Caveolin-1 enhances tissue factor pathway inhibitor exposure and function on the cell surface

Tissue factor pathway inhibitor (TFPI) blocks tissue factor-factor VIIa (TF-FVIIa) activation of factors X and IX through the formation of the TF-FVIIa-FXa-TFPI complex. Most TFPI in vivo associates with caveolae in endothelial cells (EC). The mechanism of this association and the anticoagulant role of caveolar TFPI are not yet known. Here we show that expression of caveolin-1 (Cav-1) in 293 cells keeps TFPI exposed on the plasmalemma surface, decreases the membrane lateral mobility of TFPI, and increases the TFPI-dependent inhibition of TF-FVIIa. Caveolae-associated TFPI supports the co-localization of the quaternary complex with caveolae. To investigate the significance of these observations for EC we used RNA interference to deplete the cells of Cav-1. Functional assays and fluorescence microscopy revealed that the inhibitory properties of TFPI were diminished in EC lacking Cav-1, apparently through deficient assembly of the quaternary complex. These findings demonstrate that caveolae regulate the inhibition by cell-bound TFPI of the active protease production by the extrinsic pathway of coagulation.     

Elevated Level of Fibrinogen Increases Caveolae Formation; Role of Matrix Metalloproteinase-9

The role of the inflammatory agent fibrinogen (Fg) in increased pial venular permeability has been shown previously. It was suggested that an activation of matrix metalloproteinase-9 (MMP-9) is involved in Fg-induced enhanced transcytosis through endothelial cells (ECs). However, direct link between Fg, caveolae formation, and MMP-9 activity has never been shown. We hypothesized that at an elevated level, Fg enhances formation of functional caveolae through activation of MMP-9. Male wild-type (WT, C57BL/6J) or MMP-9 gene knockout (MMP9?/?) mice were infused with Fg (4 mg/ml, final blood concentration) or equal volume of phosphate buffered saline (PBS). After 2 h, mice were sacrificed and brains were collected for immunohistochemical analyses. Mouse brain ECs were treated with 4 mg/ml of Fg or PBS in the presence or absence of MMP-9 activity inhibitor, tissue inhibitor of metalloproteinases-4 (TIMP-4, 12 ng/ml). Formation of functional caveolae was assessed by confocal microscopy. Fg-induced increased formation of caveolae, which was defined by an increased co-localization of caveolin-1 (Cav-1) and plasmalemmal vesicle-associated protein-1 and was associated with an increased phosphorylation of Cav-1, was ameliorated in the presence of TIMP-4. These results suggest that at high levels, Fg enhances formation of functional caveolae that may involve Cav-1 signaling and MMP-9 activation.     

Caveolin-1 influences vascular protease activity and is a potential stabilizing factor in human atherosclerotic disease

Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice. This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.     

Nitric oxide regulation of MMP-9 activation and its relationship to modifications of the cysteine switch

Matrix metalloproteases (MMPs) are Zn-containing endopeptidases involved in the degradation of extracellular matrix components and are typically secreted in a latent (pro-MMP) form and activated either by proteolytic or oxidative disruption of a conserved cysteine switch. Several recent studies have suggested that nitric oxide (NO) can contribute to the activation of MMPs, but the mechanisms involved are incompletely understood. We investigated the ability of NO to regulate the activation of (pro)MMP-9 using a variety of NO-donor compounds and characterized modifications of the cysteine switch using a synthetic peptide (PRCGVPDLGR) representing the cysteine switch domain of MMP-9. Among the NO-donors used, only S-nitrosocysteine (SNOC) was found to be capable of modest activation of proMMP-9, but S-nitrosoglutathione (GSNO) or the NONOates, DEA-NO, SPER-NO, or DETA-NO, were ineffective. In fact, high concentrations of DETA-NO were found to inhibit MMP-9 activity, presumably by direct interaction with the active-site Zn (2+). Analysis of chemical modifications within the Cys-containing peptide, PRCGVPDLGR, revealed rapid and transient S-nitrosylation by SNOC and GSNO, and formation of mixed disulfides and dimerized peptide as major final products. Similarly, NONOates induced transient S-nitrosylation and primarily peptide dimerization. Coordination of the peptide Cys with a synthetic Zn (2+) complex, to more closely mimic the structure of the active site in proMMP-9, reduced peptide nitrosylation and oxidation by NONOates, but enhanced peptide nitrosylation by SNOC and GSNO. Collectively, our results demonstrate that NO is incapable of directly activating proMMP-9 and that S-nitrosylation of MMP-9 propeptide by NO-donors is unrelated to their ability to regulate MMP-9 activity.     

Nitric oxide modulates capillary formation at the endothelial cell-tumor cell interface

Nitric oxide synthase expression has been documented in lung tumors, but a potential role for nitric oxide (NO) in induction of capillary formation remains to be elucidated. The purpose of this report was to characterize the direct effects of NO at the level of the tumor-endothelium interface with respect to angiogenesis. A Transwell two-compartment culture system, human endothelial cells (EC), and two human non-small cell lung cancer (CA) lines that constitutively produce NO were used to simulate the EC-tumor cell interface. Both histological types of lung CA, squamous and adenocarcinoma, induced baseline capillary formation by EC within 3 days. This process was inhibited by NO in the microenvironment because decreasing NO production with 100 microM aminoguanidine (AG) significantly increased capillary formation, whereas coincubation with 100 microM AG plus 400 microM L-arginine returned angiogenesis to baseline values. We demonstrate further that NO may exert its inhibitory effects by influencing matrix metalloproteinase expression/activity and tyrosine phosphorylation of proteins in the sprouting tips of nascent capillaries.     

Matrix metalloproteinase-2, caveolins, focal adhesion kinase and c-Kit in cells of the mouse myocardium

Matrix metalloproteinase-2 (MMP-2) may play roles at intracellular and extracellular sites of the heart in ischaemia/reperfusion injury. Caveolins (Cav-1, -2 and -3) are lipid raft proteins which play roles in cell sig-nalling. This study examined, using immunohistochemistry and two photon confocal microscopy, if MMP-2 and caveolins co-localize at the plasma membrane of cardiac cells: cardiomyocytes (CM), fibroblasts (FB) and capillary endothelial cells (CEC) in the left ventricle (LV) of the Cav-1(+/+) and Cav-1(-/-) mouse heart. In Cav-1(+/+) mouse LV MMP-2 and Cav-1 co-localized at CM plasma membranes, and at multiple locations in FB and CEC. MMP-2 co-localized with Cav-2 only at CEC. MMP-2 co-localized with Cav-3 at CM plasma membranes and Z-lines, and partially at FB and CEC. In Cav-1(-/-) LV Cav-1 and MMP-2 were absent or reduced everywhere. Cav-2 appeared at CEC despite the absence of Cav-1. Cav-3 appeared at CM plasma membranes and Z-lines, FB and CEC. Also, FAK in FB and c-Kit in interstitial Cajal-like cells (ICLC) were completely absent. By transmission electron microscopy in Cav-1(+/+), regular size caveolae (Cav) were at CEC, irregular size Cav were at CM and a few were at FB. In Cav-1(-/-) there were few Cav at CM and FB and some at CEC. To conclude, MMP-2 is closely associated with caveolins at FB and CEC as well as at CM. Also, MMP-2 is closely associated with FAK at FB and c-Kit at ICLC. Thus, Cav-1 expression is not necessary for Cav-2 expression. Cav-3 or Cav-3 with Cav-2 has the capability to make Cav.     

Microvascular hyperpermeability in caveolin-1 (-/-) knock-out mice. Treatment with a specific nitric-oxide synthase inhibitor,L-NAME,restores normal microvascular permeability in Cav-1 null mice

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (-/-) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis. Thus, a compensatory mechanism must be operating in these mice. One possible compensatory response would be an increase in the paracellular pathway, resulting in increased microvascular permeability. To test this hypothesis directly, we studied the microvascular permeability of Cav-1 null mice using a variety of complementary in vivo approaches. Radio-iodinated bovine serum albumin was injected into Cav-1-deficient mice, and its rate of clearance from the circulatory system was compared with that of wild type control mice. Our results indicate that iodinated bovine serum albumin is removed from the circulatory system of Cav-1-deficient mice at a substantially faster rate. To determine whether this defect is restricted to the paracellular movement of albumin, lungs from Cav-1-deficient mice were next perfused with the electron dense dye Ruthenium Red. Micrographs of lung endothelial cells from Cav-1-deficient mice demonstrate that the paracellular movement of Ruthenium Red is dramatically increased. In addition, electron micrographs of Cav-1-deficient lung capillaries reveal defects in tight junction morphology and abnormalities in capillary endothelial cell adhesion to the basement membrane. This defect in cell-substrate attachment is consistent with the postulated role of caveolin-1 in positively regulating integrin signaling. Because loss of caveolin-1 expression results in constitutive activation of eNOS activity, we also examined whether these increases in microvascular permeability are NO-dependent. Interestingly, treatment with l-NAME (a well established nitric-oxide synthase inhibitor) successfully reversed the microvascular hyperpermeability phenotype of Cav-1 knock-out mice. Thus, caveolin-1 plays a dual regulatory role in controlling microvascular permeability: (i) as a structural protein that is required for caveolae formation and caveolar transcytosis and (ii) as a tonic inhibitor of eNOS activity to negatively regulate the paracellular pathway.     

Effects of vascular endothelial growth factor on the lymphocyte-endothelium interactions: identification of caveolin-1 and nitric oxide as control points of endothelial cell anergy

Tumors may evade immune responses at multiple levels, including through a defect in the lymphocyte-vessel wall interactions. The angiogenic nature of endothelial cells (EC) lining tumor blood vessels may account for such anergy. In this study, we examined whether mechanisms other than down-regulation of adhesion molecules could be involved, particularly signaling pathways dependent on the caveolae platforms. To mimic the influence of the tumor microenvironment, EC were exposed to TNF-alpha and the proangiogenic vascular endothelial growth factor (VEGF). We identified a dramatic inhibition of lymphocyte adhesion on activated EC following either short or long VEGF pretreatments. We further documented that VEGF did not influence the abundance of major adhesion molecules, but was associated with a defect in ICAM-1 and VCAM-1 clustering at the EC surface. We also found that overexpression of the caveolar structural protein, caveolin-1, overcame the VEGF-mediated inhibition of adhesion and restored ICAM-1 clustering. Conversely, EC transduction with a caveolin-1 small interfering RNA reduced the TNF-alpha-dependent increase in adhesion. Finally, we identified VEGF-induced NO production by the endothelial NO synthase as the main target of the changes in caveolin-1 abundance. We found that the NO synthase inhibitor N-nitro-l-arginine methyl ester could reverse the inhibitory effects of VEGF on lymphocyte adhesion and EC cytoskeleton rearrangement. Symmetrically, a NO donor was shown to prevent the ICAM clustering-mediated lymphocyte adhesion, thereby recapitulating the effects of VEGF. In conclusion, this study provides new insights on the mechanisms leading to the tumor EC anergy vs immune cells and opens new perspectives for the use of antiangiogenic strategies as adjuvant approaches to cancer immunotherapy.     

Induction of stromelysin-1 (MMP-3) by fibroblast growth factor-2 (FGF-2) in FGF-2-/- microvascular endothelial cells requires prolonged activation of extracellular signal-regulated kinases-1 and -2 (ERK-1/2)

Basic fibroblast growth factor (FGF-2) and matrix metalloproteinases (MMPs) play key roles in vascular remodeling. Because FGF-2 controls a number of proteolytic activities in various cell types, we tested its effect on vascular endothelial cell expression of MMP-3 (stromelysin-1), a broad-spectrum proteinase implicated in coronary atherosclerosis. Endothelial cells (EC) from FGF-2-/- mice are highly responsive to exogenous FGF-2 and were therefore used for this study. The results showed that treatment of microvascular EC with human recombinant FGF-2 results in strong induction of MMP-3 mRNA and protein expression. Upregulation of MMP-3 mRNA by FGF-2 requires de novo protein synthesis and activation of the ERK-1/2 pathway. FGF-2 concentrations (5-10 ng/ml) that induce rapid and prolonged (24 h) activation of ERK-1/2 upregulate MMP-3 expression. In contrast, lower concentrations (1-2 ng/ml) that induce robust but transient (<8 h) ERK-1/2 activation are ineffective. Inhibition of ERK-1/2 activation at different times (-0.5 h to +8 h) of EC treatment with effective FGF-2 concentrations blocks MMP-3 upregulation. Thus, FGF-2 induces EC expression of MMP-3 with a threshold dose effect that requires sustained activation of the ERK-1/2 pathway. Because FGF-2 controls other EC functions with a linear dose effect, these features indicate a unique role of MMP-3 in vascular remodeling.     

Nitric oxide attenuates matrix metalloproteinase-9 production by endothelial cells independent of cGMP- or NFκB-mediated mechanisms

Cardiovascular diseases involve critical mechanisms including impaired nitric oxide (NO) levels and abnormal matrix metalloproteinase (MMP) activity. While NO downregulates MMP expression in some cell types, no previous study has examined whether NO downregulates MMP levels in endothelial cells. We hypothesized that NO donors could attenuate MMP-9 production by human umbilical vein endothelial cells (HUVECs) as a result of less NFκB activation or cyclic GMP (cGMP)-mediated mechanisms. We studied the effects of DetaNONOate (10–400 μM) or SNAP (50–400 μM) on phorbol 12-myristate 13-acetate (PMA; 10 nM)-induced increases in MMP-9 activity (by gel zymography) or concentrations (by ELISA) as well as on a tissue inhibitor of MMPs’ (TIMP)-1 concentrations (by ELISA) in the conditioned medium of HUVECs incubated for 24 h with these drugs. We also examined whether the irreversible inhibitor of soluble guanylyl cyclase ODQ modified the effects of SNAP or whether 8-bromo-cGMP (a cell-permeable analog of cGMP) influenced PMA-induced effects on MMP-9 expression. Total and phospho-NFκB p65 concentrations were measured in HUVEC lysates to assess NFκB activation. Both NO donors attenuated PMA-induced increases in MMP-9 activity and concentrations without significantly affecting TIMP-1 concentrations. This effect was not modified by ODQ, and 8-bromo-cGMP did not affect MMP-9 concentrations. While PMA increased phospho-NFκB p65 concentrations, SNAP had no influence on this effect. In conclusion, this study shows that NO donors may attenuate imbalanced MMP expression and activity in endothelial cells independent of cGMP- or NFκB-mediated mechanisms. Our results may offer an important pharmacological strategy to approach cardiovascular diseases.     

Interaction of the immunoglobulin-like cell adhesion molecule hepaCAM with caveolin-1

Subsequent to our identification of the novel immunoglobulin-like cell adhesion molecule hepaCAM, we demonstrated that hepaCAM is capable of modulating cell growth and cell–extracellular matrix interactions. In this study, we examined the localization of hepaCAM in lipid rafts/caveolae as well as the interaction of hepaCAM with the caveolar structural protein caveolin-1 (Cav-1). Our results revealed that a portion of hepaCAM resided in detergent-resistant membranes and co-partitioned with Cav-1 to low buoyant density fractions characteristic of lipid rafts/caveolae. In addition, co-localization and coimmunoprecipitation assays confirmed the association of hepaCAM with Cav-1. Deletion analysis of hepaCAM showed that the extracellular first immunoglobulin domain of hepaCAM was required for binding Cav-1. Furthermore, when co-expressed, Cav-1 induced the expression of hepaCAM as well as distributed hepaCAM to intracellular Cav-1-positive caveolar structures. Taken together, our findings indicate that hepaCAM is partially localized in the lipid rafts/caveolae and interacts with Cav-1 through its first immunoglobulin domain.     

Release of Matrix Metalloproteinases-2 and 9 by S-Nitrosylated Caveolin-1 Contributes to Degradation of Extracellular Matrix in tPA-Treated Hypoxic Endothelial Cells

Intracranial hemorrhage remains the most feared complication in tissue plasminogen activator (tPA) thrombolysis for ischemic stroke. However, the underlying molecular mechanisms are still poorly elucidated. In this study, we reported an important role of caveolin-1 (Cav-1) s-nitrosylation in matrix metalloproteinase (MMP)-2 and 9 secretion from tPA-treated ischemic endothelial cells. Brain vascular endothelial cells (bEND3) were exposed to oxygen-glucose deprivation (OGD) for 2 h before adding recombinant human tPA for 6 h. This treatment induced a significant increase of MMP2 and 9 in the media of bEND3 cells and a simultaneous degradation of fibronectin and laminin β-1, the two main components of extracellular matrix (ECM). Inhibition of MMP2 and 9 with SB-3CT completely blocked the degradation of fibronectin and laminin β-1. ODG+tPA treatment led to Cav-1 shedding from bEND3 cells into the media. Notably, OGD triggered nitric oxide (NO) production and S-nitrosylationof Cav-1 (SNCav-1). Meanwhile tPA induced activation of ERK signal pathway and stimulates the secretion of SNCav-1. Pretreatment of bEND3 cells with C-PTIO (a NO scavenger) or U0126 (a specific ERK inhibitor) significantly reduced OGD-induced S-nitrosylation of Cav-1 in cells and blocked the secretion of Cav-1 and MMP2 and 9 into the media as well as the degradation of fibronectin and laminin β-1 in OGD and tPA-treated cells. These data indicate that OGD-triggered Cav-1 S-nitrosylation interacts with tPA-induced ERK activation to augment MMP2 and 9 secretion and subsequent ECM degradation, which may account for the exacerbation of ischemic blood brain barrier damage following tPA thrombolysis for ischemic stroke.     

Shortening of microsatellite deoxy(CA) repeats involved in GL331-induced down-regulation of matrix metalloproteinase-9 gene expression

Matrix metalloproteinase-9 (MMP-9) associates with cancer cell invasion and metastasis. CL1-5 cells, a human lung adenocarcinoma cell line, expressed an elevated level of MMP-9 and exhibited a highly invasive and metastatic ability. By Matrigel assay and gelatinase zymography, the topoisomerase II poison GL331 was found to dose-dependently inhibit the invasiveness and the level of secreted MMP-9 of CL1-5 cells. Northern blot analysis indicated that cellular MMP-9 mRNA level was decreased after GL331 treatment. Furthermore, GL331-induced down-regulation of mmp-9 gene promoter was demonstrated by using a luciferase reporter gene driven by the -216 to -13 region of the mmp-9 gene promoter cloned from CL1-5 cells. By PCR amplification and gel electrophoresis, we found that GL331 caused shortening of the -216 to -13 region of the mmp-9 promoter. Direct sequencing analysis revealed that the number of d(CA) was reduced from 24 to 18 at the microsatellite d(CA) repeat region of the mmp-9 promoter. The CL1-5 cells transfected with the luciferase reporter containing 18 d(CA)s expressed only 53% of those when the reporter contained 24 d(CA)s. The promoter region of mmp-9 gene contains other positive regulatory elements, such as TRE and kappaB. We found that GL331 did not significantly influence the luciferase activity driven by TRE or kappaB. Taken together, these data suggested that GL331 inhibited MMP-9 mRNA expression at least partly through the selective induction of shortening of microsatellite d(CA) repeats. This is the first report that an anti-cancer agent can inhibit mmp-9 gene expression by inducing microsatellite DNA shortening.     

TGF-β1 facilitates MT1-MMP-mediated proMMP-9 activation and invasion in oral squamous cell carcinoma cells

Matrix metalloproteinase (MMP)-2 and MMP-9, also known as gelatinases or type IV collagenases, are recognized as major contributors to the proteolytic degradation of extracellular matrix during tumor invasion. Latent MMP-2 (proMMP-2) is activated by membrane type 1 MMP (MT1-MMP) on the cell surface of tumor cells. We previously reported that cell-bound proMMP-9 is activated by the MT1-MMP/MMP-2 axis in HT1080 cells treated with concanavalin A in the presence of exogenous proMMP-2. However, the regulatory mechanism of proMMP-9 activation remains largely unknown. Transforming growth factor (TGF)-β1 is frequently overexpressed in tumor tissues and is associated with tumor aggressiveness and poor prognosis. In this study, we examined the role of TGF-β1 on MT1-MMP-mediated proMMP-9 activation using human oral squamous cell carcinoma cells. TGF-β1 significantly increased the expression of MMP-9. By adding exogenous proMMP-2, TGF-β1-induced proMMP-9 was activated during collagen gel culture, which was suppressed by the inhibition of TGF-β1 signaling or MT1-MMP activity. This MT1-MMP-mediated proMMP-9 activation was needed to facilitate TGF-β1-induced cell invasion into collagen gel. Thus, TGF-β1 may facilitate MT1-MMP-mediated MMP-9 activation and thereby stimulate invasion of tumor cells in collaboration with MT1-MMP and MMP-2.     

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.     

The interaction of metal ions and Marimastat with matrix metalloproteinase 9

The effect of a range of metal ions on the ability of Marimastat to inhibit matrix metalloproteinase 9 (MMP-9) was examined in a fluorescence based proteolytic assay. Whilst none of the metals examined significantly affected the inhibitory ability of Marimastat, several metal ions did have a significant effect on MMP-9 activity itself. In the absence of Marimastat, Zn(II) and Fe(II) significantly inhibited MMP-9 activity at metal ion concentrations of 10 and 100 microM, respectively. In both the absence and presence of Marimastat, Cd(II) significantly inhibited MMP-9 at 100 microM. In contrast, 1 mM Co(II) significantly upregulated MMP-9 proteolytic activity.     

Caveolin-2 is a negative regulator of anti-proliferative function and signaling of transforming growth factor-β in endothelial cells

Using a combination of wild-type (WT) and caveolin-2 (Cav-2) knockout along with retroviral reexpression approaches, we provide the evidence for the negative role of Cav-2 in regulating anti-proliferative function and signaling of transforming growth factor β (TGF-β) in endothelial cells (ECs). Although, TGF-β had a modest inhibitory effect on WT ECs, it profoundly inhibited proliferation of Cav-2 knockout ECs. To confirm the specificity of the observed difference in response to TGF-β, we have stably reexpressed Cav-2 in Cav-2 knockout ECs using a retroviral approach. Similar to WT ECs, the anti-proliferative effect of TGF-β was dramatically reduced in the Cav-2 reexpressing ECs. The reduced anti-proliferative effect of TGF-β in Cav-2-positive cells was evidenced by three independent proliferation assays: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell count, and bromodeoxyuridine incorporation and correlated with a loss of TGF-β-mediated upregulation of cell cycle inhibitor p27 and subsequent reduction of the levels of hyperphosphorylated (inactive) form of the retinoblastoma protein in Cav-2 reexpressing ECs. Mechanistically, Cav-2 inhibits anti-proliferative action of TGF-β by suppressing Alk5-Smad2/3 pathway manifested by reduced magnitude and length of TGF-β-induced Smad2/3 phosphorylation as well as activation of activin receptor-like kinase-5 (Alk5)-Smad2/3 target genes plasminogen activator inhibitor-1 and collagen type I in Cav-2-positive ECs. Expression of Cav-2 does not appear to significantly change targeting of TGF-β receptors I and Smad2/3 to caveolar and lipid raft microdomains as determined by sucrose fractionation gradient. Overall, the negative regulation of TGF-β signaling and function by Cav-2 is independent of Cav-1 expression levels and is not because of changing targeting of Cav-1 protein to plasma membrane lipid raft/caveolar domains.     

Lysyl oxidase propeptide inhibits smooth muscle cell signaling and proliferation

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.     

Melanotransferrin stimulates t-PA-dependent activation of plasminogen in endothelial cells leading to cell detachment

Tissue plasminogen activator (t-PA) is an extracellular serine protease that converts the proenzyme plasminogen into the broad-spectrum substrate serine protease, plasmin. Plasmin, one of the most potent pro-angiogenic factors, is a key element in fibrinolysis, cell migration, tissue remodeling and tumor invasion. In the present investigation, we assessed the impact of the truncated form of soluble melanotransferrin (sMTf) on plasminogen activation by t-PA and subsequent endothelial cell detachment. Co-treatment of human endothelial microvessel cells with plasminogen, t-PA and sMTf significantly increased plasmin formation and activity in the culture medium. Plasmin generated in the presence of sMTf also led to a 30% reduction in fibronectin detection within cell lysates and to a 9-fold increase within the corresponding cell medium. Moreover, the presence of sMTf increases EC detachment by 6-fold compared to cells treated only with plasminogen and t-PA. Although the addition of alpha(2)-antiplasmin completely prevented plasmin formation and EC detachment, epigallocatechin gallate, GM6001 and a specific antibody directed against MMP-2 prevented cellular detachment without interfering with plasminogen activation. Overall, these data suggest that the anti-angiogenic properties of sMTf may result from local overstimulation of plasminogen activation by t-PA, thus leading to subsequent degradation of the Fn matrix and EC detachment.