Human HO-1 Research Articles

Endothelial Effects of Simultaneous Expression of Human HO-1, E5NT, and ENTPD1 in a Mouse.

The vascular endothelium is key target for immune and thrombotic responses that has to be controlled in successful xenotransplantation. Several genes were identified that, if induced or overexpressed, help to regulate the inflammatory response and preserve the transplanted organ function and metabolism. However, few studies addressed combined expression of such genes. The aim of this work was to evaluate in vivo the effects of the simultaneous expression of three human genes in a mouse generated using the multi-cistronic F2A technology. Male 3-month-old mice that express human heme oxygenase 1 (hHO-1), ecto-5'-nucleotidase (hE5NT), and ecto-nucleoside triphosphate diphosphohydrolase 1 (hENTPD1) (Transgenic) were compared to wild-type FVB mice (Control). Background analysis include extracellular nucleotide catabolism enzymes profile on the aortic surface, blood nucleotide concentration, and serum L-arginine metabolites. Furthermore, inflammatory stress induced by LPS in transgenic and control mice was used to characterize interleukin 6 (IL-6) and adhesion molecules endothelium permeability responses. Transgenic mice had significantly higher rates of extracellular adenosine triphosphate and adenosine monophosphate hydrolysis on the aortic surface in comparison to control. Increased levels of blood AMP and adenosine were also noticed in transgenics. Moreover, transgenic animals demonstrated the decrease in serum monomethyl-L-arginine level and a higher L-arginine/monomethyl-L-arginine ratio. Importantly, significantly decreased serum IL-6, and adhesion molecule levels were observed in transgenic mice in comparison to control after LPS treatment. Furthermore, reduced endothelial permeability in the LPS-treated transgenic mice was noted as compared to LPS-treated control. The human enzymes (hHO-1, hE5NT, hENTPD1) simultaneously encoded in transgenic mice demonstrated benefits in several biochemical and functional aspects of endothelium. This is consistent in use of this approach in the context of xenotransplantation.

HO-1 Protects Remnant Liver against Dysfunction after Major Hepatectomy in Humans

Objective During major resection of liver carcinoma, liver regeneration (LR) is induced by various clinical and biological factors. Heme oxygenase (HO)-1 has been found as a key inducer of LR in preclinical trial. The clinical evidence for the role of HO-1 in liver dysfunction (LD) including LR is still unknown and has been included in this study. Methods Therefore, plasma HO-1 were monitored during perioperative period in 65 patients with hepatectomy, with 35 training and 30 validation cohorts. LD were evaluated by liver function serum markers and calculation of regeneration indices, respectively. Results In the training setting, HO-1 levels were remarkably reduced after liver resection (P < 0.001) and gradually recovered within 7 days after surgery. Preoperative HO-1 specifically predicted LD during the first week after surgery (AUC: 0.757; P = 0.01). In patients with LD and complications after surgery, HO-1 levels decreased throughout the perioperative period. In addition, we had also confirmed that low levels of HO-1 (<169 ng/ml) before surgery were associated with an increase in the incidence of postoperative LD and morbidity (P = 0.007, P = 0.045), and decrease of liver regeneration (P = 0.005). And HO-1 was an independent predictor for poor clinical outcome. Conclusions We had provided the first clinical data verifying that human HO-1 was related to LD. Consequently, HO-1 levels can be used as effective clinical indicators to predict LD and clinical outcome, and can be used as intervention target before liver resection.

Viable pigs after simultaneous inactivation of porcine MHC class I and three xenoreactive antigen genes GGTA1, CMAH and B4GALNT2.

Cell surface carbohydrate antigens play a major role in the rejection of porcine xenografts. The most important for human recipients are α-1,3 Gal (Galactose-alpha-1,3-galactose) causing hyperacute rejection, also Neu5Gc (N-glycolylneuraminic acid) and Sd(a) blood group antigens both of which are likely to elicit acute vascular rejection given the known human immune status. Porcine cells with knockouts of the three genes responsible, GGTA1, CMAH and B4GALNT2, revealed minimal xenoreactive antibody binding after incubation with human serum. However, human leucocyte antigen (HLA) antibodies cross-reacted with swine leucocyte antigen class I (SLA-I). We previously demonstrated efficient generation of pigs with multiple xeno-transgenes placed at a single genomic locus. Here we wished to assess whether key xenoreactive antigen genes can be simultaneously inactivated and if combination with the multi-transgenic background further reduces antibody deposition and complement activation. Multiplex CRISPR/Cas9 gene editing and somatic cell nuclear transfer were used to generate pigs carrying functional knockouts of GGTA1, CMAH, B4GALNT2 and SLA class I. Fibroblasts derived from one- to four-fold knockout animals, and from multi-transgenic cells (human CD46, CD55, CD59, HO1 and A20) with the four-fold knockout were used to examine the effects on human IgG and IgM binding or complement activation in vitro. Pigs were generated carrying four-fold knockouts of important xenoreactive genes. In vitro assays revealed that combination of all four gene knockouts reduced human IgG and IgM binding to porcine kidney cells more effectively than single or double knockouts. The multi-transgenic background combined with GGTA1 knockout alone reduced C3b/c and C4b/c complement activation to such an extent that further knockouts had no significant additional effect. We showed that pigs carrying several xenoprotective transgenes and knockouts of xenoreactive antigens can be readily generated and these modifications will have significant effects on xenograft survival.

Pro-apoptotic effect of haem oxygenase-1 in human colorectal carcinoma cells via endoplasmic reticular stress.

Several biological effects of haem oxygenase (HO)‐1, including anti‐inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO‐1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO‐1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)‐3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT‐15, LOVO and HT‐29 cells in serum‐free (SF) conditions with increased HO‐1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP‐induced apoptosis and HO‐1 protein expression in human colon cancer cells. CoPP‐induced apoptosis of colon cancer cells was prevented by the addition of the pan‐caspase inhibitor, Z‐VAD‐FMK (VAD), and the Casp‐3 inhibitor, Z‐DEVD‐FMK (DEVD). N‐Acetyl cysteine inhibited reactive oxygen species‐generated H2O2‐induced cell death with reduced intracellular peroxide production, but did not affect CoPP‐induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO‐1 expression by those cells, and knockdown of HO‐1 protein expression by HO‐1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO4 or biliverdin, induced DNA ladders, and cleavage of Casp‐3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R‐like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM‐ and CoPP‐induced apoptosis. Increased GRP78 level and formation of the HO‐1/GRP78 complex were detected in CORM‐ and CoPP‐treated human CRC cells. A pro‐apoptotic role of HO‐1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.

Generation by somatic cell nuclear transfer of GGTA1 knockout pigs expressing soluble human TNFRI-Fc and human HO-1.

Herein, we successfully generated transgenic pigs expressing both soluble human tumor necrosis factor receptor I IgG1-Fc (shTNFRI-Fc) and human hemagglutinin (HA)-tagged-human heme oxygenase-1 (hHO-1) without Gal epitope. Healthy cloned pigs were produced by somatic cell nuclear transfer (SCNT) using the genetically modified cells. The genetic disruption of the GGTA1 genes and absence of expression of BS-IB4 lectin in tail-derived fibroblast of the SCNT-generated piglets were successfully confirmed. The expression of shTNFRI-Fc and HAhHO-1 was fully identified with protective effect against oxidative stress and apoptosis stimulation. Antibody-mediated complement-dependent cytotoxicity assay for examining the immuno-reactivity of transgenically derived pig cells showed that pigs lacking GGTA1 with the expression of double genes reduce the humoral barrier to xenotransplantation, more than pigs simply expressing double genes and the wild type. Through this approach, rapid production of a pig strain deficient in various genes may be expected to be applicable for xenotransplantation research without extensive breeding protocols.

Heme oxygenase-1 affects generation and spontaneous cardiac differentiation of induced pluripotent stem cells.

Cellular stress can influence efficiency of iPSCs generation and their differentiation. However, the role of intracellular cytoprotective factors in these processes is still not well known. Therefore, we investigated the effect of HO-1 (Hmox1) or Nrf2 (Nfe2l2), two major cytoprotective genes. Hmox1-/- fibroblasts demonstrated decreased reprogramming efficiency in comparison to Hmox1+/+ cells. Reversely, pharmacological enhancement of HO-1 resulted in higher number of iPSCs colonies. Importantly, elevated level of both p53 and p53-regulated miR-34a and 14-3-3σ was observed in HO-1-deficient fibroblasts whereas downregulation of p53 in these cells markedly increased their reprogramming efficiency. In human fibroblasts HO-1 silencing also induced p53 expression and affected reprogramming outcome. Hmox1+/+ and Hmox1-/- iPSCs similarly differentiated in vitro to cells originating from three germ layers, however, lower number of contracting cells was observed during this process in HO-1-deficient cells indicating attenuated cardiac differentiation. Importantly, silencing of Hmox1 in murine ESC using CRISPR/Cas-9 editing also impaired their spontaneous cardiac differentiation. Decreased reprogramming efficiency was also observed in Nrf2-lacking fibroblasts. Reversely, sulforaphane, a Nrf2 activator, increased the number of iPSCs colonies. However, both Nfe2l2+/+ and Nfe2l2-/- iPSCs showed similar pluripotency and differentiation capacity. These results indicate that regulation of HO-1 expression can further optimize generation and cardiac differentiation of iPSCs. © 2018 IUBMB Life, 70(2):129-142, 2018.

Vascular patterning in human heterotopic ossification

Heterotopic ossification (HO, also termed myositis ossificans) is the formation of extra-skeletal bone in muscle and soft tissues. HO is a tissue repair process gone awry, and is a common complication of surgery and traumatic injury. Medical strategies to prevent and treat HO fall well short of addressing the clinical need. Better characterization of the tissues supporting HO is critical to identifying therapies directed against this common and sometimes devastating condition. The physiologic processes of osteogenesis and angiogenesis are highly coupled and interdependent. However, few efforts have been made to document the vascular patterning within heterotopic ossification. Here, surgical pathology case files of 29 human HO specimens were examined by vascular histomorphometric analysis. Results demonstrate a temporospatial patterning of HO vascularity that depends on the "maturity" of the bony lesion. In sum, human HO demonstrates a time- and space-dependent pattern of vascularization suggesting a coupled pathophysiologic process involving the coordinate processes of osteogenesis and angiogenesis. Further imaging studies may be used to further characterize vasculogenesis within HO and whether anti-angiogenic therapies are a conceivable future therapy for this common condition.

Haem oxygenase-1 polymorphisms can affect HCV replication and treatment responses with different efficacy in humanized mice.

Enhancement of host anti-oxidant enzymes, such as haemoxygenase-1, may attenuate virus-mediated hepatocyte injury, while the induction of HO-1 by cobalt-protoporphyrin-IX (CoPP) administration, as the application of its haem degradation product biliverdin (BV), was shown to hinder HCV replication in vitro. In addition, (GT)n -repeats length in the polymorphic region of the HO-1 promoter may affect HO-1 expression and responsiveness to infection and disease severity. Aim of this study was to investigate the antiviral and hepatoprotective effects of CoPP-mediated HO-1 induction, alone or in combination with interferon alpha (peg-IFNα), in HCV-infected mice harbouring hepatocytes from donors with different HO-1-promoter polymorphisms. Upon establishment of HCV infection, CoPP, BV and peg-IFNα were given alone or in combination. Viraemia changes and intrahepatic human gene expression were determined by qRT-PCR and immunohistochemistry. CoPP administration increased human HO-1 expression and significantly reduced viraemia, although changes correlated with promoter length (Δ0.5log and Δ2log reduction with medium- and short-polymorphism respectively). Polymorphisms did not influence BV-mediated antiviral effects (Δ1log). Notably, HO-1 induction attenuated basal HCV-driven enhancement of interferon genes and pro-inflammatory cytokines, both in cells with short- or medium-polymorphisms. Moreover, simultaneous administration of CoPP and peg-IFNα reduced viraemia even stronger (median 3log), whereas 1log viraemia reduction was determined in mice receiving peg-IFNα monotherapy. Although the protective function of HO-1 could be elicited in vivo with both host polymorphisms, the strength of HO-1 induction and suppression of HCV occurred in a polymorphism-dependent manner, indicating that host-genetic determinants may affect disease progression and infection outcome.

In-Cell Enzymology To Probe His-Heme Ligation in Heme Oxygenase Catalysis.

Heme oxygenase (HO) is a ubiquitous enzyme with key roles in inflammation, cell signaling, heme disposal, and iron acquisition. HO catalyzes the oxidative conversion of heme to biliverdin (BV) using a conserved histidine to coordinate the iron atom of bound heme. This His-heme interaction has been regarded as being essential for enzyme activity, because His-to-Ala mutants fail to convert heme to biliverdin in vitro. We probed a panel of proximal His mutants of cyanobacterial, human, and plant HO enzymes using a live-cell activity assay based on heterologous co-expression in Escherichia coli of each HO mutant and a fluorescent biliverdin biosensor. In contrast to in vitro studies with purified proteins, we observed that multiple HO mutants retained significant activity within the intracellular environment of bacteria. X-ray crystallographic structures of human HO1 H25R with bound heme and additional functional studies suggest that HO mutant activity inside these cells does not involve heme ligation by a proximal amino acid. Our study reveals unexpected plasticity in the active site binding interactions with heme that can support HO activity within cells, suggests important contributions by the surrounding active site environment to HO catalysis, and can guide efforts to understand the evolution and divergence of HO function.

Efficient production of multi-modified pigs for xenotransplantation by 'combineering', gene stacking and gene editing.

Xenotransplantation from pigs could alleviate the shortage of human tissues and organs for transplantation. Means have been identified to overcome hyperacute rejection and acute vascular rejection mechanisms mounted by the recipient. The challenge is to combine multiple genetic modifications to enable normal animal breeding and meet the demand for transplants. We used two methods to colocate xenoprotective transgenes at one locus, sequential targeted transgene placement - ‘gene stacking’, and cointegration of multiple engineered large vectors - ‘combineering’, to generate pigs carrying modifications considered necessary to inhibit short to mid-term xenograft rejection. Pigs were generated by serial nuclear transfer and analysed at intermediate stages. Human complement inhibitors CD46, CD55 and CD59 were abundantly expressed in all tissues examined, human HO1 and human A20 were widely expressed. ZFN or CRISPR/Cas9 mediated homozygous GGTA1 and CMAH knockout abolished α-Gal and Neu5Gc epitopes. Cells from multi-transgenic piglets showed complete protection against human complement-mediated lysis, even before GGTA1 knockout. Blockade of endothelial activation reduced TNFα-induced E-selectin expression, IFNγ-induced MHC class-II upregulation and TNFα/cycloheximide caspase induction. Microbial analysis found no PERV-C, PCMV or 13 other infectious agents. These animals are a major advance towards clinical porcine xenotransplantation and demonstrate that livestock engineering has come of age.

Simultaneous Overexpression of Functional Human HO-1, E5NT and ENTPD1 Protects Murine Fibroblasts against TNF-α-Induced Injury In Vitro.

Several biomedical applications, such as xenotransplantation, require multiple genes simultaneously expressed in eukaryotic cells. Advances in genetic engineering technologies have led to the development of efficient polycistronic vectors based on the use of the 2A self-processing oligopeptide. The aim of this work was to evaluate the protective effects of the simultaneous expression of a novel combination of anti-inflammatory human genes, ENTPD1, E5NT and HO-1, in eukaryotic cells. We produced an F2A system-based multicistronic construct to express three human proteins in NIH3T3 cells exposed to an inflammatory stimulus represented by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine which plays an important role during inflammation, cell proliferation, differentiation and apoptosis and in the inflammatory response during ischemia/reperfusion injury in several organ transplantation settings. The protective effects against TNF-α-induced cytotoxicity and cell death, mediated by HO-1, ENTPD1 and E5NT genes were better observed in cells expressing the combination of genes as compared to cells expressing each single gene and the effect was further improved by administrating enzymatic substrates of the human genes to the cells. Moreover, a gene expression analyses demonstrated that the expression of the three genes has a role in modulating key regulators of TNF-α signalling pathway, namely Nemo and Tnfaip3, that promoted pro-survival phenotype in TNF-α injured cells. These results could provide new insights in the research of protective mechanisms in transplantation settings.

Non-coding RNA derived from the region adjacent to the human HO-1 E2 enhancer selectively regulates HO-1 gene induction by modulating Pol II binding.

Recent studies have disclosed the function of enhancer RNAs (eRNAs), which are long non-coding RNAs transcribed from gene enhancer regions, in transcriptional regulation. However, it remains unclear whether eRNAs are involved in the regulation of human heme oxygenase-1 gene (HO-1) induction. Here, we report that multiple nuclear-enriched eRNAs are transcribed from the regions adjacent to two human HO-1 enhancers (i.e. the distal E2 and proximal E1 enhancers), and some of these eRNAs are induced by the oxidative stress-causing reagent diethyl maleate (DEM). We demonstrated that the expression of one forward direction (5′ to 3′) eRNA transcribed from the human HO-1 E2 enhancer region (named human HO-1enhancer RNA E2-3; hereafter called eRNA E2-3) was induced by DEM in an NRF2-dependent manner in HeLa cells. Conversely, knockdown of BACH1, a repressor of HO-1 transcription, further increased DEM-inducible eRNA E2-3 transcription as well as HO-1 expression. In addition, we showed that knockdown of eRNA E2-3 selectively down-regulated DEM-induced HO-1 expression. Furthermore, eRNA E2-3 knockdown attenuated DEM-induced Pol II binding to the promoter and E2 enhancer regions of HO-1 without affecting NRF2 recruitment to the E2 enhancer. These findings indicate that eRNAE2-3 is functional and is required for HO-1 induction.

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1×10(5) cells) were cultured with hTNF-α (20ng/mL) and cycloheximide (10μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24h, 31.6±3.2 vs. 60.4±8.3%, respectively; p<0.05). Caspase-3/-7 activity of the shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12h, 812,452±113,078 RLU vs. 88,240±10,438 RLU, respectively; p<0.05). These results show that shTNFRI-Fc and HA-hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury. Utilization of the F2A self-cleaving peptide is a promising tool for generating multiple TG pigs for xenotransplantation.

Heme oxygenase-1 expression prevents inflammation in acute cardiac injury (P5190)

Abstract Heme oxygenase-1 (HO-1) degrades pro-oxidant heme into iron, carbon monoxide, and biliverdin. Overexpression of HO-1 prevents chronic heart failure by modulating the inflammatory response. However, when HO-1 must be induced in the course of injury to prevent cardiac failure has not been determined. We hypothesized that induction of HO-1 overexpression at the onset of acute cardiac injury prevents cardiac dysfunction through modulation of the innate immune response. We generated mice with inducible overexpression of a human HO-1 (hHO-1) transgene (MHC-HO-1 mice) by breeding mice with cardiac-specific expression of a tamoxifen (TAM)-inducible Cre recombinase (MHC-Cre mice) with mice containing an hHO-1 transgene preceded by a floxed stop signal (CBA-flox mice). Maximal induction of hHO-1 occurs 3 days after TAM administration (40 mg/kg body weight on two consecutive days) in MHC-HO-1 mice. In accord with previous reports, we found that MHC-Cre mice develop acute systolic dysfunction after TAM administration, which is characterized by severe inflammation, diffuse cardiomyocyte necrosis, and &amp;gt;80% mortality. There was an 18-fold increase in cardiac neutrophils in MHC-Cre mice treated with TAM, but the mononuclear phagocyte population was unchanged. In MHC-HO-1 mice, cardiac hHO-1 overexpression attenuated acute cardiac injury, as mortality and systolic dysfunction caused by Cre recombinase were prevented and there was no evidence of myocyte damage or cardiac inflammation.

Nrf2 activation is associated with Z-DNA formation in the human HO-1 promoter

Using a luciferase reporter assay, we previously demonstrated that a Z-DNA-forming sequence of alternating thymine–guanine repeats in the human heme oxygenase-1 gene (HO-1) promoter is involved in nuclear factor erythroid-derived 2 (NF-E2)–related factor 2 (Nrf2)-mediated HO-1 promoter activation. However, the actual Z-DNA formation in this native genomic locus has not been experimentally demonstrated. To detect Z-DNA formation in vivo, we generated a construct containing the Z-DNA-binding domain of human adenosine deaminase acting on double-stranded RNA 1 fused with enhanced green fluorescence protein, designated as the Z-probe. A chromatin immunoprecipitation assay using an anti-GFP antibody showed that the Z-probe detects the well-characterized Z-DNA formation in the CSF1 promoter. Using this detection system, we demonstrated that the glutathione-depleting agent, diethyl maleate, induced Nrf2-dependent Z-DNA formation in the HO-1 promoter, but not in the thioredoxin reductase 1 gene promoter. Moreover, a time course analysis revealed that Z-DNA formation precedes HO-1 transcriptional activation. Concurrent with Z-DNA formation, nucleosome occupancy was reduced, and the recruitment of RNA polymerase II was enhanced in the HO-1 promoter region, suggesting that Z-DNA formation enhances HO-1 gene transcription. Furthermore, Nrf2-induced BRG1 recruitment to the HO-1 promoter temporarily occurred simultaneously with Z-DNA formation. Thus, these results implicate Nrf2-dependent Z-DNA formation in HO-1 transcriptional activation and suggest the involvement of BRG1 in Z-DNA formation.

Heme oxygenase-1 attenuates epithelial-to-mesenchymal transition of human peritoneal mesothelial cells

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells has been regarded as an early mechanism of peritoneal fibrosis. A substantial and rapidly growing literature indicates that HO-1 provides the provenance for pathways that can interrupt virtually all major mechanisms of tissue injury. The effects of HO-1 expression on EMT, which plays a critical role in the development of peritoneal membrane (PM) fibrosis, are unknown and its roles in peritoneal fibrosis has not been studied, yet. A piece of human omentum obtained from consenting patients undergoing elective abdominal surgery was used for study. We treated the human peritoneal mesothelial cells (HPMCs) with high glucose solution and HO-1 inducer (hemin, 10 μmol/L). To further investigate the pure effect of HO-1 on EMT of mesothelium, gene transfer of recombinant Adenovirus-harboring human HO-1 (Adv-HO-1 gene) to HPMCs was done. Exposure of HPMCs to HG solution resulted in an increase of the expression of mesenchymal markers such as α-smooth muscle actin (α-SMA) and was associated with a decrease in the expression of epithelial markers, E-cadherin. HO-1 protein expression was decreased in the same situation. Treatment of HPMCs with HO-1 inducer, hemin showed a dosage-dependent amelioration of HG induced changes in markers of EMT with increase of expression of HO-1. Human HO-1 gene transfection resulted in a significant increase in HO-1 expression and ameliorated HG-induced changes in expression of E-cadherin and α-SMA. Taken together, our results suggest that HO-1 has a critical role in the modulation of peritoneal fibrosis, and, more important, the suppression of EMT. This study is the first to show the beneficial effect of HO-1 on reversing EMT in MC.

Generation and Characterization of Human Heme Oxygenase-1 Transgenic Pigs

Xenotransplantation using transgenic pigs as an organ source is a promising strategy to overcome shortage of human organ for transplantation. Various genetic modifications have been tried to ameliorate xenograft rejection. In the present study we assessed effect of transgenic expression of human heme oxygenase-1 (hHO-1), an inducible protein capable of cytoprotection by scavenging reactive oxygen species and preventing apoptosis caused by cellular stress during inflammatory processes, in neonatal porcine islet-like cluster cells (NPCCs). Transduction of NPCCs with adenovirus containing hHO-1 gene significantly reduced apoptosis compared with the GFP-expressing adenovirus control after treatment with either hydrogen peroxide or hTNF-α and cycloheximide. These protective effects were diminished by co-treatment of hHO-1 antagonist, Zinc protoporphyrin IX. We also generated transgenic pigs expressing hHO-1 and analyzed expression and function of the transgene. Human HO-1 was expressed in most tissues, including the heart, kidney, lung, pancreas, spleen and skin, however, expression levels and patterns of the hHO-1 gene are not consistent in each organ. We isolate fibroblast from transgenic pigs to analyze protective effect of the hHO-1. As expected, fibroblasts derived from the hHO-1 transgenic pigs were significantly resistant to both hydrogen peroxide damage and hTNF-α and cycloheximide-mediated apoptosis when compared with wild-type fibroblasts. Furthermore, induction of RANTES in response to hTNF-α or LPS was significantly decreased in fibroblasts obtained from the hHO-1 transgenic pigs. These findings suggest that transgenic expression of hHO-1 can protect xenografts when exposed to oxidative stresses, especially from ischemia/reperfusion injury, and/or acute rejection mediated by cytokines. Accordingly, hHO-1 could be an important candidate molecule in a multi-transgenic pig strategy for xenotransplantation.

Unregulated brain iron deposition in transgenic mice over‐expressing HMOX1 in the astrocytic compartment

The mechanisms responsible for pathological iron deposition in the aging and degenerating mammalian CNS remain poorly understood. The stress protein, HO-1 mediates the degradation of cellular heme to biliverdin/bilirubin, free iron, and CO and is up-regulated in the brains of persons with Alzheimer's disease and Parkinson's disease. HO-1 induction in primary astroglial cultures promotes deposition of non-transferrin iron, mitochondrial damage and macroautophagy, and predisposes cocultured neuronal elements to oxidative injury. To gain a better appreciation of the role of glial HO-1 in vivo, we probed for aberrant brain iron deposition using Perls' method and dynamic secondary ion mass spectrometry in novel, conditional GFAP.HMOX1 transgenic mice that selectively over-express human HO-1 in the astrocytic compartment. At 48 weeks, the GFAP.HMOX1 mice exhibited increased deposits of glial iron in hippocampus and other subcortical regions without overt changes in iron-regulatory and iron-binding proteins relative to age-matched wild-type animals. Dynamic secondary ion mass spectrometry revealed abundant FeO⁻ signals in the transgenic, but not wild-type, mouse brain that colocalized to degenerate mitochondria and osmiophilic cytoplasmic inclusions (macroautophagy) documented by TEM. Sustained up-regulation of HO-1 in astrocytes promotes pathological brain iron deposition and oxidative mitochondrial damage characteristic of Alzheimer's disease-affected neural tissues. Curtailment of glial HO-1 hyperactivity may limit iron-mediated cytotoxicity in aging and degenerating neural tissues.

Heme Oxygenase Gene Targeting to Adipocytes Attenuates Adiposity and Vascular Dysfunction in Mice Fed a High-Fat Diet

We examined the hypothesis that adipocyte dysfunction in mice fed a high-fat (HF) diet can be prevented by lentiviral-mediated and adipocyte specific-targeting delivery of the human heme oxygenase-1 (aP2-HO-1). A bolus intracardial injection of aP2-HO-1 resulted in expression of human HO-1 for up to 9.5 months. Transduction of aP2-HO-1 increased human HO-1 expression in fat tissues without affecting murine HO-1. In mice fed a HF diet, aP2-HO-1 transduction attenuated the increases in body weight, blood glucose, blood pressure, and inflammatory cytokines, as well as the content of both visceral and subcutaneous fat. Transduction of aP2-HO-1 increased the numbers of adipocytes of small cell size (P<0.05), insulin sensitivity (P<0.05), adiponectin levels, as well as vascular relaxation to acetylcholine compared with HF mice administered the aP2-green fluorescent protein. Adipocytes of mice fed a HF diet expressed high levels of peroxisome proliferator activator receptor, aP2, C/EBP, and Wnt5b proteins and displayed marked increases in Peg1/Mesoderm specific transcript (P<0.03). Transduction of aP2-HO-1 lowered the elevated levels of these proteins and increased Sonic hedgehog, Wnt10b, and β-catenin (P<0.05). Inhibition of HO activity by administration of tin mesoporphyrin to HF-fed mice transduced with the aP2-HO-1 reversed the decrease in Peg1/Mesoderm-specific transcript, TNFα, and MCP-1 levels. Collectively, this novel study demonstrates that adipocyte-specific overexpression of HO-1 attenuates HF-mediated adiposity and vascular dysfunction; increases insulin sensitivity; and improves adipocyte function by increasing adiponectin, Shh, and WNT10b, and by decreasing inflammatory cytokines. These effects are reversed by the HO activity inhibitor, stannous mesoporphyrin.

PP131. Regulation of heme oxygenase/carbon monoxide (ho/co) and l-arginine/nitric oxide (no) pathways by human chorionic gonadotropin (hcg) in human fetal endothelium

HO/CO and l-arginine/NO pathways are critical regulators of vascular function in early human pregnancy. HO is the rate-limiting enzyme in heme catabolism leading to the formation of biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase, carbon monoxide, and free iron. HO-1, the inducible isoform of the enzyme, is essential to promote placental function and fetal development (1). HO-1 is also required to maintain nitric oxide synthesis (2). Human chorionic gonadotropin (hCG) is the first blastocyst-derived hormonal signal playing a significant role in implantation and placentation (3). We hypothesized that hCG upregulates HO-1 and thus positively regulates early vascular events in human pregnancy. In this study we used human umbilical vein endothelial cells (HUVECs) to demonstrate that hCG up-regulates mRNA and protein abundance of HO-1. The effect of hCG on l-arginine transport and the expression of hCAT1, the main cationic amino acid transporter involved in endothelial nitric oxide synthesis were evaluated. HUVEC were isolated from normal pregnancies (approval from the Institutional Review Board) by collagenase digestion and grown (37°C, 5% CO2) in medium 199 supplemented with 20% newborn and fetal calf sera. Cells were treated with hCG (0.5-100IU/mL, 0-24hours and CoCl2 (150μM, 6hours) as positive control. Total proteins were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membranes and incubated with monoclonal anti human HO-1 or anti β-actin and respective secondary antibody conjugated with horseradish peroxidase. Protein bands were detected by enhanced chemiluminiscence. Total RNA was isolated and cDNA was obtained by reverse transcription. PCR assays were performed in a reaction mix containing 0.5μM primers, dNTPs, Taq DNA polymerase, 2 mM MgCl2. Relative abundance of HO-1, hCAT-1 and 28S were determined. l-[(3)H]Arginine transport (100μM, 2 μCi ml(-1), 37°C, 1min) were determined in HUVECs exposed (30min) to hCG (0.5-100IU/mL) and CORM-2 (CO donor). The highest level of HO-1 protein were obtained at 5IU/mL of hCG and declined at higher concentrations. hCG increased HO-1 protein abundance and maximal response was observed at 6 hours (4-fold), decreasing at 24hours exposure. These effects of hCG on HO-1 protein abundance are well correlated with changes in HO-1 mRNA regarding time of exposure and concentration of hCG. L-arginine transport and hCAT-1 mRNA were also increased (2.8-fold and 4.5-fold, respectively) by hCG. Upregulation of HO-1 as well as the increase in l-arginine transport and hCAT-1 mRNA in HUVECs are novel effects of hCG, which occur at physiological plasma concentrations of the hormone, as those found in the first weeks of pregnancy. These changes could be relevant for the maintenance of the NO synthesis in fetal and maternal vasculature, ensuring the predominance of the vasodilator tone. These effects could be also critical for vascular remodelling of uterine circulation and placental vascular development in early stages of pregnancy, the lack of which could result in abnormal placentation as described in preeclampsia and fetal growth restriction.