Mesh repair of common abdominal hernias: a review on experimental and clinical studies.

Results on hernia surgery from numerous centers confirm that tensionless repair with various meshes reduces the complication rates and the frequency of recurrences. Some evidence on incisional hernias suggests, however, that the use of mesh seems to transfer the onset of recurrences by several years. Persistent pain and other discomfort is also an unpleasant complication of otherwise successful surgery in a number of patients. Thus, improved, slowly degrading, mesh materials, with strong connective tissue-inducing action, might be more optimal for hernia surgery. Accumulating evidence also suggests that recurrent hernias appear in patients having inherited weakness of connective tissues. Numerous tissue specific collagens, in addition to the classical fibrillar I-III collagens and numerous substrate specific matrix proteinases, have recently been described in biochemical literature, and their roles as possible causes of tissue weakness are discussed.

A gene-targeting approach identifies a function for the first intron in expression of the alpha1(I) collagen gene.

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-IntDelta). The Col-IntDelta 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 Col1A1 mRNA 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 of cis-acting elements in gene regulation.

Effect of the 3'-untranslated region on the expression levels and mRNA stability of alpha 1(I) collagen gene.

Changes in the synthesis of type I collagen, a major extracellular matrix component in skin and bones, are associated with both normal growth or repair processes and with several pathological conditions such as lung fibrosis and liver cirrhosis. The expression of the alpha 1(I) collagen gene is regulated by transcriptional and post-transcriptional mechanisms. Regulation at both these levels are usually utilised when extensive changes occur in collagen synthesis. We constructed plasmids carrying the whole or partially deleted 3'-UTR sequences of the alpha 1(I) collagen gene, fused to two hGH exons and to the promoter of the alpha 1(I) collagen gene. A control plasmid contained the 3'-UTR of the hGH gene. In transient transfections into Rat-1 fibroblasts, no significant differences between plasmids were found, which suggests that although 3'-end of the gene has been shown in previous studies to contain DNaseI hypersensitive sites and to bind sequence-specific nuclear proteins it does not seem to function as a transcriptional regulator. This was further supported by the finding that TGF-beta treatment induced a 2.5-fold expression of hGH mRNA from plasmids containing collagen promoter and either hGH or alpha 1(I) collagen 3'-UTR. In stable transfections, mRNAs using the first polyadenylation site were not as stable as those transcribed from the endogenous alpha 1(I) collagen gene. We suggest that the 3'-UTR alone may not be sufficient to determine the stability of the shorter alpha 1(I) collagen mRNA species.

Synthesis of hyaluronic acid and collagen in skin fibroblasts cultured 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.

Desmosines in aneurysms of the ascending aorta (annulo-aortic ectasia).

Amino acid chromatography was used for determination of the elastin-specific amino acids desmosine and isodesmosine in acid hydrolyzates of intima-medial samples taken intraoperatively from aneurysms of human ascending aorta. Elastin concentration of the specimens was also estimated by hot alkali extraction followed by nitrogen determination of the extracted material and the insoluble residue. All patients studied had annulo-aortic ectasia i.e., dilatation of the aortic annulus and the ascending aorta. Two patients with the Marfan syndrome had low aortic elastin concentration determined by both methods. A third Marfan syndrome patient, youngest of the three, also had a slightly reduced concentration of elastin in the aorta. Aortic samples were studied from five patients who did not have the classical Marfan syndrome. Two patients of those five had decreased aortic elastin concentration. The change in elastin concentration was accompanied by high hydroxyproline/proline or hydroxylysine/lysine ratios which indicates that the proteins of the aneurysmatic aortic wall contained more collagen than the proteins of the control aortic wall. These findings point to a change in the structure or metabolism of elastin in the aortic wall in the Marfan syndrome and at least in some other patients with annulo-aortic ectasia.

Collagen in human aorta. Changes in the type III/I ratio and concentration of the reducible crosslink, dehydrohydroxylysinonorleucine in ascending aorta from healthy subjects of different age and patients with annulo-aortic ectasia.

The type III/I + III collagen ratio was studied in intima-medial samples of ascending aortas obtained from patients with the Marfan syndrome or other annulo-aortic ectasia (dilatation of the ascending aorta) and from control subjects, using electrophoretic analysis of cyanogen bromide peptides. The [3H]borohydride-reduced crosslinks of collagens were analysed by ion-exchange chromatography. Type III/I + III collagen ratios were twice as high in adult aortas as those found in skin samples of the same age. This ratio was lower in fetal and very young aortic samples and in 6-8 out of 12 pathological aortas (including one sample from a Marfan patient) when compared with adult controls. In contrast, the type III/I + III collagen ratio was high in fetal or very young skin and the values obtained from several patients did not differ from those of the control skin samples. In one pathological aorta out of six studied, the concentration of the reducible crosslink, dehydrohydroxylysinonorleucine, was higher than in controls, suggesting increased collagen synthesis or impaired maturation of collagen. These changes point to altered collagen metabolism in aortas of patients with annulo-aortic ectasia.

Fibroblast expression of collagens and proteoglycans is altered in aspartylglucosaminuria, a lysosomal storage disease.

Aspartylglucosaminuria (AGU), a lysosomal storage disease caused by deficient activity of aspartylglucosaminidase (E.C. 3.5.1.26), is characterised by progressive mental retardation and variable connective tissue signs. The ultrastructure of collagen fibrils in skin of AGU patients is abnormal and their fibroblasts synthesise reduced amounts of collagens [Näntö-Salonen et al. (1984) Lab invest. 51: 464-468]. In this work we measured the steady-state messenger RNA levels of several extracellular matrix components in skin fibroblast cultures of two patients homozygous for the most prevalent mutation (AGUFin) causing the disease in Finland. In confluent cultures the steady-state mRNA concentrations of type I and III collagens were reduced to 0.5-20% of control values. Almost as marked reduction was observed in the mRNA level of biglycan, a small interstitial proteoglycan whereas that of decorin, a closely related, collagen fibril-associated proteoglycan, was increased several-fold. Elevated decorin and decreased biglycan mRNA levels reflected the amounts of the produced corresponding proteoglycans. The differences in the mRNA levels become more pronounced with the time the cells were in culture. Fibronectin mRNA concentrations were similar in AGU and control fibroblasts. Changes in the expression and synthesis of extracellular matrix components might be related to the connective tissue symptoms of the patients.

Nuclear and cytoplasmic alpha 1 (I) collagen mRNA-binding proteins.

We have recently identified a cytoplasmic protein, alpha 1-RBF67, that specifically interacts with the conserved 3'-untranslated region of the alpha 1 (I) collagen gene. The binding activity was decreased in extracts from dexamethasone treated cells, which correlates with the known accelerated turnover of the COL1A1 RNA [Määttä, A. and Penttinen, R.P.K. (1993) Biochem. J. 295, 691-698]. Now we report that a very similar protein is present in nuclear extracts of NIH 3T3, human fibroblast and HeLa cells, which suggests that determination of cytoplasmic mRNA stability is not the sole function of the alpha 1-RBF67 activity. The binding to the RNA probe can be inhibited by annealing a DNA oligonucleotide or using excess of cold specific competitors. In UV-cross linking assays the nuclear protein has the same molecular weight (67 kDa) as the cytoplasmic one and the RNA-bound peptides generated by CNBr or V8 protease cleavage from both the cytoplasmic and the nuclear protein were identical. This protein was the only one of several nuclear collagen mRNA 3'-UTR binding proteins that was present in both nuclear and cytoplasmic extracts. In fibroblasts heparin-resistant nuclear RNA binding proteins had molecular weights of 45, 67 (alpha 1-RBF67), and 71 kDa. HeLa-cells contained an additional protein of 51 kDa and several non-specific RNA-binding proteins. The binding activity is modified by changes in the redox state, which implicates that in the nucleus the binding affinities of alpha 1(I) collagen RNA-binding protein and AP-1, a redox sensitive nuclear factor, that is important in the transcription of alpha 1(I) collagen gene, can be regulated simultaneously to the same direction.

Changes in the expression of cell surface sialoglycoproteins during transition of human monocytes into macrophages.

Cell surface sialoglycoproteins of human mononuclear phagocytes in different maturation stages were labelled by the periodate/borohydride method and separated by SDS-polyacrylamide gel electrophoresis. The main surface glycoproteins of peripheral blood monocytes had molecular masses of 115 and 95 kDa. During in vitro transition into adherent macrophages, the monocyte-characteristic surface glycoproteins disappeared. Most of the changes in the surface glycoprotein pattern occurred during the first 24 h and after 96 h the changes were completed. The major sialoglycoproteins of the macrophage cell surface had molecular masses of 130 and 55 kDa. The macrophage cell surface showed further changes when cultured in the presence of synovial fluid (10%). These results may reflect the in vivo maturation of monocytes into tissue macrophages. In synovium, tissue-derived factors may also take part in differentiation.

Highly conserved sequences in the 3'-untranslated region of the COL1A1 gene bind cell-specific nuclear proteins.

Sequencing of the 3' untranslated region (3'-UTR) of the human COL1A1 gene revealed numerous putative regulatory motifs and two highly conserved regions flanking the two polyadenylation sites. The conserved regions were separated by about 700 bp of less conserved sequences. The first region consists of almost all the 3'-UTR of the shorter (4.8 kbp) COL1A1 transcript. The second conserved domain includes a motif shared with several collagen genes. Both conserved domains bind cell-specific nuclear proteins suggesting that the 3'-UTR is important for cell specific expression of the COL1A1 gene.

Expression of extracellular matrix genes: transforming growth factor (TGF)-beta1 and ras in tibial fracture healing of lathyritic rats.

Experimental osteolathyrism, induced by dietary aminoacetonitrile (AAN), was used to study the effect of altered extracellular matrix on the expression of connective tissue components in long bone healing. AAN inhibits lysyl oxidase, which is needed for the formation of collagen cross-link precursors, and is also shown to act as a regulator of Ras. Fractured tibias in lathyritic rats develop excessive amounts of mechanically weak callus tissue with irregular cartilage and reduced glycosaminoglycan accumulation. Cartilage-specific proteins (collagen types II, IX, and X and aggrecan) were expressed temporally much wider in lathyritic calluses than in the controls, and active transcription was observed even during the fibrous and ossifying stages. Soft connective tissue was still present in 2- and 3-week-old lathyritic calluses and could explain the elevated type III collagen, biglycan, and decorin mRNA levels. Both transforming growth factor (TGF)-beta1 and c-Ha-ras, which control cell growth and differentiation, were upregulated during the cartilaginous stage. The maximal expression of TGF-beta1 preceded that of ras in osteolathyrism.

Cortisol decreases the synthesis of hyaluronic acid by human aortic smooth muscle cells in culture.

The effect of cortisol on the synthesis of glycosaminoglycans (GAGs) was studied in cultured human aortic smooth muscle cells. Cortisol, at a level slightly exceeding the physiological concentration (10(-6) M), decreased the synthesis of hyaluronic acid (HA) by 50% but had no significant effect on the synthesis of sulphated GAGs. The ratio of HA to sulphated GAGs decreased by 47%. These effects were most marked in the fraction secreted into the culture medium. Cortisol neither affected the activity of the hyaluronic acid synthesizing enzyme complex in a cell-free system nor the molecular weight distribution of hyaluronic acid. We suggest that the atherogenity of cortisol and stress may be associated with their effect on the synthesis of HA by the smooth muscle cells of the arterial wall.

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.

Polymorphisms at the Werner locus: I. Newly identified polymorphisms, ethnic variability of 1367Cys/Arg, and its stability in a population of Finnish centenarians.

The Werner syndrome gene (WRN) encodes a novel helicase of 1,432 amino acids. Homozygous mutations, all of which result in the truncation of the protein, lead to Werner syndrome. However, little is known about the role of WRN in "normal" aging. We have identified four missense polymorphisms and four conservative polymorphsims in WRN gene. A single study showed that a polymorphism at amino acid 1367 Cys(TTG)/ Arg(CTG) is associated with a variation in risk of myocardial infarction among a Japanese population. The 1367 Cys/Arg polymorphism was examined during aging in three different populations: Finnish, Mexican, and North American. The frequencies of 1367 Cys were higher than those of 1367 Arg in all the populations examined, though the frequencies varied among populations. The frequency of the 1367 Arg allele, thought to be protective against myocardial infarction in a Japanese population, was approximately three times higher in the North American and Finnish adult populations. When newborns and centenarians were compared within the Finnish population, no differences were observed in the proportions of 1367 Cys/Arg across age groups. Within the Finnish population, we confirmed a significant decrease of the APOE epsilon2 allele and an increase in the epsilon4 allele in newborn infants compared with centenarians. Thus, unlike the APOE polymorphism, there is no evidence of an association of this WRN polymorphism with longevity.

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.

Electrostimulation of rat callus cells and human lymphocytes in vitro.

Asymmetrical pulsing low voltage current was supplied via electrodes to cultured rat fracture callus cells and human peripheral blood lymphocytes. The [3H]thymidine incorporation of the callus cells and 5-[125I]iodo-2'-deoxyuridine incorporation of the lymphocytes were determined. The growth pattern of callus cells (estimated by cellular density) did not respond to electrical stimulation. However, the uptake of [3H]thymidine was increased at the early phase of cell proliferation and inhibited at later phases of proliferation. The [3H]thymidine uptake of confluent callus cell cultures did not respond to electrical stimulation. Lymphocytes reacted in a similar way; stimulated cells took up more DNA precursor than control cells at the early phase of stimulation. During cell division, induced by the mitogens phytohemagglutinin and Concanavalin-A, the uptake of DNA precursor by stimulated cells was constantly inhibited. The results suggest that electrical stimuli affect the uptake mechanisms of cell membranes. The duality of the effect seems to be dependent on the cell cycle.

Calcitonin and fracture healing. An experimental study on rats.

The effects of systemically administered calcitonin (CT) on fracture healing were analyzed in an experimental study on rats. The healing of a fracture was followed from 3 days up to 9 weeks postoperatively. Half of the rats in each age group were given daily CT 10 MRC-U/kg body wt s.c. Mechanical properties of the healing tibial fractures (tension strength) as well as various connective tissue components of the callus tissue were analyzed. No difference in the radiological or microscopical appearance of the fractures was detectable between the animals receiving CT and the controls. In the biochemical analysis matrix production as assessed from the concentrations of nitrogen, hexosamines, and hydroxyproline within the callus followed the usual lines of undistributed fracture union without any difference between the groups with and without CT. No differences could be detected in the mineralization of the callus between the specimens from animals receiving CT and those without. The tensile strength values of the fractures increased almost linearly up to 9 weeks. At 1 week the tensile strength values for fractures union in the animals without CT were approximately 50% higher, but later on no differences could be detected between the groups. These results indicate that although in the early phases of long-term CT therapy collagen synthesis may be impaired, there will be no effect on the net content of collagen or calcifying tissue in the callus or on the mechanical strength of healing fractures.

Synthesis of glycosaminoglycans and collagen in skin fibroblasts cultured from a patient with lichen myxedematosus.

A patient is described with typical skin lesions of lichen myxedematosus and IgG-type lambda paraproteinemia. Fibroblasts cultured from the skin of the patient and from the skin of control persons were used to study glycosaminoglycan and collagen synthesis; the cultures were labelled with 3H-glucosamine and 3H-proline, respectively. Fibroblasts from the patient grew to a cell density which was lower than that of the control fibroblasts. The production of glycosaminoglycans was increased in lichen myxedematosus cultures, so that the ratio of hyaluronic acid to sulphated glucosaminoglycans was higher in the patient's cultures than in control cultures. Collagen production in the patient's cultures was about half of that in control cultures, whereas the ratio of type III to type I collagen was normal.

A method for the hyaluronic acid synthetase assay in human skin.

A method for the determination of hyaluronic acid synthetase activity of human skin is described. Skin samples were crush-homogenized, and incubated with UDP-14C-glucuronic acid and UDP-N-acetylglucosamine in Tris-HCl-buffer containing MgCl2. After papain digestion of the samples, the 14C-labeled hyaluronic acid was separated in Sephadex G 50 chromatography. The radioactivity incorporated into hyaluronic acid was an expression of the activity of hyaluronic acid synthetase. This activity was found to be increased in skin biopsies obtained from psoriatic lesions and decreased after local treatment with potent corticoids.

A fibroblast cell line cultured from a hypertrophic scar displays selective downregulation of collagen gene expression: barely detectable messenger RNA levels of the pro alpha 1(III) chain of type III collagen.

The present study was designed to investigate the expression of type I, III and VI collagens by a fibroblast cell line initiated from a hypertrophic scar. The same tissue has previously been demonstrated to display markedly elevated expression of type I and III collagen mRNAs in vivo. Unexpectedly, slot-blot and Northern hybridizations revealed a barely detectable steady-state level of pro alpha 1(III) collagen chain mRNA in cultured hypertrophic scar fibroblasts. The levels of pro alpha 1(I) and alpha 2(VI) collagen chain mRNAs were essentially the same in fibroblasts cultured from hypertrophic scar and in fibroblasts cultured from normal skin. However, Northern blot analyses indicated that the ratio of 5.8 kb to 4.8 kb species of pro alpha 1(I) collagen mRNA was slightly reduced in fibroblasts originating from the hypertrophic scar compared to that in normal fibroblasts. When normal fibroblasts were incubated in conditioned medium from hypertrophic scar cultures, the expression of pro alpha 1(III) collagen chain mRNA decreased to a markedly lower level. Our studies suggest that collagen synthesis by fibroblasts in hypertrophic scars is stimulated by humoral factors which are active only in vivo. Furthermore, the results suggest that fibroblasts cultured from hypertrophic scar display a selective downregulation of different collagen genes and that this downregulation is exerted through an autocrine mechanism.