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1.  Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis 
BMC Biotechnology  2012;12:60.
Background
Many growth factors, such as bone morphogenetic protein (BMP)-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS) glycosaminoglycans (GAGs), which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS), regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo.
Results
Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1) expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™).
Conclusions
A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid significantly improved the dose-effectiveness of BMP-2 osteogenic activity for in vivo de novo bone generation when delivered together on a scaffold as a single-phase. The use of HS/CS PGs may be useful to augment GF therapeutics, and a plasmid-based approach has been shown here to be highly effective.
doi:10.1186/1472-6750-12-60
PMCID: PMC3485628  PMID: 22967000
Osteogenesis; BMP-2; Heparan sulfate; Chondroitin sulfate; Proteoglycan; TCP; Bone graft; Implant; Osteoblast; Perlecan
2.  Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan 
Biochemistry  2010;49(26):5524-5532.
Perlecan is a large multi-domain proteoglycan which is essential for normal cartilage development. In this study perlecan was localized in the pericellular matrix of hypertrophic chondrocytes in developing human cartilage rudiments. Perlecan immunopurified from medium conditioned by cultured human fetal chondrocytes was found to be substituted with heparan sulfate (HS), chondroitin sulfate (CS) and keratan sulfate (KS). Ligand and carbohydrate engagement (LACE) assays demonstrated that immunopurified chondrocyte-derived perlecan formed HS dependent ternary complexes with fibroblast growth factors (FGF) 2 and either FGFR receptors (FGFRs) 1 or 3, however these complexes were not biologically active in the BaF32 cell system. Chondrocyte-derived perlecan also formed HS dependent ternary complexes with FGF18 and FGFR3. The proliferation of BaF32 cells expressing FGFR3 was promoted by chondrocyte-derived perlecan in the presence of FGF18 and this activity was reduced by digesting the HS with either heparinase III or mammalian heparanase. These data suggest that FGF2 and 18 bind to discrete structures on the HS chains attached to chondrocyte-derived perlecan which modulate the growth factor activities. The presence and activity of mammalian heparanase may be important in the turnover of HS and subsequent signaling required for the establishment and maintenance of functional osteo-chondral junctions in long bone growth.
doi:10.1021/bi1005199
PMCID: PMC2900151  PMID: 20507176
perlecan; heparan sulfate; fibroblast growth factor; fibroblast growth factor receptor; heparanase

Results 1-2 (2)