Direct FGF-2 Gene Transfer via Recombinant Adeno-Associated Virus Vectors Stimulates Cell Proliferation, Collagen Production, and the Repair of Experimental Lesions in the Human ACL

2012 ◽  
Vol 41 (1) ◽  
pp. 194-202 ◽  
Author(s):  
Henning Madry ◽  
Dieter Kohn ◽  
Magali Cucchiarini
Keyword(s):  
Gene Transfer ◽  
Smooth Muscle Actin ◽  
Cruciate Ligament ◽  
Marker Gene ◽  
Nuclear Factor Κb ◽  
Type I ◽  
Adeno Associated Virus ◽  
Collagen Production

Background: Basic fibroblast growth factor (FGF-2) is a powerful stimulator of fibroblast proliferation and type I/III collagen production. Hypothesis: Overexpression of FGF-2 via direct recombinant adeno-associated virus (rAAV) vector–mediated gene transfer enhances the healing of experimental lesions to the human anterior cruciate ligament (ACL). Study Design: Controlled laboratory study. Methods: rAAV vectors carrying a human FGF-2 sequence or the lacZ marker gene were applied to primary human ACL fibroblasts in vitro and to intact or experimentally injured human ACL explants in situ to evaluate the efficacy and duration of transgene expression and the potential effects of FGF-2 treatment upon the proliferative, metabolic, and regenerative activities in these systems. Results: Sustained, effective dose-dependent lacZ expression was achieved in all systems tested (up to 96% ± 2% in vitro and 80%-85% in situ for at least 30 days). rAAV allowed for continuous FGF-2 production both in vitro and in the intact ACL in situ (32.7 ± 1.4 and 33.1 ± 0.8 pg/mL/24 h, respectively, ie, up to 41-fold more than in the controls at day 30; always P ≤ .001), leading to significantly and durably enhanced levels of proliferation and type I/III collagen production vis-à-vis lacZ (at least 3- and 4-fold increases at day 30, respectively; always P ≤ .001). Most notably, rAAV FGF-2 promoted a significant, long-term production of the factor in experimental ACL lesions (92.7 ± 3.9 pg/mL/24 h, ie, about 5-fold more than in the controls; P ≤ .001) associated with enhanced levels of proliferation and type I/III collagen synthesis (at least 2- and 4-fold increases at day 30, respectively; always P ≤ .001). Remarkably, the FGF-2 treatment allowed for a decrease in the amplitude of such lesions possibly because of the increased expression in contractile α–smooth muscle actin, ligament-specific transcription factor scleraxis, and nuclear factor–κB for proliferation and collagen deposition, which are all markers commonly induced in response to injury. Conclusion: Efficient, stable FGF-2 expression via rAAV enhances the healing of experimental human ACL lesions by activating key cellular and metabolic processes. Clinical Relevance: This approach has potential value for the development of novel, effective treatments for ligament reconstruction.

scholarly journals Enhanced Repair of the Anterior Cruciate Ligament by in Situ Gene Transfer: Evaluation in an in Vitro Model

2004 ◽  
Vol 10 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Arnulf Pascher ◽  
Andre F. Steinert ◽  
Glyn D. Palmer ◽  
Oliver Betz ◽  
Jean-Noel Gouze ◽  
...  
Keyword(s):  
Anterior Cruciate Ligament ◽  
Gene Transfer ◽  
In Vitro Model ◽  
Cruciate Ligament ◽  
Anterior Cruciate ◽  
Vitro Model

scholarly journals mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nozomi Igarashi ◽  
Megumi Honjo ◽  
Makoto Aihara
Keyword(s):  
Trabecular Meshwork ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Mtor Inhibitors ◽  
In Vivo Model ◽  
Type I ◽  
Fibrotic Response ◽  
Alpha Smooth Muscle Actin

AbstractWe examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.

scholarly journals An approach for the analysis of relapse and marrow reconstitution after autologous marrow transplantation using retrovirus-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 79 (10) ◽  
pp. 2694-2700 ◽  
Author(s):  
DR Rill ◽  
RC Moen ◽  
M Buschle ◽  
C Bartholomew ◽  
NK Foreman ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Marrow Transplantation ◽  
Residual Disease ◽  
Marker Gene ◽  
Autologous Bone Marrow Transplantation ◽  
Autologous Bone Marrow ◽  
Disease Recurrence ◽  
Malignant Cells

Abstract Autologous bone marrow transplantation (ABMT) is widely used as treatment for malignant disease. Although the major cause of treatment failure is relapse, it is unknown if this arises entirely because of residual disease in the patient or whether contaminating cells in the rescuing marrow contribute. Attempts to purge marrow of its putative residual malignant cells may delay hematopoietic reconstitution and are of uncertain efficacy. We now describe how retrovirus-mediated gene transfer may be used to elucidate the source of relapse after ABMT for acute myeloid leukemia and to evaluate the efficacy of purging. Clonogenic myeloid leukemic blast cells in patient marrow can be transduced with the NeoR gene-containing helper-free retrovirus, LNL6, with an efficacy of 0% to 23.5% (mean, 10.5%). Transduced colonies grow in selective media and the presence of the marker gene can be confirmed in individual malignant colonies by polymerase chain reaction. If such malignant cells remain in harvested “remission” marrow, they will therefore be marked after exposure to LNL6. Detection of the marker gene in the malignant cells present at any later relapse would be firm evidence that residual disease contributed to disease recurrence, and would permit rapid subsequent evaluation of purging techniques. The technique also marks normal marrow progenitors from patients with acute myeloblastic leukemia. These colony-forming cells can be detected in long-term marrow cultures at a frequency of 1% to 18% for up to 10 weeks after exposure to the vector. Animal models and analysis of probability tables both suggest that these levels of marking in vitro are sufficient to provide information about the mechanisms of relapse and the biology of marrow regeneration in vivo. These preclinical data form part of the basis for current clinical studies of gene transfer into marrow before ABMT.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

scholarly journals Hierarchical Coculture of Hepatocyte and Hepatic Non-Parenchymal Cells for Liver Fibrosis Studies in vitro

2020 ◽  
Author(s):  
Qiao You Lau ◽  
Fuad Gandhi Torizal ◽  
Marie Shinohara ◽  
Yasuyuki Sakai
Keyword(s):  
Growth Factor ◽  
Liver Fibrosis ◽  
Oxygen Tension ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Liver Sinusoidal Endothelial Cell ◽  
Type I ◽  
Parenchymal Cells

During chronic liver injury, inflammation leads to the development of liver fibrosis— particularly due to the activation of hepatic stellate cells (HSCs). However, the involvement of inflammatory cytokines in HSC activation is unclear. Many existing in vitro liver models do not include these non-parenchymal cells (NPCs), and hence, do not represent the physiological relevance found in vivo. Herein, we demonstrated the hierarchical coculture of primary rat hepatocytes with NPCs such as the human-derived HSC line (LX-2) and the human-derived liver sinusoidal endothelial cell line (TMNK-1). The coculture tissue had higher albumin production and hepatic cytochrome P450 3A4 activity compared to the monoculture. We then further studied the effects of stimulation by both oxygen tension and key pro-fibrogenic cytokines, such as the transforming growth factor beta (TGF-β), on HSC activation. Gene expression analysis revealed that lower oxygen tension and TGF-β1 stimulation enhanced collagen type I, III, and IV, alpha-smooth muscle actin, platelet-derived growth factor, and matrix metallopeptidase expression from LX-2 cells in the hierarchical coculture after fibrogenesis induction. This hierarchical in vitro cocultured liver tissue could, therefore, provide an improved platform as a disease model for elucidating the interactions of various liver cell types and biochemical signals in liver fibrosis studies.

scholarly journals In Vitro Packaging of Adeno-Associated Virus DNA

1998 ◽  
Vol 72 (4) ◽  
pp. 3241-3247 ◽  
Author(s):  
Xiaohuai Zhou ◽  
Nicholas Muzyczka
Keyword(s):  
Virus Particle ◽  
Mammalian Cells ◽  
Gradient Centrifugation ◽  
Marker Gene ◽  
Dnase I ◽  
Capsid Proteins ◽  
Wild Type Virus ◽  
Adeno Associated Virus ◽  
Terminal Repeats

ABSTRACT We have developed an in vitro procedure for packaging of recombinant adeno-associated virus (AAV). By using AAV replicative-form DNA as the substrate, it is possible to synthesize an infectious AAV particle in vitro that can be used to transfer a marker gene to mammalian cells. The packaging procedure requires the presence of both the AAV Rep and capsid proteins. Two kinds of in vitro products can be formed which facilitate DNA transfer. Both are resistant to heat and have a density in cesium chloride gradients that is indistinguishable from that of the in vivo-synthesized wild-type virus. This indicates that the particles formed have the appropriate protein-to-DNA ratio and a structure that shares the heat resistance of mature AAV particles. The two types of particles can be distinguished by their sensitivity to chloroform and DNase I treatment. The chloroform-resistant product is, by several criteria, an authentic AAV particle. In addition to having the correct density and being resistant to treatment with chloroform, DNase I, and heat, this particle is efficiently synthesized only if the AAV genome contains intact terminal repeats, which are known to be required for AAV packaging. It is also precipitated by a monoclonal antibody that recognizes mature virus particles but not bound by an antibody that recognizes monomeric or denatured capsid proteins. The chloroform-resistant species is not made when aphidicolin is present in the reaction mixture, suggesting that active DNA replication is required for in vitro packaging. In contrast, the chloroform-sensitive product has several features that suggest it is an incompletely assembled virus particle. It is sensitive to DNase I, does not require the presence of AAV terminal repeats, and is capable of transferring DNA that is theoretically too large to package. Sucrose gradient centrifugation of the in vitro-synthesized products reveals that the particles have sedimentation values between 60S and 110S, which is consistent with partially assembled and mature AAV particles. The in vitro packaging procedure should be useful for studying the mechanism by which a human icosahedral DNA virus particle is assembled, and it may be useful for producing recombinant AAV for gene therapy. The chloroform-sensitive particle may also be useful for transferring DNA that is too large to be packaged in mature recombinant AAV.

scholarly journals The Role of miR-21 in Osteoblasts–Osteoclasts Coupling In Vitro

Cells ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 479 ◽  
Author(s):  
Agnieszka Smieszek ◽  
Klaudia Marcinkowska ◽  
Ariadna Pielok ◽  
Mateusz Sikora ◽  
Lukas Valihrach ◽  
...  
Keyword(s):  
Cathepsin K ◽  
Nuclear Factor Κb ◽  
Tartrate Resistant Acid Phosphatase ◽  
Type I ◽  
Nuclear Factor ◽  
Paracrine Signaling ◽  
Receptor Activator ◽  
The Impact

MiR-21 is being gradually more and more recognized as a molecule regulating bone tissue homeostasis. However, its function is not fully understood due to the dual role of miR-21 on bone-forming and bone-resorbing cells. In this study, we investigated the impact of miR-21 inhibition on pre-osteoblastic cells differentiation and paracrine signaling towards pre-osteoclasts using indirect co-culture model of mouse pre-osteoblast (MC3T3) and pre-osteoclast (4B12) cell lines. The inhibition of miR-21 in MC3T3 cells (MC3T3inh21) modulated expression of genes encoding osteogenic markers including collagen type I (Coll-1), osteocalcin (Ocl), osteopontin (Opn), and runt-related transcription factor 2 (Runx-2). Inhibition of miR-21 in osteogenic cultures of MC3T3 also inflected the synthesis of OPN protein which is essential for proper mineralization of extracellular matrix (ECM) and anchoring osteoclasts to the bones. Furthermore, it was shown that in osteoblasts miR-21 regulates expression of factors that are vital for survival of pre-osteoclast, such as receptor activator of nuclear factor κB ligand (RANKL). The pre-osteoclast cultured with MC3T3inh21 cells was characterized by lowered expression of several markers associated with osteoclasts’ differentiation, foremost tartrate-resistant acid phosphatase (Trap) but also receptor activator of nuclear factor-κB ligand (Rank), cathepsin K (Ctsk), carbonic anhydrase II (CaII), and matrix metalloproteinase (Mmp-9). Collectively, our data indicate that the inhibition of miR-21 in MC3T3 cells impairs the differentiation and ECM mineralization as well as influences paracrine signaling leading to decreased viability of pre-osteoclasts.

scholarly journals Regulation of valve interstitial cell homeostasis by mechanical deformation: implications for heart valve disease and surgical repair

2017 ◽  
Vol 14 (135) ◽  
pp. 20170580 ◽  
Author(s):  
Salma Ayoub ◽  
Chung-Hao Lee ◽  
Kathryn H. Driesbaugh ◽  
Wanda Anselmo ◽  
Connor T. Hughes ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Interstitial Cell ◽  
Smooth Muscle Actin ◽  
Heart Valve Disease ◽  
Type I ◽  
Tissue Remodelling ◽  
Cell Responses ◽  
Stress States

Mechanical stress is one of the major aetiological factors underlying soft-tissue remodelling, especially for the mitral valve (MV). It has been hypothesized that altered MV tissue stress states lead to deviations from cellular homeostasis, resulting in subsequent cellular activation and extracellular matrix (ECM) remodelling. However, a quantitative link between alterations in the organ-level in vivo state and in vitro- based mechanobiology studies has yet to be made. We thus developed an integrated experimental–computational approach to elucidate MV tissue and interstitial cell responses to varying tissue strain levels. Comprehensive results at different length scales revealed that normal responses are observed only within a defined range of tissue deformations, whereas deformations outside of this range lead to hypo- and hyper-synthetic responses, evidenced by changes in α-smooth muscle actin, type I collagen, and other ECM and cell adhesion molecule regulation. We identified MV interstitial cell deformation as a key player in leaflet tissue homeostatic regulation and, as such, used it as the metric that makes the critical link between in vitro responses to simulated equivalent in vivo behaviour. Results indicated that cell responses have a delimited range of in vivo deformations that maintain a homeostatic response, suggesting that deviations from this range may lead to deleterious tissue remodelling and failure.

scholarly journals Nonrandom Transduction of Recombinant Adeno-Associated Virus Vectors in Mouse Hepatocytes In Vivo: Cell Cycling Does Not Influence Hepatocyte Transduction

2000 ◽  
Vol 74 (8) ◽  
pp. 3793-3803 ◽  
Author(s):  
Carol H. Miao ◽  
Hiroyuki Nakai ◽  
Arthur R. Thompson ◽  
Theresa A. Storm ◽  
Winnie Chiu ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Gene Transfer ◽  
Genetic Diseases ◽  
Critical Event ◽  
Dimer Formation ◽  
Adeno Associated Virus ◽  
Virus Vectors ◽  
Preclinical Trials

ABSTRACT Recombinant adeno-associated virus vectors (rAAV) show promise in preclinical trials for the treatment of genetic diseases including hemophilia. Liver-directed gene transfer results in a slow rise in transgene expression, reaching steady-state levels over a period of 5 weeks concomitant with the conversion of the single-stranded rAAV molecules into high-molecular-weight concatemers in about 5% of hepatocytes. Immunohistochemistry and RNA in situ hybridization show that the transgene product is made in about ∼5% of hepatocytes, suggesting that most rAAV-mediated gene expression occurs in hepatocytes containing the double-stranded concatemers. In this study, the mechanism(s) involved in stable transduction in vivo was evaluated. While only ∼5% of hepatocytes are stably transduced, in situ hybridization experiments demonstrated that the vast majority of the hepatocytes take up AAV-DNA genomes after portal vein infusion of the vector. Two different vectors were infused together or staggered by 1, 3, or 5 weeks, and two-color fluorescent in situ hybridization and molecular analyses were performed 5 weeks after the infusion of the second vector. These experiments revealed that a small but changing subpopulation of hepatocytes were permissive to stable transduction. Furthermore, in animals that received a single infusion of two vectors, about one-third of the transduced cells contained heteroconcatemers, suggesting that dimer formation was a critical event in the process of concatemer formation. To determine if the progression through the cell cycle was important for rAAV transduction, animals were continuously infused with 5′-bromo-2′-deoxyuridine (BrdU), starting at the time of administration of a rAAV vector that expressed cytoplasmic β-galactosidase. Colabeling for β-galactosidase and BrdU revealed that there was no preference for transduction of cycling cells. This was further confirmed by demonstrating no increase in rAAV transduction efficiencies in animals whose livers were induced to cycle at the time of or after vector administration. Taken together, our studies suggest that while virtually all hepatocytes take up vector, unknown cellular factors are required for stable transduction, and that dimer formation is a critical event in the transduction pathway. These studies have important implications for understanding the mechanism of integration and may be useful for improving liver gene transfer in vivo.

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