Critical limb ischemia (CLI), the most severe form of peripheral artery disease, is characterized by pain at rest and non-healing ulcers in the lower extremities. For patients with CLI, where the extent of atherosclerotic artery occlusion is too severe for surgical bypass or percutaneous interventions, limb amputation remains the only treatment option. Thus, cell-based therapy to restore perfusion and promote wound healing in patients with CLI is under intense investigation. Despite promising preclinical studies in animal models, transplantation of bone marrow (BM)-derived cell populations in patients with CLI has shown limited benefit preventing limb amputation. Early trials injected heterogenous mononuclear cells containing a low frequency of cells with pro-vascular regenerative functions. Most trials transferred autologous cells damaged by chronic disease that demonstrated poor survival in the ischemic environment and impaired function conferred by atherosclerotic or diabetic co-morbidities. Finally, recent preclinical studies suggest optimized blood vessel formation may require paracrine and/or structural contributions from multiple progenitor cell lineages, angiocrine-secretory myeloid cells derived from hematopoietic progenitor cells, tubule-forming endothelial cells generated by circulating or vessel-resident endothelial precursors, and vessel-stabilizing perivascular cells derived from mesenchymal stem cells. Understanding how stem cells co-ordinate the myriad of cells and signals required for stable revascularization remains the key to translating the potential of stem cells into curative therapies for CLI. Thus, combination delivery of multiple cell types within supportive bioengineered matricies may represent a new direction to improve cell therapy strategies for CLI. Stem Cells 2018;36:161-171.

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IntroductionHemophilia A is the most common severe inherited bleeding disorder. Patients with this disease are treated with recombinant or plasma-derived factor VIII (FVIII), which allows them to lead relatively normal lives. 1 In approximately 25% of treated patients, however, the development of anti-FVIII antibodies (FVIII inhibitors) severely complicates FVIII replacement therapy and significantly increases morbidity within the hemophilia population. [2][3][4][5] These antibodies neutralize the procoagulant cofactor activity of FVIII or enhance its clearance from plasma. 5 In economically developed countries, there are 2 approaches to the clinical management of FVIII inhibitors: the treatment or prevention of bleeding and long-term immune tolerance induction (ITI). Bleeding is controlled with variably effective and expensive FVIII-bypassing agents, such as recombinant (r) FVIIa and FEIBA (FVIII-inhibitor bypassing agent). In contrast, ITI is usually attempted through the administration of FVIII at a dose and frequency that depends on the ITI protocol. 6 This treatment approach is practically challenging, costly, and can take months to years to become effective. In light of the significant limitations of the current treatment options, the development of effective, rapid, and economical ITI strategies is a clinical priority.Currently, the most consistent model to study FVIII inhibitors is the hemophilia A mouse (FVIII Ϫ/Ϫ ). [7][8][9] Repeated intravenous infusion of human FVIII into hemophilia A mice results in high titer inhibitor formation. This is a CD4 ϩ T cell-dependent process that requires costimulation. [9][10][11][12] The dependence on CD4 ϩ T cells for inhibitor formation also occurs in humans. Evidence of this first came from hemophilia A patients with FVIII inhibitors who were also HIV ϩ : as patient CD4 ϩ levels declined, there was concomitant disappearance of FVIII inhibitors. 13 Therefore, therapies that blocked T-cell activation seemed to be promising candidates to prevent inhibitor formation.Indeed, Qian et al demonstrated that FVIII Ϫ/Ϫ B7.2 Ϫ/Ϫ doubleknockout mice will not develop anti-FVIII antibodies (Abs) after repeated immunization with FVIII, and that blocking the CD80-CD28 costimulatory interaction with soluble cytotoxic T lymphocyte antigen-4 (CTLA-4)-immunoglobulin (Ig) in FVIII Ϫ/Ϫ mice also prevented inhibitor formation. 10 Additional studies in FVIII Ϫ/Ϫ mice showed that blockade of the CD40-CD40L interaction with anti-CD40L monoclonal Ab (mAb) also protects against FVIII inhibitor formation. 11,12 However, costimulatory blockade must be applied with each FVIII administration to maintain tolerance, and once the blockade is removed, the protective effect is lost. As the potential health risks of long-term costimulatory blockade have not yet been determined and because many hemophilia A patients are treated frequently with FVIII and would most likely need to coadminister blockade with each infusion, this therapy is not a viable option.To reach the clinic, a therapy that induces tole...

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This study shows that tolerogenic presentation of cFVIII to the immune system can significantly reduce immunogenicity of the protein.

Previous studies have demonstrated that genetic factors play an important role in determining the likelihood of formation of anti-factor VIII (FVIII) antibodies in haemophilia A patients. We were interested in characterizing the spectrum of FVIII antibody formation and the primary and secondary immune responses after FVIII administration in two different exon 16-disrupted haemophilia A mouse strains, Balb/c and C57BL/6. Balb/c and C57BL/6 E16 haemophilia A mice were used in all experiments. Total FVIII antibodies and FVIII inhibitors were measured using ELISA and Bethesda assays respectively. T- and B-cell cytokines were quantified using ELISA and flow cytometry. FVIII antibodies, but not functional inhibitors were detectable 1 week after the first FVIII treatment in both strains. These antibodies mainly belonged to the IgM and IgA isotypes. After the fourth FVIII treatment, neutralizing anti-FVIII antibodies were detected in both mouse strains: Balb/c (mean inhibitory titer 58 BU) and C57BL/6 (mean inhibitory titer 82 BU). IgG1 levels were similar in both strains but the IgG2A and IgG2B subclasses were higher in C57BL/6 mice. The results of intracellular cytokine staining of T cells indicated that the FVIII-treated C57BL/6 mice produced more IL10 and Th1 cytokines than the FVIII-treated Balb/c mice. These studies show that C57BL/6 mice develop a stronger immune response towards FVIII than Balb/c mice. We propose that the enhanced Th1 and IL10 cytokine micro-environment induced in C57BL/6 mice is responsible for this difference. Therefore, genetic strain-dependent differences must be considered when evaluating immunological outcomes in mouse models of haemophilia A.

Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, is a widely used anticonvulsant drug that is currently undergoing clinical evaluation for anticancer therapy due to its anti-angiogenic potential. Endothelial cells (ECs) can transition into mesenchymal cells and this form of EC plasticity is called endothelial-to-mesenchymal transition (EndMT), which is widely implicated in several pathologies including cancer and organ fibrosis. However, the effect of VPA on EC plasticity and EndMT remains completely unknown. We report herein that VPA-treatment significantly inhibits tube formation, migration, nitric oxide production, proliferation and migration in ECs. A microscopic evaluation revealed, and qPCR, immunofluorescence and immunoblotting data confirmed EndMT-like phenotypic switching as well as an increased expression of pro-fibrotic genes in VPA-treated ECs. Furthermore, our data confirmed important and regulatory role played by TGFβ-signaling in VPA-induced EndMT. Our qPCR array data performed for 84 endothelial genes further supported our findings and demonstrated 28 significantly and differentially regulated genes mainly implicated in angiogenesis, endothelial function, EndMT and fibrosis. We, for the first time report that VPA-treatment associated EndMT contributes to the VPA-associated loss of endothelial function. Our data also suggest that VPA based therapeutics may exacerbate endothelial dysfunction and EndMT-related phenotype in patients undergoing anticonvulsant or anticancer therapy, warranting further investigation.

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