Therapeutic potential of ixmyelocel-T, an expanded autologous multicellular therapy for treatment of ischemic cardiovascular diseases.

INTRODUCTION: Bone marrow derived cellular therapies are an emerging approach to promoting therapeutic angiogenesis in ischemic cardiovascular disease. However, the percentage of regenerative cells in bone marrow mononuclear cells (BMMNCs) is small, and large amounts of BMMNCs are required. Ixmyelocel-T, an expanded autologous multicellular therapy, is manufactured from a small sample of bone marrow aspirate. Ixmyelocel-T contains expanded populations of mesenchymal stromal cells (MSCs) and M2-like macrophages, as well as many of the CD45+ cells found in the bone marrow. It is hypothesized that this expanded multi-cellular therapy would induce angiogenesis and endothelial repair. METHODS: A rat model of hind limb ischemia was used to determine the effects of ixmyelocel-T on blood flow recovery. To further determine the effects on endothelial cells, ixmyelocel-T was co-cultured with human umbilical vein endothelial cells (HUVEC) in non-contacting Transwell® inserts. RESULTS: Co-culture of HUVECs with ixmyelocel-T resulted secretion of a variety of pro-angiogenic factors. HUVECs stimulated by ixmyelocel-T exhibited enhanced migration, proliferation, and branch formation. Ixmyelocel-T co-culture also resulted in increased endothelial nitric oxide synthase (eNOS) expression and nitric oxide (NO) production. In tumor necrosis factor alpha (TNFα)-stimulated HUVECs, ixmyelocel-T co-culture decreased apoptosis and reactive oxygen species generation, increased super oxide dismutase activity, and decreased nuclear factor kappa B (NFκB) activation. Treatment with ixmyelocel-T in a rat model of hind limb ischemia resulted in significantly increased blood flow perfusion and capillary density, gene expression and plasma levels of the anti-inflammatory cytokine interleukin (IL)-10, plasma nitrates, plasma platelet-derived growth factor (PDGF)-BB, vascular endothelial growth factor (VEGF) expression, and significantly decreased plasma thiobarbituric acid reactive substances (TBARS). CONCLUSIONS: This work demonstrates that ixmyelocel-T interacts with endothelial cells in a paracrine manner, resulting in angiogenesis and endothelial protection. This data suggests that ixmyelocel-T could be useful for promoting of angiogenesis and tissue repair in ischemic cardiovascular diseases. In conclusion, ixmyelocel-T therapy may provide a new aspect of therapeutic angiogenesis in this patient population where expanded populations of regenerative cells might be required.

Potential beneficial effects of ixmyelocel-T in the treatment of atherosclerotic diseases.

INTRODUCTION: Advanced atherosclerotic lesions are characterized by lipid accumulation, inflammation, and defective efferocytosis. An ideal therapy should address all aspects of this multifactorial disease. Ixmyelocel-T therapy, an expanded autologous multicellular therapy showing clinical promise in the treatment of diseases associated with advanced atherosclerosis, includes a novel population of M2-like macrophages. Here, we examine the macrophages of ixmyelocel-T and determine their ability to influx modified cholesterol in an atheroprotective manner, maintaining cholesterol homeostasis and preventing cellular dysfunction and death, ultimately promoting reverse cholesterol efflux. METHODS: Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. Bone marrow mononuclear cells (BMMNCs) were produced by using density gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T via positive selection for analysis. Ixmyelocel-T and human leukemia monocyte (THP-1) cells were loaded with acetylated low-density lipoprotein (Ac-LDL) for analysis. Flow cytometry and immunofluorescence were used to examine Ac-LDL uptake, expression of cytokines was analyzed by enzyme-linked immunofluorescence assay (ELISA), and quantitative real-time PCR was used to analyze expression of cholesterol-transport genes. Both the in vitro cholesterol efflux assay and in vivo reverse cholesterol transport assay were used to examine cholesterol transport. RESULTS: Ixmyelocel-T macrophages take up acetylated low-density lipoprotein and express the scavenger receptors CD36 and scavenger receptor-B1 (SR-B1). Ixmyelocel-T did not become apoptotic or proinflammatory after lipid loading. The cholesterol transporter genes ABAC1 and ABCG1 were both statistically significantly upregulated when ixmyelocel-T macrophages were loaded with cholesterol. Ixmyelocel-T also exhibited enhanced apolipoprotein A-I (ApoAI)-mediated cholesterol efflux. In addition, in vivo reverse cholesterol-transport assay demonstrated that ixmyelocel-T was able to efflux cholesterol in this model. CONCLUSIONS: Ixmyelocel-T macrophages influx modified cholesterol, remained anti-inflammatory in the face of lipid loading and inflammatory challenge, and displayed enhanced cholesterol efflux capabilities. These combined features suggest that this autologous multicellular therapy may exert beneficial effects in atherosclerotic diseases.