Melina M. Ribeiro

Heme oxygenase-1 fused to a TAT peptide transduces and protects pancreatic β-cells

Abstract Transplantation of islets is becoming an established method for treating type 1 diabetes. However, viability of islets is greatly affected by necrosis/apoptosis induced by oxidative stress and other insults during isolation and subsequent in vitro culture. Expression of cytoprotective proteins, such as heme oxygenase-1 (HO-1), reduces the deleterious effects of oxidative stress in transplantable islets. We have generated a fusion protein composed of HO-1 and TAT protein transduction domain (TAT/PTD), an 11-aa cell penetrating peptide from the human immunodeficiency virus TAT protein. Transduction of TAT/PTD–HO-1 to insulin-producing cells protects against TNF-α-mediated cytotoxicity. TAT/PTD–HO-1 transduction to islets does not impair islet physiology, as assessed by reversion of chemically induced diabetes in immunodeficient mice. Finally, we report that transduction of HO-1 fusion protein into islets improves islet viability in culture. This approach might have a positive impact on the availability of islets for transplantation.

Nuclear Entry of Activated MAPK Is Restricted in Primary Ovarian and Mammary Epithelial Cells

Background The MAPK/ERK1/2 serine kinases are primary mediators of the Ras mitogenic signaling pathway. Phosphorylation by MEK activates MAPK/ERK in the cytoplasm, and phospho-ERK is thought to enter the nucleus readily to modulate transcription. Principal Findings Here, however, we observe that in primary cultures of breast and ovarian epithelial cells, phosphorylation and activation of ERK1/2 are disassociated from nuclear translocalization and transcription of downstream targets, such as c-Fos, suggesting that nuclear translocation is limited in primary cells. Accordingly, in import assays in vitro, primary cells showed a lower import activity for ERK1/2 than cancer cells, in which activated MAPK readily translocated into the nucleus and activated c-Fos expression. Primary cells express lower levels of nuclear pore complex proteins and the nuclear transport factors, importin B1 and importin 7, which may explain the limiting ERK1/2 import found in primary cells. Additionally, reduction in expression of nucleoporin 153 by siRNA targeting reduced ERK1/2 nuclear activity in cancer cells. Conclusion ERK1/2 activation is dissociated from nuclear entry, which is a rate limiting step in primary cells and in vivo, and the restriction of nuclear entry is disrupted in transformed cells by the increased expression of nuclear pores and/or nuclear transport factors.

Impact of Pancreatic Cold Preservation on Rat Islet Recovery and Function

Background. Islet transplantation success depends on the number and quality of islets transplanted. This study aimed at exploring the molecular mechanisms associated with cold pancreas preservation and their impact on islet cell survival and function. Methods. Rat pancreata were stored in cold University of Wisconsin preservation solution for short (3 hr; control) or long (18 hr) cold ischemia times (CIT). Results. Pancreata exposed to long CIT yielded lower islet numbers and showed reduced cellular viability; isolated islets displayed higher levels of phosphorylated stress-activated protein kinase (c-jun N-terminal Kinase and Mitogen-Activated Protein Kinase-p38), and chemokine (C-C) ligand-3, and lower levels of vascular endothelial growth factor, interleukins (IL)-9 and IL-10. Islets obtained from long-CIT pancreata were functionally impaired after transplantation. Differential proteomic expression in pancreatic tissue after CIT included increased eukaryotic translation elongation factor-1-α-1 (apoptosis related) and reduced Clade-B (serine protease inhibitor). Conclusions. Our study indicates that cold ischemia stimulates inflammatory pathways (chemokine (c-c)ligand-3, phosphorylation of c-jun N-terminal Kinase and mitogen-activated protein kinase-p38, and eukaryotic translation elongation factor-1-α-1) and decreases repair/cytoprotective pathways (IL-10, vascular endothelial growth factor, and Clade-B), all of which may negatively affect the quality and mass of islets obtained from a donor pancreas.

DELIVERY OF PROTEINS AND PEPTIDES INTO LIVE CELLS BY MEANS OF PROTEIN TRANSDUCTION DOMAINS: POTENTIAL APPLICATION TO ORGAN AND CELL TRANSPLANTATION

Proteins are primary targets in drug discovery. However, with a few rare exceptions, they are unable to cross cell membranes, a limitation that prevents the full exploitation of their therapeutic potential. Major advances have been recently made through a novel approach of protein and peptide delivery into cells known as protein transduction or protein therapy. Proteins and peptides can be directly transferred to cells when covalently linked to protein transduction domains (PTD), small peptides that can freely cross cell membranes with low lytic activity (1–3). The mechanism of cellular translocation of PTD are currently poorly understood. Most of the PTD described in the literature have a high content of basic residues. It is believed that the interaction with the negative cell membrane environment has an important role in the translocation process, and the mechanism of cell internalization may differ for each of the PTD. Several PTD have been identified in naturally occurring proteins. The most commonly studied are homeodomain transcription factors such as antennapedia (4), the herpes simplex virus type 1 protein VP22 (5), and the human immunodeficiency virus (HIV) transactivator TAT protein ( 6– 7). In addition, a new gamut of peptides with PTD capabilities have been recently identified. Some of these new peptides are derived from natural proteins, whereas others are synthetic peptides. The PTD included in these groups are described below, with emphasis on the TAT-PTD and its potential application in organ and cell transplantation.

Beneficial Effects of Ischemic Preconditioning on Pancreas Cold Preservation

Ischemic preconditioning (IPC) confers tissue resistance to subsequent ischemia in several organs. The protective effects are obtained by applying short periods of warm ischemia followed by reperfusion prior to extended ischemic insults to the organs. In the present study, we evaluated whether IPC can reduce pancreatic tissue injury following cold ischemic preservation. Rat pancreata were exposed to IPC (10 min of warm ischemia followed by 10 min of reperfusion) prior to ~18 h of cold preservation before assessment of organ injury or islet isolation. Pancreas IPC improved islet yields (964 ± 336 vs. 711 ± 204 IEQ/pancreas; p = 0.004) and lowered islet loss after culture (33 ± 10% vs. 51 ± 14%; p = 0.0005). Islet potency in vivo was well preserved with diabetes reversal and improved glucose clearance. Pancreas IPC reduced levels of NADPH-dependent oxidase, a source of reactive oxygen species, in pancreas homogenates versus controls (78.4 ± 45.9 vs. 216.2 ± 53.8 RLU/μg; p = 0.002). Microarray genomic analysis of pancreata revealed upregulation of 81 genes and downregulation of 454 genes (greater than twofold change) when comparing IPC-treated glands to controls, respectively, and showing a decrease in markers of apoptosis and oxidative stress. Collectively, our study demonstrates beneficial effects of IPC of the pancreas prior to cold organ preservation and provides evidence of the key role of IPC-mediated modulation of oxidative stress pathways. The use of IPC of the pancreas may contribute to increasing the quality of donor pancreas for transplantation and to improving organ utilization.

Effects of Pancreas Cold Ischemia on Islet Function and Quality

We used a rat model of pancreas cold preservation to assess its effects on islets. Glands were surgically retrieved and stored in University of Wisconsin (UW) solution for 3 hours (Short) or 18 hours (Long) cold ischemia time (CIT). Islet yield was significantly lower in the Long-CIT than the Short-CIT group, as well as islet recovery after overnight culture (P < .01). Islet cell viability after isolation was significantly reduced in the Long-CIT group (P < .05). Reversal of diabetes following transplantation of suboptimal islet grafts occurred earlier in the Short-CIT group than the Long-CIT. All animals in the Short-CIT group and 80% in the Long-CIT group achieved euglycemia. Freshly isolated islets showed a significant increase of JNK and p38 (P < .05) phosphorylation in Long-CIT compared with Short-CIT. Histopathological assessment of the pancreas showed a significantly higher injury score. Proteomic analysis of pancreatic tissue led to identification of 5 proteins consistently differentially expressed between Short-CIT and Long-CIT. Better understanding of the molecular pathways involved in this phenomenon will be of assistance in defining targeted interventions to improve organ use in the clinical arena.

Ischemic Preconditioning Improves Islet Recovery After Pancreas Cold Preservation

Increasing evidence supports the beneficial effects of ischemic preconditioning (IPC) of organs on subsequent ischemia. The aim of this study was to assess the effects of IPC of the pancreas on islet cell recovery after cold preservation using a rat model. The pancreas was deprived of perfusion (celiac artery and superior mesenteric artery occlusion) for 10 minutes followed by 10 minutes of reperfusion. Islet isolation was performed after 18 hours of cold ischemia. Glands undergoing IPC yielded significantly greater numbers of islets than controls. Following overnight culture, a significantly greater proportion of islets was recovered from IPC-treated pancreata. Microarray genomic analysis of pancreatic tissue revealed a significant differential expression of approximately 600 unique mRNA strands within IPC pancreata compared to only <100 unique mRNA strands within non-IPC pancreata (>2-fold change; P < .05). Proteomic analysis revealed significant differential expression of at least 5 proteins >1.5-fold change; P < .05) within the IPC vs control group. Our data indicated that IPC of the pancreas prior to cold preservation was associated with improved islet cell recovery after cold ischemia. IPC of the pancreas may represent a viable therapeutic intervention to increase islet transplantation success from a single donor and to maximize organ utilization.

Endotoxin deactivation by transient acidification.

Recombinant proteins are an important tool for research and therapeutic applications. Therapeutic proteins have been delivered to several cell types and tissues and might be used to improve the outcome of the cell transplantation. Recombinant proteins are propagated in bacteria, which will contaminate them with the lypopolysacharide endotoxin found in the outer bacterial membrane. Endotoxin could interfere with in vitro biological assays and is the major pathological factor, which must be removed or inactivated before in vivo administration. Here we describe a one-step protocol in which the endotoxin activity on recombinant proteins is remarkably reduced by transient exposure to acidic conditions. Maximum endotoxin deactivation occurs at acidic pH below their respective isoelectric point (pI). This method does not require additional protein purification or separation of the protein from the endotoxin fraction. The endotoxin level was measured both in vitro and in vivo. For in vitro assessment we have utilized Limulus Amebocyte Lysate method for in vivo the pyrogenic test. We have tested the above-mentioned method with five different recombinant proteins, including a monoclonal antibody clone 5c8 against CD154 produced by hybridomas. More than 99% of endotoxin was deactivated in all of the proteins; the recovery of the protein after deactivation varied between maximum 72.9% and minimum 46.8%. The anti-CD154 clone 5c8 activity remained unchanged as verified by the measurement of binding capability to activated lymphocytes. Furthermore, the effectiveness of this method was not significantly altered by urea, commonly used in protein purification. This procedure provides a simple and cost-efficient way to reduce the endotoxin activity in antibodies and recombinant proteins.

Expression of Heme Oxygenase-1 Mediated by A Protein Transduction Domain Protects Insulin Producing Cells from Cytokine- Induced Cytotoxicity

INTRODUCTION. Microsomal Heme Oxygenase-1 (HO-1) has been identified as a ubiquitous stress protein induced in many cell types by various stimulants such as hemolysis, inflammatory agents, oxidative stress, heat shock, and growth factors[1]. HO-1 is the enzyme that controls the degradation pathway of heme by catalyzing its oxidation into biliverdin, carbon monoxide, and iron. Although the role of HO-1 induction in oxidative stress is not completely understood, it has been shown that induction of HO-1 expression results in protection from cytokine-induced apoptosis and oxidative stress in in vitro cell culture and in various animal models, including pancreatic islet transplantation[2]. Thus, expression of HO-1 through gene therapy protocols might prove useful to reduce the deleterious effects of oxidative stress in islet isolation as well as to prevent damage in the peri-transplant period. However, viral vectors or other transfection methods available for transduction of genes into islets have limited efficacy and the presence of viral antigens (especially adenovirus) may potentially induce immunological responses against the transfected islets. Moreover, the long-term effects of genetic manipulations of islets, in particular those affecting apoptosis, may have undesirable long-term effects such as impaired mitochondrial signals regulating insulin secretion. Proteins can be directly transferred to cells when they are linked to protein transduction domains (PTDs), small peptide domains that can freely cross cell membranes[3]. In particular, proteins fused to an 11-amino acid protein transduction domain (PTD) from the human immunodeficiency virus transactivator of transcription (TAT) protein, readily transduce many cell types including pancreatic islets[4]. In this study we have characterized a functional HO-1 protein fused to a PTD METHODS. Cloning and Related Techniques. The recombinant TAT antiapoptotic fusion proteins were generated by subcloning the coding region of the murine HO-1 gene in frame with the TAT leader peptide (amino acids 47-58 YGRKKRRQRRR) in a bacterial expression generously provided by Steven Dowdy from Washington University School of Medicine, St Louis. A 6xHis-affinity tag allowed the purification of the fusion protein through affinity chromatography.

Transduction of TAT/PTD Antiapoptotic Fusion Proteins in Pancreatic Islets

INTRODUCTION. With the development of new strategies to avoid immunological rejection, transplantation of pancreatic islets has become a therapeutic reality to cure diabetes (1-2). However, despite the progress in islet isolation procedures, a single donor transplant does not provide enough islets to attain insulin independence. There is evidence that significant loss of islet cells takes place during isolation due to the triggering of apoptosis (3). There is also substantial evidence that reduction of isolation-induced apoptosis can improve the success rates of islet transplantation. The goal of this proposal is to address the needs for reduced apoptosis and improved viability of islets in conjunction with islet isolation procedures. We present in this study novel transduction methods that allow manipulation of islets to reduce apoptosis. Proteins can be directly transferred to cells when they are linked to protein transduction domains (PTDs), small peptide domains that can freely cross cell membranes. In particular, proteins fused to an 11-amino acid PTD from the human immunodeficiency virus, the TAT protein, readily diffuse across membranes and are efficiently transduced into virtually any cell type (4). The expression as well as the biological function of the TAT fusion protein are temporary without permanent modification of the cell sensitivity to apoptosis, thus avoiding undesirable long-term effects.