Engineered Vascularized Muscle Flap

One of the main factors limiting the thickness of a tissue construct and its consequential viability and applicability in vivo, is the control of oxygen ...

Technion Israel Institute of Technology

The voltage dependent anion channel (VDAC), located in the outer mitochondrial membrane, functions as a major channel allowing passage of small molecules and ions between the mitochondrial inter-membrane space and cytoplasm. Together with the adenine nucleotide translocator (ANT), which is located in the inner mitochondrial membrane, the VDAC is considered to form the core of a mitochondrial multiprotein complex, named the mitochondrial permeability transition pore (MPTP). Both VDAC and ANT appear to take part in activation of the mitochondrial apoptosis pathway. Other proteins also appear to be associated with the MPTP, for example, the 18 kDa mitochondrial Translocator Protein (TSPO), Bcl-2, hexokinase, cyclophylin D, and others. Interactions between VDAC and TSPO are considered to play a role in apoptotic cell death. As a consequence, due to its apoptotic functions, the TSPO has become a target for drug development directed to find treatments for neurodegenerative diseases and cancer. In this context, TSPO appears to be involved in the generation of reactive oxygen species (ROS). This generation of ROS may provide a link between activation of TSPO and of VDAC, to induce activation of the mitochondrial apoptosis pathway. ROS are known to be able to release cytochrome c from cardiolipins located at the inner mitochondrial membrane. In addition, ROS appear to be able to activate VDAC and allow VDAC mediated release of cytochrome c into the cytosol. Release of cytochrome c from the mitochondria forms the initiating step for activation of the mitochondrial apoptosis pathway. These data provide an understanding regarding the mechanisms whereby VDAC and TSPO may serve as targets to modulate apoptotic rates. This has implications for drug design to treat diseases such as neurodegeneration and cancer.

VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis.

We have previously shown that the anti-neoplastic agent erucylphosphohomocholine (ErPC3) requires the mitochondrial 18 kDa Translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor (PBR), to induce cell death via the mitochondrial apoptosis pathway.

Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine.

The involvement of the 18-kDa translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, in apoptosis regulation of HT29 colorectal cancer cells was studied in-vitro. In-vivo TSPO involvement in tumor growth of HT29 cells xenografted into SCID mice was studied.

The 18-kDa translocator protein, formerly known as the peripheral-type benzodiazepine receptor, confers proapoptotic and antineoplastic effects in a human colorectal cancer cell line.

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

Severe traumatic events such as burns, and cancer therapy, often involve a significant loss of tissue, requiring surgical reconstruction by means of autologous muscle flaps. The scant availability of quality vascularized flaps and donor site morbidity often limit their use. Engineered vascularized grafts provide an alternative for this need. This work describes a first-time analysis, of the degree of in vitro vascularization and tissue organization, required to enhance the pace and efficacy of vascularized muscle graft integration in vivo. While one-day in vitro was sufficient for graft integration, a three-week culturing period, yielding semiorganized vessel structures and muscle fibers, significantly improved grafting efficacy. Implanted vessel networks were gradually replaced by host vessels, coupled with enhanced perfusion and capillary density. Upregulation of key graft angiogenic factors suggest its active role in promoting the angiogenic response. Transition from satellite cells to mature fibers was indicated by increased gene expression, increased capillary to fiber ratio, and similar morphology to normal muscle. We suggest a "relay" approach in which extended in vitro incubation, enabling the formation of a more structured vascular bed, allows for graft-host angiogenic collaboration that promotes anastomosis and vascular integration. The enhanced angiogenic response supports enhanced muscle regeneration, maturation, and integration.

Improved vascular organization enhances functional integration of engineered skeletal muscle grafts.

Large soft tissue defects involve significant tissue loss, requiring surgical reconstruction. Autologous flaps are occasionally scant, demand prolonged transfer surgery, and induce donor site morbidity. The present work set out to fabricate an engineered muscle flap bearing its own functional vascular pedicle for repair of a large soft tissue defect in mice. Full-thickness abdominal wall defect was reconstructed using this engineered vascular muscle flap. A 3D engineered tissue constructed of a porous, biodegradable polymer scaffold embedded with endothelial cells, fibroblasts, and/or myoblasts was cultured in vitro and then implanted around the femoral artery and veins before being transferred, as an axial flap, with its vascular pedicle to reconstruct a full-thickness abdominal wall defect in the same mouse. Within 1 wk of implantation, scaffolds showed extensive functional vascular density and perfusion and anastomosis with host vessels. At 1 wk posttransfer, the engineered muscle flaps were highly vascularized, were well-integrated within the surrounding tissue, and featured sufficient mechanical strength to support the abdominal viscera. Thus, the described engineered muscle flap, equipped with an autologous vascular pedicle, constitutes an effective tool for reconstruction of large defects, thereby circumventing the need for both harvesting autologous flaps and postoperative scarification.

An engineered muscle flap for reconstruction of large soft tissue defects.

Abdominal wall reconstruction following extensive tissue loss is essential and can be achieved using autologous flaps. However, their use is limited due to their inadequate availability and due to post-operative donor site scarification. This work presents a step-by-step technique for fabrication of a vascularized muscle flap, to be applied in full-thickness abdominal wall defect reconstruction. Poly L-lactic acid/poly lactic-co-glycolic acid scaffolds, prepared using a salt leaching technique, were used as the supporting matrix in vitro for simultaneously seeded endothelial cells, fibroblasts and myoblasts. The cell-embedded graft was then implanted around femoral artery and vein vessels, which provided a central blood supply. Vascularization and perfusion were achieved by capillary sprouting from the main host vessel into the graft. A thick and vascularized tissue was formed within one week, and was then transferred as an autologous flap together with its main vessels, to a full-thickness abdominal wall defect. The flap remained viable after transfer and featured sufficient mechanical strength to support the abdominal viscera. Thus, this engineered muscle flap can be used as an alternative source for autologous flaps to reconstruct full-thickness abdominal wall defects.

Article	Year
VDAC activation by the 18 kDa translocator protein (TSPO), implications for apoptosis. Journal of bioenergetics and biomembranes\| PubMed ID: 18670869	2008
Ligands of the mitochondrial 18 kDa translocator protein attenuate apoptosis of human glioblastoma cells exposed to erucylphosphohomocholine. Cellular oncology : the official journal of the International Society for Cellular Oncology\| PubMed ID: 18791274	2008
The 18-kDa translocator protein, formerly known as the peripheral-type benzodiazepine receptor, confers proapoptotic and antineoplastic effects in a human colorectal cancer cell line. Pharmacogenetics and genomics\| PubMed ID: 18806692	2008
Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism f... Apoptosis : an international journal on programmed cell death\| PubMed ID: 20107899	2010
Improved vascular organization enhances functional integration of engineered skeletal muscle grafts. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21878567	2011
An engineered muscle flap for reconstruction of large soft tissue defects. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 24711414	2014
A method for constructing vascularized muscle flap. Methods (San Diego, Calif.)\| PubMed ID: 25843607	2015

Yulia Shandalov

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