Endostatin inhibits endochondral ossification.

BACKGROUND: Angiogenesis is essential for the replacement of cartilage by bone during skeletal growth and regeneration. Vascular endothelial growth factor-A (VEGF-A) is a key regulator of angiogenesis whereas endostatin, a potent inhibitor of endothelial cell proliferation and migration, is a natural antagonist of VEGF-A. The regulatory role of these peptides in angiogenesis and bone formation was investigated using adenoviral gene delivery of VEGF-A and endostatin in a mouse ectopic ossification model. METHODS: Bone formation was induced in the hamstring muscles of adult mice with native bone morphogenetic protein (BMP) extract implemented in gelatine gel together with VEGF-A and endostatin recombinant adenoviral vectors. The mice were sacrificed 1, 2, and 3 weeks after the operation and ectopic bone formation was followed radiographically and histologically. RESULTS: Significant bone formation was induced by BMP extract in all treatment groups. VEGF-A stimulated and endostatin prevented the formation of FVIII-related antigen-positive vessels as well as the number of cartilage-resorbing chondroclasts/osteoclasts. Endostatin alone or in conjugation with VEGF-A reduced bone formation. Excess of VEGF-A stimulated and endostatin reduced bone formation, respectively, at the 3-week time point. CONCLUSIONS: Our findings indicate that endostatin retards the cartilage phase in endochondral ossification which subsequently reduces bone formation in our experimental model. We conclude that bone growth and healing, which share features with ectopic bone formation, may be regulated by endostatin.

Adenoviral gene transfer restores lysyl hydroxylase activity in type VI Ehlers-Danlos syndrome.

Type VI Ehlers-Danlos syndrome is a disease characterized by disturbed lysine hydroxylation of collagen. The disease is caused by mutations in lysyl hydroxylase 1 gene and it affects several organs including the cardiovascular system, the joint and musculoskeletal system, and the skin. The skin of type VI Ehlers-Danlos syndrome patients is hyperelastic, scars easily, and heals slowly and poorly. We hypothesized that providing functional lysyl hydroxylase 1 gene to the fibroblasts in and around wounds in these patients would improve healing. In this study we tested the feasibility of transfer of the lysyl hydroxylase 1 gene into fibroblasts derived from rats and a type VI Ehlers-Danlos syndrome patient (in vitro) and into rat skin (in vivo). We first cloned human lysyl hydroxylase 1 cDNA into a recombinant adenoviral vector (Ad5RSV-LH). Transfection of human type VI Ehlers-Danlos syndrome fibroblasts (about 20% of normal lysyl hydroxylase 1 activity) with the vector increased lysyl hydroxylase 1 activity in these cells to near or greater levels than that of wild type, unaffected fibroblasts. The adenoviral vector successfully transfected rat fibroblasts producing both beta-galactosidase and lysyl hydroxylase 1 gene activity. We next expanded our studies to a rodent model. Intradermal injections of the vector to the abdominal skin of rats produced lysyl hydroxylase 1 mRNA and elevated lysyl hydroxylase 1 activity, in vivo. These data suggest the feasibility of gene replacement therapy to modify skin wound healing in type VI Ehlers-Danlos syndrome patients.

rab5 GTPase regulates adenovirus endocytosis.

Adenovirus interaction with alphav integrins is important for virus entry. We have examined the effects of adenovirus attachment on intracellular signaling in HeLa cells, with an emphasis on pathways known to be activated following integrin interaction with other ligands. We found no evidence for [Ca(2+)](c)-mediated signaling or for tyrosine phosphorylation of pp125(FAK), p130(CAS), and paxillin. However, adenovirus attachment is known to activate phosphatidylinositol-3 kinase, which in turn may regulate endocytosis via rab5 GTPase. We found that adenovirus uptake was increased by overexpression of wild-type rab5 and decreased by dominant-negative rab5. These results indicate a role for rab5 in adenovirus entry.

A compound heterozygote patient with Ehlers-Danlos syndrome type VI has a deletion in one allele and a splicing defect in the other allele of the lysyl hydroxylase gene.

We report the first deletion mutation and the first splicing defect in the lysyl hydroxylase gene in a compound heterozygote patient with Ehlers-Danlos syndrome type VI with markedly reduced lysyl hydroxylase activity. Northern analysis of the RNA isolated from skin fibroblasts of the patient demonstrated the presence of a truncated lysyl hydroxylase mRNA. PCR and sequence analysis confirmed the truncation and indicated that the cells contain two types of shortened mRNAs, one lacking the sequences corresponding to exon 16 and the other lacking that corresponding to exon 17 of the lysyl hydroxylase gene. Analysis of genomic DNA revealed deletion of the penultimate adenosine from the 3' end of intron 15 from one allele. This defect was probably responsible for the skipping of exon 16 sequences from the transcript. The other allele, inherited from the mother, contains an Alu-Alu recombination with a deletion of about 3,000 nucleotides from the gene; this abnormality explains the lack of exon 17 sequences. The identified mutations in exon 16 and exon 17 do not alter the reading frame of the transcripts.

An interaction between penton base and alpha v integrins plays a minimal role in adenovirus-mediated gene transfer to hepatocytes in vitro and in vivo.

Studies in cultured cell lines have shown that adenovirus infection involves binding of adenovirus fiber to its cell surface receptor and binding of penton base to alpha v integrins. However, much less is known about the role of these interactions in cells that are targets for adenovirus-mediated gene transfer. Earlier work showed that hepatocytes are readily infected by adenovirus, making them an attractive target for gene therapy in several diseases. We found that addition of fiber protein blocked adenovirus infection of primary cultures of hepatocytes. This suggests an important role for fiber and its receptor. However, mutation of the integrin-binding motif in penton base did not inhibit infection of hepatocytes, even though the mutation impaired infection of HeLa cells. Hepatocytes had undetectable amounts of alpha v integrins on their cell surface and showed no specific adherence to vitronectin, the natural substrate of alpha v integrins. Adenovirus with an intact penton base enhanced infection of liver following intravenous injection, but only by three-fold as compared with virus in which the integrin-binding motif was disrupted. These studies suggest that interactions between cell surface integrins and penton base are not required for adenovirus infection of hepatocytes in vitro, but the interaction enhances infection to a small degree in vivo.

Cloning of human lysyl hydroxylase: complete cDNA-derived amino acid sequence and assignment of the gene (PLOD) to chromosome 1p36.3----p36.2.

Lysyl hydroxylase (EC 1.14.11.4), an alpha 2 dimer, catalyzes the formation of hydroxylysine in collagens by the hydroxylation of lysine residues in peptide linkages. A deficiency in this enzyme activity is known to exist in patients with the type VI variant of the Ehlers-Danlos syndrome, but no amino acid sequence data have been available for the wildtype or mutated human enzyme from any source. We report the isolation and characterization of cDNA clones for lysyl hydroxylase from a human placenta lambda gt11 cDNA library. The cDNA clones cover almost all of the 3.2-kb mRNA, including all the coding sequences. These clones encode a polypeptide of 709 amino acid residues and a signal peptide of 18 amino acids. The human coding sequences are 72% identical to the recently reported chick sequences at the nucleotide level and 76% identical at the amino acid level. The C-terminal region is especially well conserved, a 139-amino-acid region, residues 588-727 (C-terminus), being 94% identical between the two species and a 76-amino-acid region, residues 639-715, 99% identical. These comparisons, together with other recent data, suggest that lysyl hydroxylase may contain functionally significant sequences especially in its C-terminal region. The human lysyl hydroxylase gene (PLOD) was mapped to chromosome 1 by Southern blot analysis of human-mouse somatic cell hybrids, to the 1p34----1pter region by using cell hybrids that contain various translocations of human chromosome 1, and by in situ hybridization to 1p36.2----1p36.3. This gene is thus not physically linked to those for the alpha and beta subunits of prolyl 4-hydroxylase, which are located on chromosomes 10 and 17, respectively.