Anders Malmström

Ripening of the human uterine cervix related to changes in collagen, glycosaminoglycans, and collagenolytic activity

Connective tissue in biopsy specimens taken from the lower part of the uterine cervix in 40 pregnant women at various gestational ages was compared to that in similar biopsy specimens from 15 nonpregnant women. The concentrations of collagen, sulfated glycosaminoglycans, and hyaluronic acid decreased during pregnancy. At the gestational age of 10 weeks, the collagen concentration was 70%, and at term 30%, of that in the nonpregnant cervix. After delivery, no further decrease was observed. The extractability of collagen increased during pregnancy, as well as during labor. Also, the water concentration increased. An increase in the collagenolytic activity was observed with advancing gestational age. The 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gin-D-Arg hydrolytic activity (collagenase) and the concentration of leukocyte elastase increased gradually by a factor of 10. The physiologic importance of the collagen was also demonstrated, since the cervical dilatation time during spontaneous labor was long in women with high concentrations of collagen and short in women with low concentrations of collagen.

Different biochemical composition of connective tissue in continent and stress incontinent women

The collagen content in biopsies from skin and ligamentum rotundum of 7 women with a long history of stress incontinence was compared with that of continent controls. The collagen was extracted with 0.5 M acetic acid, followed by digestion with pepsin and quantitated as hydroxyproline. The skin of stress incontinent women contained 40% less collagen than that of continent women. The findings for ligamentum rotundum were similar. These results suggest a deteriorated connective tissue in stress‐incontinent women and cast new light on the etiology of the disease

Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in developing bleomycin-induced pulmonary fibrosis in rats.

The development of bleomycin-induced pulmonary fibrosis in rats was studied over a period of 21 d after an intratracheal instillation of bleomycin. The expression of three small proteoglycans (biglycan, decorin, and fibromodulin), collagen III and TGF-beta 1 was studied by RNA-transfer blot analysis. The proteoglycans were also studied by SDS-polyacrylamide gel electrophoresis and Western blots. TGF-beta 1 mRNA increased threefold already on day 3 and remained elevated until day 10. After the increase of TGF-beta 1 mRNA the messages for biglycan and collagen III steadily increased to reach a maximum 10 d after bleomycin instillation. The mRNA for biglycan increased maximally fourfold and that of collagen III 2.5-fold. Decorin mRNA, in contrast to biglycan decreased and reached 20% of control on day 10. The message for fibromodulin remained constant throughout the study period. The amounts of biglycan and decorin in the tissue changed in accordance with the mRNA levels. The results corroborate and extend previous in vitro studies concerning the effect of TGF-beta 1 on the metabolism of small proteoglycans and show that these macromolecules are regulated differently also in vivo. The marked alterations of biglycan and decorin during the development of fibrosis suggests that these proteoglycans have a regulating role in this process.

Clinical outcome and changes in connective tissue metabolism after intravaginal slingplasty in stress incontinent women

The intravaginal slingplasty procedure (IVS) was carried out on 75 patients with genuine stress urinary incontinence. The main aims of the operation are to create an artificial pubourethral ligament and to tighten the suburethral vaginal wall. An important ingredient in the supportive structures of the genitourinary region is fibrous connective tissue, consisting mainly of collagen. To analyse thi component biopsies were obtained transvaginally, close to the position of the sling, both preoperatively and 2 years after surgery, from 6 patients. Collagen was analysed for concentration and extractability. Extractability by pepsin digestion was increased by 60% 2 years following surgery. Postoperative follow-up studies from 12 months to 3 years showed complete restoration of continence in 63 patients (84%) and considerable improvement in 4 others (5%). The 8 failures (9%) were all related to early rejection of the sling. The IVS procedure is an attractive surgical procedure as it necessitates minimum invasion and can be performed under local anesthesia, with a short hospital stay and sick-leave period. The enhanced collagen extractability indicates a changed metabolism, most likely induced by the implanted sling, resulting in a restoration of the elastic properties of the connective tissue.

Tissue fibrocytes in patients with mild asthma: a possible link to thickness of reticular basement membrane?

BackgroundMyofibroblasts, proposed as being derived from circulating fibrocytes, are considered to be important cells in thickening of the basement membrane in patients with asthma. We have studied the correlation of tissue fibrocyte levels to basement membrane thickness and the presence of fibrocytes in bronchoalveolar lavage fluid (BALF) in steroid-naive patients with mild asthma and controls.MethodsPatients with mild asthma (n = 9) were recruited and divided into two categories based on whether or not fibroblast-like cells could be established from BALF. Non-asthmatic healthy subjects (n = 5) were used as controls. Colocalization of the fibrocyte markers CD34, CD45RO, procollagen I, and α-smooth muscle actin (α-SMA) were identified in bronchial biopsies from patients and controls by confocal microscopy. Kruskall-Wallis method was used to calculate statistical significance and Spearman coefficient of rank correlation was used to assess the degree of association.ResultsIn patients with BALF fibroblasts, a 14-fold increase of tissue cells expressing CD34/CD45RO/α-SMA and a 16-fold increase of tissue cells expressing CD34/procollagen I was observed when compared to controls (p < 0.05). In contrast, patients without BALF fibroblasts displayed a 2-fold increase when compared to controls (p < 0.05). Fibrocytes were localized close to the basement membrane which was significantly thicker in patients with BALF fibroblasts when compared to the other two groups of subjects. Furthermore, basement membrane thickness could be correlated to the number of fibrocytes in tissue (r = 0.711). Fibroblasts-like cells were cultured from BALF where 17.6% of these cells expressed CD34, CD45RO and α-SMA.ConclusionThese findings indicate a correlation between recruited fibrocytes in tissue and thickness of basement membrane. Fibroblast progenitor cells may therefore be important in airway remodeling in steroid-naive patients with mild asthma.

Interleukin-8 is a mediator of the final cervical ripening in humans

OBJECTIVE The aim of the present study was to investigate the presence of interleukin-8 (IL-8) in the human cervix and whether the levels of interleukin-8 could be related to the ripening process during pregnancy. STUDY DESIGN Cervical biopsies were obtained in twelve term pregnant and in eight vaginally delivered women. Seven non-pregnant fertile women served as controls. After homogenisation and centrifugation, IL-8 levels were determined in the supernatant by an enzyme-immunoassay (EIA). RESULTS In women at term, the concentration of IL-8 increased six-fold from median 330 pg/ml to median 2190 pg/ml (P < 0.001). After the final cervical ripening it increased in additional 11-fold to median 26,100 pg/ml (P < 0.001). These changes are highly significant. CONCLUSION To our knowledge, this is the first time IL-8 has been identified in human cervix. Our results support the involvement of IL-8 in the connective tissue remodelling during the final cervical ripening just before onset of labour.

Changes in paraurethral connective tissue at menopause are counteracted by estrogen

OBJECTIVE To study whether the transition to menopause is accompanied by changes in the paraurethral connective tissue and if these changes are modified by estrogen replacement therapy. STUDY DESIGN Biopsies were obtained from the paraurethral tissue from 34 women; 12 menstruating, 14 postmenopausal without estrogen treatment, and 8 with estrogen treatment. Collagen concentration and collagen extractability by pepsin digestion were measured. Proteoglycan composition and concentration were analysed using Alcian blue. The mRNA levels for collagen I and III, the small proteoglycans (PGS) decorin and biglycan, and the large proteoglycan versican, were estimated. RESULTS The paraurethral biopsies consisted of fibrous connective tissue, with collagen fibers as dominating structure. Several proteoglycans were identified; versican, heparansulphate proteoglycans, biglycan and decorin. The small proteoglycan decorin represented 85% of all proteoglycans. The collagen concentration was almost doubled in postmenopausal biopsies compared to premenopausal. The collagen fibril organization was also changed with higher cross-linking after menopause whereas the amount and the composition of the proteoglycans were unchanged. The proteoglycan/collagen ratio was significantly decreased. Estrogen replacement therapy resulted in decreased collagen concentration, decreased cross-linking of the collagen and reversal of the PGS/collagen ratio to almost premenopausal level. The therapy resulted in increased levels of mRNA for collagen I and III which suggests that the changes are due to an increased turnover. CONCLUSION The decrease in estrogen levels at menopause results in a connective tissue with different qualities after menopause. Estrogen replacement therapy tends to restore the metabolism of the genitourinary connective tissue to premenopausal conditions.

Changes in the connective tissue of corpus and cervix uteri during ripening and labour in term pregnancy

The composition of the connective tissue of human cervix and corpus uteri was studied in tissue specimens from seven nonpregnant women and 14 pregnant women, delivered at term by section, to examine spontaneous cervical ripening and labour‐induced changes in both the uterine and the cervical connective tissue. The main finding in both the cervix and the corpus was a large (40–60%) decrease of the collagen concentration. The collagen extractability, obtained by pepsin digestion, was increased twofold, suggesting a change of the organization of the collagen fibrils. This reorganization process could also be demonstrated by a large increase of the collagenolytic activity demonstrated with an artificial DNP‐peptide substrate. The concentrations of sulphated glycosaminoglycans was lower in pregnant women than in non‐pregnant women. The results show that both the cervix and the corpus uteri contain substantial amounts of connective tissue components (collagen, sulphated glycosaminoglycans and hyaluronic acid) and that during ripening, reconstruction of the connective tissue components occurs in both sites. This indicates that the cervical state reflects that of the myometrium.

Different organization of collagen fibrils in stress-incontinent women of fertile age

OBJECTIVE The objective was to test the hypothesis that stress urinary incontinence in women is correlated to changes in the paraurethral connective tissue ultrastructure and metabolism. METHODS Transvaginal biopsies were obtained from the paraurethral connective tissue in women of fertile age with stress urinary incontinence and in matched continent controls. All the stress-incontinent women were characterized with urodynamic investigation. In the biopsies, collagen concentration, measured as hydroxyproline, and the degree of extraction by pepsin digestion were quantified. Proteoglycan composition and concentration were analyzed using Alcian blue precipitation, followed by electrophoretic separation and quantification. Using Northern blots mRNA levels for the collagens I and III, the small proteoglycans decorin and biglycan, and the large proteoglycan versican, were quantified. Collagen organization was examined with transmission electron microscopy and the diameters of collagen fibrils were analyzed with an interactive image analysis system (IBAS, Zeiss/Kontron). RESULTS The biochemical and morphological analyses exposed a significant difference in the paraurethral connective tissue between stress urinary incontinent women before menopause and comparable controls. The collagen concentration was almost 30% higher and the diameters of the collagen fibrils were 30% larger in the incontinent group of women. Also the organization of the collagen fibrils differed, with considerably higher cross-linking. A higher level of mRNA for collagen I and III in the incontinent group indicates that the differences can be related to an altered collagen metabolism. No change of proteoglycan amount or composition was observed, resulting in a significantly lower proteoglycan/collagen ratio in the incontinent group of women. CONCLUSION Stress urinary incontinence in fertile women is associated with a change in collagen metabolism resulting in an increased concentration of collagen and larger collagen fibrils. These alterations should result in a more rigid form of extracellular matrix, suggesting a connective tissue with impaired mechanical function.

Biosynthesis of Dermatan Sulfate CHONDROITIN-GLUCURONATE C5-EPIMERASE IS IDENTICAL TO SART2

We identified the gene encoding chondroitin-glucuronate C5-epimerase (EC 5.1.3.19) that converts d-glucuronic acid to l-iduronic acid residues in dermatan sulfate biosynthesis. The enzyme was solubilized from bovine spleen, and an ∼43,000-fold purified preparation containing a major 89-kDa candidate component was subjected to mass spectrometry analysis of tryptic peptides. SART2 (squamous cell carcinoma antigen recognized by T cell 2), a protein with unknown function highly expressed in cancer cells and tissues, was identified by 18 peptides covering 26% of the sequence. Transient expression of cDNA resulted in a 22-fold increase in epimerase activity in 293HEK cell lysate. Moreover, overexpressing cells produced dermatan sulfate chains with 20% of iduronic acid-containing disaccharide units, as compared with 5% for mock-transfected cells. The iduronic acid residues were preferentially clustered in blocks, as in naturally occurring dermatan sulfate. Given the discovered identity, we propose to rename SART2 (Nakao, M., Shichijo, S., Imaizumi, T., Inoue, Y., Matsunaga, K., Yamada, A., Kikuchi, M., Tsuda, N., Ohta, K., Takamori, S., Yamana, H., Fujita, H., and Itoh, K. (2000) J. Immunol. 164, 2565–2574) with a functional designation, chondroitin-glucuronate C5-epimerase (or DS epimerase). DS epimerase activity is ubiquitously present in normal tissues, although with marked quantitative differences. It is highly homologous to part of the NCAG1 protein, encoded by the C18orf4 gene, genetically linked to bipolar disorder. NCAG1 also contains a putative chondroitin sulfate sulfotransferase domain and thus may be involved in dermatan sulfate biosynthesis. The functional relation between dermatan sulfate and cancer is unknown but may involve known iduronic acid-dependent interactions with growth factors, selectins, cytokines, or coagulation inhibitors.