Henrik Wolff

Expression and activity of matrix metalloproteinase-2 and -9 in experimental granulation tissue

The restoration of functional connective tissue is a major goal of the wound healing process which is probably affected by matrix‐modifying enzymes. To evaluate the spatial and temporal expression of matrix metalloproteinases (MMP) MMP‐2 and MMP‐9 and to study the regulation of MMP‐2 in wound healing, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation for up to 3 months. MMP‐2 mRNA expression was seen throughout the experiment and it was highest after 2 months. MMP‐9 gene expression was low between days 8–21 and increased after 4 weeks of granulation tissue formation. Membrane‐type 1 MMP (MT1‐MMP) mRNA was upregulated early and tissue inhibitor 2 of MMP (TIMP‐2) mRNA later during wound healing. In in situ hybridization the expression of MMP‐2 mRNA was seen mostly in fibroblast‐like cells and MMP‐9 mRNA in macrophage‐like cells. MMP‐9 immunoreactivity was detected in the polymorphonuclear leukocytes and macrophage‐like cells on days 3–8. MMP‐9 proteolytic activity was observed only on days 3–8. The active form of the MMP‐2 increased up to day 14, whereafter it remained at a constant level, whereas latent MMP‐2 did not show any apparent changes during the experimental period. We conclude that MMP‐2 is important during the prolonged remodelling phase, whereas the gelatinolytic activity of MMP‐9 was demonstrated only in early wound healing, and the MMP‐9 gene is upregulated when the granulation tissue matures.

Spindle cell tumours of the pleura: a clinical, histological and comparative genomic hybridization analysis of 14 cases

We examined 14 spindle cell tumours of the pleura that were sent to a Mesothelioma Panel for re-evaluation after a primary suspicion of mesothelioma. The clinical, histological, immunohistochemical and CGH findings were investigated. Final diagnoses were eight sarcomatoid mesotheliomas (SM) and six non-mesotheliomas: two pulmonary sarcomatoid carcinomas, an epithelioid hemangioendothelioma, a malignant solitary fibrous tumour, a malignant pleural smooth muscle tumour and an extraskeletal osteosarcoma. Seven of the eight SM and two of the other six tumours presented with unilateral pleural effusion, dyspnoea, and chest pain, which are characteristic clinical findings in malignant mesothelioma. No single antibody used in the immunohistochemistry separated SM from other tumour types. The most frequently observed chromosomal losses in SM were 4q, 4p11-p13/p15, 6q and 13. Losses of 4p11-p13/p15 and 4q occurred in combination in four out of five SM with detectable chromosomal changes, but neither was found in any of the other tumours. Gain or high-level amplification of 5p was also common in SM. According to our results and literature, losses at 4p, 4q and 9p and gain at 5p are the chromosomal changes that best differentiate SM from pleural sarcomas and lung carcinomas. CGH analysis may help distinguish a cytokeratin-positive SM from a sarcomatoid carcinoma. Similarly, in the case of a cytokeratin-negative tumour, CGH analysis may disclose chromosomal changes characteristic of sarcomas or mesotheliomas.

Connective tissue growth factor and its correlation to other growth factors in experimental granulation tissue

Connective tissue growth factor (CTGF) is upregulated in a variety of fibrotic disorders, probably secondary to the activation and production of transforming growth factor-beta (TGF- g ). We have studied the expression of CTGF in a rat wound-healing model using Northern blot, in situ hybridization, and immunohistochemistry. The expression of CTGF mRNA in Northern blot and immunohistochemistry were correlated to the expression of TGF- g 1 and platelet-derived growth factor (PDGF). Northern hybridization showed the maximum expression of CTGF mRNA on day 14, whereas TGF- g 1 expression was maximal on days 7 and 14 and the time-related changes were smaller than for CTGF. PDGF A and PDGF B mRNA expressions were at maximum on day 14 and on day 21, respectively. In situ hybridization showed that fibroblast-like cells expressed CTGF most intensively, expression declining rapidly after day 14. CTGF mRNA and protein were found in blood vessel cells during the first week. In immunohistochemistry, all growth factors were expressed by fibroblast-like cells, macrophage-like cells, and blood vessels but CTGF-positive cells were fewer and were more restricted on days 5 and 7. These results demonstrate that CTGF expression together with TGF- g and PDGF are upregulated in wound healing, and CTGF expression in blood vessels suggests that CTGF is involved in angiogenesis.

Cloning of cDNA for Rat Pro α1(V) Collagen mRNA. Expression Patterns of Type I, Type III and Type V Collagen Genes in Experimental Granulation Tissue

A cDNA clone for rat pro alpha1(V) collagen mRNA was constructed using PCR amplification, with primers based on human and hamster COL5A1 gene sequences. The clone pRCVA1 is 560 nucleotides long and it encodes for the carboxy propeptide of type V procollagen. Homology shared with type I collagen sequence was 64%, with type II collagen 65% and with type III collagen 61%. To evaluate the spatial and temporal expression of type V collagen mRNA in wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 5, 8, 14, 21, 30, 59 and 84. Specific cDNA probes to pro alpha1(I), pro alpha1(III) and pro alpha1(V) collagen mRNA were used in slot blot, Northern and in situ hybridization. Type I collagen gene expression was upregulated at the initial stage of wound healing, type III collagen gene expression was constant and from the day 14 onwards type I and III collagen gene expressions were at the same level. Type V collagen gene expression was seen at every time point studied but at a considerably lower level than type I and III collagens. In situ hybridization showed that type V collagen was expressed in two different cell types. In conclusion, type V collagen was expressed in the wound healing model from at least day 5 onwards and it was synthesized by fibroblast-like and rounded cells.

Type V Collagen in Experimental Granulation Tissue

To evaluate the spatial and temporal expression of type V collagen in a wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 3, 5, 8, 14, 21, 30, 59 and 84. Acid soluble collagens were extracted and the relative amount of type V collagen was quantified by SDS-PAGE. Specific antibodies to type I, III and V collagens were used in immunohistochemistry and specific RNA probes to proalpha1(I), proalpha1(III) and proalpha1(V) collagen in in situ hybridization. Type V collagen content increased relative to type I and III collagens up to day 8 and remained at the same level for up to the three months. Type V collagen was expressed strongly in blood vessel walls as seen in immunohistochemistry. In situ hybridization showed that all of the three types of collagen were expressed mostly in fibroblast-like cells and also in rounded cells, especially type V collagen. In conclusion, type V collagen was seen in the wound healing model in increasing amounts from day 3 onwards, its localization being highly associated with blood vessels in granulation tissue and it was synthesized by fibroblast-like and rounded cells.

Biocompatibility of latex and silicone T tubes in the porcine common bile duct: an experimental study

Toxicity of materials used in indwelling drains or catheters has the potential to damage surrounding tissues. The biocompatibility of biliary T-tubes was investigated using in groups of piglets (total 30 animals). In group 1 (n=16) a choledochotomy was performed with insertion of a latex T-tube (n=6) or a silicone T-tube (n=8), or without a T-tube (n=2). In group 2 (n=14) the common bile duct (CBD) was 3/4-transected, and the lesion was sutured over a latex T-tube (n=4) or a silicone T-tube (n=5), or without a T-tube (n=5). The groups were reoperated upon after 2 and 6 weeks respectively, and the CBD was harvested for scanning electron microscopy and light microscopy. The T-tubes were examined for cell culture toxicity with a DNA synthesis ihibition test. According to the cell culture tests, latex T-tubes were toxic and silicone T-tubes nontoxic. T-tubes induced moderate to pronounced fibrosis and epithelial damage in the CBD wall, but did not induce excessive fibrosis or scarring at the site of CBD suture. In the 6-week study period, however, the grade of tissue reactions in the CBD did not correlate with the toxicity of the T-tube materials, but rather reflected a foreign body reaction and mechanical pressure. Although gross anatomical changes did not occur, neither material seemed to be completely harmless for porcine CBD wall.

Signalling initiated with CD4–TCR or TCR–TCR interactions: comparison of tyrosine phosphorylation patterns and CD45 effects

Antigen-triggered response in T cells is mediated by the T cell receptor (TCR)/CD3-complex. This signalling, however, is modulated by a number of other surface molecules. Among the most important of these is the CD4/CD8 molecule which associates with the TCR/CD3-complex and binds to the MHC complex. The molecular mechanisms involved in interactions between TCR-TCR and TCR-CD4 are not fully understood. We have earlier described an experimental model that allows us to dissect signals involving CD4-TCR interactions and those involving TCR-TCR interactions using a mouse CD4-CD8- T cell hybridoma cell-line transfected either with the TCR from a mouse T-helper 2 cell-line (D10) alone or with both the TCR and the CD4 molecule. To further characterize these two different modes of signalling in T lymphocytes we have studied the tyrosine phosphorylation patterns resulting from these interactions. In addition, we have studied the modulatory effect of the CD45 molecule on these interactions. In contrast to some earlier reports, we found that both the patterns of induced tyrosine phosphorylation and the effects of CD45 modulation were essentially similar in the CD4-TCR and the TCR-TCR signal transduction cascades. The results are consistent with a purely synergistically amplifying function for CD4 on the TCR-mediated signalling.