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Biomedical subjects

M D Layne

Publications and source records attributed to M D Layne.

At least 19 recordsLinked to original sources
PubMed2004

Pre-emptive gene therapy using recombinant adeno-associated virus delivery of extracellular superoxide dismutase protects heart against ischemic reperfusion injury, improves ventricular function and prolongs survival.

In high-risk patients, the ideal cardiovascular gene therapy requires a strategy that provides long-term protection of myocardium against episodes of ischemic/reperfusion injury. We report the development of an efficient, long-lasting pre-emptive gene therapy strategy in a rat model of ischemic-reperfusion (I/R) injury of heart. At 6 weeks prior to myocardial injury, the human extracellular superoxide dismutase (Ec-SOD) gene was delivered by direct intramyocardial injections, using a recombinant adeno-associated virus vector. Significant myocardial protection was documented by the decrease in infarct size at 24 h post I/R, improved left ventricular function at 7 weeks postinjury, and enhanced long-term survival in the SOD treated group. This concept of preinjury delivery and 'pre-emptive' gene therapy via the expression of a secreted protein that renders paracrine therapeutic action can be an effective strategy for organ protection against future injury.

Animals↗
PubMed2001

Upstream stimulatory factors regulate aortic preferentially expressed gene-1 expression in vascular smooth muscle cells.

The phenotypic modulation of vascular smooth muscle cells (VSMC) plays a central role in the pathogenesis of arteriosclerosis. Aortic preferentially expressed gene-1 (APEG-1), a VSMC-specific gene, is expressed highly in differentiated but not in dedifferentiated VSMC. Previously, we identified an E-box element in the mouse APEG-1 proximal promoter, which is essential for VSMC reporter activity. In this study, we investigated the role of upstream stimulatory factors (USF) in the regulation of APEG-1 transcription via this E-box element. By electrophoretic mobility shift assays, recombinant USF1 and USF2 homo- and heterodimers bound specifically to the APEG-1 E-box. Nuclear extracts prepared from primary cultures of rat aortic smooth muscle cells exhibited specific USF1 and USF2 binding to the APEG-1 E-box. To investigate the binding properties of USF during VSMC differentiation, nuclear extracts were prepared from the neural crest cell line, MONC-1, which differentiates into VSMC in culture. Maximal USF1 and USF2 protein levels and binding to the APEG-1 E-box occurred 3 h after the differentiation of MONC-1 cells was initiated. Co-transfection experiments demonstrated that dominant negative USF repressed APEG-1 promoter activity, and USF1, but not USF2, transactivated the APEG-1 promoter. Our studies demonstrate that USF factors contribute to the regulation of APEG-1 expression and may influence the differentiation of VSMC.

Animals↗
PubMed2001

Role of macrophage-expressed adipocyte fatty acid binding protein in the development of accelerated atherosclerosis in hypercholesterolemic mice.

Atherosclerosis is an inflammatory disease process associated with elevated levels of plasma cholesterol, especially low-density lipoproteins. The latter become trapped within the arterial wall and are oxidized and taken up by macrophages to form foam cells. This process is an initiating event for atherosclerosis. Fatty acid binding proteins (FABP) are involved in fatty acid metabolism and cellular lipid transport, and adipocyte FABP (aP2) is also expressed in macrophages. We recently generated mice lacking both apolipoprotein (Apo)E and aP2 (ApoE-/-aP2-/-) and found that these mice, compared with ApoE-/- mice, developed markedly smaller atherosclerotic lesions that contained fewer macrophages. Here we investigated the mechanism(s) responsible for this prevention of atherosclerotic lesion formation. Bone marrow transplantations were performed in ApoE-/- mice, receiving cells from either ApoE-/- or ApoE-/-aP2-/- mice. The lack of aP2 in donor marrow cells led to the development of smaller (5.5-fold) atherosclerotic lesions in the recipient mice. No differences were found in plasma cholesterol, glucose, or insulin levels between recipients of bone marrow cells from ApoE-/- or ApoE-/-aP2-/- mice. However, the expression of chemoattractant and inflammatory cytokines was decreased in macrophages from ApoE-/-aP2-/- mice compared with ApoE-/- mice, which may contribute to the decrease in atherosclerotic lesion formation. Taken together, we demonstrate the importance of macrophage aP2 in the development of atherosclerotic lesions.

Animals↗
PubMed2001

Cardiac-specific expression of heme oxygenase-1 protects against ischemia and reperfusion injury in transgenic mice.

Heme oxygenase (HO)-1 degrades the pro-oxidant heme and generates carbon monoxide and antioxidant bilirubin. We have previously shown that in response to hypoxia, HO-1-null mice develop infarcts in the right ventricle of their hearts and that their cardiomyocytes are damaged by oxidative stress. To test whether HO-1 protects against oxidative injury in the heart, we generated cardiac-specific transgenic mice overexpressing different levels of HO-1. By use of a Langendorff preparation, hearts from transgenic mice showed improved recovery of contractile performance during reperfusion after ischemia in an HO-1 dose-dependent manner. In vivo, myocardial ischemia and reperfusion experiments showed that infarct size was only 14.7% of the area at risk in transgenic mice compared with 56.5% in wild-type mice. Hearts from these transgenic animals had reduced inflammatory cell infiltration and oxidative damage. Our data demonstrate that overexpression of HO-1 in the cardiomyocyte protects against ischemia and reperfusion injury, thus improving the recovery of cardiac function.

Animals↗
PubMed2001

Exacerbation of chronic renovascular hypertension and acute renal failure in heme oxygenase-1-deficient mice.

Heme oxygenase (HO) is a cytoprotective enzyme that degrades heme (a potent oxidant) to generate carbon monoxide (a vasodilatory gas that has anti-inflammatory properties), bilirubin (an antioxidant derived from biliverdin), and iron (sequestered by ferritin). Because of properties of HO and its products, we hypothesized that HO would be important for the regulation of blood pressure and ischemic injury. We studied chronic renovascular hypertension in mice deficient in the inducible isoform of HO (HO-1) using a one kidney-one clip (1K1C) model of disease. Systolic blood pressure was not different between wild-type (HO-1(+/+)), heterozygous (HO-1(+/-)), and homozygous null (HO-1(-/-)) mice at baseline. After 1K1C surgery, HO-1(+/+) mice developed hypertension (140+/-2 mm Hg) and cardiac hypertrophy (cardiac weight index of 5.0+/-0.2 mg/g) compared with sham-operated HO-1(+/+) mice (108+/-5 mm Hg and 4.1+/-0.1 mg/g, respectively). However, 1K1C produced more severe hypertension (164+/-2 mm Hg) and cardiac hypertrophy (6.9+/-0.6 mg/g) in HO-1(-/-) mice. HO-1(-/-) mice also experienced a high rate of death (56%) within 72 hours after 1K1C surgery compared with HO-1(+/+) (25%) and HO-1(+/-) (28%) mice. Assessment of renal function showed a significantly higher plasma creatinine in HO-1(-/-) mice compared with HO-1(+/-) mice. Histological analysis of kidneys from 1K1C HO-1(-/-) mice revealed extensive ischemic injury at the corticomedullary junction, whereas kidneys from sham HO-1(-/-) and 1K1C HO-1(+/-) mice appeared normal. Taken together, these data suggest that chronic deficiency of HO-1 does not alter basal blood pressure; however, in the 1K1C model an absence of HO-1 leads to more severe renovascular hypertension and cardiac hypertrophy. Moreover, renal artery clipping leads to an acute increase in ischemic damage and death in the absence of HO-1.

Acute Kidney Injury↗
PubMed2001

Akt participation in the Wnt signaling pathway through Dishevelled.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) and the resulting stabilization of free beta-catenin are critical steps in the activation of Wnt target genes. While Akt regulates GSK3alpha/beta in the phosphatidylinositide 3-OH kinase signaling pathway, its role in Wnt signaling is unknown. Here we report that expression of Wnt or Dishevelled (Dvl) increased Akt activity. Activated Akt bound to the Axin-GSK3beta complex in the presence of Dvl, phosphorylated GSK3beta and increased free beta-catenin levels. Furthermore, in Wnt-overexpressing PC12 cells, dominant-negative Akt decreased free beta-catenin and derepressed nerve growth factor-induced differentiation. Therefore, Akt acts in association with Dvl as an important regulator of the Wnt signaling pathway.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Regulation of myogenic terminal differentiation by the hairy-related transcription factor CHF2.

We have recently cloned a novel basic helix-loop-helix factor, CHF2, that functions as a transcriptional repressor. To address its role in the regulation of myogenic terminal differentiation, we analyzed its expression pattern during C2C12 mouse myotube formation. In undifferentiated myoblasts, CHF2 is expressed at high levels. After induction of myotube formation in low serum, CHF2 expression is barely detectable at 3 days after induction. Myogenin expression, in contrast, peaks at 3 days. In transiently transfected 10T1/2 embryonic fibroblasts, CHF2 inhibited MyoD-dependent activation of the myogenin promoter in a dose-dependent fashion. Electrophoretic mobility shift analysis indicated that CHF2 inhibits the binding of the MyoD.E47 heterodimer to the E-box binding site. CHF2 also inhibited myogenic conversion of 10T1/2 cells by MyoD, as measured by skeletal myosin heavy chain protein expression. Coimmunoprecipitation analysis indicated that CHF2 forms a protein complex with MyoD. Mutational analysis of CHF2 indicated that the repression activity for both transcription and myogenic conversion mapped to a hydrophobic carboxyl-terminal region and did not require either the basic helix-loop-helix or YRPW motifs. Our data indicate that CHF2 functions as a transcriptional repressor of myogenesis by formation of an inactive heterodimeric complex with MyoD and likely plays an important role in muscle development.

Animals↗
PubMed2001

Molecular mechanisms of morning onset of myocardial infarction.

We recently isolated a novel bHLH/PAS protein, CLIF (cycle like factor), by yeast two-hybrid screening of human umbilical endothelial cell cDNA library. CLIF is preferentially expressed in endothelial and neuronal cells. Because CLIF is expressed in vascular endothelial cells and forms a heterodimer with CLOCK, the key transcription factor controlling the circadian rhythm, we hypothesized that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. Northern blot analysis of mouse organs showed circadian oscillations of PAI-I mRNA levels. In addition, the clock-related genes also showed circadian oscillation in peripheral tissues. In endothelial cells, the heterodimer of CLIF and CLOCK upregulated the PAI-1 gene expression through E-box sites. Furthermore, Period and Cryptochrome, which are negative regulators in the feedback loop of the biological clock, inhibited PAI-1 promoter activation by the CLOCK:CLIF heterodimer. These results suggest that the peripheral tissues have their own biological clock and CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. This study suggests a novel molecular mechanism of the morning onset of myocardial infarction. Here we review our recent work and literature.

ARNTL Transcription Factors↗
PubMed2001

Impaired abdominal wall development and deficient wound healing in mice lacking aortic carboxypeptidase-like protein.

Aortic carboxypeptidase-like protein (ACLP) is a member of a diverse group of proteins that contain a domain with similarity to that of the Dictyostelium discoideum protein discoidin I. The discoidin domain has been identified in mammalian milk fat globule membrane proteins, blood coagulation factors, and receptor tyrosine kinases, where it may facilitate cell aggregation, adhesion, or cell-cell recognition. Here we show that ACLP is a secreted protein that associates with the extracellular matrix (ECM). During mouse embryogenesis, ACLP is abundantly expressed in the ECM of collagen-rich tissues, including the vasculature, dermis, and the developing skeleton. We deleted the ACLP gene in mice by homologous recombination. The majority of ACLP(-/-) mice die perinatally due to gastroschisis, a severe disruption of the anterior abdominal wall and herniation of the abdominal organs. ACLP(-/-) mice that survived to adulthood developed nonhealing skin wounds. Following injury by a dermal punch biopsy, ACLP(-/-) mice exhibited deficient wound healing compared with controls. In addition, dermal fibroblasts isolated from ACLP(-/-) 18.5-day-postconception embryos exhibited a reduced proliferative capacity compared with wild-type cells. These results indicate that ACLP is an ECM protein that is essential for embryonic development and dermal wound healing processes.

Abdominal Muscles↗
PubMed2000

CLIF, a novel cycle-like factor, regulates the circadian oscillation of plasminogen activator inhibitor-1 gene expression.

The onset of myocardial infarction occurs frequently in the early morning, and it may partly result from circadian variation of fibrinolytic activity. Plasminogen activator inhibitor-1 activity shows a circadian oscillation and may account for the morning onset of myocardial infarction. However, the molecular mechanisms regulating this circadian oscillation remain unknown. Recent evidence indicates that basic helix-loop-helix (bHLH)/PAS domain transcription factors play a crucial role in controlling the biological clock that controls circadian rhythm. We isolated a novel bHLH/PAS protein, cycle-like factor (CLIF) from human umbilical vein endothelial cells. CLIF shares high homology with Drosophila CYCLE, one of the essential transcriptional regulators of circadian rhythm. CLIF is expressed in endothelial cells and neurons in the brain, including the suprachiasmatic nucleus, the center of the circadian clock. In endothelial cells, CLIF forms a heterodimer with CLOCK and up-regulates the PAI-1 gene through E-box sites. Furthermore, Period2 and Cryptochrome1, whose expression show a circadian oscillation in peripheral tissues, inhibit the PAI-1 promoter activation by the CLOCK:CLIF heterodimer. These results suggest that CLIF regulates the circadian oscillation of PAI-1 gene expression in endothelial cells. In addition, the results potentially provide a molecular basis for the morning onset of myocardial infarction.

ARNTL Transcription Factors↗
PubMed2000

Striated muscle preferentially expressed genes alpha and beta are two serine/threonine protein kinases derived from the same gene as the aortic preferentially expressed gene-1.

Aortic preferentially expressed gene (APEG)-1 is a 1.4-kilobase pair (kb) mRNA expressed in vascular smooth muscle cells and is down-regulated by vascular injury. An APEG-1 5'-end cDNA probe identified three additional isoforms. The 9-kb striated preferentially expressed gene (SPEG)alpha and the 11-kb SPEGbeta were found in skeletal muscle and heart. The 4-kb brain preferentially expressed gene was detected in the brain and aorta. We report here cloning of the 11-kb SPEGbeta cDNA. SPEGbeta encodes a 355-kDa protein that contains two serine/threonine kinase domains and is homologous to proteins of the myosin light chain kinase family. At least one kinase domain is active and capable of autophosphorylation. In the genome, all four isoforms share the middle three of the five exons of APEG-1, and they differ from each other by using different 5'- and 3'-ends and alternative splicing. We show that the expression of SPEGalpha and SPEGbeta is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiation in vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGalpha and SPEGbeta can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

Amino Acid Sequence↗
PubMed2000

Thioredoxin facilitates the induction of heme oxygenase-1 in response to inflammatory mediators.

Heme oxygenase (HO)-1 is a stress response protein that is regulated by oxidative stress. HO-1 catalyzes the generation of biliverdin, carbon monoxide, and iron from heme. Lipopolysaccharide (LPS) and interleukin (IL)-1beta induce HO-1 through the binding of nuclear proteins to AP-1 motifs in enhancer regions upstream from the transcription start site. The DNA binding activity of AP-1 proteins depends on the reduction of cysteines in their DNA-binding domains. We found that agents that disrupt free sulfhydryl groups abolish AP-1 binding activity in nuclear proteins obtained from rat aortic smooth muscle cells and macrophages stimulated with IL-1beta or LPS. Thioredoxin (TRX) may regulate the redox status of nuclear transcription factors in response to oxidative stimuli, thus we determined the role of TRX in the physiologic regulation of HO-1. TRX underwent nuclear translocation in cells stimulated with IL-1beta and LPS. We transfected macrophages with a heterologous promoter construct containing two AP-1 sites from an upstream enhancer region in the HO-1 promoter. Recombinant TRX induced promoter activity to a level analogous to that induced by LPS, and this TRX response was abolished by mutation of the AP-1 sites. An inhibitor of TRX reductase, used to prevent TRX translocation in the reduced state, decreased HO-1 induction by IL-1beta and LPS. These data provide the first evidence that TRX contributes to the induction of HO-1 by inflammatory mediators.

Animals↗
PubMed2000

Cardiovascular basic helix loop helix factor 1, a novel transcriptional repressor expressed preferentially in the developing and adult cardiovascular system.

We have cloned a cardiovascular-restricted basic helix-loop-helix factor that interacts with arylhydrocarbon receptor nuclear translocator (ARNT) in a yeast two-hybrid screen. Cardiovascular helix-loop-helix factor 1 (CHF1) is distantly related to the hairy family of transcriptional repressors. We analyzed its expression pattern during mouse embryo development. At day 8.5, the expression of CHF1 is first detected in the primitive ventricle of the primordial heart tube and persists throughout gestation. In rat hearts, this expression is down-regulated after birth, concurrent with terminal differentiation of cardiomyocytes. In the developing vasculature, CHF1 first appears in the dorsal aorta at day 9.0, which precedes the reported expression of smooth muscle cell markers, and persists into adulthood. In an in vitro system of smooth muscle cell differentiation, CHF1 mRNA was barely detectable in undifferentiated cells but was induced highly in differentiated smooth muscle cells. To determine whether CHF1 might affect the function of ARNT, we performed transfection studies. Co-transfection of CHF1 inhibited ARNT/EPAS1-dependent transcription by 85%, and this inhibition is dose-dependent. In electrophoretic mobility studies, CHF1 inhibited the binding of the ARNT/EPAS1 heterodimer to its target site. Our data suggest that CHF1 functions as a transcriptional repressor and may play an important role in cardiovascular development.

Animals↗
PubMed1999

Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain.

Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that is preferentially expressed in vascular endothelial cells. EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and, like HIF-1alpha, has been shown to bind to the HIF-1-binding site and to activate its downstream genes such as vascular endothelial growth factor (VEGF) and erythropoietin. In this report, we show that EPAS1 increased VEGF gene expression through the HIF-1-binding site. This transactivation was enhanced further by cotransfection of an aryl hydrocarbon receptor nuclear translocator expression plasmid. Deletion analysis of EPAS1 revealed a potent activation domain (amino acids 486-639) essential for EPAS1 to transactivate the VEGF promoter. We confirmed the ability of this domain to activate transcription using a Gal4 fusion protein system. Because a truncated EPAS1 protein lacking the transactivation domain at amino acids 486-639 eliminated induction of the VEGF promoter by wild-type EPAS1, the truncated protein functions as a dominant-negative mutant. Most important, infection of the cells with an adenoviral construct expressing this mutant inhibited the induction of VEGF mRNA under conditions that mimic hypoxia. Our results suggest that EPAS1 is an important regulator of VEGF gene expression. Since VEGF plays a crucial role in angiogenesis, the ability of dominant-negative EPAS1 to inhibit VEGF promoter activity raises the possibility of a novel approach to inhibiting pathological angiogenesis.

Animals↗
PubMed1999

Tumor necrosis factor-alpha and basic fibroblast growth factor differentially inhibit the insulin-like growth factor-I induced expression of myogenin in C2C12 myoblasts.

Tumor necrosis factor-alpha (TNF-alpha) plays a role in several disease states such as sepsis, cachexia, and non-insulin-dependent diabetes. TNF-alpha interferes with insulin signaling and inhibits differentiation-specific gene expression in adipose tissue and skeletal muscle. We have examined the mechanisms by which TNF-alpha, in comparison to basic fibroblast growth factor (bFGF), inhibits the insulin-like growth factor-I (IGF-I)-induced differentiation of C2C12 myoblasts. Adhesion of quiescent, suspended myoblasts to collagen in high concentrations of IGF-I (10 nM) induced these cells to proliferate during the initial 24 h postplating and in so doing transiently inhibited the expression of myogenin, an essential transcription factor controlling myoblast differentiation. Low doses of IGF-I (1 nM) were minimally mitogenic and enhanced muscle-specific gene expression. Quiescent myoblasts treated with bFGF in combination with IGF-I did not express myogenin, but expressed proliferating cell nuclear antigen and underwent DNA synthesis. In contrast, TNF-alpha in the presence or absence of 1 nM IGF-I, did not stimulate DNA synthesis in myoblasts. However, TNF-alpha inhibited myogenin mRNA and protein expression. Expression of the cyclin-dependent kinase inhibitor p21 correlated with myogenin expression and myoblast differentiation, but not with growth arrest. These results indicate that both TNF-alpha and bFGF inhibit myogenin expression but differentially influence myoblast proliferation.

Animals↗
PubMed1999

Genomic cloning and promoter analysis of aortic preferentially expressed gene-1. Identification of a vascular smooth muscle-specific promoter mediated by an E box motif.

Aortic preferentially expressed gene-1 (APEG-1) was originally identified as a 1.4-kilobase (kb) transcript preferentially expressed in differentiated vascular smooth muscle cells (VSMC). Its expression is markedly down-regulated in de-differentiated VSMC, suggesting a role for APEG-1 in VSMC differentiation. We have now determined that APEG-1 is a single-copy gene in the human, rat, and mouse genomes and have mapped human APEG-1 to chromosome 2q34. To study the molecular mechanisms regulating its expression, we characterized the genomic organization and promoter of mouse APEG-1. APEG-1 spans 4.5 kb in the mouse genome and is composed of five exons. Using reporter gene transfection analysis, we found that a 2. 7-kb APEG-1 5'-flanking sequence directed a high level of promoter activity only in VSMC. Its activity was minimal in five other cell types. A repressor region located within an upstream 685-base pair sequence suppressed the activity of this 2.7-kb promoter. Further deletion and mutation analyses identified an E box motif as a positive regulatory element, which was bound by nuclear protein prepared from VSMC. In conjunction with its flanking sequence, this E box motif confers VSMC-specific enhancer activity to a heterologous SV40 promoter. To our knowledge, this is the first demonstration of an E box motif that mediates gene expression restricted to VSMC.

Amino Acid Sequence↗
PubMed1999

Hypoxia induces severe right ventricular dilatation and infarction in heme oxygenase-1 null mice.

Heme oxygenase (HO) catalyzes the oxidation of heme to generate carbon monoxide (CO) and bilirubin. CO increases cellular levels of cGMP, which regulates vascular tone and smooth muscle development. Bilirubin is a potent antioxidant. Hypoxia increases expression of the inducible HO isoform (HO-1) but not the constitutive isoform (HO-2). To determine whether HO-1 affects cellular adaptation to chronic hypoxia in vivo, we generated HO-1 null (HO-1(-/-)) mice and subjected them to hypoxia (10% oxygen) for five to seven weeks. Hypoxia caused similar increases in right ventricular systolic pressure in wild-type and HO-1(-/-) mice. Although ventricular weight increased in wild-type mice, the increase was greater in HO-1(-/-) mice. Similarly, the right ventricles were more dilated in HO-1(-/-) mice. After seven weeks of hypoxia, only HO-1(-/-) mice developed right ventricular infarcts with organized mural thrombi. No left ventricular infarcts were observed. Lipid peroxidation and oxidative damage occurred in right ventricular cardiomyocytes in HO-1(-/-), but not wild-type, mice. We also detected apoptotic cardiomyocytes surrounding areas of infarcted myocardium by terminal deoxynucleotide transferase-mediated dUTP nick end-labeling (TUNEL) assays. Our data suggest that in the absence of HO-1, cardiomyocytes have a maladaptive response to hypoxia and subsequent pulmonary hypertension. J.Clin. Invest. 103:R23-R29 (1999).

Animals↗