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.

Targeted deletion of murine CEACAM 1 activates PI3K-Akt signaling and contributes to the expression of (Pro)renin receptor via CREB family and NF-κB transcription factors.

The carcinoembryonic antigen-related cell adhesion molecule 1 regulates insulin sensitivity by promoting hepatic insulin clearance. Mice bearing a null mutation of Ceacam1 gene (Cc1(-/-)) develop impaired insulin clearance followed by hyperinsulinemia and insulin resistance, in addition to visceral obesity and increased plasma fatty acids. Because insulin resistance is associated with increased blood pressure, we investigated whether they develop higher blood pressure with activated renal renin-angiotensin system and whether this is mediated, in part, by the upregulation of renal (pro)renin receptor (PRR) expression. Compared with age-matched wild-type littermates, Cc1(-/-) mice exhibited increased blood pressure with increased activation of renal renin-angiotensin systems and renal PRR expression. Cytoplasmic and nuclear immunostaining of phospho-PI3K p85α and phospho-Akt was enhanced in the kidney of Cc1(-/-) mice. In murine renal inner medullary collecting duct epithelial cells with lentiviral-mediated small hairpin RNA knockdown of carcinoembryonic antigen-related cell adhesion molecule 1, PRR expression was upregulated and phosphorylation of PI3K (Tyr508), Akt (Ser473), NF-κB p65 (Ser276), cAMP response element-binding protein/activated transcription factor (ATF)-1 (Ser133), and ATF-2 (Thr71) was enhanced. Inhibiting PI3K with LY294002 or Akt with Akt inhibitor VIII attenuated PRR expression. In conclusion, global null deletion of Ceacam1 caused an increase in blood pressure with increased renin-angiotensin system activation together with upregulation of PRR via PI3K-Akt activation of cAMP response element-binding protein 1, ATF-1, ATF-2, and NF-κB p65 transcription factors.

Ceacam1 deletion causes vascular alterations in large vessels.

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) promotes hepatic insulin clearance and endothelial survival. However, its role in the morphology of macrovessels remains unknown. Mice lacking Ceacam1 (Cc1-/-) exhibit hyperinsulinemia, which causes insulin resistance and fatty liver. With increasing evidence of an association among hyperinsulinemia, fatty liver disease, and atherosclerosis, we investigated whether Cc1-/- exhibited vascular lesions in atherogenic-prone aortae. Histological analysis revealed impaired endothelial integrity with restricted fat deposition and aortic plaque-like lesions in Cc1-/- aortae, likely owing to their limited lipidemia. Immunohistochemical analysis indicated macrophage deposition, and in vitro studies showed increased leukocyte adhesion to aortic wall, mediated in part by elevation in vascular cell adhesion molecule 1 levels. Basal aortic eNOS protein and NO content were reduced, in parallel with reduced Akt/eNOS and Akt/Foxo1 phosphorylation. Ligand-induced vasorelaxation was compromised in aortic rings. Increased NADPH oxidase activity and plasma 8-isoprostane levels revealed oxidative stress and lipid peroxidation in Cc1-/- aortae. siRNA-mediated CEACAM1 knockdown in bovine aortic endothelial cells adversely affected insulin's stimulation of IRS-1/PI 3-kinase/Akt/eNOS activation by increasing IRS-1 binding to SHP2 phosphatase. This demonstrates that CEACAM1 regulates both endothelial cell autonomous and nonautonomous mechanisms involved in vascular morphology and NO production in aortae. Systemic factors such as hyperinsulinemia could contribute to the pathogenesis of these vascular abnormalities. Cc1-/- mice provide a first in vivo demonstration of distinct CEACAM1-dependent hepatic insulin clearance linking hepatic to macrovascular abnormalities.

Ixmyelocel-T, an expanded multicellular therapy, contains a unique population of M2-like macrophages.

INTRODUCTION: M2 macrophages promote tissue repair and regeneration through various mechanisms including immunomodulation and scavenging of tissue debris. Delivering increased numbers of these cells to ischemic tissues may limit tissue injury and promote repair. Ixmyelocel-T is an expanded, autologous multicellular therapy cultured from bone-marrow mononuclear cells (BMMNCs). The purpose of this study was to characterize further a unique expanded population of M2-like macrophages, generated in ixmyelocel-T therapy. METHODS: Approximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. 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 with positive selection for analysis. Cell-surface phenotype was examined with flow cytometry and immunofluorescence, and expression of cytokines and chemokines was analyzed with enzyme-linked immunosorbent assay (ELISA). Quantitative real-time PCR was used to analyze expression of genes in BMMNCs, ixmyelocel-T, the CD14+ population from ixmyelocel-T, and M1 and M2 macrophages. Ixmyelocel-T was cultured with apoptotic BMMNCs, and then visualized under fluorescence microscopy to assess efferocytosis. RESULTS: Macrophages in ixmyelocel-T therapy expressed surface markers of M2 macrophages, CD206, and CD163. These cells were also found to express several M2 markers, and few to no M1 markers. After stimulation with lipopolysaccharide (LPS), they showed minimal secretion of the proinflammatory cytokines interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) compared with M1 and M2 macrophages. Ixmyelocel-T macrophages efficiently ingested apoptotic BMMNCs. CONCLUSIONS: Ixmyelocel-T therapy contains a unique population of M2-like macrophages that are characterized by expression of M2 markers, decreased secretion of proinflammatory cytokines after inflammatory stimuli, and efficient removal of apoptotic cells. This subpopulation of cells may have a potential role in tissue repair and regeneration.

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.

Susceptibility to diet-induced hepatic steatosis and glucocorticoid resistance in FK506-binding protein 52-deficient mice.

Although FK506-binding protein 52 (FKBP52) is an established positive regulator of glucocorticoid receptor (GR) activity, an in vivo role for FKBP52 in glucocorticoid control of metabolism has not been reported. To address this question, FKBP52(+/-) mice were placed on a high-fat (HF) diet known to induce obesity, hepatic steatosis, and insulin resistance. Tissue profiling of wild-type mice showed high levels of FKBP52 in the liver but little to no expression in muscle or adipose tissue, predicting a restricted pattern of FKBP52 effects on metabolism. In response to HF, FKBP52(+/-) mice demonstrated a susceptibility to hyperglycemia and hyperinsulinemia that correlated with reduced insulin clearance and reduced expression of hepatic CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1), a mediator of clearance. Livers of HF-fed mutant mice had high lipid content and elevated expression of lipogenic genes (peroxisome proliferator-activated receptor gamma, fatty acid synthase, and sterol regulatory element-binding protein 1c) and inflammatory markers (TNFalpha). Interestingly, mutant mice under HF showed elevated serum corticosterone, but their steatotic livers had reduced expression of gluconeogenic genes (phosphoenolpyruvate carboxy kinase, glucose 6 phosphatase, and pyruvate dehydrogenase kinase 4), whereas muscle and adipose expressed normal to elevated levels of glucocorticoid markers. These data suggest a state of glucocorticoid resistance arising from liver-specific loss of GR activity. Consistent with this hypothesis, reduced expression of gluconeogenic genes and CEACAM1 was observed in dexamethasone-treated FKBP52-deficient mouse embryonic fibroblast cells. We propose a model in which FKBP52 loss reduces GR control of gluconeogenesis, predisposing the liver to steatosis under HF-diet conditions attributable to a shunting of metabolism from glucose production to lipogenesis.

Mice with null mutation of Ceacam I develop nonalcoholic steatohepatitis.

Transgenic liver-specific inactivation of the carcinoembryonic antigen-related cell adhesion molecule (CEACAM1) impairs hepatic insulin clearance and causes hyperinsuline-mia, insulin resistance, elevation in hepatic and serum triglyceride levels, and visceral obesity. It also predisposes to nonalchoholic steatohepatitis (NASH) in response to a high-fat diet. To discern whether this phenotype reflects a physiological function of CEACAM1 rather than the effect of the dominant-negative transgene, we investigated whether Ceacam1 (gene encoding CEACAM1 protein) null mice with impaired insulin clearance also develop a NASH-like phenotype on a prolonged high-fat diet. Three-month-old male null and wild-type mice were fed a high-fat diet for 3 months and their NASH phenotype was examined. While high-fat feeding elevated hepatic triglyceride content in both strains of mice, it exacerbated macrosteatosis and caused NASH-characteristic fibrogenic changes and inflammatory responses more intensely in the null mouse. This demonstrates that CEACAM1-dependent insulin clearance pathways are linked with NASH pathogenesis.

Development of nonalcoholic steatohepatitis in insulin-resistant liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice.

BACKGROUND & AIMS: Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 causes hyperinsulinemia and insulin resistance, which result from impaired insulin clearance, in liver-specific S503A carcinoembryonic antigen-related cell adhesion molecule 1 mutant mice (L-SACC1). These mice also develop steatosis. Because hepatic fat accumulation precedes hepatitis, lipid peroxidation, and apoptosis in the pathogenesis of nonalcoholic steatohepatitis (NASH), we investigated whether a high-fat diet, by causing inflammation, is sufficient to induce hepatitis and other features of NASH in L-SACC1 mice. METHODS: L-SACC1 and wild-type mice were placed on a high-fat diet for 3 months, then several biochemical and histologic analyses were performed to investigate the NASH phenotype. RESULTS: A high-fat diet caused hepatic macrosteatosis and hepatitis, characterized by increased hepatic tumor necrosis factor alpha levels and activation of the NF-kappaB pathway in L-SACC1 but not in wild-type mice. The high-fat diet also induced necrosis and apoptosis in the livers of the L-SACC1 mice. Insulin resistance in L-SACC1 fed a high-fat diet increased the hepatic procollagen protein level, suggesting a role in the development of fibrosis. CONCLUSIONS: A high-fat diet induces key features of human NASH in insulin-resistant L-SACC1 mice, validating this model as a tool to study the molecular mechanisms of NASH.