Stephen G. Ball

Mesenchymal stem cell migration is regulated by fibronectin through α5β1-integrin-mediated activation of PDGFR-β and potentiation of growth factor signals

Cell migration during vascular remodelling is regulated by crosstalk between growth factor receptors and integrin receptors, which together coordinate cytoskeletal and motogenic changes. Here, we report extracellular matrix (ECM)-directed crosstalk between platelet-derived growth factor receptor (PDGFR)-β and α5β1-integrin, which controls the migration of mesenchymal stem (stromal) cells (MSCs). Cell adhesion to fibronectin induced α5β1-integrin-dependent phosphorylation of PDGFR-β in the absence of growth factor stimulation. Phosphorylated PDGFR-β co-immunoprecipitated with α5-integrin and colocalised with α5β1-integrin in the transient tidemarks of focal adhesions. Adhesion to fibronectin also strongly potentiated PDGF-BB-induced PDGFR-β phosphorylation and focal adhesion kinase (FAK) activity, in an α5β1-integrin-dependent manner. PDGFR-β-induced phosphoinositide 3-kinase (PI3K) and Akt activity, actin reorganisation and cell migration were all regulated by fibronectin and α5β1-integrin. This synergistic relationship between α5β1-integrin and PDGFR-β is a fundamental determinant of cell migration. Thus, fibronectin-rich matrices can prime PDGFR-β to recruit mesenchymal cells at sites of vascular remodelling.

Mesenchymal stem cells and neovascularization: role of platelet-derived growth factor receptors

•u2002 Introduction •u2002 The vascular endothelial growth factor/platelet‐derived growth factor super‐family of ligands and receptors ‐u2002 Vascular endothelial growth factor ligands ‐u2002 Platelet‐derived growth factor ligands ‐u2002 Vascular endothelial growth factor receptors ‐u2002 Platelet‐derived growth factor receptors •u2002 Role of platelet‐derived growth factor receptors in regulating the MSC fate ‐u2003 MSCs utilize a novel vascular endothelial growth factor/platelet‐derived growth factor receptor signalling mechanism ‐u2003 Regulation of vascular endothelial growth factor/platelet‐derived growth factor receptor signalling •u2002 MSCs and the vasculature ‐u2002 Differentiation of MSCs towards endothelial cells ‐u2002 Differentiation of MSCs towards vascular smooth muscle lineages ‐u2002 MSCs during vascular injury ‐u2002 Contribution of MSCs to vasculogenesis ‐u2002 MSCs during ischaemic myocardial tissue regeneration ‐u2002 Involvement of MSCs during tumour vasculogenesis •u2002 Summary

Inhibition of platelet-derived growth factor receptor signaling regulates Oct4 and Nanog expression, cell shape, and mesenchymal stem cell potency.

Defining the signaling mechanisms that regulate the fate of adult stem cells is an essential step toward their use in regenerative medicine. Platelet‐derived growth factor receptor (PDGFR) signaling plays a crucial role in specifying mesenchymal stem cell (MSC) commitment to mesenchymal lineages. Based on the hypothesis that selective inhibition of signaling pathways involved in differentiation may increase stem cell potency, we examined the role of PDGFR signaling in controlling the fate of human MSCs. Using a small molecular PDGFR inhibitor that induced MSCs toward a more rounded shape, expression of Oct4 and Nanog were markedly upregulated. In these PDGFR inhibitor‐treated MSCs, Oct4 and Nanog expression and cell shape were regulated by janus kinase (JAK), MAPK kinase (MEK), and epidermal growth factor receptor (EGFR) signaling. Under defined differentiation conditions, these PDGFR‐inhibited MSCs expressed definitive endodermal, ectodermal, and mesodermal markers. We also confirmed that depletion of individual PDGF receptors upregulated expression of Oct4A and Nanog. This study identifies PDGFR signaling as a key regulator of Oct4 and Nanog expression and of MSC potency. Thus, inhibiting these specific receptor tyrosine kinases, which play essential roles in tissue formation, offers a novel approach to unlock the therapeutic capacity of MSCs. STEM CELLS 2012;30:548–560

Neuropilin-1 regulates platelet-derived growth factor receptor signalling in mesenchymal stem cells.

Using human MSCs (mesenchymal stem cells) lacking VEGF (vascular endothelial growth factor) receptors, we show that the pro-angiogenic receptor neuropilin-1 associates with phosphorylated PDGFRs [PDGF (platelet-derived growth factor) receptors], thereby regulating cell signalling, migration, proliferation and network assembly. Neuropilin-1 co-immunoprecipitated and co-localized with phosphorylated PDGFRs in the presence of growth factors. Neuropilin-1 knockdown blocked PDGF-AA-induced PDGFRα phosphorylation and migration, reduced PDGF-BB-induced PDGFRβ activation and migration, blocked VEGF-A activation of both PDGFRs, and attenuated proliferation. Neuropilin-1 prominently co-localized with both PDGFRs within MSC networks assembled in Matrigel™ and in the chorioallantoic membrane vasculature microenvironment, and its knockdown grossly disrupted network assembly and decreased PDGFR signalling. Thus neuropilin-1 regulates MSCs by forming ligand-specific receptor complexes that direct PDGFR signalling, especially the PDGFRα homodimer. This receptor cross-talk may control the mobilization of MSCs in neovascularization and tissue remodelling.

Platelet-derived growth factor receptors regulate mesenchymal stem cell fate: implications for neovascularization

Importance of the field: Regulating stem cell contributions to vascularization is a challenging goal, but a fundamental aspect of regenerative medicine. Human mesenchymal stem cells retain considerable potential for adult vascular repair and regeneration therapies. They are readily obtained, rapidly proliferate in culture, display a capacity to differentiate towards endothelial or vascular smooth muscle cells, and play an important role in postnatal neovascularization in various tissue contexts. To therapeutically enhance neovascularization during ischemic disease, or inhibit neovascularization during tumorigenesis, an essential prerequisite is to determine the mechanisms which control the recruitment and differentiation of mesenchymal stem cells towards vascular cells. Areas covered in this review: In this review, we describe the current understanding of how PDGF receptors contribute prominently to neovascularization, and play a decisive role in modulating mesenchymal stem cell recruitment and differentiation towards vascular cells. We discuss PDGF receptor-based therapeutic strategies to exploit mesenchymal stem cells during vascular repair and regeneration, and to control pathological neovascularization. Take home message: PDGF receptor signaling is emerging as a critical regulatory mechanism and important therapeutic target, that critically directs the fate of mesenchymal stem cells during postnatal neovascularization.

The role of the C1 and C2 a-domains in type VI collagen assembly.

Constructs of each of the three chains of type VI collagen were generated and examined in an in vitrotranscription/translation assay supplemented with semipermeabilized cells. Each of the constructs when used in the in vitrosystem was shown to be glycosylated and to undergo intracellular assembly, the extent of which was determined by the nature of the C-terminal globular domains. All three chains containing the C1 domain formed monomers; however, the C2 domain was required for dimer and tetramer formation. In the case of the full-length α2(VI) chain, monomers, dimers, and tetramers formed in a time-dependent manner. Although the splice variant α2(VI)C2a could form monomers, it was unable to form dimers and tetramers. Similar results to the α2(VI) chain were found for the full-length α1(VI) chain, although assembly was at a slower rate. In the case of the α3(VI) chain containing both C1 and C2 domains only monomers were observed. Addition of the C3, C4, and C5 did not change this pattern. Homology modeling suggested that a 10-amino acid insertion in the C2 domain of the α3(VI) chain may interfere with dimer formation. A near full-length construct of the α3(VI) chain only formed monomers but was shown to facilitate tetramer formation in cotranslation experiments.

Density of human bone marrow stromal cells regulates commitment to vascular lineages

Mechanisms underlying the vascular differentiation of human bone marrow stromal cells (HBMSCs) and their contribution to neovascularisation are poorly understood. We report the essential role of cell density-induced signals in directing HBMSCs along endothelial or smooth muscle lineages. Plating HBMSCs at high density rapidly induced Notch signaling, which initiated HBMSC commitment to a vascular progenitor cell population expressing markers for both vascular lineages. Notch also induced VEGF-A, which inhibited vascular smooth muscle commitment while consolidating differentiation to endothelial cells with cobblestone morphology and characteristic endothelial markers and functions. These mechanisms can be exploited therapeutically to regulate HBMSCs during neovascularisation.

Collagen VI and Laminin as Markers of Differentiation of Endometrial Stroma

In women, following menstrual shedding, the endometrium regenerates under the influence of estrogen to produce a densely cellular stroma containing narrow tubular glands and small blood vessels (Fig. 22.1). In addition to cellular proliferation, this requires the deposition of a scaffolding of extracellular matrix (ECM) into the often narrow intercellular spaces. The undifferentiated stroma produces an ECM of classically mesenchymal composition: Collagens I, III, V, and VI and fibronectin have all been shown to be present (1–3), and there are periglandular deposits of tenascin (4) that appear to reflect the proliferative state of the epithelial compartment. The epithelium and blood vessels are surrounded by basement membranes containing laminin, collagen type IV, and heparan sulfate proteoglycan (HSPG) (1–3).

STEM mass mapping of type VI collagen microfibrils: Implications for chain composition and alternative splicing

Type VI collagen is a major structural interactive extracellular matrix macromolecule which forms double-beaded microfibrils in the extracellular space. These microfibrils can be isolated in native form from collagenase digest of skin fibroblast cell layers by chromatography on Sepharose CL-2B. Scanning transmission electron microscope (STEM) mass mapping showed a periodicity of 104 nm and a mass/bead of 1500 kDa. In addition, there was an uneven mass distribution along the bead. Microfibrils arise by end-to-end aggregation of tetramers which should produce microfibrils with bead mass of ≈2000 kDa and homogeneous mass distribution across the bead. Reduction in the mass across the bead implies either altered chain composition, alternative splicing or proteolytic degradation. The pattern of splicing of type VI chains in human tissues and their biological implications are poorly defined. In this study, we have examined the abundance of alternatively spliced forms of theα2(VI) andα3(VI) chains in human skin fibroblasts. BothαC2 andα2C2a variants, but not theα2C2a′ form, were expressed in these although theα2C2 form appeared more abundant. Theα3(VI) N7 domain was frequently spliced out of transcripts present in skin fibroblasts, whereas theα3(VI) N9 domain was always present. No consistent pattern of splicing was observed and splicing is unlikely to account for the reduced mass observed in type VI collagen microfibrils.