Potent Inhibition of Arterial Intimal Hyperplasia by TIMP1 Gene Transfer using AAV vectors

Seminal to the process of arterial restenosis after balloon angioplasty is extracellular matrix degradation by metalloproteinases (MMPs); activity of these proteins is strongly inhibited by the tissue inhibitors of MMPs (TIMPs). Here we exploit gene transfer using an adeno-associated virus (AAV) for TIMP1 gene delivery in a rat model of intimal hyperplasia. High-titer AAV-Timp1 efficiently transduced human coronary artery smooth muscle cells (SMCs) in vitro and inhibited the capacity of these cells to migrate through a Matrigel barrier. In injured rat carotid arteries, AAV vectors were found to transduce SMCs efficiently and to maintain transgene expression for several weeks in vivo. In AAV-Timp1-transduced animals, the intima:media ratio of injured carotids was significantly reduced by 70.5% after 2 weeks, by 58.5% after 1 month, and by 52.4% after 2 months from treatment. The decrease in intimal hyperplasia was paralleled by a significant inhibition of collagen accumulation and by increased elastin deposition in the neointima, two findings that relate to the inhibition of MMP activity. These results indicate that AAV vectors are efficient tools for delivering genes to the arterial wall and emphasize the importance of MMPs for the generation of intimal hyperplasia. Local TIMP1 gene transfer might thus represent an efficient strategy to prevent restenosis

Induction of Functional Neovascularization by Combined VEGF and Angiopoietin-1 Gene Transfer Using AAV Vectors

Vectors based on the adeno-associated virus (AAV) deliver therapeutic genes to muscle and heart at high efficiency and maintain transgene expression for long periods of time. Here we report about the synergistic effect on blood vessel formation of AAV vectors expressing the 165 aa isoform of vascular endothelial growth factor (VEGF165), a powerful activator of endothelial cells, and of angiopoietin-1 (Ang-1), which is required for vessel maturation. High titer AAV-VEGF165 and AAV-Ang-1 vector preparations were injected either alone or in combination in the normoperfused tibialis anterior muscle of rats. Long term expression of VEGF165 determined massive cellular infiltration of the muscle tissues over time, with the formation of a large set of new vessels. Strikingly, some of the cells infiltrating the treated muscles were found positive for markers of activated endothelial precursors (VEGFR-2/KDR and Tie-2) and for c-kit, an antigen expressed by pluripotent bone marrow stem cells. Expression of VEGF165 eventually resulted in the formation of structured vessels surrounded by a layer of smooth muscle cells. Presence of these arteriolae correlated with significantly increased blood perfusion in the injected areas. Co-expression of VEGF165 with angiopoietin-1-which did not display angiogenic effect per se-remarkably reduced leakage of vessels produced by VEGF165 alone

Vascular endothelial growth factor stimulates skeletal muscle regeneration in Vivo

Vascular endothelial growth factor (VEGF) is a major regulator of blood vessel formation during development and in the adult organism. Recent evidence indicates that this factor also plays an important role in sustaining the proliferation and differentiation of different cell types, including progenitor cells of different tissues, including bone marrow, bone, and the central nervous system. Here we show that the delivery of the 165-aa isoform of VEGF-A cDNA using an adeno-associated virus (AAV) vector exerts a powerful effect on skeletal muscle regeneration in vivo. Following ischemia-, glycerol-, or cardiotoxin-induced damage in mouse skeletal muscle, the delivery of AAV-VEGF markedly improved muscle fiber reconstitution with a dose-dependent effect. The expression of both VEGF receptor-1 (VEGFR-1) and VEGFR-2 was upregulated both in the satellite cells of the damaged muscles and during myotube formation in vitro; the VEGF effect was mediated by the VEGFR-2, since the transfer of PlGF, a VEGF family member interacting with the VEGFR-1, was ineffective. These results are consistent with the observation that VEGF promotes the growth of myogenic fibers and protects the myogenic cells from apoptosis in vitro and prompt a therapeutic use for VEGF gene transfer in a variety of muscular disorders

Δ133p53β isoform pro-invasive activity is regulated through an aggregation-dependent mechanism in cancer cells

International audienceAbstract The p53 isoform, Δ133p53β, is critical in promoting cancer. Here we report that Δ133p53β activity is regulated through an aggregation-dependent mechanism. Δ133p53β aggregates were observed in cancer cells and tumour biopsies. The Δ133p53β aggregation depends on association with interacting partners including p63 family members or the CCT chaperone complex. Depletion of the CCT complex promotes accumulation of Δ133p53β aggregates and loss of Δ133p53β dependent cancer cell invasion. In contrast, association with p63 family members recruits Δ133p53β from aggregates increasing its intracellular mobility. Our study reveals novel mechanisms of cancer progression for p53 isoforms which are regulated through sequestration in aggregates and recruitment upon association with specific partners like p63 isoforms or CCT chaperone complex, that critically influence cancer cell features like EMT, migration and invasion

Cyclin A2 Mutagenesis Analysis: A New Insight into CDK Activation and Cellular Localization Requirements

Cyclin A2 is essential at two critical points in the somatic cell cycle: during S phase, when it activates CDK2, and during the G2 to M transition when it activates CDK1. Based on the crystal structure of Cyclin A2 in association with CDKs, we generated a panel of mutants to characterize the specific amino acids required for partner binding, CDK activation and subcellular localization. We find that CDK1, CDK2, p21, p27 and p107 have overlapping but distinct requirements for association with this protein. Our data highlight the crucial importance of the N-terminal α helix, in conjunction with the α3 helix within the cyclin box, in activating CDK. Several Cyclin A2 mutants selectively bind to either CDK1 or CDK2. We demonstrate that association of Cyclin A2 to proteins such as CDK2 that was previously suggested as crucial is not a prerequisite for its nuclear localization, and we propose that the whole protein structure is involved

Cyclin A2, Rho GTPases and EMT

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Analysis of femoral trochanters morphology based on geometrical model

210-216This study presents morphological analysis of 20 scans of femur samples from European (Serbian) adults from trochanteric region based on the customized computer aided reverse modeling procedure. Results indicated that trochanteric region is a separate morphological unit of proximal femur, named trochanteric wedge or canoe. This new perceiving of trochanteric region seems to provide a better understanding of trochanteric wedge volume and, therefore, better trochanter fractures treatment, operation planning, implant and endoprothesis design and selection. Also, it brings a new light to anatomy of proximal femur, its biomechanics and ossification

Schematic representation of mouse Cyclin A2 cDNA with the modifications introduced in this study.

<p>All constructs were C-terminally fused to 3 Flag tags.</p

Reverse Modelling of Human Long Bones Using T-Splines - Case of Tibia

Creating a sufficiently accurate digital model of human bone geometry for a specific patient is the starting point for personalized orthopaedic surgical treatment. Such geometrical model of a particular human bone serves as a template for designing personalized bone implant, which can be an endoprostheses, fixator plate or even scaffold. Due to that role, the geometry of a human bone model has to be reusable and modifiable. Otherwise, design of the corresponding personalized implant for a particular human bone is a very difficult task. Modern reverse modelling techniques have greatly accelerated the creation of a bone geometric model. The prevailing modern approach is based on the use of non-uniform rational B-splines. However, recent research shows that the very complex topology of bio-shapes, such as human bones, can be reconstructed more easily using T-Splines. The presented approach of using T-splines in a modelling process allows creation of a bone model with important advantages regarding quality, flexibility and geometric accuracy. The process of reverse modelling starts from the cloud of points gained through computer-tomography images and uses selected referential geometric, i.e. anatomic entities as guiding elements in digital reconstruction of the particular bone geometry