Human Oral Mucosa as a Potentially Effective Source of Neural Crest Stem Cells for Clinical Practice.

We report in this study on the isolation and expansion of neural crest stem cells (NCSCs) from the epithelium of oral mucosa (OM) using reagents that are GMP-certified and FDA-approved for clinical use. Characterization analysis showed that the levels of keratins K2, K6C, K4, K13, K31, and K15-specific to OM epithelial cells-were significantly lower in the experimental NCSCs. While SOX10 was decreased with no statistically significant difference, the earliest neural crest specifier genes SNAI1/2, Ap2a, Ap2c, SOX9, SOX30, Pax3, and Twist1 showed a trend in increased expression in NCSCs. In addition, proteins of Oct4, Nestin and Noth1 were found to be greatly expressed, confirming NCSC multipotency. In conclusion, our study showed that the epithelium of OM contains NCSCs that can be isolated and expanded with clinical-grade reagents to supply the demand for multipotent cells required for clinical applications in regenerative medicine. Supported by Emmaus Medical Inc.

Thrombospondin-1 induction and VEGF reduction by proteasome inhibition.

The present study focuses on investigating the expression of thrombospondin-1 (TSP-1), a natural inhibitor of neovascularization. Immunofluorescent staining was used to detect the expression of TSP-1 in rabbit corneal tissue with vascularization induced by limbectomy. TSP-1 was detected in healthy and Cultured Autologous Oral Mucosal Epithelial Cell Sheet (CAOMECS) grafted rabbit corneas. TSP-1 was not detected in diseased corneas. Rabbit and human primary oral mucosal and corneal epithelial cells were cultured and treated with proteasome inhibitor (PI) in vitro. Changes in the expression of TSP-1, HIF-1 alpha and 2 alpha, VEGF-A, and VEGF receptor were analyzed by Western blotting. Neovascularization developed in rabbits' corneas as early as 1 month after limbectomy and was stable for at least 3 months. HIF-1 alpha and VEGF-A expression was reduced in CAOMECS grafted corneas, as compared to sham corneas. While TSP-1 expression was decreased in injured corneas, it was expressed in CAOMECS grafted corneas, but still less expressed compared to healthy corneas. PI treatment, of human oral mucosal and corneal epithelial cells increased TSP-1 expression and reduced VEGF-A expression. The results showed that TSP-1 expression was lost in injured corneal surface and that CAOMECS grafting restored TSP-1 expression to certain extent. Proteasome inhibition treatment increased TSP-1 and decreased VEGF-A expression in human oral mucosal and corneal epithelial cells. The result suggests that corneal neovascularization could be managed with the inhibition of the proteasome after CAOMECS grafting and increase corneal transparency.

Aldehyde Dehydrogenases Expression in Corneal Epithelial Cells with Limbal Stem Cell Deficiency.

PURPOSE: The purpose of the present study is to investigate the expression of aldehyde dehydrogenases (ALDHs) in rabbit corneas with limbal stem cell deficiency (LSCD) and corneas treated with cultured autologous oral mucosa epithelial cell sheet CAOMECS designed to reconstruct the ocular surface with LSCD. METHODS: New Zealand white rabbit autologous oral mucosal epithelial cells were isolated from a buccal biopsy and cultured to be grafted back onto corneas of rabbit model of LSCD. Immunofluorescent staining and Western blot analysis were used to compare the expression of ALDH1A1 and ALDH1A3 in healthy, LSCD-diseased, CAOMECS treated corneas. Human oral mucosal and corneal epithelial cells (OMECS and CECs) were cultured and treated with retinoic acid (RA) to further investigate the expression of ALDHs. RESULTS: In healthy corneas, ALDH1A1 and ALDH1A3 were markedly expressed in basal cells of corneal epithelium. In LSCD diseased corneas, ALDH1A1 and ALDH1A3 were markedly expressed in the conjunctivalized apical epithelial cells, the goblet cells, and the stroma. CAOMECS grafted corneas showed a decreased expression of ALDHs as compared to LSCD diseased corneas. Western blot analysis confirmed the up regulation of ALDH1A1 and ALDH1A3 expression in LSCD-diseased corneal epithelial cells. CAOMECS expressed low levels of ALDH1A1 and ALDH1A3, as compared to diseased CECs (D-CEC). When ALDH1A3 was up regulated by retinoic acid treatment in OMECS, Pax-6 expression was down regulated, suggesting a decrease in regenerative capacity when ALDH enzymes are up regulated. CONCLUSIONS: These findings report for the first time the up regulation of ALDH1A1 and ALDH1A3 in rabbit corneas with LSCD and document that CAOMECS grafting used to reconstruct corneal epithelium may reduce the expression levels of ALDH enzymes.

cG-CAOMECS-clinical-grade cultured autologous oral mucosal epithelial cell sheet.

The present study reports the feasibility and successful production of rabbit cG-CAOMECS, designed to reconstruct corneal epithelium of patients with bilateral limbal stem cell deficiency. To produce a safe, chemically defined and FDA compliant cG-CAOMECS, oral mucosal epithelial cells were isolated from a biopsy of rabbit buccal tissue and seeded on a cGMP-certified cell culture surface coated with GMP-grade extracellular matrix. A newly designed clinical-grade medium (KaFa™ medium) was utilized to carry out cell expansion. Detachment and harvesting of the produced cell sheet was accomplished using collagenase treatment. Live cell imaging and morphological analysis techniques were used to examine cell growth. Cells attached onto the surface and self-assembled into colony-forming units (CFUs). Microscopic examination showed that CFUs formed during the first 5 days, and basal monolayer cell sheet formed in less than 10 days. Cells expanded to form a multilayered epithelial cell sheet that was harvested after 17-19 days in culture. Immunostaining and Western blot analyses showed that deltaNp63 was expressed in the basal cells and K3/K12 was expressed in the apical cells, indicating the presence of corneal epithelial-like cells in the produced cell sheet. Adhesion molecules, E-cadherin, beta-catenin, and Cnx43 were also expressed and exhibited the epithelial integrity of the cell sheet. The expression of integrin-beta1 and beta4 confirmed that the collagenase treatment used for detaching and harvesting the cell sheet did not have adverse effects. Our results showed that the utilization of clinical-grade and FDA-approved reagents successfully supported the production of cG-CAMECS.

Clinical Trials of Limbal Stem Cell Deficiency Treated with Oral Mucosal Epithelial Cells.

The corneal surface is an essential organ necessary for vision, and its clarity must be maintained. The corneal epithelium is renewed by limbal stem cells, located in the limbus and in palisades of Vogt. Palisades of Vogt maintain the clearness of the corneal epithelium by blocking the growth of conjunctival epithelium and the invasion of blood vessels over the cornea. The limbal region can be damaged by chemical burns, physical damage (e.g., by contact lenses), congenital disease, chronic inflammation, or limbal surgeries. The degree of limbus damage is associated with the degree of limbal stem cells deficiency (partial or total). For a long time, the only treatment to restore vision was grafting part of the healthy cornea from the other eye of the patient or by transplanting a cornea from cadavers. The regenerative medicine and stem cell therapies have been applied to restore normal vision using different methodologies. The source of stem cells varies from embryonic stem cells, mesenchymal stem cells, to induced pluripotent stem cells. This review focuses on the use of oral mucosa epithelial stem cells and their use in engineering cell sheets to treat limbal stem cell deficient patients.

Vitrification and storage of oral mucosa epithelial cell sheets.

Shipping time and shipping delays might affect the quality of the stem cells based engineered "organs." In our laboratory, we have developed a limbal stem cell deficient (LSCD) rabbit model. To reverse the LSCD, we cultured oral mucosal epithelial cells for 2-3 weeks and engineered cultured autologous oral mucosa epithelial cell sheets (CAOMECS), which were grafted on the LSCD cornea. The purpose of this study was to vitrify CAOMECS and to store it until the CAOMECS can be grafted onto patients. CAOMECS were vitrified in LN2 for up to 204 days. We tested two different methods of vitrification with different solutions; however, CAOMECS were only viable when they were not stored in a vitrification solution; results were only reported from this CAOMECS. On the basis of hematoxylin and eosin staining, we showed that the CAOMECS morphology was well preserved after long-term storage in LN2 . Most of the preservation solutions maintained the CAOMECS phenotype (Ki67, proliferating cell nuclear antigen (PCNA), Beta-Catenin, ZO-1, E-Cadherin, CK3, CK4, CK13). The exception was the solution composed with ethylene glycol and Dimethyl sulfoxide (DMSO): this resulted in loss of DeltaN-p63 expression. DeltaN-p63 is an important marker for cell proliferation. The expression of proteins involved in cell-cell connection and the differentiation markers were maintained. Apoptosis was not detected in the thawed CAOMECS. We demonstrated that CAOMECS can be stored long-term in LN2 without affecting their morphology and phenotype.

Engineering, differentiation and harvesting of human adipose-derived stem cell multilayer cell sheets.

Aim: The study goals were to engineer and harvest scaffold-free undifferentiated/differentiated multilayer human adipose-derived stem cell (hADSC) cell sheets, in absence of treatment. Materials & methods: The hADSC are seeded in 35 mm culture dishes. At confluence or when multilayer cell sheets are formed, hADSC are treated with predefined differentiation culture media (adipocyte, chondrocyte and osteoblast). Results: Undifferentiated hADSC and differentiated adipocyte, osteoblast and chondrocyte hADSC multilayer cell sheets (hADSCmCS) have been harvested. Hematoxylin & eosin showed the formation of multilayer cell sheets. Undifferentiated hADSC multilayer cell sheets preserve their stem cell markers. Differentiated adipocyte, osteoblast and chondrocyte hADSCmCS expressed specific markers. Conclusion: This simple protocol opens possibilities to engineer scaffold-free hADSCm cell sheet to transplant them on damaged organs.

Corneal keratin aggresome (CKAGG) formation and clearance by proteasome activation.

PURPOSE: To understand the mechanism of corneal keratin expression and clearance in corneal epithelium with Limbal Stem Cell Deficiency (LSCD). The hypothesis is that LSCD-induced proteasome dysfunction is a contributing factor to keratin aggregation, causing corneal keratin aggresome (CKAGG) formation. METHOD: LSCD was surgically induced in rabbit corneas. LSCD corneal epithelial cells (D-CEC) were collected to investigate keratin K4 and K13 expression and CKAGG formation. Oral mucosal epithelial cells (OMECS) were isolated and cultured to study K4 and K13 expression. Cultured cells were treated with proteasome inhibitor to induce CKAGG formation. RESULTS: K4 and K13 were strongly expressed in D-CEC, with additional higher molecular weight bands of K4 and K13, suggesting CKAGG formation. Double staining of K4/K13 and ubiquitin showed co-localization of these keratins with ubiquitin in D-CEC. Proteasome inhibition also showed K4/K13 modification and accumulation in cultured OMECS, similar to D-CEC. Proteasome activation was then performed in cultured OMEC. There was no accumulation of keratins, and levels of unmodified keratins were found significantly reduced. CONCLUSION: Results showed an abnormal expression of K4 and K13 after LSCD-induced proteasome dysfunction, which coalesce to form CKAGG in Corneal Epithelial Cells (CEC). We propose that CKAGG formation may be one of the causative factors of morphological alterations in the injured corneal epithelium, and that CKAGG could potentially be cleared by enhancing proteasome activity.

Feeder Cells Free Rabbit Oral Mucosa Epithelial Cell Sheet Engineering.

The optimal cell culture method of autologous oral mucosal epithelial cell sheet is not well established for a safe transplantation on to the patients' ocular surface. Animal serum and 3T3 mouse feeder cells are currently being used to stimulate the growth of the epithelial cells. However, the use of animal compounds can have potential side effects for the patient after transplantation of the engineered cell sheet. In the present study, we focused on engineering a rabbit oral mucosal epithelial cell sheet without 3T3 mouse feeder cells using a mix of Dulbecco's Modified Eagle Medium/Bronchial Epithelial Cell Growth Medium culture media (DMEM/BEGM). Autologous oral mucosal epithelial cell sheets, engineered with DMEM/BEGM feeder cell free culture media, were compared to those cultured in presence of serum and feeder cells. Using a DMEM/BEGM mix culture media, feeder cell free culture condition, autologous oral mucosal epithelial cells reached confluence and formed a multilayered sheet. The phenotype of engineered cell sheets cultured with DMEM/BEGM were characterized and compared to those cultured with serum and feeder. Hematoxylin and eosin staining showed the formation of a similar stratified multilayer cell sheets, in both culture conditions. The expression of deltaN-p63, ABCG2, PCNA, E-cadherin, Beta-catenin, CK3, CK4, CK13, Muc5AC, was similar in both culture conditions. We demonstrated that rabbit autologous oral mucosal epithelial cell sheet can be engineered, in feeder cell free conditions. The use of the DMEM/BEGM culture media to engineer culture autologous oral mucosa epithelial cell sheet will help to identify key factors involved in the growth and differentiation of oral mucosal epithelial cells.

Proteasomes in corneal epithelial cells and cultured autologous oral mucosal epithelial cell sheet (CAOMECS) graft used for the ocular surface regeneration.

PURPOSE: This study focuses on characterizing proteasomes in corneal epithelial cells (CEC) and in cultured autologous oral mucosal epithelial cell sheets (CAOMECS) used to regenerate the ocular surface. METHODS: Limbal stem cell deficiency (LSCD) was surgically induced in rabbit corneas. CAOMECS was engineered and grafted onto corneas with LSCD to regenerate the ocular surface. RESULTS: LSCD caused an increase in inflammatory cells in the ocular surface, an increase in the formation of immunoproteasomes (IPR), and a decrease in the formation of constitutive proteasome (CPR). Specifically, LSCD-diseased CEC (D-CEC) showed a decrease in the CPR chymotrypsin-like, trypsin-like and caspase-like activities, while healthy CEC (H-CEC) and CAOMECS showed higher activities. Quantitative analysis of IPR inducible subunit (B5i, B2i, and B1i) were performed and compared to CPR subunit (B5, B2, and B1) levels. Results showed that ratios B5i/B5, B2i/B2 and B1i/B1 were higher in D-CEC, indicating that D-CEC had approximately a two-fold increase in the amount of IPR compared to CAOMECS and H-CEC. Histological analysis demonstrated that CAOMECS-grafted corneas had a re-epithelialized surface, positive staining for CPR subunits, and weak staining for IPR subunits. In addition, digital quantitative measurement of fluorescent intensity showed that the CPR B5 subunit was significantly more expressed in CAOMECS-grafted corneas compared to non-grafted corneas with LSCD. CONCLUSION: CAOMECS grafting successfully replaced the D-CEC with oral mucosal epithelial cells with higher levels of CPR. The increase in constitutive proteasome expression is possibly responsible for the recovery and improvement in CAOMECS-grafted corneas.

The Role of E-Cadherin in Maintaining the Barrier Function of Corneal Epithelium after Treatment with Cultured Autologous Oral Mucosa Epithelial Cell Sheet Grafts for Limbal Stem Deficiency.

The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) grafts was examined. CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal Stem Cell Deficiency (LSCD). E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epithelium. Beta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantly increased. ZO-1, occludin, and Cnx43 were also strongly expressed in CAOMECS. E-cadherin and beta-catenin localization at the cell membrane was reduced in LSCD corneas, while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression. LSCD corneas did not show continuous staining for ZO-1 or for Cnx43, while CAOMECS-grafted corneas showed a positive expression of ZO-1 and Cnx43. Cascade Blue® hydrazide did not pass through CAOMECS. Because E-cadherin interactions are calcium-dependent, EGTA was used to chelate calcium and disrupt cell adhesion. EGTA-treated CAOMECS completely detached from cell culture surface, and E-cadherin levels were significantly decreased. In conclusion, E cadherin high expression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane, thus promoting cellular adhesion and a functional barrier to protect the ocular surface.

Carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) for corneal epithelium reconstruction: a histological study.

This study investigates the therapeutic effects of carrier-free cultured autologous oral mucosa epithelial cell sheet (CAOMECS) transplantation for experimentally induced severe rabbit limbal stem cell deficiency (LSCD). Buccal biopsies were performed and CAOMECS were cultured and transplanted onto diseased corneas. Six-month follow-up examinations indicated that three out of four corneas with CAOMECS grafts showed a decrease in superficial vascularization, while almost all the sham corneas did not show a similar decrease. H&E staining of corneas showed that CAOMECS transplantation reduced blood vessel invasion of central cornea, reduced lymphocyte infiltration and fibrotic tissue formation. DeltaNp63 stained markedly in the grafted cornea and to a lesser extent in the sham corneas. PCNA and Ki-67 staining were much greater in the sham corneas than in the grafted and normal corneas. K3 and K13 staining demonstrated that CAOMECS transplanted corneas had much more K3- and less K13- positive cells compared to the sham corneas. Muc5AC was decreased in the central region of grafted corneas. Very little alpha-smooth muscle actin (aSMA) staining was detected in grafted corneas, while there was a greater amount of aSMA staining in sham corneas. Staining for anti-angiogenic factor TIMP -3 was also increased, and pro-angiogenic factor MMP-3 was decreased in grafted corneas compared to sham corneas. Our results indicate that CAOMECS grafts resulted in improved epithelialization of the corneal surface and decreased vascularization and fibrosis of the diseased corneas.

Bortezomib enhances fatty liver preservation in Institut George Lopez-1 solution through adenosine monophosphate activated protein kinase and Akt/mTOR pathways.

OBJECTIVES: The aim of this study is to investigate the protective mechanisms induced by bortezomib added to Institut George Lopez (IGL)-1 preservation solution to protect steatotic livers against cold ischaemia reperfusion injury and to examine whether these mechanisms occur through the activation of adenosine monophosphate activated protein kinase (AMPK), Akt/mTOR pathways. METHODS: Steatotic livers from obese rats were preserved for 24 h (at 4 °C) in IGL-1 solution with or without bortezomib (100 nM) or pretreated with AMPK inhibitor adenine 9-α-D-arabinofuranoside and preserved in IGL-1 + bortezomib. Livers were then perfused for 2 h at 37 °C. Liver injury (alanine aminotransferase/aspartate aminotransferase) and function (bile production and vascular resistance) were measured. Also, Akt/mTOR, phosphorylated AMPK (pAMPK) and apoptosis were determined by Western blot analyses. KEY FINDINGS: Bortezomib addition to IGL-1 solution significantly reduced steatotic liver injury, improved graft function and decreased liver apoptosis. These benefits were diminished by the pretreatment of obese rats with AMPK inhibitor Ara. Western blot analyses showed a significant increase in pAMPK after ischaemia and reperfusion. We also observed a significant phosphorylation of Akt in IGL-1 +bortezomib group that, in turn, induced the phosphorylation of mTOR and glycogen synthase kinase 3ß. CONCLUSIONS: Bortezomib, at low and non toxic concentration, is a promising additive to IGL-1 solution for steatotic liver preservation. Its protective effect is due to the activation of AMPK and Akt/mTOR pathways.

Proteasome inhibitors protect the steatotic and non-steatotic liver graft against cold ischemia reperfusion injury.

BACKGROUND: The dramatic shortage of organs leads to consider the steatotic livers for transplantation although their poor tolerance against ischemia reperfusion injury (IRI). Ubiquitin proteasome system (UPS) inhibition during hypothermia prolongs myocardial graft preservation. The role of UPS in the liver IRI is not fully understood. Bortezomib (BRZ) treatment at non-toxic doses of rats fed alcohol chronically has shown protective effects by increasing liver antioxidant enzymes. We evaluated and compared both proteasome inhibitors BRZ and MG132 in addition to University of Wisconsin preservation solution (UW) at low and non-toxic dose for fatty liver graft protection against cold IRI. EXPERIMENTAL: Steatotic and non-steatotic livers have been stored in UW enriched with BRZ (100 nM) or MG132 (25 µM), for 24h at 4°C and then subjected to 2-h normothermic reperfusion (37 °C). Liver injury (AST/ALT), hepatic function (bile output; vascular resistance), mitochondrial damage (GLDH), oxidative stress (MDA), nitric oxide (NO) (e-NOS activity; nitrates/nitrites), proteasome chymotrypsin-like activity (ChT), and UPS (19S and 20S5 beta) protein levels have been measured. RESULTS: ChT was inhibited when BRZ and MG132 were added to UW. Both inhibitors prevented liver injury (AST/ALT), when compared to UW alone. BRZ increased bile production more efficiently than MG132. Only BRZ decreased vascular resistance in fatty livers, which correlated with an increase in NO generation (through e-NOS activation) and AMPK phosphorylation. GLDH and MDA were also prevented by BRZ. In addition, BRZ inhibited adiponectin, IL-1, and TNF alpha, only in steatotic livers. CONCLUSION: MG132 and BRZ, administrated at low and non toxic doses, are very efficient to protect fatty liver grafts against cold IRI. The benefits of BRZ are more effective than those of MG132. This evidenced for the first time the potential use of UPS inhibitors for the preservation of marginal liver grafts and for future applications in the prevention of IRI.

Impact of altered methylation in cytokine signaling and proteasome function in alcohol and viral-mediated diseases.

Data from several laboratories have shown that ethanol (EtOH) feeding impairs many essential methylation reactions that contribute to alcoholic liver disease (ALD). EtOH is also a comorbid factor in the severity of hepatitis C virus-induced liver injury. The presence of viral proteins further exacerbates the methylation defects to disrupt multiple pathways that promote the pathogenesis of liver disease. This review is a compilation of presentations that linked the methylation reaction defects with proteasome inhibition, decreased antigen presentation, and impaired interferon (IFN) signaling in the hepatocytes and dysregulated TNFα expression in macrophages. Two therapeutic modalities, betaine and S-adenosylmethionine, can correct methylation defects to attenuate many EtOH-induced liver changes, as well as improve IFN signaling pathways, thereby overcoming viral treatment resistance.

Ethanol and hepatitis C virus suppress peptide-MHC class I presentation in hepatocytes by altering proteasome function.

BACKGROUND: Previously, we reported that exposure of hepatitis C virus (HCV) core-expressing ethanol (EtOH)-metabolizing cells to EtOH significantly suppresses proteasome activity which exists as 26S (20S and 19S) and as an unassociated 20S particle. The replacement of the constitutive proteasomal subunits with immunoproteasome (IPR) favors antigen processing. Here, we examined the effects of EtOH consumption by HCV core transgenic mice on proteasome activity in hepatocytic lysates and in partially purified 26S proteasome and the impact of these changes on antigen presentation. METHODS: HCV (-) and HCV (+) core transgenic mice were fed chow diet with or without 20% (v/v) EtOH in water for 4 weeks. Following the feeding regimen, hepatocytes were isolated and examined for chymotrypsin-like proteasome activity, oxidative stress, and the presentation of SIINFEKL-H2Kb complex. Additionally, the constitutive proteasome and IPR were purified for further analysis and identification of proteasome-interacting proteins (PIPs). RESULTS: EtOH significantly decreased proteasome activity in hepatocytes of HCV (+) mice, and this finding correlated with oxidative stress and dysregulated methylation reactions. In isolated 26S proteasome, EtOH suppressed proteasome activity equally in HCV (+) and HCV (-) mice. EtOH feeding caused proteasome instability and lowered the content of both constitutive and IPR subunits in the 20S proteasome. In addition, the level of other PIPs, PA28 and UCHL5, were also suppressed after EtOH exposure. Furthermore, in EtOH-fed mice and, especially, in HCV (+) mice, the presentation of SIINFEKL-H2Kb complex in hepatocytes was also decreased. CONCLUSIONS: Proteasomal dysfunction induced by EtOH feeding and exacerbated by the presence of HCV structural proteins led to suppression of SIINFEKL-H2Kb presentation in hepatocytes.

The use of a reversible proteasome inhibitor in a model of Reduced-Size Orthotopic Liver transplantation in rats.

Ischemia/reperfusion injury (IRI), inherent in liver transplantation (LT), is the main cause of initial deficiencies and primary non-function of liver allografts. Living-related LT was developed to alleviate the mortality resulting from the scarcity of suitable deceased grafts. The main problem in using living-related LT for adults is graft size disparity. In this study we propose for the first time that the use of a proteasome inhibitor (Bortezomib) treatment could improve liver regeneration and reduce IRI after Reduced-Size Orthotopic Liver transplantation (ROLT). Rat liver grafts were reduced by removing the left lateral lobe and the two caudate lobes and preserved in UW or IGL-1 preservation solution for 1h liver and then subjected to ROLT with or without Bortezomib treatment. Our results show that Bortezomib reduces IRI after LT and is correlated with a reduction in mitochondrial damage, oxidative stress and endoplasmic reticulum stress. Furthermore, Bortezomib also increased liver regeneration after reduced-size LT and increased the expression of well-known ischemia/reperfusion protective proteins such as nitric oxide synthase, heme oxigenase 1 (HO-1) and Heat Shock Protein 70. Our results open new possibilities for the study of alternative therapeutic strategies aimed at reducing IRI and increasing liver regeneration after LT. It is hoped that the results of our study will contribute towards improving the understanding of the molecular processes involved in IRI and liver regeneration, and therefore help to improve the outcome of this type of LT in the future.

Proteasome inhibitor treatment reduced fatty acid, triacylglycerol and cholesterol synthesis.

In the present study, the beneficial effects of proteasome inhibitor treatment in reducing ethanol-induced steatosis were investigated. A microarray analysis was performed on the liver of rats injected with PS-341 (Bortezomib, Velcade), and the results showed that proteasome inhibitor treatment significantly reduced the mRNA expression of SREBP-1c, and the downstream lipogenic enzymes, such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC), which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the rate-limiting step in fatty acid synthesis. ELOVL6, which is responsible for fatty acids long chain elongation, was also significantly downregulated by proteasome inhibitor treatment. Moreover, PS-341 administration significantly reduced the expression of acyl-glycerol-3-phosphate acyltransferase (AGPAT), and diacylglycerol acyltransferase (DGAT), enzyme involved in triacylglycerol (TAG) synthesis. Finally, PS-341 was found to downregulate the enzyme 3-hydroxy-3-methylglutaryl-CoenzymeA synthase (HMG-CoA synthase) that is responsible for cholesterol synthesis. Proteasome inhibitor was also found to play a role in intestinal lipid adsorption because apolipoproteins A (apoA-I, apoAII, apoA-IV and ApoCIII) were downregulated by proteasome inhibitor treatment, especially ApoA-II that is known to be a marker of alcohol consumption. Proteasome inhibitor treatment also decreased apobec-1 complementation factor (ACF) leading to lower level of editing and production of ApoB protein. Moreover apolipoprotein C-III, a major component of chylomicrons was significantly downregulated. However, lipoprotein lipase (Lpl) and High density lipoprotein binding protein (Hdlbp) mRNA levels were increased by proteasome inhibitor treatment. These results suggested that proteasome inhibitor treatment could be used to reduce the alcohol-enhanced lipogenesis and alcohol-induced liver steatosis. A morphologic analysis, performed on the liver of rats fed ethanol for one month and treated with PS-341, showed that proteasome inhibitor treatment significantly decreased ethanol-induced liver steatosis. SREBP-1c, FAS and ACC were increased by ethanol feeding alone, but were significantly decreased when proteasome inhibitor was administered to rats fed ethanol. Our results also show that both mRNA and protein levels of these lipogenic enzymes, up regulated by ethanol, were then downregulated when proteasome inhibitor was administered to rats fed ethanol. It was also confirmed that alcohol feeding caused an increase in AGPAT and DGAT, which was prevented by proteasome inhibitor treatment of the animal fed ethanol. Chronic alcohol feeding did not affect the gene expression of HMG-CoA synthase. However, PS341 administration significantly reduced the HMG-CoA synthase mRNA levels, confirming the results obtained with the microarray analysis. C/EBP transcription factors alpha (CCAAT/enhancer-binding protein alpha) has been shown to positively regulate SREBP-1c mRNA expression, thus regulating lipogenesis. Proteasome inhibition caused a decrease in C/EBP alpha mRNA expression, indicating that C/EBP downregulation may be the mechanism by which proteasome inhibitor treatment reduced lipogenesis. In conclusion, our results indicate that proteasome activity is not only involved in downregulating fatty acid synthesis and triacylglycerol synthesis, but also cholesterol synthesis and intestinal lipid adsorption. Proteasome inhibitor, administrated at a non-toxic low dose, played a beneficial role in reducing lipogenesis caused by chronic ethanol feeding and these beneficial effects are obtained because of the specificity and reversibility of the proteasome inhibitor used.

Proteasome inhibitor treatment in alcoholic liver disease.

Oxidative stress, generated by chronic ethanol consumption, is a major cause of hepatotoxicity and liver injury. Increased production of oxygen-derived free radicals due to ethanol metabolism by CYP2E1 is principally located in the cytoplasm and in the mitochondria, which does not only injure liver cells, but also other vital organs, such as the heart and the brain. Therefore, there is a need for better treatment to enhance the antioxidant response elements. To date, there is no established treatment to attenuate high levels of oxidative stress in the liver of alcoholic patients. To block this oxidative stress, proteasome inhibitor treatment has been found to significantly enhance the antioxidant response elements of hepatocytes exposed to ethanol. Recent studies have shown in an experimental model of alcoholic liver disease that proteasome inhibitor treatment at low dose has cytoprotective effects against ethanol-induced oxidative stress and liver steatosis. The beneficial effects of proteasome inhibitor treatment against oxidative stress occurred because antioxidant response elements (glutathione peroxidase 2, superoxide dismutase 2, glutathione synthetase, glutathione reductase, and GCLC) were up-regulated when rats fed alcohol were treated with a low dose of PS-341 (Bortezomib, Velcade(®)). This is an important finding because proteasome inhibitor treatment up-regulated reactive oxygen species removal and glutathione recycling enzymes, while ethanol feeding alone down-regulated these antioxidant elements. For the first time, it was shown that proteasome inhibition by a highly specific and reversible inhibitor is different from the chronic ethanol feeding-induced proteasome inhibition. As previously shown by our group, chronic ethanol feeding causes a complex dysfunction in the ubiquitin proteasome pathway, which affects the proteasome system, as well as the ubiquitination system. The beneficial effects of proteasome inhibitor treatment in alcoholic liver disease are related to proteasome inhibitor reversibility and the rebound of proteasome activity 72 h post PS-341 administration.

Delta-aminolevulinic dehydratase is a proteasome interacting protein.

The proteasome interacts with a large number of proteins which regulate specific cellular functions. The focus of this study is to examine the proteasome interaction with Delta-aminolevulinate dehydratase (ALAD). ALAD is involved in the heme biosynthesis pathway and was co-isolated, with the 20S proteasome using several chromatographic purification steps. The MALDI-TOF mass spectrometry analysis identified this proteasome co-isolated protein as ALAD. When the proteasome was isolated using density-gradient centrifugation, ALAD was also found in the 26S proteasome fractions. It co-isolated with the 20S more than with the 26S proteasome. Furthermore, immunoprecipitated ALAD stained positive with antibodies to proteasome subunits. These results indicate that ALAD might interact with the proteasome. It is possible that ALAD is involved in modulating proteasome activity. When purified proteasomes were incubated with ALAD it was found that ALAD changes proteasome activity in a dose dependent manner. This indicates that ALAD may play a significant role in regulating proteasome activity. The data supports the hypothesis that ALAD, an important enzyme for heme synthesis, is also important as a proteasome interacting protein.