Paradoxical rescue from ischemic lung injury by inhaled carbon monoxide driven by derepression of fibrinolysis.

Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

Heme oxygenase-1 protects against vascular constriction and proliferation.

Heme oxygenase (HO-1, encoded by Hmox1) is an inducible protein activated in systemic inflammatory conditions by oxidant stress. Vascular injury is characterized by a local reparative process with inflammatory components, indicating a potential protective role for HO-1 in arterial wound repair. Here we report that HO-1 directly reduces vasoconstriction and inhibits cell proliferation during vascular injury. Expression of HO-1 in arteries stimulated vascular relaxation, mediated by guanylate cyclase and cGMP, independent of nitric oxide. The unexpected effects of HO-1 on vascular smooth muscle cell growth were mediated by cell-cycle arrest involving p21Cip1. HO-1 reduced the proliferative response to vascular injury in vivo; expression of HO-1 in pig arteries inhibited lesion formation and Hmox1-/- mice produced hyperplastic arteries compared with controls. Induction of the HO-1 pathway moderates the severity of vascular injury by at least two adaptive mechanisms independent of nitric oxide, and is a potential therapeutic target for diseases of the vasculature.

Molecular Pathogenesis and the Possible Role of Mitochondrial Heteroplasmy in Thoracic Aortic Aneurysm.

Thoracic aortic aneurysm (TAA) is a life-threatening condition associated with high mortality, in which the aortic wall is deformed due to congenital or age-associated pathological changes. The mechanisms of TAA development remain to be studied in detail, and are the subject of active research. In this review, we describe the morphological changes of the aortic wall in TAA. We outline the genetic disorders associated with aortic enlargement and discuss the potential role of mitochondrial pathology, in particular mitochondrial DNA heteroplasmy, in the disease pathogenesis.

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).

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.

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.

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.

Endotoxin-induced mortality is related to increased oxidative stress and end-organ dysfunction, not refractory hypotension, in heme oxygenase-1-deficient mice.

BACKGROUND: Heme 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 RESULTS: To 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. CONCLUSIONS: These 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.

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.

After vascular injury, heme oxygenase-1/carbon monoxide enhances re-endothelialization via promoting mobilization of circulating endothelial progenitor cells.

BACKGROUND: Heme oxygenase-1 (HO-1), a heme degradation enzyme with multiple vasoprotective functions, is systemically induced in pathophysiological states associated with oxidative stress. OBJECTIVES: To evaluate the impact of systemic HO-1 expression on circulating endothelial progenitor cells (EPCs) and re-endothelialization after vascular injury in an animal model. METHODS: Mice received an intravenous (i.v.) injection of the adenovirus-bearing HO-1 gene (Adv-HO-1). The serum levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were determined by ELISA and gene expression examined by quantitative real-time PCR. Circulating EPCs were characterized by flow cytometry and in vitro culture. EPC recruitment and re-endothelialization in injured arteries were assessed in mice receiving GFP+-bone marrow transplantation and guide wire-induced carotid injury. The effect of carbon monoxide (CO), a byproduct from heme degradation by HO-1, was assessed by exposing mice to 250 p.p.m. CO for 2 h day(-1). RESULTS: Systemic HO-1 induction led to elevated serum levels of VEGF and SDF-1 and an increase in circulating EPCs. The re-endothelialization of denuded vessels was accelerated in mice with systemic HO-1 overexpression. A further experiment demonstrated that both EPC mobilization and re-endothelialization were significantly attenuated in mice with HO-1 deficiency. The increase in EPC mobilization and enhanced re-endothelialization was also observed in mice exposed to CO prior to carotid injury. The CO-mediated effect was associated with an increase in circulating SDF-1 but not VEGF. CONCLUSION: These findings support a vital role of HO-1 and its reaction byproduct, CO, in vascular repair through enhancing EPC mobilization.

Cloning and expression of a mammalian Na+/amino acid cotransporter with sequence similarity to Na+/glucose cotransporters.

We describe the full-length sequence and functional expression of a cDNA cloned from LLC-PK1 cells, which appears to encode a mammalian Na(+)-dependent neutral amino acid transporter with properties characteristic of system A. This sequence, designated SAAT1, is 76% identical and 89% similar in amino acid sequence to the Na(+)-dependent glucose transporter SGLT1 of the same species. A leucine zipper region was detected in both SAAT1 and SGLT1. The message for SAAT1 was a single 2.4-kilobase species in kidney, but mRNA species of 2.4 and 3.7 kilobases were observed in LLC-PK1 cells as well as in intestine. Transcripts were also found in spleen, liver, and muscle. Expression of SAAT1 in COS-7 cells resulted in increased levels of Na(+)-dependent uptake of 2-(methylamino)isobutyric acid, a specific substrate for the system A amino acid transporter. Uptake due to cDNA expression was inhibited by a range of amino acids that are transported by system A and exhibited a km of 0.8 +/- 0.2 mM. These results suggest that the system A amino acid transporter is closely related to the Na+/glucose transporter SGLT1.

Purification and reconstitution of murine mitochondrial glycerol-3-phosphate acyltransferase. Functional expression in baculovirus-infected insect cells.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial step in glycerolipid biosynthesis. We recently cloned a cDNA to a 6.8-kb mRNA, a message that can be induced dramatically by feeding a high-carbohydrate diet [Paulauskis & Sul (1988) J. Biol. Chem. 263, 7049-7054; Shin et al. (1991) J. Biol. Chem. 266, 23834-23839], and identified the open reading frame, p90, as mitochondrial GPAT [Yet et al. (1993) Biochemistry 32, 9486-9491]. To initiate characterization of mitochondrial GPAT, we purified and reconstituted the GPAT activity using phospholipids after expressing functional enzyme in Sf9 insect cells. Infection with recombinant virus containing p90 sequence resulted in high levels of GPAT expression in mitochondria, compared to noninfected cells or cells infected with the reverse orientation insertion baculovirus. There was a dramatic increase in N-ethylmaleimide-resistant mitochondrial GPAT activity. The GPAT protein was not detectable by Western blot in noninfected Sf9 cells or in cells infected with the GPAT sequence in the reverse orientation. However, in cells infected with GPAT in the correct orientation, there was a dramatic increase in the GPAT protein that was readily detectable by Coomassie staining both in total extracts and in the mitochondrial fraction. To ease the purification, we next expressed GPAT as a polyhistidine fusion protein in insect cells. The polyhistidine tag did not interfere with targeting to mitochondria or with the catalytic activity of GPAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Hormonal and nutritional control of the fatty acid synthase promoter in transgenic mice.

To study the molecular basis of tissue-specific and hormonally regulated expression of the fatty acid synthase (FAS) gene in vivo, we generated lines of transgenic mice carrying 2.1 kilobases of the 5'-flanking region (-2100 to +67) of the rat FAS gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. This reporter gene construct was strongly expressed in tissues that normally express high levels of FAS mRNA, which include liver and white adipose tissues. In contrast, CAT reporter activity was not detected in appreciable levels in lung, heart, kidney, and muscle tissues, which do not normally show significant levels of FAS activity. The relative levels of the CAT mRNA driven by the rat FAS promoter in various tissues of the transgenic animals approximated those of the endogenous mouse FAS mRNA. We also examined the hormonal and nutritional regulation of the FAS(2.1)-CAT reporter gene in transgenic mice. CAT activity was increased in both liver and white adipose tissue when fasted animals were refed a high carbohydrate, fat-free diet. These changes in CAT activity and CAT mRNA levels occurred in parallel to the changes in endogenous mouse FAS mRNA levels. On the other hand, fasting/refeeding did not change CAT activity appreciably in other tissues, such as muscle and brown adipose tissue. Administration of dibutyryl cAMP at the start of refeeding prevented an increase in CAT activity in liver. However, the cAMP effect was tissue-specific as cAMP treatment did not bring about change in CAT activity in adipose tissue. Next, to examine the effect of insulin, we made the transgenic mice insulin-deficient by streptozotocin treatment. Insulin treatment of the streptozotocin-diabetic mice increased both the CAT activity and CAT mRNA levels driven by the rat FAS promoter in liver and white adipose tissue. These changes in CAT expression by insulin paralleled those in endogenous FAS mRNA levels. Administration of glucocorticoids increased CAT activity in all tissues examined: liver, white and brown adipose tissues, lung, heart, and spleen. Overall, the first 2.1 kilobases of the 5'-flanking region of the rat FAS gene appear to contain sequence elements necessary to confer tissue-specific and hormonally regulated expression characteristic of the endogenous FAS gene.

Expression and identification of p90 as the murine mitochondrial glycerol-3-phosphate acyltransferase.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and committed step in glycerolipid biosynthesis. Mitochondrial GPAT, unlike the microsomal isozyme, prefers saturated fatty acids as a substrate. We have recently reported cloning of a cDNA to an unidentified 6.8-kb mRNA by a differential hybridization. The mRNA contains an open reading frame of 827 amino acids (p90) with 30% sequence homology in a 300 amino acid stretch to Escherichia coli GPAT. The 6.8-kb mRNA was induced dramatically when fasted mice were refed a high-carbohydrate diet. Here, we have expressed the open reading frame as trpE fusion proteins and used them to generate antibodies. The antibodies recognized a polypeptide of 90 kDa (p90) when the 6.8-kb cDNA sequence was used for in vitro transcription and translation. By Western blot analysis using these antibodies, we detected p90 in mitochondrial fractions of liver, and the p90 level was increased by refeeding. The increase in the p90 level correlated with the increase in mitochondrial GPAT activity. Moreover, p90 was not detectable in 3T3-L1 preadipocytes but markedly increased during adipose conversion. This increase was consistent with the 11-fold increase we observed in N-ethylmaleimide (NEM)-resistant mitochondrial GPAT activity during adipocyte differentiation. In addition, we have expressed p90 in CHO cells by stable transfection. The transfected genes in both correct and reverse orientations produced distinct 3.9-kb transcripts owing to the truncation of a part of the noncoding regions of the endogenous 6.8-kb mRNA before insertion into the pMSXND vector. The transfected CHO cells were treated with 2-aminopurine, an agent that increases expression of exogenous genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of Na+/glucose cotransporter (SGLT1) mRNA in LLC-PK1 cells.

The porcine kidney epithelial cell line LLC-PK1 expresses a sodium-coupled glucose cotransporter (SGLT1) together with other differentiation markers of renal proximal tubule such as trehalase and gamma-glutamyltranspeptidase. Expression is regulated by cell density and exogenous differentiation inducers such as hexamethylene bisacetamide (HMBA). Northern blot and PCR analysis of clonal cell populations indicated SGLT1 mRNA was not detectable in subconfluent cultures, but 2.2 and 3.9 kb SGLT1 mRNA species appeared after cell confluence, accompanying expression of the transport activity. SGLT1 mRNA levels were significantly increased after treatment of confluent cultures with HMBA, paralleling increases in the transport activity and immunodetectable 75 kD cotransporter subunit. SGLT1 mRNA was also increased after treatment of cultures with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), an inducer of Na+/glucose cotransport activity. The 3.9 kb SGLT1 transcript showed the largest increase after either HMBA or IBMX treatment. HMBA treatment also resulted in increased mRNA levels of two other differentiation markers--trehalase and gamma-glutamyltranspeptidase. By contrast, trehalase and gamma-glutamyltranspeptidase mRNA levels were not increased by IBMX. Regulation of Na+/glucose symporter expression by either cell density, cyclic AMP elevation, or differentiation inducer treatment occurs, at least in part, at the level of SGLT1 mRNA and can be dissociated from regulation of other differentiation markers.

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.

Suppression of interleukin-1beta-induced nitric-oxide synthase promoter/enhancer activity by transforming growth factor-beta1 in vascular smooth muscle cells. Evidence for mechanisms other than NF-kappaB.

Nitric-oxide synthases (NOS) utilize L-arginine to produce NO, a potent vasodilator that contributes to the regulation of vascular tone. We demonstrated previously that transforming growth factor (TGF)-beta1 down-regulates inducible NOS after its induction by interleukin (IL)-1beta by decreasing the rate of inducible NOS gene transcription. In the present study we transfected reporter plasmids containing various lengths of the inducible NOS 5'-flanking region into primary cultured rat aortic smooth muscle cells and stimulated the cells with IL-1beta or vehicle. IL-1beta increased the activity of the plasmid containing -1485 to +31 of the inducible NOS gene by more than 10-fold, indicating the presence of IL-1beta-responsive elements. Further deletion analysis revealed that a construct containing -234 to +31 of the inducible NOS gene contained the majority of promoter/enhancer activity after IL-1beta stimulation. Mutation of the NF-kappaB site within this region partially reduced IL-1beta-inducible activity; however, a large portion of activity remained independent of the NF-kappaB site. TGF-beta1 suppressed promoter/enhancer activity after IL-1beta stimulation, and this suppression was complete in the construct with a mutated NF-kappaB site. In addition, TGF-beta1 did not decrease the binding of nuclear proteins to the NF-kappaB site. These data suggest that the ability of TGF-beta1 to suppress inducible NOS promoter/enhancer activity occurs through a site(s) other than the NF-kappaB motif in vascular smooth muscle cells.

Induction of heme oxygenase-1 during endotoxemia is downregulated by transforming growth factor-beta1.

Heme oxygenase (HO)-1 generates CO, a gas with vasodilatory properties, during heme metabolism. HO-1 is expressed highly in vascular tissue after endotoxin stimulation, and generation of CO through the HO-1 pathway contributes to the hemodynamic compromise of endotoxic shock. Shock related to endotoxemia is an immune-mediated process that involves the generation of proinflammatory cytokines such as interleukin (IL)-1beta. Because transforming growth factor (TGF)-beta1 is a modulator of immune-mediated inflammatory responses and it blocks the hypotension of endotoxic shock, we determined whether TGF-beta1 could be used to reduce expression of HO-1 in vascular tissue and smooth muscle cells. In a rat model of endotoxic shock, lipopolysaccharide-induced HO-1 mRNA and protein expression was reduced by TGF-beta1 in highly vascularized tissue, such as heart and lung, by Northern and Western analysis. Furthermore, TGF-beta1 downregulated HO-1 mRNA after its induction by IL-1beta in vascular smooth muscle cells in culture. TGF-beta1 also decreased HO-1 but not HO-2 protein expression in these cells. TGF-beta1 decreased HO enzyme activity induced in IL-1beta treated vascular smooth muscle cells to a level not different from that in vehicle-treated cells. These studies suggest that this downregulation of HO-1 mRNA and protein expression and decrease in IL-1beta-induced HO enzyme activity may contribute to the beneficial effect of TGF-beta1 on endotoxic shock.

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.

Induction of heme oxygenase-1 expression in vascular smooth muscle cells. A link to endotoxic shock.

Endotoxic shock is a life-threatening consequence of severe Gram-negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. The production of nitric oxide (NO), through the inducible NO synthase pathway, has been implicated as a major contributor in this process. We now demonstrate that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) mRNA levels are dramatically increased in aortic tissue from rats receiving endotoxin, and this increase in vascular HO-1 message is associated with an 8.9-fold increase in HO enzyme activity in vivo. Immunocytochemical staining localizes an increase in HO-1 protein within smooth muscle cells of both large (aorta) and small (arterioles) blood vessels. Furthermore, zinc protoporphyrin IX, an inhibitor of HO activity, abrogates endotoxin-induced hypotension in rats. Studies performed in rat vascular smooth muscle cells in vitro show that the induction of HO-1 mRNA is regulated at the level of gene transcription, and this induction is independent of NO production. Taken together, these studies suggest that the up-regulation of HO-1, and the subsequent production of CO, contributes to the reduction in vascular tone during endotoxic shock.