Vitronectin modulates human mesenchymal stem cell response to insulin-like growth factor-I and transforming growth factor beta 1 in a serum-free environment

The use of animal sera for the culture of therapeutically important cells impedes the clinical use of the cells. We sought to characterize the functional response of human mesenchymal stem cells (hMSCs) to specific proteins known to exist in bone tissue with a view to eliminating the requirement of animal sera. Insulin-like growth factor-I (IGF-I), via IGF binding protein-3 or -5 (IGFBP-3 or -5) and transforming growth factor-beta 1 (TGF-beta(1)) are known to associate with the extracellular matrix (ECM) protein vitronectin (VN) and elicit functional responses in a range of cell types in vitro. We found that specific combinations of VN, IGFBP-3 or -5, and IGF-I or TGF-beta(1) could stimulate initial functional responses in hMSCs and that IGF-I or TGF-beta(1) induced hMSC aggregation, but VN concentration modulated this effect. We speculated that the aggregation effect may be due to endogenous protease activity, although we found that neither IGF-I nor TGF-beta(1) affected the functional expression of matrix metalloprotease-2 or -9, two common proteases expressed by hMSCs. In summary, combinations of the ECM and growth factors described herein may form the basis of defined cell culture media supplements, although the effect of endogenous protease expression on the function of such proteins requires investigation.

Modulation of dermal microvascular endithelial cell responses to growth factors and haemostatic factors in the presence of vitronectin

In order to effect permanent closure in burns patients suffering from full thickness wounds, replacing their skin via split thickness autografting, is essential. Dermal substitutes in conjunction with widely meshed split thickness autografts (+/- cultured keratinocytes) reduce scarring at the donor and recipient sites of burns patients by reducing demand for autologous skin (both surface area and thickness), without compromising dermal delivery at the wound face. Tissue engineered products such as Integra consist of a dermal template which is rapidly remodelled to form a neodermis, at which time the temporary silicone outer layer is removed and replaced with autologous split thickness skin. Whilst provision of a thick tissue engineered dermis at full thickness burn sites reduces scarring, it is hampered by delays in vascularisation which results in clinical failure. The ultimate success of any skin graft product is dependent upon a number of basic factors including adherence, haemostasis and in the case of viable tissue grafts, success is ultimately dependent upon restoration of a normal blood supply, and hence this study. Ultimately, the goal of this research is to improve the therapeutic properties of tissue replacements, through impregnation with growth factors aimed at stimulating migration and proliferation of microvascular endothelial cells into the donor tissue post grafting. For the purpose of my masters, the aim was to evaluate the responsiveness of a dermal microvascular endothelial cell line to growth factors and haemostatic factors, in the presence of the glycoprotein vitronectin. Vitronectin formed the backbone for my hypothesis and research due to its association with both epithelial and, more specifically, endothelial migration and proliferation. Early work using a platform technology referred to as VitroGro (Tissue Therapies Ltd), which is comprised of vitronectin bound BP5/IGF-1, aided keratinocyte proliferation. I hypothesised that this result would translate to another epithelium - endothelium. VitroGro had no effect on endothelial proliferation or migration. Vitronectin increases the presence of Fibroblast Growth Factor (FGF) and Vascular Endothelial Growth Factor (VEGF) receptors, enhancing cell responsiveness to their respective ligands. So, although Human Microvascular Endothelial Cell line 1 (HMEC-1) VEGF receptor expression is generally low, it was hypothesised that exposure to vitronectin would up-regulate this receptor. HMEC-1 migration, but not proliferation, was enhanced by vitronectin bound VEGF, as well as vitronectin bound Epidermal Growth Factor (EGF), both of which could be used to stimulate microvascular endothelial cell migration for the purpose of transplantation. In addition to vitronectin's synergy with various growth factors, it has also been shown to play a role in haemostasis. Vitronectin binds thrombin-antithrombin III (TAT) to form a trimeric complex that takes on many of the attributes of vitronectin, such as heparin affinity, which results in its adherence to endothelium via heparan sulfate proteoglycans (HSP), followed by unaltered transcytosis through the endothelium, and ultimately its removal from the circulation. This has been documented as a mechanism designed to remove thrombin from the circulation. Equally, it could be argued that it is a mechanism for delivering vitronectin to the matrix. My results show that matrix-bound vitronectin dramatically alters the effect that conformationally altered antithrombin three (cATIII) has on proliferation of microvascular endothelial cells. cATIII stimulates HMEC-1 proliferation in the presence of matrix-bound vitronectin, as opposed to inhibiting proliferation in its absence. Binding vitronectin to tissues and organs prior to transplant, in the presence of cATIII, will have a profound effect on microvascular infiltration of the graft, by preventing occlusion of existing vessels whilst stimulating migration and proliferation of endothelium within the tissue.

A chimeric vitronectin : IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells

This paper was retracted by the Journal of Stem Cells and Development on February 15, 2013.

Mechanistic investigations into interactions between IGF-I and IGFBPs and their impact on facilitating cell migration on vitronectin

Numerous studies have reported links between insulin-like growth factors (IGFs) and the extra-cellular matrix protein vitronectin (VN). We ourselves have reported that IGF-I binds to VN via IGF-binding proteins (IGFBPs) to stimulate HaCaT and MCF-7 cell migration. Here, we detail the functional evaluation of IGFBP-1, -2, -3, -4 and -6 in the presence and absence of IGF-I and VN. The data presented here, combined with our prior data on IGFBP-5, suggest that IGFBP-3, -4 and -5 are the most effective at stimulating cell migration in combination with IGF-I and VN. In addition, we demonstrate that different regions within IGFBP-3 and -4 are critical for complex formation. Furthermore, we examine whether multi-protein complexes of IGF-I and IGFBPs associated with fibronectin and collagen IV are also able to enhance functional biological responses.

Insulin-like growth factor-i : vitronectin complex-induced changes in gene expression effect breast cell survival and migration

Recent studies have demonstrated that IGF-I associates with VN through IGF-binding proteins (IGFBP) which in turn modulate IGF-stimulated biological functions such as cell proliferation, attachment and migration. Since IGFs play important roles in transformation and progression of breast tumours, we aimed to describe the effects of IGF-I:IGFBP:VN complexes on breast cell function and to dissect mechanisms underlying these responses. In this study we demonstrate that substrate-bound IGF-I:IGFBP:VN complexes are potent stimulators of MCF-7 breast cell survival, which is mediated by a transient activation of ERK/MAPK and sustained activation of PI3-K/AKT pathways. Furthermore, use of pharmacological inhibitors of the MAPK and PI3-K pathways confirms that both pathways are involved in IGF-I:IGFBP:VN complex-mediated increased cell survival. Microarray analysis of cells stimulated to migrate in response to IGF-I:IGFBP:VN complexes identified differential expression of genes with previously reported roles in migration, invasion and survival (Ephrin-B2, Sharp-2, Tissue-factor, Stratifin, PAI-1, IRS-1). These changes were not detected when the IGF-I analogue (\[L24]\[A31]-IGF-I), which fails to bind to the IGF-I receptor, was substituted; confirming the IGF-I-dependent differential expression of genes associated with enhanced cell migration. Taken together, these studies have established that IGF-I:IGFBP:VN complexes enhance breast cell migration and survival, processes central to facilitating metastasis. This study highlights the interdependence of ECM and growth factor interactions in biological functions critical for metastasis and identifies potential novel therapeutic targets directed at preventing breast cancer progression.

Hyaluronic acid : evaluation as a potential delivery vehicle for vitronectin:growth factor complexes in wound healing applications

We have previously reported that novel vitronectin:growth factor (VN:GF) complexes significantly increase re-epithelialization in a porcine deep dermal partial-thickness burn model. However, the potential exists to further enhance the healing response through combination with an appropriate delivery vehicle which facilitates sustained local release and reduced doses of VN:GF complexes. Hyaluronic acid (HA), an abundant constituent of the interstitium, is known to function as a reservoir for growth factors and other bioactive species. The physicochemical properties of HA confer it with an ability to sustain elevated pericellular concentrations of these species. This has been proposed to arise via HA prolonging interactions of the bioactive species with cell surface receptors and/or protecting them from degradation. In view of this, the potential of HA to facilitate the topical delivery of VN:GF complexes was evaluated. Two-dimensional (2D) monolayer cell cultures and 3D de-epidermised dermis (DED) human skin equivalent (HSE) models were used to test skin cell responses to HA and VN:GF complexes. Our 2D studies revealed that VN:GF complexes and HA stimulate the proliferation of human fibroblasts but not keratinocytes. Experiments in our 3D DED-HSE models showed that VN:GF complexes, both alone and in conjunction with HA, led to enhanced development of both the proliferative and differentiating layers in the DED-HSE models. However, there was no significant difference between the thicknesses of the epidermis treated with VN:GF complexes alone and VN:GF complexes together with HA. While the addition of HA did not enhance all the cellular responses to VN:GF complexes examined, it was not inhibitory, and may confer other advantages related to enhanced absorption and transport that could be beneficial in delivery of the VN:GF complexes to wounds.

Identification of vitronectin as a novel insulin-like growth factor-II binding protein

We have previously reported the presence of a 70 kDa insulin-like growth factor (IGF)-II-specific binding protein in chicken serum using Western ligand blotting approaches. In order to ascertain the identity of this 70 kDa IGF-II binding species, the protein has been purified from chicken serum using a combination of ion-exchange and gel-permeation chromatography. Interestingly, amino acid sequencing of the purified protein revealed that it has the same N-terminal sequence as chicken vitronectin (VN). The protein has the ability to specifically bind IGF-II and not IGF-I as determined by ligand blotting, cross-linking and competitive binding assay approaches. In addition, the protein binds 125I-des(l-6)-IGF-II, suggesting that the interaction with IGF-II is different to those with other characterized IGF-binding proteins. Importantly, we have ascertained that both human and bovine VN also specifically bind IGF-II. These results are particularly relevant in the light of the recent report that the urokinase-type plasminogen activator receptor, a protein that also binds VN, has been shown to associate with the cation-independent mannose-6-phosphate/GF-II receptor and suggest a possible role for IGF-II in cell adhesion and invasion.

Vitronectin – master controller or micromanager?

The concept of the cellular glycoprotein vitronectin acts as a biological ‘glue’ and key controller of mammalian tissue repair and remodelling activity is emerging from nearly 50 years of experimental in vitro and in vivo data. Unexpectedly, the vitronectin-knock-out mouse was found to be viable and to have largely normal phenotype. However, diligent observation revealed that the VN-KO animal exhibits delayed coagulation and poor wound healing. This is interpreted to indicate that vitronectin occupies a role in the earliest events of thrombogenesis and tissue repair. That role is as a foundation upon which the thrombus grows in an organised structure. In addition to closing the wound, the thrombus also serves to protect the underlying tissue from oxidation, is a reservoir of mitogens and tissue repair mediators and provides a provisional scaffold for the repairing tissue. In the absence of vitronectin (e.g. VN-KO animal) this cascade is disrupted before it begins. Our data demonstrates that a wide variety of biologically active species associate with VN. While initial studies were focused on mitogens, other classes of bioactives (e.g. glycosaminoglycans, metalloproteinases) are now also known to specifically interact with VN. Many of these interactions are long-lived, often resulting in multi-protein complexes, while others are stable for prolonged periods. Multiprotein complexes provide several advantages: prolonging molecular interaction; sustaining local concentrations, facilitating co-stimulation of cell surface receptors and thereby enhancing cellular / biological responses. We contend that these, or equivalent, multi-protein complexes mediate vitronectin functionality in vivo. It is also likely that many of the species demonstrated to associate with vitronectin in vitro, also associate with vitronectin in vivo in similar multi-protein complexes. Thus the predominant biological function of vitronectin is that of a master controller of the extracellular environment; informing, and possibly instructing cells ‘where’ to behave, ‘when’ to behave, and ‘how’ to behave (i.e. appropriately for the current circumstance).

Activation of αvβ3-Vitronectin Binding Is a Multistage Process in which Increases in Bond Strength Are Dependent on Y747 and Y759 in the Cytoplasmic Domain of β3

Integrin receptors serve as mechanical links between the cell and its structural environment. Using αvβ3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between αvβ3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the αvβ3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the β3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of αvβ3 integrin.

Vitronectin is not essential for normal mammalian development and fertility.

Vitronectin (VN) is an abundant glycoprotein present in plasma and the extracellular matrix of most tissues. Though the precise function of VN in vivo is unknown, it has been implicated as a participant in diverse biological processes, including cell attachment and spreading, complement activation, and regulation of hemostasis. The major site of synthesis appears to be the liver, though VN is also found in the brain at an early stage of mouse organogenesis, suggesting that it may play an important role in mouse development. Genetic deficiency of VN has not been reported in humans or in other higher organisms. To examine the biologic function of VN within the context of the intact animal, we have established a murine model for VN deficiency through targeted disruption of the murine VN gene. Southern blot analysis of DNA obtained from homozygous null mice demonstrates deletion of all VN coding sequences, and immunological analysis confirms the complete absence of VN protein expression in plasma. However, heterozygous mice carrying one normal and one null VN allele and homozygous null mice completely deficient in VN demonstrate normal development, fertility, and survival. Sera obtained from VN-deficient mice are completely deficient in "serum spreading factor" and plasminogen activator inhibitor 1 binding activities. These observations demonstrate that VN is not essential for cell adhesion and migration during normal mouse development and suggest that its role in these processes may partially overlap with other adhesive matrix components.

Ex vivo investigation of novel wound healing therapies and development of a 3-D human skin equivalent wound model

It has previously been found that complexes comprised of vitronectin and growth factors (VN:GF) enhance keratinocyte protein synthesis and migration. More specifically, these complexes have been shown to significantly enhance the migration of dermal keratinocytes derived from human skin. In view of this, it was thought that these complexes may hold potential as a novel therapy for healing chronic wounds. However, there was no evidence indicating that the VN:GF complexes would retain their effect on keratinocytes in the presence of chronic wound fluid. The studies in this thesis demonstrate for the first time that the VN:GF complexes not only stimulate proliferation and migration of keratinocytes, but also these effects are maintained in the presence of chronic wound fluid in a 2-dimensional (2-D) cell culture model. Whilst the 2-D culture system provided insights into how the cells might respond to the VN:GF complexes, this investigative approach is not ideal as skin is a 3-dimensional (3-D) tissue. In view of this, a 3-D human skin equivalent (HSE) model, which reflects more closely the in vivo environment, was used to test the VN:GF complexes on epidermopoiesis. These studies revealed that the VN:GF complexes enable keratinocytes to migrate, proliferate and differentiate on a de-epidermalised dermis (DED), ultimately forming a fully stratified epidermis. In addition, fibroblasts were seeded on DED and shown to migrate into the DED in the presence of the VN:GF complexes and hyaluronic acid, another important biological factor in the wound healing cascade. This HSE model was then further developed to enable studies examining the potential of the VN:GF complexes in epidermal wound healing. Specifically, a reproducible partial-thickness HSE wound model was created in fully-defined media and monitored as it healed. In this situation, the VN:GF complexes were shown to significantly enhance keratinocyte migration and proliferation, as well as differentiation. This model was also subsequently utilized to assess the wound healing potential of a synthetic fibrin-like gel that had previously been demonstrated to bind growth factors. Of note, keratinocyte re-epitheliasation was shown to be markedly improved in the presence of this 3-D matrix, highlighting its future potential for use as a delivery vehicle for the VN:GF complexes. Furthermore, this synthetic fibrin-like gel was injected into a 4 mm diameter full-thickness wound created in the HSE, both keratinocytes and fibroblasts were shown to migrate into this gel, as revealed by immunofluorescence. Interestingly, keratinocyte migration into this matrix was found to be dependent upon the presence of the fibroblasts. Taken together, these data indicate that reproducible wounds, as created in the HSEs, provide a relevant ex vivo tool to assess potential wound healing therapies. Moreover, the models will decrease our reliance on animals for scientific experimentation. Additionally, it is clear that these models will significantly assist in the development of novel treatments, such as the VN:GF complexes and the synthetic fibrin-like gel described herein, ultimately facilitating their clinical trial in the treatment of chronic wounds.

Discovery and characterization of IGFBP-mediated endocytosis in the human retinal pigment epithelial cell line ARPE-19

Insulin-like growth factor binding proteins (IGFBPs) are prime regulators of IGF-action in numerous cell types including the retinal pigment epithelium (RPE). The RPE performs several functions essential for vision, including growth factor secretion and waste removal via a phagocytic process mediated in part by vitronectin (Vn). In the course of studying the effects of IGFBPs on IGF-mediated VEGF secretion and Vn-mediated phagocytosis in the RPE cell line ARPE-19, we have discovered that these cells avidly ingest synthetic microspheres (2.0 μm diameter) coated with IGFBPs. Given the novelty of this finding and the established role for endocytosis in mediating IGFBP actions in other cell types, we have explored the potential role of candidate cell surface receptors. Moreover, we have examined the role of key IGFBP structural motifs, by comparing responses to three members of the IGFBP family (IGFBP-3, IGFBP-4 and IGFBP-5) which display overlapping variations in primary structure and glycosylation status. Coating of microspheres (FluoSpheres®, sulfate modified polystyrene filled with a fluorophore) was conducted at 37 °C for 1 h using 20 μg/mL of test protein, followed by extensive washing. Binding of proteins was confirmed using a microBCA assay. The negative control consisted of microspheres treated with 0.1% bovine serum albumin (BSA), and all test samples were post-treated with BSA in an effort to coat any remaining free protein binding sites, which might otherwise encourage non-specific interactions with the cell surface. Serum-starved cultures of ARPE-19 cells were incubated with microspheres for 24 h, using a ratio of approximately 100 microspheres per cell. Uptake of microspheres was quantified using a fluorometer and was confirmed visually by confocal fluorescence microscopy. The ARPE-19 cells displayed little affinity for BSA-treated microspheres, but avidly ingested large quantities of those pre-treated with Vn (ANOVA; p < 0.001). Strong responses were also observed towards recombinant formulations of non-glycosylated IGFBP-3, glycosylated IGFBP-3 and glycosylated IGFBP-5 (all p < 0.001), while glycosylated IGFBP-4 induced a relatively minor response (p < 0.05). The response to IGFBP-3 was unaffected in the presence of excess soluble IGFBP-3, IGF-I or Vn. Likewise, soluble IGFBP-3 did not induce uptake of BSA-treated microspheres. Antibodies to either the transferrin receptor or type 1 IGF-receptor displayed slight inhibitory effects on responses to IGFBPs and Vn. Heparin abolished responses to Vn, IGFBP-5 and non-glycosylated IGFBP-3, but only partially inhibited the response to glycosylated IGFBP-3. Our results demonstrate for the first time IGFBP-mediated endocytosis in ARPE-19 cells and suggest roles for the IGFBP-heparin-binding domain and glycosylation status. These findings have important implications for understanding the mechanisms of IGFBP actions on the RPE, and in particular suggest a role for IGFBP-endocytosis.