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* Division of Cardiology,
Molecular Biology Institute, and
Dumont-University of California, Los Angeles Transplant Center, Department of Surgery and Pathology, University of California, Los Angeles, CA 90095;
Biesecker Center and Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia and University of Pennsylvania, Philadelphia, PA 19104; and
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SangStat Medical Corporation, Fremont, CA 94555
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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HO-1 plays an important role in organ transplantation (4, 5). Soares et al. (6) provided evidence that HO-1 expression is essential for cardiac xenograft survival by demonstrating that mouse-to-rat HO-1-/-, but not HO-1+/+ grafts undergo vigorous rejection. Under conditions of HO activity inhibition, exogenous CO restored the beneficial effects of HO-1 (7). Moreover, HO-1 overexpression benefited liver (8), thyroid (9), and cardiac (10) allograft survival.
We have shown in rat models of hepatic cold ischemia, followed by ex vivo reperfusion or isotransplantation, that adenovirus or cobalt protoporphyrin (CoPP)-induced HO-1 expression resulted in better preserved hepatic architecture, decreased infiltration by T cells/macrophages, and improved animal survival (11, 12). We also observed that HO-1 provided potent cytoprotection in stringent rat cardiac (13) and renal (14) models of cold ischemia, followed by isotransplantation. In agreement with others (15), we reported on the key role of CO-dependent antiapoptotic (16) and p38 mitogen-activated protein kinase signaling (17) pathways in HO-1-mediated cytoprotection.
Pharmacological HO-1 induction has the limitation that it is not HO-1 specific (18). With the use of viral vectors, genetic manipulation has been a convenient strategy to achieve specific HO-1 overexpression, but it is often complicated by proinflammatory responses against the vector. Approaches with genetically engineered animals are required to precisely elucidate the effects and mechanisms of HO-1 action. The development of tissue-specific transgenic mice that selectively overexpress rat HO-1 in the brain (19, 20), human HO-1 in cardiomyocytes (21), and human HO-1 in smooth muscle cells (SMC) (22) has been reported. Tissue-specific HO-1 overexpression has shown to be neuroprotective during the acute period of stroke (19, 20) and cardioprotective against ischemia/reperfusion injury (23).
We now report the generation of HO-1 transgenic (Tg) mice in which the rat HO-1 gene is under the control of native rat HO-1 promoter. These Tg mice exhibited a modest HO-1 overexpression in all tissues characterized. This pattern of generalized overexpression allowed us to directly compare the role of local (graft) vs systemic (host) HO-1 in allotransplant survival. We used Tg mice as donors or recipients of MHC-incompatible hearts. Although both Tg to wild-type (WT) and WT to Tg allografts were prolonged, the beneficial effect was much more pronounced when Tg mice were used as recipients. Only grafts in Tg mice lacked evidence of immune activation (CD25) with significantly decreased CD4+ cell infiltration. This Tg mouse with generalized HO-1 overexpression provides a novel system to study mechanisms by which HO-1 induction affects allograft organ survival.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tg mice were generated by injecting C57BL/6J eggs with a 52-kb rat HO-1 construct containing 
27.7 kb of the 5' upstream region, 8.3 kb of the HO-1 gene, and 16 kb of the 3' downstream region (Fig. 1). Cloning was achieved via screening by PCR of a rat genomic P1 library (Genome Systems, St. Louis, MO) with two sets of PCR primers corresponding to the first exon (5'-GCT TCG GTG GGT TAT CTG CCG TTA T-3' and 5'-CAG TCT TAC AGG CGG GGA ATG TGA G-3') and fifth exon of the rat HO-1 gene (5'-GAG ACG CCC CGA GGA AAA TCC CAG AT-3' and 5'-CCC AAG AAA AGA GAG CCA GGC AAG AT-3'). PCR conditions were: 94°C for 5 min, 30 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 2 min, and a final 7-min 72°C extension. Three clones were identified as positive for both sets of primers, and restriction mapping revealed that two of them contained the same insert. One of the two P1 clones was digested with SfiI and subjected to preparative pulse field gel electrophoresis. The 52-kb band corresponding to the HO-1 gene and flanking regions was excised and digested with 
-agarase (Epicentre Technologies, Madison, WI) and then microinjected. Four founder lines tested positive by PCR, and three heterozygous Tg lines were established. Animals were genotyped by PCR using a set of primers specific for rat HO-1 exon 5 (5'-CCC TTC CTG TGT CTT CCT TTG-3' and 5'-ACA GCC GCC TCT ACC GAC CAC A-3'). PCR conditions were 94°C for 5 min, 30 cycles of 94°C for 30 s, 64°C for 1 min, 72°C for 2 min, and a final 7-min 72°C extension. All reactions were performed on a PerkinElmer 9700 machine (Foster City, CA).
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Cardiac transplants were performed between Tg/non-Tg (NTg) littermates (C57BL/6J; H-2b) and MHC-incompatible BALB/c (H-2d) mice (The Jackson Laboratory, Bar Harbor, ME). All animals were housed in the University of California animal facilities under specific pathogen-free conditions, and according to National Institutes of Health guidelines. Mice were anesthetized by pentobarbital, and vascularized heterotopic cardiac transplants were performed (24). Graft survival was assessed by daily palpation. Rejection was determined by cessation of the heartbeat, and verified by pathological examination.
To confirm the functional significance of HO-1, Tg recipients were treated with tin protoporphyrin (SnPP; Porphyrin Products, Logan, UT), a competitive HO activity inhibitor (7). SnPP was diluted in 100 mM NaOH to a stock solution of 50 mM and kept at -70°C until used. SnPP was administered twice i.p. (30 µM/kg) 1 day prior (day -1) and at the day of transplantation (day 0), followed by every second day thereafter (25).
Blood chemistry
Blood was collected from the retro-orbital plexus of Tg and NTg mice using heparin-coated capillaries. Total and differential cell counts, hemoglobin (Hb), hematocrit (Ht), and morphometric indexes were measured using automatic cell sorter (Celldyne; Abbott Laboratories, Abbott Park, IL). Total and direct bilirubin levels were determined (23). Carboxyhemoglobin (COHb) was determined by a blood glass analyzer (Bayer 855, Bayer, Germany).
RNA extraction/RT-PCR
Total RNA was isolated from heart, liver, spleen, kidney, and lung by the guanidine isothiocyanate acid/phenol method (26). Five micrograms of total RNA was used to synthesize first strand cDNA by the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen, San Diego, CA). The cDNA product was amplified by PCR using primers specific for the rat HO-1 exon 5 (CCC TTC CTG TGT CTT CCT TTG and ACA GCC GCC TCT ACC GAC CAC A); PCR conditions were 94°C for 5 min, 30 cycles of 94°C for 30 s, 64°C for 1 min, 72°C for 2 min, and a final 7-min 72°C extension. The RT-PCR product was separated in 1.5% (w/v) agarose gels and visualized with ethidium bromide.
Western blots
HO-1 protein levels were determined in total lysates and microsomal fractions. For total lysates, frozen tissue was homogenized in radioimmunoprecipitation assay lysis buffer (1x PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS), centrifuged at 10,000 x g for 10 min at 4°C with subsequent collection of the supernatant. For microsomal fractions, tissue was homogenized in 30 mM Tris-HCl (0.25 M sucrose, 0.15 M NaCl, pH 7.4), and centrifuged at 18,000 x g for 10 min. The supernatant fraction was then centrifuged at 100,000 x g for 60 min. Microsomal fractions were resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 2 mM MgCl2. The protein content was determined by Bio-Rad DC Protein Assay (Hercules, CA), based on the Lowry method (27). Aliquots were fractionated on 12% polyacrylamide NuPAGE Bis-Tris gels (Invitrogen) and then transferred to Hybond-ECL nitrocellulose membranes (Amersham, U.K.). Recombinant rat HO-1 protein (StressGen Biotechnologies, Victoria, BC, Canada) and preparations from HO-1-/- animals were used as positive and negative controls for HO-1 detection. Membranes were incubated with rabbit anti-HO-1 polyclonal Ab (SPA-896; 1/2000; StressGen) or goat anti-actin polyclonal Ab (sc-1616; 1/1000; Santa Cruz Biotechnology, Santa Cruz, CA), and then incubated with goat anti-rabbit IgG:HRP conjugate Ab (SAB-300; 1/5000; StressGen) or rabbit anti-goat IgG:HRP conjugate Ab (1/2000; Calbiochem, La Jolla, CA), respectively. Membranes were processed with an ECL reagent and exposed to film (Amersham).
HO activity
Tissues were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Homogenates (100 µl) were mixed with 0.8 mM NADPH, 0.8 mM glucose-6-phosphate, 1.0 U G-6-P dehydrogenase, 1 mM MgCl2, and 10 ml purified rat liver biliverdin reductase at 4°C. The reaction was initiated by the addition of hemin (final concentration 0.25 mM), followed by incubation at 37°C for 15 min. At the end of incubation period, any insoluble material was removed by centrifugation and supernatants were analyzed for bilirubin concentration. An extinction coefficient of 40 mM-1 cm-1 at A 460–530 nm was used to calculate the amount of bilirubin formed. Controls included naive samples in the absence of the NADPH generating system and all the ingredients of the reaction mixture in the absence of graft homogenates. Biliverdin reductase was purified from rat liver, as described (28).
HO-1 ELISA
Tissues were homogenized in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Flat-bottom microtiter 96-well plates were coated with 7 µg/ml anti-HO-1 mAb (OSA-111; StressGen) in PBS for 18 h. Unbound Ab was removed by washing, and remaining binding sites were blocked by incubation with a 5% BSA/PBS solution (1 h). Recombinant HO-1 (SPP-730) and tissue homogenate were diluted (0.5% BSA/0.05% Tween 20/PBS) and incubated in anti-HO-1 mAb-coated wells for 1 h. Subsequently, plates were incubated with rabbit anti-HO-1 polyclonal Ab (SPA-895; diluted 1/1000 in assay diluent; StressGen) for 30 min. Bound rabbit IgG was detected with a donkey anti-rabbit IgG-HRP conjugate (711-035-152; diluted 1/8000; The Jackson Laboratory). Unbound secondary Ab was removed by washing, and bound HRP was detected using 1 mg/ml o-phenylenediamine in substrate buffer (0.1% H2O2, 0.1 M citric acid, 0.2 M Na2HPO4, pH 5.0). The color reaction was stopped with 1 M HCl, and the OD at 490 nm was measured.
Morphology and immunohistology
Cardiac grafts were harvested at day 4, unless specified otherwise. Tissue was fixed in 10% buffered Formalin (Sigma-Aldrich, St. Louis, MO) overnight at room temperature, embedded in paraffin, coronally sectioned at 10 µm, and stained with H&E for evaluation of cell infiltrates by light microscopy. Cryostat sections of allografts were also analyzed by immunoperoxidase using mAbs to HO-1 (StressGen) and leukocyte subpopulations (BD PharMingen, San Diego, CA), as described (10, 24). Control sections were stained with isotype-matched mAbs or rabbit IgG and counterstained with hematoxylin.
Data analysis
Differences in measured variables between experimental groups were determined by Student’s t test. Values are expressed as mean ± SE, and differences were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have developed Tg mice under the control of the native rat HO-1 promoter. A rat genomic bacteriophage P1 library was screened by PCR for the HO-1 gene. Three clones were identified, and the fragment containing the promoter and HO-1 gene was excised by SfiI from one of the clones. The location of the promoter/coding region of HO-1 gene in the excised fragment was determined by restriction analysis and Southern blotting. The fragment was 52 kb long, spanning from 27.7 upstream to 16 kb downstream the HO-1 gene (Fig. 1). It was purified by pulse field gel electrophoresis, followed by -agarase digestion, and then microinjected into C57BL/6J eggs. Three transmitting Tg founder lines were established. No external phenotypic differences were observed between Tg mice and NTg littermates.
To confirm HO-1 expression at the mRNA level, RT-PCR was conducted using total RNA isolated from heart, lungs, liver, spleen, and kidneys of Tg vs NTg littermates. The PCR product was rat HO-1 specific and was detected selectively in Tg tissues (Fig. 2). We used primers that amplified a 166-bp product from rat HO-1 exon 5. Samples were treated with DNase I before first strand cDNA synthesis to avoid genomic DNA-based amplification. RNA samples from Tg mice that were not treated with reverse-transcriptase enzyme did not amplify specific band.
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6-fold in Tg line 1 vs 3-fold in Tg line 3 in comparison with NTg littermates. Hence, Tg mice exhibit a generalized HO-1 overexpression pattern. They expressed the transgene at the mRNA level in all five tissues studied, and significantly overexpressed HO-1 protein in liver (shown above), as well as in heart, spleen, and host leukocytes (shown below), as compared with NTg littermates. Our data suggest that the rat Tg was substantially more induced than the endogenous mouse HO-1 gene. Although HO-1 is crucial for iron reutilization and HO-1-/- mice are hypoferremic and anemic (29), we found no differences in serum iron, Hb, or Ht levels between Tg and NTg pairs (Table I). We also determined serum bilirubin and COHb levels, terminal products of the HO-1 enzymatic reaction. Although total bilirubin levels were comparable, Tg mice tended to show higher unconjugated bilirubin levels (p = 0.048) as compared with NTg controls (Table I). Similarly, Tg mice tended to have higher baseline COHb levels as compared with NTg littermates.
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To analyze the role of local HO-1 on allotransplant survival, hearts from C57BL/6J Tg or NTg mice were transplanted into BALB/c recipients. Whereas NTg hearts were rejected within1 wk (mean survival time ± SE = 6.5 ± 0.4 days; n = 6), Tg hearts survived 10.5 ± 0.7 days (n = 10; p = 0.0001). By using animals from different Tg lines, we ruled out a possible dysregulation of other genes due to random Tg insertion as a cause of increased survival. Indeed, as shown in Fig. 4A, donor hearts from Tg lines 1/2 had a similar survival rate (11.0 ± 1.1 days; n = 5) as donor hearts from Tg line 3 (10.0 ± 0.8 days; n = 5). As shown in Fig. 4B, HO activity (nm bilirubin/mg protein/h) in Tg hearts was 36% higher (p < 0.01) than in NTg controls (0.87 ± 0.05; n = 10, and 0.64 ± 0.08; n = 7, respectively). No differences were detected between individual Tg lines. Consistent with HO activity data, relative HO-1 protein expression measured by ELISA (ng/mg protein) in Tg hearts (0.137 ± 0.03) was significantly higher (p = 0.0001) as compared with NTg controls (n = 7–10/group), with no apparent differences between individual Tg lines.
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To evaluate the effect of host HO-1 overexpression, Tg or NTg C57BL/6J mice served as recipients of BALB/c cardiac grafts. As shown in Fig. 7A, hearts transplanted into Tg mice survived 26.8 ± 3.4 days (n = 10), significantly longer (p = 0.00009) than those transplanted into NTg controls (6.3 ± 0.1 days; n = 12). Hearts transplanted into Tg mice of lines 1 and 2 survived 36.0 ± 1.9 days (n = 5), significantly longer (p = 0.0002) than those transplanted into Tg line 3 mice (17.6 ± 2.2 days; n = 5). All five mice from line 1 (n = 3) or 2 (n = 2) survived at least 30 days, indicating that this graft prolongation resulted from HO-1 overexpression. Lower survival exhibited by Tg line 3 mice could be due to positional effects caused by random insertion of the Tg. This difference could also be due to the higher HO-1 induction displayed by Tg line 1 in comparison with Tg line 3 mice, as shown above.
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Because all donor hearts were of BALB/c origin, with no baseline differences in HO-1 expression, we then examined HO-1 levels in recipients’ spleens (Fig. 7B). HO activity (nm bilirubin/mg protein/h) in Tg animals (1.61 ± 0.12; n = 10) was at average 36% higher (p = 0.04), as compared with NTg controls (1.23 ± 0.16; n = 7). No significant differences were noted between animals from different Tg lines.
Relative HO-1 protein expression (ng/mg protein), as determined by ELISA, was also increased in Tg spleens (10.29 ± 0.9) as compared with controls (p = 0.000001; n = 7–10/group). Spleens from different Tg lines had similar HO-1 levels, implying increased HO-1 protein/HO activity in the spleen microenvironment. COHb, determined at day 4 posttransplant, was increased in Tg recipients (2.3 ± 0.4%; n = 4), as compared with NTg littermates (0.9 ± 0.3%; n = 4, p = 0.02). This indicates that increased HO-1 expression/HO activity resulted in increased CO generation and/or CO tissue levels.
Ten viable grafts were excised at day 4 for histological analysis. Cardiac allografts in NTg recipients (n = 5) showed more significant inflammatory cell infiltrates, vasculitis, and myocyte damage (coagulation necrosis, intracellular edema, cellular destruction) as compared with those transplanted into HO-1 Tg animals (n = 5) (Fig. 5, C and D). Grafts in Tg recipients that exhibited prolonged survival were also studied within 24 h of beating cessation (24, 30, 35, 37 days) and contrasted with rejecting grafts in NTg hosts (day 6). Severe cellular infiltrates with extensive interstitial edema, necrosis, fibrosis, calcification, increased vascular cell proliferation, and intravascular thrombosis were observed, confirming that in both HO-1 Tg and NTg animals grafts were ultimately rejected by a predominantly host cellular mechanism.
Immunoperoxidase staining of grafts harvested at day 4 (Fig. 8; Table II) showed that in contrast to a very low HO-1 expression observed in grafts transplanted into NTg recipients (Fig. 8c), there was marked HO-1 expression after transplantation into Tg recipients (Fig. 8d), predominantly in the infiltrating inflammatory cells. Grafts transplanted into Tg recipients exhibited a significant reduction in the number of infiltrating mononuclear cells that was characterized by a clear paucity of CD4 T cells (Fig. 8f). Although all allografts had comparable numbers of CD8+ cells (Fig. 8, g and h) and macrophages (Fig. 8, i and j), allografts in Tg mice lacked evidence of immune activation (CD25, Fig. 8l).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-myosin H chain promoter (21); or SMC specific, with human HO-1 gene under the control of SM-22 promoter (22). The use of a genome-based transgene resulted in expression levels in relation with the physiological scenario rather than fixed HO-1 levels. Under HO-1 stimulatory conditions, Tg animals still exhibited higher HO-1, as compared with NTg littermates. Furthermore, although baseline HO-1 expression did not show differences among animals from various Tg lines, stimulated HO-1 expression did, and it was Tg line dependent, as assessed in liver microsomes of hemin-treated mice. High homology between the rat and mouse HO-1 made it impossible to distinguish the Tg and endogenous HO-1 proteins because the available mAbs and polyclonal Abs recognize both. However, we were able to differentiate them at the genomic and mRNA levels.
Our results show that donor cardiac HO-1 overexpression resulted in a moderate inhibition of local inflammatory cell infiltration and a mild increase in allotransplant survival. Soares et al. (6) documented that graft HO-1 expression is required for cyclosporin A (CyA) plus cobra venom factor (CVF) treatment to abrogate rejection of mouse-to-rat cardiac xenotransplants. Under CyA plus CVF therapy, HO-1 was expressed by endothelial cells/SMC and myocytes in well-functioning grafts from HO-1+/+ mice. Our results indicate that graft HO-1 expression is also important in the allograft rejection response, and that increased local HO-1 results in prolonged cardiac transplant survival even in the absence of any concomitant immunosuppression.
The mechanism by which local HO-1 promotes graft prolongation remains to be elucidated. HO activity results in downstream products of heme catabolism with various cytoprotective functions (2, 4, 5), especially against apoptosis (6, 13, 15, 30, 31). Although increased survival caused by local HO-1 can be due to antiapoptotic and cytoprotective mechanisms, its anti-inflammatory properties should also be considered (32, 33). It has been shown that induction of HO-1 decreases endothelial cell adhesion molecules, such as P-/E-selectins (34) and ICAM-1 (35), which in turn may explain in part the decreased graft inflammatory response and somewhat decreased level of immune activation (CD25), as shown by our immunohistochemical analysis. However, local HO-1 induction only partially inhibited immune activation.
Interestingly, systemic/host HO-1 overexpression resulted in a more profound inhibition of the rejection response, resulting in long-term allograft survival, as compared with local/graft HO-1 overexpression, with comparable reduction in the total number of inflammatory infiltrates. This effect was strictly HO-1 dependent, and consistent with our previous findings (25), inhibition of HO activity by SnPP recreated vigorous acute rejection of otherwise long-term functioning cardiac allografts in Tg recipients. With Tg mice used as recipients, there was no difference in graft HO-1, so the effects were due primarily to HO-1 in recipients’ organs, most likely to be concentrated within the immune cell network. Indeed, we found significant HO-1 overexpression by graft-infiltrating host leukocytes. HO-1 overexpression detected in Tg spleens may be due to HO-1 overexpression in immune cells or it may imply HO-1 overexpression in the microenvironment in which cells interact with the host immune repertoire. Our immunohistological analysis showed diminished CD4 T cell and macrophage graft infiltration. However, it may be the absence of immune activation (CD25) resulting in a passive presence of inflammatory cells, which leads to prolonged graft survival. Indeed, local HO-1 overexpression resulted in a similar net reduction of total cell counts, but only in moderate graft survival prolongation. The lack of immune activation early posttransplant (day 4) suggests that host HO-1 overexpression blunts the alloimmune response at the graft site.
Systemically, Tg splenocytes tended to display lower proliferation indices against allogeneic stimulator splenocytes in both CD4+ and CD8+ subsets. However, splenocytes from rejecting Tg hosts showed comparable proliferation responses against donor-type Ags in MLR assay, and were able to effectively lyse target cells in a cytotoxicity assay (data not shown). This suggests that although immune activation was blunted in Tg recipients early after transplantation (day 4), it eventually ensued, leading to transplant rejection.
HO-1 activity results in the production of bilirubin that may act as an antioxidant (36) and anti-inflammatory (37) agent. By coordinated action with ferritin, HO-1 may also help to remove pro-oxidant heme groups that otherwise accumulate in the arterial wall to act in a proinflammatory fashion (38). We did not find any differences in Hb, Ht, serum iron, or bilirubin levels between Tg and NTg mice, suggesting that iron metabolism/bilirubin are not the major mechanisms of HO-1-induced protective effects. We favor the idea that increased tissue production of CO represents the major pathway responsible for HO-1 effects. Indeed, increased COHb levels in untreated Tg mice in this study became even more significant after transplantation, which may indicate higher tissue CO levels. When HO activity is inhibited, mouse hearts transplanted into CyA plus CVF-treated rats undergo widespread vascular thrombosis, extensive infiltration, and apoptosis of endothelial cells and monocytes (6). Likewise, inhibition of HO activity in our study triggered vigorous rejection of otherwise long-term surviving transplants in Tg recipients.
In summary, we have developed a new transgenic mouse with a modest generalized HO-1 overexpression that allowed us to demonstrate that although both local/graft and systemic/host HO-1 overexpression resulted in prolonged graft survival, the effect was much more profound with HO-1 overexpression in the recipient animal. The effect on graft survival was dependent on the inhibition of inflammatory cell infiltrates, and more significantly on the degree of inhibition of immune activation. Our study shows that systemic HO-1 overexpression resulted in a significant blunting of host alloreactivity in transplant recipients, of importance in considering future HO-1-inducing therapeutic regimens. Our Tg mouse represents a new tool to dissect the mechanisms by which HO-1 may interfere with the immune rejection cascade.
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Acknowledgments |
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Footnotes |
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2 J.A.A. and L.M. contributed equally to this work.
3 Current address: University of California, San Francisco Hooper Foundation, San Francisco, CA 94143-0552.
4 Current address: The First Department of Internal Medicine, Fukushima Medical University, Fukushima, Japan 960-1295.
5 Address correspondence and reprint requests to Dr. Jerzy W. Kupiec-Weglinski, The Dumont-University of California, Los Angeles Transplant Center, David Geffen School of Medicine, University of California, Los Angeles, CHS 77-120, Los Angeles, CA 90095. E-mail address: jkupiec{at}mednet.ucla.edu
6 Abbreviations used in this paper: HO-1/heme oxygenase-1; CO, carbon monoxide; COHb, carboxyhemoglobin; CoPP, cobalt protoporphyrin; CVF, cobra venom factor; CyA, cyclosporin A; Hb, hemoglobin; Ht, hematocrit; Tg, transgenic; NTg, non-Tg; SMC, smooth muscle cell; SnPP, tin protoporphyrin; WT, wild type.
Received for publication December 30, 2002. Accepted for publication May 28, 2003.
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-induced apoptosis in cultured fibroblast. Am. J. Physiol. Lung Cell. Mol. Physiol. 278:312.
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 H.-O. Pae, G.-S. Oh, B.-M. Choi, S.-C. Chae, Y.-M. Kim, K.-R. Chung, and H.-T. Chung Carbon Monoxide Produced by Heme Oxygenase-1 Suppresses T Cell Proliferation via Inhibition of IL-2 Production J. Immunol., April 15, 2004; 172(8): 4744 - 4751. [Abstract] [Full Text] [PDF]
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BALB/c). Hearts from Tg line 1/2 mice (Tg1/2; n = 5), Tg line 3 mice (Tg3; n = 5), and NTg controls (NTg; n = 6) were transplanted. Survival of Tg1/2 hearts (11.0 ± 1.1 days) or Tg3 hearts (10.0 ± 0.8 days) was significantly longer than NTg hearts (6.5 ± 0.4 days, p < 0.005). B, Cardiac HO activity (nmol bilirubin/mg protein/h). HO activity was determined in total lysates of cardiac homogenates. Assays were performed in age-matched Tg animals from lines 1 and 2 (n = 6), line 3 (n = 4), and NTg littermates (n = 7). Mean ± SE is shown.
