with type 2 diabetes mellitus and cardiovascular disease, the expression of tissue inhibitor of metalloproteinase 3 (TIMP3) is reduced in carotid atherosclerotic plaques, particularly in regions enriched with monocyte/macrophage cells. 1 TIMP3 is a secreted 24-to 27-kDa stromal protein that belongs to the TIMP family. It binds to the extracellular matrix and inhibits metalloproteases such as matrix metalloproteinase-2/9/14 and a disintegrin and a metalloprotease domain 17 (ADAM17), whose activities are increased in atherosclerotic plaques. 1,2 We previously identified TIMP3 as a modifier factor for insulin resistance and vascular inflammation in mice, and these results were partially explained by the inhibitory effect of TIMP3 on ADAM17. [3][4][5][6] In particular, analysis of metabolic and vascular tissues from different strains of Insr ϩ/Ϫ revealed that those with diabetes were characterized by deficiency for TIMP3, insulin resistance, unrestrained metalloprotease activity, and increased activation of inflammatory signals. 3 This phenotype partially reverted when the Insr ϩ/Ϫ mice were crossed with ADAM17 haploinsufficient mice or treated with a metalloprotease inhibitor. 3 Moreover, in a mouse model combining insulin resistance with nutrient overload and decreased TIMP3 activity, the loss of TIMP3 was associated with greater infiltration of F4/80 positive cells, increased monocyte chemoattractant protein-...

The tissue inhibitor of metalloproteinase (TIMP)3, a stromal protein that restrains the activity of proteases and receptors, is reduced in inflammatory metabolic disorders such as type 2 diabetes mellitus (T2DM) and atherosclerosis. We overexpressed Timp3 in mouse macrophages (MacT3) to analyze its potential antidiabetic and antiatherosclerotic effects. Transgenic mice with myeloid cells targeting overexpression of TIMP3 were generated and fed a high-fat diet for 20 weeks. Physical and metabolic phenotypes were determined. Inflammatory markers, lipid accumulation, and insulin sensitivity were measured in white adipose tissue (WAT), liver, and skeletal muscle. In a model of insulin resistance, MacT3 mice were more glucose tolerant and insulin sensitive than wild-type mice in both in vitro and in vivo tests. Molecular and biochemical analyses revealed that increased expression of TIMP3 restrained metabolic inflammation and stress-related pathways, including Jun NH2-terminal kinase and p38 kinase activation, in WAT and liver. TIMP3 overexpression in macrophages resulted in reduced activation of oxidative stress signals related to lipid peroxidation, protein carbonylation, and nitration in WAT and liver. Our data show that macrophage-specific overexpression of TIMP3 protects from metabolic inflammation and related metabolic disorders such as insulin resistance, glucose intolerance, and nonalcoholic steatohepatitis.

Obesity elicits immune cell infiltration of adipose tissue provoking chronic low-grade inflammation. Regulatory T cells (Tregs) are specifically reduced in adipose tissue of obese animals. Since interleukin (IL)-21 plays an important role in inducing and maintaining immune-mediated chronic inflammatory processes and negatively regulates Treg differentiation/activity, we hypothesized that it could play a role in obesity-induced insulin resistance. We found IL-21 and IL-21R mRNA expression upregulated in adipose tissue of high-fat diet (HFD) wild-type (WT) mice and in stromal vascular fraction from human obese subjects in parallel to macrophage and inflammatory markers. Interestingly, a larger infiltration of Treg cells was seen in the adipose tissue of IL-21 knockout (KO) mice compared with WT animals fed both normal diet and HFD. In a context of diet-induced obesity, IL-21 KO mice, compared with WT animals, exhibited lower body weight, improved insulin sensitivity, and decreased adipose and hepatic inflammation. This metabolic phenotype is accompanied by a higher induction of interferon regulatory factor 4 (IRF4), a transcriptional regulator of fasting lipolysis in adipose tissue. Our data suggest that IL-21 exerts negative regulation on IRF4 and Treg activity, developing and maintaining adipose tissue inflammation in the obesity state.