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GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Department of Clinical and Experimental Medicine, Clinica Medica 5, University and Azienda Ospedaliera of Padova, Padova, Italy (D.S., A.G.); Department of Pharmacology, New York Medical College, Valhalla, New York (N.G.A., L.Y., J.C.M.); and Center of Cardiovascular Diseases, Texas Southern University, Houston, Texas (A.O.O.)
Received July 21, 2003; accepted October 30, 2003.
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Abstract |
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; McCoubrey et al., 1992, 1997; Shibahara et al., 1993). HO-1 is a 32-kDa heat shock protein (Shibahara et al., 1987) that is inducible by noxious stimuli (Lutton et al., 1992; Neil et al., 1995; Lu et al., 1998). HO-2, a 36-kDa protein, is constitutively expressed and found in abundance in brain, testis, liver (Maines et al., 1986; Trakshel et al., 1988), and endothelium (Zakhary et al., 1996). HO-3, although related to HO-2, is a product of a different gene, with a lesser ability than HO-2 to catalyze heme degradation (McCoubrey et al., 1997).
HO-2, which is localized in endothelial cells and adventitial nerves of blood vessels (Zakhary et al., 1996), functions in circulatory regulation. Furchgott and Jothianandan (1991) reported that CO, arising from the metabolism of heme by HO, dilated blood vessels, whereas inhibition of HO by tinmesoporphyrin (SnMP) produced vasoconstriction (Kozma et al., 1999) and raised blood pressure (Johnson et al., 1995, 1996). Heme, by activating HO, has been shown to dilate the ductus arteriosus (Coceani et al., 1997). Moreover, contiguous, if not overlapping, spheres of activity within the vasculature of nitric oxide (NO) and CO were suggested by the study of Zakhary et al. (1996). The authors concluded that "the similarity of NOS and HO-2 localizations and functions in blood vessels and the autonomic nervous system implies complementary and possibly coordinated physiological roles for these two mediators". CO was shown to relax blood vessels via an endothelial-independent pathway linked to guanylate cyclase, as its effects were accompanied by the accumulation of cGMP levels in vascular smooth muscle (Christodoulides et al., 1995). Furthermore, inhibition of guanylate cyclase attenuated the vasodilator effect of CO (Wang et al., 1997). In isolated gracilis muscle arterioles of rats, which express HO-2, HO inhibitors decreased the diameter of pressurized vessels (Kozma et al., 1999), likely by inhibiting potassium channels as a result of decreased CO production, as has been reported for the thick ascending limb in response to inhibition of HO (Liu et al., 1999).
Mesenteric arteriolar vasodilatation and increased portal inflow play a major role in the development of portal hypertension in cirrhosis (Vorobioff et al., 1984; Shah et al., 1998). Various vasodilator compounds, including glucagon, adenosine, prostacyclin, NO, 
-calcitonin gene-related peptide, and adrenomedullin have been proposed as mediators of the mesenteric vascular abnormalities that occur both in cirrhosis and in prehepatic portal hypertension (Shah et al., 1998; Gatta et al., 1999). In cirrhotic rats, inhibition of NO synthesis greatly reduced mesenteric blood flow and hyporeactivity of the mesenteric circulation to vasoconstrictor agents (Sieber et al., 1993). In rats with prehepatic portal hypertension, HO inhibition modified the hyporeactivity of the mesenteric circulation to vasoconstrictors (Fernandez et al., 2001).
The present study was designed to determine whether 1) augmentation of HO contributes to mesenteric vascular abnormalities in cirrhotic rats, 2) gene transfer reproduces these alterations in normal rats, and 3) nitric oxide synthase (NOS)-NO and HO-CO systems interact in the mesenteric circulation to affect vascular tone and reactivity. To achieve these goals, the isolated perfused superior mesenteric vasculature (SMV) was studied in terms of 1) reactivity to constrictor agents in normal rats and in those with carbon tetrachloride (CCl4)-induced cirrhosis, 2) effects of inhibition of HO with SnMP in each group, and 3) effects on the SMV produced by transfection with the human HO-1 (HHO-1) gene. The participation of NO in the development of hyporeactivity of the mesenteric vasculature in cirrhotic rats was also examined. This issue was addressed by determining enhancement of the vascular responses to constrictor agents as modified by successive inhibition of CO and NO production and then reversing the order of inhibition, with NO preceding CO. We were able to identify a significant component of each system, NOS-NO and HO-CO, that contributed to the vascular hyporeactivity of cirrhotic rats, suggesting "complementary and possibly coordinated physiological roles" (Zakhary et al., 1996) for CO and NO. Our results indicate a key role for HO in the control of the mesenteric circulation, as inhibition of HO activity with SnMP normalized mesenteric perfusion pressure and restored toward normal the response of the mesenteric vasculature to vasoconstrictor agents. We also found, quite unexpectedly, an increase in HO-2, the constitutive enzyme, rather than HO-1, the inducible enzyme in the SMV in cirrhotic portal hypertension. Finally, HO activity augmented by HO-1 gene transfer decreased mesenteric perfusion pressure and reduced the reactivity of the SMV to vasoconstrictor agents.
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Materials and Methods |
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Animals. Adult male Sprague-Dawley rats (150–160 g) were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and housed in an animal facility with controlled normal day-night cycle. The rats were maintained on a standard diet of Purina chow and were allowed free access to water and food until the night before experiments. Experiments were performed in accordance with the guidelines set forth by The American Physiological Society for ethical treatment of animals.
Induction of Cirrhosis. Cirrhosis was induced in animals (
170 g b.wt.) by combined treatment with CCl4 and phenobarbital, the latter given in drinking water (Sacerdoti et al., 1991). After 1 week of phenobarbital administration, CCl4 was given by gavage, starting with a dose of 30 µl, and then increasing doses were administered according to changes in body weight. CCl4 was administered once a week and then twice a week for 8 to 10 weeks. Treatment was terminated 1 week prior to conducting experiments. Rats on phenobarbital during the time of treatment were used as controls. Free access to standard chow was allowed throughout the study. In all of the CCl4-treated rats used for study, portal pressure was measured by a Statham transducer after direct cannulation of the portal vein before the experiments; rats with portal pressure <8 mm Hg were excluded from the study.
Perfusion of Isolated Mesenteric Artery (MacGregor Preparation). The superior mesenteric artery was cannulated with a PE-60 catheter and perfused with 25 ml of warm Krebs-Henseleit buffer. After the superior mesenteric artery was isolated with its mesentery, the gut was excised near its mesenteric border. The mesenteric arterial bed was then brought in a 37°C water-jacketed container and perfused at a constant rate of 0.8 ml/min/100 g b.wt. with oxygenated (95% O2/5% CO2) Krebs-Henseleit solution (118.5 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25.0 mM NaHCO3, and 11.1 mM dextrose, pH 7.4 at 37°C). The effluent was constantly collected, and the pressure was measured by a Statham transducer.
Vascular Responses to Agonists and Antagonists. After an initial 30-min basal period, concentration-response curves were constructed with KCl, –5.22 to –3.6 log mol; PE, –9.3 to –6.3 log mol; and ET-1, –11.7 to –10.5 log mol in the presence and absence of HO inhibition with SnMP (10 µM). This concentration of SnMP does not inhibit NOS activity or activity of soluble guanylate cyclase (Meffert et al., 1994; Zakhary et al., 1996). It does, however, inhibit completely the increase in HO activity produced by either heme or SnCl2 in endothelial cells as well as smooth muscle cells (Li Volti et al., 2002). SnMP was added to the buffer 10 min before vasopressors were examined. Concentration-response curves were constructed by adding increasing bolus amounts (20–50 µl) of agonists. The maximum response was obtained within 60 s; perfusion pressure returned to baseline values in 2 to 3 min after KCl and PE and 10 to 15 min after ET-1. Increasing doses were added after return to the baseline pressure. Experiments performed to determine the differential effects of inhibition of HO and NOS in cirrhotic animals were done using single doses of PE, –7.3 log mol. After the initial equilibration period, the responses to PE were evaluated without inhibitors and then after inhibition of HO with SnMP (10 µM) (n = 6) or L-NAME (100 µM) (n = 6). After restoration of baseline conditions, the second inhibitor was added to the perfusate, and responses to the agonists were again evaluated. L-NAME was dissolved in saline. In rats transfected with the HHO-1 gene, the vasoconstrictor responses of the SMV to PE (–8 log mol) were evaluated after an equilibration period before and after inhibition of HO with SnMP (10 µM).
Western Blot Analysis. For the detection of HO-1 and HO-2 protein, microsomes were prepared from tissues as previously described (Sacerdoti et al., 1988); twenty micrograms of microsomal protein was analyzed by Coomassie staining of the gels and by immunoblot analysis of proteins transferred to nitrocellulose membrane. Briefly, membrane blots were blocked in 5% nonfat milk overnight to block nonspecific binding of antibodies. The blots were then incubated with mouse monoclonal anti-HO-1 or HO-2 antibodies (1:2000) (StressGen Biotechnologies Corp., Victoria, BC, Canada) for 1 h and washed with Tris-buffered saline containing 0.1% Tween 20 three times. The blot was subsequently incubated with rabbit anti-mouse IgG antibody (1:4000) for 1 h. The blot was washed three times with Tris-buffered saline/Tween 20, and immunoreactive proteins were visualized by means of enhanced chemiluminescence reagent and exposure to an autoradiographic film.
Microassay for Measurement of HO Activity. HO activity was measured as previously described (Chernick et al., 1989). Briefly, tissue homogenates (100 µg of protein) were incubated in a NADPH-generating system (20 µM MgCl2, 30 µM heme, and 1µCi/ml of 14C-heme). HO activity was measured as the conversion of heme to bilirubin in the presence of biliverdin reductase. 14C-bilirubin was extracted with chloroform, and radioactivity was measured. Results were expressed as counts per minute per milligram protein.
Construction of the Adenovirus Vector Containing Human HO-1 cDNA. HHO-1 cDNA with adenovirus vector was constructed using a mammalian transfection kit (Stratagene, La Jolla, CA) as previously described by Abraham et al. (1995b). Briefly, the human cDNA plasmid was constructed as follows: a Xho1-Xba1 fragment of a HHO-1 expression vector, pRc/CMVHO-1, was cloned between Xho1 and Xba1 of the pBacPAC8 to generate pBacPAC8 HHO-1. The Bg1II-BamH1 fragment of the pBacPAC8HHO was then introduced into the Bg1II site of pAdBg1II. The human embryonic cell line 293 (ATCC catalog number CRL-1573) was cotransfected with 10 µg of EcoR1-digested adenoviral HHO-1 and 1 µg of Cla1-digested dL7001DNA by calcium phosphate coprecipitation using a mammalian transfection kit. HHO-1 adenovirus construct was replicated and encapsulated into an infectious virus as described previously (Abraham et al., 1995a). The virus was concentrated by centrifugation by using Ultrafree-MC filters (Millipore Corp., Bedford, MA). The viral titers were determined by plaque assay using human embryonic 293 cells. The viral stock was stored at –80°C until use.
In Vivo Gene Transfer. Eight rats were injected intracardiacally with HHO-1 adenovirus suspension (1012 pfu/300 g b.wt.). Four rats were treated with the adenovirus without the HO-1 gene. The animals were acclimatized for 1 week prior to the start of the experiment, and experiments were performed 1 week after viral administration. Hemodynamic studies were performed as described above in six rats treated with the gene and four rats treated with the empty virus.
RNA Extraction and RT-PCR. SMV were homogenized in liquid nitrogen, and total RNA was extracted with guanine thiocynate, first purified with phenol-chloroform followed by an additional purification with chloroform and then precipitated with isopropanol (Chomczynski and Sacchi, 1987). Total RNA was resuspended in autoclaved diethyl pyrocarbonate-water and quantified using Beckman DU7400 spectrophotometer (Beckman Instruments, Fullerton, CA). To verify the quality of RNA, 10 µg of total RNA was denatured and separated by 1% agarose gel (containing 2.2 M formaldehyde) electrophoresis. The RNA was visualized under UV light with the help of ethidium bromide. Primers were designed to amplify specific fragments of the HHO-1 and/or rat HO-1. The primers used did not recognize HO-2 sequences. The RT-PCR for HHO-1 was performed by using the Advantage RT-PCR kit (BD Biosciences Clontech, Palo Alto, CA) and the primers designed to amplify a RT-PCR product with a predicted size of 555 base pairs. Briefly, 1 µg of total RNA was reverse transcribed using oligo(dT18) primer to synthesize the second strand and to obtain the cDNA. The reverse transcription was performed at 42°C for 1 h, and the reaction tubes were placed on ice. The PCR of HHO-1 was performed by adding PCR mix prepared with sense primer (5'CAGGCAGAGAATGCT GAGTTC-3'), antisense primer (5'-quenched by heating it at 95°C for 5 min and then the GATGTTGAGCAGGAACGCAGT-3'), deoxynucleoside-5'-triphosphate, Taq polymerase, 32P-dCTP, and reaction buffer. To amplify the endogenous rat HO-1, the primers were designed from the rat cDNA sequence (sense primer, 5'-TGAAGGAGGCCACCAAGGAG-3' and antisense primer, 5'-CCCCTGAGAGGTCACCCAGG-3'), which amplifies a 356-base pair fragment. RT-PCR was performed as described above.
Statistical Analysis. Results were expressed as means ± S.E.M. Concentration-response data for PE and KCl derived from each vessel were fitted separately to a logistic function by nonlinear regression, and the maximum asymptote of the curve (Rmax) and concentration of agonist producing 50% of the maximal response (EC50), which was expressed as negative log mol, were calculated using commercially available software (Prism 2.01; GraphPad Software, San Diego, CA). Concentration-response data were analyzed by two-way analysis of variance. Differences between groups were evaluated by unpaired Student's t test. The effects of inhibition of HO on the action of vasoconstrictors were evaluated by paired t test. Statistical significance was set at p < 0.05.
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Next, we examined whether inhibition of HO by SnMP (10 µM) can restore normal vasopressor responses to KCl, PE, and ET-1 that were depressed in cirrhotic animals. The vasoconstrictor responses to KCl, PE, and ET-1 were shifted to the right, the maximal response was blunted, and the EC50 for KCl was increased (Figs. 2, 3, and 4). SnMP significantly increased the response to KCl in normal (n = 8) and cirrhotic rats and restored to normal the responsiveness to KCl in the SMV (n = 8) (Fig. 2). SnMP did not affect the vasoconstrictor response to the sympathomimetic agent, PE, in normal rats (n = 8) and restored to normal the responses of the SMV to the vasoconstrictor agent in cirrhotic rats (n = 8) (Fig. 3). SnMP increased the vasoconstrictor responses of SMV to ET-1 in cirrhotic but not in normal rats (n = 8); in cirrhotic rats, the responses to ET-1 in SMV were restored to normal (n = 8) (Fig. 4). These results indicate that SnMP-induced inhibition of HO in cirrhotic rats restores toward normal the responsiveness of the SMV to vasopressor agents.
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We also examined the contributions of NO and CO to attenuating the response to PE in the SMV in view of reports that NO contributes to hyporeactivity of the SMV to pressor agents in cirrhotic rats (Sieber et al., 1993). SnMP was used to inhibit HO because of its demonstrated selectivity at the concentration employed (10 µM) and its solubility (Zakhary et al., 1996). It was important that SnMP did not inhibit NOS, as our study was directed at the vascular effects of CO in the SMV of cirrhotic rats. The relative contribution of HO-CO and NOS-NO to producing hyporeactivity of the SMV of cirrhotic rats to PE was assessed by determining the effects of inhibiting each system separately, i.e., HO-CO with SnMP and NOS-NO with L-NAME, followed by inhibition of the other system, then reversing the order of inhibition of CO and NO production (Fig. 5, A and B). After achieving a significant reduction in vascular hyporeactivity by inhibition of either HO or NOS, additional reduction was obtained on inhibiting the other gas-producing system. For example, in Fig. 5A, the constrictor response of the SMV of cirrhotic rats to PE was increased by inhibition of HO activity with SnMP and was further enhanced by inhibition of NOS activity with L-NAME. Then, the order of inhibition of HO and NOS activities was reversed in Fig. 5B. After the combined inhibition of HO and NOS, the constrictor responses of the SMV of cirrhotic rats to PE were fully restored. Moreover, the effects of either SnMP or L-NAME in restoring to normal the vasoconstrictor responses to PE (Fig. 5) in cirrhotic rats were similar, irrespective of the order of administration of SnMP or L-NAME. These findings are in accord with the findings of Thorup et al. (1999) that CO "may synergize with NO in eliciting vasorelaxation and modulating basal vascular tone" by effects on vascular mechanisms such as, for example, those served by guanylyl cyclase and cGMP.
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We examined whether responsiveness of mesenteric arteries of cirrhotic rats to vasopressor agents was associated with increased expression of HO-1 and HO-2 in this vascular bed. Western blot analysis of mesenteric blood vessels obtained from normal and cirrhotic rats (Fig. 6A) demonstrated significantly higher levels of HO-2, but not of HO-1, in vascular tissues obtained from cirrhotic rats (Fig. 6, lanes 5–8) compared with those from normal rats (Fig. 6, lanes 1–4). HO up-regulation was quantified by densitometry-based analysis of HO-1 and HO-2 proteins after blotting the ratio of each sample. As seen in the densitometry analysis (Fig. 6B), HO-2 was increased significantly in SMV obtained from cirrhotic rats when compared with those of control rats.
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To assess the effect of the increase in HO-2 protein on HO activity, cell homogenates obtained from SMVs of control and cirrhotic rats were used to measure the enzyme activity as indicated by C14 heme catabolism to C14 bilirubin (Fig. 7). Bilirubin formation was significantly increased in SMV samples obtained from cirrhotic rats compared with those obtained from control rats (p < 0.05). These findings help explain 1) the hyporeactivity of the mesenteric vasculature to vasoconstrictor agents and 2) why inhibition of HO helps restore the responsiveness to vasoconstrictor agents of the SMV obtained from cirrhotic animals.
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To examine further our hypothesis that increased HO levels in mesenteric arteries in experimental cirrhosis are responsible for vasodilation and decreased reactivity of SMV to vasoconstrictor agents, we injected eight normal rats with adenovirus containing the HHO-1 gene (1012 pfu/300 g b.wt.). These preparations of adenovirus-mediated HO-1 gene construct were shown to increase HO-1 gene expression within 7 days of infection (Abraham et al., 2000). The ability of adenovirus-mediated HHO-1 to infect mesenteric blood vessels was assessed after 7 days, and total RNA from SMV was assessed for the presence of the HHO-1 by RT-PCR and enzyme activity (Fig. 8). Positive signals were obtained for the HHO-1 gene when amplified with HHO-1 primers within 7 days of infection. Injection of adenovirus-mediated HHO-1 gene transfer resulted in the expression of HO-1 in mesenteric blood vessels. The HHO-1 mRNA signal was not detectable in mesenteric arteries infected with empty adenoviral constructs (Fig. 8, lane 2). To determine the quality of total RNA of SMVs, RT-PCR analysis was performed using GAPDH-specific primers. Amplification of GAPDH/PCR products occurred in all RNA tested (Fig. 8). HO protein in tissues obtained from rats infected with adenoviral constructs was significantly higher (1240 ± 200 versus 187 ± 13 cpm/mg protein, n = 4, p < 0.01) than in tissues obtained from rats infected with empty adenoviral constructs.
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Finally, we examined the effect of HHO-1 transfection on the mesenteric circulation. As is evident in Fig. 9, perfusion pressure and the increase in perfusion pressure of the SMV induced by PE were attenuated in animals transfected with the HHO-1 gene. Inhibition of HO with SnMP partially reversed these abnormalities. No difference was evidenced in rats treated with the empty virus. These results support our hypothesis that increased HO expression and activity in cirrhosis contribute to the aberrant responses of the SMV to vasoconstrictor agents.
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). Mesenteric arterial resistance is normally high, whereas in cirrhosis, dilatation of the mesenteric vasculature is associated with an increase in portal inflow, a major determinant of portal hypertension (Shah et al., 1998; Gatta et al., 1999). Furthermore, in experimental cirrhosis, the responsiveness of the mesenteric circulation to pressor agents is greatly impaired (Sieber et al., 1993; Gagano et al., 1997). A number of endogenous vasoactive compounds, previously enumerated, have been proposed to mediate this lack of vascular responsiveness (Shah et al., 1998; Gatta et al., 1999).
In the present study, we have shown in rats with CCl4-induced hepatic cirrhosis that the resulting attenuation of mesenteric vascular constrictor responses to diverse agonists could be reversed by inhibition of HO, highlighting the potential importance of the HO-CO system to the regulation of circulatory tone and vascular reactivity. Quite unexpectedly, the HO isoform responsible for mesenteric vascular hyporeactivity was shown to be the constitutive enzyme HO-2, not the inducible enzyme HO-1. This finding is not unique, as HO-2 levels are increased by estrogens in endothelial cells (Tschugguel et al., 2001) and by corticosterone in the brain (Weber et al., 1994). Furthermore, this finding stands in contrast to the induction of HO-1 activity in response to portal hypertension produced by ligation of the portal vein (Fernandez et al., 2001) and helps in distinguishing critical differences between these two experimental approaches, CCl4-induced cirrhosis versus portal vein constriction, to the production of altered responses of the mesenteric vasculature. For example, in contrast to CCl4-induced cirrhosis, HO inhibition in rats with ligated portal veins did not alter mesenteric vascular hyporeactivity to a 1-adrenergic agonist (Fernandez et al., 2001). Blockade of NO production was required to reverse the hyporeactivity to methoxamine in this model of portal hypertension.
We also examined potential interactions of the NOS-NO and HO-CO systems in the mesenteric vasculature in view of the demonstrated contribution of NO to splanchnic vascular hyporeactivity in cirrhotic rats (Sieber et al., 1993). Our findings suggest that these systems are complementary in the mesenteric circulation of the cirrhotic rat, which agrees with the "coordinated functions of NO and CO" proposed by Zakhary et al. (1996) and is in accord with the presence in endothelial cells of both NOS and HO (Zakhary et al., 1996) as well as the ability of each system to activate soluble guanylate cyclase and increase cGMP (Abraham et al., 2002). Alternatively, rather than overlapping spheres of activity of NO- and CO-mediated vascular effects reflecting colocalization of NOS and HO isoforms in the endothelium, segmental separation within a vasculature (eNOS predominating in large arteries and HO-2 in small arteries and microvessels) may also give rise to coordinated and complementary responses of the NOS-NO and HO-CO systems (Zakhary et al., 1996). A recent study demonstrated that despite eNOS gene deletion, mice with portal hypertension developed a hyperdynamic circulation, indicating the importance of other vasodilator systems in the production of splanchnic circulatory changes in portal hypertensive animals (Iwakiri et al., 2002).
The possibility that SnMP inhibits NOS as well as HO under the conditions of our study is unlikely, as weak inhibition of NOS with SnMP can be obtained only at a 10x higher concentration (100 µM) than that used in our study (10 µM) (Meffert et al., 1994; Zakhary et al., 1996). Furthermore, SnMP, in the concentration used in our study (10 µM), was shown to be without effect on eNOS activity in the vasculature despite colocalization of HO-2 and eNOS in the endothelium of the several vascular beds studied (Zakhary et al., 1996). This specificity of SnMP relative to HO when compared with NOS has also been reported for the central nervous system (Meffert et al., 1994). Furthermore, the data presented in Fig. 5, A and B show that prior administration of SnMP does not notably affect the action of L-NAME in augmenting the constrictor response of the SMV to PE, indicating that the activity of NOS is not, to a readily detectable degree, affected by prior treatment with SnMP under the conditions of our study. Nonetheless, an effect of NO on HO gene expression and activity may occur over the long term, as NO has been shown to induce HO-1 in endothelial cells in vitro (Hartsfield et al., 1997). On the other hand, transfection of rat endothelial cells with HHO-1, which induced a 4-fold increase in HO activity, did not affect either eNOS protein or NO production (Abraham et al., 2002).
Transfection of normal rats with HHO-1 provided additional support for the role of CO as an essential component in mechanisms that modulate reactivity of the mesenteric vasculature in experimental cirrhosis. The transfection was highly effective, and HHO-1 mRNA expression was evident in the PCR analysis of the mesenteric vasculature (Fig. 8). Furthermore, HO activity in mesenteric arteries from transfected rats was significantly increased compared with the activity in rats treated with the empty virus. Transfected rats showed hemodynamic characteristics similar to, or even more pronounced than, those of cirrhotic animals; viz., perfusion pressure and reactivity of the mesenteric vasculature to constrictor agents were decreased (Fig. 9). We have reported that adenovirus-mediated HHO-1 gene transfection was associated with a decrease in microsomal heme and release of CO (Abraham et al., 2000). Moreover, retrovirus-mediated HO gene transfer into endothelial cells, as we have shown, increases cGMP and protects against oxidant-induced cell injury (Yang et al., 1999). The increase in HO activity produced by this strategy may prove useful as a means of modifying abnormalities of regional vascular beds.
Taken together, our findings support a role for HO-CO that may synergize with NOS-NO in regulating the mesenteric circulation; they provide a conceptual basis for examining abnormalities of vascular beds in human hepatic cirrhosis. The systemic circulation, in addition to the splanchnic vasculature, is affected by hepatic cirrhosis associated with a decline in systemic vascular resistance in human subjects (Schrier et al., 1998) associated with increased CO production, the magnitude of which was correlated with the severity of the cirrhosis (De Las Heras et al., 2003). Inhibition of HO has also been reported to reverse vascular hyporeactivity to norepinephrine in cirrhotic patients (Sacerdoti et al., 2002). CO lowers blood pressure in rats by participating in the regulation of basal tone in resistance blood vessels (Johnson et al., 1995; Kozma et al., 1999). In normal rats, inhibition of HO increased blood pressure (Johnson et al., 1995), whereas in the spontaneously hypertensive rat but not in the normotensive Wistar Kyoto rat, stimulation of HO with SnCl2 decreased blood pressure (Sacerdoti et al., 1989). The present study has clinical implications: inhibition of HO may serve as a therapeutic option in cirrhotic patients, possibly improving not only portal hypertension but also systemic hemodynamics and, as a consequence, preserving renal function and reducing portal hypertension.
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ABBREVIATIONS: HO, heme-oxygenase; HO-2, constitutive heme-oxygenase; HO-1, inducible heme-oxygenase; L-NAME, NG-nitro-L-arginine methyl ester; CO, carbon monoxide; NO, nitric oxide; NOS, nitric-oxide synthase; CCl4, carbon tetrachloride; SnMP, tin-mesoporphyrin/stannous-mesoporphyrin; SMV, superior mesenteric vasculature; HHO-1, human HO-1; PE, phenylephrine; ET-1, endothelin-1; pfu, plaque-forming units; RT, reverse transcription; PCR, polymerase chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; eNOS, endothelial nitric oxide synthase.
Address correspondence to: Dr. John C. McGiff, Professor and Chairman, Department of Pharmacology, New York Medical College, Valhalla, New York 10595. E-mail: john_mcgiff{at}nymc.edu
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References |
|---|
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abraham NG, Da Silva J-L, Lavrovsky Y, Stoltz RA, Kappas A, Dunn MW, and Schwartzman ML (1995a) Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues. Investig Ophthalmol Vis Sci 36: 2202–2210.
Abraham NG, Jiang S, Yang L, Zand BA, Laniado-Schwartzman M, Marji J, Drummond GS, and Kappas A (2000) Adenoviral vector-mediated transfer of human heme oxygenase in rats virtually decrease renal heme-dependent arachidonic acid epoxygenase activity. J Pharmacol Exp Ther 293: 494–500.
Abraham NG, Lavrovsky Y, Schwartzman ML, Stoltz RA, Levere RD, Gerritsen ME, Shibahara S, and Kappas A (1995b) Transfection of the human heme oxygenase gene into rabbit coronary microvessel endothelial cells: protective effect against heme and hemoglobin toxicity. Proc Natl Acad Sci USA 92: 6798–6802.
Abraham NG, Lin JH, Dunn MW, and Schwartzman ML (1987) Presence of heme oxygenase and NADPH cytochrome P450 (c) reductase in human corneal epithelium. Investig Ophthalmol Vis Sci 28: 1464–1472.
Abraham NG, Quan S, Mieyal PA, Yang L, Burke-Wolin T, Mingone CJ, Goodman AI, Nasjletti A, and Wolin MS (2002) Modulation of cGMP by human HO-1 retrovirus gene transfer in pulmonary microvessel endothelial cells. Am J Physiol 283: L1117–L1124.
Chernick RJ, Martasek P, Levere RD, Margreiter R, and Abraham NG (1989) Sensitivity of human tissue heme oxygenase to a new synthetic metalloporphyrin. Hepatology 10: 365–369.[Medline]
Chomczynski P and Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156–159.[Medline]
Christodoulides N, Durante W, Kroll MH, and Schafer AI (1995) Vascular smooth muscle cell heme oxygenases generate guanylyl cyclase-stimulatory carbon monoxide. Circulation 91: 2306–2309.
Coceani F, Kelsey L, Seidlitz E, Marks GS, Mclaughlin BE, Vreman HJ, Stevenson DK, Rabinovitch M, and Ackerley C (1997) Carbon monoxide formation in the ductus arteriosus in the lamb: implications for the regulation of muscle tone. Br J Pharmacol 120: 599–608.[CrossRef][Medline]
De Las Heras D, Fernandez J, Gines P, Cardenas A, Ortega R, Navasa M, Barbera JA, Calahorra B, Guevara M, Bataller R, et al. (2003) Increased carbon monoxide production in patients with cirrhosis with and without spontaneous bacterial peritonitis. Hepatology 38: 452–459.[CrossRef][Medline]
Fernandez M, Lambrecht RW, and Bonkovsky HL (2001) Increased heme oxygenase activity in splanchnic organs from portal hypertensive rats: role in modulating mesenteric vascular reactivity. J Hepatol 34: 812–817.[CrossRef][Medline]
Furchgott RF and Jothianandan D (1991) Endothelium-dependent and -independent vasodilation involving cyclic GMP: relaxation induced by nitric oxide, carbon monoxide and light. Blood Vessels 28: 52–61.[Medline]
Gagano AC, Sogni P, Yang S, Cailmail S, Moreau R, Nepveux P, Couturier D, and Lebrec D (1997) Endothelial calcium-calmodulin dependent nitric oxide synthase in the in vitro vascular hyporeactivity of portal hypertensive rats. J Hepatol 26: 678–686.[CrossRef][Medline]
Gatta A, Sacerdoti D, Bolognesi M, and Merkel C (1999) Portal hypertension: state of the art. Ital J Gastroenterol Hepatol 31: 326–345.[Medline]
Hartsfield CL, Alam J, Cook JL, and Choi AM (1997) Regulation of heme oxygenase-1 gene expression in vascular smooth muscle cells by nitric oxide. Am J Physiol 273: L980–L988.
Iwakiri Y, Cadelina G, Sessa WC, and Groszmann RJ (2002) Mice with targeted deletion of eNOS develop hyperdynamic circulation associated with portal hypertension. Am J Physiol 283: G1074–G1081.
Johnson RA, Lavesa M, Askari B, Abraham NG, and Nasjletti A (1995) A heme oxygenase product, presumably carbon monoxide, mediates a vasodepressor function in rats. Hypertension 25: 166–169.
Johnson RA, Lavesa M, Deseyn K, Scholer MJ, and Nasjletti A (1996) Heme oxygenase substrates acutely lower blood pressure in hypertensive rats. Am J Physiol 271: H1132–H1138.
Kozma F, Johnson RA, Zhang F, Yu C, Tong X, and Nasjletti A (1999) Contribution of endogenous carbon monoxide to regulation of diameter in resistance vessels. Am J Physiol 276: R1087–R1094.
Liu H, Mount DB, Nasjletti A, and Wang W (1999) Carbon monoxide stimulates the apical 70-pS K+ channel of the rat thick ascending limb. J Clin Investig 103: 963–970.[Medline]
Li Volti G, Wang J, Traganos F, Kappas A, and Abraham NG (2002) Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression. Biochem Biophys Res Commun 296: 1077–1082.[CrossRef][Medline]
Lu TH, Lambrecht RW, Pepe J, Shan Y, Kim T, and Bonkovsky HL (1998) Molecular cloning, characterization and expression of the chicken heme oxygenase-1 gene in transfected primary cultures of chick embryo liver cells. Gene 207: 177–186.[CrossRef][Medline]
Lutton JD, Da Silva J-L, Moqattash S, Brown AC, Levere RD, and Abraham NG (1992) Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3b) by environmental agents. J Cell Biochem 49: 259–265.[CrossRef][Medline]
Maines MD, Trakshel GM, and Kutty RK (1986) Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible. J Biol Chem 261: 411–419.
McCoubrey WK Jr, Ewing JF, and Maines MD (1992) Human oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal. Arch Biochem Biophys 295: 13–20.[CrossRef][Medline]
McCoubrey WK Jr, Huang TJ, and Maines MD (1997) Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 247: 725–732.[Medline]
Meffert MK, Haley JE, Schuman EM, Schulman H, and Madison DV (1994) Inhibition of hippocampal heme oxygenase, nitric oxide synthase and long-term potentiation by metalloporphyrins. Neuron 13: 1225–1233.[CrossRef][Medline]
Neil TK, Abraham NG, Levere RD, and Kappas A (1995) Differential heme oxygenase induction by stannous and stannic ions in the heart. J Cell Biochem 57: 409–414.[CrossRef][Medline]
Sacerdoti D, Abraham NG, McGiff JC, and Schwartzman ML (1988) Renal cytochrome P450-dependent metabolism of arachidonic acid in spontaneously hypertensive rats. Biochem Pharmacol 37: 521–527.[CrossRef][Medline]
Sacerdoti D, Escalante B, Abraham NG, McGiff JC, Levere RD, and Schwartzman ML (1989) Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science (Wash DC) 243: 388–390.
Sacerdoti D, Escalante BA, Schwartzman ML, Abraham NG, Gatta A, and McGiff JC (1991) Renal cytochrome P-450-dependent metabolism of arachidonic acid in cirrhotic rats. J Hepatol 12: 230–235.[CrossRef][Medline]
Sacerdoti D, Mania D, Bolognesi M, Spadaro L, Tosatti A, Pierobon A, Bombonato G, and Gatta A (2002) Role of the heme oxygenase/carbon monoxide system in the alterations of cutaneous microcirculation in human cirrhosis. Hepatology 36: 517A.
Schrier RW, Arroyo V, Bernardi M, Epstein M, Henriksen JH, and Rodes J (1998) Peripheral arterial vasodilation hypothesis: a proposal for the initiation of renal sodium and water retention in cirrhosis. Hepatology 8: 1151–1157.
Shah V, Garcia-Cardena G, Sessa WC, and Groszmann RJ (1998) The hepatic circulation in health and disease: report of a single-topic symposium. Hepatology 27: 279–288.[CrossRef][Medline]
Shibahara S, Muller M, and Taguchi H (1987) Transcriptional control of rat heme oxygenase by heat shock. J Biol Chem 262: 12889–12892.
Shibahara S, Yoshizawa M, Suzuki H, Takeda K, Meguro K, and Endo K (1993) Functional analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J Biochem 113: 214–218.
Sieber CC, Lopez-Talavera JC, and Groszmann RJ (1993) Role of nitric oxide in the in vitro splanchnic vascular hyporeactivity in ascitic cirrhotic rats. Gastroenterology 104: 1750–1754.[Medline]
Thorup C, Jones CL, Gross SS, Moore LC, and Goligorsky MS (1999) Carbon monoxide induces vasodilation and nitric oxide release but suppresses endothelial NOS. Am J Physiol 277: F882–F889.
Trakshel GM, Kutty RK, and Maines MD (1988) Resolution of the rat brain heme oxygenase activity: absence of a detectable amount of the inducible form (HO-1). Arch Biochem Biophys 260: 731–739.
Tschugguel W, Stonek F, Zhegu Z, Dietrich W, Schneeberger C, Stimpfl T, Waldhoer T, Vycudilik W, and Huber JC (2001) Estrogen increases endothelial carbon monoxide, heme oxygenase 2 and carbon monoxide-derived cGMP by a receptor-mediated system. J Clin Endocrinol Metab 86: 3833–3839.
Vorobioff J, Bredfeldt JE, and Groszmann RJ (1984) Increased blood flow through the portal system in cirrhotic rats. Gastroenterology 87: 1120–1126.[Medline]
Wang R, Wang Z, and Wu L (1997) Carbon monoxide-induced vasorelaxation and the underlying mechanisms. Br J Pharmacol 121: 927–934.[CrossRef][Medline]
Weber CM, Eke BC, and Maines MD (1994) Corticosterone regulates heme oxygenase-2 and NO synthase transcription and protein expression in rat brain. J Neurochem 63: 953–962.[Medline]
Yang L, Quan S, and Abraham NG (1999) Retrovirus-mediated HO gene transfer into endothelial cells protects against oxidant-induced injury. Am J Physiol 277: L127–L133.
Zakhary R, Gaine SP, Dinerman JL, Rua M, Flavahan NA, and Snyder SH (1996) Heme oxygenase 2: endothelial and neuronal localization and role in endothelium-dependent relaxation. Proc Natl Acad Sci USA 93: 795–798.
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, p < 0.05 versus controls; 
, n = 8) and cirrhotic (
, n = 8) rats before (A) and after (B) inhibition of HO with SnMP (10 µM). 
-actin is an index of the adequacy of sample loading. B, quantitative densitometric evaluation of HO-1 and HO-2 in the SMV of control and cirrhotic rats. 
