Proteoglycan expression during transforming growth factor beta -induced keratocyte-myofibroblast transdifferentiation.

Keratocytes of the corneal stroma secrete a unique population of proteoglycan molecules considered essential for corneal transparency. In healing corneal wounds, keratocytes exhibit a myofibroblastic phenotype in response to transforming growth factor beta (TGF-beta), characterized by expression of alpha-smooth muscle actin. This study examined proteoglycan and collagen expression by keratocytes in vitro during the TGF-beta-induced keratocyte-myofibroblast transition. TGF-beta-treated primary bovine keratocytes developed myofibroblastic features, including actin stress fibers anchored to paxillin-containing focal adhesions, cell-associated fibronectin, alpha(5) integrin, and alpha-smooth muscle actin. Collagen I and III protein and mRNA increased in response to TGF-beta. Secretion of [(35)S]sulfate-labeled keratan sulfate proteoglycans decreased markedly in response to TGF-beta. Dermatan sulfate proteoglycans, however, increased in size and abundance. Protein and mRNA transcripts for normal stromal proteoglycans (lumican, keratocan, mimecan, and decorin) all decreased in response to TGF-beta, but protein expression and mRNA for biglycan, a proteoglycan present in fibrotic tissue, was markedly up-regulated. These results show that TGF-beta in vitro induces a proteoglycan expression pattern similar to that of corneal scars in vivo. This altered proteoglycan expression occurred coordinately with transdifferentiation of keratocytes to the myofibroblastic phenotype, implicating these cells as the source of fibrotic tissue in nontransparent corneal scars.

Synthesis of corneal keratan sulfate proteoglycans by bovine keratocytes in vitro.

Keratan sulfate proteoglycans (KSPGs) are the major proteoglycans of the cornea and are secreted by keratocytes in the corneal stroma. Previous studies have been able to show only transient secretion of KSPG in cell culture. In this study, cultures of bovine keratocytes were found to secrete the three previously characterized KSPG proteins into culture medium. Reactivity with monoclonal antibody I22 demonstrated substitution of these proteins with keratan sulfate chains. KSPG constituted 15% of the proteoglycan metabolically labeled with [35S]sulfate in keratocyte culture medium. This labeled KSPG contained keratan sulfate chains of 4700 Da compared to 21,000 Da for bovine corneal keratan sulfate. Labeled keratan sulfate from cultures contained nonsulfated, monosulfated, and disulfated disaccharides that were released by digestion with endo-beta-galactosidase or keratanase II. Nonsulfated disaccharides were relatively more abundant in keratan sulfate from culture than in corneal keratan sulfate. These results show that cultured bovine keratocytes maintain the ability to express all three of the known KSPG proteins, modified with keratan sulfate chains and sulfated on both N-acetylglucosamine and galactose moieties. KSPG made in vitro differs from that found in vivo in the length and sulfation of its keratan sulfate chains. The availability of cell cultures secreting corneal keratan sulfate proteoglycans provides an opportunity to examine biosynthesis and control of this important class of molecules.

Transduction of CD34-enriched human peripheral and umbilical cord blood progenitors using a retroviral vector with the Fanconi anemia group C gene.

BACKGROUND: Fanconi anemia (FA) is an autosomal recessive inherited form of bone marrow failure. FA cells are characterized by their extreme sensitivity to DNA cross-linking agents that cause DNA instability and cell death. Four genetic complementation groups for FA have been identified and the gene for the complementation C group (FACC) has been cloned. Genetic transfer of the FACC gene should provide a growth advantage in transduced hematopoietic cells. We have previously demonstrated efficient retroviral-mediated gene transduction and correction of FA(C) cell lines and peripheral blood-derived CD34+ progenitors from patients carrying mutant FACC alleles. In this report we sought to define the optimal conditions for transduction of CD34+ progenitors from mobilized peripheral blood and umbilical cord blood. METHODS: Peripheral blood hematopoietic progenitors were obtained by G-CSF mobilization followed by apheresis. Human fetal cord blood cells were obtained from full-term gestation deliveries. Cells were immunoselected for CD34 antigen expression and then incubated with recombinant retroviruses containing a selectable marker gene (neomycin). Recombinant colony stimulating factors were added to facilitate viral transduction. Cells were plated in methylcellulose and resulting hematopoietic colonies were isolated and analyzed by PCR. RESULTS: Transduction efficiency of peripheral blood progenitors (from normal individuals) using a retrovirus encoding the FACC cDNA was comparable to that of the retroviral producer G1Na.40 currently being used in clinical gene therapy marking studies. We extended our standard transduction protocol to analyze CD34+ and CD34+ CD38-subpopulations of progenitors derived from umbilical cord blood (from normal pregnancies). In addition, we tested whether FACC cDNA transduction could be improved by vector infection supported by autologous stroma. For FA(C) hematopoietic cell infection, vector supernatant transduction in the presence of recombinant human IL-3, IL-6, and SCF was found to be superior to transduction supported by autologous FA(C) patient stroma. CONCLUSIONS: We documented efficient retroviral transduction of umbilical cord blood and peripheral blood enriched for hematopoietic progenitor cells. These results suggest the feasibility of a clinical gene therapy protocol utilizing progenitor cells from both peripheral blood and umbilical cord blood.

Retroviral vector for gene therapy of X-linked severe combined immunodeficiency syndrome.

X-linked severe combined immunodeficiency syndrome (X-SCID) is a genetic disorder characterized by profound impairment of cell-mediated and humoral immunity. Affected children die of recurrent infections within 2 years of birth unless rescued by allogeneic transplantation from a suitable donor. Recently, the genetic defect responsible for X-linked SCID has been identified as a mutation in the gamma chain of the IL-2 receptor, a protein also shared by the IL-4 and IL-7 receptors and therefore now denoted the common gamma chain (gamma c). We report here the development of a high-titer amphotropic retroviral vector for transfer of gamma c. This vector was used to transfer a copy of the gamma c cDNA to murine 3T3 fibroblasts, CD34-enriched hematopoietic progenitor cells obtained from bone marrow and umbilical cord blood of normal donors, and to transplanted murine bone marrow progenitors. Murine 3T3 cells transduced by the retroviral vector were analyzed by Southern blot hybridization and Western transfer. Southern analysis confirmed the integration of unrearranged proviral DNA, and Western blot analysis demonstrated the expression of gamma c protein. CD34-enriched cells were infected with viral vectors bearing gamma c and grown in methylcellulose media. Individual colonies and pools of cells were analyzed 2 weeks later by polymerase chain reaction assay, which confirmed the proviral marking. The vector was also used to transfer a copy of the gamma c cDNA to murine bone marrow cells in a transplantation model. Infected marrow was transplanted into syngeneic Balb/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Sequence and structural implications of a bovine corneal keratan sulfate proteoglycan core protein. Protein 37B represents bovine lumican and proteins 37A and 25 are unique.

Amino acid sequence from tryptic peptides of three different bovine corneal keratan sulfate proteoglycan (KSPG) core proteins (designated 37A, 37B, and 25) showed similarities to the sequence of a chicken KSPG core protein lumican. Bovine lumican cDNA was isolated from a bovine corneal expression library by screening with chicken lumican cDNA. The bovine cDNA codes for a 342-amino acid protein, M(r) 38,712, containing amino acid sequences identified in the 37B KSPG core protein. The bovine lumican is 68% identical to chicken lumican, with an 83% identity excluding the N-terminal 40 amino acids. Location of 6 cysteine and 4 consensus N-glycosylation sites in the bovine sequence were identical to those in chicken lumican. Bovine lumican had about 50% identity to bovine fibromodulin and 20% identity to bovine decorin and biglycan. About two-thirds of the lumican protein consists of a series of 10 amino acid leucine-rich repeats that occur in regions of calculated high beta-hydrophobic moment, suggesting that the leucine-rich repeats contribute to beta-sheet formation in these proteins. Sequences obtained from 37A and 25 core proteins were absent in bovine lumican, thus predicting a unique primary structure and separate mRNA for each of the three bovine KSPG core proteins.

Arterial lumican. Properties of a corneal-type keratan sulfate proteoglycan from bovine aorta.

A glycoprotein reactive with antibodies against corneal keratan sulfate proteoglycan (KSPG) was purified 300-fold from extracts of bovine aorta using DEAE ion-exchange, gel-filtration, hydrophobic interaction, and reverse-phase chromatographic separations. The intact glycoprotein was 70-80 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Deglycosylation with endo-beta-galactosidase and N-glycanase reduced the size to 48 and 37 kDa, respectively, similar to the large isoforms of corneal KSPG. N-terminal amino acid sequence of the arterial KSPG was identical with lumican, the 37B isoform of corneal KSPG, and the arterial KSPG reacted with an antibody to synthetic peptide duplicating this sequence. Arterial KSPG and corneal lumican displayed identical tryptic maps. Arterial lumican contains fucose and mannose in amounts similar to corneal KSPG, but galactose, glucosamine, and sulfate were reduced compared to KSPG from cornea. Treatment of arterial lumican with endo-beta-galactosidase released 8-9 mol of glucosamine and galactose per mol of protein as oligosaccharides. These eluted as neutral, nonsulfated oligosaccharides on high pH anion-exchange chromatography. The size of arterial lumican was not altered by glycosidases having specificity for sulfated keratan sulfate, nor was the charge of the lumican molecule altered by digestion with endo-beta-galactosidase. These data show arterial lumican to be a glycoprotein containing unsulfated lactosaminoglycan chains. Abundance of low sulfate lumican in many tissues indicates that this protein occurs predominantly as a glycoprotein rather than as the more widely studied, highly sulfated proteoglycan present in the cornea.

Unique glycosylation of three keratan sulfate proteoglycan isoforms.

Recent work demonstrates isoforms of bovine corneal keratan sulfate proteoglycan containing structurally unique core proteins of 25 and 37 kDa (Funderburgh, J., and Conrad, G. (1990) J. Biol. Chem. 265, 8297-8303). In the current study, two forms (37A and 37B) of the 37-kDa protein were separated by ion-exchange chromatography after removal of keratan sulfate with endo-beta-galactosidase. Keratan sulfate linkage sites in core proteins were labeled with UDP-[3H]galactose using galactosyltransferase. Labeled proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analyzed by tryptic digestion and reversed-phase chromatography. The 37A protein has three keratan sulfate-linkage sites, and the 37B and 25-kDa proteins each contain one linkage site. Reversed-phase tryptic maps of the three proteins differed in total peptide profile and in glycosylated peptides labeled with periodate-[3H]-NaBH4. Tryptic mapping of the two 37-kDa isoforms after deglycosylation showed differences in total tryptic peptides, in peptides labeled with [14C]iodoacetic acid, and in peptides recognized by antibodies to a mixture of the 37-kDa cores. Antibody to a synthetic peptide with N-terminal sequence obtained from mixed 37-kDa cores reacted exclusively with the 37B isoform. These results show that bovine corneal keratan sulfate proteoglycan has three different core proteins each with distinct glycosylation and unique primary structure.