Biomedical subjects

B R Olsen

Publications and source records attributed to B R Olsen.

At least 19 recordsLinked to original sources

PubMed2002

Delivery of plasmid DNA to articular chondrocytes via novel collagen-glycosaminoglycan matrices.

Our primary objective was to fabricate a porous gene-supplemented collagen-glycosaminoglycan (GSCG) matrix for sustained delivery (over a period of several weeks) of plasmid DNA to articular chondrocytes when implanted into cartilage lesions. The specific aims of this in vitro study were to determine the release kinetics profiles of plasmid DNA from the GSCG matrices, and to determine the ability of the released plasmid DNA to transfect adult canine articular chondrocytes. In particular, we evaluated the effects of two variables, cross-linking treatment and the pH at which the DNA was incorporated into the matrices, on the amount of the plasmid DNA that remained bound to the GSCG matrices after passive (nonenzymatic) leaching and on the expression of a reporter gene in articular chondrocytes grown in the GSCG matrices. Collagen-glycosaminoglycan matrices were synthesized without cross-linking, and by three cross-linking treatments: dehydrothermal (DHT) treatment, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) treatment, and exposure to ultraviolet (UV) radiation. The plasmid DNA was incorporated into the collagen-glycosaminoglycan matrices in solutions at pH 2.5 or 7.5. Transmission electron microscopy studies revealed plasmid DNA bound to the walls of the porous GSCG matrices. In general, the GSCG matrices fabricated at pH 2.5 retained a larger fraction of the initial DNA load after 28 days of incubation in Tris-EDTA buffer. The passive, solvent-mediated release of the plasmid DNA from the GSCG matrices showed a biphasic pattern consisting of a faster, early release rate over the initial 8 hr of leaching followed by a slower, late release rate that was relatively constant over the subsequent 28 days of leaching. Electrophoretic analyses revealed that the plasmid DNA released from the GSCG matrices fabricated at pH 2.5 had been linearized and/or degraded; whereas the plasmid DNA leached from the GSCG matrices prepared with a DNA solution at pH 7.5 was primarily supercoiled and linear. Plasmid DNA released from all GSCG matrix formulations was able to generate luciferase reporter gene expression in monolayer-cultured chondrocytes transfected with the aid of a commercial lipid reagent, and in chondrocytes cultured in the GSCG matrices without the aid of a supplemental transfection reagent. Luciferase expression in chondrocyte-seeded GSCG constructs was evident throughout the culture period (28 days), with the EDC and UV cross-linked matrices prepared at pH 7.5 providing the highest transgene expression levels. We conclude that released plasmid DNA continually transfected canine articular chondrocytes seeded into GSCG matrices in vitro for a 4-week period as evidenced by luciferase reporter gene expression. Thus, GSCG matrices can be fabricated to provide sustained release of plasmid DNA carrying a potential therapeutic gene.

Animals↗

PubMed2001

LDL receptor-related protein 5 (LRP5) affects bone accrual and eye development.

In humans, low peak bone mass is a significant risk factor for osteoporosis. We report that LRP5, encoding the low-density lipoprotein receptor-related protein 5, affects bone mass accrual during growth. Mutations in LRP5 cause the autosomal recessive disorder osteoporosis-pseudoglioma syndrome (OPPG). We find that OPPG carriers have reduced bone mass when compared to age- and gender-matched controls. We demonstrate LRP5 expression by osteoblasts in situ and show that LRP5 can transduce Wnt signaling in vitro via the canonical pathway. We further show that a mutant-secreted form of LRP5 can reduce bone thickness in mouse calvarial explant cultures. These data indicate that Wnt-mediated signaling via LRP5 affects bone accrual during growth and is important for the establishment of peak bone mass.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Intravital microscopy reveals novel antivascular and antitumor effects of endostatin delivered locally by alginate-encapsulated cells.

The current study describes new, antivascular, and antitumor effects of human endostatin. A novel system for continuous, localized delivery of antiangiogenic compounds to brain tumors was used. The delivery system was composed of endostatin-producing 293 cells encapsulated into immuno-isolating sodium alginate. Intravital multifluorescence microscopy was used to assess vascular and antitumor effects of endostatin in C6 glioma spheroids implanted into an ectopic as well as an orthotopic setting. Analysis of total and functional vascular density, microvascular diameters, vessel perfusion, tumor growth, and tumor cell migration were performed repetitively. Tumor growth was reduced by 35% in treated animals. It was of interest that tumor cell invasion into the surrounding tissue was also inhibited. The total vascular density was reduced by 67.6%, perfusion by 67%, and vessel diameters by 37%. This resulted in a significant reduction in tumor perfusion, although the vessel permeability was not influenced. We have demonstrated that human endostatin not only reduces total vascular density, as shown previously, but also greatly reduces the functionality and the diameters of the vessels. Furthermore, we show that this therapeutic approach also inhibits tumor cell invasion, thus supporting the hypothesis that tumor angiogenesis and invasion represent two interrelated processes. Finally, this work further confirms the new therapeutic concept using alginate cell-encapsulation technology for the localized delivery of therapeutic compounds to central nervous system malignancies.

Alginates↗

PubMed2001

A nonsense mutation in MSX1 causes Witkop syndrome.

Witkop syndrome, also known as tooth and nail syndrome (TNS), is a rare autosomal dominant disorder. Affected individuals have nail dysplasia and several congenitally missing teeth. To identify the gene responsible for TNS, we used candidate-gene linkage analysis in a three-generation family affected by the disorder. We found linkage between TNS and polymorphic markers surrounding the MSX1 locus. Direct sequencing and restriction-enzyme analysis revealed that a heterozygous stop mutation in the homeodomain of MSX1 cosegregated with the phenotype. In addition, histological analysis of Msx1-knockout mice, combined with a finding of Msx1 expression in mesenchyme of developing nail beds, revealed that not only was tooth development disrupted in these mice, but nail development was affected as well. Nail plates in Msx1-null mice were defective and were thinner than those of their wild-type littermates. The resemblance between the tooth and nail phenotype in the human family and that of Msx1-knockout mice strongly supports the conclusions that a nonsense mutation in MSX1 causes TNS and that Msx1 is critical for both tooth and nail development.

Adult↗

PubMed2001

Autosomal dominant craniometaphyseal dysplasia is caused by mutations in the transmembrane protein ANK.

Craniometaphyseal dysplasia (CMD) is a rare skeletal disorder characterized by progressive thickening and increased mineral density of craniofacial bones and abnormally developed metaphyses in long bones. Linkage studies mapped the locus for the autosomal dominant form of CMD to an approximately 5-cM interval on chromosome 5p, which is defined by recombinations between loci D5S810 and D5S1954. Mutational analysis of positional candidate genes was performed, and we describe herein three different mutations, in five different families and in isolated cases, in ANK, a multipass transmembrane protein involved in the transport of intracellular pyrophosphate into extracellular matrix. The mutations are two in-frame deletions and one in-frame insertion caused by a splicing defect. All mutations cluster within seven amino acids in one of the six possible cytosolic domains of ANK. These results suggest that the mutated protein has a dominant negative effect on the function of ANK, since reduced levels of pyrophosphate in bone matrix are known to increase mineralization.

Amino Acid Sequence↗

PubMed2001

A double-deletion mutation in the Pitx3 gene causes arrested lens development in aphakia mice.

The recessive aphakia (ak) mouse mutant is characterized by bilateral microphthalmia due to a failure of lens morphogenesis. We fine-mapped the ak locus to the interval between D19Umi1 and D19Mit9, developed new polymorphic markers, and mapped candidate genes by construction of a BAC contig. The Pitx3 gene, known to be expressed in lens primordia, shows zero recombination with the ak mutation on our intersubspecific intercross panel representing 1170 meioses. A recent report described a deletion in the intergenic region between Gbf1 and Pitx3 as the possible ak mutation. Our results differ in that we find not only the distant intergenic deletion, but also a much larger deletion directly in the Pitx3 gene, eliminating exon 1 and extending into intron 1 and the promoter region. Pitx3 transcript levels are severely reduced in ak/ak mice from E11.5 to newborn (5 +/- 1% of the wildtype levels at E13.5), while an involvement of the flanking Gbf1 and Cig30 genes in the aberrant lens development is highly unlikely based on expression analysis. We conclude that the ak mutation consists of two deletions, the larger of which removes part of Pitx3, indicating a crucial role of this gene in early lens development.

Acetyltransferases↗

PubMed2001

Clinical genetics of familial keloids.

BACKGROUND: Keloids are proliferative fibrous growths that result from an excessive tissue response to skin trauma. Most keloids occur sporadically, but some cases are familial. However, the genetics of keloid formation have only rarely been documented, and the mode of inheritance is not known. OBJECTIVE: To elucidate the clinical genetic characteristics of keloid wound-healing disorder. OBSERVATIONS: We studied the clinical and genetic characteristics of 14 pedigrees with familial keloids. The ethnicity of these families is mostly African American (n = 10), but also white (n = 1), Japanese (n = 2), and African Caribbean (n = 1). The pedigrees account for 341 family members, of whom 96 displayed keloids. Of the affected family members, 36 are male and 60 are female. The age of onset varies from early childhood to late adulthood. There is variable expression of keloids within the same families: some affected members have only minor earlobe keloids, whereas others have very severe keloids affecting large areas of the body. In the described pedigrees, 7 individuals are obligate unaffected carriers, revealing nonpenetrance in about 6.8% of keloid gene carriers. Syndromes associated with keloids, namely Rubinstein-Taybi and Goeminne syndrome, were not found in these families. Additionally, linkage to the gene loci of these syndromes and X-chromosomal linkage were excluded. CONCLUSIONS: The pattern of inheritance observed in these families is consistent with an autosomal dominant mode with incomplete clinical penetrance and variable expression. This is the most comprehensive collection of keloid families described to date, and it allows for the first time the elucidation of the clinical genetic characteristics of the familial form of this wound-healing disorder.

Adolescent↗

PubMed2001

The mechanism of palatal clefting in the Col11a1 mutant mouse.

The occurrence of cleft palate in mutant mice offers an opportunity to understand the possible role of specific genes in palatogenesis. Here, cleft palate in mice carrying the chondrodysplasia (cho) defect, which consists of an autosomal-recessive mutation in the collagen gene Col11a1, was investigated. The proposed cause of cleft palate in cho homozygous mice is failure of the palatal shelves to adhere and make contact due to mandibular growth abnormalities. Another cause of cleft palate that has recently been demonstrated in other animal models is failure of the midline epithelial seam forming between the shelves to undergo epithelial-mesenchymal transformation (EMT). The present strategy to test the likelihood of this second possibility was to culture the unfused cho/cho palatal shelves at different stages of development to see if they were capable of adhering and undergoing EMT in vitro. By using carboxydichlorofluorescein succinimidyl ester to trace the fate of the medial-edge epithelium (MEE), it was shown that cho/cho palates have full potential for MEE adherence and EMT up to embryonic day 17.5/18.5, when epithelia keratinize before birth, preventing the adherence of both the normal and homozygous palatal shelves. Thus, the major effect of the mutant collagen gene on the palate is likely to be via mandibular growth disruption. The possibility that unfused palatal shelves in other clinical syndromes can adhere and undergo EMT if brought into contact at appropriate times before birth has important therapeutic implications.

Animals↗

PubMed2001

Mouse models of abnormal skeletal development and homeostasis.

Studies of a number of mouse mutations with skeletal defects have contributed significantly to the understanding of bone development and homeostasis. In many cases, such mutants are also genetic models of disorders in humans, characterized by reduced bone mass (osteoporosis), increased bone mass (osteopetrosis), or abnormalities in endochondral ossification (chondrodysplasias).

Animals↗

PubMed2001

Tissue specific regulation of VEGF expression during bone development requires Cbfa1/Runx2.

Vascular endothelial growth factor (VEGF) is a critical regulator of angiogenesis during development, but little is known about the factors that control its expression. We provide the first example of tissue specific loss of VEGF expression as a result of targeting a single gene, Cbfa1/Runx2. During endochondral bone formation, invasion of blood vessels into cartilage is associated with upregulation of VEGF in hypertrophic chondrocytes and increased expression of VEGF receptors in the perichondrium. This upregulation is lacking in Cbfa1 deficient mice, and cartilage angiogenesis does not occur. Finally, over-expression of Cbfa1 in fibroblasts induces an increase in their VEGF mRNA level and protein production by stimulating VEGF transcription. The results demonstrate that Cbfa1 is a necessary component of a tissue specific genetic program that regulates VEGF during endochondral bone formation.

3T3 Cells↗

PubMed2001

Expression of frizzled genes in mouse costochondral chondrocytes.

Protein products of frizzled genes are cell membrane receptors for Wnt proteins that play multiple roles during development. We examined the expression of nine frizzled genes in mouse chondrocytes, and detected transcripts of six of the nine genes. We also cloned the entire cDNA of mouse frizzled-1 and compared its cDNA sequence and the cysteine-rich and transmembrane domains of its translated product to sequences of frizzled-1 from C. elegans, Drosophila, chicken and human. We used the T31 Mouse/Hamster radiation hybrid panel to map the mouse frizzled-1 to mouse chromosome 5 (5 cM from the centromere), and frizzled-9 to mouse chromosome 5 (74 cM from the centromere).

Amino Acid Sequence↗

PubMed2001

The role of collagen-derived proteolytic fragments in angiogenesis.

Basement membrane molecules and fragments derived from them are regulators of biological activities such as cell growth, differentiation and migration. This review describes proteolytically derived fragments from the non-collagenous (NC1) domain at the C-terminus of the basement membrane collagens type IV, XV and XVIII, which have been implicated as regulators of angiogenesis. Endostatin is an endogenous collagen XVIII/NC1 derivative, inhibiting endothelial cell proliferation and migration in vitro and tumor-growth in vivo. A homologous NC1 domain fragment of type XV collagen has anti-angiogenic activity as well. Furthermore, NC1 domain fragments of the most abundant basement membrane collagen, type IV collagen, have been shown to inhibit induced vessel growth.

Animals↗

PubMed2001

Local endostatin treatment of gliomas administered by microencapsulated producer cells.

We describe a technique for the treatment of malignant brain tumors based on local delivery of the anti-angiogenic protein endostatin from genetically engineered cells encapsulated in ultrapure sodium alginate. Alginate consists of L-guluronic and D-mannuronic acid, which in the presence of divalent cations forms an extended gel network, in which cells reside and remain immunoisolated, when implanted into the rat brain. Here, we show that endostatin-transfected cells encapsulated in alginate maintain endostatin secretion for at least four months after intracerebral implantation in rats. During the implantation period 70% of the encapsulated cells remained viable, as opposed to 85% in in vitro-cultured capsules. Rats that received transplants of BT4C glioma cells, together with endostatin-producing capsules (0.2 microg/ml per capsule), survived 84% longer than the controls. The endostatin released from the capsules led to an induction of apoptosis, hypoxia, and large necrotic avascular areas within 77% of the treated tumors, whereas all the controls were negative. The encapsulation technique may be used for many different cell lines engineered to potentially interfere with the complex microenvironment in which tumor and normal cells reside. The present work may thus provide the basis for new therapeutic approaches toward brain tumors.

Alginates↗

PubMed2001

Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism.

Cherubism (MIM 118400) is an autosomal dominant inherited syndrome characterized by excessive bone degradation of the upper and lower jaws followed by development of fibrous tissue masses, which causes a characteristic facial swelling. Here we describe seven mutations in the SH3-binding protein SH3BP2 (MIM 602104) on chromosome 4p16.3 that cause cherubism.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Linkage disequilibrium narrows locus for venous malformation with glomus cells (VMGLOM) to a single 1.48 Mbp YAC.

Venous malformations with glomus cells are localised cutaneous lesions of vascular dysmorphogenesis. They are usually sporadic, but sometimes familial. Using five families, we mapped the locus, VMGLOM, to chromosome 1p21-p22. In order to refine this locus, spanning 4-6 Mbp, we then studied seven additional families. They exhibited linkage to VMGLOM and the combined lod score for all 12 families was 18.41 at theta = 0.0 for marker D1S188. We found a distinct haplotype shared by seven families, comprising seven alleles which are rare in the general population (P < 0.01). This indicates that the haplotype is identical by descent in all seven families, and hence the locus can be refined by inferring ancestral crossovers. Using this approach, we position the causative gene between two markers on the same non-chimeric YAC of 1.48 Mbp, a feasible size for positional cloning. As there is no known gene involved in vasculogenesis and/or angiogenesis in this YAC, the identification of the causative gene is likely to reveal a novel regulator or vascular development.

Alleles↗

PubMed2001

Gene for the human transmembrane-type protein tyrosine phosphatase H (PTPRH): genomic structure, fine-mapping and its exclusion as a candidate for Peutz-Jeghers syndrome.

Mutations in the serine/threonine kinase STK11 lead to Peutz-Jeghers syndrome (PJS) in a subset of affected individuals. Significant evidence for linkage to a second potential PJS disease locus on 19q13.4 has previously been described in one PJS family (PJS07). In the current study, we investigated this second locus for PJS gene candidates. We mapped the main candidate gene in this region, the gene for the transmembrane-type protein tyrosine phosphatase H (PTPRH), within 15 kb telomeric to the marker D19S880. We determined its genomic structure, and performed mutation analysis of all exons and the exon-intron junctions of the PTPRH gene in the PJS07 family. No disease causing mutation was identified in PTPRH in affected individuals, suggesting the existence of an as yet not identified gene on 19q13.4 as a second PJS gene.

Chromosomes, Human, Pair 19↗

PubMed2001

Clonality and altered behavior of endothelial cells from hemangiomas.

Hemangioma, the most common tumor of infancy, is a benign vascular neoplasm of unknown etiology. We show, for the first time to our knowledge, that endothelial cells from proliferating hemangioma are clonal, and we demonstrate that these hemangioma-derived cells differ from normal endothelial cells in their rates of proliferation and migration in vitro. Furthermore, migration of hemangioma endothelial cells is stimulated by the angiogenesis inhibitor endostatin, unlike the inhibition seen with normal endothelial cells. We conclude that hemangiomas constitute clonal expansions of endothelial cells. This is consistent with the possibility that these tumors are caused by somatic mutations in one or more genes regulating endothelial cell proliferation.

Alleles↗

PubMed2001

The role of collagen II and cartilage fibril-associated molecules in skeletal development.

OBJECTIVE: The extracellular matrix (ECM) of hyaline cartilage contains an elaborated collagen fibrillar network, which is essential for the mechanical stability and the proper function of the tissue. Cartilage collagen fibrils consist of collagen II, the quantitatively minor collagens IX and XI, and several non-collagenous fibril-associated proteins. To understand the role some of these molecules in skeletal development, we have generated transgenic mouse strains harboring ablated genes for collagens II and IX, and matrilin-1. DESIGN: Mice lacking collagen II, collagen IX and matrilin-1 have been established earlier in our laboratory using standard techniques. To determine the consequences of the null mutations we used skeletal staining, histochemical and immunohistochemical assays, in situ hybridization and ultrastructural analysis. RESULTS: Transgenic mice deficient in collagen II (Col2a1-/-) die at birth and display a severely malformed skeleton characterized by abnormal endochondral ossification and impaired intervertebral disc development. Mice lacking collagen IX (Col9a1-/-) are viable and develop an osteoarthritis-like phenotype in knee joints between 9-12 months of age. To test the possibility that the reduction in collagen II content has an influence on the onset of degenerative changes of articular cartilage, we have generated mice, which are heterozygous for the collagen II null mutation and homozygous for the collagen IX null mutation. Col2a1+/- Col9a1-/- mice show no accelerated development of osteoarthritis compared with the collagen IX knockout animals. Finally, mice lacking matrilin-1, a non-collagenous glycoprotein that binds to both collagen fibrils and aggrecan, develop normally without detectable abnormalities in their skeleton. CONCLUSIONS: Our transgenic mouse strains carrying null mutations in genes encoding cartilage ECM proteins demonstrate that these proteins have different roles during skeletal development. Collagen II is important for cartilage formation, collagen IX for cartilage maintenance and matrilin-1 is redundant.

Animals↗