Published online before print June 22, 2006, doi: 10.1161/01.ATV.0000233359.74484.77
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© 2006 American Heart Association, Inc.
Vascular Biology |
Establishment of a Functionally Active Collagen-Binding Vascular Endothelial Growth Factor Fusion Protein In Situ
From the Department of Regenerative Medicine (T.I., M.E., M.W., Y.I., K.T., H.I., H.M., T.T, T.A.), Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, Japan and Stem Cell Translational Research, Institute of Biomedical Research; Innovation/RIKEN Center for Developmental Biology (T.A.), Chuo-ku, Kobe, Hyogo, Japan.
Correspondence to Takayuki Asahara or Tetsuya Ishikawa, Department of Regenerative Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa 259-1193 Japan. E-mail Asa777{at}aol.com or tecchan@is.icc.u-tokai.ac.jp
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Abstract |
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Objective— Tissue regeneration requires both growth factor and extracellular matrix such as collagen, serving as a scaffold for cell growth. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain (FNCBD) and vascular endothelial growth factor (VEGF) 121, and investigated its properties.
Methods and Results— FNCBD-VEGF121 specifically bound to gelatin and type I, II, III, IV, and V collagen. This collagen-bound FNCBD-VEGF121 captured soluble VEGF receptor 2 (VEGFR-2)/Fc chimeric protein. Cell growth-promoting activity of FNCBD-VEGF121 was almost identical to that of VEGF121. The VEGF fusion protein significantly enhanced the expression of VEGFR-2 (71.6±0.8%) on endothelial progenitor cells (EPCs) derived from umbilical cord blood. Expectably, the collagen-bound VEGF fusion protein not only promoted the growth of endothelial cells (ECs) but also induced the expression of VEGFR-2 (63.7±0.8%) on non-adherent cells expanded from bone marrow CD34+ cells. Moreover, the VEGF fusion protein enhanced sprout formation of ECs in a matrigel model. In vivo experiments revealed that FNCBD-VEGF121 had local effects but not systemic effect on EPC mobilization.
Conclusions— These results suggest that FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates both ECs and EPCs in situ, supplying a vascular regeneration niche.
Tissue regeneration requires both growth factor and extracellular matrix. We established FNCBD-VEGF121, consisting of the fibronectin collagen-binding domain and vascular endothelial growth factor 121. FNCBD-VEGF121 stably maintains an optimally high and local concentration of VEGF with collagen matrix and stimulates cellular activity in situ, supplying a vascular niche regeneration.
Key Words: collagen • endothelium • growth substance • proteins
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Introduction |
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Vascular endothelial growth factor (VEGF) expression in situ promotes the migration and proliferation of endothelial cells (ECs) and angiogenesis.1 Previous studies suggest the feasibility of VEGF to treat arterial diseases such as restenosis and ischemia.2–6 During embryogenesis, VEGF is an initial determinant of hemangioblast differentiation into endothelial progenitor cells (EPCs).7–10 Recently, we isolated EPCs from peripheral blood and bone marrow, and demonstrated that the progenitor cells contributed to postnatal vasculogenesis via incorporation into sites of physiological and pathological neovascularization in vivo.11–13 VEGF is one of key regulators for EPCs in postnatal neovascularization.14–16 Transplantation of EPCs successfully enhances vascular regeneration by in situ differentiation and proliferation within ischemic organs.17–19 In ex vivo EPC enrichment, cytokine or growth factor may be critical for in vitro differentiation in culture media after isolation of mononuclear cells (MNCs) from blood or bone marrow. Indeed, similar MNC culture with other cytokines such as granulocyte-macrophage colony stimulating factor and tumor necrosis factor-
induces the differentiation of progenitors into dendritic cells,20,21 whereas VEGF inhibits dendritic cell maturation and preferentially promotes endothelial lineage differentiation.22,23 More recently, we reported a collagen-binding growth factor fusion protein consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain (FNCBD). This fusion protein stably binds to collagen materials, and then exerts its growth factor activity.24 The fibronectin moiety with high collagen affinity enhances the effective local concentration of the growth factor fusion protein at the site of administration in the extracellular collagen matrix that is exposed on catheter-injured arteries or localized in intramuscular tissues.25 VEGF is the most important growth factor for neovascularization via angiogenesis and postnatal vasculogenesis in the therapeutic application. This growth factor, especially VEGF121 with no heparin-binding property, exhibits not only limited target specificity and short retention times in the tissues but also instability because of higher molecular weight and dimmer when exposed to heat, acid, or protease, although EGF is relatively resistant against them. It is of interest to clarify whether collagen-binding VEGF121 fusion protein with our methodology has the potential to maintain an optimally high and local concentration with collagen matrix and stimulate both ECs and EPCs in situ.
In this study, we investigated the properties of recombinant FNCBD-VEGF121 fusion protein consisting of VEGF121 and FNCBD.
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Methods |
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Please see http://atvb.ahajournals.org.
HCAEC and EPC Culture With FNCBD-VEGF121
Cell growth-promoting activity was examined by WST-1 assay (Dojindo, Tokyo, Japan).26 Please see http://atvb.ahajournals.org
Peripheral Blood Nuclear Cell Isolation and Its Scattergram Analysis
Peripheral blood nuclear cells were analyzed by fluorescence-activated-cell sorter (FACS). Cells were segregated into lymphocyte-size (LS) and monocyte-size (MS) fractions by gating light scatter analysis as previously described.14
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Results |
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FNCBD-VEGF121 Binds to Gelatin and Type I, II, III, IV, and V Collagens
We assayed collagen-binding property of the fusion protein. Figure 1 shows that FNCBD-VEGF121 specifically bound to collagens in a dose-dependent manner, whereas it did not bind to blocking proteins that was considered as nonspecific binding. FNCBD-VEGF121 showed the highest affinity to type III collagen and gelatin, medium affinity to type I collagen, and the lowest to type II, IV, and V collagen. In contrast, no binding of VEGF121 was observed to any type of collagen. These results indicate that FNCBD-VEGF121 has the property to bind to major collagen types, preferentially to type III collagen that is known to play a major role in tissue remodeling after injury. This finding suggests that our recombinant fusion protein would be optimally delivered to remodeling tissues rich in type III collagen.
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Collagen-Bound FNCBD-VEGF121 Captures VEGFR-2/Fc Chimeric Protein
We investigated whether the fusion protein had the ability to associate with VEGF receptor. Figure 2 shows that collagen-bound FNCBD-VEGF121 captured soluble VEGFR-2 /Fc chimeric protein in a dose-dependent manner, whereas it had no action on blocking proteins. In contrast, VEGF121 did not trap soluble VEGFR-2 in the wells, because it had no affinity to collagens and blocking proteins. These findings encourage our hypothesis that FNCBD-VEGF121 can retain an optimally high and local concentration of VEGF with collagen matrix and may stimulate both ECs and EPCs in situ.
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FNCBD-VEGF121 Promotes HCAEC Growth
Cell growth-promoting activity of FNCBD-VEGF121 was examined using HCAECs. Figure 3 shows that the dose-response curve of FNCBD-VEGF121 was similar to that of VEGF121 in a WST-1 colorimetric assay. The result indicates that FNCBD-VEGF121 has functionally intact VEGF activity without impairment caused by fusion. Taken together, we demonstrated that FNCBD-VEGF121 was a bifunctional fusion protein that has both collagen binding property and VEGF activity.
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FNCBD-VEGF121 Induces the Differentiation of MNCs into EPCs
We used MNCs from umbilical cord blood in this assay, because abundant EPCs can be differentiated from a relatively smaller volume of the blood.27 MNCs were seeded in dishes coated with human fibronectin and maintained in the media with FNCBD-VEGF121 or control buffer. After 8 days in the culture with FNCBD-VEGF121, most of adherent cells showed a spindle-shaped EPC morphology (supplemental Figure I, available online at http://atvb.ahajournals.org). EPCs became a flat-shaped EC-like appearance at day 12 (supplemental Figure I) and expressed EPC markers,11,17,28 including VEGFR-2 (71.6±0.8%), endoglin (86.1±2.4%), VE-cadherin (7.6±1.3%), VEGFR-1 (92.7±0.6%), CXCR4 (46.6±0.4%), and CD31 (36.8±2.1%), but not lymphocytic markers, CD5 (0.5±0.2%) and CD19 (0.2±0.2%), as shown in Figure 4. Data represent the mean±SD of duplicate analyses. Especially, FNCBD-VEGF121 significantly enhanced the expression of VEGFR-2 on EPCs compared with that of control buffer (71.6±0.8% versus 32.7±0.1%, P<0.01), although a concentration and quality of lot of fetal bovine serum highly affected the expression (not shown). These results indicate that functionally intact FNCBD-VEGF121 contributes to the differentiation of cord blood MNC subpopulation into EPCs.
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FNCBD-VEGF121 Promotes HCAEC Growth Via Collagen Binding and Enhances Sprout Formation of ECs in a Matrigel Model
Cell growth-promoting activity of the fusion protein was assayed using HCAECs. Collagen-coated wells were incubated with FNCBD-VEGF121 or VEGF121. After washing the wells, cells were seeded into wells and cultured for 3 days and then the activity was examined. Figure 5 shows that FNCBD-VEGF121 substantially stimulated the growth of HCAECs in a dose-dependent manner after binding to collagen-coated wells. On the contrary, unfused VEGF had no effect, because it was washed out with buffer. These results indicate that the fusion protein exerts its growth factor activity as a collagen-associated VEGF. Likewise, FNCBD-VEGF121, which remained bound to collagen for 7 days, had cell growth-promoting activity almost comparable to that of the corresponding fusion proteins that had been bound at day 7 (supplemental Figure II). ECs, seeded on semi-solid Matrigel with FNCBD-VEGF121 exhibited a higher rate of migration, invasion of extracellular matrix, and differentiation into multicellular capillary-like structure (sprout formation), whereas ECs on Matrigel treated with VEGF121 did not effectively form a network of capillary-like structure (supplemental Figure III).
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Collagen-Bound FNCBD-VEGF121 Induces the Expression of VEGFR-2 on Expanded CD34+ Cells in Situ
To avoid the affect of fetal bovine serum in the assay, we used the cells that were expanded from CD34+ cells in a serum-free culture system. In vitro-expanded cells were seeded into the collagen-coated wells that had treated with FNCBD-VEGF121 or VEGF121. After cell culture for 7 days, FACS analysis was performed on cultured cells. The cells, treated with FNCBD-VEGF121, showed a statistically higher expression of VEGFR-2 (63.7±0.8%), although the cells remained to be suspended in the media. In contrast, the cells in VEGF121-treated wells expressed the receptor in a lower extend (27.7±0.7%). These results indicate that collagen-associated FNCBD-VEGF121 enhances the expression of VEGFR-2 on expanded cells (Figure 6).
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FNCBD-VEGF121 Targets Connective Tissue in Interstitial Space
To investigate the property of FNCBD-VEGF121 in vivo, the localization of administered molecule in injured or remote tissues was compared with nonfused VEGF121 and VEGF165. VEGF121, VEGF165, FNCBD-VEGF121, or control buffer was injected into injured tibialis anterior muscles that comprised remodeling connective tissues rich in type III collagen. FNCBD-VEGF121 targeted the connective tissues, as revealed by the fluorescent immunostaining against human FNCBD (supplemental Figure IV-A) or human VEGF (supplemental Figure IV-B). Staining of rat laminin supported that FNCBD-VEGF121 was delivered to the interstitial spaces of tibialis anterior muscle (supplemental Figure IV-A and IV-B). Numerous proliferating interstitial cells were detected in remodeling connective tissue with fluorescent immunostaining against PCNA (supplemental Figure IV-C), suggesting that FNCBD-VEGF stimulated pre-existing ECs or tissue stem cells in the interstitial spaces of skeletal muscle fibers. On the contrary, VEGF was not stained when administered 70 nM (supplemental Figure IV-D) or 700 nM (not shown) VEGF 121, 70 nM (supplemental Figure IV-E) or 700 nM (supplemental Figure IV-F) VEGF 165, or control buffer into the injured muscles. Likewise, any nonfused VEGF protein was not detected in the muscle of the contralateral hindlimbs (not shown). The observations implicated that the functionally active VEGF fusion protein performed specific targeting to remodeling connective tissue comprising collagens in the interstitial spaces of the muscle, whereas nonfused VEGF was systemically diffused away or degraded.
FNCBD-VEGF121 Does Not Mobilize EPCs
The systemic effects of FNCBD-VEGF121 administration were investigated in a mouse hindlimb ischemia model. Immediately after operative ligation of femoral artery, athymic nude mice (n=18) received an intramuscular injection of FNCBD-VEGF121, VEGF121, VEGF165, or control buffer. To evaluate the effect of each VEGF on EPC kinetics, FACS analysis was performed to identify the mobilized cellular population, as it was previously reported that EPC population of monocyte-size (MS) fraction exhibited more differentiated EPC development than that of lymphocyte-size (LS) fraction.14 The cells were detected in both the LS, lower side-angle light scatter, and MS, higher side-angle light scatter, cell fraction. Supplemental Figure VA shows light scatter dot plots that the increase of mononuclear cell population in peripheral blood was more prominent in MS fraction than in LS fraction 4 days after VEGF121 or VEGF165 administration compared with FNCBD-VEGF121 or control buffer administration. We observed a significant increase in the ratio of MS/LS with VEGF121 or VEGF165 when compared with that of FNCBD-VEGF121 or control buffer as shown in supplemental Figure VB. These results suggest that FNCBD-VEGF121 did not have systemic effect on mobilization of EPCs because it was not delivered to the remote sites such as bone marrow.
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Discussion |
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We demonstrated that FNCBD-VEGF121 stably maintained an optimally high and local accumulation of VEGF with collagen matrix and stimulated both ECs and EPCs in situ. It would be essential to deliver an optimal dose of therapeutic VEGF in situ for neovascularization via angiogenesis and postnatal vasculogenesis by ECs and EPCs. However, the most of recent works demonstrating the potential of VEGF therapy to increase neovascularization has demonstrated the systemic effect of VEGF on EPC mobilization. Locally delivered FNCBD-VEGF121 does not promote EPC mobilization as other delivery techniques. Aicher et al demonstrated that tissue distribution of systemically transplanted EPCs were monitored in athymic nude rats.29 A small proportion of radiolabeled EPCs were detected in a targeted organ, myocardium, and most of the cells were distributed to other organs. This implies that local accumulation of EPCs would be more safe and effective strategy than systemic dispersion or uncontrolled mobilization of EPCs in clinical cell therapy. Considering complicated systemic effect on undesired neovascularization in case of arthritis, diabetic retinopathy, tumor growth, and metastasis, FNCBD-VEGF121 would open a novel and challenging clinical option for growth factor delivery or cell transplantation against ischemic cardiovascular diseases.
Our and other previous studies have demonstrated the applicability of gene transfer using VEGF and/or EPCs to treat vascular lesion.30–37 Likewise, it is of interest to investigate whether the gene therapy using a gene encoding FNCBD-VEGF121 has the potential to maintain an optimally high and local accumulation of VEGF for a longer period.
Recently, myogenic and endothelial cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen.38 It is noteworthy that these skeletal muscle-derived CD34+/45– (Sk-34) cells are new candidates of adult stem cells that are distinct from satellite cells, side-population (SP) cells, and bone marrow-derived stem cells.38,39 These findings suggest that Sk-34 cells reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal vasculogenesis and skeletal muscle growth. It is expected that our collagen-binding growth factor would be delivered to interstitial spaces of skeletal muscle and stimulate those adult stem cells in situ for tissue regeneration such as neovascularization and new fiber formation of skeletal muscle.
Stimulation of EphB4 receptors with ephrinB2/Fc chimeric protein resulted in dose- and time-dependent phosphorylation of Akt in human microvascular ECs.40 Those cells possessed abundant EphB4 receptors with no endogenous ephrinB2 expression. EphB4 receptor activation with ephrinB2/Fc chimera increased proliferation and nitrite levels increased, indicating increased nitric oxide production. Signaling of EC growth appears to be mediated by a PI3K/Akt/endothelial nitric oxide synthase/protein kinase G/mitogen-activated protein kinase cascade. EphB4 receptor stimulation with ephrinB2/Fc chimera also increased migration and increased activation of both matrix metalloproteinase (MMP)-2 and MMP-9. Their studies demonstrated that EphB4 receptor with ephrinB2 fusion protein stimulated migration and proliferation of ECs. The chimeric ephrinB2 ligand was soluble and bound to the surface of plastic dishes. It stimulated the EphB4 receptor in the solid phase, as ephrinB2 ligand was a trans-membrane protein and it was insoluble in its natural state. Therefore it is possible that collagen-binding ephrinB2 fusion protein as well as ephrinB2/Fc chimera might play a role in angiogenesis, as it is a collagen-bound form in situ. It would also strengthen our concept to clarify the feasibility of such chimeric proteins.
Notch signaling is a known regulator of cell fate in numerous developmental systems and on hematopoietic stem cells (HSC). The hematopoietic system is maintained by HSC. A rare population of HSC undergoes self-renewal as well as continuously produces progeny that differentiate into the various hematopoietic lineages. Activation of endogenous Notch signaling in human cord blood derived CD34+CD38– (a putative enriched population of HSC) cells with the immobilized extracellular domain of the Notch ligand, Delta-1 (Delta-1/Fc chimeric protein) inhibited myeloid differentiation and induced a 100-fold increase in the number of CD34+ cells compared with a soluble truncated form of Delta-1 in a serum-free culture system.41 Thus, the immobilized ligand without its trans-membrane domain and intracellular domain could function with those stem cells as if it were a native membrane-bound form of Delta-1. This implies that an immobilized collagen-binding fusion protein consisting of the extracellular domain of Delta-1 and FNCBD might be also active on expansion culture of the stem cells.
Our fusion protein with specific protease recognition site might be useful as a modulator of an artificial stem cells "niche," the in vivo regulatory microenvironment where stem cells reside. Stem cells in bone marrow exist in a quiescent state or are instructed to differentiate and mobilize to circulation with specific signals. MMP-9 (MMP-9), induced in BM cells, causes shedding (release) of soluble SCF (sSCF), permitting the transfer of c-Kit+ stem/progenitors from the quiescent to proliferative niche.42 Similarly, Release of VEGF as well as sSCF by any protease might enable stem cells to translocate to a permissive vascular niche favoring differentiation and reconstitution of the stem cell storage.
In conclusion, we established a functionally active collagen-binding VEGF fusion protein in situ, and suggested that a variety of fusion proteins with our methodology might stimulate their corresponding receptors via collagen binding.
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Acknowledgments |
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We are grateful to T. Kitajima and H. Terai for preparation of recombinant proteins. Cord blood samples were supplied by the Tokai University Cord Blood Bank.
Source of Funding
This study was supported by a grant-in-aid for a Research Grant of the Science Frontier Program from the Ministry of Education, Science, Sports, and Culture of Japan.
Disclosures
None.
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Footnotes |
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Original received January 29, 2006; final version accepted May 4, 2006.
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