Macrophage fatty acid oxidation inhibits atherosclerosis progression.

Atherosclerosis is a chronic disorder of the vessel wall. One key regulator of disease progression is lipid handling in macrophages. However, the role of macrophage mitochondrial-dependent fatty acid ß-oxidation (FAO) in atherosclerosis is not well defined. To address this, we focused on carnitine palmitoyltransferase (CPT) 1 and 2, which play an essential role in the transport of long chain fatty acids (FAs) into the mitochondria. Using conditional alleles of these mitochondrial enzymes, we have generated myeloid-specific Cpt1a and Cpt2 knockout mutants (CPT1a M-KO and CPT2 M-KO). In culture, macrophages derived from CPT1a and CPT2 M-KO mice have impaired FAO, enhanced expression of the CD36 scavenger receptor, increased uptake of oxidized low-density lipoprotein (oxLDL), and augmented transformation into cholesterol-rich foam cells. In line with these in vitro observations, in the atherosclerosis-susceptible apolipoprotein E (ApoE) KO background, CPT2 M-KO mice demonstrated augmented atherosclerosis, accompanied by increased accumulation of aortic macrophages with elevated CD36 expression. These data suggest that macrophage FAO is athero-protective and that augmenting FAO may potentially slow atherosclerotic progression.

Sonic hedgehog signaling regulates the mammalian cardiac regenerative response.

Certain organisms, including zebrafish, are capable of complete cardiac regeneration in response to injury. This response has also been observed in newborn mice, although in this case, the regenerative capacity is lost at approximately one week of age. The mechanisms regulating this short temporal window of cardiac regeneration in mice are not well understood. Here, we show that sonic hedgehog (Shh) signaling modulates the neonatal mouse regenerative response. In particular, we demonstrate that following apical resection of the heart on postnatal day 1, mice activate Shh ligand expression and downstream signaling. This response is largely absent when surgery is performed on non-regenerative, postnatal day 7 pups. Furthermore, an enhanced cardiac regeneration response was detected in ptch heterozygous mice which have a genetically-based constitutive increase in Shh signaling. We further show that Shh ligand is produced in the myocardium by non-myocytes and appears to regulate cardiomyocyte proliferation, as well as the recruitment of monocytes/macrophages to the regenerating area. Finally, we demonstrate that a small molecule activator of Shh signaling promotes heart regeneration, whereas an inhibitor of Shh signaling impairs the regenerative response. Together, these results implicate Shh signaling as a regulator of mammalian heart regeneration and suggest that modulating this pathway may lead to new potential therapies for cardiovascular diseases.

The In Vivo Biology of the Mitochondrial Calcium Uniporter.

The identification of the molecular composition of the mitochondrial calcium uniporter has allowed for the genetic manipulation of its components and the creation of various in vivo genetic models. Here, we review the initial attempts to modulate the expression of components of the calcium uniporter in a range of organisms from plants to mammals. This analysis has confirmed the strict requirement for the uniporter for in vivo mitochondrial calcium uptake and for maintaining mitochondrial calcium homeostasis. We further discuss the physiological effects following genetic manipulation of the uniporter on tissue bioenergetics and the threshold for cell death. Finally, we analyze the limited information regarding the role of various uniporter components in human disease.

Measuring In Vivo Mitophagy.

Alterations in mitophagy have been increasingly linked to aging and age-related diseases. There are, however, no convenient methods to analyze mitophagy in vivo. Here, we describe a transgenic mouse model in which we expressed a mitochondrial-targeted form of the fluorescent reporter Keima (mt-Keima). Keima is a coral-derived protein that exhibits both pH-dependent excitation and resistance to lysosomal proteases. Comparison of a wide range of primary cells and tissues generated from the mt-Keima mouse revealed significant variations in basal mitophagy. In addition, we have employed the mt-Keima mice to analyze how mitophagy is altered by conditions including diet, oxygen availability, Huntingtin transgene expression, the absence of macroautophagy (ATG5 or ATG7 expression), an increase in mitochondrial mutational load, the presence of metastatic tumors, and normal aging. The ability to assess mitophagy under a host of varying environmental and genetic perturbations suggests that the mt-Keima mouse should be a valuable resource.

The NAD-dependent deacetylase SIRT2 is required for programmed necrosis.

Although initially viewed as unregulated, increasing evidence suggests that cellular necrosis often proceeds through a specific molecular program. In particular, death ligands such as tumour necrosis factor (TNF)-α activate necrosis by stimulating the formation of a complex containing receptor-interacting protein 1 (RIP1) and receptor-interacting protein 3 (RIP3). Relatively little is known regarding how this complex formation is regulated. Here, we show that the NAD-dependent deacetylase SIRT2 binds constitutively to RIP3 and that deletion or knockdown of SIRT2 prevents formation of the RIP1-RIP3 complex in mice. Furthermore, genetic or pharmacological inhibition of SIRT2 blocks cellular necrosis induced by TNF-α. We further demonstrate that RIP1 is a critical target of SIRT2-dependent deacetylation. Using gain- and loss-of-function mutants, we demonstrate that acetylation of RIP1 lysine 530 modulates RIP1-RIP3 complex formation and TNF-α-stimulated necrosis. In the setting of ischaemia-reperfusion injury, RIP1 is deacetylated in a SIRT2-dependent fashion. Furthermore, the hearts of Sirt2(-/-) mice, or wild-type mice treated with a specific pharmacological inhibitor of SIRT2, show marked protection from ischaemic injury. Taken together, these results implicate SIRT2 as an important regulator of programmed necrosis and indicate that inhibitors of this deacetylase may constitute a novel approach to protect against necrotic injuries, including ischaemic stroke and myocardial infarction.

Atg7 modulates p53 activity to regulate cell cycle and survival during metabolic stress.

Withdrawal of nutrients triggers an exit from the cell division cycle, the induction of autophagy, and eventually the activation of cell death pathways. The relation, if any, among these events is not well characterized. We found that starved mouse embryonic fibroblasts lacking the essential autophagy gene product Atg7 failed to undergo cell cycle arrest. Independent of its E1-like enzymatic activity, Atg7 could bind to the tumor suppressor p53 to regulate the transcription of the gene encoding the cell cycle inhibitor p21(CDKN1A). With prolonged metabolic stress, the absence of Atg7 resulted in augmented DNA damage with increased p53-dependent apoptosis. Inhibition of the DNA damage response by deletion of the protein kinase Chk2 partially rescued postnatal lethality in Atg7(-/-) mice. Thus, when nutrients are limited, Atg7 regulates p53-dependent cell cycle and cell death pathways.

Oncogene-induced senescence results in marked metabolic and bioenergetic alterations.

Oncogene-induced senescence (OIS) is characterized by permanent growth arrest and the acquisition of a secretory, pro-inflammatory state. Increasingly, OIS is viewed as an important barrier to tumorgenesis. Surprisingly, relatively little is known about the metabolic changes that accompany and therefore may contribute to OIS. Here, we have performed a metabolomic and bioenergetic analysis of Ras-induced senescence. Profiling approximately 300 different intracellular metabolites reveals that cells that have undergone OIS develop a unique metabolic signature that differs markedly from cells undergoing replicative senescence. A number of lipid metabolites appear uniquely increased in OIS cells, including a marked increase in the level of certain intracellular long chain fatty acids. Functional studies reveal that this alteration in the metabolome reflects substantial changes in overall lipid metabolism. In particular, Ras-induced senescent cells manifest a decline in lipid synthesis and a significant increase in fatty acid oxidation. Increased fatty acid oxidation results in an unexpectedly high rate of basal oxygen consumption in cells that have undergone OIS. Pharmacological or genetic inhibition of carnitine palmitoyltransferase 1, the rate-limiting step in mitochondrial fatty acid oxidation, restores a pre-senescent metabolic rate and, surprisingly, selectively inhibits the secretory, pro-inflammatory state that accompanies OIS. Thus, Ras-induced senescent cells demonstrate profound alterations in their metabolic and bioenergetic profiles, particularly with regards to the levels, synthesis and oxidation of free fatty acids. Furthermore, the inflammatory phenotype that accompanies OIS appears to be related to these underlying changes in cellular metabolism.

BRCA1 is an essential regulator of heart function and survival following myocardial infarction.

The tumour suppressor BRCA1 is mutated in familial breast and ovarian cancer but its role in protecting other tissues from DNA damage has not been explored. Here we show a new role for BRCA1 as a gatekeeper of cardiac function and survival. In mice, loss of BRCA1 in cardiomyocytes results in adverse cardiac remodelling, poor ventricular function and higher mortality in response to ischaemic or genotoxic stress. Mechanistically, loss of cardiomyocyte BRCA1 results in impaired DNA double-strand break repair and activated p53-mediated pro-apoptotic signalling culminating in increased cardiomyocyte apoptosis, whereas deletion of the p53 gene rescues BRCA1-deficient mice from cardiac failure. In human adult and fetal cardiac tissues, ischaemia induces double-strand breaks and upregulates BRCA1 expression. These data reveal BRCA1 as a novel and essential adaptive response molecule shielding cardiomyocytes from DNA damage, apoptosis and heart dysfunction. BRCA1 mutation carriers, in addition to risk of breast and ovarian cancer, may be at a previously unrecognized risk of cardiac failure.

Wnt signaling regulates hepatic metabolism.

The contribution of the Wnt pathway has been extensively characterized in embryogenesis, differentiation, and stem cell biology but not in mammalian metabolism. Here, using in vivo gain- and loss-of-function models, we demonstrate an important role for Wnt signaling in hepatic metabolism. In particular, ß-catenin, the downstream mediator of canonical Wnt signaling, altered serum glucose concentrations and regulated hepatic glucose production. ß-Catenin also modulated hepatic insulin signaling. Furthermore, ß-catenin interacted with the transcription factor FoxO1 in livers from mice under starved conditions. The interaction of FoxO1 with ß-catenin regulated the transcriptional activation of the genes encoding glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK), the two rate-limiting enzymes in hepatic gluconeogenesis. Moreover, starvation induced the hepatic expression of mRNAs encoding different Wnt isoforms. In addition, nutrient deprivation appeared to favor the association of ß-catenin with FoxO family members, rather than with members of the T cell factor of transcriptional activators. Notably, in a model of diet-induced obesity, hepatic deletion of ß-catenin improved overall metabolic homeostasis. These observations implicate Wnt signaling in the modulation of hepatic metabolism and raise the possibility that Wnt signaling may play a similar role in the metabolic regulation of other tissues.

Xanthine oxidoreductase depletion induces renal interstitial fibrosis through aberrant lipid and purine accumulation in renal tubules.

Xanthine oxidoreductase (XOR) is an enzyme responsible for purine degradation, reactive oxygen species production, and adipogenesis. XOR gene-disrupted (XOR(-/-)) mice demonstrate renal failure and early death within several months. The aim of this study was to elucidate the mechanism of renal damage in XOR(-/-) mice and to determine the physiological role of XOR in the kidney. Histological analysis revealed that renal tubular damage in XOR(-/-) mice was accompanied by deposition of crystals and lipid-rich substances. Triglyceride content in renal homogenates was significantly increased in XOR(-/-) mice. The level of lipogenesis-related gene expression was comparable in XOR(+/+) and XOR(-/-) mice, whereas the expression of adipogenesis-related gene expression was significantly elevated in XOR(-/-) mice. Urinary excretions of xanthine and hypoxanthine were markedly elevated in XOR(-/-) mice. Immunohistochemical analysis, Western blotting, and real time RT-PCR revealed that various markers of fibrosis, inflammation, ischemia, and oxidative stress were increased in XOR(-/-) mice. Finally, we demonstrate that primary renal epithelial cells from XOR(-/-) mice are more readily transformed to myofibroblasts, which is a marker of increased epithelial mesenchymal transition. These results suggest that XOR gene disruption induced the depletion of uric acid and the accumulation of triglyceride-rich substances, xanthine, and hypoxanthine in the renal tubules. We believe that these changes contribute to a complex cellular milieu characterized by inflammation, tissue hypoxia, and reactive oxygen species production, ultimately resulting in renal failure through increased renal interstitial fibrosis.

Bmi1 regulates mitochondrial function and the DNA damage response pathway.

Mice deficient in the Polycomb repressor Bmi1 develop numerous abnormalities including a severe defect in stem cell self-renewal, alterations in thymocyte maturation and a shortened lifespan. Previous work has implicated de-repression of the Ink4a/Arf (also known as Cdkn2a) locus as mediating many of the aspects of the Bmi1(-/-) phenotype. Here we demonstrate that cells derived from Bmi1(-/-) mice also have impaired mitochondrial function, a marked increase in the intracellular levels of reactive oxygen species and subsequent engagement of the DNA damage response pathway. Furthermore, many of the deficiencies normally observed in Bmi1(-/-) mice improve after either pharmacological treatment with the antioxidant N-acetylcysteine or genetic disruption of the DNA damage response pathway by Chk2 (also known as Chek2) deletion. These results demonstrate that Bmi1 has an unexpected role in maintaining mitochondrial function and redox homeostasis and indicate that the Polycomb family of proteins can coordinately regulate cellular metabolism with stem and progenitor cell function.

Augmented Wnt signaling in a mammalian model of accelerated aging.

The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.

Human mesenchymal stem cells exert potent antitumorigenic effects in a model of Kaposi's sarcoma.

Emerging evidence suggests that both human stem cells and mature stromal cells can play an important role in the development and growth of human malignancies. In contrast to these tumor-promoting properties, we observed that in an in vivo model of Kaposi's sarcoma (KS), intravenously (i.v.) injected human mesenchymal stem cells (MSCs) home to sites of tumorigenesis and potently inhibit tumor growth. We further show that human MSCs can inhibit the in vitro activation of the Akt protein kinase within some but not all tumor and primary cell lines. The inhibition of Akt activity requires the MSCs to make direct cell-cell contact and can be inhibited by a neutralizing antibody against E-cadherin. We further demonstrate that in vivo, Akt activation within KS cells is potently down-regulated in areas adjacent to MSC infiltration. Finally, the in vivo tumor-suppressive effects of MSCs correlates with their ability to inhibit target cell Akt activity, and KS tumors engineered to express a constitutively activated Akt construct are no longer sensitive to i.v. MSC administration. These results suggest that in contrast to other stem cells or normal stromal cells, MSCs possess intrinsic antineoplastic properties and that this stem cell population might be of particular utility for treating those human malignancies characterized by dysregulated Akt.

Xanthine oxidoreductase is an endogenous regulator of cyclooxygenase-2.

Xanthine oxidoreductase (XOR) is the enzyme responsible for the final step in purine degradation resulting in the generation of uric acid. Here we have generated mice deficient in XOR. As expected, these animals lack tissue XOR activity and have low to undetectable serum levels of uric acid. Although normal at birth, XOR-/- mice fail to thrive after 10 to 14 days, and most die within the first month. The cause of death appears to be a form of severe renal dysplasia, a phenotype that closely resembles what has been observed previously in cyclooxygenase-2 (COX-2)-deficient mice. We further demonstrate that in the first month of life, a period in which the mouse kidney is undergoing rapid maturation and remodeling, wild-type mice exhibit an approximately 30-fold increase in renal XOR activity, with a corresponding induction of COX-2 expression. In contrast, during this same period, XOR-/- animals fail to augment renal COX-2 expression. Finally, we show that in vitro and in vivo, uric acid can stimulate basal COX-2 expression. These results demonstrate that XOR activity is an endogenous physiological regulator of COX-2 expression and thereby provide insight into previous epidemiological evidence linking elevated serum uric levels with systemic hypertension and increased mortality from cardiovascular diseases. In addition, these results suggest a novel molecular link between cellular injury and the inflammatory response.

Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A.

Werner syndrome is a hereditary disease characterized by cancer predisposition, genetic instability, and the premature appearance of features associated with normal aging. At the molecular level this syndrome has been related to mutations in the Werner helicase, a member of the RecQ family of DNA helicases which are required to maintain genomic stability in cells. Here we show by a yeast two-hybrid screen that the Werner helicase can directly interact with the regulatory subunit (RIbeta) of cAMP protein kinase A (PKA). We confirm that this interaction occurs in vivo. Interestingly, serum withdrawal causes a redistribution of the Werner helicase within the nucleus of mammalian cells. Raising intracellular cAMP levels or increased expression of the regulatory but not the catalytic subunit of PKA inhibits this nuclear redistribution stimulated by serum deprivation. These results suggest that similar to lower organisms, gene products linked to genomic instability and aging may be directly regulated by growth factor-sensitive, PKA-dependent pathways.

Oxidants painting the cysteine chapel: redox regulation of PTPs.

Growth factors and cytokines appear to stimulate the intracellular production of reactive oxygen species (ROS). Evidence suggests that this alteration in the cellular redox state is essential for downstream signaling, but the precise mechanism has remained elusive. A new study now demonstrates that ligand-stimulated intracellular hydrogen peroxide can specifically and reversibly regulate the activity of protein tyrosine phosphatases.