Regulation of membrane type-1 matrix metalloproteinase activity and intracellular localization in clinical thoracic aortic aneurysms

Objective

Membrane type-1 matrix metalloproteinase (MT1-MMP) is elevated during thoracic aortic aneurysm (TAA) development in mouse models, and plays an important role in the activation of matrix metalloproteinase (MMP)-2 and the release of matrix- bound transforming growth factor-β. In this study, we tested the hypothesis that MT1-MMP is subject to protein kinase C (PKC)–mediated regulation, which alters intracellular trafficking and activity with TAAs.

Differential regulation of matrix metalloproteinase activities in abdominal aortic aneurysms

BACKGROUND: The increased synthesis of matrix metalloproteinases (MMPs) by aortic smooth muscle cells (SMCs) is thought to be involved in the etiopathogenesis of abdominal aortic aneurysms (AAAs), but the functional regulation and the activation states of these MMPs remain unclear. In this study, we assessed the expression levels and the functional regulation of several MMPs in the pathogenesis of AAAs. METHODS: Human healthy aorta and AAA specimens were homogenized, and the proteolytic activities of MMP-2 and MMP-9 and of the macrophage metalloelastase (MMP-12) were assessed with zymography. Protein expression of MMP-1, MMP-12, membrane-type 1 MMP (MT1-MMP), tissue inhibitor of MMP 1 (TIMP-1), TIMP-2, TIMP-3, alpha-actin, and beta-actin was analyzed with electrophoresis on sodium dodecyl sulfate gels and immunoblotting. RESULTS: MMP-1, MMP-9, and MMP-12 zymogen levels and proteolytic activities were increased in AAAs when compared with healthy aorta. A severe reduction in alpha-actin--positive vascular SMCs was observed in all the AAA specimens and was correlated with an increase in TIMP-3 but not TIMP-1 or TIMP-2 potential activities. Although pro--MMP-2 activity was decreased, the extent of activated MMP-2 remained unaffected in the AAAs. In accordance with this result, a highly activated MT1-MMP form was also observed in AAAs. CONCLUSION: These data suggest that chronic aortic wall inflammation is mediated by macrophage infiltration, which may account for the destruction of medial elastin, as reflected by SMC down regulation, through increased levels of active MMP-1 and MMP-12. Moreover, altered MT1-MMP proteolytic turnover and differential regulation of TIMP expression in AAAs suggest that tight regulatory mechanisms are involved in the molecular regulation of MMP activation processes in the pathogenesis of AAAs.     

Increased matrix metalloproteinase 2 expression in vascular smooth muscle cells cultured from abdominal aortic aneurysms

OBJECTIVES: Recent evidence has implicated matrix metalloproteinase 2 (MMP-2) in the pathogenesis of aneurysms. The aim of this study was to examine MMP-2 production and expression by aortic smooth muscle cells (SMCs) and dermal fibroblasts derived from patients with abdominal aortic aneurysms (AAAs). METHODS: Aortic SMCs and dermal fibroblasts were cultured from patients with AAAs or from age-matched controls with atherosclerosis. The production of MMP and tissue inhibitor of metalloproteinase into culture media was analyzed with the use of gelatin zymography, Western blotting, and enzyme-linked immunosorbent assay. Gene expression was analyzed with Northern blotting. RESULTS: All cells studied constitutively produced MMP-2. Aortic SMCs cultured from aneurysmal tissue expressed MMP-2 protein and messenger RNA at a significantly higher level than SMCs from controls (P =.008). Dermal fibroblasts from patients with AAAs expressed MMP-2 at a similar level to controls. In both cell types, tissue inhibitor of metalloproteinase 2 and membrane type 1-MMP were expressed at similar levels. CONCLUSIONS: These data suggested that the regulation of MMP-2 gene expression was altered in the aortic SMCs of patients with aneurysms, but this finding was not repeated in other mesenchymal tissue.     

The matrix metalloproteinase inhibitor BB-94 limits expansion of experimental abdominal aortic aneurysms

Purpose: Matrix metalloproteinases (MMPs) are proteolytic enzymes that can degrade the extracellular matrix of the aortic wall and lead to the formation of abdominal aortic aneurysms (AAAs). MMP inhibitors are a class of drugs that were developed to inhibit the activity of these proteolytic enzymes and are currently being studied as a way to control inflammatory diseases and cancer metastases. In this project, BB-94 (also known as batimastat), a specific inhibitor of MMPs, was evaluated for its ability to control aneurysmal growth in an experimental AAA model. Methods: Experimental AAAs were created in a standard rat model by perfusing elastase into an isolated segment of aorta. The rats then were randomized to postoperatively undergo treatment daily with the MMP inhibitor BB-94 or the carrier control solution. Measurements of the aortic diameter were made at the time of initial surgery and at the time of death on postoperative day 7. Aortic tissue was obtained for histologic examination, elastin evaluation, and MAC 1-α antibody staining to evaluate the inflammatory response. Results: The rats that underwent treatment with BB-94 had significantly less aneurysmal dilatation and a 113% increase in aortic size, as compared with the control rats that had a 157% increase (P = .026). Histologic examination of the harvested aortas and grading of the elastin content showed a significantly greater elastin preservation in those rats that were treated with BB-94 as compared with the control rats (P = .036). MAC 1-α antibody staining showed an attenuation of the inflammatory response in the group of rats that underwent treatment with BB-94. Morphologic examination also revealed that the control of the inflammatory response correlated with the areas of elastin preservation. Conclusion: MMP inhibition with BB-94 limited the expansion of AAAs in this rat model. BB-94 appears to work not only as a direct pharmacologic inhibitor of MMPs but also as an interference with the inflammatory response seen in AAAs. Control of the inflammatory response was an unexpected result and may be related to the alterations in feedback mechanisms that are related to extracellular matrix degradation. Because this class of drugs is presently being developed to control the MMP inflammatory response seen with arthritis, these drugs also may ultimately serve as a pharmacologic treatment for patients with AAAs. (J Vasc Surg 1999;29:130-9.)     

基质金属蛋白酶在腹主动脉瘤组织中的表达

目的探查基质金属蛋白酶类（MMPs）在腹主动脉瘤（AAA）组织中产生的源泉。方法采用间质胶原酶（MMP-1）和明胶酶以MMP-9）的mRNA探针在20例AAA组织及4例正常人腹主动脉组织的切片上行原位杂交实验结果MMP-1及MMP-9在巨噬细胞、平滑肌细胞和淋巴细胞均有表达,其中巨噬细胞的MMPs表达强烈。结论MMPs在AAA的形成和扩张中发挥重要作用,炎性细胞是产生MMPS的主要源泉,并影响问质细胞的MMPS表达。     

Collagen types and matrix protein content in human abdominal aortic aneurysms

Deficiencies of total collagen, type III collagen, and elastin have been proposed to explain aneurysm formation. Infrarenal aortas were collected from 19 patients (age 70 +/- 7 years) undergoing operative repair of abdominal aortic aneurysms (diameter 7 +/- 2 cm) and from 13 autopsies (age 63 +/- 17 years) of patients without aneurysm disease (controls). Wall thickness and collagen and elastin concentration were determined in full-thickness aorta. Collagen types I and III were measured after digestion with cyanogen bromide, which solubilized nearly 90% of total collagen for typing. Cyanogen bromide peptides were separated by sequential carboxymethylcellulose and agarose chromatography and quantified by peak area measurement with computerized image analysis. Histologic examination revealed prominent inflammatory cell infiltration and deficient, fragmented elastin in the aneurysms. Aortic wall thickness was similar in aneurysms and in control specimens. In the aneurysms, collagen was increased (37% +/- 16% vs 24% +/- 5%; p less than 0.05) and elastin was decreased (1% +/- 1% vs 12% +/- 7%; p less than 0.001), expressed as a percentage of delipidized, decalcified dry weight. Collagen type I accounted for 74% +/- 4% of aneurysm and 73% +/- 4% of control collagen solubilized for typing, and collagen type III accounted for 26% +/- 4% of aneurysm and 27% +/- 4% of control collagen solubilized for typing. Neither patients with a family history of aneurysms nor those without a history of aneurysms had collagen type III deficiency. Atherosclerotic abdominal aortic aneurysms are associated with an inflammatory process and may result from elastin degradation and not a deficiency of type III collagen.     

Differing patterns of matrix metalloproteinase expression in patients with ascending aortic aneurysms and bicuspid aortic valves

Purpose: Abnormal matrix metalloproteinase (MMP) expression contributes to the development of infrarenal abdominal aortic aneurysms. Recent data have suggested that MMP-2 and MMP-9 may also play a role in thoracic aortic disease. We sought to determine whether the presence of a bicuspid aortic valve (BAV) had an impact on the pattern of MMP expression in ascending aortic aneurysms. Methods: Intraoperative samples of ascending aorta were obtained from 19 patients with ascending aortic aneurysms, 8 (42%) of which also had a BAV. Control samples of ascending aorta were obtained from 6 patients undergoing coronary artery bypass or organ donation/transplantation. None of the patients had aortic dissection or Marfan syndrome. Immunohistochemistry was used to identify expression of MMP-2 and MMP-9 within the aortic wall. Results: The frequency of MMP-2 expression did not differ between BAV patients (5/8, 63%) and non-BAV aneurysm patients (9/11, 82%; p = 0.6); MMP-2 expression was absent in the control group (0/6; p = 0.03 vs. BAV and 0.002 vs. non-BAV). MMP-9 expression, however, occurred rarely in both the BAV groups (1/8, 13%) and the control group (1/6, 17%; p = 1.0); positive MMP-9 staining was substantially more common in the non-BAV patients (7/11, 64%) than in the BAV group (p = 0.06). Conclusions: Ascending aortic aneurysms are associated with increased MMP-2 expression, regardless of the presence of a BAV. Increased MMP-9 expression, however, primarily occurred in aneurysms without a BAV. Variations in MMP expression patterns among different types of thoracic aortic aneurysms warrant further investigation.     

Role of matrix metalloproteinase inhibitors in preventing abdominal aortic aneurysm

Abdominal aortic aneurysm (AAA) is a significant health problem in the United States, with approximately 30,000 repair operations annually. Treatment of AAA is associated with more than 150,000 hospital admissions per year. The development of AAA is characterized by destruction of the elastic media of the aortic wall. A large body of evidence suggests that a group of enzymes called matrix metalloproteinases (MMPs) plays a significant role in the destruction of extracellular matrix in the aortic wall. MMP inhibition has, therefore, been viewed as an alternative pharmacotherapeutic approach to slow down the development and progression of small AAAs, thus reducing the need for surgical intervention.     

基质金属蛋白酶及抑制因子与腹主动脉瘤临床病理特点的相关性研究

目的：研究基质金属酶（MMP）-2、MMP-9及抑制因子TIMP-1在腹主动脉瘤中的表达及与临床病理特征之间的关系。方法：应用免疫组化PV-9000通用型二步法对70例腹主动脉瘤和15例正常腹主动脉标本中的MMP-2、MMP-9及TIMP-1表达进行检测。结果：腹主动脉瘤组织中MMP-2和MMP-9蛋白表达阳性率明显高于正常腹主动脉组织,TIMP-1蛋白表达阳性率和正常腹主动脉没有统计学差异,（X^2=0.103,P=0.991）;MMP-2蛋白的表达与腹主动脉瘤的直径呈负相关（X^2=13.785,P=0.032）,MMP-9蛋白的表达与患者临床症状,腹主动脉瘤直径、破裂有相关性,（P〈0.05）,TIMP-1蛋白表达阳性率与临床病理特征无相关性（X^2=0.103,P=0.991）。结论：腹主动脉瘤组织中MMP高表达和TIMP的相对弱表达在腹主动脉瘤发生、发展过程中起重要作用,MMP-9可以预测腹主动脉瘤的自然病程从而作为腹主动脉瘤手术治疗的指征之一。     

Cyclooxygenase-2 expression and its association with increased angiogenesis in human abdominal aortic aneurysms

Although the mechanism whereby non-steroidal anti-inflammatory drugs may reduce abdominal aortic aneurysm (AAA) development is unknown, one potential route is via inhibition of the cyclooxygenase (COX) enzyme. Despite the fact that evidence from animal models suggests a role for the COX-2 isoform in promotion of AAA development, only very limited data exist on COX-2 expression in human AAAs. Semiquantitative immunohistochemistry for COX-2 was performed on a series of formalin-fixed, paraffin-embedded human AAAs (n = 49). Associated clinicopathological data, including the degree of inflammatory cell infiltration and neorevascularization, were obtained. COX-2 protein was detected in 46 of 49 (94%) human AAAs. Expression of COX-2 protein varied widely between AAAs. COX-2 protein localized to cells in the inflammatory infiltrate with a morphology characteristic of macrophages. COX-2 expression increased with the extent of inflammatory cell infiltration (P < 0.001) and with the degree of AAA neorevascularization (P < 0.001). Logistic regression analysis identified neorevascularization (P < 0.001) as the only significant independent predictor of COX-2 positivity in human AAAs. COX-2 protein is present at increased levels in the majority of human AAAs and is expressed by mononuclear cells in the inflammatory cell infiltrate. Promotion of angiogenesis by COX-2 may play a role in AAA development.     

Suppression of experimental abdominal aortic aneurysms by systemic treatment with a hydroxamate-based matrix metalloproteinase inhibitor (RS 132908)

BACKGROUND: Abdominal aortic aneurysms (AAAs) are associated with chronic inflammation, disruption of medial elastin, and increased local production of elastolytic matrix metalloproteinases (MMPs). The purpose of this study was to investigate how treatment with a hydroxamate-based MMP antagonist (RS 132908) might affect the development of experimental AAAs. METHODS: Male Wistar rats underwent intraluminal perfusion of the abdominal aorta with 50 units of porcine pancreatic elastase followed by treatment for 14 days with RS 132908 (100 mg/kg/day subcutaneously; n = 8) or with vehicle alone (n = 6). The external aortic diameter (AD) was measured in millimeters before elastase perfusion and at death, with AAA defined as an increase in AD (DeltaAD) of at least 100%. Aortic wall elastin and collagen concentrations were measured with assays for desmosine and hydroxyproline, and fixed aortic tissues were examined by light microscopy. RESULTS: AAAs developed in all vehicle-treated rats, with a mean AD (+/- SE) that increased from 1.60 +/- 0.03 mm before perfusion to 5.98 +/- 1.02 mm on day 14 (DeltaAD = 276.4 +/- 67.7%). AAAs developed in only five of eight animals (62.5%) after MMP inhibition, with a mean AD that increased from 1.56 +/- 0.05 mm to 3.59 +/- 0.34 mm (DeltaAD = 128.1 +/- 18.7%; P <.05, vs vehicle). The overall inhibition of aortic dilatation attributable to RS 132908 was 53.6 +/- 6.8%. Aortic wall desmosine fell by 85.4% in the vehicle-treated rats (1210.6 +/- 87.8 pmol/sample to 176.7 +/- 33.4 pmol/sample; P <.05) but only by 65.6% in the animals treated with RS 312908 (416.2 +/- 120.5 pmol/sample). In contrast, hydroxyproline was not significantly affected by either elastase perfusion or drug treatment. Microscopic examination revealed the preservation of pericellular elastin and a greater degree of fibrocollagenous wall thickening after MMP inhibition, with no detectable difference in the extent of inflammation. CONCLUSIONS: Systemic MMP inhibition suppresses aneurysmal dilatation in the elastase-induced rodent model of AAA. Consistent with its direct inhibitory effect on various MMPs, RS 132908 promotes the preservation of aortic elastin and appears to enhance a profibrotic response within the aortic wall. Hydroxamate-based MMP antagonists may therefore be useful in the development of pharmacologic approaches to the suppression of AAAs.     

The plasma level of matrix metalloproteinase 9 may predict the natural history of small abdominal aortic aneurysms. A preliminary study.

OBJECTIVES: increased levels of various proteinases have been detected in abdominal aortic aneurysms (AAA) and are assumed to cause the degradation of the aortic wall. To determine whether systemic measurement of these proteinases and their inhibitors may predict the natural cause of AAA. METHODS AND MATERIAL: serum (S) and plasma (P) samples were obtained from 121 men following the diagnosis of a small AAA (3-5 cm) at population screening. Annual control scans were performed to check for expansion. Circulating levels of elastase-alpha 1 antitrypsin-complexes, alpha 1 antitrypsin, matrix metalloproteinase (MMP) 2 & 9, tissue-inhibitor-matrixproteinase 1 & 2, procollagen III-N-terminal-propeptide, and elastin-peptides were measured in a random group of 36 men. RESULTS: alpha 1 antitrypsin was significantly and positively associated with expansion. Similarly, P-MMP9 levels were significantly associated with size and expansion. There was a difference between median serum and plasma values, probably because of secretion from platelets. CONCLUSION: P-MMP9 and P-alpha 1 antitrypsin may predict the natural history of AAA.