2.1. HUVECs Cultivation on Type I Collagen and Fibronectin
The primary purpose of this study is to explore the respective effect of soluble and bound VEGF on endothelial cells. Therefore, instead of using the commercial matrix (such as Matrigel®), which already contains growth factors, pure collagen type I and fibronectin were used to make the in-house biogel with a definite combination. Type I collagen as the primary substrate provided the endothelial cells with attachment sites when the cells moved. Fibronectin attached to the collagen surface provided cell attachment and binding sites for soluble VEGF initially contained in the culture medium to form bound VEGF. The cell suspension was added to a 12-well plate coated with collagen with or without fibronectin. The initial number of endothelial cells was 80,000 cells per well, and the cell distribution images were recorded every 6 h.
The endothelial cells were cultivated under four conditions, considering whether fibronectin was coated on the collagen and whether VEGF was added to the culture medium. The endothelial cells in the four cases formed the network to different extents. The results are as shown in Figure 1. In the two cases with no VEGF but with or without fibronectin addition (−F−V and +F−V), HUVECs started the morphological change from round to elongated shape at about 6 h, and the change became evident at 12 h. The cells in these two cases were found to undergo a rapid cell death process that the number of adherent endothelial cells continually decreased. At 24 h of incubation, only a small number of cells remained in the sight field. It is worth mentioning that the cells could express the morphological change even without VEGF added.
Figure 1. HUVECs morphological forms and cellular networks in different cell culture conditions: without fibronectin (−F), with fibronectin (+F), without VEGF (−V), and with VEGF (+V). Black arrows in the enlarged photos indicate the cells with morphological change into the elongated shape, while white arrows indicate the cells still in the original cobblestone shape.
Under the culture condition without fibronectin but with VEGF (−F+V), many cells with morphological changes appeared at 6 h. However, the cell network could not ultimately form afterward. In the +F+V case, the beginning of morphological change occurred a bit later than in the –F+V case. The originally round-shaped cells became elongated, and the dividing boundary for adjacent cells became blurred at 12 h. These characteristics were more evident subsequently, which shows that VEGF stimulated the endothelial cells continuously. The cell-free area in the visual field became apparent as a result of cell aggregation. The cell colony evolved in the direction of forming the capillary-like network.
2.2. Effects of Soluble and Bound VEGF
Comparison between the –F+V and +F+V cases, as shown in Figure 1, reveals that the endothelial cells begin morphological changes earlier; however, a cell network could not form afterward if the biogel had no fibronectin. In contrast, although the cell commenced morphological change somewhat later in +F+V, the cells continued to migrate and aggregate into a cellular network. This result indicated that soluble and bound VEGF might have different effects on cell behavior. However, in addition to binding VEGF, fibronectin could bind cells through its integrin-binding domain. To clarify whether it was the cell-binding or VEGF-binding to the fibronectin responsible for the different cell behaviors, we conducted a series of experiments using the synthetic VEGF-blocking-peptide (VBP). The sequences of VBP were designed according to the fibronectin-binding domain of VEGF (exon-7). This peptide was added to the fibronectin-coating wells before adding the HUVEC suspension to shield the growth factor-binding domain of fibronectin. Because the bound VEGF was prevented from forming, the added VEGF would remain in the soluble form.
The VPB shielding effect was assessed using fluorescence immunoassay, where a FITC-conjugated anti-VEGF antibody was used to monitor the VEGF. Results are as shown in Figure 2 for four different cell culture conditions: without VBP (-P), with VBP (+P), without VEGF (-V), and with VEGF (+V). The darker fluorescence response in the case of +P+V than in –P+V indicated that VBP could avoid VEGF from biding fibronectin. The two subsets of -P-V and +P-V showed that even in the cases with no adding VEGF in the culture medium, the fluorescence response could still be detected, albeit the responses were weaker than the counterpart cases of -P+V and +P+V, respectively. The results indicated that HUVECS could secrete VEGF. This autocrine signaling explained why HUVECs presented a morphological change in the cases without adding VEGF from outside. However, because the cells experienced rapid cell death in the two cases without VEGF added, adding VEGF was essential to cell viability to prevent the cell un-attachment and cell death during the network developing process.
Figure 2. Bound VEGF location after 6 h of incubation in different cell culture conditions: without VBP (−P), with VBP (+P), without VEGF (−V), and with VEGF (+V). A fluorescent signal from the primary antibody against VEGF was detected on the cell membrane and biogel surface. The VBP shielding effect avoided VEGF from binding biogel resulting in the darker VEGF field in both the +P−V and +P+V cases.
We further tested the VBP effect on cells by changing dose usage. The number of junctions quantitatively indicated the integrity and complexity of the capillary-like network. The results are displayed in Figure 3, which shows HUVECs could differentiate into a network and established an average of 140 junctions per view field for the cases with no or adding a small amount of VBP. The junction number dramatically decreased with the peptide concentration larger than 0.0125 ng/μL, but the cells kept attaching to the biogel. The results showed that the usage of VBP interfered with the regulating effect of VEGF and affected the integrity of network structure but not the cell adhesion.
Figure 3. The effect of VBP dosage on the cell network integrity. The average number of network junctions per view field served as a quantitative indicator of network integrity. Cell cords recognized by ImageJ software are shown, where the pink lines present the segments (connections between two junctions), the green lines the branches (connections between junction and node), and the blue lines the isolated elements (connections between two nodes). Data were collected after 24 h of cell culture and averaged with three repeated experiments.
Based on the optimum culturing environment (+F+V), as shown in Figure 1, we tested on HUVECs the stimulating effect of soluble and bound VEGF, respectively, by adding 400 μL DPBS per well with 0.125 ng/μL dissolved VBP. The cell culture images are shown in Figure 4. In the -P situation, i.e., without VBP addition, HUVECs showed the change in morphology and subsequently formed the capillary-like network. In the +P situation, i.e., with VBP addition, HUVECs could not ultimately create the cell network, albeit the cells presenting morphological change earlier than the -P case.
Figure 4. HUVECs distribution with and without VBP addition. In the -P case, which means without the VBP addition, HUVECs showed the change in morphology at 12 h with some cells remaining in the cobblestone shape and subsequently formed the capillary-like networks at 18 h. In the +P case, which means with the VBP addition, most HUVECs showed the morphological change at 12 h; however, the network could not form afterward.
More details about the network integrity and complexity are summarized in Figure 5. The number of junctions, the number of segments, and the total segment length were measured as representative indicators. If no VBP was added (−P+V), these three values remain high for all the observation times, indicating that a cell network was developed to a high degree. In contrast, with VBP added (+P+V), these three values decreased significantly after 18 h, indicating no cell network could ultimately form with VBP added. Because adding VBP (+P) prevented VEGF binding onto fibronectin, the added VEGF would remain soluble. The soluble VEGF was shown incapable of sustaining the network formation.
Figure 5. Junction number, segment number, and total segment length of the capillary-like network. The higher value of these three indicators, the more integrity of the network. All experiments were repeated five times. Data are presented as mean SD. The Student’s t-test was performed for statistical evaluation: * = p < 0.05, ** = p < 0.01, and ns = non-significant. The asterisk above the -P+V charts presents the p-value between the −P+V and +P+V cases at the same observation time.