Renin inhibition by aliskiren prevents atherosclerosis progression: comparison with irbesartan, atenolol, and amlodipine.

Hypertension is associated with increased risk of cardiovascular diseases. Antihypertensive treatment, particularly blockade of the renin-angiotensin system, contributes to prevent atherosclerosis-mediated cardiovascular events. Direct comparison of different antihypertensive treatments on atherosclerosis and particularly plaque stabilization is sparse. ApoE(-/-) mice with vulnerable (2-kidney, 1-clip renovascular hypertension model) or stable (1-kidney, 1-clip renovascular hypertension model) atherosclerotic plaques were used. Mice were treated with aliskiren (renin inhibitor), irbesartan (angiotensin-receptor blocker), atenolol (beta-blocker), or amlodipine (calcium channel blocker). Atherosclerosis characteristics were assessed. Hemodynamic and hormonal parameters were measured. Aliskiren and irbesartan significantly prevented atherosclerosis progression in 2-kidney, 1-clip mice. Indeed, compared with untreated animals, plaques showed thinner fibrous cap (P<0.05); smaller lipid core (P<0.05); decreased media degeneration, layering, and macrophage content (P<0.05); and increased smooth muscle cell content (P<0.05). Interestingly, aliskiren significantly increased the smooth muscle cell compared with irbesartan. Despite similar blood pressure lowering, only partial plaque stabilization was attained by atenolol and amlodipine. Amlodipine increased plaque smooth muscle cell content (P<0.05), whereas atenolol decreased plaque inflammation (P<0.05). This divergent effect was also observed in 1-kidney, 1-clip mice. Normalizing blood pressure by irbesartan increased the plasma renin concentration (5932+/-1512 ng/mL per hour) more than normalizing it by aliskiren (16085+/-5628 ng/mL per hour). Specific renin-angiotensin system blockade prevents atherosclerosis progression. First, evidence is provided that direct renin inhibition mediates atherosclerotic plaque stabilization. In contrast, beta-blocker and calcium channel blocker treatment only partially stabilize plaques differently influencing atherogenesis. Angiotensin II decisively mediates plaque vulnerability. The plasma renin concentration measurement by an indirect method did not confirm the excessive increase of plasma renin concentration reported in the literature during aliskiren compared with irbesartan or amlodipine treatment.

Heterogeneity in endothelium-derived nitric oxide-mediated relaxation of different sized pulmonary arteries of newborn lambs.

Endothelium-derived nitric oxide (EDNO) plays a pivotal role in regulating pulmonary circulation. To determine whether there is a heterogeneity in EDNO-mediated responses of different sized pulmonary vessels, we studied small and large isolated pulmonary arteries of newborn lambs (diameter, 0.4-0.7 and 1.5-2.5 mm, respectively). The isometric tension of vessel rings were recorded while suspended in organ chambers filled with modified Krebs-Ringer bicarbonate solution (95% O2-5% CO2, 37 degrees C). In vessels preconstricted with norepinephrine, acetylcholine and bradykinin induced a greater relaxation of small pulmonary arteries than of large pulmonary arteries. Acetylcholine, bradykinin, and nitric oxide also induced a greater increase in cGMP content in small arteries than in large ones. The responses to acetylcholine and bradykinin were endothelium-dependent and inhibited by nitro-L-arginine, an inhibitor of nitric oxide synthase. In vessels without endothelium, the response to nitric oxide was inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, an inhibitor of soluble guanylate cyclase. The activity of soluble guanylyl cyclase of small arteries was greater than that of large arteries under basal conditions and after stimulation with S-nitroso-N-acetylpenicillamine, a nitric oxide donor. These results demonstrate that heterogeneity exists in EDNO-mediated relaxation of small and large pulmonary arteries in newborn lambs. A difference in the soluble guanylate cyclase activity of vascular smooth muscle may have contributed to this phenomenon.

New insight in aetiopathogenesis of aortic diseases.

BACKGROUND: Knowledge in the aetiopathogeny of aortic disease helps to characterise aortic lesions better and determine the risk of evolution and therapeutic strategies as well. This article focusses on aneurysms and dissections, and excludes causes related to infection, systemic inflammatory diseases and trauma. METHODS AND RESULTS: The biomedical literature of the past 10 years has been reviewed here. Aortic diseases are heterogeneous along the aorta as far as their genetic determinants, contribution of smooth muscle cells, inflammation and thrombus formation are concerned. Degradation of extracellular matrix by proteases causing aortic disease is a 'terminal' event, modulated by genetic background, haemodynamic strain, cellular events and thrombus formation. New genetic determinants of aortic disease have been identified. Proteases degrading the aortic wall are derived from a variety of cell types in addition to macrophages, including neutrophils on the luminal thrombus, mesenchymal and endothelial cells in the wall. Smooth muscle cells contribute to aortic wall homeostasis against inflammation and proteolysis. The degradation of the wall is followed by, or paralleled with, a failure of aortic reconstruction. CONCLUSIONS: Aortic diseases are diverse, and involve a multiplicity of biological systems in the vascular wall and at the interface with blood. Future research needs to unravel distinct cellular and molecular mechanisms causing the clinical events, in particular, dissection, expansion of already formed aneurysms and rupture.

Ultrahigh dose-rate FLASH irradiation increases the differential response between normal and tumor tissue in mice.

In vitro studies suggested that sub-millisecond pulses of radiation elicit less genomic instability than continuous, protracted irradiation at the same total dose. To determine the potential of ultrahigh dose-rate irradiation in radiotherapy, we investigated lung fibrogenesis in C57BL/6J mice exposed either to short pulses (≤ 500 ms) of radiation delivered at ultrahigh dose rate (≥ 40 Gy/s, FLASH) or to conventional dose-rate irradiation (≤ 0.03 Gy/s, CONV) in single doses. The growth of human HBCx-12A and HEp-2 tumor xenografts in nude mice and syngeneic TC-1 Luc(+) orthotopic lung tumors in C57BL/6J mice was monitored under similar radiation conditions. CONV (15 Gy) triggered lung fibrosis associated with activation of the TGF-β (transforming growth factor-β) cascade, whereas no complications developed after doses of FLASH below 20 Gy for more than 36 weeks after irradiation. FLASH irradiation also spared normal smooth muscle and epithelial cells from acute radiation-induced apoptosis, which could be reinduced by administration of systemic TNF-α (tumor necrosis factor-α) before irradiation. In contrast, FLASH was as efficient as CONV in the repression of tumor growth. Together, these results suggest that FLASH radiotherapy might allow complete eradication of lung tumors and reduce the occurrence and severity of early and late complications affecting normal tissue.

MRI visualized neo-intimal dissection and co-localization of novel apoptotic markers apolipoprotein C-1, ceramide and caspase-3 in a Watanabe hyperlipidemic rabbit model

PURPOSE: Apoptotic arterial wall vascular smooth muscle cell death is known to contribute to plaque vulnerability and rupture. Novel apoptotic markers like apolipoprotein C-I have been implicated in apoptotic human vascular smooth muscle cell death via recruiting a neutral sphingomyelinase (N-SMase)-ceramide pathway. In vivo relevance of these observations in an animal model of plaque rupture has not been shown. METHODS AND RESULTS: Using Watanabe rabbits, we investigated three different groups (group 1, three normal Watanabe rabbits; group 2, six Watanabe rabbits fed with high cholesterol diet for 3 months; group 3, five Watanabe rabbits with similar diet but additional endothelial denudation). We followed progression of atherosclerosis to pharmacologically induced plaque rupture non-invasively using novel 3D magnetic resonance Fast-Field-Echo angiography (TR=7.2, TE=3.6 ms, matrix=512 x 512) and Fast-Spin-Echo vessel wall imaging methods (TR=3 heart beats, TE=10.5 ms, matrix=304 x 304) on 1.5 T MRI. MRI provided excellent image quality with good MRI versus histology vessel wall thickness correlation (r=0.8). In six animals of group 2/3 MRI detected neo-intimal dissection in the abdominal aorta which was accompanied by immuno-histochemical demonstration of concomitant aforementioned novel apoptotic markers, previously implicated in the apoptotic smooth muscle cell death in vitro. CONCLUSIONS: Our studies suggest a potential role for the signal transduction pathway involving apolipoprotein C-I for in vivo apoptosis and atherosclerotic plaque rupture visualized by MRI.

Increased connexin43 expression in human sphenous veins in culture is associated with intimal hyperplasia

Résumé Objectif : L'hyperplasie intimale est un processus de remodelage vasculaire qui apparaît après une lésion vasculaire. Les mécanismes impliqués dans l'hyperplasie intimale sont la prolifération, la dédifférentiation et la migration des cellules musculaires lisses depuis la média vers l'espace sous-intimal. Nous avons émis l'hypothèse que les jonctions communicantes de type gap, qui coordonnent certains processus physiologiques tels que la croissance et la différentiation cellulaire, pouvaient participer au développement de l'hyperplasie intimale. Méthodes : Des segments de veines saphènes humaines prélevées chirurgicalement lors de pontages, ont été ouverts longitudinalement avec la surface luminale placée vers le haut et maintenus en culture pendant 14 jours. Des fragments veineux ont été préparés pour une évaluation histologique, pour des mesures de l'épaisseur de la néointima, et pour des analyses immunocytochimiques de l'ARN messager ainsi que des protéines. Résultats : Parmi les 4 connexines (Cxs 37, 40, 43 et 45) qui forment les jonctions communicantes dans les veines, nous avons focalisé notre étude sur l'expression des Cxs 43 et 40; nous avons démontré que la Cx43 est exprimée dans les cellules musculaires lisses et les cellules endothéliales alors que la Cx40 est uniquement présente dans l'endothélium. Après 14 jours en culture, des analyses histomorphométriques ont montré une augmentation significative de l'épaisseur de l'intima démontrant la présence d'hyperplasie intimale. Une analyse temporelle a révélé une augmentation progressive de la Cx43 jusqu'à une augmentation maximale de six à huit fois au niveau de l'ARN messager et des protéines après 14 jours en culture. Au contraire, l'expression de la Cx40 n'était pas modifiée. Des analyses par immunofluorescence ont montré également une augmentation de la Cx43 dans les membranes des cellules musculaires lisses de la média. Le développement de l'hyperplasie intimale in vitro est diminué en présence de fluvastatin et cette diminution est associée à une réduction de l'expression de la Cx43. Conclusions : Ces données démontrent que la Cx43 est augmentée in vitro pendant le processus d'hyperplasie intimale et que la fluvastatin prévient cette induction. Ces résultats suggèrent un rôle crucial joué par la communication intercellulaire impliquant la Cx43 dans la veine humaine durant le développement de l'hyperplasie intimale. Abstract Objective: Intimal hyperplasia is a vascular remodelling process that occurs after a vascular injury. The mechanisms involved in intimal hyperplasia are proliferation, dedifferentiation, and migration of medial smooth muscle cells towards the subintimal space. We postulated that gap junctions, which coordinate physiologic processes such as cell growth and differentiation, might participate in the development of intimal hyperplasia. connexin43 (Cx43) expression levels may be altered in intimal hyperplasia, and we therefore evaluated the regulated expression of Cx43 in human saphenous veins in culture in the presence or not of fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity. Methods: Segments of harvested human saphenous veins, obtained at the time of bypass graft, were opened longitudinally with the luminal surface uppermost and maintained in culture for 14 days. Vein fragments were then processed for histologic examination, neointimal thickness measurements, immunocytochemistry, RNA, and proteins analysis. Results: Of the four connexins (Cx37, 40, 43, and 45), we focused on Cx43 and Cx40, which we found by real-time polymerase chain reaction to be expressed in the saphenous vein because they are the predominant connexins expressed by smooth muscle cells and endothelial cells. Afrer 14 days of culture, histomorphometric analysis showed a significant increase in the intimal thickness as observed during the process of intimal hyperplasia. Atime-course analysis revealed a progressive upregulation of Cx43 to reach a maximal increase of sixfold to eightfold at both transcript and protein levels after 14 days in culture. In contrast, the expression of Cx40, abundantly expressed in the endothelial cells, was not altered. Immunofluorescence showed a large increase in Cx43 within smooth muscle cell membranes of the media layer. The development of intimal hyperplasia in vitro was decreased in presence of fluvastatin and was associated with reduced Cx43 expression. Conclusions: These data show that Cx43 is increased in vitro during the process of intimal hyperplasia and that fluvastatin could prevent this induction, supporting a critical role for Cx43-mediated gap-junctional communication in the human vein during the development of intimal hyperplasia. (J Vasc Surg 2005;41:1043-52.)

Simple method for detection of MYH11 DNA rearrangements in patients with inv(16)(p13q22) and acute myeloid leukemia.

The pericentric inversion on chromosome 16 [inv(16)(p13q22)] and related t(16;16)(p13;q22) are recurrent aberrations associated with acute myeloid leukemia (AML) M4 Eo. Both abberations result in a fusion of the core binding factor beta (CBFB) and smooth muscle myosin heavy chain gene (MYH11). A selected genomic 6.9-kb BamHl probe detects MYH11 DNA rearrangements in 18 of 19 inv(16)/t(16;16) patients tested using HindIII digested DNA. The rearranged fragments were not detectable after remission in two cases tested, while they were present after relapse in one of these two cases tested.

Connexins 43 and 26 are differentially increased after rat bladder outlet obstruction.

To evaluate the regulation of connexin expression by fluid pressure, we have studied the effects of elevated transmural urine pressure on Connexin43 (Cx43) and Cx26. We chose to focus on these two proteins out of the five connexins (Cx26, 43, 40, 37, and 45) which we found by RT-PCR to be expressed in the rat bladder, since in situ hybridization and immunofluorescence showed that Cx43 is the predominant connexin expressed by smooth muscle cells (SMC), whereas Cx26 is abundantly expressed only in the latter cell type. To evaluate whether these connexins are affected by changes in transmural urine pressure, we used a rat model of bladder outlet obstruction, in which a ligature is placed around the urethra. Under conditions of increased fluid pressure due to urine retention, we observed that the expression of both Cx43 and Cx26 increased at both transcript and protein levels, reaching a maximum 7-9 h after the ligature. Further analysis revealed that these changes were accounted for by a fourfold increase in Cx43 mRNA of SMC but not urothelial cell and by a fivefold increase in Cx26 mRNA of urothelium. Scrape-loading of propidium iodide showed that the latter change was paralleled by a twofold increase in coupling between urothelial cells. The data show that Cx43 and Cx26 are differentially regulated during bladder outlet obstruction and contribute to the response of the bladder wall to increased voiding pressure, possibly to control its elasticity.

Human cardiospheres as a source of multipotent stem and progenitor cells.

Cardiospheres (CSs) are self-assembling multicellular clusters from the cellular outgrowth from cardiac explants cultured in nonadhesive substrates. They contain a core of primitive, proliferating cells, and an outer layer of mesenchymal/stromal cells and differentiating cells that express cardiomyocyte proteins and connexin 43. Because CSs contain both primitive cells and committed progenitors for the three major cell types present in the heart, that is, cardiomyocytes, endothelial cells, and smooth muscle cells, and because they are derived from percutaneous endomyocardial biopsies, they represent an attractive cell source for cardiac regeneration. In preclinical studies, CS-derived cells (CDCs) delivered to infarcted hearts resulted in improved cardiac function. CDCs have been tested safely in an initial phase-1 clinical trial in patients after myocardial infarction. Whether or not CDCs are superior to purified populations, for example, c-kit(+) cardiac stem cells, or to gene therapy approaches for cardiac regeneration remains to be evaluated.

A collagen-poly(lactic acid-co-ɛ-caprolactone) hybrid scaffold for bladder tissue regeneration.

Scaffold materials should favor cell attachment and proliferation, and provide designable 3D structures with appropriate mechanical strength. Collagen matrices have proven to be beneficial scaffolds for tissue regeneration. However, apart from small intestinal submucosa, they offer a limited mechanical strength even if crosslinking can enhance their mechanical properties. A more cell-friendly way to increase material strength is to combine synthetic polymer meshes with plastic compressed collagen gels. This work describes the potential of plastic compressed collagen-poly(lactic acid-co-ɛ-caprolactone) (PLAC) hybrids as scaffolds for bladder tissue regeneration. Human bladder smooth muscle and urothelial cells were cultured on and inside collagen-PLAC hybrids in vitro. Scaffolds were analyzed by electron microscopy, histology, immunohistochemistry, and AlamarBlue assay. Both cell types proliferated in and on the hybrid, forming dense cell layers on top after two weeks. Furthermore, hybrids were implanted subcutaneously in the backs of nude mice. Host cell infiltration, scaffold degradation, and the presence of the seeded bladder cells were analyzed. Hybrids showed a lower inflammatory reaction in vivo than PLAC meshes alone, and first signs of polymer degradation were visible at six months. Collagen-PLAC hybrids have potential for bladder tissue regeneration, as they show efficient cell seeding, proliferation, and good mechanical properties.

Connexin26 is regulated in rat urothelium by the scaffold protein IB1/JIP-1.

Proper function of the wall of bladder requires gap junctional communication for coordinating the responses of smooth muscle (SMC) and urothelial cells exposed to urine pressure. In the rat bladder, Cx43 is expressed by SMC and urothelial cells, whereas Cx26 expression is restricted to the epithelium. We used a model of bladder outlet obstruction, in which a ligature is placed around the urethra to increase voiding pressure. Increased fluid pressure was associated with increased Cx43 and Cx26 mRNA expression and with the activation of a signaling cascade including the transcription factor c-Jun, which is a component of the AP-1 complex. The signaling pathway of the c-Jun NH2 terminal kinase (JNK) requires the presence of the scaffold protein Islet-Brain1/c-Jun amino-terminal kinase Interacting Protein-1 (IB1/JIP-1). Under stress conditions resulting from urine retention, we have found a reduced content of IB1/JIP-1 in urothelial cells, which in turn induced a drastic increase of JNK and AP-1 binding activities. The stress-induced activation of JNK was prevented by overexpressing IB1/JIP-1, using a viral gene transfer approach, a condition which also resulted in a decrease in Cx26 mRNA. The data show that: 1) mechanical stress of urothelial cells activates in vivo JNK, as a consequence of a regulated expression of IB1/JIP-1 and 2) that urothelial Cx26 may be directly regulated by the AP-1 complex.

SOST expression is restricted to the great arteries during embryonic and neonatal cardiovascular development.

Spatial-temporal regulation of bone morphogenetic protein (BMP) and Wnt activity is essential for normal cardiovascular development, and altered activity of these growth factors causes maldevelopment of the cardiac outflow tract and great arteries. In the present study, we show that SOST, a Dan family member reported to antagonize BMP and Wnt activity, is expressed within the medial vessel wall of the great arteries containing smooth muscle cells. The ascending aorta, aortic arch, brachiocephalic artery, common carotids, and pulmonary trunk were all associated with SOST expressing smooth muscle cells, while the heart itself, including the valves, and more distal arteries, that is, pulmonary arteries, subclavian arteries, and descending aorta, were negative. SOST was expressed from embryonic day 15.5 up to the neonatal period. SOST expression, however, did not correspond with inhibition of Smad-dependent BMP activity or beta-catenin-dependent Wnt activity in the great arteries. Activity of both signaling pathways was already down-regulated before induction of SOST expression.

Gene deletion of dopamine beta-hydroxylase and alpha1-adrenoceptors demonstrates involvement of catecholamines in vascular remodeling.

In vitro studies have shown that stimulation of alpha1-adrenoceptors (ARs) directly induces proliferation, hypertrophy, and migration of arterial smooth muscle cells and adventitial fibroblasts. In vivo studies confirmed these findings and showed that catecholamine trophic activity becomes excessive after experimental balloon injury and contributes to neointimal growth, adventitial thickening, and lumen loss. However, past studies have been limited by selectivity of pharmacological agents. The aim of this study, in which mice devoid of norepinephrine and epinephrine synthesis [dopamine beta-hydroxylase (DBH-/-)] or deficient in alpha1-AR subtypes expressed in murine carotid (alpha1B-AR-/- and alpha1D-AR-/-) were used, was to test the hypothesis that catecholamines contribute to wall hypertrophy after injury. At 3 wk after injury of wild-type mice, lumen area and carotid circumference increased significantly, and hypertrophy of media and adventitia was in excess of that needed to restore circumferential wall stress to normal. In DBH-/- and alpha1B-AR-/- mice, increases in lumen area, circumference, and hypertrophy of the media and adventitia were reduced by 50-91%, resulting in restoration of wall tension to nearly normal (DBH-/-) or normal (alpha1B-AR-/-). In contrast, in alpha1D-AR-/- mice, increases in lumen area, circumference, and wall hypertrophy were unaffected and wall thickening remained in excess of that required to return tension to normal. When examined 5 days after injury, proliferation and leukocyte infiltration were inhibited in DBH-/- mice. These studies suggest that the trophic effects of catecholamines are mediated primarily by alpha1B-ARs in mouse carotid and contribute to hypertrophic growth after vascular injury.

Acid-sensing channels in human bladder: expression, function and alterations during bladder pain syndrome.

Purpose: To examine the possible role of H+-activated acid-sensing ion channels (ASICs) in pain perception we characterized their expression in bladder dome biopsies of Bladder Pain Syndrome (BPS) patients and controls, in cultured human urothelium and in urothelial TEU-2 cells.Materials and Methods: Cold cut biopsies from the bladder dome were obtained in 8 asymptomatic controls and 28 patients with symptoms of BPS. ASIC expression was analyzed by QPCR and immunofluorescence. The channel function was measured by electrophysiology.Results: ASIC1a, ASIC2a and ASIC3 mRNAs were detected in human bladder. Similar amounts of ASIC1a and -3 were detected in detrusor smooth muscle, whereas in urothelium ASIC3 levels were higher than -1a. ASIC2a mRNA levels were lower than either -1a or -3 in both layers. ASIC currents were measured in TEU-2 cells and in primary cultures of human urothelium, and ASIC expression was confirmed by QPCR. Differentiation of TEU-2 cells caused an up-regulation of ASIC2a and ASIC3, and a down-regulation of ASIC1a mRNAs. BPS patients showed an up-regulation of ASIC2a and -3 mRNA, whereas ASIC1a remained unchanged. In contrast, the mRNA levels of TRPV1 were down-regulated during BPS. All differences were statistically significant (p<0.05)Conclusions: Several different ASIC subunits are expressed in human bladder and TEU-2 cells, where their levels are regulated during urothelial differentiation. An up-regulation of ASIC2a and -3 in BPS suggests their involvement in increased pain and hyperalgesia. A down-regulation of TRPV1 mRNA levels might indicate a different regulatory mechanism, controlling its expression in human bladder.

The role of MMP-9 and its fibronectin-like domain in tumor growth & invasion : certainties, controversies & discrepancies

Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-g revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-g in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-g activity in Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-9 revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-9 in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-9 activity in those cells and therefore prevent MMP-9-induced activation of TGF-b, which results in increased invasion. Curiously, xenografts of SW480 colorectal adenocarcinoma cells stably expressing the FN domain of MMP-9 displayed reduced growth at both the primary (subcutaneous) injection site and the lungs of NOD/SCID mice, in experimental metastasis assays, whilst the same cells overexpressing wt MMP-9 showed enhanced growth and dissemination. Gelatin zymography of conditioned medium revealed that these effects may be due to the FN domain, which displaces MMP-9 from SW480 cell surface. These observations suggest a dual role of MMP-9 and its FN domain in primary tumor growth and metastasis, underscoring the notion that the effect of MMP-9 on tumor cells may depend on the cell type and highlighting possible protective effects of MMPs in tumor progression.