Hepatocellular heme oxygenase-1: a potential mechanism of erythropoietin-mediated protection after liver ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

Suppressor of cytokine signaling expression with increasing severity of murine hepatic ischemia-reperfusion injury.

BACKGROUND/AIMS: Preservation of function requires tight regulation of the cellular events initiated when hepatic ischemia is followed by reperfusion (IR). One important mechanism modulating the cytokine-directed response to injury is Suppressors of Cytokine Signaling. SOCS1 and SOCS3 ensure appropriate intensity and duration of cytokine signaling through negative feedback on JAK-STAT signaling. The contribution of SOCS1 and SOCS3-mediated regulation to the evolution of hepatic IR injury is unknown. METHODS: C57Blk6 mice were subjected to mild (20 min) or severe (90 min) hepatic ischemia. Liver was analyzed for cytokine and SOCS1/3 induction as well as JAK-STAT activation at intervals after reperfusion. RESULTS: Tnf, Il-1beta, and Il-6 expression paralleled increasing injury severity. Despite early phosphorylation of both STAT1 and STAT3 after severe injury, only nuclear translocation of activated STAT3, suggesting that the induction of target genes through JAK-STAT after IR is predominantly via STAT3. Socs3 was expressed across the injury spectrum while Socs1 was induced only in the face of severe IR injury. Severe IR in Il-6 deficient mice confirmed that Il-6, acting via STAT3, serves as a primary inducer of both regulatory mechanisms. CONCLUSIONS: Under the influence of IL-6-mediated STAT3 signaling, Socs1 serves as a complimentary regulatory mechanism when Socs3 is insufficient to limit cytokine-mediated inflammation after hepatic IR.

Orthotopic transplantation of intestinal mucosal organoids in rodents.

BACKGROUND: Orthotopic transplantation of intestinal mucosal organoids that contain putative mucosal stem cells serves as an important step toward implementing intestinal gene therapy and treatment for malabsorption syndromes in animals and humans. We hypothesized that intestinal mucosal organoids can be transplanted along the axis of the small bowel giving rise to a neomucosa expressing proteins of its donor origin. METHODS: Epithelial organoids were harvested from neonatal mice or rat small intestine with the use of a combination of enzymatic digestion with dispase and collagenase, and gravity sedimentation. In adult syngeneic recipients, a 7-cm segment of midjejunum was isolated, leaving its vascular pedicle intact. The remaining proximal and distal segments were anastomosed to restore intestinal continuity. The isolated segments were randomly subjected to surgical or chemical mucosectomy with a chelator solution for 30, 45, or 60 minutes and then compared. Histologic examination was used to confirm the presence of enterocytes, goblet cells, enteroendocrine cells, and Paneth cells in the neomucosal segments. To confirm the presence of ileal bile acid transporter (IBAT) gene message and function, we measured sodium-dependent bile acid uptake and IBAT-messenger RNA. Immunohistochemical examination using anti-IBAT antibodies was performed to demonstrate the expression of IBAT in the neomucosal segments. Experiments were repeated in a murine model transgenic for the green fluorescent protein to verify donor origin of the engrafted mucosa expressing IBAT. RESULTS: The area of peak IBAT function was found to be located in the terminal ileum. Organoid units harvested from this region were capable of generating a small-bowel neoileal mucosa after being seeded into the jejunum. This mucosa was histologically confirmed to differentiate into all 4 intestinal lineages and to express IBAT signal, confirming its donor-derived origin. Optimal engraftment of mucosa expressing the IBAT protein was found in isolated jejunal segments debrided for 45 minutes. Sodium-dependent bile acid uptake was 5-fold higher in the neoileum, compared with the jejunum. IBAT-mRNA levels in the neoileum were 18-100-fold higher than those in the jejunum. Areas of green fluorescent protein-positive mucosa stained positively with anti-IBAT antibody in adjacent sections, suggesting that the regenerated mucosa is from transplanted ileal stem cells. CONCLUSIONS: Orthotopic transplantation of epithelial organoids containing ileal stem cells was used to generate a neoileal mucosa that expressed all 4 intestinal lineages along with a new zone of active bile acid uptake and IBAT expression in a recipient jejunal segment.

Engraftment of mucosal stem cells into murine jejunum is dependent on optimal dose of cells.

BACKGROUND: Transplantation of intestinal mucosal stem cells is an important step in the development of intestinal gene therapy and treatment of intestinal mucosal diseases. We hypothesized that engraftment rates increase proportionally with increasing doses of seeded stem cells and increasing jejunal débridment. MATERIALS AND METHODS: Intestinal mucosal organoids were harvested from neonatal mice carrying a green-fluorescent protein (GFP) transgene and transplanted into adult GFP(-) mice (n = 66). In recipients, two jejunal segments (1.5 cm) were isolated with their blood circulation left intact with anastomosis of the distal and proximal segments to restore continuity. Debridement of native enterocytes was performed by perfusing luminally with ethylene diamine tetraacetc acid solutions for 20 min. A total of 5,000, 10,000, or 25,000 organoids were then seeded. Three weeks later, cross sections (n = 398) of the segments were evaluated for the presence of GFP(+) neomucosa using fluorescence microscopy. Additional segments were débrided for 30 and 40 min (n = 83). Other conditions were not tested because of the rate of high mortality in these experiments. RESULTS: The group seeded with 10,000 organoid units at 20 min showed the highest engraftment of GFP(+) epithelium. Engraftment was improved by increasing débridment times at this seeding density. Overall mortality was 70%. CONCLUSIONS: These findings suggest that there is an optimal seeding density of stem cell clusters for enhanced engraftment in this model. Mortality prohibited complete testing of all combinations of seeding density and debridement times.

Comparison of polyester scaffolds for bioengineered intestinal mucosa.

INTRODUCTION: Biodegradable polyester scaffolds have proven useful for growing neointestinal tissue equivalents both in vitro and in vivo. These scaffolds allow cells to attach and grow in a 3-dimensional space while nutrient flow is maintained throughout the matrix. The purpose of this study was to evaluate different biopolymer constructs and to determine mucosal engraftment rates and mucosal morphology. HYPOTHESIS: We hypothesized that different biopolymer constructs may vary in their ability to provide a good scaffolding onto which intestinal stem cell organoids may be engrafted. STUDY DESIGN: Eight different microporous biodegradable polymer tubes composed of polyglycolic acid (PGA), polylactic acid, or a combination of both, using different fabrication techniques were seeded with intestinal stem cell clusters obtained from neonatal rats. Three different seeded polymer constructs were subsequently placed into the omentum of syngeneic adult recipient rats (n = 8). Neointestinal grafts were harvested 4 weeks after implantation. Polymers were microscopically evaluated for the presence of mucosal growth, morphology, scar formation and residual polymer. RESULTS: Mucosal engraftment was observed in 7 out of 8 of the polymer constructs. A maximal surface area engraftment of 36% (range 5-36%) was seen on nonwoven, randomly entangled, small fiber PGA mesh coated with aerosolized 5% poly-L-lactic acid. Villous and crypt development, morphology and created surface area were best on PGA nonwoven mesh constructs treated with poly-L-lactic acid. Electrospun microfiber PGA had poor overall engraftment with little or no crypt or villous formation. CONCLUSION: Intestinal organoids can be engrafted onto biodegradable polyester scaffoldings with restitution of an intestinal mucosal layer. Variability in polymer composition, processing techniques and material properties (fiber size, luminal dimensions and pore size) affect engraftment success. Future material refinements should lead to improvements in the development of a tissue-engineered intestine.

Treatment of bile acid malabsorption using ileal stem cell transplantation.

BACKGROUND: We hypothesized that ileal stem cell clusters transplanted into a segment of jejunum can be used to treat bile acid malabsorption. STUDY DESIGN: In adult Lewis rats, a 15-cm segment of jejunum was isolated with its blood circulation left intact and partially stripped of enterocytes using luminal high-velocity perfusions with 3mmol/L ethylenediamine tetra-acetic acid solutions. Continuity was restored by anastomosing the proximal and distal gut. Ileal stem cell clusters were harvested from neonatal Lewis rats and transplanted into the stripped segments to generate a "neoileum." After 4weeks, recipients underwent resection of the native ileum, and the isolated neoileum was anastomosed in its place. After an additional 4weeks, a 48-hour stool collection was performed. The engrafted segment was harvested for taurocholate uptake studies, ileal bile acid transporter (IBAT) protein by immunohistomorphometry, and IBAT mRNA quantitation by reverse transcription polymerease chain reaction. Data were analyzed by ANOVA/t-test. Rats undergoing ileectomy, jejunectomy, or sham operations served as controls. RESULTS: Total bile acid loss in the stool was markedly lower in rats with a neoileum compared with rats with an ileectomy (p < 0.001). Total taurocholate uptake was notably increased in the neoileum compared with the jejunum (p < 0.001). IBAT protein signal intensity was considerably higher in the neoileum compared with jejunum (p < 0.001). IBAT mRNA amounts in the neoileal group were comparable with those in normal rat ileum and were considerably higher (p = 0.003) than in the jejunum. CONCLUSIONS: Ileal stem cell clusters were used to establish a new zone of bile acid uptake and IBAT expression in a jejunal segment. This neoileum eliminated loss of bile acids in the stool after ileectomy. This is the first time that transplantation of intestinal stem cell clusters has been shown to correct a clinical malabsorption syndrome.

Identification of optimal harvest sites of ileal stem cells for treatment of bile acid malabsorption in a dog model.

Ileal mucosal stem cells expressing the sodium-dependent ileal bile acid transporter (IBAT) have been successfully transplanted into the jejunum of rodents in projects aimed at creating a "neoileum" to treat bile acid malabsorption. To find optimal harvest sites for a dog model of stem cell transplantation, the exact location of peak IBAT expression in the donor ileum needs to be known. We therefore mapped IBAT function, IBAT mRNA, and IBAT protein in the ileum of Beagle dogs (N=3). Mucosal samples were taken every 5 cm in the ileum and every 20 cm in the jejunum of each dog. Sodium-dependent (active) and sodium-independent (passive) taurocholate uptake rates were measured using a standardized everted sleeve technique. IBAT mRNA concentrations were determined by semiquantitative reverse transcriptase-polymerase chain reaction and IBAT protein concentrations by fluorometric immunohistochemical analysis. The small bowel measured 208+/-17 cm (mean+/-standard error of the mean). Active and passive uptake rates were found to follow distinct distribution curves. Significant active uptake was seen only at the terminal 50 cm and peaked at 479+/-176 pM/mm(2). Depending on location, active uptake accounted for approximately half of the total uptake. IBAT mRNA and protein distributions corroborated uptake curves. The terminal 10 to 50 cm of ileum has the highest bile acid uptake capacity. This short segment appears to be the most promising donor site for ileal stem cell transplants to create a "neoileum" in dogs.