Autologous Umbilical Cord Blood-Derived Mononuclear Cell Therapy Promotes Cardiac Proliferation and Adaptation in a Porcine Model of Right Ventricle Pressure Overload.

Congenital heart diseases, including single ventricle circulations, are clinically challenging due to chronic pressure overload and the inability of the myocardium to compensate for lifelong physiological demands. To determine the clinical relevance of autologous umbilical cord blood-derived mononuclear cells (UCB-MNCs) as a therapy to augment cardiac adaptation following surgical management of congenital heart disease, a validated model system of right ventricular pressure overload due to pulmonary artery banding (PAB) in juvenile pigs has been employed. PAB in a juvenile porcine model and intramyocardial delivery of UCB-MNCs was evaluated in three distinct 12-week studies utilizing serial cardiac imaging and end-of-study pathology evaluations. PAB reproducibly induced pressure overload leading to chronic right ventricular remodeling including significant myocardial fibrosis and elevation of heart failure biomarkers. High-dose UCB-MNCs (3 million/kg) delivered into the right ventricular myocardium did not cause any detectable safety issues in the context of arrhythmias or abnormal cardiac physiology. In addition, this high-dose treatment compared with placebo controls demonstrated that UCB-MNCs promoted a significant increase in Ki-67-positive cardiomyocytes coupled with an increase in the number of CD31+ endothelium. Furthermore, the incorporation of BrdU-labeled cells within the myocardium confirmed the biological potency of the high-dose UCB-MNC treatment. Finally, the cell-based treatment augmented the physiological adaptation compared with controls with a trend toward increased right ventricular mass within the 12 weeks of the follow-up period. Despite these adaptations, functional changes as measured by echocardiography and magnetic resonance imaging did not demonstrate differences between cohorts in this surgical model system. Therefore, this randomized, double-blinded, placebo-controlled pre-clinical trial establishes the safety of UCB-MNCs delivered via intramyocardial injections in a dysfunctional right ventricle and validates the induction of cardiac proliferation and angiogenesis as transient paracrine mechanisms that may be important to optimize long-term outcomes for surgically repaired congenital heart diseases.

Dysregulated ribonucleoprotein granules promote cardiomyopathy in RBM20 gene-edited pigs.

Ribonucleoprotein (RNP) granules are biomolecular condensates-liquid-liquid phase-separated droplets that organize and manage messenger RNA metabolism, cell signaling, biopolymer assembly, biochemical reactions and stress granule responses to cellular adversity. Dysregulated RNP granules drive neuromuscular degenerative disease but have not previously been linked to heart failure. By exploring the molecular basis of congenital dilated cardiomyopathy (DCM) in genome-edited pigs homozygous for an RBM20 allele encoding the pathogenic R636S variant of human RNA-binding motif protein-20 (RBM20), we discovered that RNP granules accumulated abnormally in the sarcoplasm, and we confirmed this finding in myocardium and reprogrammed cardiomyocytes from patients with DCM carrying the R636S allele. Dysregulated sarcoplasmic RBM20 RNP granules displayed liquid-like material properties, docked at precisely spaced intervals along cytoskeletal elements, promoted phase partitioning of cardiac biomolecules and fused with stress granules. Our results link dysregulated RNP granules to myocardial cellular pathobiology and heart failure in gene-edited pigs and patients with DCM caused by RBM20 mutation.

Ultrasound Shear Wave Elastography as a Measure of Porcine Hepatic Disease in Right Heart Dysfunction: A Pilot Study.

Patients with congenital heart disease with a pressure-overloaded right ventricle can develop liver disease and would benefit from non-invasive diagnostic modalities such as ultrasound shear wave elastography (US SWE). We sought to investigate the ability of US SWE to measure dynamic changes in liver stiffness with an acute fluid bolus in an animal model. Three piglets underwent surgical intervention to create a pressure-overloaded right ventricle and, 12 wk later, underwent US SWE, both pre- and post-intravenous infusion of a saline bolus. Ultrasound measures of shear modulus, velocity and attenuation were taken to characterize hepatic mechanical properties. Liver stiffness exhibited a dynamic component that increased after fluid bolus, although not reaching statistical significance with our small sample size, and these changes were greater in more diseased livers. US SWE may provide a promising non-invasive method for assessing dynamic changes in hydration status and degree of liver disease.

Quantification of Etoposide Hypersensitivity: A Sensitive, Functional Method for Assessing Pluripotent Stem Cell Quality.

Human induced pluripotent stem cells (hiPSC) hold great promise in diagnostic and therapeutic applications. However, translation of hiPSC technology depends upon a means of assessing hiPSC quality that is quantitative, high-throughput, and can decipher malignant teratocarcinoma clones from normal cell lines. These attributes are lacking in current approaches such as detection of cell surface makers, RNA profiling, and/or teratoma formation assays. The latter remains the gold standard for assessing clone quality in hiPSCs, but is expensive, time-consuming, and incompatible with high-throughput platforms. Herein, we describe a novel method for determining hiPSC quality that exploits pluripotent cells' documented hypersensitivity to the topoisomerase inhibitor etoposide (CAS No. 33419-42-0). Based on a study of 115 unique hiPSC clones, we established that a half maximal effective concentration (EC50) value of <300 nM following 24 hours of exposure to etoposide demonstrated a positive correlation with RNA profiles and colony morphology metrics associated with high quality hiPSC clones. Moreover, our etoposide sensitivity assay (ESA) detected differences associated with culture maintenance, and successfully distinguished malignant from normal pluripotent clones independent of cellular morphology. Overall, the ESA provides a simple, straightforward method to establish hiPSC quality in a quantitative and functional assay capable of being incorporated into a generalized method for establishing a quality control standard for all types of pluripotent stem cells. Stem Cells Translational Medicine 2017;6:1829-1839.

Modeling structural and functional deficiencies of RBM20 familial dilated cardiomyopathy using human induced pluripotent stem cells.

Dilated cardiomyopathy (DCM) is a leading cause of heart failure. In families with autosomal-dominant DCM, heterozygous missense mutations were identified in RNA-binding motif protein 20 (RBM20), a spliceosome protein induced during early cardiogenesis. Dermal fibroblasts from two unrelated patients harboring an RBM20 R636S missense mutation were reprogrammed to human induced pluripotent stem cells (hiPSCs) and differentiated to beating cardiomyocytes (CMs). Stage-specific transcriptome profiling identified differentially expressed genes ranging from angiogenesis regulator to embryonic heart transcription factor as initial molecular aberrations. Furthermore, gene expression analysis for RBM20-dependent splice variants affected sarcomeric (TTN and LDB3) and calcium (Ca(2+)) handling (CAMK2D and CACNA1C) genes. Indeed, RBM20 hiPSC-CMs exhibited increased sarcomeric length (RBM20: 1.747 ± 0.238 µm versus control: 1.404 ± 0.194 µm; P < 0.0001) and decreased sarcomeric width (RBM20: 0.791 ± 0.609 µm versus control: 0.943 ± 0.166 µm; P < 0.0001). Additionally, CMs showed defective Ca(2+) handling machinery with prolonged Ca(2+) levels in the cytoplasm as measured by greater area under the curve (RBM20: 814.718 ± 94.343 AU versus control: 206.941 ± 22.417 AU; P < 0.05) and higher Ca(2+) spike amplitude (RBM20: 35.281 ± 4.060 AU versus control:18.484 ± 1.518 AU; P < 0.05). ß-adrenergic stress induced with 10 µm norepinephrine demonstrated increased susceptibility to sarcomeric disorganization (RBM20: 86 ± 10.5% versus control: 40 ± 7%; P < 0.001). This study features the first hiPSC model of RBM20 familial DCM. By monitoring human cardiac disease according to stage-specific cardiogenesis, this study demonstrates RBM20 familial DCM is a developmental disorder initiated by molecular defects that pattern maladaptive cellular mechanisms of pathological cardiac remodeling. Indeed, hiPSC-CMs recapitulate RBM20 familial DCM phenotype in a dish and establish a tool to dissect disease-relevant defects in RBM20 splicing as a global regulator of heart function.

Human umbilical cord blood-derived mononuclear cells improve murine ventricular function upon intramyocardial delivery in right ventricular chronic pressure overload.

INTRODUCTION: Stem cell therapy has emerged as potential therapeutic strategy for damaged heart muscles. Umbilical cord blood (UCB) cells are the most prevalent stem cell source available, yet have not been fully tested in cardiac regeneration. Herein, studies were performed to evaluate the cardiovascular safety and beneficial effect of mononuclear cells (MNCs) isolated from human umbilical cord blood upon intramyocardial delivery in a murine model of right ventricle (RV) heart failure due to pressure overload. METHODS: UCB-derived MNCs were delivered into the myocardium of a diseased RV cardiac model. Pulmonary artery banding (PAB) was used to produce pressure overload in athymic nude mice that were then injected intramyocardially with UCB-MNCs (0.4×10^6 cells/heart). Cardiac functions were then monitored by telemetry, echocardiography, magnetic resonance imaging (MRI) and pathologic analysis of heart samples to determine the ability for cell-based repair. RESULTS: The cardio-toxicity studies provided evidence that UCB cell transplantation has a safe therapeutic window between 0.4 to 0.8 million cells/heart without altering QT or ST-segments or the morphology of electrocardiograph waves. The PAB cohort demonstrated significant changes in RV chamber dilation and functional defects consistent with severe pressure overload. Using cardiac MRI analysis, UCB-MNC transplantation in the setting of PAB demonstrated an improvement in RV structure and function in this surgical mouse model. The RV volume load in PAB-only mice was 24.09±3.9 compared to 11.05±2.09 in the cell group (mm3, P-value<0.005). The analysis of pathogenic gene expression (BNP, ANP, Acta1, Myh7) in the cell-transplanted group showed a significant reversal with respect to the diseased PAB mice with a robust increase in cardiac progenitor gene expression such as GATA4, Kdr, Mef2c and Nkx2.5. Histological analysis indicated significant fibrosis in the RV in response to PAB that was reduced following UCB-MNC's transplantation along with concomitant increased Ki-67 expression and CD31 positive vessels as a marker of angiogenesis within the myocardium. CONCLUSIONS: These findings indicate that human UCB-derived MNCs promote an adaptive regenerative response in the right ventricle upon intramyocardial transplantation in the setting of chronic pressure overload heart failure.

Safety and feasibility for pediatric cardiac regeneration using epicardial delivery of autologous umbilical cord blood-derived mononuclear cells established in a porcine model system.

Congenital heart diseases (CHDs) requiring surgical palliation mandate new treatment strategies to optimize long-term outcomes. Despite the mounting evidence of cardiac regeneration, there are no long-term safety studies of autologous cell-based transplantation in the pediatric setting. We aimed to establish a porcine pipeline to evaluate the feasibility and long-term safety of autologous umbilical cord blood mononuclear cells (UCB-MNCs) transplanted into the right ventricle (RV) of juvenile porcine hearts. Piglets were born by caesarean section to enable UCB collection. Upon meeting release criteria, 12 animals were randomized in a double-blinded fashion prior to surgical delivery of test article (n=6) or placebo (n=6). The UCB-MNC (3×10(6) cells per kilogram) or control (dimethyl sulfoxide, 10%) products were injected intramyocardially into the RV under direct visualization. The cohorts were monitored for 3 months after product delivery with assessments of cardiac performance, rhythm, and serial cardiac biochemical markers, followed by terminal necropsy. No mortalities were associated with intramyocardial delivery of UCB-MNCs or placebo. Two animals from the placebo group developed local skin infection after surgery that responded to antibiotic treatment. Electrophysiological assessments revealed no arrhythmias in either group throughout the 3-month study. Two animals in the cell-therapy group had transient, subclinical dysrhythmia in the perioperative period, likely because of an exaggerated response to anesthesia. Overall, this study demonstrated that autologous UCB-MNCs can be safely collected and surgically delivered in a pediatric setting. The safety profile establishes the foundation for cell-based therapy directed at the RV of juvenile hearts and aims to accelerate cell-based therapies toward clinical trials for CHD.

Inhibition of DNA topoisomerase II selectively reduces the threat of tumorigenicity following induced pluripotent stem cell-based myocardial therapy.

The advent of induced pluripotent stem cell (iPSC) technology creates new opportunities for transplant-based therapeutic strategies. The potential for clinical translation is currently hindered by the risk of dysregulated cell growth. Pluripotent stem cells reprogrammed by three-factor (Sox2, Klf, and Oct4) and four-factor (Sox2, Klf, Oct4, and c-Myc) strategies result in the capacity for teratogenic growth from residual pluripotent progeny upon in vivo transplantation. However, these pluripotent stem cells also have a stage-specific hypersensitivity to DNA-damaging agents that may allow separation of lineage-specific therapeutic subpopulation of cells. We aimed to demonstrate the selective effect of DNA topoisomerase II inhibitor, etoposide, in eliminating pluripotent cells in the early cardiac progenitor population thus decreasing the effect of teratoma formation. Immunodeficient murine hearts were infarcted and received implantation of a therapeutic dose of cardiac progenitors derived from partially differentiated iPSCs. Etoposide-treated cell implantation reduced mass formation in the intracardiac and extracardiac chest cavity compared with the same dose of iPSC-derived cardiac progenitors in the control untreated group. In vivo bioluminescence imaging confirmed the localization and engraftment of transplanted cells in the myocardium postinjection in both groups. Comparatively, the equivalent cell population without etoposide treatment demonstrated a greater incidence and size of teratoma formation. Hence, pretreatment with genotoxic etoposide significantly lowered the threat of teratogenicity by purging the contaminating pluripotent cells, establishing an adjunctive therapy to further harness the clinical value of iPSC-derived cardiac regeneration.

Maternal obesity affects gene expression and cellular development in fetal brains.

OBJECTIVES: Female rat neonates reared on a high carbohydrate (HC) milk formula developed chronic hyperinsulinemia and adult-onset obesity (HC phenotype). Furthermore, we have shown that fetal development in the HC intrauterine environment (maternal obesity complicated with hyperinsulinemia, hyperleptinemia, and increased levels of proinflammatory markers) resulted in increased levels of serum insulin and leptin in term HC fetuses and the spontaneous transfer of the HC phenotype to the adult offspring. The objectives of this study are to identify changes in global gene expression pattern and cellular development in term HC fetal brains in response to growth in the adverse intrauterine environment of the obese HC female rat. METHODS: GeneChip analysis was performed on total RNA obtained from fetal brains for global gene expression studies and immunohistochemical analysis was performed on fetal brain slices for investigation of cellular development in term HC fetal brains. RESULTS: Gene expression profiling identified changes in several clusters of genes that could contribute to the transfer of the maternal phenotype (chronic hyperinsulinemia and adult-onset obesity) to the HC offspring. Immunohistochemical analysis indicated diminished proliferation and neuronal maturation of stem-like cells lining the third ventricle, hypothalamic region, and the cerebral cortex in HC fetal brains. DISCUSSION: These results suggest that maternal obesity during pregnancy could alter the developmental program of specific fetal brain cell-networks. These defects could underlie pathologies such as metabolic syndrome and possibly some neurological disorders in the offspring at a later age.

Apoptotic susceptibility to DNA damage of pluripotent stem cells facilitates pharmacologic purging of teratoma risk.

Pluripotent stem cells have been the focus of bioengineering efforts designed to generate regenerative products, yet harnessing therapeutic capacity while minimizing risk of dysregulated growth remains a challenge. The risk of residual undifferentiated stem cells within a differentiated progenitor population requires a targeted approach to eliminate contaminating cells prior to delivery. In this study we aimed to validate a toxicity strategy that could selectively purge pluripotent stem cells in response to DNA damage and avoid risk of uncontrolled cell growth upon transplantation. Compared with somatic cell types, embryonic stem cells and induced pluripotent stem cells displayed hypersensitivity to apoptotic induction by genotoxic agents. Notably, hypersensitivity in pluripotent stem cells was stage-specific and consistently lost upon in vitro differentiation, with the mean half-maximal inhibitory concentration increasing nearly 2 orders of magnitude with tissue specification. Quantitative polymerase chain reaction and Western blotting demonstrated that the innate response was mediated through upregulation of the BH3-only protein Puma in both natural and induced pluripotent stem cells. Pretreatment with genotoxic etoposide purged hypersensitive pluripotent stem cells to yield a progenitor population refractory to teratoma formation upon transplantation. Collectively, this study exploits a hypersensitive apoptotic response to DNA damage within pluripotent stem cells to decrease risk of dysregulated growth and augment the safety profile of transplant-ready, bioengineered progenitor cells.

The induction of angiogenesis by cerium oxide nanoparticles through the modulation of oxygen in intracellular environments.

Angiogenesis is the formation of new blood vessels from existing blood vessels and is critical for many physiological and pathophysiological processes. In this study we have shown the unique property of cerium oxide nanoparticles (CNPs) to induce angiogenesis, observed using both in vitro and in vivo model systems. In particular, CNPs trigger angiogenesis by modulating the intracellular oxygen environment and stabilizing hypoxia inducing factor 1α endogenously. Furthermore, correlations between angiogenesis induction and CNPs physicochemical properties including: surface Ce(3+)/Ce(4+) ratio, surface charge, size, and shape were also explored. High surface area and increased Ce(3+)/Ce(4+) ratio make CNPs more catalytically active towards regulating intracellular oxygen, which in turn led to more robust induction of angiogenesis. Atomistic simulation was also used, in partnership with in vitro and in vivo experimentation, to reveal that the surface reactivity of CNPs and facile oxygen transport promotes pro-angiogenesis.

Vascular endothelial growth factor A (VEGF-A) induces endothelial and cancer cell migration through direct binding to integrin {alpha}9{beta}1: identification of a specific {alpha}9{beta}1 binding site.

Integrin α9ß1 mediates accelerated cell adhesion and migration through interactions with a number of diverse extracellular ligands. We have shown previously that it directly binds the vascular endothelial growth factors (VEGF) A, C, and D and contributes to VEGF-induced angiogenesis and lymphangiogenesis. Until now, the α9ß1 binding site in VEGF has not been identified. Here, we report that the three-amino acid sequence, EYP, encoded by exon 3 of VEGF-A is essential for binding of VEGF to integrin α9ß1 and induces adhesion and migration of endothelial and cancer cells. EYP is specific for α9ß1 binding and neither requires nor activates VEGFR-2, the cognate receptor for VEGF-A. Following binding to EYP, integrin α9ß1 transduces cell migration through direct activation of the integrin signaling intermediates Src and focal adhesion kinase. This interaction is biologically important because it mediates in vitro endothelial cell tube formation, wound healing, and cancer cell invasion. These novel findings identify EYP as a potential site for directed pharmacotherapy.

Modulation of ozone-sensitive genes in alpha-tocopherol transfer protein null mice.

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP-/-) have a systemic alpha-tocopherol (AT) deficiency, with their lung AT levels being < 10% of those in AT-replete ATTP(+/+) mice when fed a standard rodent chow diet. ATTP(+/+) and ATTP(-/-) mice (4 wk old male mice, n = 16 per group) were fed a standard diet (35 IU AT/kg diet) for 8-12 wk, exposed 6 h/day for 3 days to either to O(3) (0.5 ppm) or filtered air, then sacrificed. No significant differences in plasma or lung AT concentrations were observed in response to this level of O(3) exposure. Lung genomic responses of the lungs to O(3) were determined using Affymetrix 430A 2.0 arrays containing over 22,600 probe sets representing 14,000 well-characterized mouse genes. As compared with filtered air exposure, O(3) exposure resulted in 99 genes being differentially expressed in ATTP(-/-) mice, as compared to 52 differentially expressed genes in ATTP(+/+) mice. The data revealed an O(3)-induced upregulation of genes related to cell proliferation/DNA repair and inflammatory-immune responses in both ATTP(+/+) and ATTP(-/-) mice, with the expression of 22 genes being common to both, whereas 30 and 77 genes were unique to ATTP(+/+) and ATTP(-/-) mice, respectively. The expressions of O(3) sensitive genes-Timp1, Areg, Birc5 and Tnc-were seen to be further modulated by AT status. The present study reveals AT modulation of adaptive response of lung genome to O(3) exposure.

Vascular endothelial growth factors C and D induces proliferation of lymphangioleiomyomatosis cells through autocrine crosstalk with endothelium.

Lymphangioleiomyomatosis (LAM) is a potentially fatal lung disease characterized by nodules of proliferative smooth muscle-like cells. The exact nature of these LAM cells and their proliferative stimuli are poorly characterized. Herein we report the novel findings that the lymphangiogenic vascular endothelial growth factors (VEGF) C and D induce LAM cell proliferation through activation of their cognate receptor VEGF-R3 and activation of the signaling intermediates Akt/mTOR/S6. Furthermore, we identify expression of the proteoglycan NG2, a marker of immature smooth muscle cells, as a characteristic of LAM cells both in vitro and in human lung tissue. VEGF-C-induced LAM cell proliferation was in part a result of autocrine stimulation that resulted from cross talk with lymphatic endothelial cells. Ultimately, these findings identify the lymphangiogenic VEGF proteins as pathogenic growth factors in LAM disease and at the same time provide a novel pharmacotherapeutic target for a lung disease that to date has no known effective treatment.

Sarcolipin and ubiquitin carboxy-terminal hydrolase 1 mRNAs are over-expressed in skeletal muscles of alpha-tocopherol deficient mice.

The transcriptome of ataxic muscles from alpha-tocopherol transfer protein deficient (ATTP-KO), 23-month old, mice was compared with that of their normal littermates. Genes encoding sarcolipin (sln) and ubiquitin carboxyl-terminal hydrolase (uchl1) were over-expressed (> or =10-fold) in ataxic muscles. SLN is a 3.2 kDa membrane protein that binds to sarcoplasmic reticulum calcium ATPase, regulates Ca(+ +) transport and muscle relaxation-contraction cycles. UCHL1 is a 24.8 kDa member of proteosome proteins; it is over-expressed in myofibrillar myopathy and is associated with neurodegenerative diseases. Furthermore, six additional transcripts, three encoding thin-filament proteins and three encoding Ca(+ +) sensing proteins that participate in contraction-relaxation cycle, and eight transcripts that encode members of lysosomal proteins were also over-expressed in ataxic muscles. These observations suggest that chronic alpha-tocopherol (AT) deficiency activates critical genes of muscle contractility and protein degradation pathways, simultaneously. The magnitude of induction of sln and uchl1 was lower in asymptomatic, 8-month old, ATTP-KO mice and in 8-month old mice fed an AT-depleted diet. These studies suggest sln and uchl1 genes as novel targets of AT deficiency and may offer molecular correlates of well documented descriptions of neuromuscular dysfunctions in AT-deficient rodents. Since the neuromuscular deficits of ATTP-KO mice appear to be similar to those of patients with ATTP mutations, it is suggested that over-expression of sln and uchl1 may also contribute to AT-sensitive ataxia in humans.

Mice lacking alpha-tocopherol transfer protein gene have severe alpha-tocopherol deficiency in multiple regions of the central nervous system.

Ataxia with vitamin E deficiency is caused by mutations in alpha-tocopherol transfer protein (alpha-TTP) gene and it can be experimentally generated in mice by alpha-TTP gene inactivation (alpha-TTP-KO). This study compared alpha-tocopherol (alpha-T) concentrations of five brain regions and of four peripheral organs from 5 months old, male and female, wild-type (WT) and alpha-TTP-KO mice. All brain regions of female WT mice contained significantly higher alpha-T than those from WT males. alpha-T concentration in the cerebellum was significantly lower than that in other brain regions of WT mice. These sex and regional differences in brain alpha-T concentrations do not appear to be determined by alpha-TTP expression which was undetectable in all brain regions. All the brain regions of alpha-TTP-KO mice were severely depleted in alpha-T. The concentration of another endogenous antioxidant, total glutathione, was unaffected by gender but was decreased slightly but significantly in most brain regions of alpha-TTP-KO mice. The results show that both gender and the hepatic alpha-TTP, but not brain alpha-TTP gene expression are important in determining alpha-T concentrations within the brain. Interestingly, functional abnormality (ataxia) develops only very late in alpha-TTP-KO mice in spite of the severe alpha-tocopherol deficiency in the brain starting at an early age.

Lactate sensitive transcription factor network in L6 cells: activation of MCT1 and mitochondrial biogenesis.

We hypothesized that in addition to serving as a fuel source and gluconeogenic precursor, lactate anion (La-) is a signaling molecule. Therefore, we screened genome-wide responses of L6 cells to elevated (10 and 20 mM) sodium-La- added to buffered, high-glucose media. Lactate increased reactive oxygen species (ROS) production and up-regulated 673 genes, many known to be responsive to ROS and Ca2+. The induction of genes encoding for components of the mitochondrial lactate oxidation complex was confirmed by independent methods (PCR and EMSA). Specifically, lactate increased monocarboxylate transporter-1 (MCT1) mRNA and protein expression within 1 h and cytochrome c oxidase (COX) mRNA and protein expression in 6 h. Increases in COX coincided with increases in peroxisome proliferator activated-receptor gamma coactivator-1alpha (PGC1alpha) expression and the DNA binding activity of nuclear respiratory factor (NRF)-2. We conclude that the lactate signaling cascade involves ROS production and converges on transcription factors affecting mitochondrial biogenesis.

Genome wide responses of murine lungs to dietary alpha-tocopherol.

Alpha-tocopherol (alpha-T) may affect biological processes by modulating mRNA concentrations. This study screened the responses of approximately 15,000 lung mRNAs to dietary alpha-T in mice. The lung was chosen as the target organ because it is subjected to cyclical variations in oxidant and inflammatory stressors and alpha-T has been implicated in their modulations. The analysis identified approximately 400 mRNAs sensitive to alpha-T status of lungs determined by dietary alpha-T. The female lung transcriptome appears to be more sensitive to the alpha-T status than that of the male lungs. Here, we focus on the induction of 13 cytoskeleton genes by dietary alpha-T because they were similarly induced in the male and the female lungs. Their inductions were confirmed by quantitative-real-time-polymerase chain reaction (qRT-PCR). Immunohistochemical analyses of three of the encoded proteins suggest that they are expressed in lung vasculature and alveolar regions. The data suggest that the lung alpha-T status may modulate cytoarchitecture of lungs.