Accommodated organs can survive in the presence of anti-organ antibodies and complement. Heme oxygenase-1 (HO-1) is essential to ensure accommodation in concordant xenotransplant models. However, whether induction of HO-1 over-expression could protect porcine endothelial cells (PECs) against human xenoantibodies and complement-mediated lysis and induce an in vitro accommodation is still unknown. The SV40-immortalized porcine aorta-derived endothelial cell line (iPEC) was pre-incubated with 20, 50, or 80 μmol/L of cobalt-protoporphyrins IX (CoPPIX) for 24 h, and the HO-1 expression in iPECs was analyzed by using Western blotting. CoPPIX-treated or untreated iPECs were incubated with normal human AB sera, and complement-dependent cytotoxicity (CDC) was measured by both flow cytometry and lactate dehydrogenase (LDH) release assay. In vitro treatment with CoPPIX significantly increased the expression of HO-1 in iPECs in a dose-dependent manner. Over-expression of HO-1 was successfully achieved by incubation of iPECs with either 50 or 80 μmol/L of CoPPIX. However, HO-1 over-expression did not show any protective effects on iPECs against normal human sera-mediated cell lysis. In conclusion, induction of HO-1 over-expression alone is not enough to protect PECs from human xenoantibodies and complement-mediated humoral injury. Additionally, use of other protective strategies is needed to achieve accommodation in pig-to-primate xenotransplantation.
Animals ; Antibodies, Heterophile ; immunology ; Cell Line ; Complement System Proteins ; immunology ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; immunology ; Heme Oxygenase-1 ; metabolism ; Humans ; Protoporphyrins ; pharmacology ; Swine ; Up-Regulation ; drug effects

To explore the role of heme oxygenase (HO)-1 experimental system in dachengqitang (DD) ameliorating ALI induced by lipopolysaccharide (LPS) in mice. Seventy-five male Kunming mice were randomly divided into control group (normal saline was instilled intratracheally(50 microL/per mouse), LPS group (LPS was instilled intratracheally to replicate ALI model), DD + LPS group, DD + LPS + ZnPP (ZnPP, HO-1 specific inhibitor) group and the DD group. Mice were killed at 6 h after administration. Lung indexes were tested; lung histomorphological changes were observed under microscope, and neutrophils (PMN) number and protein content of bronchoalveolar lavage fluid (BALF) were measured; HO-1 mRNA and protein expression in lung tissue were detected by RT-PCR and Western blot. The results showed that intratracheal instillation of LPS in mice can cause significant morphological changes in lung tissue. Both PMN numbers and protein content in BALF were increased. meanwhile the expressions of HO-1 mRNA and protein in lung tissue were increased. Pretreated with DD and then intratracheally instillated LPS coulde ameliorat lung tissue injury, reduced PMN BALF number and protein content, but increase HO-1 mRNA and protein expression in the lung tissue when compared with LPS. HO-1 inhibitor ZnPP coulde inhibite the ameliorative effect of DD. The results suggest that the ameliorative effect of DD on ALI induced by LPS in mice were related with upregulation HO-1 mRNA and protein.
Acute Lung Injury ; chemically induced ; prevention & control ; Animals ; Blotting, Western ; Bronchoalveolar Lavage Fluid ; chemistry ; cytology ; Gene Expression Regulation, Enzymologic ; drug effects ; Heme Oxygenase-1 ; antagonists & inhibitors ; genetics ; metabolism ; Leukocyte Count ; Lipopolysaccharides ; Lung ; drug effects ; enzymology ; pathology ; Male ; Mice ; Neutrophils ; cytology ; drug effects ; Phytotherapy ; methods ; Plant Extracts ; pharmacology ; Proteins ; metabolism ; Protoporphyrins ; pharmacology ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Treatment Outcome

OBJECTIVETo investigate the effect of heme oxygenase/carbon monoxide (HO-1/CO) system on lipid deposition at aortic intima and the mechanism involved in hyperlipidemic rabbits.

METHODSTotally 32 rabbits, were divided into four groups. One group as control. Three groups for the following treatments: 1.5% cholesterol ration (Ch group, n = 8); 1.5% cholesterol ration plus HO-1 inducer hemin (Hm group, n = 8); and instead of hemin, the HO-1 inhibitor, zinc protoporphyrin IX (Zn group, n = 8) was given by injection into the abdominal cavity. Experiments were lasted for 12 weeks. Rabbit aortas were then isolated as the samples for histopathologic and ultrastructural examination. The protein expressions of HO-1 and endothelin-1 (ET-1) were investigated by immunohistochemical staining and Western blot analysis.

RESULTSComparing with the Ch group, rabbits of the Hm group showed a remarkably less extent of lipid deposition at the aortic intima [(17.9 ± 3.0)% vs (54.0 ± 4.2)%], and rabbits of the Zn group had a marked extent of lesion development [(61.1 ± 3.5)%]. Lipid deposition, endothelial damage and neo-intimal formation were less severe in rabbits of the Hm group than those in the Zn or Ch group, respectively. Comparing with the control group, rabbits of the Ch group showed a significant decrease of aortic NO production and cNOS activity. However, there were an enhancement of CO production and HO-1 activity (P < 0.01). Compared with Ch group, rabbits of the Hm group showed a remarkable elevation of aortic HO activity and CO production, whereas rabbits of the Zn group showed a marked decrease of both parameters. Compared with the Ch group, rabbits of the Hm group demonstrated a marked reduction of aorta ET-1 expression, whereas Zn group had a significantly higher ET-1 expression.

CONCLUSIONSModulation of HO-1/CO system may improve vascular endothelial function and inhibit smooth muscle cell proliferation in hypercholesterolemic rabbits, likely through a compensatory mechanism and a reduction of ET-1 expression, eventually leading to an inhibition of atherosclerotic plaque development.

Animals ; Aorta ; metabolism ; pathology ; Carbon Monoxide ; metabolism ; Cholesterol ; pharmacology ; Endothelin-1 ; metabolism ; Enzyme Inhibitors ; pharmacology ; Heme Oxygenase-1 ; antagonists & inhibitors ; metabolism ; Hemin ; pharmacology ; Hyperlipidemias ; metabolism ; pathology ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Plaque, Atherosclerotic ; metabolism ; pathology ; prevention & control ; Protoporphyrins ; pharmacology ; Rabbits ; Tunica Intima ; metabolism ; pathology

BACKGROUNDThe hepatopulmonary syndrome (HPS) is a severe vascular complication in lungs resulting in systemic hypoxemia in patients with cirrhosis and portal hypertension. The underlying structural change in HPS is intrapulmonary vasodilation, which can lead to impaired oxygenation of pulmonary venous blood. It has been demonstrated that the heme oxygenase-1/carbon monoxide (HO-1/CO) system plays an important role in the control of vascular tone. The aim of this study was to further investigate the role of HO-1 in the pathogenesis of HPS in animal model.

METHODSTotally 35 rats were divided into liver cirrhosis, zinc protoporphyrin IX (ZnPP), cobalt protoporphyrin (CoPP) and sham groups. Biliary cirrhosis was established in the first three groups by bile duct ligation. Rats in the ZnPP and CoPP groups received once intraperitoneal injection of ZnPP and CoPP, respectively, 24 hours before sample collection. Expression of HO-1 mRNA in lung was detected by reverse-transcription polymerase chain reaction, while protein expression was determined by immunohistochemical analysis. Hematoxylin and eosin staining was performed to confirm the presence of liver cirrhosis and intrapulmonary vasodilation. Arterial blood gases, mean arterial pressure and portal vein pressure were also measured. Analysis of variance or Wilcoxon statistical methods were used to determine statistical significance.

RESULTSCompared with the sham group, the cirrhotic group demonstrated increased expression of pulmonary HO-1 mRNA and protein (P < 0.01). The level of arterial carboxyhemoglobin (COHb), alveolar-arterial oxygen gradient (A-aPO2), mean arterial pressure, portal vein pressure (P < 0.05, respectively), and intrapulmonary vasodilation were also significantly increased. Compared with the cirrhotic group, CoPP treatment increased pulmonary HO-1 mRNA and protein expression, the level of A-aPO2 (P < 0.05 respectively), COHb (P < 0.01), and intrapulmonary vasodilation, while ZnPP treatment decreased pulmonary HO-1 mRNA and protein expression, the level of COHb (P < 0.05 respectively), and intrapulmonary vasodilation, without obvious alteration of mean arterial pressure and portal vein pressure.

CONCLUSIONIncreased pulmonary HO-1 expression is an important contributor to the development of experimental HPS.

Animals ; Enzyme Inhibitors ; therapeutic use ; Heme Oxygenase-1 ; genetics ; metabolism ; Hemodynamics ; Immunohistochemistry ; Liver Cirrhosis ; enzymology ; genetics ; Lung ; drug effects ; metabolism ; Male ; Protoporphyrins ; therapeutic use ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction

Erythropoietic protoporphyria (EPP) is a rare disorder of heme biosynthesis caused by mutations in the gene encoding the enzyme ferrochelatase. In EPP, deficient ferrochelatase activity leads to the excessive production and biliary excretion of protoporphyrin (PP). The major clinical features of EPP are photosensitivity and hepatobiliary disease that may progress to severe liver disease, that are caused by the toxicity of PP. EPP-related liver disease has been treated medically or surgically including liver transplantation. We described a 20-year-old male with severe liver disease who was diagnosed with EPP based on clinical and laboratory findings. He was treated with cholestyramine resin. Six months after the treatment, he was doing well without any abdominal pain or photosensitivity.
Bilirubin/blood ; Cholestyramine Resin/*therapeutic use ; Edema/complications ; Erythema/complications ; Ferrochelatase/genetics/metabolism ; Humans ; Liver Diseases/complications/*diagnosis/pathology ; Male ; Protoporphyria, Erythropoietic/complications/*diagnosis/drug therapy ; Protoporphyrins/metabolism ; Young Adult

This study was aimed to investigate the effects of cobalt protoporphyrin (CoPP) on hyperexpression of heme oxygenase-1 (HO-1) and secretion of IL-10 in bone marrow-derived mesenchymal stem cells (BMMSC). The BMMSC were isolated from rats and cultured, then were randomly divided into 4 groups, and were induced in culture medium with CoPP of 0, 25, 50, and 75 µmol/L respectively. The expressions of CD34, CD45, CD44 and CD71 in BMMSC were detected by flow cytometry; the osteoblast and adipocyte induction of BMMSC was determined by alizarin red and oil red staining respectively; the proliferation status of BMMSC was assayed by MTT method; the HO-1 expression in test groups was detected by RT-PCR; the optimal concentration of CoPP for induction of HO-1 expression was screened. The ELISA kit was used to detect the HO-1 levels in each groups. The results showed that the expressions of CD34, CD45 in BMMSC were negative while the expressions of CD44 and CD71 in BMMSC were positive. After induced by osteoblast and adipocyte inductor, BMMSC were positive for alizarin red staining and oil red staining. Various concentrations of CoPP had no significant impact on cell proliferation. When the CoPP concentration of 50 µmol/L could induce strong expression of HO-1, expression level in test groups was significantly higher than that in control group, meanwhile the IL-10 secretion was significantly higher than that in control group. It is concluded that CoPP has no ability to promote cell proliferation, 50 µmol/L is the optimum concentration for up-regulation of CoPP-induced BMMSC HO-1 expression, the up-regulation of HO-1 expression can increased IL-10 secretion significantly.
Animals ; Bone Marrow Cells ; metabolism ; Cells, Cultured ; Heme Oxygenase (Decyclizing) ; metabolism ; Interleukin-10 ; secretion ; Mesenchymal Stromal Cells ; metabolism ; Protoporphyrins ; pharmacology ; Rats ; Rats, Wistar ; Up-Regulation

OBJECTIVETo investigate the protective effect of overexpressed heme oxygenase-1 (HO-1) in hypoxia and the correlation between HO-1 overexpressoin and hypoxia inducible factor-1alpha (HIF-1alpha) in human hepatoma HepG2 cells.

METHODSThe expressions of HO-1 and HIF-1alpha mRNA as well as the protein were detected by RT-PCR and Western blotting, respectively. MTT assay was used to examine the relative cell survival rate. The total superoxide dismutase (SOD) activity was examined with a SOD kit.

RESULTSHypoxia induced overexpression of HO-1 gene in HepG2 cells at transcriptional and translational levels. The relative survival rate of HepG2 cells under hypoxia was significantly decreased after the HO-1 protein overexpression was inhibited by ZnPPIX (P < 0.01). The total SOD activity of cells was significantly increased after cells were treated by hypoxia for 16 hours (P < 0.05), while decreased significantly by HO-1 inhibitor ZnPPIX treatment (P < 0.01). HIF-1alpha was upregulated under hypoxia. In addition, the HO-1 overexpression under hypoxia was decreased by HIF-1alpha inhibitor, while the HIF-1alpha expression level under hypoxia was not significantly changed after HO-1 expression was inhibited by ZnPPIX.

CONCLUSIONThe overexpression of HO-1 in hypoxic HepG2 cells is HIF-1alpha-dependent or at least partly HIF-1alpha-dependent. The relative survival rate of hypoxic hepatoma cells was significantly decreased by HO-1 inhibitor treatment. The results of this study may offer new thought and drug target for the therapy of human hepatoma in the future.

Cell Hypoxia ; Cell Survival ; Heme Oxygenase-1 ; antagonists & inhibitors ; metabolism ; Hep G2 Cells ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Protoporphyrins ; pharmacology ; RNA, Messenger ; metabolism ; Superoxide Dismutase ; metabolism

OBJECTIVETo investigate the effects of CO release inhibitor zinc protoporphyria IX (ZnPP IX) and NO release inhibitor L-NAME on the content of cGMP in the penile tissue of rats.

METHODSThirty Wistar rats were randomly divided into a normal control, a ZnPP IX, and an L-NAME group, given saline (1 ml/kg/d), ZnPP IX (45 micromol/kg/d) and L-NAME (50 mg/kg/d), respectively, for 7 days. Then all the rats were killed, homogenate made from their penile tissues and detected for the contents of NOS, NO, CO and cGMP.

RESULTSThe contents of CO, NOS, NO and cGMP were all reduced in both the ZnPP IX and L-NAME groups as compared with the control group (P < 0.05).

CONCLUSIONZnPP IX and L-NAME can reduce the concentrations of CO and NO in the penile tissues of rats, and consequently the content of cGMP.

Animals ; Carbon Monoxide ; antagonists & inhibitors ; metabolism ; Cyclic GMP ; metabolism ; Male ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; antagonists & inhibitors ; metabolism ; Penis ; drug effects ; metabolism ; Protoporphyrins ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo study the protective effect of islet xenograft and its possible mechanism of high expression of heme oxygenase-1 (HO-1) in donor pancreas islet induced by cobalt protoporphyrin (CoPP).

METHODSMale SD rats and C57BL/6 mouse were used as donors and recipients respectively. Donors were divided into 3 groups according to different pretreatment 24 hours before donation: control group (injected intraperitoneally with NaCl), induce group [injected intraperitoneally with cobalt-protoporphyrin (CoPP)], block group (injected intraperitoneally with CoPP and zinc protoporphyrin simultaneously). A modified approach was used for islet isolation.Recipients were rendered diabetic by intraperitoneal injection of streptozotocin. Islets were transplanted into mouse subrenal capsule. Postoperative mouse glycemia were monitored daily and normoglycemia time was compared among each group. The receptor mouse serum IL-10 was detected by ELISA approach, and real-time PCR was used to check the expression of IL-10 mRNA in islet graft tissues. The graft tissues were observed for the lymphocyte infiltration after HE staining.

RESULTSDiabetes mice accepted islets untreated, induced or blocked maintained the euglycemia for (9.3 +/- 1.4), (16.3 +/- 1.5) and (9.7 +/- 1.0) d respectively. The xeno-islets presented HO-1 over-expression survived much longer than that absent (P < 0.05), it was no significance between control group and block group (P > 0.05). The mouse islet serum IL-10 content after induction was (73.0 +/- 9.7) pg/ml, significantly higher than (30.6 +/- 3.9) pg/ml of the untreated group and (32.1 +/- 5.9) pg/ml of the blocked group (P < 0.05), there was no difference between control group and block group (P > 0.05). Moreover, the IL-10 mRNA expression up-regulated statistic significantly in HO-1 induced islet xeno-graft. Pathological examination showed that the graft lymphocyte infiltration of the induced group was obviously less serious than the other two groups.

CONCLUSIONSThe higher expression of HO-1 induced by CoPP in vivo would significantly prolong graft survival time and its mechanism could be related to immune modulation of IL-10.

Animals ; Diabetes Mellitus, Experimental ; metabolism ; pathology ; surgery ; Graft Survival ; Heme Oxygenase-1 ; drug effects ; metabolism ; Interleukin-10 ; metabolism ; Islets of Langerhans ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL ; Pancreas Transplantation ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Subrenal Capsule Assay ; Transplantation, Heterologous

OBJECTIVETo examine the effect of angiotensin-converting enzyme inhibitor (ACEI) on hydrogen peroxide (H(2)O(2))-induced decrease in contraction of isolated rataortic rings, and to investigate its mechanisms.

METHODSThe thoracic aortic rings with endothelium of male Sprague-Dawley rats were mounted on a bath system. Isometric contractions of aortic rings were measured.

RESULT(1) After incubation with captopril (an ACEI with sulfhydryl groups) or perindoprilate (an ACEI without sulfhydryl groups), the decrease in contraction response to PE was prevented in arteries which were pretreated with 300 micromol/L H(2)O(2). (2) Captopril enhanced the HO-1 activity of thoracic aorta. After inhibition of HO-1 activity by ZnPP IX, the protection effect of captopril was abrogated. Hemin (an inducer of HO-1) and bilirubin (a product of HO-1) could mimic the antioxidative effect of captopril. (3) Both L-NAME (an inhibitor of NOS) and methylene blue (an inhibitor of GC) could abolish the protective effect of captopril. (4) SNAP could protect aortic rings against H(2)O(2) attack, and ZnPP IX could cancel the effect of SNAP.

CONCLUSIONBoth ACEI with or without sulfhydryl groups could prevent the H(2)O(2) induced decrease in contraction responses to PE in intact aortic rings. The increase of NO and activation of HO-1 might be involved in the mechanism.

Angiotensin-Converting Enzyme Inhibitors ; pharmacology ; Animals ; Antioxidants ; pharmacology ; Aorta, Thoracic ; drug effects ; metabolism ; physiology ; Bilirubin ; pharmacology ; Captopril ; pharmacology ; Heme Oxygenase-1 ; metabolism ; Hemin ; pharmacology ; Hydrogen Peroxide ; pharmacology ; In Vitro Techniques ; Male ; Methylene Blue ; pharmacology ; NG-Nitroarginine Methyl Ester ; pharmacology ; Nitric Oxide ; metabolism ; Penicillamine ; analogs & derivatives ; pharmacology ; Protoporphyrins ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Vasoconstriction ; drug effects