Main Extracellular Matrix Proteins Research Articles

Reconstruction of the human nipple-areolar complex: a tissue engineering approach.

Introduction: Nipple-areolar complex (NAC) reconstruction after breast cancer surgery is challenging and does not always provide optimal long-term esthetic results. Therefore, generating a NAC using tissue engineering techniques, such as a decellularization-recellularization process, is an alternative option to recreate a specific 3D NAC morphological unit, which is then covered with an in vitro regenerated epidermis and, thereafter, skin-grafted on the reconstructed breast. Materials and methods: Human NACs were harvested from cadaveric donors and decellularized using sequential detergent baths. Cellular clearance and extracellular matrix (ECM) preservation were analyzed by histology, as well as by DNA, ECM proteins, growth factors, and residual sodium dodecyl sulfate (SDS) quantification. In vivo biocompatibility was evaluated 30days after the subcutaneous implantation of native and decellularized human NACs in rats. In vitro scaffold cytocompatibility was assessed by static seeding of human fibroblasts on their hypodermal side for 7days, while human keratinocytes were seeded on the scaffold epidermal side for 10days by using the reconstructed human epidermis (RHE) technique to investigate the regeneration of a new epidermis. Results: The decellularized NAC showed a preserved 3D morphology and appeared white. After decellularization, a DNA reduction of 98.3% and the absence of nuclear and HLA staining in histological sections confirmed complete cellular clearance. The ECM architecture and main ECM proteins were preserved, associated with the detection and decrease in growth factors, while a very low amount of residual SDS was detected after decellularization. The decellularized scaffolds were in vivo biocompatible, fully revascularized, and did not induce the production of rat anti-human antibodies after 30days of subcutaneous implantation. Scaffold in vitro cytocompatibility was confirmed by the increasing proliferation of seeded human fibroblasts during 7days of culture, associated with a high number of living cells and a similar viability compared to the control cells after 7days of static culture. Moreover, the RHE technique allowed us to recreate a keratinized pluristratified epithelium after 10days of culture. Conclusion: Tissue engineering allowed us to create an acellular and biocompatible NAC with a preserved morphology, microarchitecture, and matrix proteins while maintaining their cell growth potential and ability to regenerate the skin epidermis. Thus, tissue engineering could provide a novel alternative to personalized and natural NAC reconstruction.

Preventing Aortic Aneurysm Initiation And Expansion By Inducing Immune Tolerance To Aortic Specific Antigens Embedded In Biodegradable Poly(lactide-co-glycolide) Nanoparticles

Abdominal aortic aneurysm (AAA) is a life-threatening disease diagnosed in over 200,000 Americans each year. There are currently no effective strategies to prevent AAA expansion and avoid the need for surgery. Our prior work has shown that patients with AAA have elevated plasma levels of elastin fragments, anti-elastin antibodies, and elevated levels of Th17 cells sensitized to elastin degradation products (EDPs), the sum of which suggests that AAA is the result of an autoimmune response targeting elastin, the main extra-cellular matrix protein in the aorta.

Extracellular Matrix Biomimetic Hydrogels, Encapsulated with Stromal Cell-Derived Factor 1, Improve the Composition of Foetal Tissue Grafts in a Rodent Model of Parkinson's Disease.

Clinical studies have provided evidence for dopamine (DA) cell replacement therapy in Parkinson’s Disease. However, grafts derived from foetal tissue or pluripotent stem cells (PSCs) remain heterogeneous, with a high proportion of non-dopaminergic cells, and display subthreshold reinnervation of target tissues, thereby highlighting the need to identify new strategies to improve graft outcomes. In recent work, Stromal Cell-Derived Factor-1 (SDF1), secreted from meninges, has been shown to exert many roles during ventral midbrain DA development and DA-directed differentiation of PSCs. Related, co-implantation of meningeal cells has been shown to improve neural graft outcomes, however, no direct evidence for the role of SDF1 in neural grafting has been shown. Due to the rapid degradation of SDF1 protein, here, we utilised a hydrogel to entrap the protein and sustain its delivery at the transplant site to assess the impact on DA progenitor differentiation, survival and plasticity. Hydrogels were fabricated from self-assembling peptides (SAP), presenting an epitope for laminin, the brain’s main extracellular matrix protein, thereby providing cell adhesive support for the grafts and additional laminin–integrin signalling to influence cell fate. We show that SDF1 functionalised SAP hydrogels resulted in larger grafts, containing more DA neurons, increased A9 DA specification (the subpopulation of DA neurons responsible for motor function) and enhanced innervation. These findings demonstrate the capacity for functionalised, tissue-specific hydrogels to improve the composition of grafts targeted for neural repair.

Integrated Cells and Collagen Fibers Spatial Image Analysis.

Modern technologies designed for tissue structure visualization like brightfield microscopy, fluorescent microscopy, mass cytometry imaging (MCI) and mass spectrometry imaging (MSI) provide large amounts of quantitative and spatial information about cells and tissue structures like vessels, bronchioles etc. Many published reports have demonstrated that the structural features of cells and extracellular matrix (ECM) and their interactions strongly predict disease development and progression. Computational image analysis methods in combination with spatial analysis and machine learning can reveal novel structural patterns in normal and diseased tissue. Here, we have developed a Python package designed for integrated analysis of cells and ECM in a spatially dependent manner. The package performs segmentation, labeling and feature analysis of ECM fibers, combines this information with pre-generated single-cell based datasets and realizes cell-cell and cell-fiber spatial analysis. To demonstrate performance and compatibility of our computational tool, we integrated it with a pipeline designed for cell segmentation, classification, and feature analysis in the KNIME analytical platform. For validation, we used a set of mouse mammary gland tumors and human lung adenocarcinoma tissue samples stained for multiple cellular markers and collagen as the main ECM protein. The developed package provides sufficient performance and precision to be used as a novel method to investigate cell-ECM relationships in the tissue, as well as detect structural patterns correlated with specific disease outcomes.

Sos1 Modulates Extracellular Matrix Synthesis, Proliferation, and Migration in Fibroblasts.

Non-reversible fibrosis is common in various diseases such as chronic renal failure, liver cirrhosis, chronic pancreatitis, pulmonary fibrosis, rheumatoid arthritis and atherosclerosis. Transforming growth factor beta 1 (TGF-β1) is involved in virtually all types of fibrosis. We previously described the involvement of Ras GTPase isoforms in the regulation of TGF-β1-induced fibrosis. The guanine nucleotide exchange factor Son of Sevenless (Sos) is the main Ras activator, but the role of the ubiquitously expressed Sos1 in the development of fibrosis has not been studied. For this purpose, we isolated and cultured Sos1 knock-out (KO) mouse embryonic fibroblasts, the main extracellular matrix proteins (ECM)-producing cells, and we analyzed ECM synthesis, cell proliferation and migration in the absence of Sos1, as well as the role of the main Sos1-Ras effectors, Erk1/2 and Akt, in these processes. The absence of Sos1 increases collagen I expression (through the PI3K-Akt signaling pathway), total collagen proteins, and slightly increases fibronectin expression; Sos1 regulates fibroblast proliferation through both PI3K-Akt and Raf-Erk pathways, and Sos1-PI3K-Akt signaling regulates fibroblast migration. These study shows that Sos1 regulates ECM synthesis and migration (through Ras-PI3K-Akt) and proliferation (through Ras-PI3K-Akt and Ras-Raf-Erk) in fibroblasts, and describe for the first time the role of the Sos1-Ras signaling axis in the regulation of cellular processes involved in the development of fibrosis.

Decellularized Human Chorion Membrane as a Novel Biomaterial for Tissue Regeneration.

Although some placenta-derived products are already used for tissue regeneration, the human chorion membrane (HCM) alone has been poorly explored. In fact, just one study uses decellularized HCM (dHCM) with native tissue architecture (i.e., without extracellular matrix (ECM) suspension creation) as a substrate for cell differentiation. The aim of this work is to fully characterize the dHCM for the presence and distribution of cell nuclei, DNA and ECM components. Moreover, mechanical properties, in vitro biological performance and in vivo biocompatibility were also studied. Our results demonstrated that the HCM was successfully decellularized and the main ECM proteins were preserved. The dHCM has two different surfaces, the reticular layer side and the trophoblast side; and is biocompatible both in vitro and in vivo. Importantly, the in vivo experiments demonstrated that on day 28 the dHCM starts to be integrated by the host tissue. Altogether, these results support the hypothesis that dHCM may be used as a biomaterial for different tissue regeneration strategies, particularly when a membrane is needed to separate tissues, organs or other biologic compartments.

Cell attachment effects of collagen nanoparticles on crosslinked electrospun nanofibers.

Since collagen is naturally a main extracellular matrix protein, it has been applied widely in skin's tissue engineering scaffolds to mimics the characteristics of extracellular matrix for proper transplantation of living cells. However, there are challenges that come with application of this natural polymer such as high solubility in aqueous environments which requires further consideration such as chemically cross-linking in order to stabilization. But these treatments also affect its functionality and finally cellular behaviors on scaffold. In this research we evaluated the suitability of collagen nanofibers versus collagen nanoparticles for cell adhesion and viability on glutaraldehyde cross-linked scaffolds. Appling a dual-pump electrospining machine a blend PCL-Gelatin from one side and collagen nanofibers or collagen nanoparticles from the other side were collected on the collector. The fabricated scaffolds were characterized by scanning electron microscopy, contact angle, and mechanical analysis. The cell viability, adhesion and morphology were studied respectively using MTT assay, hoechst staining and scanning electron microscopy. The results indicated significantly improvement of cell viability, adhesion and better spreading on scaffolds with collagen nanoparticles than collagen nanofibers. It seems changes in surface morphology, viscoelastic moduli and swelling ability following cross-linking with glutaraldehyde in scaffold with collagen nanoparticles are still favorable for cellular proliferation. Based on these results, in the case of glutaraldehyde cross-linking, application of collagen nanoparticles rather than collagen nanofibers in tissue regeneration scaffolds will better mimic the extracellular matrix characteristics; and preserve the viability and adhesion of seeded cells.

Characterization of adhesion properties of the cardiomyocyte integrins and extracellular matrix proteins using atomic force microscopy.

Integrins are transmembrane adhesion receptors that play important roles in the cardiovascular system by interacting with the extracellular matrix (ECM). However, direct quantitative measurements of the adhesion properties of the integrins on cardiomyocyte (CM) and their ECM ligands are lacking. In this study, we used atomic force microscopy (AFM) to quantify the adhesion force (peak force and mean force) and binding probability between CM integrins and three main heart tissue ECM proteins, ie, collagen (CN), fibronectin (FN), and laminin (LN). Functionalizing the AFM probes with ECM proteins, we found that the peak force (mean force) was 61.69 ± 5.5 pN (76.54 ± 4.0 pN), 39.26 ± 4.4 pN (59.84 ± 3.6 pN), and 108.31 ± 4.2 pN (129.63 ± 6.0 pN), respectively, for the bond of CN-integrin, FN-integrin, and LN-integrin. The binding specificity between CM integrins and ECM proteins was verified by using monoclonal antibodies, where α10 - and α11 -integrin bind to CN, α3 - and α5 -integrin bind to FN, and α3 - and α7 -integrin bind to LN. Furthermore, adhesion properties of CM integrins under physiologically high concentrations of extracellular Ca2+ and Mg2+ were tested. Additional Ca2+ reduced the adhesion mean force to 68.81 ± 4.0 pN, 49.84 ± 3.3 pN, and 119.21 ± 5.8 pN and binding probability to 0.31, 0.34, 0.40 for CN, FN, and LN, respectively, whereas Mg2+ caused very minor changes to adhesion properties of CM integrins. Thus, adhesion properties between adult murine CM integrins and its main ECM proteins were characterized, paving the way for an improved understanding of CM mechanobiology.

Advanced glycation end products as a source of artifacts in immunoenzymatic methods

The most abundant proteins in the arteries are those of extracellular matrix, ie. collagen and elastin. Due to their long half-lifes these proteins have an increased chance to undergo glycation. The aim of this study was to determine relationship between the content of the main extracellular matrix proteins and the advanced glycation end products (AGEs) in arteries. In this study 103 fragments of aorta were analyzed by ELISA and immunobloting for the content of collagens type I, III and IV and elastin and the content of advanced glycation end-products (AGE). A negative correlation between the content of collagens type III and IV and AGE (r = −0,258, p = 0,0122, and a weak negative correlation between collagen type III and age of the sample donor (r = 0,218, p = 0,0262) were demonstrated. This result comes as a surprise and it contradicts an intuitive assumption that with more glycation substrate, i.e. matrix proteins, more AGE products are expected. We have concluded that the results of the ELISA tests must have been influenced by the glycation. As a consequence, either modified protein molecules were not being recognized by the antibodies, or the glycation, and formation of crosslinks have blocked access of the antibodies to the antigen. It will conceal the effect of the linear dependence between the result (absorbance/densitometry) from the quantity of protein to which the antibody is directed.

Decellularized bovine cotyledons may serve as biological scaffolds with preserved vascular arrangement.

Technically produced scaffolds are common to establish transplantable tissues for regenerative medicine, but also biological ones that are closer to the natural condition become of interest. Placentas are promising, because they represented available, complete organs with rich extracellular matrix (ECM) and well-developed vasculature that easily could build anastomoses to a host's organ. Only placentas from larger animal models such as the bovine meet the dimensions large enough for most organs but are not adequately described yet. We here studied the nature of the ECM in 27 natural and decellularized bovine cotyledons, that is, the fetal part of the placentomes, by means of histology, immunohistochemistry, and electron microscopy. Successful decellularization was done by perfusion with 0.01%, 0.1%, and 0.5% sodium dodecyl sulfate each and subsequent immersion in 1% Triton X-100, resulting in a removal of cells and DNA, whereas the structure of the allantochorionic surface and villi was preserved. Although some fibres disappeared, also the arrangement of the main ECM proteins was largely similar before and after decellularization: Along the larger vessels, a densely packed network of thick fibres occurred, organized in layers without cells or spaces in between. Collagen IV, fibronectin, and laminin contributed to those areas. In contrast, collagen I and III characterized the meshwork of medium-sized and thin fibres in the mesenchyme, respectively. In conclusion, decellularized bovine cotyledons indeed had characteristics of a biological scaffold and provide an interesting alternative to develop large-scale scaffolds with complex vascular architecture for tissue engineering purposes.

Bioengineering a Human Face Graft: The Matrix of Identity.

During the last decade, face allotransplantation has been shown to be a revolutionary reconstructive procedure for severe disfigurements. However, offer to patients remains limited due to lifelong immunosuppression. To move forward in the field, a new pathway in tissue engineering is proposed. Our previously reported technique of matrix production of a porcine auricular subunit graft has been translated to a human face model. 5 partial and 1 total face grafts were procured from human fresh cadavers. After arterial cannulation, the specimens were perfused using a combined detergent/polar solvent decellularization protocol. Preservation of vascular patency was assessed by imaging, cell and antigen removal by DNA quantification and histology. The main extracellular matrix proteins and associated cytokines were evaluated. Lip scaffolds were cultivated with dermal, muscle progenitor and endothelial cells, either on discs or in a bioreactor. Decellularization was successful in all facial grafts within 12 days revealing acellular scaffolds with full preservation of innate morphology. Imaging demonstrated a preservation of the entire vascular tree patency. Removal of cells and antigens was confirmed by reduction of DNA and antigen markers negativation. Microscopic evaluation revealed preservation of tissue structures as well as of major proteins. Seeded cells were viable and well distributed within all scaffolds. Complex acellular facial scaffolds were obtained, preserving simultaneously a cell-friendly extracellular matrix and a perfusable vascular tree. This step will enable further engineering of postmortem facial grafts, thereby offering new perspectives in composite tissue allotransplantation.

Diverse expression of N-acetylglucosaminyltransferase V and complex-type β1,6-branched N-glycans in uveal and cutaneous melanoma cells

Although both uveal (UM) and cutaneous (CM) melanoma cells derive from the transformed melanocytes, their biology varies significantly in several aspects. Malignant transformation is frequently associated with alternations in cell glycosylation, in particular those concerning branched complex-type N-glycans. These changes occur principally in β1,4-N-acetylglucosaminyltransferase III (GnT-III) that catalyzes the synthesis of glycans with bisected N-acetylglucosamine (GlcNAc) and β1,6-N-acetylglucosaminyltransferase V (GnT-V) that is involved in forming β1,6-branched antenna in complex-type glycans. We searched for the reasons of a different behavior of CM and UM cells in the expression of GnT-III and GnT-V and their oligosaccharide products. Our study showed that UM cells have more β1,6-branched glycans than CM cells, what results from a higher expression of MGAT5 gene encoding GnT-V. The higher β1,6-branching of glycans in UM may contribute to their higher potential to migrate on fibronectin and weaker binding to main extracellular matrix proteins, observed in our previous studies.

A material-based platform to modulate fibronectin activity and focal adhesion assembly.

We present a detailed characterization of fibronectin (FN) adsorption and cell adhesion on poly(ethyl acrylate) (PEA) and poly(methyl acrylate) (PMA), two polymers with very similar physicochemical properties and chemical structure, which differ in one single methyl group in the lateral chain of the polymer. The globular solution conformation of FN was retained following adsorption onto PMA, whereas spontaneous organization of FN into protein (nano) networks occurred on PEA. This distinct distribution of FN at the material interface promoted a different availability, measured via monoclonal antibody binding, of two domains that facilitated integrin binding to FN: FNIII10 (RGD sequence) and FNIII9 (PHSRN synergy sequence). The enhanced exposure of the synergy domain on PEA compared to PMA triggered different focal adhesion assemblies: L929 fibroblasts showed a higher fraction of smaller focal plaques on PMA (40%) than on PEA (20%). Blocking experiments with monoclonal antibodies against FNIII10 (HFN7.1) and FNIII9 (mAb1937) confirmed the ability of these polymeric substrates to modulate FN conformation. Overall, we propose a simple and versatile material platform that can be used to tune the presentation of a main extracellular matrix protein (FN) to cells, for applications than span from tissue engineering to disease biology.

Fibronectin and stem cell differentiation – lessons from chondrogenesis

The extracellular matrix (ECM) is an intricate network of proteins that surrounds cells and has a central role in establishing an environment that is conducive to tissue-specific cell functions. In the case of stem cells, this environment is the stem cell niche, where ECM signals participate in cell fate decisions. In this Commentary, we describe how changes in ECM composition and mechanical properties can affect cell shape and stem cell differentiation. Using chondrogenic differentiation as a model, we examine the changes in the ECM that occur before and during mesenchymal stem cell differentiation. In particular, we focus on the main ECM protein fibronectin, its temporal expression pattern during chondrogenic differentiation, its potential effects on functions of differentiating chondrocytes, and how its interactions with other ECM components might affect cartilage development. Finally, we discuss data that support the possibility that the fibronectin matrix has an instructive role in directing cells through the condensation, proliferation and/or differentiation stages of cartilage formation.

473 EFFECTIVENESS OF TREATMENT OF TENDINITIS AND PLANTAR FASCIITIS BY TENDOACTIVE™

IL-1β, stimulated with IL-1β and Tendoactive® or pre-stimulated with Tendoactive® followed by co-treatment with Tendoactive® and IL-1β. Cell viability, adhesion, proliferation and the production of ECM were analysed with light microscopy and transmission electron microscopy (TEM). Immunofluorescence was used to evaluate production of type I collagen, the main extracellular matrix protein produced by tenocytes. We also studied the expression of the signal transduction and adhesion molecule β1integrin.Western Blotting (WB) was performed to evaluate the expression of apoptotic and inflammatory markers (metalloprotease1 (MMP-1), cyclooxygenase-2 (Cox-2) and caspase-3). Results: Tendoactive® had a potent stimulatory effect on human tenocyte proliferation and ECM production and was also able to suppress the catabolic, apoptotic and inflammatory effects induced by IL-1β in human tenocytes. This was demonstrated by the suppression of IL-1β-induced expression of MMP-1, Cox-2 and caspase-3 and up-regulation of type I collagen and β1-integrin. Conclusions: The results presented suggest that this formulation inhibits catabolic and inflammatory processes in an in vitro model of tendonitis.Tendoactive® may therefore be used on prophylaxis and treatment of tendinopathies to stimulate tendon healing, regeneration and repair.

474 A DOSE-DEPENDENT IMPACT OF A STANDARDIZED POWDER MADE FROM HIPS OF A ROSE SUBSPECIES ( ROSA canina) ON WOMAC PAIN SCORES IN PATIENTS WITH OSTEOARTHRITIS OF THE HIP AND/OR KNEE

IL-1β, stimulated with IL-1β and Tendoactive® or pre-stimulated with Tendoactive® followed by co-treatment with Tendoactive® and IL-1β. Cell viability, adhesion, proliferation and the production of ECM were analysed with light microscopy and transmission electron microscopy (TEM). Immunofluorescence was used to evaluate production of type I collagen, the main extracellular matrix protein produced by tenocytes. We also studied the expression of the signal transduction and adhesion molecule β1integrin.Western Blotting (WB) was performed to evaluate the expression of apoptotic and inflammatory markers (metalloprotease1 (MMP-1), cyclooxygenase-2 (Cox-2) and caspase-3). Results: Tendoactive® had a potent stimulatory effect on human tenocyte proliferation and ECM production and was also able to suppress the catabolic, apoptotic and inflammatory effects induced by IL-1β in human tenocytes. This was demonstrated by the suppression of IL-1β-induced expression of MMP-1, Cox-2 and caspase-3 and up-regulation of type I collagen and β1-integrin. Conclusions: The results presented suggest that this formulation inhibits catabolic and inflammatory processes in an in vitro model of tendonitis.Tendoactive® may therefore be used on prophylaxis and treatment of tendinopathies to stimulate tendon healing, regeneration and repair.

A TLR4/MD2 fusion protein inhibits LPS-induced pro-inflammatory signaling in hepatic stellate cells

Activated hepatic stellate cells (HSCs) play a key role in hepatic fibrogenesis. In injured liver they are the main extracellular matrix protein producing cell type and further perpetuate hepatic injury by secretion of pro-inflammatory mediators. Since LPS-mediated signaling through toll-like receptor 4 (TLR4) has been identified as key fibrogenic signal in HSCs we aimed to test TLR4 as potential target of therapy via ligand-binding soluble receptors. Incubation of human HSCs with a fusion protein between the extracellular domain of TLR4 and MD2 which binds LPS inhibited LPS-induced NFκB and JNK activation. TLR4/MD2 abolished LPS-induced secretion of IL-6, IL-8, MCP1, and RANTES in HSCs. In addition, TLR4/MD2 fused to human IgG-Fc neutralized LPS activity. Since TLR4 mutant mice are resistant to liver fibrosis, the TLR4/MD2 soluble receptor might represent a new therapeutic molecule for liver fibrogenesis in vivo.

Egr-1 Regulates Expression of the Glial Scar Component Phosphacan in Astrocytes after Experimental Stroke

Ischemic brain injury causes tissue damage and neuronal death. The deficits can often be permanent because adult neurons fail to regenerate. One barrier to neuronal regeneration is the formation of the glial scar, a repair mechanism that is otherwise necessary to seal off necrotic areas. The process of gliosis has been well described, but the mechanisms regulating the robust production of scar components after injury remain poorly understood. Here we show that the early growth response 1 transcriptional factor (Egr-1, also called Krox24, Zif268, and NGFI-A) is expressed in astrocytes in the ventricular wall, corpus callosum, and striatum of normal mouse brain. After experimental stroke caused by permanent occlusion of the middle cerebral artery, Egr-1 was expressed long term in reactive astrocytes that accumulate around the injury site. Gain- and loss-of-function studies in primary astrocytes indicated that Egr-1 regulates the transcription of chondroitin sulfate proteoglycans genes, the main extracellular matrix proteins of the glial scar. Egr-1 bound to a site within the phosphacan promoter and transactivated its expression. Egr-1-deficient mice accumulated lower levels of phosphacan RNA and protein than wild-type mice after stroke, but there were no measurable differences in neurite outgrowth toward the infarct area between the two groups. Our findings suggest that Egr-1 is an important component of the transcriptional network regulating genes involved in gliosis after ischemic injury.

Smooth Muscle α-Actin Deficiency in Myofibroblasts Leads to Enhanced Renal Tissue Fibrosis

Myofibroblasts are a major source of proinflammatory cytokines and extracellular matrix in progressive tissue fibrosis leading to chronic organ failure. Myofibroblasts are characterized by de novo expression of smooth muscle alpha-actin (SMalphaA), which correlates with the extent of disease progression, although their exact role is unknown. In vitro cultured myofibroblasts from kidney of SMalphaA knock-out mice demonstrate significantly more prominent cell motility, proliferation, and type-I procollagen expression than those of wild-type myofibroblasts. These pro-fibrotic properties are suppressed by adenovirus-mediated SMalphaA re-expression, accompanied by down-regulation of focal adhesion proteins. In interstitial fibrosis model, tissue fibrosis area, proliferating interstitial cell number, and type-I procollagen expression are enhanced under SMalphaA deficiency. In mesangioproliferative glomerulonephritis model, cell proliferation in the mesangial area is also enhanced in SMalphaA knock-out mice. Adenoviral SMalphaA introduction into renal interstitium obviously ameliorates tissue fibrosis in interstitial fibrosis model. These results indicate that SMalphaA suppresses the pro-fibrotic properties of myofibroblasts, highlighting the significance of smooth muscle-related proteins in moderating chronic organ fibrosis under pathological conditions.

RNA Interference against Urokinase in Hepatocellular Carcinoma Xenografts in Nude Mice

The serine protease urokinase-type plasminogen activator (u-PA) is overexpressed in hepatocellular carcinoma (HCC) and its expression level is inversely correlated with the patients’ survival. The purpose of this study was to examine the effects of vector-based RNA interference (RNAi) of u-PA on the growth of human HCC xenografts in nude mice in order to investigate the role of u-PA in human HCC. Our results showed that the subcutaneous injection of small interfering RNAs (siRNA) u-PA SKHep1C3 stable transfected cells (pS siRNA u-PA) led to a growth delay in xenograft development, compared to those generated from empty vector; the molecular characterization of nodules (carried out by PCR, RT-PCR and immunohistochemical analysis) revealed the presence of plasmid DNA, the u-PA gene expression knockdown, at both mRNA and protein levels, giving evidence of a long-term and target-specific inhibition by vector-based RNAi 11 weeks after cell inoculation. We further studied the effects of u-PA downmodulation on extracellular matrix (ECM) proteins evaluating the expression and organization of fibronectin (FN; one of the main ECM proteins). Immunohistochemical and immunofluorescence analysis of FN revealed FN fibrils in pS siRNA u-PA xenografts and in pS siRNA u-PA cells, thus identifying the FN fibril organization as a downstream effect of u-PA knockdown in this system.