Koji Maemura

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).

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.

Endotoxin-Induced Mortality Is Related to Increased Oxidative Stress and End-Organ Dysfunction, Not Refractory Hypotension, in Heme Oxygenase-1–Deficient Mice

BackgroundHeme oxygenase (HO)-1 is an enzyme that degrades heme to generate CO (a vasodilatory gas), iron, and the potent antioxidant bilirubin. A disease process characterized by decreases in vascular tone and increases in oxidative stress is endotoxic shock. Moreover, HO-1 is markedly induced in multiple organs after the administration of endotoxin (lipopolysaccharide [LPS]) to mice. Methods and ResultsTo determine the role of HO-1 in endotoxemia, we administered LPS to mice that were wild-type (+/+), heterozygous (±), or homozygous null (−/−) for targeted disruption of HO-1. LPS produced a similar induction of HO-1 mRNA and protein in HO-1+/+ and HO-1+/− mice, whereas HO-1−/− mice showed no HO-1 expression. Four hours after LPS, systolic blood pressure (SBP) decreased in all the groups. However, SBP was significantly higher in HO-1−/− mice (121±5 mm Hg) after 24 hours, compared with HO-1+/+ (96±7 mm Hg) and HO-1+/− (89±13 mm Hg) mice. A sustained increase in endothelin-1 contributed to this SBP response. Even though SBP was higher, mortality was increased in HO-1−/− mice, and they exhibited hepatic and renal dysfunction that was not present in HO-1+/+ and HO-1+/− mice. The end-organ damage and death in HO-1−/− mice was related to increased oxidative stress. ConclusionsThese data suggest that the increased mortality during endotoxemia in HO-1−/− mice is related to increased oxidative stress and end-organ (renal and hepatic) damage, not to refractory hypotension.

Thy-1, a Novel Marker for Angiogenesis Upregulated by Inflammatory Cytokines

We identified the cell surface glycoprotein Thy-1 on the endothelium of newly formed blood vessels in four models of angiogenesis in adult rats. Anti-Thy-1 staining showed that Thy-1 was upregulated in adventitial blood vessels after balloon injury to the carotid artery. Preabsorption with a rat Thy-1-Ig fusion construct eliminated all immunoreactivity and thus confirmed the specificity of the Thy-1 staining. Thy-1 was also expressed in the endothelium of small blood vessels formed after tumor implantation in the cornea, in periureteral vessels formed after ligation of the renal artery, and in small blood vessels of the uterus formed during pregnancy. In contrast with its expression during adult angiogenesis, Thy-1 was not expressed in the endothelium of blood vessels during embryonic angiogenesis. In vitro, the inflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha upregulated Thy-1 mRNA by 8- and 14-fold, respectively. Vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor-beta, and platelet-derived growth factor-BB had no effect on Thy-1 mRNA. Thus, Thy-1 appears to be a marker of adult but not embryonic angiogenesis. The upregulation of Thy-1 by cytokines but not growth factors indicates the importance of inflammation in the pathogenesis of adult angiogenesis.

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-1α (HIF-1α) and, like HIF-1α, 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.

Characterization of the Mouse Aortic Carboxypeptidase-Like Protein Promoter Reveals Activity in Differentiated and Dedifferentiated Vascular Smooth Muscle Cells

The dedifferentiation and proliferation of vascular smooth muscle cells (VSMCs) contribute to the formation of vascular lesions. In this study, the regulation of aortic carboxypeptidase-like protein (ACLP) expression in VSMCs was investigated. After mouse carotid injury, the expression of ACLP increases in the dedifferentiated VSMCs of the neointima in a pattern that differs from that of smooth muscle &agr;-actin. To better understand the regulation of ACLP in VSMCs, we characterized the 21-exon mouse ACLP gene and 5′-flanking region and examined its promoter activity. In transient transfection assays, 2.5 kb of the ACLP 5′-flanking sequence directed high levels of luciferase reporter activity in primary cultured rat aortic smooth muscle cells, and this activity was not dependent on serum response factor. We identified a positive element between base pairs −156 and −122 by analysis of 5′ deletion and mutant constructs. By use of electrophoretic mobility shift assays with rat aortic smooth muscle cell nuclear extracts, Sp1 and Sp3 transcription factors bound to this region, and transfection assays in D.Mel.2 cells revealed that both Sp1 and Sp3 transactivated the ACLP promoter. Transgenic mice harboring the −2.5-kb ACLP promoter upstream from a nuclear-targeted LacZ gene were generated, and expression was detected in the VSMCs of large blood vessels, arterioles, and veins. Interestingly, ACLP promoter–LacZ reporter activity increased within the neointimal VSMCs of injured carotid vessels, consistent with the expression of the endogenous ACLP protein. The ACLP promoter may provide a novel tool to target gene expression to dedifferentiated VSMCs.

Molecular Cloning, Characterization, and Promoter Analysis of the Mouse Crp2/SmLim Gene PREFERENTIAL EXPRESSION OF ITS PROMOTER IN THE VASCULAR SMOOTH MUSCLE CELLS OF TRANSGENIC MICE

Several members of the LIM protein family have important roles in development and differentiation. We recently isolated a rat cDNA encoding a new member of this family, CRP2/SmLIM, that contains two LIM domains and is expressed preferentially in vascular smooth muscle cells (VSMC). To study the molecular mechanisms that regulate VSMC-specific transcription of theCrp2/SmLim gene, we cloned the cDNA and gene of mouseCrp2/SmLim. Mouse Crp2/SmLim is a single copy gene of six exons and five introns spanning approximately 20 kilobases of genomic DNA. By 5′-rapid amplification of cDNA ends and S1 nuclease protection assay, we determined that the transcription start site is an A residue 80 base pairs 5′ of the translation initiation codon. A TATA-like sequence is located 27 base pairs 5′ of the transcription start site, and there are potentialcis-acting elements (GATA, Sp1, AP-2, E box, CCAC box, and GArC motif) in the 5′-flanking sequence. In transient transfection assays in rat aortic smooth muscle cells in primary culture, 5 kilobases of the Crp2/SmLim 5′-flanking sequence generated a high level of luciferase reporter gene activity. By deletion analysis and gel mobility shift assay, we found that the region between bases −74 and −39 of this 5 kilobase DNA fragment binds Sp1 and confers basal promoter activity in the Crp2/SmLim gene. In vitro, the 5-kilobase fragment was active in multiple cell types.In vivo, however, the 5-kilobase fragment directed high level expression of the lacZ reporter gene preferentially in the VSMC of transgenic mice, indicating the presence of VSMC-specific element(s) in this fragment.

Regulation of Smooth Muscle Cell Differentiation by AT-Rich Interaction Domain Transcription Factors Mrf2α and Mrf2β

Despite the importance of vascular smooth muscle cells in the regulation of blood vessel function, the molecular mechanisms governing their development and differentiation remain poorly understood. Using an in vitro system whereby a pluripotent neural crest cell line (MONC-1) can be induced to differentiate into smooth muscle cells, we isolated a cDNA fragment that was robustly induced during this differentiation process. Sequence analysis revealed high homology to a partial cDNA termed modulator recognition factor 2 (Mrf2). Because the full-length cDNA has not been reported, we cloned the full-length Mrf2 cDNA by cDNA library screening and 5 rapid amplification of cDNA ends and identified two isoforms of Mrf2 (alpha [3.0 kb] and beta [3.7 kb]) that differ in the N-terminus but share the DNA-binding domain. Protein homology analysis suggests that Mrf2 is a member of the AT-rich interaction domain family of transcription factors, which are known to be critically involved in the regulation of development and cellular differentiation. Mrf2alpha and Mrf2beta are highly induced during in vitro differentiation of MONC-1 cells into smooth muscle cells, and Mrf2alpha is expressed in adult mouse cardiac and vascular tissues. To define the function of Mrf2, we overexpressed both isoforms in 3T3 fibroblast cells and observed an induction of smooth muscle marker genes, including smooth muscle alpha-actin and smooth muscle 22alpha. Furthermore, Mrf2alpha and Mrf2beta retarded cellular proliferation. These data implicate Mrf2 as a novel regulator of smooth muscle cell differentiation and proliferation.

Striated Muscle Preferentially Expressed Genes α and β 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)α and the 11-kb SPEGβ 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 SPEGβ cDNA. SPEGβ 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 SPEGα and SPEGβ is developmentally regulated in the striated muscle during C2C12 myoblast to myotube differentiationin vitro and cardiomyocyte maturation in vivo. This developmental regulation suggests that both SPEGα and SPEGβ can serve as sensitive markers for striated muscle differentiation and that they may be important for adult striated muscle function.

Molecular Mechanisms of Morning Onset of Myocardial Infarction

Abstract: 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‐1 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.