Self-assembly Perlecan For Constructed Materials Of Enhancing Angiogenesis

The extracellular matrix (ECM) consists of a complex mixture of proteins and glycoproteins serving multiple functions. During tissue repair,the ECM serves as a platform to supply growth factors that modulate such diverse processes as angiogenesis,migration,proliferation,differentiation,and wound healing. These characteristics of ECM provide opportunities to improve biological activities of materials used for tissue engineering and wide-range of clinical therapies. In development of new class biomaterials,the strategy of immobilizing ECM components with basic materials was applied in many cases. Hyaluronic acid (HA) is a promising candidate for the development of novel biosynthetic biomaterials for local delivery of cells and bioactive factors because of its unique physicochemical properties and its excellent biocompatibility and biodegradability. In its natural setting,HA is the only non-sulfated glycosaminoglycan (GAG) present in the ECM of vertebrates. Consisting of repeating disaccharide units (β-l,4-D-glucuronic acid-β-l, 3-N-acetyl-D-glucosamine) with an overall molecular weight between 100 and 5000 kDa, HA is present in all connective tissues as a major and constituent of ECM. In the ECM of connective tissues,HA forms a scaffold for binding other protein constitutes, such as aggrecan, and plays important roles in maintaining tissue morphologic organization, preserving extracellular space and transporting ions, solutes and nutrients. Based on its properties, HA is potential to combine with other ECM molecules for the development of new class biomimetic materials.Perlecan,a heparan sulfate proteoglycan (HSPG),has a wide range of biological activities and functions in ECM including modulation of proliferation, angiogenesis, morphogenesis and tissue regeneration. Perlecan has a protein core of approximately 400 kDa and consists of five distinct domains. Through domainⅠor V that decorated with heparan sulfate (HS) and chondroitin sulfate (CS) chains, Perlecan can bind to heparin-binding growth factors (HBGFs), such as fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF), and functions as a ligand reservoir for storage and release of HBGFs. In addition, Perlecan also can protect these growth factors from inactivation by proteolytic digestion,functions as a coreceptor for FGF-2, and regulates FGF activity through its HS chains. Because HS benefits to HBGFs, HS or heparin decorated biomaterials are under study,and used to delivery HBGFs. In addition,Perlecan domainⅠor HS can bind to natural collagenⅠfibrils,which is involved in ECM assembly. The association of Perlecan with collagenⅠdepends on HS chains and collagenⅠtriple-helical configuration. Thus, Perlecan domainⅠ/collagenⅠfibrils complex was used as matrix to improve,or modify biomaterials to associate HBGFs.Angiogenesis is an essential feature of tissue remodeling and wound healing and tissue regeneration,and has long been a desired therapeutic approach to improve clinical outcomes of conditions typified by ischemia. VEGF has demonstrated the ability to generate new blood vessels,and sustained delivery of VEGF has been proven necessary to generate blood vessels as demonstrated by implantable, controlled release devices. According to properties HA, Perlecan and collagenⅠ, we hypothesized that Perlecan would bind both VEGF-165 and collagenⅠfibrils in a self-assembling fashion, and Perlecan/collagenⅠfibrils (P/C) complex can improve HA hydrogel's bioactivity in sustained delivery of VEGF, and consequently enhances VEGF-induced angiogenesis. In this investigation, we examined the interaction of Perlecan with VEGF-165 and collagenⅠfibrils, and further evaluated VEGF-165 anchored in, or released from different HA hydrogels, HA hydrogel and"hybrid"HA hydrogel incorporated with P/C complex. Finall, the ability of hydrogels in enhancing angiogenic process was assessed with aortic ring culture model. Collectively, the finding suggested that P/C complex can improve VEGF-165 immobilization, sustain release of the growth factor and promote VEGF-induced angiogenesis in HA hydrogel decorated with the complex. The increase in activity of the"hybrid"HA hydrogel most likely reflects the ability of Perlecan to bind, concentrate,and retain the growth factor in microinvironment of the hydrogel culture.1. Preperation of PerlecanThe EHS tumor was maintained in the right or left hind leg muscle of C57BLmice following injection of tumor cells as previously described . The tumors were usually maintained in the mice hind legs for 3-4 weeks usually attaining a growth of approximately 3 4 grams. The animals were sacrificed by lethal injection of Nembutal (0.50 ml of 50 mg/ml solution per mouse),before the tumor tissue reached an approximate weight of 4 grams. EHS tumor tissue was harvested from the mice as previously described. All extraction steps (described below) were carried out by agitation using a rotary shaker at 600 rpm. The tumor tissue (50 grams at a time) was routinely minced and extracted with 2.5 tissue volumes of 50 mM Tris-HCl (pH 7.5),3.4 M NaCl, containing a protease inhibitor cocktail including 10 mM EDTA,10 mM NEM, 10 mM 6-aminohexanoic acid, 5.0 mM benzamidine-HCl, and 1 mM PMSF. The supernatants were collected following centrifugation at 17,000. times.g for 30 minutes. This high salt extraction was repeated and the resulting pellet obtained was extracted with 2.5 tissue volumes of 4M guanidine-HCl, 0.5% (w/v) CHAPS, 50 mM Tris-HCl (pH 7.5) containing a protease inhibitor cocktail (as described above) for 3 hours at 4.degree. C. The supernatants were again collected following centrifugation at 17, 000.times.g for 30 minutes. Additional extractions of the remaining pellet were achieved using 1.5 tissue volumes with guanidine-HCl (as described above),first for 2 hours,and then overnight. The guanidine extracts were then pooled and applied (80 mls at a time) to Sephacryl S-400 columns (4.8.times.28 cm). Samples were then eluted using a urea buffer containing 7M urea, 0.2M NaCl, 0.1% (w/v) CHAPS, 50 mM Tris-HCl (pH 8.0) containing protease inhibitors as described above. The void volume fractions (believed to contain Perlecan) from the Sephacryl S-400 column were then pooled and supplemented with 0.5% Triton X-100 (v/v) and applied to a 50 ml DEAE-Sephacel column packed in a 60 ml plastic syringe equilibrated with urea buffer. Contaminant proteins and non-PGs were removed by first washing the column with 3 column volumes of urea buffer containing 1% Triton X-100, followed by 3 column volumes of urea buffer containing 0.25M NaCl and 1% Triton X-100,and then 3 column volumes of urea buffer containing 0.25 M NaCl without Triton X-100. Bound PGs were then eluted with 5 column volumes of urea buffer containing 1M NaCl or 3M NaCl. The 3M NaCl elutions were used to determine whether tightly bound PGs not eluted with 1M NaCl still remained bound to the column.PGs obtained from the DEAE-Sephacel column were first loaded onto a Sephacryl S-500 column (2.6.times.60 cm) in order to try to separate the 220 kDa PG from Perlecan (700-800 kDa). In a preliminary study, we found the Sephacryl S-500 column was not able to properly separate the 220 kDa PG from Perlecan (both were found in the void volume fractions), due to the self-aggregating ability of the 220 kDa PG. Even the use of dissociating buffers including, a) 4M guanidine-HCl, 0.5% CHAPS, and 50 mM Tris-HCl (pH 7.5), b) 7M urea, 0.2M NaCl, 1% SDS (w/v), and 50 mM Tris-HCl (pH 8.0) and c) 7M urea, 0.2M NaCl, 0.1% CHAPS, and 50 mM Tris-HCl (pH 8.0), were not able to prevent the aggregation of the 220 kDa PG. Separation of the 220 kDa PG from Perlecan was finally achieved using a Sephacryl S-1000 column (5.times.95 cm) under associating conditions (1M urea buffer containing 50 mM Tris-HCl and protease inhibitors including 1.4 mM EDTA,1.4 mM NEM, 1.4 mM 6-aminohexanoic acid, 0.7 mM benzamidine, and 0.14 mM PMSF(pH 8.0).PGs eluted from DEAE-Sephacel were loaded (50 mls per run) onto the Sephacryl S-1000 column (as described above). PGs eluted from the Sephacryl S-1000 column were monitored by SDSPAGE analysis in order to assess the purity of Perlecan and other HSPGs produced. For this analysis,100μl aliquots of each 60 ml fraction was precipitated with 4 volumes of absolute ethanol by cooling on dry ice for 1 hour, and then centrifuged on a microcentrifuge at 12,000.times.g for 20 minutes, and run on SDS-PAGE as described below. Pooled fractions (from 5 separate runs) containing Perlecan (Kav=0.29-0.54) were then concentrated on and eluted from a 15 ml DEAE-Sephacel column with 3M NaCl, and rechromatographed onto the Sephacryl S-1000 (as described above). Usually three passes through the Sephacryl S-1000 column gave high quality Perlecan preparations free from any other contaminating HSPGs (i.e. 220 kDa PG) or other PGs. The Perlecan fractions were pooled and concentrated onto a 10 ml DEAE-Sephacel column and ethanol precipitated (as described above). The resulting pellets were dissolved in 3 5 ml of double distilled water and extensively dialyzed against double distilled water until the conductivity of the end product contained very little to no salt (2-3μmhos) as measured using a digital conductivity meter. The final Perlecan product was then freeze-dried and stored. The final purity of the Perlecan preparations were further assessed by Alcian blue staining,Coomassie Blue staining, silver staining, and a series of Western blots (as described below) 2. Binding of VEGF-165 to PerlecanThe method of dot blot was employed to test the interaction between Perlecan and VEGF-165 as described by Yang. Briefly,3μg of Perlecan in 100μl of Phosphate Buffered Saline (PBS) was blotted on nitrocellulose membrane. After blocked with 5% (w/v) fat-free milk powder in 0.05% (v/v) Tween 20 in PBS for 2 hours, 100ng of recombined human VEGF-165 was added into each well of the blotting apparatus. The membrane was washed with 0.05% (v/v) Tween 20 in PBS to eliminate unbinding VEGF-165 from the membrane, and subsequently incubated with biotinylated rabbit anti-VEGF antibody for 1 hour at room temperature. After incubated with Neutr-Avidin horseradish peroxidase conjugated (NeutrAvidinTM- HRP),the binding of FGF-2 to Plnwas evaluated by densitometry and expressed as individual density values (IDVs).3. Preparation of collagenⅠfibrils and binding of collagenⅠfibrils to PerlecanTo immobilize collagen into plastic plates,direct coating method was used as described previously . Each well of 96-well microplates was incubated with 20μg of collagenⅠfibrils, or gelatin (denatured collagen) or acid dispersion (collagenⅠmonomer) in 100μl for 24 h at 37℃. Control wells were coated with 20μg of BSA in PBS. After rinsing with PBS,the coated 96-well plates were stored at 40℃for future use. In addition, collagen coating efficicncy was determined by measuring hydroxyproline content of the coated well surfaces.All collagen forms used gave similar coating efficiencies Perlecan was biotinylated with Sulfo-NHS-LC-Biotin using EZ-Link? Sulfo- NHS-LC-Biotinylation Kit (Pierce Biotechology,Inc. Rockford,IL. USA), according to the instructions provided by manufacturer. Purification of biotinylated Perlecan was performed by applying Dextran Desalting Column (MW cutoff = 5000). Biotin incorporation in Perlecan was determined by HABA method as described before.The association of biotinylated Perlecan with collagenⅠfibrils in microplates was determined by binding of NeutrAvidin conjugated horseradish peroxidase (NeutrAvidin-HRP). To determine the characteristic of binding interaction between biotinylated Perlecan and collagenⅠfibrils, 100μl of biotinylated Perlecan in blocking buffer was added at increasing concentration (0-56μg/ml) to each well of a 96-well microplate and incubated for 2 hours at room temperature. The bound biotinylated Perlecan was evaluated by incubation of NeutrAvidm-HRP (0.1μg/ml) for 30 min following washing with PBS. The wells finally were incubated with 200μl of TMB solution followed by washing with PBS. The reaction was stopped with 200μl of 2M sulfuric acid. The optical density was measured at 450 nm. For other substrates, gelatin, collagenⅠmonomer and BSA, the same assay was employed to assess interactions of biotinylated Perlecan with them.4. Effect of collagenⅠfibrils to Bio-Perlecan adding PerlecanTo determine if Perlecan constitute contributes binding of biotinylated Perlecan to collagenⅠfibrils, the biotinylated Perlecan binding of to collagenⅠfibrils was evaluated further by competitive binding of natural Perlecan (un-biotinylated Perlecan). In the assay, 3μg of biotinylated Perlecan was added to each collagenⅠfibril-coated well in the presence of increasing molar ratios of Perlecan/biotinylated Perlecan (from 0 to 32). The association of biotinylated Perlecan with collagenⅠfibrils was measured as described above.5. Effect of collagenⅠfibrils to bio-Perlecan adding heparan sulfateTo investigate to what extent the protein and GAG constituents of Perlecan mediated interactions with collagenⅠfibril, soluble heparan sulfate (HS) was used to compete for biotinylated Perlecan (3μg/well) binding to collagenⅠfibrils by increasing concentration of HS (0-480μg/ml)6. Perlecan binding assay for different collagenThe association of biotinylated Perlecan with collagenⅠfibrils, gelatin,collagenⅠmonomer and BSA in microplates was determined by binding of NeutrAvidin conjugated horseradish peroxidase (NeutrAvidin-HRP). To determine the characteristic of binding interaction between biotinylated Perlecan and collagenⅠfibrils, 100μl of biotinylated Perlecan in blocking buffer was added at increasing concentration (0-56μg/ml) to each well of a 96-well microplate and incubated for 2 hours at room temperature. The bound biotinylated Perlecan was evaluated by incubation of NeutrAvidm-HRP (0.1μg/ml) for 30 min following washing with PBS. The wells finally were incubated with 200μl of TMB solution followed by washing with PBS. The reaction was stopped with 200μl of 2M sulfuric acid. The optical density was measured at 450 nm.7. Binding of VEGF-165 to Perlecan/collagenⅠfibrils (P/C) complexTo prepare Perlecan/collagenⅠ(P/C) complex, 600μg of Perlecan was added to 2ml of collagenⅠfibrils suspension (3.0 mg/ml). After 3 hours incubation at room temperature, the collagenⅠfibrils suspension was centrifuged, and the supenatant was removed. Three times washing was taken with PBS to remove unbound Perlecan from collagenⅠfibrils. For solid binding assay,20μg of collagenⅠfibrils associated with Perlecan (P/C complex) was coated on each well with same way as other substrates to immobilize P/C complex to microplate. Solid phase binding and ELISA-based assay was used to assess VEGF-165 associated with different substrates as described before. After immobilizing substrates (collagenⅠfibrils , P/C complex , gelatin and col1agenⅠmonomer) into 96-well microplates and blocking with 3% (w/v) BSA in PBS, VEGF (20 ng) in blocking buffer was added to each well and incubated for 2 hours at room temperature. After washing three times with PBS, anti-VEGF antibody conjugated to HRP and colorimetric reagents of the VEGF Quantikine ELISA Kit (R&D System, Inc. Minneapolis, MN) were used to identify the VEGF associated with these substrates, according to manufacturer's instructions.8. Preparation of HA hydrogel and"hybrid"HA hydrogel containing P/C complex and binding of VEGF-165 to hydrogels HA with molecular weights of 490 kD was used in the experiment. HA derivatives were synthesized following the method described previously. Briefly, HA-adipic dihydrazide (HA-ADH) was prepared by reacting HA with a 30-fold molar excess of adipic dihydrazide in the presence of l-elhyl-3-carbodiimide (EDC) and 1- hydroxybenzotriazole (HOBt) at pH 6.8 and room temperature. HA-aldehyde (HA-CHO) was prepared by reacting HA with an equi-molar sodium periodate. The reaction was terminated by adding ethylene glycol. The products (HA-ADH and HA-CHO) were purified by exhaustive dialysis, lyophilized and stored at 4℃.To prepare cross-linked hyaluronic acid hydrogels , one volume of HA-ADH solution(15 mg/ml) was mixed with one volume of HA-CHO solution (15 mg/ml) in room temperature. To prepare"hybrid"HA hydrogel containing P/C complex,2 mg of P/C complex was initially added into either one of two solutions,HA-ADH or HA-CHO,and then they was mixed well to form 0.4 ml of hydrogel. The concentration of P/C complex is equal to collagenⅠfibrils concentration calculated. Same final HA concentration (30 mg/ml) was reached for either HA hydrogel or"hybrid"HA hydrogel in our experiment.To investigate the binding of VEGF-165 to various hydrogels, an ELISA-based assay was employed as described previously with some modification. Before binding assay, 0.4 ml of hydrogel was formed in each well of 24-well cell culture insert (BD FalconTM Cell Culture Inserts, pore size = 8.0μm). After blocking with 3% (w/v) BSA in PBS,HA hydrogels and"hybrid"HA hydrogels containing P/C complex were incubated with VEGF-165 (100 ng/ml) with constant rotary agitation for 2h at room temperature, and then washed 3 times with PBS on shaker to remove unbound VEGF-165. VEGF-165 binding to hydrogels was measured with biotinylated anti-VEGF antibody (1:800, R&D sysem Inc. Minneapolis, MN, USA). Next,the biotinylated anti-VEGF antibody was identified by NeutrAvidin-HRP (0.1μg/ml). Each hydrogel was further reacted with 0.5 ml of TMB substrate to produce color. The hydrogels stained by color reagent were dried with a Kaydry wiper (Kimberly-Clark, Co, Roswell, GA, USA) to stop the reaction and immediately photographed. In addition, after mixed with same volume of stop buffer,the color reactant solution was transferred to wells of 96-wel1 microplate for absorbance measurement at 450 nm.9. Quantification of VEGF-165 releaseThe release kinetics of VEGF-165 from either HA hydrogels or"hybrid"HA hydrogels containing P/C complex were measured using a sandwich ELISA. 0.4 ml of hydrogel was formed in each cell culture insert (pore size = 8.0μm) after adding HA-ADH solution and HA-CHO solution with 1/1 volume ratio (for HA hydrogel), or further adding P/C complex (for"hybrid"HA hydrogel), as described above. Before gel had set,30 ng of VEGF-165 was added into either one of two solutions, HA-ADH or HA-CHO, and subsequently mixed with other solution with pipette tip. The cell culture inserts containing hydrogels were placed in 12-well tissue culture plate (Nalge-Nunc International), and subsequently 4.0 ml of EBM was added into each well. The 12-well tissue culture plates was moved into incubator (37℃,5% CO2) with constant rotary agitation (100 rpm). The release buffer (EBM) was retrieved at 0.5, 1, 2, 8, 12, 16 and 20 days. The content of VEGF-165 in the release buffer was determined with VEGF Quantikine ELISA Kit,according to the manufacturer's instruction, and the percent of VEGF-165 released from the substrates were calculated in our experiment.10. Mouse aortic ring outgrowth assayThe mouse aorta model was used to assess the characterization of angiogenesis induced by VEGF as described by other authors with some modification. Briefly, thoracic aortas were dissected from the posterior mediastinum of eight-week old C57BL/6 mice (purchased from Expcrimental Animal Center,The Forth Military Medical University,China) and placed in serum-free endothelial basal medium (EBM,Clonetics,San Diego,CA,USA) immediately. After cleaned of blood and fibro- adipose tissue under a dissecting microscope,the aotas were cross-cut into 1mm-long rings with Noyes scissors (Roboz Surgical Instrument Co. Rockville, MA, USA). These rings were rinsed, and soaked in EBM for use.To embedding aortic ring into hydrogels, 30μl of hydrogel was coated initially on the bottom of 4-well tissue culture dishes (Nalge Nunc Intemational) to form a uniform thin disc of approximately 5 mm in diameter. After gel formation,the aortic ring was placed on the top of coated hydrogel with the luminal axis of each ring parallel to the bottom of thc tissuc cu1ture dish. Each ring was then covered with 30μl of hydrogel. Once the gel had set, 0.4 ml of serum-free EBM was added to each well. Aortic rings embedded in hydrogel were cultured in incubator ((37℃, 5% CO2), The EBM was changed every two days. For VEGF stimulation, aortic rings embedded in different hydrogel were incubated with 0.3ml of VEGF-165 (60 ng/ml) in EBM for 2 hours before serum-free EBM was added or changed. The rings hydrogels cultures un-treated with VEGF-165 were used as controls in our experiment.To quantify angiogenic spouting,images were captured with a Nikon digital camera(DXMI200F) mounted on a Nikon microscope (SMZ 1500). Software of Zeiss LSM. Imagine Browser (version 3,2,0,115) was used to measure the area covered by Microvellels(mm2) as published methods with some modifications. Images of aortic rings sprouting were analyzed by manually encircling outgrowth area. Mean area was camputered from triplicated samples of outgrowths.11. Implantation studiesThe study was performed with the approval and according to the guidelines of the Institutional Animal Care and Use Committee of Fourth Military Medical University. Nude mice were anesthetized upon intraperitoneal injection of a mixture of ketelar (80 mg/kg) and xylazine (12 mg/kg) and the dorsal skin was sterilized by 75% alcohol. The scaffolds (four scaffolds per mouse) were implanted subcutaneously in both side of the dorsal area. After 1, 2, 4 weeks,the implanted scaffolds were explanted, fixed in formalin, paraffin-embedded, sectioned into 5-mm slices, and immunostained with antibodies against CD31, according to the manufacturer's instructions.