When mature adipocytes are subjected to an in vitro dedifferentiation strategy referred to as ceiling culture, these mature adipocytes can revert to a more primitive phenotype and gain cell proliferative ability. We refer to these cells as dedifferentiated fat (DFAT) cells. In the present study, we examined the multilineage differentiation potential of DFAT cells. DFAT cells obtained from adipose tissues of 18 donors exhibited a fibroblast-like morphology and sustained high proliferative activity. Flow cytometric analysis revealed that DFAT cells comprised a highly homogeneous cell population compared with that of adipose-derived stem/stromal cells (ASCs), although the cell-surface antigen profile of DFAT cells was very similar to that of ASCs. DFAT cells lost expression of mature adipocytes marker genes but retained or gained expression of mesenchymal lineage-committed marker genes such as peroxisome proliferator-activated receptor gamma (PPARgamma), RUNX2, and SOX9. In vitro differentiation analysis revealed that DFAT cells could differentiate into adipocytes, chondrocytes, and osteoblasts under appropriate culture conditions. DFAT cells also formed osteoid matrix when implanted subcutaneously into nude mice. In addition, clonally expanded porcine DFAT cells showed the ability to differentiate into multiple mesenchymal cell lineages. These results indicate that DFAT cells represent a type of multipotent progenitor cell. The accessibility and ease of culture of DFAT cells support their potential application for cell-based therapies.

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The family of vascular endothelial growth factors (VEGFs) currently includes VEGF-A, -B, -C, -D, -E, and placenta growth factor (PlGF). Several of these factors, notably VEGF-A, exist as different isoforms, which appear to have unique biological functions. The VEGF family proteins bind in a distinct pattern to three structurally related receptor tyrosine kinases, denoted VEGF receptor-1, -2, and -3. Neuropilins, heparan-sulfated proteoglycans, cadherins, and integrin alphavbeta3 serve as coreceptors for certain but not all VEGF proteins. Moreover, the angiogenic response to VEGF varies between different organs and is dependent on the genetic background of the animal. Inactivation of the genes for VEGF-A and VEGF receptor-2 leads to embryonal death due to the lack of endothelial cells. Inactivation of the gene encoding VEGF receptor-1 leads to an increased number of endothelial cells, which obstruct the vessel lumen. Inactivation of VEGF receptor-3 leads to abnormally organized vessels and cardiac failure. Although VEGF receptor-3 normally is expressed only on lymphatic endothelial cells, it is up-regulated on vascular as well as nonvascular tumors and appears to be involved in the regulation of angiogenesis. A large body of data, such as those on gene inactivation, indicate that VEGF receptor-1 exerts a negative regulatory effect on VEGF receptor-2, at least during embryogenesis. Recent data imply a positive regulatory role for VEGF receptor-1 in pathological angiogenesis. The VEGF proteins are in general poor mitogens, but binding of VEGF-A to VEGF receptor-2 leads to survival, migration, and differentiation of endothelial cells and mediation of vascular permeability. This review outlines the current knowledge about the signal transduction properties of VEGF receptors, with focus on VEGF receptor-2.

Vascular endothelial growth factor receptor-2 (VEGFR-2) activation by VEGF-A is essential in vasculogenesis and angiogenesis. We have generated a pan-phosphorylation site map of VEGFR-2 and identified one major tyrosine phosphorylation site in the kinase insert (Y951), in addition to two major sites in the C-terminal tail (Y1175 and Y1214). In developing vessels, phosphorylation of Y1175 and Y1214 was detected in all VEGFR-2-expressing endothelial cells, whereas phosphorylation of Y951 was identified in a subset of vessels. Phosphorylated Y951 bound the T-cell-specific adapter (TSAd), which was expressed in tumor vessels. Mutation of Y951 to F and introduction of phosphorylated Y951 peptide or TSAd siRNA into endothelial cells blocked VEGF-A-induced actin stress fibers and migration, but not mitogenesis. Tumor vascularization and growth was reduced in TSAd-deficient mice, indicating a critical role of Y951-TSAd signaling in pathological angiogenesis

Previous studies have shown that the adaptor protein Shb is involved in receptor tyrosine kinase signaling. In this study, we demonstrate that Shb is phosphorylated in an Src-dependent manner upon vascular endothelial growth factor (VEGF) stimulation using porcine aortic endothelial cells expressing the human VEGF receptor 2 (VEGFR-2) (KDR). In co-immunoprecipitation experiments, we could detect an interaction between Shb and the VEGFR-2 in human telomerase-immortalized microvascular endothelial cells. Furthermore, in a glutathione S-transferase pull-down assay, the Src homology 2 domain of Shb was shown to interact with phosphorylated tyrosine 1175 in the C-terminal tail of VEGFR-2. VEGFinduced Shb phosphorylation was lost in porcine aortic endothelial cells expressing a chimeric murine VEGFR-2 (Flk-1) with a mutation at the corresponding position. Shb expression was specifically decreased by 80%, in a transient manner, by using the short interfering RNA technique. Reduced Shb expression led to a loss of stimulation of phosphatidylinositol 3-kinase, phosphorylation of focal adhesion kinase at tyrosine 576, the generation of focal adhesions, and stress fiber formation in response to VEGF. Furthermore, we show that VEGF-induced migration is inhibited in Shb short interfering RNA-treated cells. Our data demonstrate that Shb is important for VEGF signaling in endothelial cells. This is achieved by Shb binding to tyrosine 1175 in the VEGFR-2, which regulates VEGF-induced formation of focal adhesions and cell migration, of which the latter occurs in a phosphatidylinositol 3-kinase-dependent manner.