Mapping of the secretome of primary isolates of mammalian cells, stem cells and derived cell lines

Within a mammalian organism, the interaction among cells both at short and long distances is mediated by soluble factors released by cells into the extracellular environment. The secreted proteins may involve extracellular matrix proteins, proteinases, growth factors, protein hormones, immunoregulatory cytokines, chemokines or other bioactive molecules that have a direct impact on target cell phenotype. Stem cells of mesenchymal, adipose, neural and embryonic origin, fibroblast feeder cells as well as primary isolates of astrocytes, endothelial and muscle cells have recently become targets of intensive secretome profiling with the search for proteins regulating cell survival, proliferation, differentiation or inflammatory response. Recent advances and challenges of the stem cell and primary cell secretome analysis together with the most relevant results are discussed in this review.     

Inhibition of fibroblast chemotaxis by superoxide dismutase

Superoxide radicals are known to be important mediators in chronic inflammatory and fibrotic processes, in which accumulation of fibroblasts is thought to play a major role in the pathogenetic events. The enzyme superoxide dismutase removes these radicals by a catalytic reaction. Chemotactic response of human fibroblasts and fibrosarcoma-derived cells (HT-1080) to fibroblast conditioned medium, fibronectin and platelet-derived growth factor was inhibited in a dose-dependent manner in the presence of superoxide dismutase, while random migration, cell proliferation, cell viability and synthesis of collagen and non-collagenous proteins was not altered. In contrast, phorbol myristate acetate, an inducer of superoxide generation, stimulated the chemotactic movement of fibroblasts to the attractants. Evidence for the formation of superoxide is provided by the reduction of tetrazolium salt by activated fibroblasts which could be inhibited by superoxide dismutase. Thus, it is concluded that superoxide in small amounts is involved in the mechanism of fibroblast chemotaxis. Superoxide dismutase may, therefore, reduce fibroblast migration into sites of injury or inflammation.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

Human fibroblast-derived growth factor is a mitogen and chemoattractant for endothelial cells

Human fibroblasts were found to produce a potent mitogen and chemoattractant for fetal bovine aortic endothelial cells. Homogenates from AG1523 and AG1518 foreskin, CCD18Lu lung, and CCD18Co colon fibroblasts produced half-maximal stimulation of endothelial cell growth at concentrations of 1-7 micrograms/ml. The factor was purified from large-scale cultures of the CCD18Co fibroblasts using cation exchange chromatography and heparin-Sepharose chromatography. Such preparations were mitogenic for endothelial cells in vitro at concentrations of about 5-10 ng/ml, and promoted chemotaxis at 0.1-1 ng/ml. Heparinase treatment of the cells prevented the chemotactic response. These properties suggest that the factor may be related to fibroblast growth factor.     

Endothelial cell migration in stable gradients of vascular endothelial growth factor A and fibroblast growth factor 2: effects on chemotaxis and chemokinesis

Gradients of secreted signaling proteins guide growing blood vessels during both normal and pathological angiogenesis. However, the mechanisms by which endothelial cells integrate and respond to graded distributions of chemotactic factors are still poorly understood. We have in this study investigated endothelial cell migration in response to hill-shaped gradients of vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2) using a novel microfluidic chemotaxis chamber (MCC). Cell migration was scored at the level of individual cells using time-lapse microscopy. A stable gradient of VEGFA165 ranging from 0 to 50 ng/ml over a distance of 400 microm was shown to strongly induce chemotaxis of endothelial cells of different vascular origin. VEGFA121, unable to bind proteoglycan and neuropilin coreceptors, was also shown to induce chemotaxis in this setup. Furthermore, a gradient of FGF2 was able to attract venular but not arterial endothelial cells, albeit less efficiently than VEGFA165. Notably, constant levels of VEGFA165, but not of FGF2, were shown to efficiently reduce chemokinesis. Systematic exploration of different gradient shapes led to the identification of a minimal gradient steepness required for efficient cell guidance. Finally, analysis of cell migration in different regions of the applied gradients showed that chemotaxis is reduced when cells reach the high end of the gradient. Our findings suggest that chemotactic growth factor gradients may instruct endothelial cells to shift toward a nonmigratory phenotype when approaching the growth factor source.     

CYR61 stimulates human skin fibroblast migration through Integrin alpha vbeta 5 and enhances mitogenesis through integrin alpha vbeta 3, independent of its carboxyl-terminal domain

CYR61, an angiogenic factor and a member of the CCN protein family, is an extracellular matrix-associated, heparin-binding protein that mediates cell adhesion, promotes cell migration, and enhances growth factor-stimulated cell proliferation. CYR61 induces angiogenesis and promotes tumor growth in vivo and is expressed in dermal fibroblasts during cutaneous wound healing. It has been demonstrated recently that adhesion of primary skin fibroblasts to CYR61 is mediated through integrin alpha(6)beta(1) and cell surface heparan sulfate proteoglycans, resulting in adhesive signaling and up-regulation of matrix metalloproteinases 1 and 3. CYR61 is composed of four discrete structural domains that bear sequence similarities to the insulin-like growth factor-binding proteins, von Willebrand factor type C repeat, thrombospondin type 1 repeat, and a carboxyl-terminal (CT) domain that resembles cysteine knots found in some growth factors. In this study, we show that a CYR61 mutant (CYR61DeltaCT) that has the CT domain deleted is unable to support adhesion of primary human skin fibroblasts but is still able to stimulate chemotaxis and enhance basic fibroblast growth factor-induced mitogenesis similar to wild type. In addition, fibroblast migration to CYR61 is mediated through integrin alpha(v)beta(5) but not integrins alpha(6)beta(1) or alpha(v)beta(3). Furthermore, we show that CYR61 binds directly to purified integrin alpha(v)beta(5) in vitro. By contrast, CYR61 enhancement of basic fibroblast growth factor-induced DNA synthesis is mediated through integrin alpha(v)beta(3), a known receptor for CYR61 that mediates CYR61-dependent cell adhesion and chemotaxis in vascular endothelial cells. Thus, CYR61 promotes primary human fibroblast adhesion, migration, and mitogenesis through integrins alpha(6)beta(1), alpha(v)beta(5), and alpha(v)beta(3), respectively. Together, these findings establish CYR61 as a novel ligand for integrin alpha(v)beta(5) and show that CYR61 interacts with distinct integrins to mediate disparate activities in a cell type-specific manner.     

Pigment epithelium-derived factor exerts opposite effects on endothelial cells of different phenotypes

The anti-angiogenic activity of pigment epithelium-derived factor (PEDF) has recently been discovered on the basis of its inhibition of ischemia-induced retinal neovascularization in an animal model of retinopathy of the premature. Moreover PEDF inhibits the migration and proliferation of various endothelial cells maintained in culture with FGF(2). Since vascular endothelial growth factor (VEGF) is the main angiogenic factor expressed in hypervascularized retinas, we investigated the functions of PEDF on retinal endothelial cells whose angiogenic phenotype is controlled or not by long term exposure to VEGF as observed in human pathologies such as diabetic retinopathy. Here, we observed that PEDF exerts opposite effects on endothelial cells depending on their phenotype. We determined that when PEDF inhibits endothelial cell growth, it inhibits VEGF-induced MAPK activation. However, in endothelial cells cultured with VEGF, PEDF has a synergistic action on cell proliferation with VEGF, and this corresponds to increased MAPK activation.     

Inhibitory effects of a bacteria-derived sulfated polysaccharide against basic fibroblast growth factor-induced endothelial cell growth and chemotaxis

The effects of sulfated polysaccharides on the growth and chemotaxis of endothelial cells promoted by basic fibroblast growth factor (bFGF), a heparin-binding growth factor, and epidermal growth factor (EGF), a non-heparin-growth factor, were examined. The binding abilities of these two growth factors to D-gluco-galactan sulfate (DS-4152) were the same as to heparin. DS-4152 inhibited the growth and chemotaxis of the cells stimulated by bFGF, and prevented the binding of bFGF to the cells at both its low and high affinity binding sites: the former and the latter are heparin-like molecules and receptor proteins for bFGF, respectively. However, DS-4152 affected neither the binding of EGF to endothelial cells nor the proliferation and chemotaxis of the cells stimulated by the factor. Heparin also inhibited the binding of bFGF to low affinity binding sites to the same degree as DS-4152, but had little effect on the binding of bFGF to high affinity sites and no effects on bFGF-induced endothelial cell growth. Chondroitin sulfate A prevented neither the binding of bFGF to both sites of the cells nor bFGF-induced cell proliferation. We thus concluded that the inhibitory effects of DS-4152 against the growth and chemotaxis of endothelial cells induced by bFGF might be due to the prevention of bFGF binding to its receptor proteins resulting from the binding of DS-4152 to bFGF. © 1993 Wiley-Liss, Inc.     

Age-related expression of PEDF/EPC-1 in human endometrial stromal fibroblasts: implications for interactive senescence

Aging is the major risk factor for many cancers, and age-related changes in the tissue microenvironment can facilitate tumor growth. This study uses human endometrial cells to begin to test the hypothesis that age-related changes in pigment epithelium-derived factor/early population doubling cDNA-1 (PEDF/EPC-1) levels create an environment that is more permissive to tumor growth. Endometrial stromal fibroblasts (ESF) are the predominant cell type in the human endometrium and exert regulatory control over the glandular epithelial cells, which are the source of most tumors. As ESF age in vitro, their ability to regulate appropriate growth and differentiation of epithelial cells declines. Endometrial epithelial cells in primary culture expressed relatively low levels of PEDF/EPC-1 mRNA. In contrast, early passage quiescent ESF from adult donors produce higher levels of the 1.5-kb PEDF/EPC-1 mRNA and 50-kDa secreted protein than epithelial cells. As ESF age in vitro the relative abundance of PEDF/EPC-1 mRNA declines, as does the level of PEDF/EPC-1 protein secreted into cell culture medium. Treatment with PEDF/EPC-1 protein had no effect on ESF proliferation but did inhibit anchorage-dependent and anchorage-independent proliferation of endometrial carcinoma cells in a dose- and time-dependent manner. These findings imply that an age-related loss of PEDF/EPC-1 expression by ESF could eliminate a negative regulator of cancer cell growth and, thereby, contribute to the age-related increase in cancer incidence.     

Fibroblast nemosis induces angiogenic responses of endothelial cells

Increasing evidence points to a central link between inflammation and activation of the stroma, especially of fibroblasts therein. However, the mechanisms leading to such activation mostly remain undescribed. We have previously characterized a novel type of fibroblast activation (nemosis) where clustered fibroblasts upregulated the production of cyclooxygenase-2, secretion of prostaglandins, proteinases, chemotactic cytokines, and hepatocyte growth factor (HGF), and displayed activated nuclear factor-κB. Now we show that nemosis drives angiogenic responses of endothelial cells. In addition to HGF, nemotic fibroblasts secreted vascular endothelial growth factor (VEGF), and conditioned medium from spheroids promoted sprouting and networking of human umbilical venous endothelial cells (HUVEC). The response was partly inhibited by function-blocking antibodies against HGF and VEGF. Conditioned nemotic fibroblast medium promoted closure of HUVEC and human dermal microvascular endothelial cell monolayer wounds, by increasing the motility of the endothelial cells. Wound closure in HUVEC cells was partly inhibited by the antibodies against HGF. The stromal microenvironment regulates wound healing responses and often promotes tumorigenesis. Nemosis offers clues to the activation process of stromal fibroblasts and provides a model to study the part they play in angiogenesis-related conditions, as well as possibilities for therapeutical approaches desiring angiogenesis in tissue.     

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.     

Alkaline phosphatase dual‐binding sites for collagen dictate cell migration and microvessel assembly in vitro

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro‐ or microvasculature) play a role in the formation of microvessel‐like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor‐β1, and interleukin‐8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel‐like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins’ aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.     

Secretome analysis of human bone marrow derived mesenchymal stromal cells

As an essential cellular component of the bone marrow (BM) microenvironment mesenchymal stromal cells (MSC) play a pivotal role for the physiological regulation of hematopoiesis, in particular through the secretion of cytokines and chemokines. Mass spectrometry (MS) facilitates the identification and quantification of a large amount of secreted proteins (secretome), but can be hampered by the false-positive identification of contaminating proteins released from dead cells or derived from cell medium. To reduce the likelihood of contaminations we applied an approach combining secretome and proteome analysis to characterize the physiological secretome of BM derived human MSC. Our analysis revealed a secretome consisting of 315 proteins. Pathway analyses of these proteins revealed a high abundance of proteins related to cell growth and/or maintenance, signal transduction and cell communication thereby representing key biological functions of BM derived MSC on protein level. Within the MSC secretome we identified several cytokines and growth factors such as VEGFC, TGF-β1, TGF-β2 and GDF6 which are known to be involved in the physiological regulation of hematopoiesis. By comparing the peptide patterns of secretomes and cell lysates 17 proteins were identified as candidates for proteolytic processing. Taken together, our combined MS work-flow reduced the likelihood of contaminations and enabled us to carve out a specific overview about the composition of the secretome from human BM derived MSC. This methodological approach and the specific secretome signature of BM derived MSC may serve as basis for future comparative analyses of the interplay of MSC and HSPC in patients with hematological malignancies.     

Inhibition of angiogenesis by tissue inhibitor of metalloproteinase

Matrix proteases play a critical role in cell invasion and migration, including the process of angiogenesis. The ability of specific factors to induce angiogenic responses correlates with their stimulation of matrix protease synthesis and release. Using an in vivo angiogenesis assay, the endothelial cell response to known angiogenic factors, basic fibroblast growth factor (bfGF) and adipocyte conditioned medium, was blocked by an inhibitor of matrix metalloproteinase activity, TIMP-1. The TIMP effect was mediated, at least in part, through the inhibition of endothelial cell migration, as determined by the ability of TIMP to block chemotaxis in a Boyden chamber assay. These results indicate that the inhibition of migration is a direct effect on the endothelial cells and does not require accessory cells. An additional observation was that the RNA levels for TIMP were significantly reduced in differentiated adipocytes, compared to undifferentiated F442A controls. Therefore, the acquisition of an angiogenic phenotype may involve not only the induction of positive factors, but also the suppression of angiogenesis inhibitors. © 1994 Wiley-Liss, Inc.     

Pigment Epithelium-Derived Factor Is a Survival Factor for Cerebellar Granule Cells in Culture

Abstract: Pigment epithelium-derived factor (PEDF), purified from human fetal retinal pigment epithelium cell culture medium, was shown to potentiate the differentiation of human Y-79 retinoblastoma cells. To investigate potential neurotrophic effects of PEDF on neurons other than those of retinal derivation, we used cultures of cerebellar granule cells. The number of cerebellar granule cells was significantly larger in the presence of PEDF, as demonstrated by an assay for viable cells that uses 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H -tetrazolium, inner salt, conversion, by cell count, and by immunocytochemistry. The effect of PEDF showed a dose-response relationship, with a larger effect in chemically defined medium than in serum-containing medium [ED₅₀ = 30 ng/ml (0.70 n M ) in chemically defined medium and 100 ng/ml (2.3 n M ) in serum-containing medium]. PEDF had no effect on incorporation of bromodeoxyuridine (cell proliferation) or on neurofilament content (neurite outgrowth) measured by an enzyme-linked immunoadsorbent assay. These results demonstrate that PEDF has a neurotrophic survival effect on cerebellar granule cells in culture and suggest the possibility that it may affect other CNS neurons as well.     

Prostaglandin E(2) inhibits fibroblast chemotaxis

Fibroblasts are the major source of extracellular connective tissue matrix, and the recruitment, accumulation, and stimulation of these cells are thought to play important roles in both normal healing and the development of fibrosis. Prostaglandin E(2) (PGE(2)) can inhibit this process by blocking fibroblast proliferation and collagen production. The aim of this study was to investigate the inhibitory effect of PGE(2) on human plasma fibronectin (hFN)- and bovine bronchial epithelial cell-conditioned medium (BBEC-CM)-induced chemotaxis of human fetal lung fibroblasts (HFL1). Using the Boyden blind well chamber technique, PGE(2) (10(-7) M) inhibited chemotaxis to hFN 40.8 +/- 5.3% (P < 0.05) and to BBEC-CM 49.7 +/- 11.7% (P < 0.05). Checkerboard analysis demonstrated inhibition of both chemotaxis and chemokinesis. The effect of PGE(2) was concentration dependent, and the inhibitory effect diminished with time. Other agents that increased fibroblast cAMP levels, including isoproterenol (10(-5) M), dibutyryl cAMP (10(-5) M), and forskolin (3 x 10(-5) M) had similar effects and inhibited chemotaxis 54.1, 95.3, and 87.0%, respectively. The inhibitory effect of PGE(2) on HFL1 cell chemotaxis was inhibited by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, which suggests a cAMP-dependent effect mediated by PKA. In summary, PGE(2) appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response after injury.     

PEDF regulates vascular permeability by a γ-secretase-mediated pathway

Increased vascular permeability is an inciting event in many vascular complications including diabetic retinopathy. We have previously reported that pigment epithelium-derived factor (PEDF) is able to inhibit vascular endothelial growth factor (VEGF)-induced angiogenesis through a novel γ-secretase-dependent pathway. In this study, we asked whether inhibition of VEGF-induced permeability by PEDF is also γ-secretase-mediated and to dissect the potential mechanisms involved. Vascular permeability was assessed in vitro by measuring transendothelial resistance and paracellular permeability to dextran and in vivo by following leakage of intravenous FITC-labelled albumin into the retina in the presence or absence of VEGF and PEDF in varying combinations. Experiments were undertaken in the presence or absence of a γ-secretase inhibitor. To assess junctional integrity immunohistochemistry for the adherens junction (AJ) proteins, VE-cadherin and β-catenin, and the tight junction (TJ) protein, claudin-5 was undertaken using cultured cells and flat mount retinas. Protein expression and the association between AJ proteins, VEGF receptors (VEGFRs) and γ-secretase constituents were determined by immunoprecipitation and Western Blot analysis. In selected experiments the effect of hypoxia on junctional integrity was also assessed. PEDF inhibition of VEGF-induced permeability, both in cultured microvascular endothelial cell monolayers and in vivo in the mouse retinal vasculature, is mediated by γ-secretase. PEDF acted by a) preventing dissociation of AJ and TJ proteins and b) regulating both the association of VEGF receptors with AJ proteins and the subsequent phosphorylation of the AJ proteins, VE-cadherin and β-catenin. Association of γ-secretase with AJ proteins appears to be critical in the regulation of vascular permeability. Although hypoxia increased VEGFR expression there was a significant dissociation of VEGFR from AJ proteins. In conclusion, PEDF regulates VEGF-induced vascular permeability via a novel γ-secretase dependent pathway and targeting downstream effectors of PEDF action may represent a promising therapeutic strategy for preventing or ameliorating increased vascular permeability.     

Fibronectin-induced protein carboxy-O-methylation in aortic endothelial cells

In a previous report (Bowersox, J.C. and Sorgente, N. (1982) Cancer Res. 42, 2547–2551) we demonstrated that the glycoprotein fibronectin is a chemoattractant for vascular endothelial cells. In probing the mechanisms by which fibronectin induces endothelial cell chemotaxis, we have discovered that the carboxy-O-methylation of cellular proteins is stimulated by fibronectin. By measuring the incorporation of l-[methyl-³H]methionine into alkali-labile, [³H]methyl ester linkages, we determined that fibronectin stimulated protein carboxy-O-methylation in aortic endothelial cells in a time- and concentration-dependent manner; the greatest stimulation occurred at 100 μg/ml fibronectin (approx. 35% above controls). When inhibitors of carboxymethylation were added, fibronectin-induced stimulation of protein methylation did not occur. Furthermore, inhibitors of methylation prevented the chemotaxis of endothelial cells in response to fibronectin. These data support our hypothesis that fibronectin mediates endothelial cell chemotaxis, such as that occurring during neovascularization. As carboxy-O-methylation of cell proteins is also effected by fibronectin, transmethylation reactions may be an important component of endothelial cell chemotaxis.