Type IV collagen,type IV collagenase activity and ability of cell proliferation in human thyroid tumours

BACKGROUND: The processes of malignant tumour invasion and metastasis are known to include the destruction of cell stroma and vascular basement membrane. It has been suggested that type IV collagenase degrades type IV collagen, a main component of the basement membrane. METHODS: In our study, type IV collagenase activity in human thyroid tumours was measured by the Liotta method. The degree of destruction of diseased regions of thyroid tumours was immunohistochemically determined by anti-type IV collagen antibody staining. Cell proliferation in the tumours was estimated using anti-proliferating cell nuclear antigen (PCNA) and epidermal growth factor receptor (EGFR). RESULTS: T4 thyroid carcinomas with higher type IV collagenase activity and very weak type IV discontinuous immunostaining for type IV collagen of follicular basement membranes, exhibited many PCNA or EGFR positive cells. In benign tumours, normofollicular- or macrofollicular-type tumours with low type IV collagenase activity showed few PCNA and EGFR positive cells and intact type IV collagen of basement membranes, as seen in normal thyroids. Conversely, an atypical adenoma with higher type IV collagenase activity showed many PCNA and EGFR positive cells and weak type IV discontinuous immunostaining for type IV collagen, as in thyroid carcinomas. CONCLUSION: These findings suggest that staining for type IV collagen and type IV collagenase activity reflect the ability of cell proliferation, and help predict the aggressiveness of invasion and metastasis in human thyroid tumours.     

胰腺星状细胞超微结构的在体实验研究

目的 在慢性胰腺炎动物模型,观察在体胰腺星状细胞的超微结构特点.方法 Sprague-Dawley大鼠随机分为实验组(n=12)和对照组(n=12).实验组给予16周高脂饮食诱导建立大鼠胰腺纤维化动物模型,对照组常规喂养.用HE和天狼猩红饱和苦味酸染色法观察胰腺组织病理学改变,免疫组化法识别胰腺组织中活化态及非活化态胰腺星状细胞,之后进一步通过透射电镜和免疫电镜方法对胰腺星状细胞的超微结构进行观察,最终对周细胞和泡心细胞进行形态学鉴别.结果 病理形态学观察表明,长期高脂饮食可以诱导大鼠胰腺组织发生纤维化改变.实验组胰腺组织中活化态与非活化态胰腺星状细胞共存,细胞数量都较正常组明显增加.结论 胰腺星状细胞在体超微结构特征的研究将为进一步研究其功能奠定形态学基础.     

Type IV collagenase activity and cavernous sinus invasion in human pituitary adenomas

Summary Pituitary adenomas are regarded generally as benign tumours, but some of them can invade the cavernous sinus. On the other hand, matrix metalloproteinase-9 (MMP-9), which is a type IV collagenase, recently has been found to be expressed in matastases and to be related to the invasiveness of various malignant tumours including brain tumours. In order to investigate some characteristic features of pituitary adenomas which invade the cavernous sinus, we examined immunohistological studies for MMP-9 in seven pituitary adenomas for type IV collagen in a dura mater and assayed for type IV collagenase activity in seven adenomas using type IV collagen labelled with fluorescein isothiocyaniate (FITC).We found immunopositive adenoma cells for MMP-9 in all invasive adenoma and immunopositive spindle like cells for type IV collagen in the dura mater. All three invasive adenomas had high levels of type IV collagenase activity (0.57–0.72 U/ml), but the four adenomas which did not invade the cavernous sinus had low levels of type IV collagenase activity (0.0–0.10 U/ml).These results suggest that the level of type IV collagenase activity in a pituitary adenoma may be related to its ability to invade the cavernous sinus.     

New direct assay method of type IV collagenase in tissue homogenate and biochemical role of collagenase against type I, and IV collagens to the invasion of the stomach and lung cancer

In order to study the role of both collagenases against type I and type IV collagen (type I and type IV collagenase) with regard to tumor invasion and metastasis, the activities of both collagenases in tissue homogenate in each 40 cases of stomach and lung cancers were investigated. The direct assay method of type IV collagenase in tissue was established through modification of Liotta's method. In stomach cancer, the part of advancing front of cancer showed the highest activity of type I collagenase. The adjacent mucosa to cancer also showed high activity of type IV collagenase. The cancer tissues that had the remarkable finding of vascular invasion of cancer cells showed high activity of type IV collagenase. In lung cancer, the correlation between the size of cancer mass and activity of type I collagenase was shown. Squamous cell carcinoma in comparison to adenocarcinoma had higher activity of type I collagenase and poor activity of type IV collagenase. These results suggested that the activity of type I collagenase might participate in local invasion and the activity of type IV collagenase might be associated with vascular invasion of cancer through disruption of basement membrane and they could be one of the useful biochemical tumor marker to represent the growth and metastatic pattern.     

Pancreatic stellate cell activation and MMP production in experimental pancreatic fibrosis

BACKGROUND: The early events in pancreatic fibrosis are poorly understood. We examined the production of collagen and matrix metalloproteinases as well as the activation of pancreatic stellate cells in a rodent model of pancreatic fibrosis. MATERIALS AND METHODS: Pancreatitis was induced in rats by hyperstimulation with cerulein (50 microg/kg/day ip) and concurrent pancreatic duct obstruction (SHOP model) for 96 h (n = 48). Sham animals were injected with saline and underwent laparotomy and manipulation of the pancreas with no duct obstruction (n = 28). Rats were sacrificed daily for 18 days. Serial pancreatic sections were stained with H&E [histology], trichrome [collagen], and alpha smooth muscle actin (alpha-SMA) antibodies [activated stellate cells]. Total pancreatic matrix metalloproteinase (MMP)-2 and 9 were determined by gelatin zymography. MMP-1 production was examined using Western blotting. RESULTS: There were occasional alpha-SMA-positive cells in the pancreatic parenchyma of normal and sham animals. Within 48 h of pancreatitis induction in SHOP animals, histologic evidence of pancreatic inflammation was present, and stellate cells (alpha-SMA-positive cells) appeared surrounding pancreatic acini. The appearance of these cells was followed by collagen deposition in the same area. MMP-1 and 2 proteins increased significantly during pancreatitis while MMP-9 did not. The pancreatic architecture returned to normal by 18 days after the induction of pancreatitis. CONCLUSION: Acute pancreatic inflammation results in stellate cell activation and collagen deposition in the same area. Collagen is then resorbed at a time when MMP-1 and 2 peak. The fibrosis of acute pancreatic inflammation in this model completely resolves with restoration of normal architecture.     

Expression of the type IV collagenase system during mouse kidney development and tubule segmentation.

Type IV collagenases matrix metalloproteinase-2 (MMP2) and MMP9 and their related proteins, MT1-MMP, tissue inhibitor of metalloproteinases 1 (TIMP1), TIMP2, and TIMP3, are expressed during kidney morphogenesis and nephrogenesis, but the renal ontogeny of these proteins is only partially known, and their persistence in the adult remains controversial. Their expression was analyzed from early metanephric stages to adulthood by Western blot semiquantitative analysis; laser confocal microscopy of whole-mount kidneys; and a two-step immunoperoxidase labeling procedure using specific markers of proximal tubule (megalin), ascending limb of Henle's loop (Tamm Horsfall protein), and collecting duct (Dolichos biflorus agglutinin lectin). By Western blot, all antigens were detected at day 11.5, peaked at day 16.5, and persisted in the adult at lower levels, although MMP2 was less modulated. All antigens were expressed in metanephric mesenchyme at embryonic day 11.5 and became concentrated in neural cell adhesion molecule-positive-induced mesenchymal cells at day 12.5. Only MT1-MMP and to a lesser extent MMP2 were detected in the ureter bud. At day 16.5, all antigens predominated in the cytoplasm of the proximal tubule, except TIMP1, which was mostly expressed in the ascending limb of Henle's loop and distal tubule. During tubule segmentation, components of the type IV collagenase system showed both spatial and temporal regulation. The distribution of gelatinases was not strictly superimposable to that of their natural inhibitors TIMP, especially for MMP9 and TIMP1. All components persisted in specific segments of the adult renal tubule, where MMP9, MMP2, and MT1-MMP showed an apical expression, suggesting that substrates for these enzymes should be in the tubule lumen or in the apical cell domain and not in the extracellular matrix. These results suggest that a regulated balance of gelatinase activity is required during kidney organogenesis and that gelatinases continue to play a role in adult renal tubule physiology.     

1型血管紧张素Ⅱ受体拮抗剂抑制大鼠胰腺纤维化形成

目的探讨1型血管紧张素Ⅱ受体(AT1)拮抗剂洛沙坦对大鼠实验性胰腺纤维化形成的抑制作用。方法胰管内注射2％三硝基苯磺酸(TNBS)诱导大鼠胰腺纤维化模型。于制模后第2天，治疗组给予洛沙坦(10mg／kg体重)灌胃，每日1次，对照组给予等容积的无菌蒸馏水。于制模后3，7，14，21．28d分别处死两组大鼠(每时点各6只)，并留取血清和胰腺组织。通过HE染色和Van Gieson(V-G)染色观察胰腺组织病理学改变和细胞外基质胶原纤维分布。分别应用放射免疫法和酶动力法测定血清透明质酸(HA)和淀粉酶。胰腺组织AT1受体蛋白和mRNA表达分别采用免疫组化和逆转录-聚合酶链式反应(RT-PCR)方法。结果洛沙坦可抑制TNBS诱导的大鼠胰腺纤维化形成，降低胰腺组织炎症细胞浸润、腺泡细胞坏死及纤维化程度，并且能降低血清HA和淀粉酶水平、下调AT1受体基因和蛋白的表达。结论1型血管紧张素Ⅱ受体拮抗剂对TNBS诱导的大鼠胰腺纤维化形成具有抑制作用，表明肾素-血管紧张素系统(RAS)在慢性胰腺炎胰腺纤维化的发生发展过程中起重要的介导调节作用。     

Increased collagenase activity in early aneurysmal dilatation

Increased collagenase activity has been implicated as a basic abnormality in aortic aneurysm formation. We studied a localized aneurysmal change, poststenotic dilatation, and its relation to collagenase and elastase activity of the aortic wall. Cynomolgus monkeys underwent midthoracic aortic coarctation to produce poststenotic dilatation. Serial angiography showed that poststenotic dilatation was minimal or absent at 10 days, just discernible at 3 months, and prominent at 6 months. At the 3-month time interval, collagenase activity in the region of the poststenotic dilatation increased twofold compared with the same region in aortas from animals without poststenotic dilatation (p less than 0.05). There was no change in aortic elastase activity. These data indicate that collagenolysis and aneurysmal dilatation may be induced by local modifications of pressure and/or flow. Increased collagenase activity associated with abdominal aortic aneurysms may not represent an intrinsic metabolic defect but rather a response to altered hemodynamic conditions.     

Decreased sympathochromaffin activity in IDDM

Catecholamines released from the sympathochromaffin system produce metabolic changes similar to those of diabetes mellitus. However, increased sympathochromaffin activity does not appear to be a feature of insulin-dependent diabetes mellitus (IDDM), although physiologic catecholamine increments may contribute to short-term metabolic derangements under some conditions. Increased glycemic sensitivity to epinephrine is a feature of IDDM but is the result of the inability to secrete insulin rather than of increased cellular sensitivity to catecholamines. Absolute insulin deficiency results in increased metabolic (glycemic, lipolytic, and ketogenic) sensitivity to catecholamines. More generalized hypersensitivity occurs in diabetic autonomic neuropathy. However, the clinical relevance of these alterations in sensitivity remains to be established. On the other hand, decreased sympathochromaffin activity is common and causes considerable morbidity and some mortality in people with diabetes. In addition to increased sensitivity to catecholamines, decreased sympathochromaffin activity results in the clinical syndromes of postural hypotension, hypoglycemia unawareness, defective glucose counterregulation, or a combination of these. The latter two syndromes cause an increased frequency of severe iatrogenic hypoglycemia, at least during intensive therapy of IDDM. Thus, decreased rather than increased sympathochromaffin activity often complicates IDDM. Clearly, ways to prevent, correct, or compensate for this component of diabetic autonomic neuropathy must be learned before diabetes can be managed effectively and safely in all patients who suffer from the disease until diabetes mellitus is eradicated.     

Evaluation of basement membrane components and the 72 kDa type IV collagenase in serous tumors of the ovary.

We studied the distribution of the basement membrane components laminin and type IV collagen in 46 serous tumors of the ovary, including a group of low malignant potential tumors with microinvasion. The findings were correlated with the expression of the 72 kDa type IV collagenase, an enzyme that initiates the degradation of type IV collagen and consequently may play a role in the process of invasion. Benign cystadenomas and tumors of low malignant potential without microinvasion showed a continuous basement membrane; whereas invasive carcinomas, peritoneal implants, and lymph node metastasis had frequent disruptions and extensive areas without basement membrane components. Early invasion in tumors of low malignant potential was characterized by focal disruptions in basement membranes and complete absence of laminin and type IV collagen around single or clusters of microinvasive cells. Type IV collagenase was negative or minimally expressed in cystadenomas, whereas in invasive carcinomas and metastasis the reactivity was moderate to intense. Microinvasive cells in tumors of low malignant potential were strongly positive. The collagenase IV was also localized in cell clusters elsewhere in the tumors where the basement membrane was still preserved. These cells had a similar morphology to that of the microinvasive cells. We conclude that detection of basement membrane components may be useful in recognizing early invasion in this group of ovarian neoplasms. The correlation between progressive anomalies of the basement membrane and expression of type IV collagenase suggests that this enzyme functions directly in the degradation of basement membrane components and facilitates the invasive process.     

Morphological changes in pancreatic fragments prepared for transplantation by collagenase treatment

Canine pancreases were excised, minced, mechanically chopped, and incubated with collagenase in a manner similar to that used routinely in the preparation of mixed-cell pancreatic autografts. The resultant pancreatic fragments were studied by light and electron microscopy after various periods of collagenase incubation. As a control, pancreatic tissue was studied immediately after organ excision, immediately before addition of collagenase, and after various periods of incubation in balanced salt solution without collagenase. The mincing and chopping procedures alone induced a large population of highly aberrant, severely vacuolated acinar cells within the fragments. This vacuolation was caused by massive dilation of the cisternae of the rough endoplasmic reticulum. Subsequent incubation in collagenase solution, which appeared to destroy the aberrant cells in a selective manner, led to a remnant population with much improved acinar cell morphology. Incubation in balanced salt solution without collagenase resulted in a progressive increase in vacuolated cell incidence until abnormal pancreatic acinar cells dominated the tissue. The findings suggest that collagenase treatment may facilitate mixed-cell pancreatic transplantation by culling degenerative acinar cells from the grafted cell population, thereby reducing the likelihood of autodigestion at the transplant site. The extensive acinar cell destruction mediated by the collagenase may also be responsible for the release of a previously described hypotensive factor (pancreatic shock factor) into the graft infusion.     

A pathological study of experimental chemonucleolysis with collagenase

This study was undertaken to examine and quantitate the chemical injury and repair of the dog nucleus pulposus in collagenase-induced chemonucleolysis. Thirty three adult mongrel dogs were used into which the purified collagenase was injected at a rate of 200 units. The dogs were sacrificed after roentgenography. Disc specimens were stained primarily with safranin-O for microscopic study, and fresh tissues were used for electron microscopic study. For immunohistochemical analysis, paraffin sections were reacted with anti-bovine types I, II and III collagen antibodies. Two weeks after the injection, the height of the injected disc was reduced by about 50% and there was no sign of recovery for 52 weeks. There was a profound loss of safranin-O stainability in the nucleus pulposus, annulus fibrosus and cartilagenous endplate. By 8 weeks, regeneration of the nucleus pulposus began and chondrocytes appeared from the junctional area. The regenerated nucleus pulposus was stained well with safranin-O and consisting largely of type II collagen immunohistochemically. In the regenerated nucleus pulposus, collagen fibers and proteoglycans were reconstructed in a net-work with hyaluronic acids.     

Decreased vascular density in mouse pancreatic islets after transplantation

An adequate revascularization is crucial for islet survival and function after transplantation. Previous studies have suggested that islet revascularization is concluded within 14 days after transplantation. We investigated if the vascular density of transplanted islets and endogenous pancreatic islets differs. Cultured islets were syngeneically transplanted into the kidney, liver, or spleen of C57BL/6 mice. One month later, the graft-bearing organ was removed, and histological specimens were prepared and stained for endothelium with the lectin Bandeiraea simplicifolia. Pancreata from nontransplanted control animals were prepared similarly. Uniform staining of endothelium within the grafts and endogenous islets was obtained. The vascular density was markedly decreased in transplanted islets at all implantation sites, but preferentially in islets implanted into the spleen. The vascular density in the connective tissue surrounding the transplanted islets was very high compared with that of graft intra-islet capillaries. A much lower vascular density was detected in connective tissue surrounding implanted microspheres of a size similar to the islets, which suggests that the islets per se induced blood vessel formation in their vicinity. We conclude that the vascular density in revascularized transplanted islets is markedly decreased compared with endogenous islets. This has potential implications for islet graft metabolism and function.     

细菌性颅内动脉瘤72-和92-KDa IV型胶原酶及其组织抑制因子mRNA的原位杂交

目的　探讨细菌性颅内动脉瘤 (BIAs)的发病机理。方法　用地高辛标记的 72 和92 KDaIV型胶原酶 (MMP 2和MMP 9)及其组织抑制因子 (TIMP) 1、TIMP 2反义RNA探针与BIAs(n =2 )和正常脑动脉组织 (n =6 )进行原位杂交 ,定位产生它们的细胞。并用正义RNA探针进行阴性对照。结果　 2份感染性颅内动脉瘤标本中均见MMP 9mRNA阳性杂交信号。且在内膜 ,尤其是相当于内弹力层部位 ,阳性杂交信号密集存在。动脉瘤壁内弹力层消失。阳性杂交信号定位于单核细胞、成纤维细胞和平滑肌细胞。正常脑动脉标本中未发现MMP 9mRNA阳性杂交信号。在BIAs和正常脑动脉组织中未发现MMP 2、TIMP 1和TIMP 2的mRNA阳性杂交信号。结论　感染因素引起炎性细胞浸润 ,表达MMP 9mRNA增多 ,导致内弹力层断裂、消失 ,甚至动脉壁全层结构的破坏 ,从而引发BIAs形成。     

慢性胰腺纤维化研究的新热点

实质纤维化是慢性胰腺炎的特征。静息型胰腺星状细胞受转化生长因子β（TGF-β）刺激后转化激活,导致细胞外基质大量生成并最终发展为纤维化。Smad能正性或者负性调节TGF-β信号传导,深入研究其机制有助于明确纤维化的成因,指导慢性胰腺炎的治疗。     

Type I, type III, and type IV collagenolytic enzyme activity in lung cancer tissue]

To investigate the changes of collagen metabolism in patients with lung cancer, activities of types I, III, and IV collagenolytic enzymes in specimens obtained from lung cancer tissue and pathomorphological changes of the corresponding specimens were analyzed. And the comparison of the biochemical and morphological changes were evaluated. Nine resected pulmonary carcinomas were used and 5 samples were obtained from each carcinoma tissue. In each sample, types I, III, and IV enzyme activities were measured and its histopathological examination was done. Enzyme activities of 5 samples from the same carcinoma tissue were considerably different in all subtype of collagenolytic enzymes. The relationship between each collagenolytic enzyme activity of these samples and their histopathological findings are as follows: 1) No relationship is found between the amount of carcinoma cells in cancer tissue and enzyme activity in any subtype, 2) increase in the fibrosis in cancer tissue is accompanied by decrease in type I collagenolytic enzyme activity, 3) the substantial increase in inflammatory cells coexisting with carcinoma is accompanied by increase in types I and III collagenolytic enzyme activities. Moreover, type III collagenolytic enzyme activity elevates significantly when inflammatory cells infiltrate among carcinoma cells.     

人间质胶原酶基因高效表达对体外肝纤维化的影响

目的 观察人间质胶原酶(MMP1)基因高效表达对体外肝纤维化的影响。方法 利用DNA重组技术构建了pcDNA3-MMP1真核表达重组质粒。经脂质体包裹后,转染张氏传代人肝细胞,Western blot检测 MMP1蛋白表达量。在体外乙醇诱导纤维化培养条件下,抗生蛋白链菌素-生物素复合物标志(LSAB)免疫组织化学法检测肝细胞周围Ⅰ、Ⅲ型胶原生成状况,图象分析仪半定量分析胶原纤维的相对含量(RCC)。结果 成功构建真核表达质粒pcDNA3-MMP1,检测RCC 显示未转染组为132.6±5.7,转染组为118.8±4.8;统计学分析表明未转染组明显高于转染组(P<0.01)。结论 本研究证明重组质粒pcDNA3-MMP1能在体外致纤维化培养条件下拮抗肝细胞间质过多胶原产生。     

Repetitive mechanical stretch increases extracellular collagenase activity in vaginal fibroblasts

OBJECTIVES: The objectives were 1) to determine whether human vaginal fibroblasts are mechanosensitive and 2) to study the impact of mechanical stretch on these cells in the presence and absence of hormones. METHODS: Fibroblasts obtained from biopsies of full thickness vagina of 3 women were cyclically biaxially stretched at a magnitude of 8 and 16% for 72 hours with or without 17-β-estradiol plus progesterone. Culture media was collected and total collagenase activity was measured in duplicate using a fluorogenic substrate degradation assay. Data were analyzed at the 0.05 level of significance using Student t-test. RESULTS: Cells remained 90% viable throughout the experiments. Relative to the controls, hormonal treatment alone decreased collagenase activity (P=0.008). In the presence of mechanical stretch and in the absence of hormones, collagenase activity was increased (8% elongation, P=0.04; 16% elongation, P=0.001, respectively). The increase in collagenase activity was linearly correlated with magnitude (P<0.001). In the presence of hormones, the increase in enzyme activity by mechanical stretch was suppressed to baseline control levels (P=0.46). There was no difference in suppression by hormones by magnitude (P=0.48). CONCLUSIONS: Vaginal connective tissue fibroblasts are mechanosensitive with increased collagenase activity in the presence of stretch. This degradative behavior is inhibited in the presence of hormones. The data provide a mechanism by which events that induce vaginal stretch may lead to progression of pelvic organ prolapse, particularly, in the absence of hormones. Further studies are needed to determine whether these events lead to tissue with inferior mechanical properties.