Matrix metalloproteinases and their tissue inhibitors in the lesions of cardiac and pulmonary sarcoidosis: an immunohistochemical study

The pathogenesis of the tissue damage and fibrosis in sarcoidosis is poorly understood. The matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) must be considered in this regard, because they control the lysis of connective tissue components. Immunohistochemical studies (peroxidase and dual labeling for confocal microscopy) of reactivity for MMP-1, MMP-2, MMP-3, MMP-7, MMP-9, and the 4 membrane-type-MMPs were made on tissues from patients with cardiac (n = 4) and pulmonary (n = 5) sarcoidosis. The granulomas were histochemically similar in both organs. The multinucleated giant cells (MGCs) showed moderate reactivity for MMP-1 and MMP-9 and variable reactivity for MMP-2 and MMP-3; in addition, they showed colocalization of MT-1-MMP, which activates MMP-2. The reactivity of epithelioid cells (ECs) was moderate for MMP-2 and mild for other MMPs. Macrophages showed weaker reactivity for MMPs than did MGCs and ECs. All 3 types of cells showed very low reactivity for TIMPs. Staining for type IV collagen showed focal damage to the basement membranes of cardiac myocytes and pulmonary alveoli near the granulomas. The cells in sarcoid granulomas contain an abundance of MMPs and a paucity of TIMPs. The MGCs also contain MT-1-MMP and thus can activate MMP-2 in the granulomas. The MMPs can cause damage to adjacent cardiac myocytes and pulmonary alveoli, leading to the interstitial fibrosis produced by sarcoidosis.     

Immunohistochemical study of metalloproteinases and their tissue inhibitors in the lungs of patients with diffuse alveolar damage and idiopathic pulmonary fibrosis.

Immunohistochemical and confocal microscopic studies of the localization of matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen were made in lung tissues from patients with normal pulmonary histology (n = 3), diffuse alveolar damage (n = 14), and idiopathic pulmonary fibrosis (n = 12). Pretreatment with pepsin revealed otherwise undetectable MMP- and TIMP-immunoreactive sites. In normal lung, MMP-2, MMP-9, TIMP-1, and TIMP-2 were localized in ciliated cells, endothelial cells, pneumocytes, macrophages, and smooth muscle cells; fibroblasts showed a strong reaction only for MMP-2. Only TIMP-2 showed co-localization with type IV collagen. Myofibroblasts and epithelial cells expressed increased reactivity for MMPs and TIMPs in both disorders. The reactivities for MMPs and TIMPs were stronger in diffuse alveolar damage. MMP-2 showed focal co-localization in capillary endothelial and disrupted epithelial basement membranes, suggesting activation of collagenolysis. A protective effect against this lysis was suggested by the extensive co-localization of TIMP-2 with type IV collagen and fibrillar collagens. Alveolar buds showed increased reactivity for MMPs and TIMPs in their lining epithelial cells, myofibroblasts, and their basement membranes; however, their matrices were mostly unreactive. These findings emphasize the complexity of the roles of MMPs and TIMPs in collagen turnover in diffuse alvcolar damage and idiopathic pulmonary fibrosis.     

Expression of Matrix Metalloproteinases in Invasive Pulmonary Adenocarcinoma with Bronchioloalveolar Component and Atypical Adenomatous Hyperplasia

To investigate the association between the expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) and the clinicopathological features in lepidic and invasive components of adenocarcinoma of the lung, we performed immunostaining for type IV collagen, various MMPs, and TIMPs in 27 cases of invasive adenocarcinomas and 5 cases of atypical adenomatous hyperplasia of alveolar epithelial cells (AAH). Mean extent of lepidic growth was 61% and the survival was significantly better in cases with 50% or more lepidic component. The preservation of type IV collagen in lepidic areas correlated inversely with lymphatic or vascular invasion (P = 0.02 and 0.002, respectively). Five-year survival was reduced in cases showing destruction of type IV collagen (P = 0.004) or expression of MMP-2 (P = 0.008) in lepidic areas. MMP-2 co-localized with MT-1-MMP (its activating enzyme) and TIMP-2 in neoplastic cells. Reactivity for other MMPs and TIMPs did not correlate with destruction of type IV collagen or prognosis. Type IV collagen was preserved in all cases of AAH. MMP-2, but not MT-1-MMP, was expressed in two of the five cases of AAH. Immunostaining for type IV collagen MMP-2 is useful in evaluating the prognosis of the lung.     

不同组织微环境中人胃癌异种移植瘤侵袭性及基质金属蛋白酶谱表达的差异

目的 探讨组织微环境对癌细胞侵袭性影响机制中基质金属蛋白酶表达的意义。方法 取人胃腺癌组织移植于裸小鼠皮下,成瘤后进行皮下和腹腔内传代接种,形态学观察2处异种移植瘤侵袭性的不同并用免疫组织化学链霉素抗生物素蛋白-过氧化物酶法检测基质金属蛋白酶(MMP)-2、MMP-7、MMP-9、MMP-13、TM1-MMP、TM2-MMP、TM3-MMP 7种MMPs在瘤组织中的表达。结果 人胃癌裸小鼠皮下异种移植瘤呈膨胀性生长,侵袭性不明显;除MMP-7外,其他6种MMPs在皮下移植瘤细胞及间质中均无表达。腹腔内移植瘤呈侵袭性生长、纤维间质增多,多种MMPs均在侵袭前沿的肿瘤细胞及间质中表达。同一瘤株来源的人胃癌细胞在裸小鼠不同组织环境中所呈现的侵袭性及MMPs表达差异均有显著性。结论 (1)肿瘤细胞与相邻的间质细胞之间存在相互诱导作用,组织环境对肿瘤侵袭表型可有决定性的影响。(2)MMPs的表达与肿瘤细胞生长方式及侵袭性有密切联系;肿瘤侵袭前沿的间质细胞产生的MMPs也可能参与肿瘤细胞的侵袭过程。     

Role for activation of matrix metalloproteinases in the pathogenesis of pulmonary lymphangioleiomyomatosis

BACKGROUND: Matrix metalloproteinases (MMPs) have been shown to be involved in the pathogenesis of the destructive pulmonary lesions in patients with lymphangioleiomyomatosis (LAM); in the present report, the activation of these enzymes is examined. OBJECTIVE: To evaluate the role of MMPs and their activating enzymes, immunohistochemical and confocalmicroscopic techniques were used to localize alpha-smooth muscle actin (alpha-SMA), HMB-45, proliferating cell nuclear antigen (PCNA), MMP-2, membrane-type 1 MMP (MT1-MMP), MT2-MMP, and MT3-MMP in lung tissues from 10 women with LAM. Tissue samples were obtained from 5 patients before treatment and in 5 patients after hormone treatment (progesterone and/or tamoxifen citrate). RESULTS: Staining for alpha-SMA and MMP-2 was present in all the abnormal smooth muscle cells (LAM cells) in both groups. The percentages of PCNA-, MMP-2-, or MT1-MMP-positive LAM cells were much higher in the untreated group than in the treated group, whereas the percentages of HMB-45-reactive LAM cells were similar in both groups. The reactions for MT1-MMP and PCNA were preferentially localized in small spindle-shaped LAM cells; the reaction for HMB-45 was found in large epithelioid LAM cells. Many of the PCNA-positive cells were also positive for MT1-MMP. Staining for MT2-MMP and MT3-MMP was negative. CONCLUSIONS: This study demonstrates an association between cellular proliferation and the presence of MT1-MMP in LAM cells. The activation of MMP-2 by MT1-MMP may play an important role in the destruction of lung tissue in this disorder.     

Enhanced expression of vascular endothelial growth factor in pulmonary plexogenic arteriopathy due to congenital heart disease

Congenital heart disease (CHD) leading to increased pulmonary blood pressure and flow is an important cause of pulmonary plexogenic arteriopathy (PPA). This type of arteriopathy tends to progress to an irreversible stage, hallmarked histologically by the emergence of a number of characteristic lesions, which include concentric laminar intimal proliferation and fibrosis, and plexiform lesions. The pathogenesis of these lesions, which connote a very poor prognosis, is not well understood. Since endothelial cell proliferation has been demonstrated in these lesions, it was hypothesized that vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, might play a role in their pathogenesis. Thirty-nine patients with various types of CHD, who underwent cardiac catheterization and subsequent cardiac surgery, were studied prospectively. On the basis of a detailed assessment of the type of cardiac defect, the haemodynamic abnormalities, and the histopathological features evident from open lung biopsies, taken in all instances, patients were histologically grouped into cases with moderate PPA (n=18), advanced PPA (n=7), pulmonary congestive vasculopathy (PCV, n=5), and controls lacking pulmonary hypertension or increased pulmonary blood flow (n=4). Five patients were excluded from analysis because of inadequate sample size or quality. The presence of VEGF was assessed immunohistochemically using standard procedures and was correlated with haemodynamic and histological data. Immunoreactive VEGF was detected in pulmonary arterial smooth muscle cells and endothelial cells in 13 out of 34 cases and was more frequent and more pronounced in patients with the histological lesions of advanced PPA than in those with moderate PPA (p<0.01). VEGF positivity was particularly prominent in the lesions characteristic of advanced PPA. No difference in VEGF expression was observed between controls, PVC, and moderate PPA cases. Measured haemodynamic parameters did not differ significantly between VEGF-positive and VEGF-negative cases. We conclude that VEGF may play a role in the angioproliferative changes of advanced PPA.     

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.     

Role of metalloproteinases MMP-9 and MT1-MMP in CXCL12-promoted myeloma cell invasion across basement membranes

Malignant plasma cells in multiple myeloma home to the bone marrow (BM), accumulate in different niches and, in late disease, migrate from the BM into blood. These migratory events involve cell trafficking across extracellular matrix (ECM)-rich basement membranes and interstitial tissues. Metalloproteinases (MMP) degrade ECM and facilitate tumour cell invasion. The chemokine CXCL12 is expressed in the BM, and it was previously shown that it triggers myeloma cell migration and activation. In the present work we show that CXCL12 promotes myeloma cell invasion across Matrigel-reconstituted basement membranes and type I collagen gels. MMP-9 activity was required for invasion through Matrigel towards CXCL12, whereas TIMP-1, a MMP-9 inhibitor that we found to be expressed by myeloma and BM stromal cells, impaired the invasion. In addition, we show that the membrane-bound MT1-MMP metalloproteinase is expressed by myeloma cells and contributes to CXCL12-promoted myeloma cell invasion across Matrigel. Increase in MT1-MMP expression, as well as induction of its membrane polarization by CXCL12 in myeloma cells, might represent potential mechanisms contributing to this invasion. CXCL12-promoted invasion across type I collagen involved metalloproteinases different from MT1-MMP. These data indicate that CXCL12 could contribute to myeloma cell trafficking in the BM involving MMP-9 and MT1-MMP activities.     

Expression of matrix metalloproteinases in benign and malignant follicular thyroid lesions

AIMS: To examine expression of matrix metalloproteinases (MMPs) and related proteins in follicular thyroid lesions (FTLs) and to determine their usefulness for differential diagnosis of FTLs, particularly between minimally invasive carcinoma and adenoma. METHODS AND RESULTS: Six widely invasive follicular carcinomas (WIFCs), 15 minimally invasive follicular carcinomas (MIFCs), 19 follicular adenomas (FAs) and 10 adenomatous goitres (AGs) were analysed immunohistochemically for MMP-1, MMP-2, MMP-7, MMP-9, membrane-type 1-MMP (MT1-MMP) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2). MMP-1 was positive in all FTLs. MMP-2 and MMP-7 were positive in more than 80% of WIFC and MIFC cases, whereas they were negative in all FA and AG cases except one MMP-2+ FA (P < 0.001). MMP-9 stained positive significantly more in MIFC than FA or AG cases (P < 0.05, respectively). The positivity of MT1-MMP and TIMP-2 was different among some of the FTLs, but with no significant difference between MIFC and FA cases. In-situ hybridization of MMP-2 and MMP-7 mRNA in selected cases demonstrated the expression of these enzymes in the tumour cells as well as in some stromal cells. CONCLUSIONS: Our results confirm MMP expression mainly in malignant FTLs and suggest that MMP-2 and MMP-7 may be useful markers to distinguish MIFC from FA.     

Protein kinase C activation by phorbol ester increases in vitro invasion through regulation of matrix metalloproteinases/tissue inhibitors of metalloproteinases system in D54 human glioblastoma cells

To elucidate possible mechanisms of phorbol 12-myristate 13-acetate (PMA) induced in vitro invasiveness of glioblastoma cells, we examined expression levels of membrane-type 1 matrix metalloproteinase (MT1-MMP), MMP-2, MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 using Western blotting and gelatin zymography assay, and found that PMA induced the secretion of MMP-9, activated MMP-2 proenzyme to fully active form of 59 kDa, down-regulated the TIMP-1 and TIMP-2 secretion, and increased MT1-MMP on the cell surface. However, PKC inhibitor Go 6983 reversed all of these effects brought about by PMA. We, therefore, conclude the activation of PKC by PMA in these cells plays a critical role in the regulation of MMPs/TIMPs system, which has a major role in tumor invasion and metastasis.     

Immunohistochemical examination of plexiform-like complex vascular lesions in the lungs of broiler chickens selected for susceptibility to idiopathic pulmonary arterial hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a disease of unknown cause that is characterized by elevated pulmonary arterial pressure and pulmonary vascular resistance, and by extensive vascular remodelling. In human IPAH patients, remodelling of the pulmonary vasculature results in the formation of plexiform lesions in the terminal pulmonary arterioles. Various molecules are expressed in the human plexiform lesions, including alpha smooth muscle actin, von Willebrand factor, vascular endothelial growth factor, vascular endothelial growth factor receptor type 2, hypoxia inducible factor-1α, survivin, tenascin, collagen, fibronectin, and various immune/inflammatory cells such as, cytotoxic lymphocytes, B lymphocytes, MHC class II cells, and monocytes/macrophages are also present. Plexiform lesions rarely develop in the lungs of laboratory animals, but plexiform-like complex vascular lesions (CVL) do develop spontaneously in the lungs of broiler chickens from an IPAH-susceptible line. To examine angioproliferative and immune-system-related activities associated with CVL in broiler lungs, paraformaldehyde-fixed, paraffin-embedded lung sections from 8-week-old to 24-week-old broiler chickens were stained immunohistochemically using monoclonal or polyclonal antibodies specific for angioproliferative molecules and immune/inflammatory cells. The CVL in the lungs of broiler chickens exhibited positive staining for both angioproliferative molecules and immune/inflammatory cells. These observations combined with the close histological resemblance of broiler CVL to the plexiform lesions of human IPAH patients further validates chickens from our IPAH-susceptible line as an excellent animal model of spontaneous plexogenic arteriopathy.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in bronchial squamous preinvasive lesions

Metalloproteinases and their inhibitors are known to play an important role in the extracellular matrix remodeling associated with preinvasive lesions and invasive carcinomas; however, little is known about their role in early lung carcinoma. Immunohistochemical studies were made of the reactivity of bronchial squamous preneoplastic lesions from cigarette smokers, including basal cell hyperplasia, squamous metaplasia, dysplasia, carcinoma in situ, and invasive squamous cell carcinoma for matrix metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and type IV collagen in 13 patients. Staining for type IV collagen disclosed discontinuities in basement membranes from basal cell hyperplasia to dysplasia, progressing to destruction in carcinoma in situ and invasive carcinoma. Reactivity for MMP-9 was mild in basal cell hyperplasia and squamous metaplasia, increasing in carcinoma in situ and invasive carcinoma. In contrast, reactivity for MMP-1 was strong in basal cell hyperplasia and squamous metaplasia, decreasing in carcinoma in situ and invasive carcinoma. Some neoplastic cells in carcinoma in situ and invasive carcinoma were MMP-3 positive. Staining for MMP-2 and TIMP-1 was moderate to strong in all squamous preinvasive lesions. Confocal microscopy showed MMP-9-positive cells passing through fragmented basement membranes in which type IV collagen and MMP-9 were colocalized. Type IV collagen colocalized with MMP-2 in all lesions and with TIMP-1 in basal cell hyperplasia and squamous metaplasia. The inverse relationships between the reactivity for MMP-1 and MMP-9 with progression of bronchial squamous preinvasive lesions suggest important roles for these MMPs in basement membrane remodeling in these lesions.     

E6/E7 oncoproteins of high risk HPV-16 upregulate MT1-MMP,MMP-2 and MMP-9 and promote the migration of cervical cancer cells

Background: E6 and E7 of high risk human papillomavirus 16 (HPV16) were reported to correlate with the cervical cancer (CC). And the presence of matrix metalloproteinases (MMPs) has also been indicated to be associated with CC. Methods: The present study investigated the expression of MMPs (MT1-MMP, MMP-2 and MMP-9) in CC cells with HPV16-E6/E7 oncoprotein(s) negative or positive, and then determined the regulation of HPV16-E6/E7 oncoproteins on the expression of MMPs (MT1-MMP, MMP-2 and MMP-9) and the migration of cervical cancer Caski and SiHa cells with RNAi technology. Results: It was demonstrated that the overexpression or the knockdown of HPV16-E6/E7 promoted or reduced MT1-MMP, MMP-2 and MMP-9 in CC cells. And the HPV16-E6, -E7 or -E6E7 influenced the migration of CC cells. The overexpression or the knockdown of them promoted or inhibited the migration of C33A or Caski/SiHa cells. Moreover, the chemical inhibition of MMP-2 or MMP-9 significantly reduced the migration of CC Caski or SiHa cells. Conclusions: Our results demonstrated that the E6-HPV16 or E7-HPV16 promoted the activity of MMP-2/9, and contributed to the migration of cervical cells.     

The ultrastructure of plexogenic pulmonary arteriopathy

The lungs from 16 cases of plexogenic pulmonary arteriopathy obtained at heart-lung transplantation, half of which had primary pulmonary hypertension, were examined by electron microscopy. From these the probable pathogenesis of pulmonary arterial intimal fibrosis in plexogenic pulmonary arteriopathy was deduced. The earliest detectable change was migration of smooth muscle cells from the media, through the internal elastic lamina into the intima. These cells collected beneath the endothelium and lost many of their myofilaments to become myofibroblasts. They were associated with ground substance but scanty collagen fibrils. As the quantity of interstitial collagen increased, the myofibroblasts reverted to a muscular structure, became elongated, and assumed a regular, circumferential orientation. This later stage coincided with the development of plexiform lesions. At both early and later stages, the muscular pulmonary arteries were contracted but not markedly so, and muscular evaginations were not seen. On the other hand, the cellular intimal proliferations developed early and were occlusive. This suggests that occlusion of small pulmonary arterial vessels by myofibroblasts may be at least as important as vasoconstriction in the early elevation of the pulmonary vascular resistance in primary pulmonary hypertension.     

Acute Trypanosoma cruzi infection in mouse induces infertility or placental parasite invasion and ischemic necrosis associated with massive fetal loss

Production of matrix-degrading proteases, particularly matrix metalloproteinases (MMPs), by endothelial cells is a critical event during angiogenesis, the process of vessel neoformation that occurs in normal and pathological conditions. MMPs are known to be highly regulated at the level of synthesis and activation, however, little is known about the regulation of MMP secretion by endothelial cells. We found that cultured human umbilical vein endothelial cells shed vesicles (300 to 600 nm) originating from localized areas of the cell plasma membrane, as revealed by ultrastructural analysis. Normal and reverse zymography, Western blot, and immunogold analyses of the vesicles showed two gelatinases, MMP-2 and MMP-9, in both the active and proenzyme forms, the MT1-MMP proenzyme located on the external side of the vesicle membrane and the two inhibitors TIMP-1 and TIMP-2. Serum and the angiogenic factors, fibroblast growth factor-2 and vascular endothelial growth factor, stimulated the shedding of MMPs as vesicle components. Shedding the vesicle was rapid, as it was already completed after 4 hours. Addition of shed vesicles to human umbilical vein endothelial cells resulted in autocrine stimulation of invasion through a layer of reconstituted basement membrane (Matrigel) and cord formation on Matrigel. We conclude that endothelial cells shed MMP-containing vesicles and this may be a mechanism for regulating focalized proteolytic activity vital to invasive and morphogenic events during angiogenesis.     

Arterial enlargement in response to high flow requires early expression of matrix metalloproteinases to degrade extracellular matrix

This study investigated the effects of high flow and shear stress on the expression of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-2 (TIMP-2) during flow-induced arterial enlargement using a model of arteriovenous fistula (AVF) creation on the carotid artery with the corresponding jugular vein in Japanese white male rabbits. Flow increased 8-fold 7 days after AVF. Endothelial cells (EC) and smooth muscle cells (SMC) proliferated with internal elastic lamina (IEL) degradation in response to high flow and shear stress. Expression of MMP-2 mRNA peaked at 2 days (1700-fold) and maintained high level expression. MMP-9 mRNA gave a 10.8-fold increase within 2 days and decreased later. Their proteins were detected in EC and SMC. Membrane type-1-MMP (MT1-MMP) mRNA increased 121-fold at 3 days and maintained high expression. TGF-beta1 was increased after AVF. Two-peak up-regulation of Egr-1 mRNA was recognized at 1 and 5 days of AVF. These results suggest that high flow and shear stress can mediate EC and SMC to express MMP-2 and MMP-9, which degrade cell basement membranes and IEL to induce arterial enlargement. The disproportional increase in MT1-MMP and TIMP-2 might contribute to MMP-2 activation. Egr-1 and TGF-beta1 might play important roles in this process.     

CD44 expression in plexiform lesions of idiopathic pulmonary arterial hypertension

Plexiform lesions in pulmonary arteries are a characteristic histological feature for idiopathic pulmonary arterial hypertension (IPAH). The pathogenesis of the plexiform lesion is not fully understood, although it may be related to endothelial cell dysfunction and local inflammation. CD44 is a cell adhesion molecule and it is also involved in angiogenesis, endothelial cell proliferation and migration. The expression of CD44 was examined in lung plexiform lesions obtained from patients with IPAH (IPAH group, n= 7) and pulmonary arterial hypertension associated with atrial septal defect (ASD-PAH group, n= 4). Expression of CD44 was detected in 49 out of 52 plexiform lesions (93%) from all patients in the IPAH group, whereas 31 plexiform lesions obtained from the ASD-PAH group lacked CD44 positivity by immunohistochemistry. In the IPAH group, CD44 was localized in the endothelial cells of microvessels within plexiform lesions and activated T cells in and around the lesions. Furthermore, T cell infiltration and endothelial cell proliferation activity were prominent in the plexiform lesions of the IPAH group, compared to those of the ASD-PAH group. These findings suggest that CD44 and activated T cell infiltration play an important role in the development of plexiform lesions particularly in IPAH.     

Three-Dimensional Reconstruction of Pulmonary Arteries in Plexiform Pulmonary Hypertension Using Cell-Specific Markers : Evidence for a Dynamic and Heterogeneous Process of Pulmonary Endothelial Cell Growth

The plexiform lesions of severe pulmonary hypertension (PH) are complex vascular structures composed primarily of endothelial cells. In this study, we use immunohistochemical markers to identify the various cell layers of pulmonary vessels and to identify different endothelial cell phenotypes in pulmonary arteries affected by severe PH. Our computerized three-dimensional reconstructions of nine vessels in five patients with severe PH demonstrate that plexiform (n = 14) and concentric-obliterative (n = 6) lesions occur distal to branch points of small pulmonary arteries. And, whereas plexiform lesions occur as solitary lesions, concentric-obliterative lesions appear to be only associated with, and proximal to, plexiform structures. The endothelial cells of plexiform lesions express intensely and uniformly the vascular endothelial growth factor (VEGF) receptor KDR and segregate phenotypically into cyclin-kinase inhibitor p27/kip1-negative cells in the central core of the plexiform lesion and p27/kip1-positive cells in peripheral areas adjacent to incipient blood vessel formation. Using immunohistochemistry and three-dimensional reconstruction techniques, we show that plexiform lesions are dynamic vascular structures characterized by at least two endothelial cell phenotypes. Plexiform arteriopathy is not merely an end stage or postthrombotic change--it may represent one stage in an ongoing, angiogenic endothelial cell growth process.     

The metalloproteinase inhibitor TIMP-2 is down-regulated by androgens in LNCaP prostate carcinoma cells

Tissue inhibitors of metalloproteinases (TIMPs) have been shown to perform several biological functions in tumor promotion, principally by their action of inhibiting matrix metalloproteinases (MMPs) at different steps of the metastatic process. In particular, TIMP-2 is involved in a functional complex with the membrane-type 1 (MT1) MMP to convert the secreted MMP-2 progelatinase into the fully active proteolytic enzyme. We used the human, androgen-sensitive prostate carcinoma cell line LNCaP in coculture with the human osteosarcoma cell line OHS to experimentally address the possibility of androgen-dependent regulatory effects on the functional MT1-MMP/TIMP-2/MMP-2 complex upon interaction between prostate carcinoma and osteoblastic cells in metastasis of prostate cancer to bone. In the LNCaP cells a gradual, time-dependent decline in TIMP-2 mRNA expression was observed in the presence of the synthetic androgen analogue R1881 (100 nM), reaching ∼25% of the control level after 48 h of incubation. Consistent with this, the accumulation of secreted TIMP-2 in media from R1881-treated cells was significantly inhibited already after 3 h. Neither MMP-2 gelatinolytic activity nor expression of MT1-MMP was detected in LNCaP cells. In contrast, the OHS cells showed membrane-associated MT1-MMP expression as well as MMP-2 secretion. However, R1881 treatment of the LNCaP/OHS coculture model did not seem to change the overall proteolytic activity of the MT1-MMP/TIMP-2/MMP-2 complex. Hormonal control of TIMP-2 expression in prostate carcinoma cells has not been previously reported, but whether such regulation has any functional role in the development of osteoblastic metastases in prostate cancer is still unclear. This revised version was published online in July 2006 with corrections to the Cover Date.