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Promotion of Angiogenesis by an Artificial Extracellular Matrix Protein Containing the Laminin-1-Derived IKVAV Sequence Makiko Nakamura,† Kumiko Yamaguchi,‡ Masayasu Mie,† Makoto Nakamura,§ Keiichi Akita,‡ and Eiry Kobatake*,† Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Graduate School of Medicine and Dentistry, Tokyo Medical and Dental University, and Department of Biomedical Engineering, University of Toyama. Received March 20, 2009; Revised Manuscript Received July 7, 2009
The laminin-1-derived IKVAV sequence is known for its angiogenic function. We previously developed artificial extracellular matrix (ECM) proteins containing the IKVAV sequence. They were designed to have collagenbinding activity and active functional units that promote network formation of vascular endothelial cells. The resultant fusion protein, called EREI2CBD, was confirmed to bind to collagen type I and promote tubular network formation of endothelial cells cultured in collagen gel in Vitro. In this study, EREI2CBD was applied to the chick chorioallantoic membrane (CAM) assay to investigate in ViVo angiogenic activity. The CAM assay results showed that EREI2CBD caused the number and area of vascular branches to be increased. The constructed fusion protein and the engineering strategy of designing multifunctional ECM proteins support current tissue engineering techniques.
INTRODUCTION In recent years, much attention has been focused on tissue engineering techniques to provide artificial tissue substitutes as an alternative for donor tissue and organs. Scaffold material is one of the essential factors for tissue engineering to support cell attachment and provide necessary signals for cell growth and organization. In a previous study, we developed artificial extracellular matrix (ECM) proteins designed to have a stable structural unit and active functional units that promote cell attachment and network formation of vascular endothelial cells (1). To achieve that purpose, we constructed new fusion proteins with multiple activities of (i) cell adhesion, (ii) promoting tubular network formation, (iii) structural support, and (iv) affinity to collagen. As a functional part of these fusion proteins, the Ile-Lys-ValAla-Val (IKVAV) peptide sequence, located in the laminin R1 chain, was chosen as it has a variety of activities including promoting cell adhesion, neurite outgrowth (2), and angiogenesis (3, 4). An elastin-derived (APGVGV) motif was also adapted to produce a stable matrix protein. This polypeptide module had been studied in our laboratory as an artificial matrix protein and enabled the short active peptides such as IKVAV or GRGDS to work efficiently as a scaffold protein (5-7). The resultant protein was illustrated in Figure 1, shown as EREI2. The EREI2 fusion protein was constructed with recombinant DNA technology to conjugate the functional peptide sequences and the rigid poly (APGVGV) module. EREI2 was designed to be two repeats of (APGVGV)12, RGD, (APGVGV)12, and the IKVAV sequence because the activity of the smaller molecule (EREI) was shown to be less than EREI2 (1). As the GRGDS-IKVAV conjugated peptide was reported to have stronger cell-adhesive property than the IKVAV sequence (8), * Author for correspondence. 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.: +81-45-924-5760; Fax: +81-45-924-5779; E-mail:
[email protected]. † Tokyo Institute of Technology. ‡ Tokyo Medical and Dental University. § University of Toyama.
Figure 1. Construction of artificial ECM. (A) E12 means 12 repeats of the elastin-derived APGVGV sequence. R and I indicate the GRGDS sequence derived from fibronectin and the laminin-derived CSRARKQAASIKVAVSADR sequence, respectively. Collagen-binding domain (CBD) was derived from fibronectin and fused to the C-terminus of the engineered protein. CBD and CBD-fused EREI2 had a histidine tag on their N-terminus.
GRGDS sequence was incorporated into EREI2 to enhance the interaction between cells and the scaffold. Furthermore, we introduced collagen-binding activity into the designed ECMs in order to enable the accumulation of ECM onto specific scaffold materials. Since collagen is biocompatible and is easily processed into several shapes such as membrane, gel, or sponge, it is considered to be a useful material for tissue engineering. Collagen-immobilized forms of engineered ECMs have the potential to provide a safe and functional angiogenesis model for enhancing the activity of necessary signals locally and effectively. Here, we focused on the collagen-binding domain (CBD) of fibronectin (9). CBD sequences are widely used as fusion partners for chimera proteins, as they provide the option for localized application on collagen (10-12). The resultant collagen-specific, immobilized fusion ECM, called EREI2CBD, promoted angiogenic properties in a collagen type I gel model (1). Therefore, EREI2CBD promoted capillary formation of endothelial cells and revealed its potential to produce multifunctional artificial ECM. However, the in ViVo angiogenesis process was dissimilar to an in Vitro collagen gel model, and therefore, the application of EREI2CBD under in ViVo conditions required further investigation. In the present study, the angiogenic activity of EREI2CBD was determined using a chick chorioallantoic membrane (CAM) assay. The CAM assay is a simple technique for investigating
10.1021/bc900126b CCC: $40.75 2009 American Chemical Society Published on Web 08/18/2009
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angiogenic behavior in ViVo (13-15). EREI2CBD was immobilized on a permeable collagen membrane and the membrane was applied onto CAMs with a glass coverslip. Angiogenic activity, that is, growth of capillary vessels, was then investigated.
EXPERIMENTAL PROCEDURES Materials. The artificial fusion ECMs, EREI2, EREI2CBD, and CBD were prepared as described in a previous study (1). The 19-mer peptide, CSRARKQAASIKVAVSADR, which we refer to as the IKVAV peptide, was synthesized by a solidphase method using Fmoc. Permeable atelocollagen membrane (MEN-01) was purchased from Koken Co. Ltd. (Tokyo, Japan). All other chemicals were of analytical grade. Preparation of Artificial Proteins Immobilized on a Collagen Membrane. Permeable atelocollagen membrane was cut into appropriate sizes, soaked with phosphate buffered saline (PBS), and put onto 13-mm-diameter glass coverslips (Thermo Fisher Scientific Inc.). After drying at room temperature, 2 µg of artificial proteins in PBS was added onto the membrane and dried again at room temperature. For the experiment described in the Results section “Concentration-dependent angiogenic behavior of EREI2CBD”, EREI2CBD was applied at 0.5-2 µg. Negative control samples contained 20 µL of PBS only. Membranes adhering onto coverslips were stored at 4 °C until use. Determination of Diffusion of CBD-Fused ECMs from the Collagen Membrane. Atelocollagen membranes immobilized with artificial proteins such as EREI2 and EREI2CBD were prepared as described above. Membrane-adhering coverslips were placed into each well of 24-well culture plates (Falcon), and 300 µL of PBS was added. After 3 h of incubation at 37 °C, 150 µL of supernatant PBS was sampled. Whole protein concentrations desorbed into bulk PBS were determined with the micro BCA protein assay kit (Pierce). Concentrations were calculated after subtraction of negative control membranes soaked with PBS only. Each assay was repeated three times. Chick Chorioallantoic Membrane Assay. The chick chorioallantoic membrane (CAM) assay was performed using embryonated eggs. On embryonic day 3, 4 mL of ovalbumin was removed from each egg. After opening windows on embryonic day 10, glass coverslips with the protein-treated atelocollagen membrane were placed membrane side face-down onto the surface of the CAM and photographed. Two days later, glass coverslips were cut off together with egg membranes and photographed after washing with PBS. The mortality rate of the embryos supplied with each sample was calculated as the ratio of dead embryos to the sum of three repeated experiments. Quantitative evaluation of vessel areas was performed by determining the number of black pixels per field using Scion image software (Scion Corp.) and by manually counting the number of vessel branches. These numbers were calculated from the whole area of glass coverslips pictured at 0 and 48 h. Each assay was repeated three times using 8 to 12 eggs for each treatment. Statistical Evaluation. Statistical significance of the results was determined using Student’s t test. A value of P < 0.05 was considered to be significant. Mean ( standard deviation (s.d.) values of three experiments are presented.
RESULTS Immobilization of CBD-Fused ECM to a Collagen Membrane. The previously constructed artificial ECM proteins (1) are illustrated in Figure 1. The EREI2 fusion protein consisted of two repeats of (APGVGV)12, RGD, (APGVGV)12, and the IKVAV sequence. Fibronectin-derived collagen-binding domain (CBD) and CBD-fused EREI2 were designed to immobilize the
Figure 2. Determination of diffusion of ECMs from the atelocollagen membrane. Two micrograms of constructed ECMs were air-dried onto atelocollagen membranes and incubated in PBS for 3 h at 37 °C. Results show the diffused protein concentration into PBS. Each experiment was repeated 3 times. Error bars represent the standard deviation of the mean. **P < 0.01. Table 1. Mortality Rate of Chick Embryos Treated with Engineered Proteins PBS IKVAV pep. CBD EREI2CBD
amount (µg)
mortality rate
2 2 0.5 1 2 5
0/33 0/26 1/26 2/33 1/29 0/31 8/30
fusion construct onto the collagen-based material. CBD-fused ECM and CBD also contain a histidine tag sequence (His6) at their N terminals to aid purification. To confirm the localization of CBD-fused ECM on collagen membranes, EREI2 and EREI2CBD were fixed onto the membrane by air-drying, and then the amount diffused from the membranes was determined after 3 h of incubation in PBS. The results are shown in Figure 2. A higher protein concentration was detected in PBS from the EREI2-immobilized membrane than that of EREI2CBD, suggesting that more EREI2CBD remained fixed onto the collagen membrane than EREI2. We then confirmed EREI2CBD binding to the collagen membrane and determined its potential to function locally under in ViVo conditions. Concentration-Dependent Angiogenic Behavior of EREI2CBD. For the assessment of angiogenic behavior of EREI2CBD, serial amounts were immobilized on the collagen membrane and applied to the CAM assay. The CAM assay was performed as described in previous reports with some modifications for the collagen membrane (16, 17). The mortality rate of chick embryos supplemented with EREI2CBD is shown in Table 1. Since the negative control PBS samples survived the 2 day incubation, it was confirmed that our CAM assay protocol itself did not influence chick embryo survival. Table 1 also indicates that EREI2CBD did not significantly affect embryo mortality at concentrations ranging from 0.5 µg to 2 µg. In contrast, when 5 µg of EREI2CBD was applied, nearly 30% of embryos died after 2 days. The mortality table shows that high amounts of EREI2CBD could not be applied to our CAM assay and 5 µg of EREI2CBD was the limit for chick embryo survival. All subsequent experiments on capillary vessel formation were performed at concentrations ranging from 0.5 µg to 2 µg. In our present study, angiogenesis was evaluated with two indexes, the vessel area and the number of vessel branches. The results are shown in Figure 3. Increases of vessel area in chick embryos were calculated from the black pixel ratio at 48 h compared to 0 h (Figure 3A). This indicated that 0.5 and 2 µg of EREI2CBD significantly increased vessel area. Vascular
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Throughout the results, the angiogenic activity of EREI2CBD was confirmed. Although CBD showed some angiogenic properties such as increased vessel area, EREI2CBD was superior to CBD in promoting vessel sprouting. Therefore, the CBD-fused form of EREI2 is a promising approach for promotion of angiogenesis.
DISCUSSION
Figure 3. Angiogenic behavior of EREI2CBD. EREI2CBD was airdried at the indicated concentrations onto atelocollagen membranes and applied to the CAM assay. Zero means the control sample with PBS alone. (A) Quantitative evaluation of vessel areas by determining the number of black pixels per field. The number of black pixels was calculated within the whole area of glass coverslips imaged at 0 and 48 h. Results show the ratio of pixel counts at 0 to 48 h. (B) The number of vessel branches after 48 h of incubation. Each experiment was repeated 3 times. Error bars represent the standard deviation of the mean. NS, not significant; *P < 0.05, **P < 0.01; ***P < 0.001 compared to the PBS control sample (referred to as zero).
sprouting, an important factor for the angiogenic process, was evaluated by counting the number of vessel branches. The number of branches after 2 days was increased in a concentration-dependent manner and was significantly increased in the case of 2 µg EREI2CBD (Figure 3B). Therefore, EREI2CBD promoted vascular sprouting in an angiogenesis assay. From the results of Table 1 and Figure 3, the angiogenic properties of EREI2CBD were confirmed in the CAM assay. The optimal amount of EREI2CBD was determined to be 2 µg, as this concentration did not significantly affect survival of the chick embryos but induced angiogenic activity. In the subsequent experiments, 2 µg of EREI2CBD was applied for further determination of angiogenic ability. Determination of the Angiogenic Activity of EREI2CBD. The angiogenic activity of EREI2CBD was compared using the same amounts of CBD protein or IKVAV synthetic peptide. Two micrograms of CBD protein or IKVAV synthetic peptide were shown to be harmless to chick embryos (Table 1). Figure 4A indicated that the capillary vessel network was more complex when EREI2CBD was applied to CAM. The morphology of vessels was similar in PBS and IKVAV peptide treated CAMs. CBD caused an increase of vascular thickness, but EREI2CBD showed the greatest increase in the number of vascular branches. Quantitative results of angiogenic activity are shown in Figure 4B,C, and these corresponded to the above observations. The vessel areas of CBD- or EREI2CBD-treated CAMs were greatly increased compared to PBS and IKVAV peptide-treated CAMs (Figure 4B). CBD also increased the number of vascular branches compared to PBS. EREI2CBD further increased vascular branching, and this was significantly more than CBDtreated CAMs (Figure 4C).
In the present study, we have determined the angiogenic properties of an artificial multifunctional extracellular matrix (ECM) protein. This novel ECM protein, referred to as EREI2CBD, was previously constructed by fusing functional peptide sequences derived from existing ECM proteins (1). We arranged two well-known active sequences with (APGVGV)12 to construct ECMs that had strong cell-adhesive properties and the ability to induce cellular network signals. The sequences we focused on were GRGDS and CSRARKQAASIKVAVSADR. The latter IKVAV-containing sequence was reported to promote endothelial cell migration and tubular network formation (3, 4) through the interaction of an unknown receptor on the cellular surface. EREI2CBD also had a collagen-binding domain (CBD) fused to its C-terminus, and the CBD part enabled the fusion protein to be immobilized onto collagen. Since fixed forms of functional proteins such as growth factors could function locally, they have been recently reported to work more efficiently than their free forms (12). Furthermore, collagen is a well-known biocompatible material that has given satisfactory results for tissue regeneration (18, 19). Therefore, we focused on the collagen-immobilized form of the designed fusion protein for construction of an efficiently functioning ECM protein. We used atelocollagen-based material as it is free of immunogenicity as terminus telopeptides have been digested (20, 21). The CAM assay was performed with an EREI2CBD-immobilized atelocollagen membrane for assessment of angiogenesis in ViVo. Collagen-specific binding of EREI2CBD was confirmed by an ELISA assay using collagen type I-coated microplates in our previous study (1). Here, it was determined whether EREI2CBD could be fixed onto collagen membranes for in ViVo applications. By determining the amount of atelocollagen diffused from the membrane into PBS (Figure 2), it was revealed that more EREI2CBD remained on the membrane than EREI2. After 3 h of incubation, only 0.6 µg of EREI2CBD (30% of the applied amount) was detected in PBS while 1.8 µg of EREI2 (90% of the applied amount) was released. The diffusion rate of EREI2CBD was much lower than EREI2, and EREI2CBD was confirmed to be immobilized onto the atelocollagen membrane. Therefore, the CBD part of the constructed ECM functioned as an anchor to atelocollagen-based material, and EREI2CBD was determined to have the potential to work efficiently in its localized form. Next, EREI2CBD was applied to an in ViVo angiogenic model, the CAM assay, and the optimized amount to evaluate its angiogenic ability was determined. The CAM assay is a wellknown angiogenic model (13-15). Kleinman et al. introduced the CAM assay for assessment of anticancer drugs such as functional peptides (16, 17). Along with these previous reports, we examined the angiogenic properties of EREI2CBD using the CAM assay. Our first concern was biocompatibility of EREI2CBD. The designed fusion protein, EREI2CBD, was produced by means of recombinant DNA technology and E. coli fermentation. According to the related work reported by Urry et al. (22), high levels of purification were required so that E. coli-expressed recombinant proteins could be used in ViVo. They tried to remove trace amounts of E. coli proteins by a series of purification steps. In contrast (22), EREI2CBD was purified with a simple single step. The mortality table (Table 1) indicated that a high concentration of EREI2CBD was fatal
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Figure 4. Angiogenic activity of EREI2CBD. Two micrograms of IKVAV synthetic peptide, CBD, or EREI2CBD were air-dried onto atelocollagen membranes and applied to the CAM assay. (A) Morphologies of capillary vessels treated with each sample. The squares indicate the position of the atelocollagen membrane. Each photo was pictured after 48 h of incubation. (B) Quantitative evaluation of vessel areas. The number of black pixels was calculated within the whole areas of glass coverslips imaged at 0 and 48 h. Results show the ratio of pixel counts at 0 to 48 h. (C) The number of vessel branches after 48 h incubation. Each experiment was repeated 3 times. Error bars represent the standard deviation of the mean. NS, not significant; *P < 0.05, **P < 0.01; ***P < 0.001 compared to the PBS sample.
to chick embryos, which might be due to remaining harmful substances derived from E. coli cell lysates such as endotoxins (23). Table 1 showed that EREI2CBD should be applied at concentrations less than 5 µg. In the future, high levels of purity are required before pharmaceutical use of EREI2CBD. To determine the angiogenic behavior of EREI2CBD, two semiquantitative methods were used (Figure 3). One was to determine the number of black pixels per field, a value that represented the vessel area of samples. The other method was to count the number of capillary vessel branches to evaluate the development of a vascular network. From the results of Figure 3B, the increase in the number of vessel branches was dependent on EREI2CBD concentration. EREI2CBD promoted
cardiovascular sprouting and generation of neovessels. The laminin-1-derived IKVAV sequence included in EREI2CBD has been reported to affect endothelial migration, which is the first important process of angiogenesis (3, 4). Therefore, the CAM assay results suggested that EREI2CBD could retain the angiogenic properties of the functional peptide sequence in in ViVo conditions. Furthermore, promotion of vessel sprouting caused an increase in vessel area, especially when 2 µg of EREI2CBD was added (Figure 3A). Figure 3 results showed the angiogenic function of EREI2CBD, and 2 µg was applied to all subsequent experiments. To further analyze EREI2CBD angiogenic activity, EREI2CBD was compared to the IKVAV synthetic peptide and CBD protein
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(Figure 4). For the IKVAV peptide, no effect was observed in either vessel area or the number of branches compared to a PBS control. Previous reports showed angiogenic activity of the IKVAV sequence using concentrations of 200 µg (3, 4), while only 2 µg was used in our present study. Therefore, the concentration of synthetic IKVAV peptide was too low to affect angiogenic activity. Since short IKVAV peptide sequences included in EREI2CBD were stably supported with a rigid (APGVGV)12 scaffold and localized onto a collagen membrane, they may interact with the cellular surface more efficiently. Furthermore, the poly(APGVGV) sequence itself was reported to promote cell migration and may enhance the angiogenic effect of the IKVAV peptide (24, 25). Therefore, the EREI2CBD peptide was superior to synthetic IKVAV peptides for inducing angiogenesis, and our collagen-binding artificial ECMs were functional at lower concentrations than synthetic peptides, as expected. In contrast to the IKVAV peptide, CBD itself showed some angiogenic effects compared with the PBS control. Vessel area was increased by the CBD protein at the same ratio as EREI2CBD, although the increase of vessel branches by CBD was inferior to that of EREI2CBD. CBD was determined to have cell adhesive activity and promoted tube formation of endothelial cells in Vitro in our previous study (1). Those results suggested that CBD increased the thickness of capillary vessels through promotion of endothelial cell attachment and migration. The CBD sequence adopted here was Ala260-Trp 599 of fibronectin (9), and recently, Schor et al. reported that some Ile-Gly-Asp (IGD) peptide motifs included in this CBD sequence had cell adhesive ability and motogenic activity (26, 27). These reports correspond with our results and suggest that CBD plays some role in the cellular signaling pathway in addition to its collagen-binding property. Further analysis is required to determine the angiogenic function of CBD. However, our experiments (Figure 4) revealed that the angiogenic activities of CBD did not compete with EREI2CBD. Rather, EREI2CBD was superior to CBD in increasing the number of vessel branches, which suggests a specific effect for the designed ECM domain EREI2 that includes the IKVAV sequence. In conclusion, the artificially designed fusion ECM protein, EREI2CBD, was shown to have angiogenic activity using an in ViVo CAM assay. EREI2CBD could be immobilized onto atelocollagen membranes and increase vessel area, especially the number of capillary vessel branches. Our design strategy for constructing fusion proteins produced stable, functional ECM proteins that worked more efficiently than synthetic peptides. Atelocollagen-based material and collagen-binding engineered ECM proteins hold potential for generating functional tissues by tissue engineering.
ACKNOWLEDGMENT This work was supported by Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows, and the Ministry of Education, Culture, Sports, Science & Technology (MEXT), Japan.
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