Nicholas Lehman

Multipotent adult progenitor cells prevent macrophage-mediated axonal dieback and promote regrowth after spinal cord injury.

Macrophage-mediated axonal dieback presents an additional challenge to regenerating axons after spinal cord injury. Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to ameliorate this detrimental inflammation-related process has not been investigated. Using an in vitro model of axonal dieback as well as an adult rat dorsal column crush model of spinal cord injury, we found that multipotent adult progenitor cells (MAPCs) can affect both macrophages and dystrophic neurons simultaneously. MAPCs significantly decrease MMP-9 (matrix metalloproteinase-9) release from macrophages, effectively preventing induction of axonal dieback. MAPCs also induce a shift in macrophages from an M1, or “classically activated” proinflammatory state, to an M2, or “alternatively activated” antiinflammatory state. In addition to these effects on macrophages, MAPCs promote sensory neurite outgrowth, induce sprouting, and further enable axons to overcome the negative effects of macrophages as well as inhibitory proteoglycans in their environment by increasing their intrinsic growth capacity. Our results demonstrate that MAPCs have therapeutic benefits after spinal cord injury and provide specific evidence that adult stem cells exert positive immunomodulatory and neurotrophic influences.

Phospholipase D2-derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR

The product of phospholipase D (PLD) enzymatic action in cell membranes, phosphatidic acid (PA), regulates kinases implicated in NADPH oxidase activation, as well as the mammalian target of rapamy‐cin (mTOR) kinase. However, other protein targets for this lipid second messenger must exist in order to explain other key PA‐mediated cellular functions. In this study, PA was found to specifically and saturably bind to and activate recombinant and immunoprecipitated endogenous ribosomal S6 kinase (S6K) with a stoichi‐ometry of 94:1 lipid/protein. Polyphosphoinositides PI4‐P and PI4, 5P2 and cardiolipin could also bind to and activate S6K, albeit with different kinetics. Conversely, PA with at least one acyl side chain saturated (10:0) was ineffective in binding or activating the enzyme. Transfection of COS‐7 cells with a wild‐type myc‐(pcDNA)‐PLD2 construct resulted in high PLD activity, concomitantly with an increase in ribosomal p70S6K enzyme activity and phosphorylation in T389 and T421/S424 as well as phosphorylation of p70S6Ks natural substrate S6 protein in S235/S236. Overexpression of a lipase inactive mutant (K758R), however, failed to induce an increase in both PLD and S6K activity or phosphorylation, indicating that the enzymatic activity of PLD2 (i.e., synthesis of PA) must be present to affect S6K. Neither inhibiting mTOR kinase activity with rapamycin nor silencing mTOR gene expression altered the augmentative effect of PLD2 exerted on p70S6K activity. This finding indicates that PA binds to and activates p70S6K, even in the absence of mTOR. Lastly, COS‐7 transfection with PLD2 changed the pattern of subcellular expression, and a colocalization of S6K and PLD2 was observed by immunofluorescence microscopy. These results show for the first time a direct (mTOR‐independent) participation of PLD in the p70S6K pathway and implicate PA as a nexus that brings together cell phospholipases and kinases.—Lehman, N., Ledford, B., Di Fulvio, M., Frondorf, K., McPhail, L. C., Gomez‐Cambronero, J. Phospholipase D2‐derived phosphatidic acid binds to and activates ribosomal p70 S6 kinase independently of mTOR. FASEB J. 21, 1075–1087 (2007)

The elucidation of novel SH2 binding sites on PLD2.

Our laboratory has recently reported that the enzyme phospholipase D2 (PLD2) exists as a ternary complex with PTP1b and the growth factor receptor bound protein 2 (Grb2). Here, we establish the mechanistic underpinnings of the PLD2/Grb2 association. We have identified residues Y169 and Y179 in the PLD2 protein as being essential for the Grb2 interaction. We present evidence indicating that Y169 and Y179 are located within two consensus sites in PLD2 that mediate an SH2 interaction with Grb2. This was demonstrated with an SH2-deficient GSTGrb2 R86K mutant that failed to pull-down PLD2 in vitro. In order to elucidate the functions of the two neighboring tyrosines, we created a new class of deletion and point mutants in PLD2. Phenylalanine replacement of Y169 (PLD2 Y169F) or Y179 (PLD2 Y179F) reduced Grb2 binding while simultaneous mutation completely abolished it. The role of the two binding sites on PLD2 was found to be functionally nonequivalent: Y169 serves to modulate the activity of the enzyme, whereas Y179 regulates total tyrosine phosphorylation of the protein. Interestingly, binding of Grb2 to PLD2 occurs irrespectively of lipase activity, since Grb2 binds to catalytically inactive PLD2 mutants. Finally, PLD2 residues Y169 and Y179 are necessary for the recruitment of Sos, but only overexpression of the PLD2 Y179F mutant resulted in increased Ras activity, p44/42Erk phosphorylation and enhanced DNA synthesis. Since Y169 remains able to modulate enzyme activity and is capable of binding to Grb2 in the PLD2 Y179F mutant, we propose that Y169 is kept under negative regulation by Y179. When this is released, Y169 mediates cellular proliferation through the Ras/MAPK pathway.

Percutaneous adventitial delivery of allogeneic bone marrow-derived stem cells via infarct-related artery improves long-term ventricular function in acute myocardial infarction.

Acute myocardial infarction (AMI) results in ischemic damage and death of cardiomyocytes and loss of vasculature. Stem cell therapy has emerged as a potentially promising strategy for maximizing cardiac function following ischemic injury. Issues of cell source, delivery, and quantification of response have challenged development of clinically viable strategies. In this study we investigate the effects of a well-defined bone marrow-derived allogeneic cell product delivered by catheter directly to the myocardium via the infarctrelated vessel on global and regional measures of left ventricular (LV) function in a porcine model of anterior wall myocardial infarction. Multipotent adult progenitor cells (MAPCs) were derived and expanded from the bone marrow of a donor Yorkshire pig. Anterior wall myocardial infarction (AMI) was induced by 90 min of mid-LAD occlusion using a balloon catheter. Two days after AMI was induced, either vehicle (Plasma Lyte-A, n = 7), low-dose (20 million, n = 6), or high-dose (200 million, n = 6) MAPCs were delivered directly to the myocardium via the infarct-related vessel using a transarterial microsyringe catheter-based delivery system. Echocardiography was used to measure LV function as a function of time after AMI. Animals that received low-dose cell treatment showed significant improvement in regional and global LV function and remodeling compared to the high-dose or control animals. Direct myocardial delivery of allogeneic MAPCs 2 days following AMI through the vessel wall of the infarct-related vessel is safe and results in delivery of cells throughout the infarct zone and improved cardiac function despite lack of long-term cell survival. These data further support the hypothesis of cell-based myocardial tissue repair by a paracrine mechanism and suggest a clinically translatable strategy for delivering cells at any time after AMI to modulate cardiac remodeling and function.

Molecular Imaging of the Paracrine Proangiogenic Effects of Progenitor Cell Therapy in Limb Ischemia

Background— Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPCs) promotes recovery of blood flow through the recruitment of proangiogenic monocytes. Methods and Results— Hind-limb ischemia was produced in mice by iliac artery ligation, and MAPCs were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPCs indicated that cells survived for 1 week. Contrast-enhanced ultrasound on days 3, 7, and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX3CR-1-positive monocytes were significantly higher in MAPC-treated mice than in the control groups at days 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold-greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or tumor necrosis factor-&agr;–treated cremaster muscle demonstrated that MAPCs migrate to perimicrovascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14+ monocytes was 10-fold greater in response to MAPC-conditioned than basal media. Conclusions— In limb ischemia, MAPCs stimulate the recruitment of proangiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond the MAPC lifespan, suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

Development of a surrogate angiogenic potency assay for clinical-grade stem cell production

BACKGROUND AIMS Clinical results from acute myocardial infarction (AMI) patients treated with MultiStem®, a large-scale expanded adherent multipotent progenitor cell population (MAPC), have demonstrated a strong safety and benefit profile for these cells. The mechanism of benefit with MAPC treatment is a result, in part, of its ability to induce neovascularization through trophic support. Production of clinical-grade stem cell products requires the development of lot-release criteria based on potency assays that directly reflect the fundamental mechanistic pathway underlying the therapeutic response to verify manufacturing process consistency and product potency. METHODS AND RESULTS Using an in vitro endothelial tube formation assay, a potency assay has been developed that reflects MAPC pro-angiogenic activity. Serum-free conditioned media collected from MAPC culture induced endothelial tube formation. A proteomic survey of angiogenic factors produced by the cells in vitro revealed candidate factors linked to angiogenic potency. Three cytokines, chemokine (C-X-C motif) ligand 5 (CXCL5), interleukin 8 (IL-8) and vascular endothelial growth factor (VEGF), were required for this angiogenic activity. Depletion of any of these factors from the media prevented tube formation, while adding back increasing amounts of these cytokines into the depleted serum-free conditioned media established the lower limits of each of the cytokines required to induce angiogenesis. CONCLUSIONS A necessary threshold of angiogenic factor expression was established using an in vitro angiogenesis assay. By correlating the levels of the cytokines required to induce tube formation in vitro with levels of the factors found in the spent media from manufacturing production runs, detection of these factors was identified as a surrogate potency assay with defined pass/fail criteria.

Human multipotent adult progenitor cells transcriptionally regulate fucosyltransferase VII

BACKGROUND AIMS Targeted recruitment of leukocytes to sites of inflammation is a crucial event in normal host defense against pathogens, and attachment to and rolling on activated endothelial cells is a prerequisite first step for eventual leukocyte extravasation into sites of inflammation. These key events are mediated by interactions between glycosylated ligands expressed on leukocytes and selectins expressed on activated endothelium. Cell surface expression of selectin ligands on leukocytes is regulated by the rate-limiting enzyme fucosyltransferase VII (Fut7), and in its absence extravasation of leukocytes is severely inhibited. Multipotent adult progenitor cells (MAPCs) are an adherent cell population isolated from adult bone marrow. Intravenous administration of MAPCs provided functional improvement in multiple pre-clinical models of injury or disease, but the mechanisms by which these outcomes were achieved remain poorly understood. METHODS In vitro cell analysis studies including fluorescence-activated cell sorting, messenger RNA analysis, T-cell proliferation assays and endothelial cell binding assays were performed. RESULTS The in vitro cell analysis studies characterized the ability of MAPCs to secrete factors that transcriptionally attenuate expression of Fut7 in T cells, blocking the terminal fucosylation event in the biosynthesis of selectin ligands and reducing T-cell binding to endothelial cells. CONCLUSIONS This study presents the first example of a distinct regulatory mechanism involving transcriptional down-regulation of Fut7 by MAPCs that could modulate the trafficking behavior of T cells in vivo.