Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

Therapeutic angiogenesis may be applied in medical conditions to promote stimulation of angiogenesis. Angiogenesis is a multistep process, which includes endothelial cell proliferation, migration, and tube formation, which is mediated by various angiogenic polypeptides. Thus, studies that elucidate the cellular mechanisms involved in these processes are necessary to develop novel therapeutic strategies. This study investigated the in vitro effects of the pro-angiogenic factors, insulin-like growth factor-1 (IGF-1) and/or chemokine (CC motif) ligand 2 (CCL2), on endothelial cells. Flow cytometry analysis showed that IGF-1 and CCL2 treatment did not interfere with IGF-1 receptor (IGF-1R) expression, but CCL2 treatment increased CCL2 receptor (CCR2) expression. Immunofluorescence analysis revealed that the IGF-1/CCL2 combination induced a greater increase in fibronectin deposition, but the treatments did not alter the expression of the fibronectin receptors, CD49e and CD44. The interaction of fibronectin with cytokines demonstrated that IGF-1/CCL2 promoted changes in intermediate F-actin remodeling that may result in increased endothelial cell adhesion and cell migration mediated by fibronectin. Furthermore, IGF-1/CCL2 stimulated endothelial cells, grown on fibronectin, to form capillary-like structures and intercellular lumina with greater luminal area. These data suggest that IGF-1/CCL2 combination and a fibronectin matrix may contribute to the angiogenesis process to stimulate adhesion, migration, and tube formation by endothelial cells as a result of F-actin remodeling.


Introduction
The endothelium is a monolayer of cells lining the interior of the blood and lymphatic vessels. This cellular layer is attached to the basal membrane and participates in the exchange of materials between blood and tissues. Endothelial cells have essential activities in the control of vascular functions and play an important role in the formation of new blood vessels and penicillin, and 100 U/mL streptomycin (all from Invitrogen, Carlsbad, CA, USA) and were cultured at 37°C in a fully humidified atmosphere flushed with 5% CO 2 .

Proliferation assay
tEnd.1 cells (3 × 10 4 ) were seeded in 6-well culture plates in RPMI 1640 complete medium for 16 h for cellular adhesion. After this period, the cells were washed with phosphate buffered saline (PBS) and were treated with recombinant mouse insulin-like growth fator-1 (IGF-1) (Sigma-Aldrich, St Louis, MO, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 8 h. After treatment, cells were counted using a hemocytometer.

Immunocytochemistry
After treatment with IGF-1 and/or CCL2 for 24 h, cells were subjected to an indirect immunofluorescence assay as previously described [31]. Samples were washed with PBS (Sigma-Aldrich), followed by treatment with 1% bovine serum albumin (BSA) (Sigma-Aldrich). The tEnd.1 cells were incubated with a primary anti-FN antibody (rabbit, 1:50; Sigma-Aldrich) for 1 h at room temperature. After additional washes, cells were incubated with the secondary antibody, goat anti-rabbit-FITC conjugated (1:200, Sigma-Aldrich) for 45 min at room temperature. Immunostained samples were analyzed by fluorescence microscopy (Nikon Eclipse 50i; Nikon Instruments Inc., Chicago, IL, USA). Negative controls, in which primary antibodies were replaced by unrelated immunoglobulins or in which the secondary antibody was used alone, did not generate any significant immunolabeling. Quantitative fluorescence analyses were performed by transforming specific staining in pixels and by dividing the total pixel numbers by the area analyzed, obtaining the numbers of pixels/μm 2 .

Cytoskeleton staining assay
After treatment with IGF-1 and/or CCL2 for 24 h, 2 × 10 3 cells were seeded in a 24-well plate with round glass coverslips previously coated with 10 μg/mL of FN (Sigma-Aldrich) or 10 μg/mL of BSA and incubated for 16 h. Cells were washed with PBS, fixed, and permeabilized for 5 min with 4% paraformaldehyde in PHEM buffer (60mM PIPES, 2mM HEPES, 10mM EGTA, and 2mM MgCl 2 , Sigma-Aldrich) containing 0.5% Triton X-100 and 5% sucrose (Sigma-Aldrich). Post-fixation was performed for 15 min with the same buffer without Triton X-100. After washing, tEnd.1 cells were stained with phalloidin-Alexa 488 (Molecular Probes, Eugene, OR, USA) for 1 h. Cell spreading was estimated by the area occupied by the cell, using the Image J software (NIH, Bethesda, MD, USA). A confocal inverted microscope (LSM-510, Zeiss, Göttingen, Germany) was used for observation with a 63× objective.

Cellular adhesion assay
In a 96-well plate previously coated with 0.1% BSA or 10 μg/mL FN, 5 × 10 4 cells treated with IGF-1 and/or CCL2 for 24 h were added in each well to measure the cellular adhesion. Non-adherent cells were washed away 1 h later. Adherent cells were fixed with formaldehyde and stained with crystal violet (Sigma-Aldrich). Spectrophotometer readings were taken at an absorbance of 540 nm [32].

Endothelial migration assay
Migration of tEnd.1 was assessed using a transwell system, which consists of inserts with polycarbonate membranes having a diameter of 10 mm and a pore size of 8.0 μm (Corning Costar, Cambridge, MA, USA). A suspension of 2 × 10 5 tEnd.1 cells was added to the upper chamber of inserts previously coated with 0.1% BSA or 10 μg/mL FN, and IGF-1 and/or CCL2 were used as chemotactic factors. After 6 h of migration, migrant cells in the bottom chamber were fixed, stained, and counted using methods previously described [33].

Cellular morphological analysis
tEnd.1 cells (5 × 10 3 ) were cultured on round slides in 24-well plates. The culture was treated with IGF-1 and/or CCL2 for 24 h in RPMI 1640 with 2% FBS. Cells were then fixed with methanol and stained with Giemsa. Subsequently, the coverslips were mounted on permanent slides and analyzed by light microscopy. Photographs were obtained by using a Nikon DS-Ri1 camera coupled to a Nikon Eclipse 50i microscope (Nikon Instruments Inc.).

Capillary-like network formation assay
The ability of tEnd.1 cells to form capillary-like structures was evaluated on surfaces coated with 0.1% BSA or 10 μg/mL FN, as described previously [34] with some modifications. The FN coating was prepared on round glass coverslips in a 24-well plate and the cells were plated. On the fourth day, the culture medium was changed and treatment was renewed. On the eighth day, cells were fixed and stained, and the coverslips were mounted on permanent slides and analyzed by light microscopy. Luminal area and the formation of capillary-like structures were measured by DP2-BSW software (version: Olympus Soft Imaging Solution GmbH, Munster, Germany).

Statistical analysis
The data obtained were analyzed using one-way or two-way ANOVA, followed by Bonferroni's post-test. The values are presented as the mean ± standard error of the mean (SEM) and considered significant when p 0.05.

CCL2 increased CCR2 expression in endothelial cells
IGF-1 and CCL2 concentrations were determined on the basis of the murine thymic endothelioma cell line (tEnd.1) proliferation and cell viability. A significant increase in cell number was observed in presence of IGF-1 at 10, 50, and 100 ng/mL (Fig. 1A). Treatment with 10 ng/mL of CCL2 significantly stimulated endothelial cell viability (Fig. 1B). The influence of IGF-1 and CCL2 on the biological properties of endothelial cells is mediated via their respective receptors. The effect of IGF-1 and/or CCL2 on the expression of their respective receptors was analyzed by flow cytometry. tEnd.1 cells expressed both receptors. A high percentage of cells expressed IGF-1R (82% ± 0.156%) and a lower percentage of cells expressed CCR2 (11% ± 0.433%) (Fig. 1C). IGF-1 and/or CCL2 treatment did not interfere with the percentage of cells that expressed IGF-1R. However, the percentage of cells that expressed CCR2 increased significantly (73%) after treatment with CCL2 alone than that of the untreated control, IGF-1, and combined IGF-1/CCL2 treated cells (Fig. 1C).
IGF-1/CCL2 combination augmented fibronectin deposition by tEnd.1 cells tEnd.1 cells expressed IGF-1 and CCL2 receptors and could induce matrix deposition. As a major structural component of resistant vessels, the extracellular matrix (ECM) plays a substantial role in the maintenance of vessel integrity [35]. Therefore, endothelial cell properties are not only regulated by cytokines, but also in conjunction with ECM molecules. The effect of IGF-1 and/or CCL2 on ECM deposition was determined by FN immunostaining. Qualitative analysis showed that IGF-1 and/or CCL2 treatment increased FN deposition ( Fig. 2A). This was confirmed by quantitative fluorescence intensity, which demonstrated a significant increase in FN accumulation. In addition, FN deposition after IGF-1/CCL2 combination treatment was significantly higher than in the control and single treatments (Fig. 2B). The interaction of FN with cell surface receptors, usually through binding of the α5β1 integrin receptor, induces FN activation [36]. Therefore, the effect of IGF-1 and/or CCL2 on the expression of FN receptors CD49e (α5/VLA5) and CD44 in tEnd.1 cells was analyzed by flow cytometry. The results indicated that a high percentage of cells expressed CD49e (97% ± 0.092%) and CD44 (99% ± 0.026%) (Fig. 2C). However, IGF-1 and/or CCL2 treatment did not alter the percentage of cells expressing these receptors as compared to the untreated control cells (Fig. 2C).

IGF-1/CCL2 combination promoted F-actin cytoskeleton organization
To investigate whether cytokines and FN interact to stimulate actin cytoskeleton organization, the effect of IGF-1 and/or CCL2 on F-actin was determined on BSA-or FN-coated surfaces, followed by direct staining with phalloidin. Ours results showed that F-actin cytoskeleton on FN matrix was more spreading (2.2×) than that of the cells grown on BSA without treatment. Moreover, IGF/CCL2 treatment of cells grown on the FN matrix increased the cell number (1.6×) and induced larger (2.6×) lamellipodia than those of the cells grown on BSA coating (Fig. 3A). IGF-1 treatment resulted in a more elongated cytoskeleton, while IGF-1/CCL2 combination treatment increased number and area of lamellipodia.

IGF-1/CCL2 combination increased tEnd.1 cell adhesion and promoted migration
Intermediate levels of cytoskeletal linkage proteins are associated with maximal migration [37]. Thus, we evaluated whether the cytoskeletal organization promoted by the IGF-1/CCL2 combination treatment of tEnd.1 cells grown on the FN matrix would affect their adhesion and migration. The adhesion capacity was determined through 1 h adherence on BSA or FN-coating surfaces after treatment with IGF-1 and/or CCL2. tEnd.1 cells presented a lower adherence to the BSA-coated surface and only the IGF-1/CCL2 combination increased tEnd.1 cell adhesion (Fig. 3B). However, all treatments significantly increased the adhesion of tEnd.1 cells on the FN-coated surface. Furthermore, CCL2 and the IGF-1/CCL2 combination significantly increased adhesion as compared to the control and IGF-1 treatment alone (Fig. 3B). To assess the effect of IGF-1/CCL2 combination on cell motility in vitro, transwell chambers were coated with BSA or FN and cell migration was determined by the number of migrating cells that adhered to the bottom of the transwell membrane after chemotactic IGF-1 and/or CCL2 treatment (Fig. 3C). The chemotactic response of tEnd.1 cells was significant only when cells were   IGF-1 and Chemokine on Endothelial Cells stimulated by the IGF-1/CCL2 combination on the BSA-coated surface and this response was higher than that of the single treatments (Fig. 3D). However, tEnd.1 cells significantly migrated after stimulation with all treatments on the FN-coated surface as compared to the control (Fig. 3D). In addition, cells stimulated with the IGF-1/CCL2 combination showed a peak of migration that was statistically significant compared to the control group and single treatments, showing a synergistic effect of IGF-1 and CCL2 on cell migration (Fig. 3D).

IGF-1/CCL2 stimulated intra-and intercellular lumen formation by tEnd.1 cells
Because the IGF-1/CCL2 combination stimulated cell migration, we investigated whether cytokines could modulate tubulogenesis in vitro. The morphological analysis of tEnd.1 cells demonstrated that the combined IGF-1/CCL2 treatment for 24 h promoted intracellular lumen formation in the absence of ECM (Fig. 4A). When cells remained in culture for a longer period (8 days) on BSA-or FN-coated surfaces, they showed the ability to form more complex structures similar to the capillaries (Fig. 4B). The number of capillary-like structures formed after treatment with IGF-1 and/or CCL2 was statistically higher than the untreated cells grown on both BSA-or FN-coated surfaces (Fig. 4C). The luminal area of capillary-like structures was measured and only cells treated with the IGF-1/CCL2 combination showed a significant increase in luminal area in cells grown on BSA-coated slides, whereas luminal area was significantly increased by all treatments in cells grown on FN-coated slides (Fig. 4D).

Discussion
Therapeutic angiogenesis refers to the beneficial application of angiogenesis stimulation in medicine. This therapy may be achieved by administering pro-angiogenic polypeptides and can be employed in wound healing, fracture repair, reconstructive surgery, and collateral vessel formation [38]. Furthermore, the combination of growth factors could have important implications for the treatment of severe arterial insufficiency in patients with diseases that are not amenable to direct revascularization [39]. This study demonstrated that a combination of IGF-1 and CCL2 could induce the migration of tEnd.1 cells in vitro, contributing to tubulogenesis and lumen formation with greater average area on a FN matrix.
As previously observed, in human umbilical vein endothelial cells [20,21,40], tEnd.1 cells expressed IGF-1 and CCL2 receptors. However, tEnd.1 cells express more IGF-1 receptors than CCR2. Low CCR2 expression in tEnd.1 cells was also demonstrated in bEnd.3 cells in which the level of expression of CCR2 mRNA under resting conditions might be the result of specific in vitro conditions, which have been shown to be critical for the downregulation of CCR2 in monocytes or macrophages [41,23]. CCL2 alone significantly stimulated CCR2 expression, while IGF-1/CCL2 treatment did not affect CCR2 expression. Yet, it is conceivable that the mechanisms controlling the expression of this molecule in tEnd.1 cells are different depending on the stimulus. Thus, more studies on intracellular signaling are necessary to explain the mechanisms of interaction between IGF-1/CCL2 in endothelial cells.
The interaction between cells and extracellular components is essential in determining cellular behaviors in tissues [42]. Several ECM compounds act on the endothelial function together with cytokines present in the matrix. The IGF-1 and/or CCL2 effect on increased FN deposition was also shown in bEnd.3 cells and epithelial cells [23,43]. However, IGF-I did not affect FN production in human corneal epithelial cells (HCECs) [44]. Differences in FN expression after IGF-1 treatment could be justified by the accumulation and/or binding of FN molecules to the FN receptors on the cell surface [45]. Considering that the expression of FN receptors was not affected, it will be useful to evaluate the expression of integrin subunit αv, which, through integrin αvβ3, promotes recycling to focal contacts required for persistent migration [46,47] and tyrosine phosphorylation of focal adhesion kinase, which plays an important role in the regulation of cell morphology and in promoting cell migration events [48,49].
Integrin-ligand binding triggers actin cytoskeleton organization at specific sites on the surface membrane to facilitate cell movement or maintain tissue stability [50]. The interaction between the ECM and IGF-1 or CCL2 on the cytoskeleton of tEnd.1 cells cultured on a FN-rich matrix was similar to that observed in previous studies in epithelial cells and bEnd.3 cells [51]. The F-actin reorganization promoted by IGF-1/CCL2 association induced more changes in tEnd.1 cells, stimulating active cytoskeleton reorganization and elongated configuration, to stimulate the formation of microspikes, i.e., very short filopodia almost completely embedded in the cell cortex or leading edge [52]. This F-actin remodeling likely affected the adhesion and had an effect on tEnd.1 cell migration on the FN matrix. A significant peak of migration was observed when tEnd.1 cells were treated with IGF and CCL2, which probably means a change in the cell behavior. The maximal migration may be justified by active changes during cytoskeleton remodeling because lamellipodia and filopodia are essential for cell motility and substrate adhesion [53]. In addition, elongated cytoskeleton configuration mimics the plane configuration, which increases sensitivity to specific growth factors during vasodilatation [54,4].
Angiogenesis is defined as the formation of new blood vessels from pre-existing vessels via sprouting [55]. Sprouting endothelial cells assemble into solid cords, which undergo tubulogenesis to form vessels with a central lumen [56,57]. In this study, we showed that IGF-1/ CCL2 combination treatment of tEnd.1 cells led to intracellular lumina and coalescent vacuoles, driving vascular lumen formation. Previous studies have demonstrated that intracellular and intercellular fusion of endothelial vacuoles drives vascular lumen formation [58,59]. IGF-1 and CCL2 also possess the ability to incorporate vascular networks [16,23]. However, the luminal area of capillary-like structures on FN matrix was accentuated by the IGF-1/CCL2 combination treatment, as previously described for the combination of VEGF and basic fibroblast growth factor on angiogenesis. The combination of growth factors stimulated significantly greater and more rapid augmentation of collateral circulation, resulting in superior hemodynamic improvement [39]. In addition, when used together, IGF-1 and VEGF exerted complementary therapeutic effects in post-infarction heart failure [27].
Potential angiogenic activity of IGF-1 associated with CCL2 in the presence of FN matrix indicates new properties for pro-angiogenic peptides employed in therapeutic angiogenesis. This underscores the importance of further studies to elucidate the possible mechanisms involved in the combined effect of IGF-1 and CCL2 on endothelial cells.