Differential roles of angiogenic chemokines in endothelial progenitor cell-induced angiogenesis.

This study aimed to analyze the role of endothelial progenitor cell (EPC)-derived angiogenic factors and chemokines in the multistep process driving angiogenesis with a focus on the recently discovered macrophage migration inhibitory factor (MIF)/chemokine receptor axis. Primary murine and murine embryonic EPCs (eEPCs) were analyzed for the expression of angiogenic/chemokines and components of the MIF/CXC chemokine receptor axis, focusing on the influence of hypoxic versus normoxic stimulation. Hypoxia induced an upregulation of CXCR2 and CXCR4 but not CD74 on EPCs and triggered the secretion of CXCL12, CXCL1, MIF, and vascular endothelial growth factor (VEGF). These factors stimulated the transmigration activity and adhesive capacity of EPCs, with MIF and VEGF exhibiting the strongest effects under hypoxia. MIF-, VEGF-, CXCL12-, and CXCL1-stimulated EPCs enhanced tube formation, with MIF and VEGF exhibiting again the strongest effect following hypoxia. Tube formation following in vivo implantation utilizing angiogenic factor-loaded Matrigel plugs was only promoted by VEGF. Coloading of plugs with eEPCs led to enhanced tube formation only by CXCL12, whereas MIF was the only factor which induced differentiation towards an endothelial and smooth muscle cell (SMC) phenotype, indicating an angiogenic and differentiation capacity in vivo. Surprisingly, CXCL12, a chemoattractant for smooth muscle progenitor cells, inhibited SMC differentiation. We have identified a role for EPC-derived proangiogenic MIF, VEGF and MIF receptors in EPC recruitment following hypoxia, EPC differentiation and subsequent tube and vessel formation, whereas CXCL12, a mediator of early EPC recruitment, does not contribute to the remodeling process. By discerning the contributions of key angiogenic chemokines and EPCs, these findings offer valuable mechanistic insight into mouse models of angiogenesis and help to define the intricate interplay between EPC-derived angiogenic cargo factors, EPCs, and the angiogenic target tissue.

Levels of macrophage migration inhibitory factor and glucocorticoids in chronic wound patients and their potential interactions with impaired wound endothelial progenitor cell migration.

Macrophage migration inhibitory factor (MIF), a structurally and functionally unique pleiotropic mediator in inflammation and immune processes, was identified decades ago. There is now strong evidence that MIF promotes revascularization and is involved in wound healing processes. However, its exact role in wound healing is still a matter of debate. A cohort of 33 patients was recruited, including 14 patients with acute and 19 patients with chronic wounds. Both serum and wound fluid samples were collected from each patient, and MIF and cortisol concentrations were determined. To functionally underscore MIF's potential role in wound revascularization, a chemotaxis assay was adapted to test whether and to what extent serum samples and wound fluids of each group promote the chemotactic migration of endothelial progenitor cells (EPCs). MIF serum levels were significantly higher in chronic wound patients than in acute wound patients. Wound exudates of chronic wounds, however, contained a significantly lower concentration of MIF. In chronic wound patients, EPC migration might be delayed, as suggested by in vitro chemotaxis experiments. Despite the overall descriptive nature of this study, we conclude that MIF is correlated with occurrence of chronic wound. The increased MIF levels in the serum of chronic wound patients might be due to MIF's systemic effect of its proinflammatory activities, while its locally decreased levels in chronic wound exudates might be responsible for impaired recruitment of EPCs. Additional prospective data and detailed in vivo models are needed for a more comprehensive understanding of the role of MIF in chronic wound healing.

Hypoxia-induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment.

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non-cognate ligand of the CXC-family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine-like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC-derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia-induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non-classical mechanism and originated from pre-formed MIF stores. Early hypoxia-triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia-inducible factor (HIF)-1α-induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF-1α-induced de novo synthesis of MIF. To functionally investigate the role of hypoxia-inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL(+) KDR(+) CD31(+) EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose-dependent bell-shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia-conditioned HUVECs, an effect that was completely abrogated by anti-MIF- or anti-CXCR4-antibodies. Thus, hypoxia-induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia-induced myocardial damage.

The effect of cross-linking of collagen matrices on their angiogenic capability.

The poor vascularization rate of matrices following cell invasion is considered to be one of the main shortcomings of scaffolds used in tissue engineering. In the past decade much effort has been directed towards enhancing the angiogenic potential of biomaterials. A great many studies have appeared reporting about enhancement of vascularization by immobilizing angiogenic factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-2 (FGF-2). We have also tried to achieve this goal by modifying collagen matrices by covalent incorporation of heparin into the matrices and loading them with VEGF. We and others have observed that loading angiogenic factors to heparinized materials markedly increases angiogenic capacity. In the present paper we also investigated the angiogenic properties of collagen matrices which were only cross-linked, i.e. in the absence of heparin. The angiogenic capacity of the modified matrices was evaluated using the chorioallantoic membrane assay. Differences in angiogenic potential were deduced from macroscopic and microscopic analyses of the chorioallantoic membrane, as well as from dry weight changes. Cross-linked only matrices and matrices both cross-linked and heparinized appeared to show a significantly larger angiogenic potential than unmodified matrices. As previously observed, loading VEGF to these matrices further stepped up angiogenic potential. Quite surprisingly, cross-linking had a substantial impact on angiogenic potential. In terms of magnitude, this effect was similar to the effect of loading VEGF to heparinized matrices. Both modification procedures resulted in an increase of average pore size within the collagen matrices, and this observation may explain the more rapid invasion of mouse fibroblasts into cross-linked and heparinized matrices. Form changes of the implants were also monitored during the in vivo contacts: cross-linked and heparinized matrices showed far better resistance against contraction, as compared to unmodified matrices. Results from the chorioallantoic membrane assay experiments were compared with data obtained from rat model experiments, which confirmed the results from the chorioallantoic membrane assay. This relatively simple assay was again shown to be extremely helpful in evaluating and predicting the angiogenic capabilities of biomaterials for use in tissue engineering and wound healing.

Macrophage migration inhibitory factor (MIF) promotes fibroblast migration in scratch-wounded monolayers in vitro.

MIF was recently redefined as an inflammatory cytokine, which functions as a critical mediator of diseases such as septic shock, rheumatoid arthritis, atherosclerosis, and cancer. MIF also regulates wound healing processes. Given that fibroblast migration is a central event in wound healing and that MIF was recently demonstrated to promote leukocyte migration through an interaction with G-protein-coupled receptors, we investigated the effect of MIF on fibroblast migration in wounded monolayers in vitro. Transient but not permanent exposure of primary mouse or human fibroblasts with MIF significantly promoted wound closure, a response that encompassed both a proliferative and a pro-migratory component. Importantly, MIF-induced fibroblast activation was accompanied by an induction of calcium signalling, whereas chronic exposure with MIF down-regulated the calcium transient, suggesting receptor desensitization as the underlying mechanism.

The impact of proteinase-induced matrix degradation on the release of VEGF from heparinized collagen matrices.

The in vivo application of engineered matrices in human wound healing processes is often hampered by the slow rate of vascularization. Therefore much research is directed towards enhancing the angiogenic properties of such matrices. One approach for enhancing the vascularization is the incorporation of angiogenic growth factors. Recently, we and others have reported on immobilizing such factors into collagen matrices either by covalent attachment or by physical binding to covalently incorporated heparin. Especially the latter procedure has been shown to lead to substantial increase rates in vascularization in in vivo experiments. The increases have been proposed to depend on the sustained release of the incorporated angiogenic growth factors from the heparinized collagen matrices. In this paper, we report on investigations to study the release of vascular endothelial growth factor (VEGF) from collagen matrices under conditions which mimic potential in vivo situations. Relevant proteinase concentrations were deduced from in vitro experiments in which we evaluated the secretion of selected matrix metalloproteinases from fibroblasts in contact with collagen. The release of VEGF from non-modified, cross-linked and heparinized collagen matrices in the absence and in the presence of varying concentrations of proteinases was then determined by ELISA and liquid scintillation counting. The release behaviour appears to be controlled by both the presence of heparin and the levels of proteinases applied. Experiments with matrices containing radioactively labelled heparin suggest that VEGF release results from the consecutive and simultaneous release of three species of VEGF molecules that differ in their binding affinities to the differently modified collagen matrices. The species binding specifically to heparin most likely accounts for the previously observed increases in angiogenic potential between loading VEGF to non-heparinized and heparinized collagen matrices.

In vitro behavior of a porous TiO2/perlite composite and its surface modification with fibronectin.

In this study, we introduce a porous composite material, termed "Ecopore", and describe in vitro investigation of the material and its modification with fibronectin. The material is a sintered compound of rutile TiO2 and the volcanic silicate perlite with a macrostructure of interconnecting pores. It is both inexpensive and easy to manufacture. We first investigated Ecopore for corrosion and leaching of elements in physiological saline. The corrosion supernatants did not contain critical concentrations of toxic trace elements. In an in vitro model, human primary osteoblasts (HOB) were cultured directly on Ecopore. HOB grew on the composite as well as on samples of its single constituents, TiO2 and perlite glass, and remained vital, but cellular spreading was less than on tissue culture plastic. The pro-inflammatory cytokines IL-1 and TNF-alpha were below detection limits in HOB culture supernatants, whereas IL-6 was detectable on a low level. To enhance cellular attachment and growth, the surface of the composite was modified by etching, functionalization with aminosilane and coupling of fibronectin. This modification greatly enhanced the spreading of HOB, indicated by vital staining and Sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) metabolism assays. HOB grew on the entire visible surface of porous fibronectin-modified composite, expressing alkaline phosphatase, a mature osteoblast marker. We conclude that Ecopore is non-toxic and sustains HOB growth, cellular spreading being improvable by coating with fibronectin. The composite may be usable in the field of bone substitution.

Macrophage migration inhibitory factor is a potential inducer of endothelial progenitor cell mobilization after flap operation.

BACKGROUND: Endothelial progenitor cells (EPCs) promote angiogenesis and play an important role in tissue revascularization and wound healing. Yet, the exact stimuli and mechanisms for the mobilization remain understood poorly. Macrophage migration inhibitory factor (MIF), which is a structurally unique pleiotropic cytokine, has been suggested to play a role in EPC recruitment and thus was a target of this study. METHODS: This study included 20 patients who underwent flap operation. Subjects were divided into 3 groups according to the pattern of flap applied. The number of circulating EPCs and serum levels of MIF or CXCL12 were determined at different time intervals. In vitro chemotaxis experiments using Transwell devices were carried out to test whether MIF promotes the chemotactic migration of EPCs. To underscore functionally the chemotactic potential of MIF toward EPCs in flap patients, the chemotactic effects of serum samples from all groups were also examined in the presence and absence of monoclonal anti-macrophage migration inhibitory factor and anti-CXCL12 antibodies on EPC recruitment using in vitro migration chambers. RESULTS: In flap patients, the number of circulating EPCs and serum levels of MIF but not CXCL12 serum levels were increased markedly compared with preoperative levels at day 1 after operation, especially in the group of free microvascular flaps. Serum levels of CXCL12 in the flap patients were increased only significantly compared with the healthy control group. An analysis between EPCs and MIF revealed a significant correlation, whereas no correlation was observed for CXCL12. MIF exerted a dose-dependent, prochemotactic effect on isolated human EPCs, and serum samples from all flap patients promoted EPC migration. Importantly, this effect was blocked partially by anti-macrophage migration inhibitory factor and to a weaker extent by anti-CXCL12 antibodies. CONCLUSION: We conclude that MIF plays an important role in the mobilization of EPCs, which is dependent on the degree of ischemia. Enhancement by MIF of chemotactic EPCs migration in vitro underpins its proposed in vivo function.

Circulating fibrocytes--biology and mechanisms in wound healing and scar formation.

Fibrocytes were first described in 1994 as fibroblast-like, peripheral blood cells. These bone marrow-derived mesenchymal progenitor cells migrate into regions of tissue injury. They are unique in their expression of hematopoietic and monocyte lineage markers and extracellular matrix proteins. Several studies have focused on the specific role of fibrocytes in the process of wound repair and tissue regeneration. We discuss herein the biology and mechanistic action of fibrocytes in wound healing, scar formation, and maintenance of tissue integrity. Fibrocytes synthesize and secrete different cytokines, chemokines, and growth factors, providing a wound milieu that supports tissue repair. They further promote angiogenesis and contribute to wound closure via pathways involving specific cytokines, leukocyte-specific protein-1, serum amyloid P, and adenosine A(2A) receptors. Fibrocytes are involved in inflammatory fibrotic processes in such diseases as systemic fibrosis, atherosclerosis, asthma, hypertrophic scarring, and keloid formation. Accumulating literature has emphasized the important role of fibrocytes in wound healing and fibrosis. Detailed mechanisms nevertheless remain to be investigated to elucidate the full therapeutic potential of fibrocytes in the treatment of fibrosing disorders and the enhancement of tissue repair.

Improving the angiogenic potential of collagen matrices by covalent incorporation of Astragalus polysaccharides.

The high degree of degradation and the low angiogenic capabilities of temporary tissue substitutes still represent a major challenge in the field of tissue engineering. In an attempt to meet some of these challenges we covalently incorporated Astragalus polysaccharides, a plant extract with angiogenic properties, into collagen matrices. This contribution aims at developing a three-dimensional scaffold for temporarily covering tissue defects in tissue engineering and wound healing e.g. third degree burn wounds. Collagen matrices were modified by incorporating Astragalus polysaccharides (Ap) by means of covalent cross-linking with the watersoluble carbodiimide EDC. Matrices with different Ap/EDC ratios were prepared. After intensively washing of collagen matrices, the Ap modified and non-modified collagen matrices were exposed to the chorioallantoic membrane or implanted into subcutaneous pockets of rats. The number of capillaries in the chorioallantoic membrane in the vicinity of the samples, the hemoglobin contents within the explants and the hydroxyproline contents in the tissues attached to the explants were enhanced. Immunohistochemical evaluation of the explants revealed an increase in the recruitment of CD34+-cells in the modified matrices, indicative of improved angiogenic capabilities. To explore the underlying mechanisms, human umbilical vascular endothelial cells (HUVECs) were exposed to varying concentrations of Ap, collagen I and combinations thereof. The proliferative and chemotactic activities of HUVECs, as well as the protein expression of integrin αV, were strongly enhanced. The modification of collagen matrices with Astragalus polysaccharides of Ap with the cross-linking agent EDC leads to matrices with an increased angiogenic potential. The angiogenic capabilities of the modified collagen matrices appeared to depend on the Ap to EDC ratio. The presented results demonstrate that the incorporation of Astragalus polysaccharides into collagen matrices is an interesting and promising alternative for making wound dressings more angiogenic and improving their capabilities for covering tissue defects.

Macrophage migration inhibitory factor-A potential diagnostic tool in severe burn injuries?

Serum macrophage migration inhibitory factor (MIF) and procalcitonin (PCT) concentrations as well as leucocyte numbers were evaluated in a retrospective study with 23 patients with severe burn injuries. The MIF and PCT concentrations as well as the number of leucocytes (LEU) were monitored over a period of 5 days. The total body surface area (TBSA) and sepsis-related organ failure assessment (SOFA) scores were also evaluated. The MIF, PCT concentrations and leucocyte counts were profoundly increased in all patients with severe burn wounds. At the time of admission into the intensive care unit, no significant differences were observed for the MIF and PCT levels between patients with a TBSA<60% (Group 1) and patients with a TBSA>60% (Group 2). After 48 h, however, the MIF and PCT levels reached very high levels in a subgroup of the patients, whereas these levels became normal again in other subgroups. The group of patients with a TBSA>60% was, therefore, subdivided in three groups (subgroups 2a-c). The MIF and PCT data pairs in these subgroups appeared to correlate in an inhomogeneous manner. These levels in the subgroup 2a (i.e., lethal within 5 days) were strongly elevated over those observed in Group 1 (TBSA<60%) and highly increased concentrations of both MIF and PCT correlated with lethal outcome. The combined determination of MIF and PCT might, therefore, be useful to discriminate between post-burn inflammation and systemic inflammatory response syndrome (SIRS) or sepsis with lethal outcome.

Modification of collagen matrices for enhancing angiogenesis.

The vascularization of engineered tissues in many cases does not keep up with the ingrowth of cells. Nutrient and oxygen supply are not sufficient, which ultimately leads to the death of the invading cells. The enhancement of the angiogenic capabilities of engineered tissues therefore represents a major challenge in the field of tissue engineering. The immobilization of angiogenic growth factors may be useful for enhancing angiogenesis. The most potent angiogenic growth factor specific to endothelial cells, vascular endothelial growth factor (VEGF), occurs in several splice variants. The variant with 165 amino acids both has a high angiogenic activity and a high affinity for heparin. We therefore incorporated heparin molecules into collagen matrices by covalently cross-linking them to amino functions on the collagen. Physical binding of VEGF to the heparin may then prevent a rapid clearance from the implant, while the release rate may become coupled to the degradation of the collagen matrix. The modified matrices were characterized by determination of the extent of the heparin immobilization, the in vitro degradation rate by collagenase. For testing the angiogenic properties, non-modified and heparinized collagen specimens were--either loaded with VEGF or non-loaded--subcutaneously implanted on the back of rats. Specimens were explanted after varying periods of implantation, the dry weights and the hemoglobin contents, as well as immunostained histological sections were evaluated: heparinized collagen matrices loaded with VEGF are vascularized to a substantially higher extent as compared to non-modified matrices.