Phase I trial: the use of autologous cultured adipose-derived stroma/stem cells to treat patients with non-revascularizable critical limb ischemia.

BACKGROUND AIMS: Non-revascularizable critical limb ischemia (CLI) is the most severe stage of peripheral arterial disease, with no therapeutic option. Extensive preclinical studies have demonstrated that adipose-derived stroma cell (ASC) transplantation strongly improves revascularization and tissue perfusion in ischemic limbs. This study, named ACellDREAM, is the first phase I trial to evaluate the feasibility and safety of intramuscular injections of autologous ASC in non-revascularizable CLI patients. METHODS: Seven patients were consecutively enrolled, on the basis of the following criteria: (i) lower-limb rest pain or ulcer; (ii) ankle systolic oxygen pressure <50 or 70 mm Hg for non-diabetic and diabetic patients, respectively, or first-toe systolic oxygen pressure <30 mm Hg or 50 mm Hg for non-diabetic and diabetic patients, respectively; (iii) not suitable for revascularization. ASCs from abdominal fat were grown for 2 weeks and were then characterized. RESULTS: More than 200 million cells were obtained, with almost total homogeneity and no karyotype abnormality. The expressions of stemness markers Oct4 and Nanog were very low, whereas expression of telomerase was undetectable in human ASCs compared with human embryonic stem cells. ASCs (10(8)) were then intramuscularly injected into the ischemic leg of patients, with no complication, as judged by an independent committee. Trans-cutaneous oxygen pressure tended to increase in most patients. Ulcer evolution and wound healing showed improvement. CONCLUSIONS: These data demonstrate the feasibility and safety of autologous ASC transplantation in patients with objectively proven CLI not suitable for revascularization. The improved wound healing also supports a putative functional efficiency.

The nucleophosmin-anaplastic lymphoma kinase oncogene interacts, activates, and uses the kinase PIKfyve to increase invasiveness.

NPM-ALK is a chimeric tyrosine kinase detected in most anaplastic large cell lymphomas that results from the reciprocal translocation t(2,5)(p23;q35) that fuses the N-terminal domain of nucleophosmin (NPM) to the catalytic domain of the anaplastic lymphoma kinase (ALK) receptor. The constitutive activity of the kinase is responsible for its oncogenicity through the stimulation of several downstream signaling pathways, leading to cell proliferation, migration, and survival. We demonstrated previously that the high level of phosphatidylinositol 5-phosphate measured in NPM-ALK-expressing cells is controlled by the phosphoinositide kinase PIKfyve, a lipid kinase known for its role in vesicular trafficking. Here, we show that PIKfyve associates with NPM-ALK and that the interaction involves the 181-300 region of the oncogene. Moreover, we demonstrate that the tyrosine kinase activity of the oncogene controls PIKfyve lipid kinase activity but is dispensable for the formation of the complex. Silencing or inhibition of PIKfyve using siRNA or the PIKfyve inhibitor YM201636 have no effect on NPM-ALK-mediated proliferation and migration but strongly reduce invasive capacities of NPM-ALK-expressing cells and their capacity to degrade the extracellular matrix. Accordingly, immunofluorescence studies confirm a perturbation of matrix metalloproteinase 9 localization at the cell surface and defect in maturation. Altogether, these results suggest a role for PIKfyve in NPM-ALK-mediated invasion.

Cutting edge: Dok-1 and Dok-2 adaptor molecules are regulated by phosphatidylinositol 5-phosphate production in T cells.

Downstream of tyrosine kinase (Dok) proteins Dok-1 and Dok-2 are involved in T cell homeostasis maintenance. Dok protein tyrosine phosphorylation plays a key role in establishing negative feedback loops of T cell signaling. These structurally related adapter molecules contain a pleckstrin homology (PH) domain generally acting as a lipid/protein-interacting module. We show that the presence of this PH domain is necessary for the tyrosine phosphorylation of Dok proteins and their negative functions in T cells. We find that Dok-1/Dok-2 PH domains bind in vitro to the rare phosphoinositide species, phosphatidylinositol 5-phosphate (PtdIns5P). Dok tyrosine phosphorylation correlates with PtdIns5P production in T cells upon TCR triggering. Furthermore, we demonstrate that PtdIns5P increase regulates Dok tyrosine phosphorylation in vivo. Together, our data identify a novel lipid mediator in T cell signaling and suggest that PH-PtdIns5P interactions regulate T cell responses.

PtdIns5P protects Akt from dephosphorylation through PP2A inhibition.

Phosphatidylinositol 5-phosphate (PtdIns5P), the most recently discovered phosphoinositide, has been proposed to play a role as a lipid mediator of intracellular signaling. We have previously shown that PtdIns5P generated by IpgD, an effector of the causative agent of dysentery Shigella flexneri, activates the PI 3-kinase/Akt pathway. Here, we demonstrate that PtdIns5P is able to protect Akt from dephosphorylation. This effect is not due to inhibition of the phosphoinositide phosphatase regulating PtdIns(3,4,5)P(3) levels PTEN but rather to PtdIns5P-induced phosphorylation and subsequent inhibition of the catalytic subunit of PP2A phosphatases. These data shed light on a new mechanism used by S. flexneri bacteria to sustain Akt activation to increase survival of the host cells during bacterial replication.

Shigella flexneri infection generates the lipid PI5P to alter endocytosis and prevent termination of EGFR signaling.

The phosphoinositide metabolic pathway, which regulates cellular processes implicated in survival, motility, and trafficking, is often subverted by bacterial pathogens. Shigella flexneri, a bacterium that causes dysentery, injects IpgD, a phosphoinositide phosphatase that generates the lipid phosphatidylinositol 5-phosphate (PI5P), into host cells, thereby activating the phosphoinositide 3-kinase-Akt survival pathway. We show that epidermal growth factor receptor (EGFR) is required for PI5P-dependent activation of Akt in infected HeLa cells or cells ectopically expressing IpgD. Cells treated with PI5P had increased numbers of early endosomes with activated EGFR, no detectable EGFR in the late endosomal or lysosomal compartments, and prolonged EGFR signaling. Endosomal recycling and retrograde pathways were spared, indicating that the effect of PI5P on the degradative route to the late endocytic compartments was specific. Thus, we identified PI5P, which was enriched in endosomes, as a regulator of vesicular trafficking that alters growth factor receptor signaling by impairing lysosomal degradation, a property used by S. flexneri to favor survival of host cells.

Matrix metalloproteinase-9 is upregulated in nucleophosmin-anaplastic lymphoma kinase-positive anaplastic lymphomas and activated at the cell surface by the chaperone heat shock protein 90 to promote cell invasion.

Many anaplastic large cell lymphomas (ALCL) express the chimeric oncogene NPM-ALK, which drives malignant transformation and invasion. In this study, we show that NPM-ALK expression increases matrix metalloproteinase-9 (MMP-9) expression. Accordingly, we found that 100% of a large panel of ALK(+) ALCL biopsies examined were also MMP-9(+), in contrast to only 36.3% of ALK(-) tumors. Mechanistic studies revealed that Rac1 drove MMP-9 secretion. The MMP inhibitor GM6001 and MMP-9 blocking antibodies abolished the invasiveness of NPM-ALK(+) cells. Interestingly, the hyaluronan receptor CD44 acted as a docking surface for MMP-9 and the chaperone heat shock protein 90 on the cell surface, where MMP-9 was cleaved and activated. Membrane-associated MMP-9 was localized to invadopodia, which display a strong gelatinase activity. Taken together, our observations strengthen the concept that chaperones have a major extracellular role in the regulation of protein activation status, and reveal new factors that are crucial for spreading and invasion of ALK(+) ALCL. They also point out new factors crucial for ALK(+) ALCL.