Synthesis of type I and III collagen, expression of fibronectin and matrix metalloproteinases-1 and -13 in hernial sac of patients with inguinal hernia

Since many years the importance of a weakness of the soft tissue for the development of hernias is discussed controversially. The tensile strength of the tissue is supposed to depend largely on the varying proportion of type I collagen with its high tensile strength and the immature type III collagen. Their relation is regulated by several collagenases, mainly matrix metalloproteinases-1 and -13 (MMP-1 and MMP-13), whereas fibronectin plays a key role for the adherence of cells within the extracellular matrix. The aim of this study was to investigate whether an alteration in type I and type III collagen synthesis, amounts of MMP-1 and MMP-13 and the expression of fibronectin were associated with the development of inguinal hernia. We analysed the hernial sac of patients with indirect (n = 9) and direct (n = 7) inguinal hernias and peritoneum in controls (n = 7) by immunohistochemistry and Western blot analysis. The results showed that the ratio of relative amount of I/III collagen was markedly decreased in patients with either indirect or direct hernias as compared with controls (p < 0.001) with a concomitant increase in type III collagen synthesis. MMP-13 was expressed neither in the hernial sac nor in the peritoneum of the controls, but the positive reactions of MMP-1 were found in the surface of the subserosa of the hernial sac in patients with indirect or direct hernias without any difference compared to controls. Furthermore, the relative amount of fibronectin in patients with either indirect or direct hernias is not significantly different from controls (p > 0.05). In regard to the known alterations of the collagen metabolism in fascia and skin of hernia patients the changed collagen I/III ratio with its increase of type III collagen in hernial sacs support the presence of a systemic disturbance of collagen metabolism. The absence of changes of the expression of collagenases (MMP-1, MMP-13) and the constant levels of fibronectin underline the central role of collagen synthesis for the development of indirect or direct hernias.     

Collagen distribution and expression of matrix metalloproteinases 1 and 13 in patients with anastomotic leakage after large-bowel surgery

PURPOSE: Despite improved surgical techniques, anastomotic leakage is still a serious complication in colorectal surgery, resulting in increased morbidity and mortality. Based on recent studies showing a disturbance of the collagen synthesis in hernia patients, this pilot study was designed to find out whether there are any hints of disorders concerning the collagen metabolism in patients developing anastomotic leakage after colonic surgery. METHODS: Ten samples of colonic tissue from patients who developed anastomotic dehiscence after surgery for colonic cancer were compared with 14 specimens from patients with uncomplicated anastomotic healing. All samples were obtained at the primary operation. We performed a sirius red test for the overall collagen content and immunhistochemical studies facing differentiation between collagen I and III and the expression of MMP-1 and MMP-13. RESULTS: In the bowel sections from patients developing anastomotic leakage, decreased levels of collagen I and III, and a smaller amount of overall collagen were found. The ratio of collagen I:III was similar in both groups. While MMP-1 was expressed in all samples, there was a difference concerning the expression of MMP-13. In only 6 of 14 patients with uncomplicated healing was MMP-13 present, while in 9 of 10 patients developing a leakage. CONCLUSIONS: Our data may indicate the presence of an individual disturbance of the collagen metabolism in patients developing a leakage after colorectal surgery. Further studies are underway to define more precisely whether a preexisting impairment of wound healing could be a risk factor for anastomotic leakage.     

Collagen I/III and matrix metalloproteinases (MMP) 1 and 13 in the fascia of patients with incisional hernias.

The late appearance ofincisional hernias several years after laparotomy and the high recurrence rates after operation strongly imply the presence of a disorder of the connective tissue, although a specific defect in patients with incisional hernias has not yet been identified. In the present study we used both immunohistochemistry and Western blot analysis to evaluate the ratio of collagen I and III and the expression of the metalloproteinases (MMP) 1 and 13 in the fascia of patients with incisional or recurrent incisional hernias. Samples of healthy skin or stable skin scar in patients without hernias served as controls. Altogether, our data indicated a significantly decreased ratio of collagen I/III in the fascia of patients with incisional hernias and recurrent incisional hernias. Furthermore, in these patients the expression of MMP-1 was decreased compared to the controls, whereas MMP-13 could not be detected in any fascia sample, with or without hernias present. For the first time, our results give evidence of the existence of a possible collagen disorder in these patients. The decreased ratio ofcollagen I/III is explainable due to a relative increase of collagen type III, which is known to be characterized by thin fibril diameters and lowered mechanical strength. The altered collagen ratio might be the result of the decreased activity of MMP-1, whereas the absent MMP-13 expression did not seem to modify the scar formation. Thus, our data indicate the presence of collagen metabolic disorders in patients with incisional hernias and recurrent incisional hernias. Furthermore, these results might explain the poor results of a mesh-free hernia repair, which again builds up scar tissue of inadequate collagen composition and strength.     

Renal expression of matrix metalloproteinases in human ANCA-associated glomerulonephritis.

BACKGROUND: Expression of matrix metalloproteinases (MMPs) by infiltrating and intrinsic renal cells is increased in inflammatory conditions, and may correlate with disease activity of glomerulonephritis. We analysed renal expression of MMPs, tissue inhibitor of metalloproteinase-1 (TIMP-1) and markers of neutrophil and monocyte infiltration in renal biopsies of patients with active anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis. METHODS: Immunohistochemical expression of MMP-2, -3, -9, TIMP-1, the neutrophil- and monocyte-derived MMP activators cathepsin G, neutrophil elastase and myeloperoxidase (MPO), and the monocyte marker CD14 was determined in renal biopsies of active proteinase 3 (PR3)-ANCA (n = 7) and MPO-ANCA (n = 6) associated glomerulonephritis, and in normal renal tissue (n = 4). Double labelling experiments of MMPs and TIMP-1 were performed with MPO and CD68, labelling neutrophils and macrophages. RESULTS: MMP-2-, MMP-3-, MMP-9- and TIMP-1-positive cells were detected in ANCA-associated glomerulonephritis in glomeruli with active inflammation (cellular crescents or fibrinoid necrosis), only occasionally in normal appearing glomeruli, and not in sclerotic glomeruli and positive cells were found in the tubulo-interstitium. MMPs and TIMP-1 were expressed predominantly by MPO-and CD68-positive cells. In normal renal tissue, no expression was detected, with the exception of weak mesangial staining for MMP-2. In ANCA-associated glomerulonephritis, glomerular MMP-2, -9 and TIMP-1 correlated with glomerular cathepsin G expression, while the number of MMP-9-expressing cells per glomerulus correlated with the percentage of crescentic glomeruli. Tubulo-interstitial expression of MMPs correlated with all markers of neutrophil and monocyte infiltration, and interstitial MMP-9 and TIMP-1 expression correlated with renal function at the time of renal biopsy. CONCLUSIONS: Expression of glomerular and interstitial MMP-2, -3, -9 and TIMP-1 is increased in active ANCA-associated glomerulonephritis and correlates with inflammatory activity.     

Type I collagen content is increased in lungs of patients with adult respiratory distress syndrome.

Collagen in lung tissue was examined from patients with adult respiratory distress syndrome, from patients who did not have this disease but required mechanical ventilation and oxygen treatment, and from patients without overt lung disease. Cyanogen bromide peptide mapping techniques were used to determine the ratio of type I to type III collagen present in these lungs. In the fibrotic lungs from patients with adult respiratory distress syndrome a shift was found in the ratio of type I to type III from the normal value of 2:1 to a mean value of 3.4:1. In patients with normal lungs and those with other lung diseases collagen type ratios were normal. Our data suggest that (i) changes in lung collagen of patients with adult respiratory distress syndrome resemble those previously described in patients with idiopathic pulmonary fibrosis, although the changes occur much more rapidly in the former; (ii) the increased content of collagen in lungs of patients with adult respiratory distress syndrome shown by others is predominantly of type I collagen; and (iii) the stimulus to the lung to produce excess type I collagen relative to type III is not solely of iatrogenic origin--that is, resulting from oxygen or ventilator treatment.     

Immunoelectron microscopy of osteonectin and type I collagen in osteoblasts and bone matrix

Summary The pathway of production, secretion, and extracellular deposition of type I collagen and osteonectin was studied by immunoelectron microscopy using respective polyclonal antibodies. Protein A gold and immunogold methods yielded to similar results in human callus tissue used as a model of bone formation. The intracellular distribution of osteonectin in active osteoblasts is found as a faint immunolabeling of vesicular Golgi fields and some lamellae of rough endoplasmic reticulum. A more intensive labeling occurs in opaque cytoplasmic vesicles pointing to the process of secretion as some of the vesicles are connected with the basal cell membrane. Our type I collagen antibody did not label the respective intracellular compartments. Extracellulary, the type I collagen antibody showed a continuous labeling from the immature subcellular osteoid to the mineralized bone. Osteonectin antibodies were bound first to the deeper layer of osteoid maturation with intensity increasing below the mineralization front. Osteonectin is thought to be associated with mineralization of bone matrix.     

Aberrant esophageal HLA-DR expression in a high percentage of patients with Crohn's disease.

Esophageal histology is not well studied in patients with Crohn's disease (CD). We, therefore, analyzed the histologic and immunohistologic appearance of esophageal mucosa in CD. Biopsy specimens taken from the esophagus of 57 consecutive patients with known CD of the large and/or small bowel, of 200 Crohn's-free controls, of 15 cases with ulcerative colitis, and of 5 cases with viral esophagitis were evaluated. In controls, most patients had either HLA-DR negative esophageal epithelium or showed focal or diffuse basal staining. HLA-DR expression of all epithelial layers (transepithelial staining) was observed in only four (2%) control subjects, in one case with herpes esophagitis, but not in patients with ulcerative colitis. In contrast, transepithelial HLA-DR expression was found in 19 (33%) patients with CD (p < 0.0001). In CD patients, it was associated with a significantly increased epithelial content in T-cells (CD3+, TIA-1+, granzyme B+), B-cells (CD79a+), natural killer cells (CD57+), and macrophages (CD68+). There was no correlation with either histological findings elsewhere in the upper gastrointestinal tract or with laboratory findings, symptoms, CDAI, or medication. Transepithelial esophageal HLA-DR expression is common in CD. Immunohistochemistry may prove useful in supporting the histologic diagnosis of CD in staging procedures, for initial diagnosis as well as in doubtful cases.     

Expression of the extracellular matrix proteins collagen I, collagen III and fibronectin and matrix metalloproteinase-1 and -13 in the skin of patients with inguinal hernia

Although abnormal collagen metabolism has been ascribed an important role in the high recurrence rates after surgical hernia repair, knowledge on tissue sampled in the region affected by inguinal hernias is poor. In the present study, we determined collagen type I and type III in the skin of adult patients with indirect and direct inguinal hernias by both immunohistochemistry and Western blot analysis. In addition, we quantified the immunohistochemical expression of fibronectin and matrix metalloproteinase (MMP)-1 and -13. The results indicated that the ratio of collagen type I/III was significantly decreased in the skin of patients with either indirect (n = 9) or direct hernia (n = 7), with a concomitant increase in collagen type III (p < 0.001 vs. controls, n = 7, without affection of the inguinal region). There was no significant difference between patients with indirect and direct hernia (p > 0.05). MMP-13 was not expressed in any of the skin samples investigated, whereas MMP-1 was found in the epidermis. Fibronectin was predominantly detected at the epidermal-dermal junction. MMP-1, MMP-13 and fibronectin levels were significantly different between patients and controls (p > 0. 05). We conclude that in contrast to the unchanged expression of fibronectin and MMP-1 and MMP-13, the decreased ratios of collagen tpye I/III with the basically increased amount of collagen type III could be of significant importance for the pathophysiology of hernias. The specific ratio collagen I/III probably reflects the altered structural integrity and mechanical stability of the connective tissue in both indirect and direct hernias. Moreover, our findings stress that hernias should be regarded as the manifestation of a systemic disease in the inguinal region with a genetic background, explaining the high recurrence rates after repeated suture repair, as well as the usefulness of surgical meshes in this clinical setting.     

Up-regulation of extracellular matrix proteoglycans and collagen type I in human crescentic glomerulonephritis

BACKGROUND: The pathogenesis of crescentic glomerulonephritis (CGN) involves cellular migration and proliferation in the urinary space, frequently followed by fibrous organization. Extracellular matrix proteoglycans (PGs) may regulate these events via effects on cellular migration, interactions with growth factors, including transforming growth factor-beta (TGF-beta), and control of collagen fibrillogenesis. The expression of PG in human CGN is unknown. METHODS: Renal tissues from 18 patients with CGN were examined immunohistochemically for versican, decorin, biglycan and collagen type I, and were compared with morphologically normal tissues from six tumor nephrectomies. Synthesis of decorin, biglycan, and procollagen type I mRNAs was evaluated by in situ hybridization. RESULTS: Versican was strongly expressed in cellular crescents and periglomerular areas, whereas decorin and biglycan accumulated in collagen type I-enriched regions, including fibrocellular and fibrous crescents, and interstitial fibrosis. PG and collagen type I accumulation colocalized with myofibroblasts in crescents, periglomerular areas, and interstitium. CONCLUSIONS: The temporal and spatial patterns of expression demonstrated in this study provide evidence to support pathogenic roles for PG in the evolution of CGN. Based on known biological properties of this molecule, versican may facilitate migration of cells in developing crescents. Decorin and biglycan may contribute to progression of CGN, perhaps via interactions with collagen type I in the remodeled extracellular matrix.     

RECK expression in osteosarcoma: correlation with matrix metalloproteinases activation and tumor invasiveness.

Osteosarcoma is a malignant tumor of bone characterized by its high metastatic potential. For the development of metastasis, activation of matrix metalloproteinases (MMPs) is required. A novel MMPs inhibitor, reversion inducing cysteine rich protein with Kazal motifs (RECK), is known to down-regulate MMPs and suppress the invasive and metastatic potential in many tumor-derived cell lines and some types of tumors. The expression of RECK and its role in tumor invasiveness have never been studied in osteosarcoma. We examined RECK mRNA expression and MMPs activation in osteosarcoma using quantitative real time PCR, gelatin zymography, invasion assay, and transfection experiments. RECK was expressed but down-regulated in osteosarcoma cells. Activation of pro-MMP-2 was observed in all samples, whereas activation of MMP-2 and pro-MMP-9 was detected in only 11% and 7% of the samples, respectively. MMP-9 was not activated in any of the samples. The level of RECK expression was inversely correlated with pro-MMP-2 activation, and overexpression of RECK by transfection resulted in decreased pro-MMP-2 activation and reduced tumor invasiveness. These findings suggest that RECK plays an important role in the invasiveness of osteosarcoma.     

Circulating levels of matrix metalloproteinases and tissue inhibitors of metalloproteinases in patients with incisional hernia

Incisional hernia formation is a common complication to laparotomy and possibly associated with alterations in connective tissue metabolism. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are closely involved in the metabolism of the extracellular matrix. Our aim was to study serum levels of multiple MMPs and TIMPs in patients with and without incisional hernia. Out of 305 patients who underwent laparotomy, 79 (25.9%) developed incisional hernia over a median follow‐up period of 3.7 years. Pooled sera from a subset (n = 72) of these patients were screened for MMP‐1, MMP‐2, MMP‐3, MMP‐7, MMP‐8, MMP‐9, MMP‐10, MMP‐12, MMP‐13, TIMP‐1, TIMP‐2, and TIMP‐4 using a multiplex sandwich fluorescent immunoassay supplemented with gelatin zymography. The screening indicated differences in serum MMP‐9 and TIMP‐1 levels. Consequently, MMP‐9 and TIMP‐1 levels were measured in serum in the whole patient cohort with enzyme‐linked immunosorbent assay. There were no significant differences in either MMP‐9 (p = 0.411) or TIMP‐1 (p = 0.679) levels between hernia and hernia‐free patients. MMP‐9 was significantly increased in smokers compared with nonsmokers (p = 0.016). In conclusion, a possible involvement of MMPs and TIMPs in the pathogenesis of incisional hernia formation was not reflected systemically.     

Biomechanical regulation of type I collagen gene expression in ACLs in organ culture.

In this study, an ex vivo organ culture system that allows the application of controlled loads to the anterior cruciate ligament (ACL) was designed and used to characterize the influence of a step input in mechanical load on gene expression. A procedure for isolating bone-ACL-bone (B-ACL-B) complexes from rat knees was developed. After harvest and 24 hour culture, B-ACL-B complexes exhibited percentages of viability similar to that in intact ACLs (approximately 90%). Application of a physiologically relevant load of 5 N (superimposed on a I N tare load) resulted in changes in levels of mRNA encoding type I collagen. While levels of type I collagen mRNA significantly increased 32+/-13% (mean +/- standard errors of the mean (SEM)) over controls within the first hour of loading, levels decreased significantly to 44+/-9% of control after 2 h. Displacements induced by the 5 N load were measured by video dimensional analysis. Calculated axial strains of 0.141+/-0.034 were achieved rapidly during the first hour and remained essentially unchanged thereafter. These results demonstrate the feasibility of maintaining ligaments in organ culture and illustrate the time course expression of type I collagen following the application of a mechanical load.     

Urinary type IV collagen excretion reflects renal morphological alterations and type IV collagen expression in patients with type 2 diabetes mellitus

AIM: We tried to establish the significance of quantifying urinary type IV collagen (IV-C) excretion for the evaluation of renal involvement of type 2-diabetic patients. METHODS: Twenty patients (13 males and 7 females; age range 31 to 69 years) with type 2 diabetes mellitus had undergone renal biopsy and relationship between the severity of morphological alteration, IV-C expression and urinary IV-C excretion were examined. RESULTS: Urinary IV-C excretion significantly correlated with mesangial expansion score (p = 0.49, p < 0.05) and tubulointerstitial injury score (p = 0.56, p < 0.05). Furthermore, urinary IV-C excretion significantly correlated with both glomerular (r = 0.56, p < 0.01) and tubulointerstitial IV-C expression areas. Urinary protein excretion also correlated with mesangial expansion score and tubulointerstitial injury score. However, it did not correlate with the expression of IV-C in the kidney. CONCLUSIONS: These results suggest that urinary IV-C excretion reflects the pathogenetic process of diabetic nephropathy, which urinary protein excretion alone cannot do sufficiently. It can be concluded that urinary IV-C excretion could be a more useful marker for the evaluation of renal involvement of type 2-diabetic patients.     

Measurements and clinical usefulness of carboxyterminal propeptide of type I collagen and cross-linked carboxyterminal telopeptide of type I collagen in patients with prostate cancer]

Carboxyterminal propeptide of type I collagen (P1CP) and cross-linked carboxyterminal telopeptide of type I collagen (1CTP) are known as parameters of bone metastasis in patients with prostate cancer (PCa). We measured the serum P1CP and 1CTP levels in 52 PCa patients and evaluated the clinical usefulness of these serum markers. Both serum levels of P1CP and 1CTP were significantly higher in patients with extent of disease (EOD) grade 2 or 3 bone metastases than in patients without bone metastasis. Thus, P1CP and 1CTP are not as useful at first detection of bone metastases as bone scintigram. On the other hand, in the patients who indicated high serum levels of P1CP or 1CTP before initial treatment, the changes in the concentrations of these markers may be helpful in evaluating the response to treatment or the progression of disease. Our results suggest that P1CP and 1CTP are useful markers for monitoring the metastatic burden in the bone of PCa patients, but the efficacy is limited in high EOD grade cases.     

Increases in type III collagen gene expression and protein synthesis in patients with inguinal hernias.

OBJECTIVE: The aim of this study was to determine if alterations in fibrillar collagen synthesis were associated with the development of inguinal hernias. SUMMARY BACKGROUND DATA: Previous work has suggested that alterations in connective tissue accumulation may play a functional role in the development of inguinal hernias. In particular, several investigators have suggested that alterations in collagen synthesis, causally related to connective disorders such as osteogenesis imperfecta, may also be responsible for the inguinal herniation that is markedly increased in such patients. This study was undertaken therefore to study collagen synthesis in patients with inguinal hernia in the absence of any other connective tissue disease. METHODS: Skin fibroblasts from 9 patients with hernias and 15 control individuals were radiolabeled with 3H-proline. Trypsin-chymotrypsin-resistant type I and III collagens were isolated and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and recovery was quantified by laser densitometry. Steady-state levels of alpha 1(I) and alpha 1(III) procollagen mRNAs were also determined by northern and slot-blot hybridization analysis. RESULTS: The alpha 1(I)/alpha 1(III) collagen ratios were shown to be 6.3 +/- 0.34 in fibroblasts from control individuals and 3.0 +/- 0.25 in fibroblasts from patients with inguinal hernias. This statistically significant difference (p < 0.0001) was caused by an increase in the secretion of alpha 1(III) procollagen from the fibroblasts of patients with hernias. A concomitant increase in the steady-state levels of alpha 1(III) procollagen mRNA was observed in total RNA isolated from the patients' fibroblasts. CONCLUSIONS: A constitutive and systemic increase in type III collagen synthesis may result in reduced collagen fibril assembly in the abdominal wall, eventually leading to the development of herniation. Although it is not yet clear what genetic factors are responsible for the elevation in type III collagen synthesis in patients with hernias, this study represents the first attempt to define individuals with an abnormality in collagen production that may be specifically related to herniation. A clearer understanding of the possible genetic factors that influence the pathophysiology of this disease will be important to improve the treatment of patients in whom inguinal hernias develop.     

Anovaginal and rectovaginal fistula in patients with Crohn's disease.

Between 1971 and 1991, details of 67 women with perianal Crohn's disease were recorded prospectively using the Cardiff classification. Two groups were identified according to the presence (n = 29) or absence (n = 38) of anorectal Crohn's fistula involving the vagina. Patients in both groups were of a similar age and had had Crohn's disease for a similar period before diagnosis of perianal involvement. The incidence of associated perianal lesions, superficial ulcers, cavitating ulcers, other fistulas and strictures was not significantly different between the two groups. A greater proportion of patients with anorectal-vaginal fistulation (n = 15) had distal intestinal Crohn's disease (rectal or contiguous colorectal) compared with women with no vaginal fistulation (n = 14). A range of therapies was used to manage women with perianal Crohn's disease, from local surgery to a defunctioning stoma and/or proctectomy. Only 13 of 38 women with perianal Crohn's disease but no vaginal fistula required a defunctioning stoma or proctectomy, whereas 18 of 29 with anorectal-vaginal fistulation underwent these procedures (P < 0.05). A vaginal fistula has a considerable adverse effect on the outcome of perianal Crohn's disease.     

Presence of Chlamydia pneumoniae in abdominal aortic aneurysms is not associated with increased activity of matrix metalloproteinases.

OBJECTIVE: to test the hypothesis that the presence of Chlamydia pneumoniae (C. pneumoniae) in the wall of abdominal aortic aneurysms (AAA) is associated with increased activity of matrix metalloproteinase (MMP)-2 and/or MMP-9.DESIGN: case-control study. MATERIAL AND METHODS: in a series of 40 patients with AAA > or =5cm in maximal cross-sectional diameter, C. pneumoniae-DNA was identified in the aneurysm wall by nested PCR in 14 (35%) patients. Another 14 C. pneumoniae-DNA-negative AAA patients from the same series, matched for gender and aneurysm diameter, were used as controls. In each group there were 7 asymptomatic (aAAA) and 7 ruptured (rAAA) aneurysms. MMP-2 and -9 activity was estimated in AAA wall biopsies by gelatin zymography. RESULTS: patients with a C. pneumoniae-DNA-positive aneurysm wall specimen showed an over-all lower activity of MMP-2 and MMP-9 (pro- and active enzyme) compared to the C. pneumoniae-DNA negative patients. However, there were no statistically significant differences in MMP activity between the two groups of patients with aAAA. Among patients with rAAA both pro-MMP-9 (p=0,026) and active-MMP-9 (p=0.007) were significantly lower in C. pneumoniae-DNA-positive patients compared to C. pneumoniae-DNA-negative patients, whereas there were no significant differences in pro-MMP-2 or active-MMP-2. CONCLUSION: this preliminary study does not support the hypothesis that the presence of C. pneumoniae in the AAA wall is associated with increased activity of MMP-2 and MMP-9.