Ex vivo allotransplantation engineering: Delivery of mesenchymal stem cells prolongs rejection-free allograft survival.

Current pharmacologic regimens in transplantation prevent allograft rejection through systemic recipient immunosuppression but are associated with severe morbidity and mortality. The ultimate goal of transplantation is the prevention of allograft rejection while maintaining recipient immunocompetence. We hypothesized that allografts could be engineered ex vivo (after allotransplant procurement but before transplantation) by using mesenchymal stem cell-based therapy to generate localized immunomodulation without affecting systemic recipient immunocompetence. To this end, we evaluated the therapeutic efficacy of bone marrow-derived mesenchymal stem cells in vitro and activated them toward an immunomodulatory fate by priming in inflammatory or hypoxic microenvironments. Using an established rat hindlimb model for allotransplantation, we were able to significantly prolong rejection-free allograft survival with a single perioperative ex vivo infusion of bone marrow-derived mesenchymal stem cells through the allograft vasculature, in the absence of long-term pharmacologic immunosuppression. Critically, transplanted rats rejected a second, nonengineered skin graft from the same donor species to the contralateral limb at a later date, demonstrating that recipient systemic immunocompetence remained intact. This study represents a novel approach in transplant immunology and highlights the significant therapeutic opportunity of the ex vivo period in transplant engineering.

The HSPGs Syndecan and Dallylike bind the receptor phosphatase LAR and exert distinct effects on synaptic development.

The formation and plasticity of synaptic connections rely on regulatory interactions between pre- and postsynaptic cells. We show that the Drosophila heparan sulfate proteoglycans (HSPGs) Syndecan (Sdc) and Dallylike (Dlp) are synaptic proteins necessary to control distinct aspects of synaptic biology. Sdc promotes the growth of presynaptic terminals, whereas Dlp regulates active zone form and function. Both Sdc and Dlp bind at high affinity to the protein tyrosine phosphatase LAR, a conserved receptor that controls both NMJ growth and active zone morphogenesis. These data and double mutant assays showing a requirement of LAR for actions of both HSPGs lead to a model in which presynaptic LAR is under complex control, with Sdc promoting and Dlp inhibiting LAR in order to control synapse morphogenesis and function.

Drosophila enabled promotes synapse morphogenesis and regulates active zone form and function.

BACKGROUND: Recent studies of synapse form and function highlight the importance of the actin cytoskeleton in regulating multiple aspects of morphogenesis, neurotransmission, and neural plasticity. The conserved actin-associated protein Enabled (Ena) is known to regulate development of the Drosophila larval neuromuscular junction through a postsynaptic mechanism. However, the functions and regulation of Ena within the presynaptic terminal has not been determined. METHODS: Here, we use a conditional genetic approach to address a presynaptic role for Ena on presynaptic morphology and ultrastructure, and also examine the pathway in which Ena functions through epistasis experiments. RESULTS: We find that Ena is required to promote the morphogenesis of presynaptic boutons and branches, in contrast to its inhibitory role in muscle. Moreover, while postsynaptic Ena is regulated by microRNA-mediated mechanisms, presynaptic Ena relays the output of the highly conserved receptor protein tyrosine phosphatase Dlar and associated proteins including the heparan sulfate proteoglycan Syndecan, and the non-receptor Abelson tyrosine kinase to regulate addition of presynaptic varicosities. Interestingly, Ena also influences active zones, where it restricts active zone size, regulates the recruitment of synaptic vesicles, and controls the amplitude and frequency of spontaneous glutamate release. CONCLUSION: We thus show that Ena, under control of the Dlar pathway, is required for presynaptic terminal morphogenesis and bouton addition and that Ena has active zone and neurotransmission phenotypes. Notably, in contrast to Dlar, Ena appears to integrate multiple pathways that regulate synapse form and function.

Direct observation demonstrates that Liprin-alpha is required for trafficking of synaptic vesicles.

Axonal transport is required for the elaboration and maintenance of synaptic morphology and function. Liprin-alphas are scaffolding proteins important for synapse structure and electrophysiology. A reported interaction with Kinesin-3 (Kif1a) suggested Liprin-alpha may also be involved in axonal transport. Here, at the light and ultrastructural levels, we discover aberrant accumulations of synaptic vesicle markers (Synaptotagmin and Synaptobrevin-GFP) and clear-core vesicles along Drosophila Liprin-alpha mutant axons. Analysis of presynaptic markers reveals reduced levels at Liprin-alpha synapses. Direct visualization of Synaptobrevin-GFP transport in living animals demonstrates a decrease in anterograde processivity in Liprin-alpha mutants but also an increase in retrograde transport initiation. Pull-down assays reveal that Liprin-alpha interacts with Drosophila Kinesin-1 (Khc) but not dynein. Together, these findings suggest that Liprin-alpha promotes the delivery of synaptic material by a direct increase in kinesin processivity and an indirect suppression of dynein activation. This work is the first to use live observation in Drosophila mutants to demonstrate the role of a scaffolding protein in the regulation of bidirectional transport. It suggests the synaptic strength and morphology defects linked to Liprin-alpha may in part be due to a failure in the delivery of synaptic-vesicle precursors.

In Vivo Imaging of Reactive Oxygen Species in a Murine Wound Model.

The generation of reactive oxygen species (ROS) is a hallmark of inflammatory processes, but in excess, oxidative stress is widely implicated in various pathologies such as cancer, atherosclerosis and diabetes. We have previously shown that dysfunction of the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2)/ Kelch-like erythroid cell-derived protein 1 (Keap1) signaling pathway leads to extreme ROS imbalance during cutaneous wound healing in diabetes. Since ROS levels are an important indicator of progression of wound healing, specific and accurate quantification techniques are valuable. Several in vitro assays to measure ROS in cells and tissues have been described; however, they only provide a single cumulative measurement per sample. More recently, the development of protein-based indicators and imaging modalities have allowed for unique spatiotemporal analyses. L-012 (C13H8ClN4NaO2) is a luminol derivative that can be used for both in vivo and in vitro chemiluminescent detection of ROS generated by NAPDH oxidase. L-012 emits a stronger signal than other fluorescent probes and has been shown to be both sensitive and reliable for detecting ROS. The time lapse applicability of L-012-facilitated imaging provides valuable information about inflammatory processes while reducing the need for sacrifice and overall reducing the number of study animals. Here, we describe a protocol utilizing L-012-facilitated in vivo imaging to quantify oxidative stress in a model of excisional wound healing using diabetic mice with locally dysfunctional Nrf2/Keap1.

Ex Vivo Major Histocompatibility Complex I Knockdown Prolongs Rejection-free Allograft Survival.

BACKGROUND: Widespread application of vascularized composite allotransplantation (VCA) is currently limited by the required lifelong systemic immunosuppression and its associated morbidity and mortality. This study evaluated the efficacy of ex vivo (after procurement but before transplantation) engineering of allografts using small interfering RNA to knockdown major histocompatibility complex I (MHC-I) and prolong rejection-free survival. METHODS: Endothelial cells (ECs) were transfected with small interfering RNA targeted against MHC-I (siMHC-I) for all in vitro experiments. MHC-I surface expression and knockdown duration were evaluated using quantitative polymerase chain reaction (qPCR) and flow cytometry. After stimulating Lewis recipient cytotoxic lymphocytes (CTL) with allogeneic controls or siMHC-I-silenced ECs, lymphocyte proliferation, CTL-mediated and natural killer-mediated EC lysis were measured. Using an established VCA rat model, allografts were perfused ex vivo with siMHC-I before transplantation. Allografts were analyzed for MHC-I expression and clinical/histologic evidence of rejection. RESULTS: Treatment with siMHC-I resulted in 80% knockdown of mRNA and 87% reduction in cell surface expression for up to 7 days in vitro (P < 0.05). Treatment of ECs with siMHC-I reduced lymphocyte proliferation and CTL-mediated cytotoxicity (77% and 50%, respectively, P < 0.01), without increasing natural killer-mediated cytotoxicity (P = 0.66). In a rat VCA model, ex vivo perfusion with siMHC-I reduced expression in all tissue compartments by at least 50% (P < 0.05). Knockdown prolonged rejection-free survival by 60% compared with nonsense-treated controls (P < 0.05). CONCLUSIONS: Ex vivo siMHC-I engineering can effectively modify allografts and significantly prolong rejection-free allograft survival. This novel approach may help reduce future systemic immunosuppression requirements in VCA recipients.

Targeted protection of donor graft vasculature using a phosphodiesterase inhibitor increases survival and predictability of autologous fat grafts.

BACKGROUND: Fat grafting is limited by unpredictable long-term graft retention. The authors postulate that injury to the donor-derived microvasculature during harvest and subsequent ischemia may account for this clinical variability. They examined the use of the U.S. Food and Drug Administration-approved phosphodiesterase-5 inhibitor sildenafil citrate to protect graft microvasculature and its role in revascularization and survival. METHODS: Inguinal fat of donor Tie2/LacZ mice was infiltrated with sildenafil or saline, harvested, and transplanted onto the dorsa of recipient FVB mice. Additional donor mice were perfused with intraarterial trypsin to inactivate the fat graft microvasculature before harvest and transplantation. Differences in graft revascularization, perfusion, volume of retention, and biochemical changes were assessed. RESULTS: Surviving fat grafts were characterized by exclusively donor-derived vasculature inosculating with the recipient circulation at the graft periphery. Inactivation of donor-derived microvasculature decreased early graft perfusion and led to nearly total graft loss by 8 weeks. Sildenafil attenuated vascular ischemic injury, consistent with reductions in VCAM-1 and SDF1α expression at 48 hours and 4-fold increases in microvasculature survival by 2 weeks over controls. Compared with controls, targeted sildenafil treatment improved early graft perfusion, doubled graft retention at 12 weeks (83 percent versus 39 percent; p < 0.05), ultimately retaining 64 percent of the original graft volume by 24 weeks (compared to 4 percent; p < 0.05) with superior histologic features. CONCLUSIONS: Fat graft vascularization is critically dependent on maintenance of the donor microvasculature. Sildenafil protects the donor microvasculature during transfer and revascularization, increasing long-term volume retention. These data demonstrate a rapidly translatable method of increasing predictability and durability of fat grafting in clinical practice.