Biochemical and pharmacological characterization of different recombinant acid alpha-glucosidase preparations evaluated for the treatment of Pompe disease.

Pompe disease results in the accumulation of lysosomal glycogen in multiple tissues due to a deficiency of acid alpha-glucosidase (GAA). Enzyme replacement therapy for Pompe disease was recently approved in Europe, the U.S., Canada, and Japan using a recombinant human GAA (Myozyme, alglucosidase alfa) produced in CHO cells (CHO-GAA). During the development of alglucosidase alfa, we examined the in vitro and in vivo properties of CHO cell-derived rhGAA, an rhGAA purified from the milk of transgenic rabbits, as well as an experimental version of rhGAA containing additional mannose-6-phosphate intended to facilitate muscle targeting. Biochemical analyses identified differences in rhGAA N-termini, glycosylation types and binding properties to several carbohydrate receptors. In a mouse model of Pompe disease, glycogen was more efficiently removed from the heart than from skeletal muscle for all enzymes, and overall, the CHO cell-derived rhGAA reduced glycogen to a greater extent than that observed with the other enzymes. The results of these preclinical studies, combined with biochemical characterization data for the three molecules described within, led to the selection of the CHO-GAA for clinical development and registration as the first approved therapy for Pompe disease.

Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices.

Fibroplasia and angiogenesis are essential components of tissue repair when substantial tissue has been lost at a site of injury. Platelets and monocyte/macrophages accumulate at these sites and release a variety of growth factors that are thought to initiate and sustain the repair. Often the involved tissue contracts, a process that can markedly reduce the amount of fibroplasia and angiogenesis necessary for the reestablishment of organ integrity. Such tissue contraction occurs over hours or days, a much slower time course than the rapid, reversible contraction of muscle tissue. Fibroblasts, which are rich in f-actin bundles, appear to be responsible for wound contraction. However, the signals that stimulate contraction are not known. Using cultured fibroblasts, which are also rich in f-actin bundles, we demonstrate the platelet and monocyte isoforms of platelet-derived growth factor (PDGF; AB and BB) but not PDGF-AA, can stimulate fibroblasts to contract collagen matrix in a time course similar to that of wound contraction. In addition, PDGF appears to be the predominant fibroblast/collagen gel contraction activity released from platelets. Vasoactive agonists known to stimulate smooth and striated muscle contraction do not stimulate fibroblast-driven collagen gel contraction.

TGF-beta in the central nervous system: potential roles in ischemic injury and neurodegenerative diseases.

The Transforming Growth Factor-betas (TGF-beta) are a group of multifunctional proteins whose cellular sites of production and action are widely distributed throughout the body, including the central nervous system (CNS). Within the CNS, various isoforms of TGF-beta are produced by both glial and neural cells. When evaluated in either cell culture or in vivo models, the various isoforms of TGF-beta have been shown to have potent effects on the proliferation, function, or survival of both neurons and all three glial cell types, astrocytes, microglia and oligodendrocytes. TGF-beta has also been shown to play a role in several forms of acute CNS pathology including ischemia, excitotoxicity and several forms of neurodegenerative diseases including multiple sclerosis, Parkinson's disease, AIDS dementia and Alzheimer's disease.

Inhibition and promotion of differentiated-like phenotype of a human lung carcinoma in athymic mice by natural and recombinant forms of transforming growth factor-beta.

Both structurally related forms of transforming growth factor-beta (TGF-beta types I and II) are potent inhibitors of tumor cell growth in vitro and can also modulate the differentiation of some cells in culture. In this study, we describe the effects of natural and recombinant TGF-betas on the growth and differentiation of a xenograft of human lung adenocarcinoma A549 in male athymic BALB/c mice. Subcutaneous, peritumoral injection of both forms of TGF-beta inhibited, in a dose-dependent manner, the growth of established human lung tumors. Histologically, tumors inhibited by TGF-beta appeared more differentiated, as judged by reduced mitotic activity and a predominance of highly specialized mucus-secreting goblet-like cell types. These findings suggest that TGF-betas can be useful in the development of novel, perhaps less cytotoxic, cancer therapeutic strategies.

Aging does not lessen the effectiveness of TGFbeta2-enhanced bone regeneration.

Controversy exists over the potency of bone healing in the aged skeleton, and there is concern that enhancement of bone regeneration after use of bone-stimulating growth factors may not be effective in the aged. In this study, 30 skeletally mature beagles (1-2 or 10-12 years old) had titanium implants placed bilaterally in the proximal humerus for a period of 4 weeks in a model of intramembranous bone regeneration. A bony defect made at the time of surgery created a 3-mm gap between the implant surface and the host bone. Some of the implants were treated with recombinant human TGFbeta2 (rhTGFbeta2) at various does (0.32-35 microg per implant), and some served as paired controls. The dose response was similar in young and old animals. The most effective dose, 35 microg, led to a 3-fold increase in the volume fraction of new bone within the gap in both the young (p = 0.001) and old (p = 0.002) animals. At this dose, there was a 5-fold increase in osteoblast surface. While age did not significantly affect the quantity of new bone formed as assessed by backscatter scanning electron microscopy, the older animals had thinner regenerated trabeculae that tended to be spaced more closely than the younger animals. Coupled with the finding that the increase in osteoid was greater in the old animals compared with the young animals, these qualitative differences suggest that there may have been a slight delay in the rate or a defect of mineralization in the old animals.

Preparation of [3H]collagen for studies of the biologic fate of xenogenic collagen implants in vivo.

Reduction of a commercially available, pepsin-solubilized, bovine dermal collagen (Vitrogen 100) with sodium [3H]borohydride provided radiolabeled collagen preparations with specific activities ranging from 7.1-12.0 muCi/mg collagen. These specific activities were 2-3 times greater than those obtained by reduction of intact rat tail tendon collagen under similar conditions. The alpha, beta, and higher aggregate components of type I collagen were radiolabeled as well as the alpha component of a small amount of type III collagen present in the samples. Fractionation of cyanogen bromide peptides showed that alpha 1(I)CB7, alpha 1(I)CB8, and alpha 2(I)CB3,5 were the predominant peptides labeled by this procedure. Amino acid analysis indicated that the majority of the radioactivity was in reducible cross-links, precursors of these cross-links, and in hexosyllysine residues. Reconstitution experiments comparing this radiolabeled collagen with nonlabeled collagen showed them to be indistinguishable. Bacterial collagenase digestion of this reconstituted fibrillar collagen in both a lightly cross-linked (glutaraldehyde 0.0075%) and noncross-linked form provided evidence that digestion of labeled and nonlabeled collagens proceeded at similar rates. Thus, labeling did not change the properties of the collagen. Cross-linking made the preparation refractory to proteolytic degradation. Injection of fibrillar collagen preparations, spiked with radiolabeled collagen, into the guinea pig dermis followed by quantitation of the amount of radioactivity recovered from implant sites as a function of time, indicated that the lightly cross-linked samples also were more resistant to degradation in vivo than the noncross-linked preparation. The half-life of noncross-linked collagen was about 4 days while that of the cross-linked collagen was about 25 days. These degradation rates were much faster than observed for similar, nonlabeled samples injected into the dermis of humans, presumably due to a higher metabolic activity in the guinea pig dermis.

Inhibition of connective tissue formation in dermal wounds covered with synthetic, moisture vapor-permeable dressings and its reversal by transforming growth factor-beta.

An investigation of synthetic, adherent, moisture vapor-permeable dressings (SAM) on dermal wounds healing by secondary intent has yielded the novel observation that SAM dressings severely inhibited the deposition of granulation tissue and subsequent collagenous tissue when compared with air-exposed wounds in mouse and guinea pig systems. Repair tissue was quantitated histomorphometrically in full-thickness wounds covered with SAM or left air-exposed for periods up to 3 weeks. Early in healing, mouse wounds left open to the atmosphere formed a scab which overlay a large volume of granulation tissue derived from two sources, one lateral, and the other deep and centrally located. In contrast, SAM-covered wounds contained only a small amount of granulation tissue which was derived solely from lateral sources. Granulation tissue was replaced by fibrous connective tissue over time, and this was always less in SAM-covered wounds. Deposition of large amounts of connective tissue in air-exposed wounds was associated with significant polymorphonuclear and mononuclear cell infiltrates, while the lack of granulation tissue formation in SAM-covered sites was associated with reduced inflammation. Dressing-induced inhibition of connective tissue could be partially reversed by treatment with transforming growth factor-beta form 1 or 2. Deposition of granulation tissue in large lenticular wounds in guinea pig skin, but not in 6-mm punch wounds, was also inhibited when the wounds were covered with SAM, and the morphology of air-exposed and SAM-covered wounds was similar to that in mice. SAM-covered wounds in mice and guinea pigs may be useful as models of chronic non-healing wounds.

Characterization of the microheterogeneity of recombinant primate prolactin: implications for posttranslational modifications of the hormone in vivo.

Recombinant baboon and monkey prolactins were expressed in murine C127 cells. The hormones were purified from the conditioned media of these cells using a combination of cation, anion, and gel filtration chromatographies. This purification scheme provided approximately a 20-fold purification of the proteins with a 40% cumulative yield. Sodium dodecyl sulfate gel electrophoresis of the purified hormones in conjunction with Coomassie blue staining and immunoblotting procedures revealed three major prolactin-related bands with molecular weights corresponding to Mr 16,000, 23,000, and 27,000. Based on these analyses the samples were judged to be greater than 90% pure. Amino terminal sequence analysis of the purified baboon and monkey hormones provided three distinct prolactin-related sequences for each preparation. The predominant sequence corresponded to the predicted amino terminal sequences of the hormones which began with leucine at position 1. Two minor sequences, individually representing approximately 10-20% of the total population, were also identified; one starting at position 11 and the other at position 133. Carbohydrate compositional analysis of the proteins suggested that greater than 50% of the population were glycosylated with a fucosylated complex oligosaccharide. Analysis of the specific bioactivity of the recombinant hormones in the Nb2 cell proliferation assay showed them to be comparable to the NIH and WHO human pituitary-derived standards.

Sequence homologies between nucleotide binding regions of CFTR and G-proteins suggest structural and functional similarities.

Sequence homology between the alpha-subunits of G-proteins and other GTP-binding proteins and certain regions within the nucleotide binding domains (NBDs) of cystic fibrosis transmembrane conductance regulator (CFTR) indicates that these protein structures may be similar. A sequence alignment of the NBDs of CFTR and NBDs from other membrane transporters, forms the basis of a structural model. This model predicts that one of the conserved sequences GGQR, within which a number of CF mutations occur, forms part of the nucleotide binding pocket and serves as an ON/OFF conformational switch as observed in GTP binding proteins. Furthermore, based on subtle sequence differences between the first and second NBDs of CFTR and from structure-activity data, we suggest that the nucleotide binding site environments of the two NBDs are different.

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro.

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.

Enhanced proliferation and differentiation of human articular chondrocytes when seeded at low cell densities in alginate in vitro.

Dedifferentiated human articular chondrocytes exhibited a wide variation in their capacity to proliferate and redifferentiate in an alginate suspension culture system. The greatest extent of proliferation and redifferentiation was seen to be dependent on the formation of clonal populations of chondrocytes and correlated inversely with the initial cell seeding density. Redifferentiating chondrocytes seeded at low density (1 x 10(4) cells/ml alginate) compared with chondrocytes that were seeded at high density (1 x 10(6) cells/ml alginate) showed a nearly 3-fold higher median increase in cell number. a 19-fold greater level of type-II collagen mRNA expression, a 4-fold greater level of aggrecan mRNA expression, and a 6-fold greater level of sulfated glycosaminoglycan deposition at 4 weeks of culture. Matrix molecules from low-density cultures were assembled into chondrocyte-encapsulated, spherical extracellular matrices that were readily visualized in sections from 12-week cultures stained with antibodies against types I and II collagen and aggrecan. Ultrastructural analysis of 12-week low-density cultures confirmed the presence of thin collagen fibrils throughout the matrix.

Osteogenesis in rats with an inductive bovine composite.

Subcutaneous (S.C.) implantation of allogeneic demineralized bone matrix in rats results in endochondral bone formation. In contrast, implants of bovine demineralized bone matrix in rat S.C. tissue show inconsistent cartilage and bone formation, presumably due to an intense inflammatory reaction at the implant site. To overcome this response, a partially purified bone inducing extract was prepared from bovine bone by a series of steps that included demineralization, guanidine/HCl extraction, gel filtration, and cation exchange chromatography. To develop a carrier, the inactive guanidine/HCl-extracted matrix was then trypsinized to remove the inflammatory and immunogenic components, thus yielding a predominantly collagenous matrix. Bovine composites were prepared by combining different amounts of the bone inducing extract with a carrier that consisted of the trypsinized bone matrix and purified soluble bovine dermal collagen. Subcutaneous implantation of the composite preparation resulted in dose-dependent endochondral bone formation in rats. The inductive activity and the low-level inflammatory response were comparable to allogeneic implants.

Locally delivered rhTGF-beta2 enhances bone ingrowth and bone regeneration at local and remote sites of skeletal injury.

The purposes of the present study were to determine if recombinant human transforming growth factor-beta-2 (rhTGF-beta2) enhances bone ingrowth into porous-coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3-mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF-beta2 in buffer at a dose per implant of 1.2 microg (n = 6), 12 microg (n = 7), or 120 microg (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non-TGF-beta treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15%, of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF-beta treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 microg/implant group (a 2.2-fold increase over the paired internal control (P = 0.004) and a 4-fold increase over the external control (P < 0.001)). The TGF-beta treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 microg groups (1.8-fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8-fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9-fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7-fold increase. P = 0.008). Thus, application of rhTGF-beta2 to a porous-coated implant-stimulated local bone ingrowth and gap healing in a weakly dose-dependent manner and stimulated bone regeneration in the 3-mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor.

Alternative delivery of keratinocytes using a polyurethane membrane and the implications for its use in the treatment of full-thickness burn injury.

The Epicel ASAProgram service generates autologous keratinocyte grafts used for the closure of full-thickness wounds in moderately and severely burned patients. The manufacturing process used to generate Epicel service autografts (ESA) is based upon the keratinocyte co-culture technique described by Rheinwald and Green which employs murine Swiss 3T3/J2 fibroblasts as feeder cells. Recently, a technique has been described that employs a polyurethane wound dressing, HydroDerm (HD, Innovative Technologies, Ltd), as a delivery vehicle for cultured keratinocytes intended for autologous grafting. We have examined the practical feasibility of this technique and report on testing the ability of HD to support keratinocyte growth and epithelium formation in vitro, at the air-liquid interface (ALI), and in vivo, after grafting to full-thickness wounds created on the backs of athymic (Swiss Nu/Nu) mice. The results demonstrate that keratinocytes grow on the HD dressing in Gibco SFM at a rate that is approximately 15 per cent of that observed when cells are cultivated on tissue culture (TC) plastic using standard techniques, yet the cells retain their proliferative capacity and form an epithelium in vitro when cultivated at the ALI on a dermal substrate. Keratinocyte-seeded HD membranes were also transferred to full-thickness wounds in athymic mice. Animals grafted with cells seeded to HD developed human epithelium, as revealed by species-specific detection of involucrin and evolved a normal attachment to the wound substratum, as demonstrated through the expression of dermally opposed laminin and alpha 6 beta 4 integrin. The ability of keratinocytes to maintain proliferative potential after seeding onto HD and their ability to form a properly oriented epithelium in vitro and in vivo suggests that this wound dressing may be useful as a vehicle for autologous keratinocyte grafting and help to provide earlier epithelial coverage to the burned patient. However, because of the slow proliferation rate of keratinocytes on HydroDerm, timely graft delivery would be best achieved by combining cell expansion via the Rheinwald and Green culture system, followed by the seeding of cells onto HydroDerm in a reduced calcium medium for subsequent autologous grafting.

Transgenic expression of a variant of human tissue-type plasminogen activator in goat milk: purification and characterization of the recombinant enzyme.

A glycosylation variant of human tissue-type plasminogen activator (tPA) designated longer-acting tissue-type plasminogen activator (LAtPA) was extensively purified from the milk of a transgenic goat by a combination of acid fractionation, hydrophobic interaction chromatography and immunoaffinity chromatography. This scheme provided greater than 8,000-fold purification of the protein, a cumulative yield of 25% and purity greater than 98% as judged by SDS gel electrophoresis. SDS gel electrophoresis revealed that the transgenic enzyme was predominantly the "two chain" form of the protease. The specific activity of the purified transgenic protein, based on the average of the values obtained for three different preparations, was 610,000 U/mg as judged by amidolytic activity assay. This was approximately 84% of the value observed for the recombinant enzyme produced in mouse C127 cells. Analysis of the transgenic protein indicated that it had a significantly different carbohydrate composition from the recombinant enzyme produced in C127 cells. Molecular size analysis of the oligosaccharides from the transgenic and C127 cell-derived LAtPA preparations confirmed their differences and showed that the mouse cell-derived preparation contained larger, complex-type N-linked oligosaccharide structures than the material produced in goat mammary tissue.

The utility of collagen-based vehicles in delivery of growth factors for hard and soft tissue wound repair.

Bovine demineralized bone powder and reconstituted bovine dermal collagen have been effectively utilized during the past several years to deliver a variety of growth factors in animal models of hard and soft tissue wound repair. Bone morphogenetic proteins have been delivered in a demineralized bone powder matrix to promote ectopic bone formation in the rat subcutaneous model with the objective of studying the process of endochondral bone formation and evaluating the utility of such factors in promoting repair of hard tissue defects. Reconstituted bovine dermal collagen gels and sponges, including composites of collagen and heparin, have been utilized to deliver growth factors such as platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-beta) and fibroblast growth factor (FGF) to study their effects in subcutaneous and incisional models of dermal wound repair. The results of these experimental animal studies have provided convincing evidence that the rheological properties, biocompatibility and resorbable nature of type I collagen make it an excellent delivery vehicle for evaluation of a variety of growth factors in human clinical studies of hard and soft tissue would repair.

In vitro phosphorylation of type I collagen by cyclic AMP-dependent protein kinase.

Both the triple-helical and denatured forms of nonfibrillar bovine dermal type I collagen were tested as substrates for the catalytic subunit of cAMP-dependent protein kinase in an in vitro reaction. Native, triple-helical collagen was not phosphorylated, but collagen that had been thermally denatured into individual alpha chains was a substrate for the protein kinase. Catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured collagen to between 3 to 4 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Pepsin-solubilized and intact collagens were phosphorylated similarly, as long as each was in a nonhelical conformation. The first 2 mol of phosphate incorporated into type I collagen by the protein kinase were present in the alpha 2(I) chain. The alpha 1(I) chain was only phosphorylated during long incubations in which the stoichiometry exceeded 2 mol of phosphate/mol of (alpha 1(I)2 alpha 2(I). Phosphoserine was the only phosphoamino acid identified in collagen that had been phosphorylated to any degree by the protein kinase. The 2 mol of phosphate incorporated into the alpha 2(I) chain were localized to the alpha 2(I)CB4 cyanogen bromide fragment. The catalytic subunit of cAMP-dependent protein kinase phosphorylated denatured pepsin-solubilized collagen with a Km of 8 microM and a Vmax of approximately 0.1 mumol/min/mg of enzyme. Denatured, but not triple-helical, type I collagen was also phosphorylated by cGMP-dependent protein kinase, although it was a poorer substrate for this enzyme than for the cAMP-dependent protein kinase. Collagen was not a substrate for phospholipid-sensitive Ca2+-dependent protein kinase. These results suggest the potential for nascent alpha chains of type I collagen to be susceptible to phosphorylation by cAMP-dependent protein kinase in vivo prior to triple-helix formation. Such a phosphorylation of collagen could be relevant to the action of cAMP to increase the intracellular degradation of newly synthesized collagen.