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1.  Androgen receptor promotes sex-independent angiogenesis in response to ischemia and is required for activation of vascular endothelial cell growth factor receptor signaling 
Circulation  2013;128(1):60-71.
Background
Hypoandrogenemia is associated with an increased risk of ischemic diseases. Since actions of androgens are exerted through androgen receptor (AR) activation, we studied hind limb ischemia in AR knockout (KO) mice to elucidate the role of AR in response to ischemia.
Methods and Results
Both male and female ARKO mice exhibited impaired blood flow recovery, more cellular apoptosis and a higher incidence of autoamputation after ischemia. In ex vivo and in vivo angiogenesis studies, AR-deficient vascular endothelial cells showed reduced angiogenic capability. In ischemic limbs of ARKO mice, reductions in the phosphorylation of the Akt protein kinase and endothelial nitric oxide synthase (eNOS) were observed despite a robust increase in hypoxia-inducible factor 1α and vascular endothelial cell growth factor (VEGF) gene expression. In in vitro studies, siRNA-mediated ablation of AR in vascular endothelial cells blunted VEGF-stimulated phosphorylation of Akt and eNOS. Immunoprecipitation experiments documented an association between AR and kinase insert domain protein receptor (KDR) that promoted the recruitment of downstream signaling components.
Conclusion
These results document a physiological role of AR in gender-independent angiogenic potency and provide evidence for a novel cross-talk between androgen/AR signaling and VEGF/KDR signaling pathways.
doi:10.1161/CIRCULATIONAHA.113.001533
PMCID: PMC3933182  PMID: 23723256
androgen receptor; angiogenesis; KDR; Akt; eNOS
2.  Lipidomic analysis of the liver identifies changes of major and minor lipid species in adiponectin deficient mice 
Adiponectin protects from hepatic fat storage but adiponectin deficient mice (APN−/−) fed a standard chow do not develop liver steatosis. This indicates that other pathways might be activated to compensate for adiponectin deficiency. An unbiased and comprehensive screen was performed to identify hepatic alterations of lipid classes in these mice. APN−/− mice had decreased hepatic cholesteryl esters while active SREBP2 and systemic total cholesterol were not altered. Upregulation of cytochromes for bile acid synthesis suggests enhanced biliary cholesterol excretion. Analysis of 37 individual fatty acid species showed reduced stearate whereas total fatty acids were not altered. Total amount of triglycerides and phospholipids were equally abundant. A selective increase of monounsaturated phosphatidylcholine and phosphatidylethanolamine which positively correlate with hepatic and systemic triglycerides with the latter being elevated in APN−/− mice, was identified. Stearoyl-CoA desaturase 1 (SCD1) is involved in the synthesis of monounsaturated fatty acids and despite higher mRNA expression enzyme activity was not enhanced. Glucosylceramide postulated to contribute to liver damage was decreased.
This study demonstrates that adiponectin deficiency is associated with hepatic changes in lipid classes in mice fed a standard chow which may protect from liver steatosis.
doi:10.1016/j.yexmp.2012.03.008
PMCID: PMC3907090  PMID: 22465357
Lipid profiling; Liver; Adiponectin deficiency; Hepatic gene expression
4.  A major X-linked locus affects kidney function in mice 
Molecular genetics and genomics : MGG  2012;287(11-12):845-854.
Chronic kidney disease is a common disease with increasing prevalence in the western population. One common reason for chronic kidney failure is diabetic nephropathy. Diabetic nephropathy and hyperglycemia are characteristics of the mouse inbred strain KK/HlJ, which is predominantly used as a model for metabolic syndrome due to its inherited glucose intolerance and insulin resistance. We used KK/HlJ, an albuminuria-sensitive strain, and C57BL/6J, an albuminuria-resistant strain, to perform a quantitative trait locus (QTL) cross to identify the genetic basis for chronic kidney failure. Albumin-creatinine-ratio (ACR) was measured in 130 F2 male offspring. One significant QTL was identified on chromosome (Chr) X and four suggestive QTLs were found on Chrs 6, 7, 12, and 13. Narrowing of the QTL region was focused on the X-linked QTL and performed by incorporating genotype and expression analyses for genes located in the region. From the 485 genes identified in the X-linked QTL region, a few candidate genes were identified using a combination of bioinformatic evidence based on genomic comparison of the parental strains and known function in urine homeostasis. Finally, this study demonstrates the significance of the X chromosome in the genetic determination of albuminuria.
doi:10.1007/s00438-012-0720-x
PMCID: PMC3508201  PMID: 23011808
chronic kidney failure; QTL; genetic cross; ACR; diabetic nephropathy
5.  Sca1-Derived Cells Are a Source of Myocardial Renewal in the Murine Adult Heart 
Stem Cell Reports  2013;1(5):397-410.
Summary
Although the mammalian heart is one of the least regenerative organs in the body, recent evidence indicates that the myocardium undergoes a certain degree of renewal to maintain homeostasis during normal aging. However, the cellular origin of cardiomyocyte renewal has remained elusive due to lack of lineage tracing experiments focusing on putative adult cardiac precursor cells. We have generated triple-transgenic mice based on the tet-cre system to identify descendants of cells that have expressed the stem cell marker Sca1. We found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart. Our results reveal a source of cells for cardiomyocyte renewal and provide a possible explanation for the limited contribution of Sca1-derived cells to myocardial repair under pathological conditions.
Graphical Abstract
Highlights
•Sca1pos cells continuously generate cardiomyocytes during adult life•Some Sca1pos cells are tightly associated with cardiomyocytes•Sca1pos-derived cells show limited clonal expansion•Pressure overload moderately increases the number of Sca1-derived cardiomyocytes
To understand the renewal of cardiomyocytes, Uchida, Braun, and colleagues followed the fates of Sca1-positive cells in the heart. By utilizing triple-transgenic mice, they found a significant and lasting contribution of Sca1-derived cells to cardiomyocytes during normal aging. Ischemic damage and pressure overload resulted in increased differentiation of Sca1-derived cells to the different cell types present in the heart.
doi:10.1016/j.stemcr.2013.09.004
PMCID: PMC3841250  PMID: 24286028
6.  Mitofusins 1 and 2 are Essential for Postnatal Metabolic Remodeling in Heart 
Circulation research  2012;111(8):1012-1026.
Rationale
At birth, there is a switch from placental to pulmonary circulation and the heart commences its aerobic metabolism. In cardiac myocytes, this transition is marked by increased mitochondrial biogenesis and remodeling of the intracellular architecture. The mechanisms governing the formation of new mitochondria and their expansion within myocytes remain largely unknown. Mitofusins (Mfn-1 and Mfn-2) are known regulators of mitochondrial networks but their role during perinatal maturation of the heart has yet to be examined.
Objective
Determine the significance of mitofusins, during early postnatal cardiac development.
Methods and Results
We genetically inactivated Mfn-1 and Mfn-2 in mid-gestational and postnatal cardiac myocytes using a loxP/Myh6-cre approach. At birth, cardiac morphology and function of double-knockout (DKO) mice are normal. At that time, DKO mitochondria increase in numbers, appear to be spherical and heterogeneous in size but exhibit normal electron-density. By postnatal day 7, the mitochondrial numbers in DKO myocytes remain abnormally expanded and many lose matrix components and membrane organization. In this context, DKO mice develop dilated cardiomyopathy (DCM). This leads to a rapid decline in survival and all DKO mice perish before 16 days of age. Gene expression analysis of DKO hearts shows that mitochondria biogenesis genes are down regulated, the mitochondrial DNA is reduced and so are mitochondrially-encoded transcripts and proteins. Furthermore, mitochondrial turnover pathways are dysregulated.
Conclusions
Our findings establish that Mfn-1 and Mfn-2 are essential in mediating mitochondrial remodeling during postnatal cardiac development, a time of dramatic transitions in the bioenergetics and growth of the heart.
doi:10.1161/CIRCRESAHA.112.274142
PMCID: PMC3518037  PMID: 22904094
Cardiac growth; mitochondria; biogenesis; mtDNA; p62
7.  Plasma adiponectin and mortality in critically ill subjects with acute respiratory failure 
Critical care medicine  2010;38(12):2329-2334.
Objective
Adiponectin, an anti-inflammatory cytokine produced by adipose tissue, has been shown to modulate survival in animal models of critical illness. We examined the association between plasma adiponectin and clinical outcomes in critically ill patients with acute respiratory failure.
Design
Secondary analysis of a single-center, randomized controlled trial.
Setting
Medical intensive care unit of a university-based, tertiary medical center.
Patients
One hundred seventy-five subjects with acute respiratory failure enrolled in randomized, controlled pilot trial of trophic vs. full-caloric enteral feeding (EDEN pilot study).
Interventions
None.
Measurements and Main Results
Adiponectin measured within 48 hrs of respiratory failure (Apn1) was inversely correlated with body mass index (r = −0.25, p = .007) and was higher in females (median, 12.6 µg/mL; interquartile range, 7.6–17.1) than males (9.45 µg/mL; 6.2–14.2; p = .02). Adiponectin increased at day 6 (Apn1: 11.4 µg/mL [6.6 –15.3] vs. Apn6: 14.1 µg/mL [10.3–18.6], p < .001). This increase was significant only in survivors (Δ adiponectin in survivors: 3.9 ± 6 µg/mL, n = 80, p < .001 vs. Δ in nonsurvivors: 1.69 ± 4.6 µg/mL, n = 14, p =.19). Higher Apn1 was significantly associated with 28-day mortality (odds ratio 1.59 per 5-µg/mL increase; 95% confidence interval 1.15–2.21; p = .006). No measured demographic, clinical, or cytokine covariates, including interleukin-6, interleukin-8, interleukin-10, interleukin-1β, interleukin-12, tumor necrosis factor-α, and interferon-γ, were confounders or effect modifiers of this association between adiponectin and mortality.
Conclusions
Independent of measured covariates, increased plasma adiponectin levels measured within 48 hrs of respiratory failure are associated with mortality. This finding suggests that factors derived from adipose tissue play a role in modulating the response to critical illness.
doi:10.1097/CCM.0b013e3181fa0561
PMCID: PMC3703623  PMID: 20890191
adiponectin; respiratory insufficiency; critical illness; biologic markers; epidemiology; translational research
8.  Defective Autophagy and mTORC1 Signaling in Myotubularin Null Mice 
Molecular and Cellular Biology  2013;33(1):98-110.
Autophagy is a vesicular trafficking pathway that regulates the degradation of aggregated proteins and damaged organelles. Initiation of autophagy requires several multiprotein signaling complexes, such as the ULK1 kinase complex and the Vps34 lipid kinase complex, which generates phosphatidylinositol 3-phosphate [PtdIns(3)P] on the forming autophagosomal membrane. Alterations in autophagy have been reported for various diseases, including myopathies. Here we show that skeletal muscle autophagy is compromised in mice deficient in the X-linked myotubular myopathy (XLMTM)-associated PtdIns(3)P phosphatase myotubularin (MTM1). Mtm1-deficient muscle displays several cellular abnormalities, including a profound increase in ubiquitin aggregates and abnormal mitochondria. Further, we show that Mtm1 deficiency is accompanied by activation of mTORC1 signaling, which persists even following starvation. In vivo pharmacological inhibition of mTOR is sufficient to normalize aberrant autophagy and improve muscle phenotypes in Mtm1 null mice. These results suggest that aberrant mTORC1 signaling and impaired autophagy are consequences of the loss of Mtm1 and may play a primary role in disease pathogenesis.
doi:10.1128/MCB.01075-12
PMCID: PMC3536306  PMID: 23109424
9.  Obligatory participation of macrophages in an angiopoietin 2-mediated cell death switch 
Development (Cambridge, England)  2007;134(24):4449-4458.
Macrophages have a critical function in the recognition and engulfment of dead cells. In some settings, macrophages also actively signal programmed cell death. Here we show that during developmentally scheduled vascular regression, resident macrophages are an obligatory participant in a signaling switch that favors death over survival. This switch occurs when the signaling ligand angiopoietin 2 has the dual effect of suppressing survival signaling in vascular endothelial cells (VECs) and stimulating Wnt ligand production by macrophages. In response to the Wnt ligand, VECs enter the cell cycle and in the absence of survival signals, die from G1 phase of the cell cycle. We propose that this mechanism represents an adaptation to ensure that the macrophage and its disposal capability are on hand when cell death occurs.
doi:10.1242/dev.012187
PMCID: PMC3675770  PMID: 18039971
Macrophage; Angiopoietin; Wnt; Programmed cell death; Vascular regression; Cell cycle
10.  What can adiponectin say about left ventricular function? 
Heart (British Cardiac Society)  2009;96(5):331-332.
doi:10.1136/hrt.2009.178590
PMCID: PMC3673540  PMID: 19934101
11.  Akt1–Mediated Skeletal Muscle Growth Attenuates Cardiac Dysfunction and Remodeling After Experimental Myocardial Infarction 
Circulation. Heart failure  2011;5(1):116-125.
Background
It is appreciated that aerobic endurance exercise can attenuate unfavorable myocardial remodeling following myocardial infarction. In contrast, little is known about the effects of increasing skeletal muscle mass, typically achieved through resistance training, on this process. Here, we utilized transgenic (TG) mice that can induce the growth of functional skeletal muscle by switching Akt1 signaling in muscle fibers to assess the impact of glycolytic muscle growth on post-myocardial infarction cardiac remodeling.
Methods and Results
Male-noninduced TG mice and their nontransgenic littermates (control) were subjected to left anterior coronary artery ligation. Two days after surgery, mice were provided doxycycline in their drinking water to activate Akt1 transgene expression in a skeletal muscle-specific manner. Myogenic Akt1 activation led to diminished left ventricular dilation and reduced contractile dysfunction compared with control mice. Improved cardiac function in Akt1 TG mice was coupled to diminished myocyte hypertrophy, decreased interstitial fibrosis, and increased capillary density. ELISA and protein array analyses demonstrated that serum levels of proangiogenic growth factors were upregulated in Akt1 TG mice compared with control mice. Cardiac eNOS was activated in Akt1 TG mice after myocardial infarction. The protective effect of skeletal muscle Akt activation on cardiac remodeling and systolic function was abolished by treatment with the eNOS inhibitor l-NAME.
Conclusions
Akt1–mediated skeletal muscle growth attenuates cardiac remodeling after myocardial infarction and is associated with an increased capillary density in the heart. This improvement appears to be mediated by skeletal muscle to cardiac communication, leading to activation of eNOS-signaling in the heart.
doi:10.1161/CIRCHEARTFAILURE.111.964783
PMCID: PMC3670415  PMID: 22135402
angiogenesis; exercise training; myocardial infarction; nitric oxide synthase; remodeling heart failure
12.  Adiponectin upregulates hepatocyte CMKLR1 which is reduced in human fatty liver 
Chemokine-like receptor 1 (CMKLR1) ligands chemerin and resolvin E1 are suggested to have a role in non-alcoholic fatty liver disease (NAFLD). Here, expression of CMKLR1 in liver cells and NAFLD was studied. CMKLR1 was detected in primary human hepatocytes (PHH), Kupffer cells, bile-duct cells and hepatic stellate cells. In human and rodent fatty liver and in fibrotic liver of mice fed a methionine–choline deficient diet CMKLR1 was reduced. Hepatocytes are the major cells in the liver and effects of adipokines, cytokines and lipids on CMKLR1 in PHH were analyzed. Increased cellular triglyceride or cholesterol content, lipopolysaccharide, IL-6, TNF and leptin did not influence CMKLR1 levels in PHH whereas profibrotic TGFβ tended to reduce CMKLR1. Adiponectin strongly upregulated CMKLR1 mRNA and protein in PHH and hepatic CMKLR1 when injected into wild type mice. Further, CMKLR1 was suppressed in the liver of adiponectin deficient mice. These data indicate that low CMKLR1 in NAFLD may partly result from reduced adiponectin activity.
doi:10.1016/j.mce.2011.10.032
PMCID: PMC3670424  PMID: 22118966
Adipokine; Hepatic steatosis; Chemerin receptor; Liver
13.  Impact of a Single Intracoronary Administration of Adiponectin on Myocardial Ischemia/Reperfusion Injury in a Pig Model 
Background
Adiponectin plays a protective role in the development of obesity-linked disorders. We demonstrated that adiponectin exerts beneficial actions on acute ischemic injury in mice hearts. However, the effects of adiponectin treatment in large animals and its feasibility in clinical practice have not been investigated. This study investigated the effects of intracoronary administration of adiponectin on myocardial ischemia-reperfusion (I/R) injury in pigs.
Methods and Results
The left anterior descending coronary artery was occluded in pigs for 45 minutes and then reperfused for 24 hours. Recombinant adiponectin protein was given as a bolus intracoronary injection during ischemia. Cardiac functional parameters were measured by a manometer-tipped catheter. Apoptosis was evaluated by terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining. Tumor necrosis factor-α and interleukin-10 transcripts were analyzed by real-time polymerase chain reaction. Serum levels of derivatives of reactive oxygen metabolites and biological antioxidant potential were measured. Adiponectin protein was determined by immunohistochemical and Western blot analyses. Intracoronary administration of adiponectin protein led to a reduction in myocardial infarct size and improvement of left ventricular function in pigs after I/R. Injected adiponectin protein accumulated in the I/R-injured heart. Adiponectin treatment resulted in decreased tumor necrosis factor-α and increased interleukin-10 mRNA levels in the myocardium after I/R. Adiponectin-treated pigs had reduced apoptotic activity in the I/R-injured heart and showed increased biological antioxidant potential levels and decreased derivatives of reactive oxygen metabolite levels in the blood stream after I/R.
Conclusions
These data suggest that adiponectin protects against I/R injury in a preclinical pig model through its ability to suppress inflammation, apoptosis, and oxidative stress. Administration of intracoronary adiponectin could be a useful adjunctive therapy for acute myocardial infarction.
doi:10.1161/CIRCINTERVENTIONS.109.872044
PMCID: PMC3668696  PMID: 20332381
adiponectin; myocardial infarction; reperfusion
14.  The FOXO3a Transcription Factor Regulates Cardiac Myocyte Size Downstream of AKT Signaling* 
The Journal of biological chemistry  2005;280(21):20814-20823.
Although signaling mechanisms inducing cardiac hypertrophy have been extensively studied, little is known about the mechanisms that reverse cardiac hypertrophy. Here, we describe the existence of a similar Akt/forkhead signaling axis in cardiac myocytes in vitro and in vivo, which is regulated by insulin, insulin-like growth factor (IGF), stretch, pressure overload, and angiotensin II stimulation. FOXO3a gene transfer prevented both IGF and stretch-induced hypertrophy in rat neonatal cardiac myocyte cultures in vitro. Transduction with FOXO3a also caused a significant reduction in cardiomyocyte size in mouse hearts in vivo. Akt/FOXO signaling regulated the expression of multiple atrophy-related genes “atrogenes,” including the ubiquitin ligase atrogin-1 (MAFbx). In cardiac myocyte cultures, transduction with constitutively active Akt or treatment with IGF suppressed atrogin-1 mRNA expression, whereas transduction with FOXO3a stimulated its expression. FOXO3a transduction activated the atrogin-1 promoter in both cultured myocytes and mouse heart. Thus, in cardiomyocytes, as in skeletal muscle, FOXO3a activates an atrogene transcriptional program, which retards or prevents hypertrophy and is down-regulated by multiple physiological and pathological stimuli of myocyte growth.
doi:10.1074/jbc.M500528200
PMCID: PMC3632436  PMID: 15781459
15.  Akt Promotes Survival of Cardiomyocytes In Vitro and Protects Against Ischemia-Reperfusion Injury in Mouse Heart 
Circulation  2000;101(6):660-667.
Background
IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1–regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo.
Methods and Results
IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1–mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1–induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury.
Conclusions
These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.
PMCID: PMC3627349  PMID: 10673259
myocytes; apoptosis; Akt; ischemia; reperfusion
16.  Akt Mediates Cytoprotection of Endothelial Cells by Vascular Endothelial Growth Factor in an Anchorage-dependent Manner* 
The Journal of biological chemistry  1999;274(23):16349-16354.
Regulation of endothelial cell apoptosis is a critical modulator of normal and pathological angiogenesis. In this study, we examined the role of the protein kinase Akt/PKB in endothelial cell survival in response to growth factor and matrix attachment signals. Vascular endothelial growth factor(VEGF)-induced cytoprotection of endothelial cell monolayers correlated with the wortmannin-sensitive induction of Akt activity. Transfection of an adenovirus expressing a dominant-negative Akt mutant decreased endothelial cell viability in the presence of VEGF. Conversely, adenoviral transduction of wild-type Akt facilitated the cell survival effects of VEGF, whereas transduction of constitutively active Akt conferred endothelial cell survival in the absence of VEGF. Constitutively active Akt also conferred survival to endothelial cells in suspension culture, whereas stimulation with VEGF did not. In suspension cultures, VEGF stimulation was unable to activate Akt, and Akt protein levels were repressed in cells undergoing anoikis. These data suggest that cross-talk between growth factor- and anchorage-dependent signaling pathways are essential for Akt activation and endothelial cell survival.
PMCID: PMC3624707  PMID: 10347193
17.  The Polyphenols Resveratrol and S17834 prevent the Structural and Functional Sequelae of Diet-Induced Metabolic Heart Disease in Mice 
Circulation  2012;125(14):1757-1764.
Background
Diet-induced obesity is associated with metabolic heart disease characterized by left ventricular (LV) hypertrophy and diastolic dysfunction. Polyphenols such as resveratrol (RSV) and the synthetic flavonoid derivative S17834 exert beneficial systemic and cardiovascular effects in a variety of settings including diabetes and chronic hemodynamic overload.
Methods and Results
We characterized the structural and functional features of a mouse model of diet-induced metabolic syndrome, and used the model to test the hypothesis that the polyphenols prevent myocardial hypertrophy and diastolic dysfunction. Male C57BL/6J mice were fed a normal diet or a diet high in fat and sugar (HFHS) with or without concomitant treatment with S17834 or RSV for up to 8 months. HFHS diet-fed mice developed progressive LV hypertrophy and diastolic dysfunction with preservation of systolic function in association with myocyte hypertrophy and interstitial fibrosis. In HFHS-fed mice there was increased myocardial oxidative stress with evidence of oxidant-mediated protein modification via tyrosine nitration and 4-OH-2-nonenol (HNE) adduction. HFHS-fed mice also exhibited increases in plasma fasting glucose, insulin and HOMA-IR indicative of insulin resistance. Treatment with S17834 or RSV prevented LV hypertrophy and diastolic dysfunction. For S17834, these beneficial effects were associated with decreases in oxidant-mediated protein modifications and hyper-insulinemia, and increased plasma adiponectin.
Conclusions
RSV and S17834 administered concurrently with a HFHS diet prevent the development of LV hypertrophy, interstitial fibrosis and diastolic dysfunction. Multiple mechanisms may contribute to the beneficial effects of the polyphenols including a reduction in myocardial oxidative stress and related protein modifications, amelioration of insulin resistance and increased plasma adiponectin. The polyphenols RSV and S17834 may be of value in the prevention of diet-induced metabolic heart disease.
doi:10.1161/CIRCULATIONAHA.111.067801
PMCID: PMC3354628  PMID: 22388319
left ventricular hypertrophy; diastolic dysfunction; 4-OH-2-nonenol; metabolic syndrome; oxidative stress
18.  Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy 
Cell  2004;117(3):399-412.
Summary
Skeletal muscle atrophy is a debilitating response to fasting, disuse, cancer, and other systemic diseases. In atrophying muscles, the ubiquitin ligase, atrogin-1 (MAFbx), is dramatically induced, and this response is necessary for rapid atrophy. Here, we show that in cultured myotubes undergoing atrophy, the activity of the PI3K/AKT pathway decreases, leading to activation of Foxo transcription factors and atrogin-1 induction. IGF-1 treatment or AKT overexpression inhibits Foxo and atrogin-1 expression. Moreover, constitutively active Foxo3 acts on the atrogin-1 promoter to cause atrogin-1 transcription and dramatic atrophy of myotubes and muscle fibers. When Foxo activation is blocked by a dominant-negative construct in myotubes or by RNAi in mouse muscles in vivo, atrogin-1 induction during starvation and atrophy of myotubes induced by glucocorticoids are prevented. Thus, forkhead factor(s) play a critical role in the development of muscle atrophy, and inhibition of Foxo factors is an attractive approach to combat muscle wasting.
PMCID: PMC3619734  PMID: 15109499
19.  NADPH Oxidase 4 Promotes Endothelial Angiogenesis Through eNOS Activation 
Circulation  2011;124(6):731-740.
Background
Reactive Oxygen Species (ROS) serve signaling functions in the vasculature, and hypoxia has been associated with increased ROS production. NADPH oxidase 4 (Nox4) is an ROS-producing enzyme that is highly expressed in the endothelium, yet its specific role is unknown. We sought to determine the role of Nox4 in the endothelial response to hypoxia.
Methods and Results
Hypoxia induced Nox4 expression both in vitro and in vivo and overexpression of Nox4 was sufficient to promote endothelial proliferation, migration, and tube formation. To determine the in vivo relevance of our observations, we generated transgenic mice with endothelial-specific Nox4 overexpression using the VE-cadherin promoter (VECad-Nox4 mice). In vivo, the VECad-Nox4 mice had accelerated recovery from hind limb ischemia and enhanced aortic capillary sprouting. Because endothelial nitric oxide synthase (eNOS) is involved in endothelial angiogenic responses and eNOS is activated by ROS, we probed the effect of Nox4 on eNOS. In cultured ECs overexpressing Nox4 we observed a significant increase in eNOS protein expression and activity. To causally address the link between eNOS and Nox4 we crossed our transgenic Nox4 mice with eNOS-/- mice. Aorta from these mice did not demonstrate enhanced aortic sprouting and VECad-Nox4 mice on the eNOS-/- background did not demonstrate enhanced recovery from hind limb ischemia.
Conclusions
Collectively, we demonstrate that augmented endothelial Nox4 expression promotes angiogenesis and recovery from hypoxia in an eNOS-dependent manner.
doi:10.1161/CIRCULATIONAHA.111.030775
PMCID: PMC3589548  PMID: 21788590
NADPH oxidase 4; Reactive Oxygen Species; Endothelium; Angiogenesis; Endothelial Nitric Oxide Synthase
20.  Airway Delivery of Soluble Factors from Plastic-Adherent Bone Marrow Cells Prevents Murine Asthma 
Asthma affects an estimated 300 million people worldwide and accounts for 1 of 250 deaths and 15 million disability-adjusted life years lost annually. Plastic-adherent bone marrow–derived cell (BMC) administration holds therapeutic promise in regenerative medicine. However, given the low cell engraftment in target organs, including the lung, cell replacement cannot solely account for the reported therapeutic benefits. This suggests that BMCs may act by secreting soluble factors. BMCs also possess antiinflammatory and immunomodulatory properties and may therefore be beneficial for asthma. Our objective was to investigate the therapeutic potential of BMC-secreted factors in murine asthma. In a model of acute and chronic asthma, intranasal instillation of BMC conditioned medium (CdM) prevented airway hyperresponsiveness (AHR) and inflammation. In the chronic asthma model, CdM prevented airway smooth muscle thickening and peribronchial inflammation while restoring blunted salbutamol-induced bronchodilation. CdM reduced lung levels of the TH2 inflammatory cytokines IL-4 and IL-13 and increased levels of IL-10. CdM up-regulated an IL-10–induced and IL-10–secreting subset of T regulatory lymphocytes and promoted IL-10 expression by lung macrophages. Adiponectin (APN), an antiinflammatory adipokine found in CdM, prevented AHR, airway smooth muscle thickening, and peribronchial inflammation, whereas the effect of CdM in which APN was neutralized or from APN knock-out mice was attenuated compared with wild-type CdM. Our study provides evidence that BMC-derived soluble factors prevent murine asthma and suggests APN as one of the protective factors. Further identification of BMC-derived factors may hold promise for novel approaches in the treatment of asthma.
doi:10.1165/rcmb.2010-0391OC
PMCID: PMC3361359  PMID: 21903873
cellular therapy; bone marrow stromal cells; hypersensitivity; paracrine
21.  Thiazolidinediones reduce pathological neovascularization in ischemic retina via an adiponectin-dependent mechanism 
Objective
The insulin-sensitizing agents referred to as thiazolidinediones (TZDs) possess anti-atherogenic and anti-inflammatory actions that contribute to protection against diabetic macrovascular complications. However, little is known about the effects of TZDs on retinal microvessel disorders. Here, we investigated whether TZDs modulate retinal vessel formation in a mouse model of oxygen- retinopathy.
Methods and Results
Neonatal mice were subjected to ischemia-induced retinopathy to produce pathological neovascular tuft formation. Pioglitazone (10 mg/kg/day), rosiglitazone (10 mg/kg/day) or vehicle was given by gavage once a day from postnatal day 7 (P7) to P17. Systemic treatment of wild-type (WT) mice with TZDs led to a significant decrease in pathological retinal neovascularization during ischemia compared with vehicle treatment, which was accompanied by increased plasma levels of the fat-derived hormone adiponectin. In contrast to WT mice, TZDs had no effects on ischemia-induced pathological retinal vessel formation in adiponectin-knockout (APN-KO) mice. Pioglitazone reduced tumor necrosis factor (TNF)-α expression in ischemic retina in WT mice but not in APN-KO mice. Furthermore, pioglitazone increased plasma adiponectin levels in TNF-α-KO mice but did not affect ischemia-induced pathological retinal neovascularization in this strain.
Conclusions
These data show that TZDs attenuate pathological retinal microvessel formation through adiponectin-mediated modulation of TNF-α production.
doi:10.1161/ATVBAHA.109.198465
PMCID: PMC3552615  PMID: 19910632
pioglitazone; adiponectin; neovascularization; ischemia; angiogenesis
22.  Therapeutic Impact of Follistatin-Like 1 on Myocardial Ischemic Injury in Preclinical Models 
Circulation  2012;126(14):1728-1738.
Background
Acute coronary syndrome is a leading cause of death in developed countries. Follistatin-like 1 (FSTL1) is a myocyte-derived secreted protein that is upregulated in the heart in response to ischemic insult. Here, we investigated the therapeutic impact of FSTL1 on acute cardiac injury in small and large preclinical animal models of ischemia/reperfusion and dissected its molecular mechanism.
Methods and Results
Administration of human FSTL1 protein significantly attenuated myocardial infarct size in a mouse or pig model of ischemia/reperfusion, which was associated with a reduction of apoptosis and inflammatory responses in the ischemic heart. Administration of FSTL1 enhanced the phosphorylation of AMP-activated protein kinase in the ischemia/reperfusion–injured heart. In cultured cardiac myocytes, FSTL1 suppressed apoptosis in response to hypoxia/reoxygenation and lipopolysaccharide-stimulated expression of proinflammatory genes through its ability to activate AMP-activated protein kinase. Ischemia/reperfusion led to enhancement of bone morphogenetic protein-4 expression and Smad1/5/8 phosphorylation in the heart, and FSTL1 suppressed the increased phosphorylation of Smad1/5/8 in ischemic myocardium. Treating cardiac myocytes with FSTL1 abolished the bone morphogenetic protein-4 –stimulated increase in apoptosis, Smad1/5/8 phosphorylation, and proinflammatory gene expression. In cultured macrophages, FSTL1 diminished lipopolysaccharide-stimulated expression of proinflammatory genes via activation of AMP-activated protein kinase and abolished bone morphogenetic protein-4 – dependent induction of proinflammatory mediators.
Conclusions
Our data indicate that FSTL1 can prevent myocardial ischemia/reperfusion injury by inhibiting apoptosis and inflammatory response through modulation of AMP-activated protein kinase– and bone morphogenetic protein-4 – dependent mechanisms, suggesting that FSTL1 could represent a novel therapeutic target for post-myocardial infarction, acute coronary syndrome.
doi:10.1161/CIRCULATIONAHA.112.115089
PMCID: PMC3548325  PMID: 22929303
apoptosis; inflammation; ischemia; myocytes; cardiac; reperfusion
23.  Adiponectin attenuates LPS-induced acute lung injury through suppression of endothelial cell activation1 
Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN’s role in the early development of ALI to lipopolysaccharide (LPS) was investigated. Intra-tracheal (i.t.) LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN−/−) mice compared to wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN−/− mice as early as 4 hours after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 hours after LPS administration showed increased pro-inflammatory gene expression (e.g. IL-6) only in endothelial cells of APN−/− mice when compared to wt mice. Direct effects on lung endothelium were demonstrated by APN’s ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient (T-cad−/−) mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after i.t. LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g. obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium.
doi:10.4049/jimmunol.1100426
PMCID: PMC3253176  PMID: 22156343
Adiponectin; acute lung injury; endothelium; T-cadherin
24.  Identification of Follistatin-Like 1 by Expression Cloning as an Activator of the Growth Differentiation Factor 15 Gene and a Prognostic Biomarker in Acute Coronary Syndrome 
Clinical chemistry  2012;58(8):1233-1241.
BACKGROUND
Growth differentiation factor 15 (GDF15) is a stress-responsive cytokine and biomarker that is produced after myocardial infarction and that is related to prognosis in acute coronary syndrome (ACS). We hypothesized that secreted proteins that activate GDF15 production may represent new ACS biomarkers.
METHODS
We expressed clones from an infarcted mouse heart cDNA library in COS1 cells and assayed for activation of a luciferase reporter gene controlled by a 642-bp fragment of the mouse growth differentiation factor 15 (GDF15) gene promoter. We measured the circulating concentrations of follistatin-like 1 (FSTL1) and GDF15 in 1369 patients with ACS.
RESULTS
One cDNA clone that activated the GDF15 promoter–luciferase reporter encoded the secreted protein FSTL1. Treatment with FSTL1 activated GDF15 production in cultured cardiomyocytes. Transgenic production of FSTL1 stimulated GDF15 production in the murine heart, whereas cardiomyocyte-selective deletion of FSTL1 decreased production of GDF15 in cardiomyocytes, indicating that FSTL1 is sufficient and required for GDF15 production. In ACS, FSTL1 emerged as the strongest independent correlate of GDF15 (partial R2 = 0.26). A total of 106 patients died of a cardiovascular cause during a median follow-up of 252 days. Patients with an FSTL1 concentration in the top quartile had a 3.7-fold higher risk of cardiovascular death compared with patients in the first 3 quartiles (P < 0.001). FSTL1 remained associated with cardiovascular death after adjustment for clinical, angiographic, and biochemical variables.
CONCLUSIONS
Our study is the first to use expression cloning for biomarker discovery upstream of a gene of interest and to identify FSTL1 as an independent prognostic biomarker in ACS.
doi:10.1373/clinchem.2012.182816
PMCID: PMC3539794  PMID: 22675198
25.  Complement C1q Activates Canonical Wnt Signaling and Promotes Aging-Related Phenotypes 
Cell  2012;149(6):1298-1313.
SUMMARY
Wnt signaling plays critical roles in development of various organs and pathogenesis of many diseases, and augmented Wnt signaling has recently been implicated in mammalian aging and aging-related phenotypes. We here report that complement C1q activates canonical Wnt signaling and promotes aging-associated decline in tissue regeneration. Serum C1q concentration is increased with aging, and Wnt signaling activity is augmented during aging in the serum and in multiple tissues of wild-type mice, but not in those of C1qa-deficient mice. C1q activates canonical Wnt signaling by binding to Frizzled receptors and subsequently inducing C1s-dependent cleavage of the ectodomain of Wnt coreceptor low-density lipoprotein receptor-related protein 6. Skeletal muscle regeneration in young mice is inhibited by exogenous C1q treatment, whereas aging-associated impairment of muscle regeneration is restored by C1s inhibition or C1qa gene disruption. Our findings therefore suggest the unexpected role of complement C1q in Wnt signal transduction and modulation of mammalian aging.
doi:10.1016/j.cell.2012.03.047
PMCID: PMC3529917  PMID: 22682250

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