Increased amounts of elastic fibers have been associated to the pathological remodeling of tissue [ 26 ]. Moreover, fibrous septa particularly rich in cross-linked elastic fibers are known to be more resistant to matrix metalloproteinases MMPs -mediated degradation, thus contributing to fibrosis irreversibility [ 27 ]. Therefore, these proteins might represent multiple targets to develop therapeutic strategies to facilitate liver cirrhosis reversion. Fig 5 recapitulates the HCV-associated liver fibrotic molecular stage-specific hallmarks observed here, and notably, emphasizes the match between the METAVIR-based histological evaluation and the molecular dynamic changes observed.
In conclusion, this study contributes to the knowledge of human ECM composition in liver fibrosis progression.
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However, we believe that this should be considered as a starting point, as many issues on the ECM structure of fibrotic liver still remain to be addressed. What is the intra- and intermolecular cross-linking profile? What cross-links have a role in inhibiting ECM degradation? What is the cellular origin of the described proteins? What kind of post-transcriptional and post-translational regulations occur in the pathological deposition of the ECM molecules? We believe that the presented data will support future studies to address these issues. Clinicopathological features of Fibrotic Patients and Label free proteomic analysis.
We are deeply grateful to Ms. We would like to thank Prof. This study is dedicated to the memory of my friend Stefano, who fought against liver cancer. Conceived and designed the experiments: C. Mancone A. Performed the experiments: C. Montaldo C. Analyzed the data: C. Baiocchini C. Mancone MT. Wrote the paper: C. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract Chronic liver damage leads to pathological accumulation of ECM proteins liver fibrosis. Introduction The extracellular matrix ECM is a critical component of the human liver microenvironment.
Histological analyses Each human biopsy sample was evaluated histologically according to the METAVIR grading system for disease stage and grade of necroinflammatory activity.
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Results Liver ECM scaffold isolation by tissue decellularization ECM isolation by immersion and agitation in decellularization agents has been applied for a wide variety of tissue specimens, including dermis, skeletal muscles, peripheral nerves and heart valves [ 8 ]. Download: PPT. Characterization of the decellularized liver ECM scaffold To investigate the effects of decellularization protocol on the extracellular matrix network, histological investigations were performed. Fig 2. Histological and ultrastructural analysis of decellularized human liver ECM scaffold.
Fig 3. Fig 4. Fig 5. Schema summarizing the ECM protein components that are differentially regulated in the transitions among the fibrotic stages. Discussion Obtaining a comprehensive description of the ECM molecular composition in human liver health and disease has so far been hampered by the difficulty to isolate decellularized hepatic ECM scaffolds.
F2 With the exception of a slight increase for collagen types I and III, the transition from portal to moderate fibrosis is characterized more by ECM remodeling than by its increase. F3 The main efforts to manage HCV-related liver disease are concentrated in developing anti-fibrotic strategies to counteract the transition from moderate to severe fibrosis. F4 The entire liver architecture is lost when fibrosis advances to cirrhosis. Supporting Information. S1 Fig.
Supplementary Fig 1. S2 Fig. Supplementary Fig 2. Histological analysis of decellularized murine hepatic lobe.
S1 File. Supplementary Materials and Methods. S1 Table. Supplementary Table I. Proteomic Results from one human decellularized liver biopsy with low fibrosis.
S2 Table. Supplementary Table II. S3 Table. Supplementary Table III. Acknowledgments We are deeply grateful to Ms. Author Contributions Conceived and designed the experiments: C. References 1. Hynes RO. The extracellular matrix: not just pretty fibrils. Science ; — View Article Google Scholar 2.
An extracellular matrix microarray for probing cellular differentiation. Nat Methods ;2: — Wells RG.
Research foci Cell and Gene Therapy
The role of matrix stiffness in regulating cell behavior. Hepatology ; — Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix. Nat Med ; — Proteomic analysis of extracellular matrix from the hepatic stellate cell line LX-2 identifies CYR61 and Wnt-5a as novel constituents of fibrotic liver. J Proteome Res ; — Extracellular matrix signatures of human primary metastatic colon cancers and their metastases to liver. BMC Cancer ; An overview of tissue and whole organ decellularization processes.
Peroxisome ProliferatorActivated Receptor Y. Control of the Hepatic Stellate Cell Phenotype. Major Intracellular Signaling Pathways. Polypeptide Growth Factor Receptors.
Gene therapy in the vasculature and the kidney | Nefrología
Transforming Growth Factor3 Receptor Superfamily. Seven Transmembrane Domain Receptors. Tumor Necrosis Factor Receptor Superfamily. Other Cytokine Receptors. Transcriptional Regulation of Matrix Metalloproteinases. For example, it has been extremely difficult to achieve transduction of renal glomerular cells with many of the agents under investigation.
It has become clear recently that gene transfer into vessels can be achieved ex vivo under high hydrostatic pressure 9, and that physical injection of DNA on gold particles delivered under high pressure from a gene gun can be achieved For instance, systemic factors, which include, among others, abnormalities in cholesterol metabolism can increase the risk of, and accelerate the rate of progression of atherosclerosis. Systemic gene therapy has beem used in a number of animal models to reduce serum cholesterol concentrations, and formation of atherosclerotic lesions As noted above, systemic infusion of adenoviral vectors leads to transient, high level expression in liver.
Similarly, apolipoprotein E reconstitution in apoE deficient mice has been achieved using adenoviral vectors, and overexpression of human apoAI has been shown to raise plasma HDL levels in mice and hamsters. Systemic gene therapy with using adenoviral vector mediated expression of the fibrinolytic tissue type plasminogen activator t-PA , has also met success in mice In patients with renal disease, the lack of erythropoietin was a major cause of morbidity, until it was overcome in the last decade this problem has been overcome by recombinant erythropoeitin therapy.
Recent trials in animals have shown that long-term expression of erythropoietin can be a achieved with peritoneal adenovirus based gene transfer and with transfer of the erythropoietin cDNA in stably transfected cells If such constructs could be introduced into venous endothelium under a hypoxia-regulated promoter, it could even be possible to obtain long-term and appropriately regulated expression of this gene in dialysis patients.
The advantages of local vs. First, potential untoward effects at other locations and in other tissues are minimized, and second, as concentration of vector is often limiting, gene transfer in a confined region can be achieved more easily. Another approach has been to overexpress, locally, normal endothelial cell proteins known to inhibit smooth muscle cell proliferation.
Introduction of antisense oligonucleotides which inhibit synthesis of proteins involved in cellcycle regulation in the blood vessel wall has met with considerable success in the rat arterial balloon injury model Antisense oligonucleotides against cdc2 kinase, c-myc, c-myb and proliferating cell nuclear antigen PCNA all demonstrated reduction of neointima formation, when compared to areas in the same vessel not treated with the oligonucleotide 13, Though inhibition of growth factor or growth factor receptor synthesis might also be considered as a potential strategy to inhibition of proliferation, the enormous redundancy and interplay between growth factors and other mitogens makes inhibition of their common downstream mitogenic effector system more attractive as a therapy.
Nevertheless, the antisense strategy has been successful in inhibiting TGF-1 mediated glomerular matrix deposition in the anti thy 1. The transcription factor binding decoy approach, using HJV-liposome mediated gene transfer, has also been used with success in the balloon injury model. Decoys that bind the transcription factor E2F, important in the activation of several cell-cycle control proteins. The presence of the DNA decoy capable of binding E2F leads to inhibition of proliferation in injured arteries 4, and similarly inhibition of mesangial cells proliferation in the anti thy 1.
Viral gene transfer techniques are also being used to alter the vascular response to injury. Studies in which proteins that normally function to inhibit cell proliferation have been introduced. For instance, the retinoblastoma gene product Rb , a tumor suppresser protein that arrests cell cycle progression, inhibited local cell proliferation and subsequent neointima formation, when transferred into rat carotid artery using a replication deficient adenoviral vector, after balloon injury Similarly, adenoviral transfer of the cDNA encoding the antiproliferative transcription factor gax, inhibited vessel stenosis in the rabbit iliac artery Exogenous genes that render cells susceptible to toxic drugs have also been used to prevent vascular smooth muscle proliferation.
Nabel used adenoviral gene transfer to introduce the herpesvirus thymidine kinase cDNA into porcine arteries that had been subjected to balloon injury Thymidine kinase converts the nucloside analog gancyclovir, through phosphorylation, into active drug which inhibits DNA elongation and therefore kills dividing cells. Local expression of thymidine kinase significantly inhibited cell proliferation at the site of injury and subsequent neointima formation in animals given gancylcovir.
Extracellular Matrix and The Liver
It is generally difficult to achieve gene delivery into most cells at the site of injury in a blood vessel. Both, enhanced blood vessel reactivity to acetylcholine and reduced proliferation of neointimal cells was shown. A large number of other targets in the vascular response to injury have been subjected to interruption or enhancement using gene transfer techniques. In addition, gene therapy is being applied to vein bypass grafts, which are notorious for rapid development of stenosis, and in the field of transplantation, where accelerated arteriosclerosis of vessels within the transplant can lead to organ failure.
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One of the principal obstacles to be overcome in the field of vascular gene therapy is the efficient delivery of the gene of interest to the target cell population. Transfer efficiency can be augmented when the endothelial cell barrier is breached, as is common in the balloon injury model. However, efficient gene transfer in vein grafts, for instance, and in blood vessels of organs to be transplanted, is still problematic.
In many cases, effective endothelial cell gene transfer can be achieved, but medial smooth muscle and other underlying cells are transduced only if the endothelium is first disrupted in some way. Since endothelial cell injury itself elicits a remodeling response in veins and large vessels, this approach may be counterproductive. Another problem concerns the efficiency of gene transfer, already discussed above.
For adenoviral vectors, uptake of the vector can itself elicit an immune response, which can limit the length of transgene expression, and can stimulate a local inflammatory response that can, itself, lead to blood vessel remodeling. For AAV, gene transfer into some cells is much more efficient than into other cells, owing, presumably, to the lack of expression of the appropriate viral receptor by some cells.
These include ischemia and wound healing. In humans with myocardial infarction, it has been shown that local injection of recombinant FGF-2, an angiogenic growth factor stimulated microvessel growth Translation Protein 2. Splicing Fig. Genes consist of translated exons and untranslated introns, promoter and downstream elements sequences.