Background— Bone marrow cell therapy is reported to contribute to collateral formation through cell incorporation into new or remodeling vessels. However, the possible role of a paracrine contribution to this effect is less well characterized. Methods and Results— Murine marrow-derived stromal cells (MSCs) were purified by magnetic bead separation of cultured bone marrow. The release of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), placental growth factor (PlGF), and monocyte chemoattractant protein-1 (MCP-1) was demonstrated by analysis of MSC conditioned media (MSC-CM). MSC-CM enhanced proliferation of endothelial cells and smooth muscle cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partly attenuated these effects. Balb/C mice (n=10) underwent distal femoral artery ligation, followed by adductor muscle injection of 1×10 6 MSCs 24 hours later. Compared with controls injected with media (n=10) or mature endothelial cells (n=8), distal limb perfusion improved, and mid-thigh conductance vessels increased in number and total cross-sectional area. MSC injection improved limb function and appearance, reduced the incidence of auto-amputation, and attenuated muscle atrophy and fibrosis. After injection, labeled MSCs were seen dispersed between muscle fibers but were not seen incorporated into mature collaterals. Injection of MSCs increased adductor muscle levels of bFGF and VEGF protein compared with controls. Finally, colocalization of VEGF and transplanted MSCs within adductor tissue was demonstrated. Conclusions— MSCs secrete a wide array of arteriogenic cytokines. MSCs can contribute to collateral remodeling through paracrine mechanisms.

Abstract-We recently demonstrated that marrow stromal cells (MSCs) augment collateral remodeling through release of several cytokines such as VEGF and bFGF rather than via cell incorporation into new or remodeling vessels. The present study was designed to characterize the full spectrum of cytokine genes expressed by MSCs and to further examine the role of paracrine mechanisms that underpin their therapeutic potential. Normal human MSCs were cultured under normoxic or hypoxic conditions for 72 hours. The gene expression profile of the cells was determined using Affymetrix GeneChips representing 12 000 genes. A wide array of arteriogenic cytokine genes were expressed at baseline, and several were induced Ͼ1.5-fold by hypoxic stress. The gene array data were confirmed using ELISA assays and immunoblotting of the MSC conditioned media (MSC CM ). MSC CM promoted in vitro proliferation and migration of endothelial cells in a dose-dependent manner; anti-VEGF and anti-FGF antibodies only partially attenuated these effects. Similarly, MSC CM promoted smooth muscle cell proliferation and migration in a dose-dependent manner. Using a murine hindlimb ischemia model, murine MSC CM enhanced collateral flow recovery and remodeling, improved limb function, reduced the incidence of autoamputation, and attenuated muscle atrophy compared with control media. These data indicate that paracrine signaling is an important mediator of bone marrow cell therapy in tissue ischemia, and that cell incorporation into vessels is not a prerequisite for their effects.

Epidermal growth factor receptor (EGFR) and insulin-like growth factor

mTOR-generated signals play critical roles in growth of leukemic cells by controlling mRNA translation of genes that promote mitogenic responses. Despite extensive work on the functional relevance of rapamycin-sensitive mTORC1 complexes, much less is known on the roles of rapamycin-insensitive (RI) complexes, including mTORC2 and RI-mTORC1, in BCR-ABL-leukemogenesis. We provide evidence for the presence of mTORC2 complexes in BCR-ABL-transformed cells and identify phosphorylation of 4E-BP1 on Thr37∕46 and Ser65 as RI-mTORC1 signals in primary chronic myelogenous leukemia (CML) cells. Our studies establish that a unique dual mTORC2∕mTORC1 inhibitor, OSI-027, induces potent suppressive effects on primitive leukemic progenitors from CML patients and generates antileukemic responses in cells expressing the T315I-BCR-ABL mutation, which is refractory to all BCR-ABL kinase inhibitors currently in clinical use. Induction of apoptosis by OSI-027 appears to negatively correlate with induction of autophagy in some types of BCR-ABL transformed cells, as shown by the induction of autophagy during OSI-027-treatment and the potentiation of apoptosis by concomitant inhibition of such autophagy. Altogether, our studies establish critical roles for mTORC2 and RI-mTORC1 complexes in survival and growth of BCR-ABL cells and suggest that dual therapeutic targeting of such complexes may provide an approach to overcome leukemic cell resistance in CML and Phþ ALL.mRNA translation | cell proliferation | cellular signaling | kinase | OSI-027

Overexpression and enhanced activation of the epidermal growth factor receptor (EGFR) is frequently observed in human carcinomas. Inhibitors of EGFR signaling have shown clinical utility; however, understanding response at the molecular level is important to define patient subsets most likely to benefit, as well as to support the rational design of drug combinations. Pancreatic and colorectal tumor cell lines insensitive to EGFR inhibition were those that had lost or mutated the epithelial junction constituents E-cadherin and ;-catenin, had lost homotypic adhesion, and often gained proteins associated with an epithelial to mesenchymal -like transition, such as vimentin, zeb1, or snail. In matched pairs of colorectal tumor cells, the epithelial lines showed an average 7-fold greater sensitivity than mesenchymal-like lines. In human pancreatic and colorectal tumor tissues, gain of mesenchymal characteristics and loss of epithelial characteristics correlated with advancing tumor stage. These data indicate an especially sensitive patient subset as well as a rationale for the combination of EGFR antagonists with agents that affect the epithelial to mesenchymal -like transition process as a mechanism to enhance sensitivity for more advanced mesenchymal-like tumors. [Mol Cancer Ther 2007;6(2):532 -41]