Cell Surface Plasminogen Activation Research Articles

#2510 Urokinase plasminogen activator receptor upregulation in lupus nephritis associates with distinct interferon hub genes in stressed podocytes

Abstract Background and Aims Systemic Lupus Erythematosus (SLE) is a complex autoimmune disease characterized by immune system dysfunction, leading to inflammation and damage in various organs and tissues. Renal involvement known as lupus nephritis is one of the most severe complications occurring in 60% of patients with SLE, significantly impacting patient morbidity and mortality. Understanding the intricate relationship between SLE and renal manifestations is crucial for effective management and improved patient outcomes. Recently, the cell surface urokinase plasminogen activator receptor (uPAR) has emerged as a significant player in the pathogenesis of lupus nephritis. However, the molecular mechanisms through which uPAR influences lupus nephritis remain an active area of investigation. Therefore, we here pursued a transcriptome array-based approach to identify molecular patterns in association with uPAR in lupus nephritis. Method PLAUR mRNA expression levels (encoding uPAR, reporter ID: 210845_s_at, platform: Affymetrix Human Genome U133 Plus 2.0 Array, altCDF v10) were extracted from microdissected control kidneys (glomerular compartment: n = 14) and lupus nephritis (glomerular: n = 32, tubulointerstitial: n = 32). Pathway analysis was performed for gene enrichment associated with PLAUR mRNA expression with a correlation threshold of ≥0.5 by using reactome (https://reactome.org), pathways with a predefined entities value of P ≤ .001 were included. Data was validated in cultured podocytes stressed with Adriamycin, protein expression profiling was performed by mass spectrometry in combination with stable isotope labelling by amino acids in cell culture (SILAC). Results We here identified a predominant glomerular expression of uPAR in lupus nephritis that was increased as compared to healthy controls. While glomerular uPAR expression was predominantly associated with interferon signaling, none of type I or type II interferons were induced. Interestingly, we identified distinct interferon hub genes that were associated with uPAR in lupus nephritis and enriched in stressed podocytes (GBP2: P < .0001, IFI30: P = .0006, IFIT1: P < .0001, IFITM3: P = .0308). Finally, uPAR and associated interferon hub genes correlated with proteinuria in lupus nephritis. Conclusion The therapeutic implications of targeting uPAR in lupus nephritis are promising, and ongoing research endeavors are aimed at unraveling the complexities of uPAR as a potential diagnostic marker and therapeutic target for this challenging renal pathology. We here expand our current knowledge and link uPAR upregulation in lupus nephritis to distinct interferon hub genes in stressed podocytes.

Proteomics Reveals Cell-Surface Urokinase Plasminogen Activator Receptor Expression Impacts Most Hallmarks of Cancer.

While metastasis is the primary cause of colorectal cancer (CRC) mortality, the molecular mechanisms underpinning it remains elusive. Metastasis is propagated through driver oncogene/suppressor gene mutations, accompanied by passenger mutations and underlying genomic instability. To understand cancer biology, a unifying framework called the "hallmarks of cancer" (HoCs) has been developed, which organizes cell biological alterations under ten key hallmarks. Underlying these HoCs, genome instability generates mutational diversity that is amplified by inflammation. Recognizing how critical cancer cell-surface proteins influence, these HoCs have been proposed to accelerate precision medicine therapeutic development. A moderate decrease (43%↓) in HCT116 cell surface urokinase plasminogen activator receptor (uPAR) expression mitigates against many HoCs driven by these cell's KRAS and PIK3CA mutational signature. Comprehensive proteomics (whole cell lysis with two membrane protein enrichments) coupled with ingenuity pathway analysis (IPA) demonstrates that uPAR negates essential pathways across the HoC spectrum, particularly those associated with metastasis, resisting cell death, and sustaining proliferation, and parallels Cancer Hallmarks Analytics Tool analysis. Decreasing uPAR predominantly alters metastasis-related and uPAR-interactome protein expression (e.g., EGFR, caveolin, vitronectin, integrin β4). Collectively, it is demonstrated that uPAR is a lynchpin protein capable of regulating several HoC pathways in a classical CRC mutational background.

Deficiency of plasminogen receptor, Plg‐RKT, causes defects in plasminogen binding and inflammatory macrophage recruitment in vivo

Background Plg-RKT is a novel integral membrane plasminogen receptor that binds plasminogen via a C-terminal lysine exposed on the cell surface and promotes plasminogen activation on the cell surface by both tissue plasminogen activator and urokinase plasminogen activator. Objectives To evaluate the role of Plg-RKT in vivo we generated Plg-RKT-/- mice using a homologous recombination technique. Methods We characterized the effect of Plg-RKT deletion on reproduction, viability, health and spontaneous thrombosis and inflammation. Results Plg-RKT-/- mice were viable and fertile. Survival of Plg-RKT-/- mice and Plg-RKT+/+ littermates was not significantly different. However, quite strikingly, all pups of Plg-RKT-/- females died within 2 days of birth, consistent with a lactation defect in Plg-RKT-/- mothers. Additionally, there was a significant effect of Plg-RKT deficiency on the growth rates of female, but not male, mice. In experimental peritonitis studies, Plg-RKT-/- mice exhibited a marked defect in macrophage recruitment. As a contributing mechanism, the capacity of Plg-RKT-/- macrophages for plasminogen binding was markedly decreased. Conclusions These studies demonstrate that Plg-RKT is required for plasminogen binding and macrophage migration in vivo. In addition, Plg-RKT deficiency is not compatible with survival of the species, due to the death of all offspring of Plg-RKT-/- females. This new mouse model will be important for future studies aimed at delineating the role of cell surface plasminogen activation in challenge and disease models in vivo.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis.

BackgroundThrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis.MethodsTo assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM.ResultsInhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa.ConclusionsTaken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process.

The Urokinase-type Plasminogen Activator and the Generation of Inhibitors of Urokinase Activity and Signaling

Urokinase (uPA) was originally identified in human urine for its ability to catalyse the transformation of plasminogen into its active form, plasmin which degrades fibrin and extracellular matrix components. Two major, functionally independent regions have been identified in the uPA molecule: a non-catalytic N-terminal region (residues 1-135) and a large catalytic region (residues 159-411) spaced by the "connecting peptide" (residues 136-158). Binding of uPA to its specific surface receptor (uPAR) amplifies cell surface plasminogen activation, thus enhancing pericellular proteolysis. The uPAR, linked to the lipid bilayer via a glycosylphosphatidylinositol anchor, mediates signaling through the assembly of a multiprotein complex with transmembrane receptors, like the integrins, EGFR, GPCRs. Receptor engagement with uPA results in a variety of cell responses, including increased proliferation, survival, migration and invasion. These responses may be enhanced by the concomitant binding of the uPA "connecting peptide" region to αvβ5 integrin, thus favoring uPAR-integrin association. Receptors engaged with uPA exhibit a high affinity for vitronectin, stimulating cell adhesion. The uPA/uPAR system is regarded as one of the key systems driving tumour invasion and metastases. Different strategies to prevent the activity of the protease, as well as the interactions of uPAR with integrins and GPCRs have been designed. Many preclinical studies are ongoing and, at least, two uPA-related compounds have reached Phase II clinical trials. The aim of this review is to provide a comprehensive picture of the functionally relevant interactions, together with a description of the promising compounds and strategies to control uPA activity and signaling in human pathologies.

Intraocular Properties of a Repository Urokinase Receptor Antagonist Å36 Peptide in Rabbits

Purpose: To evaluate the intraocular properties of Å36, a peptide that directly antagonizes the cell surface urokinase receptor and so prevents pericellular urokinase plasminogen activator activity.Methods: A total of 41 rabbits were used. The toxicity study tested three doses of Å36: 1 mg/ eye, 0.3 mg/eye, and 0.1 mg/eye. At 2 and 12 weeks, eyes were evaluated by ERG and histology. Pharmacokinetics were studied in rabbit eyes with the dose of 1 mg/eye in two different formulations: a micronized preparation and a non-micronized formulation. Eyes were enucleated at months 1, 2, 3, 4, and 5. Vitreous, retina, and choroid were collected separately for active Å36 analysis.Results: We did not find ocular toxicity with low and medium doses. At the highest dose, there was a transient toxicity at 2 weeks but was not notable at 3 months. The target choroid concentration of Å36 was chosen as ≥100 nM. The micronized formulation at months 1, 2, and 3 combined, showed variable levels in the choroid giving 5/10 (50%) of the therapeutic level; the non-micronized formulation at months 4 and 5 combined, gave 6/7 (86%) of the therapeutic level, although this difference was not statistically significant.Conclusion: Å36 appears to be long lasting; the non-micronized formulation of Å36 gave concentrations above therapeutic level in the choroid at months 4 and 5. Optimization of the formulation of Å36, particularly the particle size, may result in a promising new compound for exudative age-related macular degeneration treatment.

Identification of a receptor‐induced binding site (RIBS) in plasminogen induced by its interaction with cells

When plasminogen is bound to cells its activation is markedly enhanced compared to the reaction in solution, a key step in regulation of thrombolysis and cell migration. To study the mechanism of cell‐surface plasminogen activation we developed a monoclonal antibody (mAb 51) that reacts with cell‐associated plasminogen in blood in the presence of a high molar excess of soluble plasminogen. Thus, mAb 51 detects receptor‐induced binding sites (RIBS) induced in plasminogen upon its interaction with cells. The objective of our study was to define the RIBS epitope in plasminogen recognized by mAb 51. Plasminogen was denatured, reduced and alkylated and then digested with trypsin. Using western blotting and a specific Mab 51‐based ELISA for plasminogen, we established the concentration of trypsin that resulted in maximal digestion of plasminogen, while still retaining reactivity with mAb 51. The plasminogen digest was applied to a mAb 51 immunoaffinity column. MALDI analysis of the eluate yielded a single peak with a mass of 3,076 m/z. In LC‐MS‐MS, a peak with the same mass was obtained and assigned to a peptide corresponding to K177‐Y154, spanning a domain linking plasminogen kringles 1 and 2. Our data suggest that this domain becomes accessible when plasminogen interacts with cells. Thus, this conformational change detected by mAb 51 is likely to contribute to the ability of plasminogen to be activated on the cell surface.

Proteomics-based discovery of a novel, structurally unique, and developmentally regulated plasminogen receptor, Plg-RKT, a major regulator of cell surface plasminogen activation

AbstractActivation of plasminogen, the zymogen of the primary thrombolytic enzyme, plasmin, is markedly promoted when plasminogen is bound to cell surfaces, arming cells with the broad spectrum proteolytic activity of plasmin. In addition to its role in thrombolysis, cell surface plasmin facilitates a wide array of physiologic and pathologic processes. Carboxypeptidase B-sensitive plasminogen binding sites promote plasminogen activation on eukaryotic cells. However, no integral membrane plasminogen receptors exposing carboxyl terminal basic residues on cell surfaces have been identified. Here we use the exquisite sensitivity of multidimensional protein identification technology and an inducible progenitor cell line to identify a novel differentiation-induced integral membrane plasminogen receptor that exposes a C-terminal lysine on the cell surface, Plg-RKT (C9orf46 homolog). Plg-RKT was highly colocalized on the cell surface with the urokinase receptor, uPAR. Our data suggest that Plg-RKT also interacts directly with tissue plasminogen activator. Furthermore, Plg-RKT markedly promoted cell surface plasminogen activation. Database searching revealed that Plg-RKT mRNA is broadly expressed by migratory cell types, including leukocytes, and breast cancer, leukemic, and neuronal cells. This structurally unique plasminogen receptor represents a novel control point for regulating cell surface proteolysis.

Enhanced Cell-Associated Fibrinolytic Pathway but Not Coagulation Pathway Activity Contributes to Motility in Metastatic Breast Cancer Cells.

Abstract 3170Poster Board III-105Activation of tumor cell-associated coagulation and fibrinolytic pathways occurs in many different malignant and metastatic disease processes, including breast cancer. It has been proposed that coagulation and fibrinolytic pathway activation highjack host hemostatic mechanisms and facilitate the metastatic process. However, depending on tumor cell gene expression, tumor microenvironment, and tissue of origin, it is not fully understood whether the coagulation pathway or fibrinolytic pathway, alone or together in concert, contributes to the metastatic phenotype. To characterize and compare the coagulation and fibrinolytic pathways of normal and metastatic cells, we utilized the MCF-10 family of breast cell lines. The MCF-10 family of breast cell lines was originally derived from a woman with benign fibrocystic breast disease, which led to the near normal immortalized MCF-10A cells. After transformation with T24-Ha-ras, more aggressive cell lines were derived, ultimately leading to the metastatic MCF-10CA1 cell line. Using an in vitro modified-Boyden chamber model, MCF-10CA1 cells were significantly more motile than MCF-10A cells both at baseline using BSA as the chemoattractant, and when using EGF as the chemoattractant. Thus, as predicted by properties previously reported in the literature, the metastatic MCF-10CA1 cells were substantially more motile than the near normal parental cell line, MCF-10A. Both cell types supported similar rates of factor Xa generation, plasma thrombin generation, and fibrin formation. Using laser scanning confocal microscopy, we observed that MCF-10A cells produced a stable fibrin network, whereas MCF-10CA1 cells lysed the surrounding fibrin network within 24 hours of network formation. Importantly, fibrin located proximal to (within 10 microns of) the MCF-10CA1 cell surface lysed significantly faster than fibrin located 100 microns from the surface. These results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. A cell-surface plasminogen activation assay showed that MCF-10CA1 cells supported substantially increased plasmin generation rates compared to MCF-10A cells, providing a mechanism for the increased fibrinolytic activity of these cells towards the fibrin network. Metastatic MCF-10CA1 cells expressed significantly increased levels of urokinase (uPA) and decreased levels of plasminogen activator inhibitor-1 (PAI-1) compared to the MCF-10A cells. Blocking uPA activity with an active-site directed-inhibitor (amiloride) decreased MCF-10CA1 cell motility to essentially the same level as MCF-10A cells. Inhibiting the phosphatidylinositol 3-kinase/Akt signaling axis of MCF-10CA1 cells with LY294002 similarly decreased cell surface plasminogen activation activity and cell motility. Collectively, these results suggest that the tumor-associated fibrinolytic pathway is a key distinguishing feature between the metastatic MCF10-CA1 cells and normal MCF-10A cells. Our results support continued investigation of urokinase inhibition, either by directly blocking uPA activity or by down-regulating uPA expression, as an attractive adjunctive therapeutic target to reduce metastatic breast cancer. DisclosuresNo relevant conflicts of interest to declare.

Urokinase-type plasminogen activator receptor

Pure human cellular receptor for urokinase plasminogen activator (u-PAR) and forms thereof, including a truncated water-soluble form produced by cleaving or removing a glycosyl-phosphatidylinositol anchor, are provided, as is cDNA for u-PAR. Cell surface plasminogen activation, believed to play an important role in tissue remodelling, particularly during cancer invasion, is inhibited by antibodies against u-PAR and by inactivated analogues of u-PA. In situ hybdridization showed presence of u-PAR mRNA in the invasion front of colon carcinomas, proving the diagnostic utility of the cDNA. Water-soluble u-PAR is useful for inhibition of u-PA catalyzed plasminogen activation in solution.

Homocysteine-mediated thrombosis and angiostasis in vascular pathobiology.

The mechanisms by which homocysteine contributes to atherothrombosis are complex and their in vivo relevance uncertain. In this issue of the JCI, Jacovina and colleagues report a unique in vivo mechanism by which homocysteine may contribute to vascular disease (see the related article beginning on page 3384). This group had previously reported that homocysteine impairs endothelial cell surface plasminogen activation by posttranslationally modifying annexin A2, the coreceptor for plasminogen and tissue plasminogen activator. They now show that an annexin A2-deficient mouse rendered hyperhomocysteinemic by dietary means has impaired fibrinolysis, perivascular fibrin persistence, and attenuated angiogenesis (angiostasis). Potential mechanisms by which homocysteine-dependent changes in endothelial phenotype link thrombosis to angiostasis are reviewed and their relationship to homocysteine-dependent vascular disease considered.

Plasminogen activator inhibitors regulate cell adhesion through a uPAR‐dependent mechanism

Binding of type-1 plasminogen activator inhibitor (PAI-1) to cell surface urokinase (uPA) promotes inactivation and internalization of adhesion receptors (e.g., urokinase receptor (uPAR), integrins) and leads to cell detachment from a variety of extracellular matrices. In this report, we begin to examine the mechanism of this process. We show that neither specific antibodies to uPA, nor active site inhibitors of uPA, can detach the cells. Thus, cell detachment is not simply the result of the binding of macromolecules to uPA and/or of the inactivation of uPA. We further demonstrate that another uPA inhibitor, protease nexin-1 (PN-1), also stimulates cell detachment in a uPA/uPAR-dependent manner. The binding of both inhibitors to uPA leads to the specific inactivation of the matrix-engaged integrins and the subsequent detachment of these integrins from the underlying extracellular matrix (ECM). This inhibitor-mediated inactivation of integrins requires direct interaction between uPAR and those integrins since cells attached to the ECM through integrins incapable of binding uPAR do not respond to the presence of either PAI-1 of PN-1. Although both inhibitors initiate the clearance of uPAR, only PAI-1 triggers the internalization of integrins. However, cell detachment by PAI-1 or PN-1 does not depend on the endocytosis of these integrins since cell detachment was also observed when clearance of these integrins was blocked. Thus, PAI-1 and PN-1 induce cell detachment through two slightly different mechanisms that affect integrin metabolism. These differences may be important for distinct cellular processes that require controlled changes in the subcellular localization of these receptors.

Inhibition of Micrometastatic Prostate Cancer Cell Spread in Animal Models By 213Bilabeled Multiple Targeted α Radioimmunoconjugates

To investigate the therapeutic potential of 213Bilabeled multiple targeted alpha-radioimmunoconjugates for treating prostate cancer (CaP) micrometastases in mouse models. PC-3 CaP cells were implanted s.c., in the prostate, and intratibially in NODSCID mice. The expression of multiple tumor-associated antigens on tumor xenografts and micrometastases was detected by immunohistochemistry. Targeting vectors were two monoclonal antibodies, and a plasminogen activator inhibitor type 2 that binds to cell surface urokinase plasminogen activator, labeled with 213Bi using standard methodology. In vivo efficacy of multiple alpha conjugates (MTAT) at different activities was evaluated in these mouse models. Tumor growth was monitored during observations and local regional lymph node metastases were assessed at the end of experiments. The take rate of PC-3 cells was 100% for each route of injection. The tumor-associated antigens (MUC1, urokinase plasminogen activator, and BLCA-38) were heterogeneously expressed on primary tumors and metastatic cancer clusters at transit. A single i.p. injection of MTAT (test) at high and low doses caused regression of the growth of primary tumors and prevented local lymph node metastases in a concentration-dependent fashion; it also caused cancer cells to undergo necrosis and apoptosis. Our results suggest that MTAT can impede primary PC-3 CaP growth at three different sites in vivo through induction of apoptosis, and can prevent the spread of cancer cells and target lymph node micrometastases in a concentration-dependent manner. MTAT, by targeting multiple antigens, can overcome heterogeneous antigen expression to kill small CaP cell clusters, thus providing a potent therapy for micrometastases.

Control of prostate cancer spheroid growth using 213 Bi-labeled multiple targeted α radioimmunoconjugates

Micrometastasis is a major problem for prostate cancer (CaP) patients. Our study investigated the therapeutic potential of multiple targeted alpha-therapy (MTAT) in the treatment of CaP micrometastases (spheroids) using (213)Bi-labeled multiple targeted alpha-radioimmunoconjugates. The expression of multiple tumor-associated antigens (TAAs) on frozen sections of human fresh CaP tissues and spheroids cultured from DU 145 and LNCaP-LN3 CaP cell lines was detected by immunohistochemistry and flow cytometry. Targeting vectors were two monoclonal antibodies (MAbs), and plasminogen activator inhibitor type 2 (PAI2) that binds to cell surface urokinase plasminogen activator (uPA). These vectors were labeled with (213)Bi using standard methodology. DU 145 and LNCaP-LN3 spheroids were incubated with different activities of test and control alpha-conjugates (ACs), and spheroid growth was measured for volume change and growth delay over a 50-day period using light microscopy. TAAs were expressed heterogeneously on frozen sections from human CaP tissues and CaP spheroids. MTAT combining three ACs (one-third dose of each) with an activity of 6.4 MBq/ml completely targeted small DU 145 and LNCaP-LN3 spheroids (diameter <100 microm) and slightly regressed the growth of medium spheroids (180-200 microm); MTAT with 2.2 or 4.8 MBq/ml activities delayed the growth of tumor spheroids. Our results suggest that the cytotoxicity of MTAT to CaP spheroids is highly dependent on antigenic expression, concentration of radioactivity and spheroid size. MTAT may be a potent therapeutic agent for micrometastases, effectively targeting small CaP cell clusters, and overcoming the heterogeneous expression of targeted antigens.

Role of the Yersinia pestis plasminogen activator in the incidence of distinct septicemic and bubonic forms of flea-borne plague.

Yersinia pestis is transmitted by fleas and causes bubonic plague, characterized by severe local lymphadenitis that progresses rapidly to systemic infection and life-threatening septicemia. Here, we show that although flea-borne transmission usually leads to bubonic plague in mice, it can also lead to primary septicemic plague. However, intradermal injection of Y. pestis, commonly used to mimic transmission by fleabite, leads only to bubonic plague. A Y. pestis strain lacking the plasmid-encoded cell-surface plasminogen activator, which is avirulent by intradermal or s.c. injection, was able to cause fatal primary septicemic plague at low incidence, but not bubonic plague, when transmitted by fleas. The results clarify a long-standing uncertainty about the etiology of primary septicemic plague and support an evolutionary scenario in which plague first emerged as a flea-borne septicemic disease of limited transmissibility. Subsequent acquisition of the plasminogen activator gene by horizontal transfer enabled the bubonic form of disease and increased the potential for epidemic spread.

Stimulation of cell surface plasminogen activation by membrane-bound melanotransferrin: A key phenomenon for cell invasion

The activation of plasminogen at the cell surface is a crucial step in cell migration and invasion. In the present study, the effect of membrane-bound melanotransferrin (mMTf), also known as human melanoma antigen p97, on cell surface plasminogen binding and activation was investigated by using Chinese Hamster Ovary (CHO) cells transfected with full-length melanotransferrin (MTf) cDNA and SK-MeL-28 melanoma cells. The expression of mMTf in CHO increased cell surface plasminogen binding by about 2-fold. In addition, application of the monoclonal antibody L235 against MTf as well as truncated, soluble MTf (sMTf) abolished plasminogen binding to MTf-transfected and SK-MeL-28 cells, indicating that mMTf is a potential cell surface plasminogen receptor. Moreover, mMTf expression in CHO cells stimulates plasminogen activation at the cell surface by about 2.5-fold. In addition to the induced binding and activation of plasminogen, cell motility, migration and invasion were about 3-fold higher in CHO cells expressing mMTf. Both monoclonal antibody L235 and truncated sMTf inhibited mMTf-stimulated CHO cell motility, migration and invasion. Overall, our results indicate a key role for mMTf in cell surface plasminogen binding and in activation processes involved during cell migration and invasion.

Plasminogen receptors: the sine qua non of cell surface plasminogen activation.

Localization of plasminogen and plasminogen activators on cell surfaces promotes plasminogen activation and serves to arm cells with the broad spectrum proteolytic activity of plasmin. Cell surface proteolysis by plasmin is an essential feature of physiological and pathological processes requiring extracellular matrix degradation for cell migration including macrophage recruitment during the inflammatory response, tissue remodeling, wound healing, tumor cell invasion and metastasis and skeletal myogenesis. Cell associated plasmin on platelets and endothelial cells is optimally localized for promotion of clot lysis. In more recently recognized functions that are likely to be independent of matrix degradation, cell surface-bound plasmin participates in prohormone processing as well as stimulation of intracellular signaling. This issue of Frontiers in Bioscience on Plasminogen Receptors encompasses chapters focusing on the kinetics of cell surface plasminogen activation and the regulation of plasminogen receptor activity as well as the contribution of plasminogen receptors to the physiological and pathophysiological processes of myogenesis, muscle regeneration and cancer. The molecular identity of plasminogen receptors is cell-type specific, with distinct molecular entities providing plasminogen receptor function on different cells. This issue includes chapters on the well studied plasminogen receptor functions.

Attenuation of neointimal vascular smooth muscle cellularity in atheroma by plasminogen activator inhibitor type 1 (PAI-1).

Rupture of vulnerable atheroma often underlies acute coronary syndromes. Vulnerable plaques exhibit a paucity of vascular smooth muscle cells (VSMCs) in the cap. Therefore, decreased VSMC migration into the neointima may predispose to vulnerability. The balance between cell surface plasminogen activator activity and its inhibition [mediated primarily by plasminogen activator inhibitor type 1 (PAI-1)] modulates migration of diverse types of cells. We sought to determine whether increased expression of PAI-1 would decrease migration of VSMCs in vitro and neointimal cellularity in vivo in apolipoprotein E knockout (ApoE(-/-)) mice fed a high-fat diet. Increased vessel wall expression of PAI-1 in transgenic mice was induced with the SM22alpha promoter. VSMC migration through Matrigel in vitro was quantified with laser scanning cytometry. Expression of PAI-1 was increased threefold in the aortic wall of SM22-PAI transgene-positive mice. Neointimal cellularity of vascular lesions was decreased by 26% (p=0.01; n=5 each) in ApoE(-/-) mice with the SM22-PAI transgene compared with ApoE(-/-) mice. VSMCs explanted from transgene-positive mice exhibited twofold greater expression of PAI-1 and their migration was attenuated by 27% (p=0.03). Accordingly, increased expression of PAI-1 protein by VSMCs reduces their migration in vitro and their contribution to neointimal cellularity in vivo.

N-Acetyl-Cysteine Promotes Angiostatin Production and Vascular Collapse in an Orthotopic Model of Breast Cancer

The antioxidant N-acetyl-cysteine (NAC) has been shown to be chemopreventive in clinical studies, and in recent studies, has shown promise in preventing tumor progression. Although the effects of NAC on tumorigenesis have been associated with decreased angiogenesis, the mechanism of the anti-angiogenic activity has not been determined. In the following study, we describe a novel mechanism whereby NAC therapy blocks MDA-MB-435 breast carcinoma cell proliferation and metastasis in an in vivo tumorigenic model. Athymic nude mice bearing MDA-MB-435 xenografts were treated with systemic NAC daily for 8 weeks. NAC treatment resulted in endothelial cell apoptosis and reduction of microvascular density within the core of the tumor leading to significant tumor cell apoptosis/necrosis. Angiostatin accumulated in tumors from NAC-treated but not control animals. Additional studies using a vascular endothelial growth factor-dependent chicken chorioallantoic membrane angiogenic assay recapitulated NAC-induced endothelial apoptosis and coordinate production of angiostatin, a potent endothelial apoptotic factor. In vitro studies showed angiostatin was formed in endothelial cultures in a vascular endothelial growth factor- and NAC-dependent manner, a process that requires endothelial cell surface plasminogen activation. These results suggest that systemic NAC therapy promotes anti-angiogenesis through angiostatin production, resulting in endothelial apoptosis and vascular collapse in the tumor.