Influence of receptor activator of nuclear factor kappa B on human aortic valve myofibroblasts

Calcific aortic valve stenosis, the main heart valve disease in the elderly, is based on progressive calcification and fibrous thickening of the valve. Several reports addressed the pathogenesis of tissue calcification in this disorder, but few data exist on the molecular mechanisms of the fibrosis and remodeling of the extracellular matrix. The cytokine "receptor activator of nuclear factor kappa B ligand" (RANKL), is expressed in stenotic aortic valves and involved in valvular calcification during calcific aortic valve stenosis. The present study aimed to assess the influence of RANKL on the molecular mechanisms of connective tissue remodeling. In an established cell culture model of primary human aortic valve myofibroblasts, stimulation with RANKL increased cell proliferation as compared to medium alone. Matrix metalloproteinase (MMP)-1 was detectable time-dependently in conditioned media from RANKL-stimulated cells, but absent in media from control cells. MMP-1 activity was increased by RANKL, as measured by collagenase activity assay. Zymography showed an increase in active MMP-2 in RANKL-stimulated cells. These results support the concept that MMPs are involved in the connective tissue remodeling during calcific aortic valve stenosis. RANKL might regulate this process by promoting cell proliferation and MMP expression and activation.     

HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis

Calcific aortic stenosis (CAS) is the most common valvular disease in Western countries. Histological findings in patients with CAS extremely resemble those of atherosclerosis and include accumulation and modification of lipoproteins, inflammation, extracellular matrix remodeling, and calcification. Angiogenesis is another prominent feature of CAS; however, there is only a limited amount of data available regarding the mechanisms behind the pathological neovascularization of a structure that is originally avascular. The present study aims to identify the molecular basis that regulates blood vessel growth in stenotic aortic valves, focusing on the role of HIF-1α and VEGF pathway. A total of 19 native degenerating aortic valves obtained at valve replacement surgery have been processed for Western blot, immunohistochemical, morphometric, and ultrastructural analyses. First, we have demonstrated the adverse ECM remodeling and the significant thickening of the leaflet also showing that HIF-1α and VEGF are significantly upregulated in the stenotic valves, are locally produced and colocalize with angiogenesis and areas of calcification. Next, we have characterized, for the first time to the best of our knowledge, the morphological features of the neovasculature evidencing the presence of intact blood vessels in close proximity to the mineralized zones. These results suggest that the complex structural remodeling of the matrix might reduce oxygen availability in the valve cusp contributing to the stabilization of HIF-1α that in turn induces a metabolic adaptation through the upregulation of VEGF and the formation of new blood vessels not only to overcome the hypoxic state but also to sustain the calcification process.     

Overexpression of transforming growth factor-beta 1 in the valvular fibrosis of chronic rheumatic heart disease

For the purpose of determining the pathogenic role of transforming growth factor-beta1 (TGF-beta 1) in the mechanism of chronic rheumatic heart disease, we evaluated the expression of TGF-beta 1, proliferation of myofibroblasts, and changes in extracellular matrix components including collagen and proteoglycan in 30 rheumatic mitral valves and in 15 control valves. High TGF-beta 1 expression was identified in 21 cases (70%) of rheumatic mitral valves, whereas only 3 cases (20%) of the control group showed high TGF-beta 1 expression (p<0.001). Additionally, increased proliferation of myofibroblasts was observed in the rheumatic valves. High TGF-beta1 expression positively correlated with the proliferation of myofibroblasts (p=0.004), valvular fibrosis (p<0.001), inflammatory cell infiltration (p=0.004), neovascularization (p=0.007), and calcification (p<0.001) in the valvular leaflets. The ratio of proteoglycan to collagen deposition inversely correlated with TGF-beta 1 expression in mitral valves (p=0.040). In conclusion, an ongoing inflammatory process, the expression of TGF-beta 1, and proliferation of myofibroblasts within the valves have a potential role in the valvular fibrosis, calcification, and changes in the extracellular matrix that lead to the scarring sequelae of rheumatic heart disease.     

Regulation of matrix metalloproteinases and their inhibitors in the left ventricular myocardium of patients with aortic stenosis

Aortic stenosis (AS) results in myocyte and extracellular matrix remodeling in the human left ventricle (LV). The myocardial renin-angiotensin system is activated and collagens I and III and fibronectin accumulate. We determined the yet unknown regulation of enzymes that control collagen turnover, i.e., LV matrix metalloproteinases (MMP) and their tissue inhibitors (TIMPs) in human AS. We compared LV samples from AS patients undergoing elective aortic valve replacement (n=19) with nonused donor hearts with normal LV function (controls, n=12). MMP-2, MMP-9, MT1-MMP, and extracellular matrix metalloproteinase inducer (EMMPRIN), TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA were quantitated by real-time RCR. MMP-1, MMP-2, MMP-3, TIMP-3, TIMP-4, and EMMPRIN protein were measured by immunoblotting and MMP-9 and TIMP-1 protein by ELISA. Gelatinolytic MMP-2 and MMP-9 activity was measured by zymography. MMP-2 was increased in AS at mRNA, protein, and activity levels (131%, 193%, and 138% of controls). MMP-3 protein (308%) and EMMPRIN mRNA and protein were also upregulated (171% and 200%). In contrast, MMP-1 (37%) and MMP-9 mRNA, protein, and activity (26%, 21%, and 52%) were downregulated. MMP-9 activity was inversely correlated with LV size. TIMP-1 mRNA and protein were decreased (55% and 73%). In contrast, TIMP-2 mRNA (358%), TIMP-3 mRNA and protein (145% and 249%) were increased. TIMP-4 mRNA was not altered, but TIMP-4 protein was upregulated to 350%. Changes were similar in AS patients with normal and impaired LV ejection fraction. The dysregulation of myocardial MMPs and TIMPs in human AS starts at an early disease stage when LV function is still normal. In spite of upregulation of some MMPs the balance between MMP and TIMP is shifted towards MMP inhibition in human AS and may contribute to collagen accumulation.     

Systemic and local levels of fetuin-A in calcific aortic valve stenosis

Calcific aortic valve stenosis, the most frequent heart valve disorder in developed countries, is an actively regulated process with similarities to bone formation. Fetuin-A has recently been identified as a potent circulating inhibitor of calcification. While several studies involving patients with end-stage renal disease have shown an association between low serum fetuin-A and cardiovascular calcification, nothing is known about fetuin-A serum levels in non-renal patients with calcific aortic valve stenosis. Furthermore, while fetuin-A has been localized in calcified areas of atherosclerotic arteries, data about fetuin-A deposition in stenotic aortic valves are unavailable at present. Serum fetuin-A levels were determined in patients with (n=31) and without (n=28) calcified aortic valve stenosis by ELISA. Creatinine and CRP levels were determined and glomerular filtration rate (GFR) was calculated by the MDRD formula. Immunohistochemistry for fetuin-A was performed on human calcified stenotic (n=14) and control (n=8) aortic valves using a monoclonal antibody. Serum fetuin-A levels were lower in patients with calcific aortic stenosis as compared to the control group (1.41+/-0.33 versus 1.57+/-0,27 mg/dl; p=0.046). This difference was particularly evident in individuals with a normal GFR >or=60 ml/min (1.36+/-0.24 versus 1.63+/-0.27 mg/dl; p=0.007). Furthermore, specific staining of fetuin-A was found in stenotic valves but not in healthy control valves. The data suggest a role of fetuin-A in the pathogenesis calcific aortic valve stenosis independently of the renal function and support the concept that mechanisms of calcium homeostasis are involved in the development of calcific aortic stenosis.     

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.     

Plasmin induces apoptosis of aortic valvular myofibroblasts

Previous studies have described remodelling of the extracellular substratum by matrix metalloproteinases (MMPs) in aortic valves. However, involvement of the fibrinolytic system has not yet been analysed. We hypothesized that plasminogen and plasminogen activator(s) are present in aortic valves and that plasminogen activation could induce the degradation of adhesive proteins and apoptosis of the valvular myofibroblasts. We employed ELISA, western blotting, fibrin‐agar zymography, and immunochemistry to detect components of the plasminogen activation system, in samples of aortic valves and valvular myofibroblasts in primary culture. Using myofibroblast cultures, real‐time measurement of plasminogen activation was performed in the absence and presence of inhibitors (amiloride, ε‐aminocaproic acid, and an MMP inhibitor); the degradation of fibronectin was visualized on western blots; and the apoptotic process was assessed by detection of phosphatidylserine exposure (binding of FITC‐annexin V) and DNA fragmentation (TUNEL and ELISA). We demonstrate that a time‐ and plasminogen concentration‐dependent generation of plasmin occurs on the surface of cultured valvular myofibroblasts expressing both u‐PA and t‐PA. Only u‐PA appears to activate plasminogen as t‐PA is essentially found in complex with PAI‐1. Plasmin‐dependent degradation of pericellular proteins, such as fibronectin, leads to cell detachment and apoptosis. In conclusion, various proteins of the fibrinolytic system are synthesized in vitro by cultured myofibroblasts from aortic valves, leading to plasmin‐dependent cell detachment‐induced apoptosis, a biological process named anoikis. The presence of plasminogen in aortic valves suggests that this process may be operating in vivo and may participate in valvular tissue remodelling, as also suggested by the finding of apoptotic cells in valvular tissue. This is the first demonstration of the presence and potential role of enzymes of the fibrinolytic system in aortic valves. Copyright © 2009 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Immunohistochemical study of endothelin-1 and matrix metalloproteinases in plexogenic pulmonary arteriopathy

The matrix metalloproteinases (MMPs) and endothelin-1, a potent vasoconstrictor and mitogen for smooth muscle cells, have been shown to be involved in the pathogenesis of various vascular disorders. However, the expression of endothelin-1 and the activation of MMPs have not been fully evaluated in plexogenic pulmonary arteriopathy (PPA). Immunohistochemical and confocal microscopic studies were conducted to evaluate the reactivity of lung tissue from six patients with pulmonary hypertension for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, factor VIII, endothelin-1, various types of MMPs (MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9), membrane type-MMPs (MT-MMPs), tissue inhibitors of MMPs (TIMPs), and type IV collagen. Four major arterial morphological abnormalities were recognized in PPA: muscularization of pulmonary arterioles, onion-skin lesions, cellular and mature plexiform lesions, and atheromas in elastic pulmonary arteries. Reactivity for MMP-2 and MT-1-MMP was found in endothelial cells and, to a lesser extent, in myofibroblasts proliferating in various lesions of PPA. Increased expression of endothelin-1 was observed in the latter cells and in endothelial cells. Some myofibroblasts were positive for MMP-3 and MMP-7 in the vascular lesions except for mature plexiform lesions. MMP-1, MMP-9 and TIMP-2 tended to be positive only in the atheromatous lesions. Staining for type IV collagen showed focal thinning and discontinuities of the endothelial basement membrane in plexiform lesions. This study demonstrates colocalization of MMP-2 with MT-1-MMP and increased expression of endothelin-1 in various arterial lesions of PPA. These changes may play important roles in the remodeling of arterial structures, particularly of basement membranes, in this disorder.     

Expression of metalloproteinases and its inhibitor in later stage of rabbit neointima development

Neointima formation after arterial de-endothelialization refers not only to smooth muscle cell (SMC) migration and proliferation, but also involves extracellular matrix (ECM) metabolism. Most studies regarding the role of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in neointima have focused on the early phase of vascular remodeling. In this study, we examined the expression of MMP and TIMP in rabbit aortic neointima at a relatively late stage of lesion development, between 4 and 12 weeks after initial de-endothelialization. Northern blot analysis revealed expression of steady-state MMP-9 mRNA was increased up to the 4th week and MMP-2 mRNA to the 12th week after de-endothelialization. In situ hybridization shown that MMP positive cells were predominantly distributed in arterial neointima. Expression of TIMP-1 mRNA was continuously up-regulated up to the 12th week and TIMP-1 positive cells, primarily SMCs, were also localized to the neointimal tissue. Alteration at mRNA level was accompanied by that at protein level, as assessed by SDS-PAGE zymography for MMPs and immunoblotting for TIMP-1. The profile of alteration at protein level correlated well with that at mRNA level. These data suggest that synthesis of MMPs and TIMP is a prolonged process and arterial SMC is a major source of MMP production in arterial neointima. Enhanced synthesis of MMPs and TIMPs at late stage of neointimal development may contribute to arterial ECM metabolism.     

Matrix Metalloproteinase-2 Is Associated with Tenascin-C in Calcific Aortic Stenosis

We previously showed that the expression of tenascin (TN-C), an extracellular matrix glycoprotein found in developing bone and atherosclerotic plaque, and matrix metalloproteinase-2 (MMP-2) are coordinated and interdependent in cultured vascular smooth muscle cells. In this study, we hypothesized that TN-C and MMP-2 are mechanistically involved in the pathobiology of calcific aortic stenosis. Human calcific aortic stenosis cusps demonstrated immunohistochemically prominent deposition of TN-C, MMP-2, and alkaline phosphatase activity, as well as MMP-2 gelatinolytic activity. Although far lesser amounts of TN-C were noted in several of the grossly non-calcified valve cusps, MMP-2 and AP were never detected. Further, when aortic valve interstitial cells (both sheep and human) were cultivated on collagen supplemented with TN-C, both MMP-2 mRNA expression and MMP-2 gelatinolytic activity (both pro and active forms), were up-regulated compared to control. These observations support the view that accumulation of first TN-C and then MMP-2 are associated with progression of calcification. The residual presence of these proteins in severe calcifications is indicative of their involvement in the pathogenesis.     

Molecular mechanisms underlying the onset of degenerative aortic valve disease

Morbidity from degenerative aortic valve disease is increasing worldwide, concomitant with the ageing of the general population and the habitual consumption of diets high in calories and cholesterol. Immunohistologic studies have suggested that the molecular mechanism occurring in the degenerate aortic valve resembles that of atherosclerosis, prompting the testing of HMG CoA reductase inhibitors (statins) for the prevention of progression of native and bioprosthetic aortic valve degeneration. However, the effects of these therapies remain controversial. Although the molecular mechanisms underlying the onset of aortic valve degeneration are largely unknown, research in this area is advancing rapidly. The signaling components involved in embryonic valvulogenesis, such as Wnt, TGF-β₁, BMP, and Notch, are also involved in the onset of aortic valve degeneration. Furthermore, investigations into extracellular matrix remodeling, angiogenesis, and osteogenesis in the aortic valve have been reported. Having noted avascularity of normal cardiac valves, we recently identified chondromodulin-I (chm-I) as a crucial anti-angiogenic factor. The expression of chm-I is restricted to cardiac valves from late embryogenesis to adulthood in the mouse, rat, and human. In human degenerate atherosclerotic valves, the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinases and angiogenesis is observed in the area of chm-I downregulation. Gene targeting of chm-I resulted in VEGF expression, angiogenesis, and calcification in the aortic valves of aged mice, and aortic stenosis is detected by echocardiography, indicating that chm-I is a crucial factor for maintaining normal cardiac valvular function by preventing angiogenesis. The present review focuses on the animal models of aortic valve degeneration and recent studies on the molecular mechanisms underlying the onset of degenerative aortic valve disease.     

Role of metalloproteinases and tissue inhibitors of metalloproteinases during cardiopulmonary bypass in rats

Matrix metalloproteinases (MMPs) and the tissue inhibitors of matrix metalloproteinases (TIMPs) regulate matrix remodeling in the heart. Changes in synthesis and release of MMPs and TIMPs are observed after extracorporeal circulation (ECC). Thus, MMPs and TIMPs are supposed to be involved in ECC-mediated cardiac dysfunction. The aim was to examine the role of MMPs and TIMPs in ECC-mediated cardiac dysfunction. Extracorporeal circulation was instituted in rats for 60 min at a flow rate of 120 ml/kg/min. Three groups (n = 10) were studied: group CAO: 60 min ECC without aortic cross-clamping, group CAC: 60 min ECC including 30 min aortic cross-clamping (crystalloid Inzolen(?) cardioplegia), and group CAB: 60 min ECC including 30 min aortic cross-clamping (blood cardioplegia). Left ventricular (LV) function was measured with conductance catheter. Matrix metalloproteinase-activity was determined by zymography and TIMP activity was determined by reverse zymography. Gene expression of MMPs and TIMPs was determined by real-time polymerase chain reaction. Sixty minutes after weaning from bypass, there was a preserved LV function in the CAO and CAB group and an impaired LV function in the CAC group. We observed an increased myocardial activity and an increased myocardial messenger RNA expression of MMP-2, MMP-9, TIMP-1, and TIMP-4 in all ECC groups, when compared with sham animals. With regard to enzyme activity, there was an imbalance of MMP/TIMP ratio leading to an increased activity of MMP in the CAC group. In terms of gene expression, there was an imbalance of MMP-2/TIMP-4 ratio leading to an increased expression of MMP-2 in the CAC group. MMP-2 contributes to myocardial reperfusion injury in this in vivo model of ECC with cardioplegic arrest.     

Cronic non-rheumatic valvular heart disease. An autopsy study.

The frequency of chronic non-rheumatic valvular heart disease in Iceland was investigated via autoposies performed from November 1965 through December 1974. During this period, about 12.400 Icelanders died at the age of 16 years and older and 28.8 per cent of these were included in the study. At autopsy, males outnumbered females by 2:1. The frequency of calcific aortic stenosis was found to be 3.63 per cent and the prevalence was calculated to be 3.17 per cent among males and 4.50 per cent among females. Calcific aortic stenosis in tricuspid valves was more frequent in females and calcific aortic stenosis in bicuspid valves was more frequent in males. Among the hearts with calcific aortic stenosis, 70.8 per cent were found to have normally tricuspid valves, 25.4 per cent bicuspid valves and 3.8 per cent tricuspid valves with an unicommissural fusion. In 0.59 per cent of the hearts the aortic valve was either bicuspid or had an uncommissural fusion without the features of calcific stenosis. However, a functional stenosis was suggested by the increased weight of most of these hearts. The frequency of bicuspid aortic valves was 1.2 per cent with a prevalence in males of 1.54 per cent and in females 0.50 per cent. A calcified mitral annulus was found in 1.98 per cent of the hearts and in most, it was either associated with calcific aortic stenosis in a tricuspid valve, or it was a single valvular disease. Rheumatic valvular disease was found in 1.08 per cent of the heart examined.     

Matrix metalloproteinase expression in nonrheumatic aortic stenosis.

BACKGROUND: Nonrheumatic aortic stenosis (NAS) is considered to be a degenerative process characterized by valve thickening, fibrocalcific masses, collagen disarray, and an inflammatory infiltrate. The matrix metalloproteinases (MMPs) are a family of zinc- and calcium-dependent enzymes produced by inflammatory cells that are capable of degrading collagen, elastin, and proteoglycans. This study sought to test the hypothesis that MMPs are involved in the pathogenesis of NAS. METHODS AND RESULTS: Aortic values were obtained from nine patients with NAS undergoing valve replacement and from four patients without NAS during autopsy. Microscopic analysis of NAS specimens revealed variable areas of calcium deposits, fibrosis, and an extensive cellular infiltrate consisting of macrophages, lymphocytes, and fibroblasts. Control aortic valves demonstrated normal architecture, a predominance of fibroblasts, occasional scattered macrophages, and no lymphocytes. Immunohistochemical staining with antibodies to MMP-1, -2, -3 and -9 revealed expression of each enzyme in macrophages, lymphocytes, and fibroblasts of all NAS patients. MMP-1, -2 and -3 were expressed by resident fibroblasts and macrophages in normal valves, but to a lesser degree. MMP-9 was not identified in normal valves. CONCLUSIONS: The current study confirms an inflammatory infiltrate composed of macrophages and lymphocytes in NAS. Additionally, the increased expression of MMP-1, -2, and -3, along with the unique expression of MMP-9 in NAS valve leaflets was documented. These findings are consistent with the hypothesis that NAS is associated with chronic inflammation and that the increased expression of MMPs may contribute to the pathogenesis of this disease process.     

Coexpression of cyclooxygenase-2 and matrix metalloproteinases in human aortic atherosclerotic lesions

Inflammation appears to have a major role in the development of atherosclerosis. Cyclooxygenase-2 (COX-2) is involved in the inflammatory response via the generation of prostanoids that, in turn, are involved in the production of matrix metalloproteinases (MMPs). This study aimed to investigate atherosclerosis in human aortas for in situ tissue distribution of COX-2, MMPs including MMP-9 and membrane type 1 MMP (MT1-MMP), and tissue inhibitor of metalloproteinase-2 (TIMP-2). Immunohistochemical studies were performed on atherosclerotic lesions of aortas from patients with aortic aneurysms (n = 4) and dissections (n = 3) by using antibodies to COX-2, MMP-9, MT1-MMP, and TIMP-2. Control tissues were obtained from traumatically dissected aortas (n = 2). All specimens from diseased aortas had atherosclerotic lesions ranging from fatty streak to atheromatous plaques. In control, there was no expression of COX-2, MMP-9, and MT1-MMP in all aortic layers. Immunoreactivity for COX-2 was predominantly noted in macrophages and smooth muscle cells (SMCs) of the intima including atherosclerotic plaque itself and the medial layer of the plaque base, as well as in SMCs and endothelial lining of the vasa vasorum in the adventitia. Immunoreactivity for MMP-9 and MT1-MMP was found in the same distribution as that of COX-2. Additionally, the expression of TIMP-2 increased in relation to MMP-9 expression. This study demonstrates that COX-2 is coexpressed with MMP-9 and MT1-MMP, not only by macrophages and SMCs in atherosclerotic lesions, but also in endothelial lining of the vasa vasorum of human aortas. Thus, vascular inflammatory reactions may influence extracellular matrix remodeling by coactivation of MMPs in the development of atherosclerosis and, in turn, the progression of disease.     

Distribution patterns of matrix metalloproteinase (MMP)-2 and -9 and their inhibitors (TIMP-1 and TIMP-2) in the human decidua during early pregnancy

Early human trophoblast shows dramatic invasive properties during early pregnancy. A tightly-regulated activation of matrix metalloproteinases (MMPs) is considered to be of critical importance for the control of trophoblast invasion. The aim of the present study was to determine MMP-2, MMP-9, TIMP-1 and TIMP-2 protein expression in decidual endometrium during the first trimester of pregnancy (22-42 days post coitus) with special attention to their expression patterns in endometrial compartments. Cytokeratin staining applied to adjacent sections was used to identify epithelial and trophoblast cells. We observed that MMP-2, particularly in the fourth week, appeared to be expressed more strongly in extravillous trophoblasts (EVTs) and vascular endothelial cells in the first trimester of pregnancy. Therefore, MMP-2 is likely to be the primary mediator in invasion of the trophoblast into the decidual endometrium, as well as in vascular remodeling and angiogenesis in the first trimester of pregnancy. The high expression of TIMP-1 and TIMP-2 in EVTs and glandular epithelium suggests that a restricted and balanced expression of these molecules is important for matrix remodeling and controlled trophoblast invasion during placentation. We conclude that (1) MMP-2 and MMP-9 and their inhibitors TIMP-1, and TIMP-2 determine the invasive behavior of trophoblast into the endometrium, and in particular, (2) MMP-2 may be the key regulator of trophoblast invasion in early human pregnancy.