Risto Penttinen

Diminished Callus Size and Cartilage Synthesis in α1β1 Integrin-Deficient Mice during Bone Fracture Healing

Integrins mediate cell adhesion to extracellular matrix components. Integrin α1β1 is a collagen receptor expressed on many mesenchymal cells, but mice deficient in α1 integrin (α1-KO) have no gross structural defects. Here, the regeneration of a fractured long bone was studied in α1-KO mice. These mice developed significantly less callus tissue than the wild-type (WT) mice, and safranin staining revealed a defect in cartilage formation. The mRNA levels of nine extracellular matrix genes in calluses were evaluated by Northern blotting. During the first 9 days the mRNA levels of cartilage-related genes, including type II collagen, type IX collagen, and type X collagen, were lower in α1-KO mice than in WT mice, consistent with the reduced synthesis of cartilaginous matrix appreciated in tissue sections. Histological observations also suggested a diminished number of chondrocytes in the α1-KO callus. Proliferating cell nuclear antigen staining revealed a reduction of mesenchymal progenitors at the callus site. Although, the number of mesenchymal stem cells (MSCs) obtained from WT and α1-KO whole marrow was equal, in cell culture the proliferation rate of the MSCs of α1-KO mice was slower, recapitulating the in vivo observation of reduced callus cell proliferation. The results demonstrate the importance of proper collagen-integrin interaction in fracture healing and suggest that α1 integrin plays an essential role in the regulation of MSC proliferation and cartilage production.

Polymorphisms at the Werner locus: II. 1074Leu/Phe, 1367Cys/Arg, longevity, and atherosclerosis

Werner syndrome (WS) is a progeroid syndrome caused by autosomal recessive null mutations at the WRN locus. The WRN gene encodes a nuclear protein of 180 kD that contains both exonuclease and helicase domains. WS patients develop various forms of arteriosclerosis, particularly atherosclerosis, and medial calcinosis. The most common cause of death in Caucasian subjects with WS is myocardial infarction. Previous studies have identified specific polymorphisms within WRN that may modulate the risk of atherosclerosis. Population studies of the 1074Leu/Phe and 1367Cys/Arg polymorphisms were undertaken to evaluate the role of WRN in atherogenesis. Frequencies of the 1074Leu/Phe polymorphisms in Finnish and Mexican populations revealed an age-dependent decline of 1074Phe/Phe genotype. In Mexican newborns, but not in Finnish newborns, the 1074Leu/Phe and 1367Cys/ Arg polymorphisms were in linkage disequilibrium. Among coronary artery disease subjects, there was a tendency for the 1074Phe allele to be associated with coronary stenosis in a gene dose-dependent manner. Furthermore, the 1367Arg/Arg genotype predicted a lower degree of coronary artery occlusion, as measured by NV50, when compared to the 1367Cys/Cys or 1367Cys/Arg genotypes. However, these tendencies did not achieve statistical significance. Samples from Mexican patients with ischemic stroke showed a trend of haplotype frequencies different from that in a control group of Mexican adults. These data support the hypothesis that WRN may mediate not only WS, but may also modulate more common age-related disorders and, perhaps, a basic aging process.

A Gene-Targeting Approach Identifies a Function for the First Intron in Expression of the α1(I) Collagen Gene

ABSTRACT The role of the first intron of the Col1A1 gene in the regulation of type I collagen synthesis remains uncertain and controversial despite numerous studies that have made use of transgenic and transfection experiments. To examine the importance of the first intron in regulation of the gene, we have used the double-replacement method of gene targeting to introduce, by homologous recombination in embryonic stem (ES) cells, a mutated Col1A1 allele (Col-IntΔ). The Col-IntΔ allele contains a 1.3-kb deletion within intron I and is also marked by the introduction of a silent mutation that created an XhoI restriction site in exon 7. Targeted mice were generated from two independently derived ES cell clones. Mice carrying two copies of the mutated gene were born in the expected Mendelian ratio, developed normally, and showed no apparent abnormalities. We used heterozygous mice to determine whether expression of the mutated allele differs from that of the normal allele. For this purpose, we developed a reverse transcription-PCR assay which takes advantage of the XhoI polymorphism in exon 7. Our results indicate that in the skin, and in cultured cells derived from the skin, the intron plays little or no role in constitutive expression of collagen I. However, in the lungs of young mice, the mutated allele was expressed at about 75% of the level of the normal allele, and in the adult lung expression was decreased to less than 50%. These results were confirmed by RNase protection assays which demonstrated a two- to threefold decrease in Col1A1mRNA in lungs of homozygous mutant mice. Surprisingly, in cultured cells derived from the lung, the mutated allele was expressed at a level similar to that of the wild-type allele. Our results also indicated an age-dependent requirement for the intact intron in expression of the Col1A1 gene in muscle. Since the intron is spliced normally, and since the mutant allele is expressed as well as the wild-type allele in the skin, reduced mRNA stability is unlikely to contribute to the reduction in transcript levels. We conclude that the first intron of the Col1A1 gene plays a tissue-specific and developmentally regulated role in transcriptional regulation of the gene. Our experiments demonstrate the utility of gene-targeting techniques that produce subtle mutations for studies ofcis-acting elements in gene regulation.

Synthesis of hyaluronic acid and collagen in skin fibroblasts cultures from patients with osteogenesis imperfecta

Collagen and hyaluronic acid syntheses were studied in skin fibroblast cultures from patients with osteogenesis imperfecta and age-matched controls by labeling the cultures either with [3H]proline and separating the collagenous proteins with DEAE- and CM-cellulose chromatographies, or double-labeling the cultures with [3H]glucosamine and [14C]glycine and separating radioactive hyaluronic acid from glycoproteins and sulphated proteoglycans by DEAE-cellulose chromatography. The activities of the cell layer hyaluronate synthesizing enzyme complex (hyaluronate synthetase) were also determined. The osteogenesis imperfecta cultures were classified into three variants on the basis of type III collagen synthesis. Type III collagen amounted to approx. 40--50% from total collagen in the first variety and approx. 25--30% in the second variety. No difference was noted in the ratio of type III collagen to total collagen in the third variety in comparison with control cultures. The radioactivities of 3H-labeled hyaluronic acid in DEAE-cellulose chromatograms were compared with those of the 14C-labeled proteins. The ratios ranged 9.2--17.3 in the cultures from the patients and 4.6--8.8 in the control cultures. Hyaluronate synthetase activities were 1.3--2.0-fold higher in the osteogenesis imperfecta cells than in their controls. Increased hyaluronic acid synthesis in skin fibroblasts correlated with the severity of the disease but not with the increase in type III collagen synthesis.

Development of peritoneal fibrosis occurs under the mesothelial cell layer.

This study was carried out in order to find out which part of the peritoneal wall reacts toward silica and produces peritoneal fibrosis. Colloidal silica was injected into the peritoneal cavity of rats to induce chemical peritonitis and frozen sections of the peritoneal wall were stained with specific antibodies toward type I and III collagens and fibronectin. A massive proliferation of granulation tissue was observed between the submesothelial and muscular layers within 48 hr visualized by prominent fibronectin staining. Type III collagen formed lamellar-like structures in the newly formed granulation tissue. The connective tissue reaction was extended into the underlying muscular tissue. Three weeks after silica injection the reactive granulation tissue exceeded the original peritoneum three- to fourfold in thickness. At this stage it contained extended fibrillar structures oriented perpendicular to the surface or muscular layers of the peritoneum. Type I collagen antibody was bound to the superficial cell layer in the control samples and in the early peritonitis whereas the entire granulation tissue was evenly stained at 3 weeks. Type III collagen antibody was bound to the surface layer of the peritoneum, granulation tissue, and perimysial connective tissue throughout the healing period. The results indicate that the peritoneal fibrotic process occurs under the thin peritoneal lining cell layer and on the surface of the muscle layer.

Collagens in Neurofibromas and Neurofibroma Cell Cultures

Neurofibromas contain approximately 30-50% collagen of their lipid-free dry weight, which is about half of the value of skin but approximately twice that described for peripheral nerve endoneurium. Immunohistochemical stainings indicate that neurofibromas contain types I, III, IV, and V collagens and fibronectin. Most of the neurofibroma cells are type IV collagen and S-100 protein positive, which provides immunohistochemical evidence that neurofibromas are mostly composed of Schwann cell-like cells. The proteoglycan/collagen ratio is 4 to 10 times higher in the neurofibromas than in the surrounding dermal tissue. This would explain the typical soft consistency of the neurofibromas and may contribute to a favorable milieu for tumor growth. Pure fibroblastic cell cultures are obtained from neurofibromas after repeated passages. The cultured cells synthesized type I and III collagens and fibronectin, indicating that these cells are important in the production of the fibrous connective tissue proteins in neurofibromas.

NF1 Gene Expression in Mouse Fracture Healing and in Experimental Rat Pseudarthrosis

Neurofibromatosis type 1 (NF1) is an inherited disease with an incidence of about 1:3000 worldwide. Approximately half of all patients with NF1 present osseous manifestations, which can vary from mild to severely debilitating changes such as congenital pseudarthrosis. In the present study, fracture healing of mouse tibia was followed and specimens were collected 5, 9, 14, and 22 days postoperatively. Experimental pseudarthrosis of rat was followed up to 15 weeks postoperatively. In situ hybridization and immunohistochemistry were used to demonstrate expression of NF1 tumor suppressor and phosphorylated p44/42 mitogen-activated protein kinase (MAPK), an indicator of the Ras-MAPK pathway. The results showed that ossified callus was formed in mouse fracture 22 days after the operation. The final outcome of rat pseudarthrosis was detected 9 weeks after the operation, presenting abundant cartilaginous callus at the pseudarthrosis. NF1 gene expression was noted in the maturing and in the hypertrophic cartilages during normal mouse fracture healing, and in rat pseudarthrosis. Phosphorylated p44/42 MAPK was detected in a subpopulation of the hypertrophic chondrocytes in both models. Furthermore, positive labeling for NF1 mRNA and protein was detected in endothelium in both the pseudarthrosis and in the fracture. In conclusion, NF1 gene expression and function are needed for normal fracture healing, possibly restraining excessive Ras-MAPK pathway activation.

Healing of experimental fractures in the denervated limbs of the rat.

The effects of denervation on healing of experimental fractures of the tibia after sciatic nerve section or spinal cord transsection or both were studied in rats. In addition to normal weight-bearing controls, non-weight-bearing controls were prepared by means of experimental hip luxations. The healing of fractures was monitored by tensile strength measurements, and by X-ray studies up to two months. The tensile strengths of the fractures with peripheral nerve lesion or with spinal cord trauma were significantly higher than those of the respective weight-bearing or non-weight-bearing control fractures at the early phase of healing. Osseous union of denervated fractures was radiographically evident earlier or at least at the same time as in the controls. Denervation, however, caused irregularities in the radiopacity and increased the length of fracture callus. These results confirm the clinical observations on enhanced healing of fractures in extremities of patients with spinal cord trauma.

Fate of Bone Marrow-Derived Stromal Cells after Intraperitoneal Infusion or Implantation into Femoral Bone Defects in the Host Animal

The fate of intraperitoneally injected or implanted male rat bone marrow-derived stromal cells inside female sibling host animals was traced using Y-chromosome-sensitive PCR. When injected intraperitoneally, Y-chromosome-positive cells were found in all studied organs: heart muscle, lung, thymus, liver, spleen, kidney, skin, and femoral bone marrow with a few exceptions regardless of whether they had gone through osteogenic differentiation or not. In the implant experiments, expanded donor cells were seeded on poly(lactide-co-glycolide) scaffolds and grown under three different conditions (no additives, in osteogenic media for one or two weeks) prior to implantation into corticomedullar femoral defects. Although the impact of osteogenic in vitro cell differentiation on cell migration was more obvious in the implantation experiments than in the intraperitoneal experiments, the donor cells stay alive when injected intraperitoneally or grown in an implant and migrate inside the host. However, when the implants contained bioactive glass, no signs of Y-chromosomal DNA were observed in all studied organs including the implants indicating that the cells had been eliminated.

Enhanced fibroblast collagen production by a macrophage-derived factor (CEMF).

Abstract A basic fraction with pl values of 10.0–10.4 was isolated from macrophage culture medium. This fraction stimulated the production of collagen into fibroblast culture medium but inhibited the production of other proteins. Both changes were strongly dependent on the concentration of the factor. As revealed by SDS-PAGE the production of type I collagen was especially stimulated. The specific enhancement of fibroblast collagen production was also observed in the presence of serum proteins. The collagen synthesis enhancing macrophage-derived factor preparation (CEMF) contained three proteins, one major (Mr 23 kD) and two minor (Mr 49 and 71 kD) fractions in SDS-PAGE. When prelabeled fibroblast medium was exposed to CEMF a protein with Mr of 350 kD or greater was converted to a smaller size. This change was inhibited by EDTA but not by serum proteinase inhibitors.