Accessing 2-substituted piperidine iminosugars by organometallic addition/intramolecular reductive amination: aldehyde vs. nitrone route.

A dual synthetic strategy to afford 2-substituted trihydroxypiperidines is disclosed. The procedure involved Grignard addition either to a carbohydrate-derived aldehyde or to a nitrone derived thereof, and took advantage of an efficient ring-closure reductive amination strategy in the final cyclization step. An opposite diastereofacial preference was demonstrated in the nucleophilic attack to the two electrophiles, which would finally produce the same piperidine diastereoisomer as the major product. However, use of a suitable Lewis acid in the Grignard addition to the nitrone allowed reversing the selectivity, giving access to 2-substituted piperidines with the opposite configuration at C-2.

Type II CRISPR/Cas9 approach in the oncological therapy.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a prokaryotic adaptable immune mechanism used by many bacteria and archaea to protect themselves from foreign nucleic acids. This complex system can recognize and cut non-self DNA in order to provide the prokaryotic organisms a strong defense against foreign viral or plasmid attacks and make the cell immune from further assaults. Today, it has been adapted to be used in vitro and in vivo in eukaryotic cells to perform a complete and highly selective gene knockout or a specific gene editing. The ease of use and the low cost are only two features that have made it very popular among the scientific community and the possibility to be used as a clinical treatment in several genetic derived pathologies has rapidly spread its fame worldwide. However, CRISPR is still not fully understood and many efforts need to be done in order to make it a real power tool for the human clinical treatment especially for oncological patients. Indeed, since cancer originates from non-lethal genetic disorders, CRISPR discovery fuels the hope to strike tumors on their roots. More than 4000 papers regarding CRISPR were published in the last ten years and only few of them take in count the possible applications in oncology. The purpose of this review is to clarify many problematics on the CRISPR usage and highlight its potential in oncological therapy.

u-PAR expression in cancer associated fibroblast: new acquisitions in multiple myeloma progression.

BACKGROUND: Multiple Myeloma (MM) is a B-cell malignancy in which clonal plasma cells progressively expand within the bone marrow (BM) as effect of complex interactions with extracellular matrix and a number of microenvironmental cells. Among these, cancer-associated fibroblasts (CAF) mediate crucial reciprocal signals with MM cells and are associated to aggressive disease and poor prognosis. A large body of evidence emphasizes the role of the urokinase plasminogen activator (u-PA) and its receptor u-PAR in potentiating the invasion capacity of tumor plasma cells, but little is known about their role in the biology of MM CAF. In this study, we investigated the u-PA/u-PAR axis in MM-associated fibroblasts and explore additional mechanisms of tumor/stroma interplay in MM progression. METHODS: CAF were purified from total BM stromal fraction of 64 patients including monoclonal gammopathy of undetermined significance, asymptomatic and symptomatic MM, as well as MM in post-treatment remission. Flow cytometry, Real Time PCR and immunofluorescence were performed to investigate the u-PA/u-PAR system in relation to the level of activation of CAF at different stages of the disease. Moreover, proliferation and invasion assays coupled with silencing experiments were used to prove, at functional level, the function of u-PAR in CAF. RESULTS: We found higher activation level, along with increased expression of pro-invasive molecules, including u-PA, u-PAR and metalloproteinases, in CAF from patients with symptomatic MM compared to the others stages of the disease. Consistently, CAF from active MM as well as U266 cell line under the influence of medium conditioned by active MM CAF, display higher proliferative rate and invasion potential, which were significantly restrained by u-PAR gene expression inhibition. CONCLUSIONS: Our data suggest that the stimulation of u-PA/u-PAR system contributes to the activated phenotype and function of CAF during MM progression, providing a biological rationale for future targeted therapies against MM.

Endothelial Progenitor Cells in Sprouting Angiogenesis: Proteases Pave the Way.

Sprouting angiogenesis consists of the expansion and remodelling of existing vessels, where the vascular sprouts connect each other to form new vascular loops. Endothelial Progenitor Cells (EPCs) are a subtype of stem cells, with high proliferative potential, able to differentiate into mature Endothelial Cells (ECs) during the neovascularization process. In addition to this direct structural role EPCs improve neovascularization, also secreting numerous pro-angiogenic factors able to enhance the proliferation, survival and function of mature ECs, and other surrounding progenitor cells. While sprouting angiogenesis by mature ECs involves resident ECs, the vasculogenic contribution of EPCs is a high hurdle race. Bone marrowmobilized EPCs have to detach from the stem cell niche, intravasate into bone marrow vessels, reach the hypoxic area or tumour site, extravasate and incorporate into the new vessel lumen, thus complementing the resident mature ECs in sprouting angiogenesis. The goal of this review is to highlight the role of the main protease systems able to control each of these steps. The pivotal protease systems here described, involved in vascular patterning in sprouting angiogenesis, are the matrix-metalloproteinases (MMPs), the serineproteinases urokinase-type plasminogen activator (uPA) associated with its receptor (uPAR) and receptorassociated plasminogen/plasmin, the neutrophil elastase and the cathepsins. Since angiogenesis plays a critical role not only in physiological but also in pathological processes, such as in tumours, controlling the contribution of EPCs to the angiogenic process, through the regulation of the protease systems involved, could yield new opportunities for the therapeutic prospect of efficient control of pathological angiogenesis.

Endothelial cells and normal breast epithelial cells enhance invasion of breast carcinoma cells by CXCR-4-dependent up-regulation of urokinase-type plasminogen activator receptor (uPAR, CD87) expression.

Here we show the increase of invasion of three breast cancer cell lines (8701-BC, MDA-MB-231 and SKBR3) upon long-term co-incubation with culture medium of normal microvascular endothelial cells (MVEC) and normal breast epithelial cells (HB2). The enhancement of invasion relied on the interaction of microvascular endothelial cell and normal breast epithelial cell CXCL12 (SDF1) chemokine, whose expression by breast cancer cells was very low, with the cognate CXCR4 receptor of malignant cells, which resulted in over-expression of the urokinase-type plasminogen activator receptor (uPAR) on their surfaces. uPAR over-expression, showed by RT-PCR and Western blotting, was paralleled by increased urokinase-type plasminogen activator (uPA) partitioning on the cell surface with respect to the fluid phase, as demonstrated by zymography. Long-term interaction of SDF1 with CXCR4 stimulated sustained activation of JNK phosphorylation. Blocking antibodies to CXCR4 were able to block the endothelial/epithelial cell-dependent enhancement of invasion, as well as to inhibit SDF1-CXCR4-dependent JNK phosphorylation and uPAR over-expression of malignant cells. We suggest that acquisition of the angiogenic phenotype by breast cancer cells triggers an amplification loop, in which endothelial cells and normal breast epithelial cells of the tumour cooperate to provide facilitated routes to cell invasion and metastasis and to enhance the aggressive phenotype of cancer cells.

Piascledine modulates the production of VEGF and TIMP-1 and reduces the invasiveness of rheumatoid arthritis synoviocytes.

BACKGROUND: In rheumatoid arthritis (RA), hypertrophy of the synovial membrane generates a tumour-like pannus that invades the joint cavity and erodes cartilage and bone. Invasion of the extracellular matrix (ECM) is accompanied by angiogenesis, in which vascular endothelial growth factor (VEGF) and tissue inhibitors of metalloproteinases (TIMPs), produced by synoviocytes lining the pannus, have a primary role. Piascledine (PSD) is used in the treatment of osteoarthritis and has anti-inflammatory effects in vitro. OBJECTIVE: To study the effects of PSD on levels of VEGF and TIMP-1 and chemoinvasion in RA synoviocytes and healthy controls. METHODS: The effects of PSD 5, 10, and 20 microg/mL were evaluated, with/without interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNFalpha) 20 ng/mL, on synoviocytes. The levels of VEGF and TIMP-1 were assayed in the culture medium by enzyme-linked immunosorbent assay (ELISA). Chemoinvasion was measured by the Boyden chamber invasion assay. RESULTS: RA synoviocytes treated with PSD showed, compared to basal, lower levels of VEGF (41080+/-830 vs. 79210+/-920 pg/106 cells, p<0.001) and increased levels of TIMP-1 (23540+/-93.2 vs. 12860+/-42.9 ng/106 cells, p<0.001). PSD decreased dose-dependently IL-1beta and TNFalpha induced migration. CONCLUSIONS: In RA synoviocytes, and also to a lesser extent in control cells, PSD modulates VEGF and TIMP-1 and decreases chemoinvasion. PSD might have a role in the treatment of RA synovitis controlling invasiveness.

Systemic sclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavage of the endothelial cell urokinase receptor.

Failure of endothelial cells to develop new vessels in response to hypoxia is a distinctive feature of systemic sclerosis (SSc) in the avascular phase. We have previously shown that SSc endothelial cells over-express matrix metalloproteinase-12 (MMP-12), which blocks angiogenesis by cleavage of the endothelial urokinase-type plasminogen activator receptor (uPAR). In the present study, we have investigated whether over-expression of MMP-12 and of angiostatic factors, or hypo-expression of angiogenic factors by SSc fibroblasts, contributes to impaired angiogenesis in SSc. Dermal fibroblasts were isolated from healthy subjects (N-Fb) and patients with diffuse SSc (SSc-Fb). Angiogenesis of target normal human microvascular endothelial cells (H-MVECs) was assayed by Matrigel invasion, cell proliferation, and capillary morphogenesis. uPAR cleavage and MMP-12 activity were evaluated by western blotting. We show that the over-expression of MMP-12 by SSc-Fb determines uPAR cleavage in H-MVECs. Conditioned medium from SSc-Fb impaired H-MVEC proliferation, invasion, and capillary morphogenesis. Anti-MMP-12 antibodies restored such impairment. Altered expression of angiostatic/angiogenic factors, including transforming growth factor beta1, did not account for SSc-Fb-dependent impairment of angiogenesis. The over-expression of MMP-12 by both SSc-Fb and SSc endothelial cells indicates that MMP-12 over-production may have a critical pathogenic role in SSc-associated vascular alterations.

Raloxifene reduces urokinase-type plasminogen activator-dependent proliferation of synoviocytes from patients with rheumatoid arthritis.

Extracellular fibrinolysis, controlled by the membrane-bound fibrinolytic system, is involved in cartilage damage and rheumatoid arthritis (RA) synovitis. Estrogen status and metabolism seem to be impaired in RA, and synoviocytes show receptors for estrogens. Our aims in this study were to evaluate in healthy and RA synoviocytes the effects of Raloxifene (RAL), a selective estrogen receptor modulator (SERM), on: proliferation; the components of the fibrinolytic system; and chemoinvasion. The effects of RAL were studied in vitro on synoviocytes from four RA patients and four controls. Proliferation was evaluated as cell number increase, and synoviocytes were treated with 0.5 microM and 1 microM RAL with and without urokinase-plasminogen activator (u-PA) and anti-u-PA/anti-u-PA receptor (u-PAR) antibodies. Fibrinolytic system components (u-PA, u-PAR and plasminogen activator inhibitor (PAI)-1) were assayed by ELISA with cells treated with 0.5 microM and 1 microM RAL for 48 h. u-PA activity was evaluated by zymography and a direct fibrinolytic assay. U-PAR/cell and its saturation were studied by radioiodination of u-PA and a u-PA binding assay. Chemoinvasion was measured using the Boyden chamber invasion assay. u-PA induced proliferation of RA synoviocytes was blocked by RAL (p < 0.05) and antagonized by antibodies alone. The inhibitory effect of RAL was not additive with u-PA/u-PAR antagonism. RA synoviocytes treated with RAL showed, compared to basal, higher levels of PAI-1 (10.75 +/- 0.26 versus 5.5 +/- 0.1 microg/10(6) cells, respectively; p < 0.01), lower levels of u-PA (1.04 +/- 0.05 versus 3.1 +/- 0.4 ng/10(6) cells, respectively; p < 0.001), and lower levels of u-PAR (11.28 +/- 0.22 versus 23.6 +/- 0.1 ng/10(6) cells, respectively; p < 0.001). RAL also significantly inhibited u-PA-induced migration. Similar effects were also shown, at least partially, in controls. RAL exerts anti-proliferative and anti-invasive effects on synoviocytes, mainly modulating u-PAR and, to a lesser extent, u-PA and PAI-1 levels, and inhibiting cell migration and proliferation.

Deflazacort modulates the fibrinolytic pattern and reduces uPA-dependent chemioinvasion and proliferation in rheumatoid arthritis synoviocytes.

OBJECTIVE: Extracellular fibrinolysis, controlled by the cell-associated fibrinolytic system (urokinase plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor type-1, PAI-1), is involved in cartilage damage generation and in rheumatoid arthritis (RA) synovitis. Since steroids reduce the rate of radiological progression of RA, we planned to evaluate in healthy and RA synoviocytes the effects of the steroid deflazacort on uPA, uPAR and PAI-1 expression, and subsequent phenotypic modifications in terms of uPA/uPAR-dependent invasion and proliferation. METHODS: uPA, uPAR and PAI-1 levels were studied by ELISA, RT-PCR (uPAR) and zymography (uPA) in synoviocytes from four RA patients and four healthy controls. Chemoinvasion was assessed by the Boyden chamber invasion assay, using Matrigel as the invasion substrate. Proliferation was evaluated by cell counting. Both invasion and proliferation were measured upon treatment with deflazacort 5 muM with or without parallel stimulation with uPA 500 ng/ml or in the presence of monoclonal anti-uPA and anti-uPAR antibodies. RESULTS: Invasion and proliferation of RA synoviocytes require a proper functional balance of the fibrinolytic system. Both deflazacort and monoclonal antibodies against uPA and uPAR reduced expression and activity of the system, thus inhibiting invasion and proliferation. In RA synoviocytes, deflazacort induced higher PAI-1 and lower uPA and uPAR levels, as well as a decrease in uPA enzymatic activity. The levels of uPAR mRNA were concomitantly reduced, as was uPA-induced chemoinvasion. All these effects were also shown in controls, though to a lesser extent. CONCLUSIONS: Deflazacort might control RA synovial proliferation and invasion by differential modulation of single members of the fibrinolytic system.

Rheumatoid synovial fibroblasts constitutively express the fibrinolytic pattern of invasive tumor-like cells.

OBJECTIVE: In rheumatoid arthritis (RA) the synovial membrane proliferates and invades the underlying tissues. The cell-associated fibrinolytic system (urokinase-type plasminogen activator, uPA; uPA receptor, uPAR; plasminogen activator inhibitor-type 1, PAI-1) is pivotal in cell invasion and proliferation. For this reason, the expression and the role of such enzymatic system was investigated in synovial fibroblasts (SF) of normal and RA patients. METHODS: In SF obtained from RA patients and control subjects, uPA, uPAR and PAI-1 were measured by ELISA of cell lysates and culture medium and by RT-PCR of mRNAs. uPA was also studied by zymography. Proliferation was measured by cell counting and cell invasion with the Boyden chamber. RESULTS: RA-SF over-express uPAR and PAI-1 and are more prone than the normal counterpart to spontaneous and uPA-challenged invasion and proliferation, which are counteracted by antagonists of the fibrinolytic system. CONCLUSIONS: RA-SF display the fibrinolytic pattern and behaviour of invasive tumor-like cells. Antagonists of the fibrinolytic system are able to revert growth and invasion of both normal and RA-SF.

Effects of blocking urokinase receptor signaling by antisense oligonucleotides in a mouse model of experimental prostate cancer bone metastases.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Cell invasion is affected by differential expression of the urokinase plasminogen activator/urokinase plasminogen activator receptor system in muscle satellite cells from normal and dystrophic patients.

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti-u-PA and anti-u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA-induced invasion, as well as in u-PA-induced proliferation.

Regulation of urokinase/urokinase receptor interaction by heparin-like glycosaminoglycans.

We show here that the interaction between the urokinase-type plasminogen activator and its receptor, which plays a critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin (glucosamine-NSO(3)(6-OSO(3))-iduronic acid(2-OSO(3))) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit affinity for urokinase plasminogen activator and confer specificity on urokinase/urokinase receptor interaction. Cell surface clearance of heparan sulfate reduces the affinity of such interaction with a parallel decrease of specific urokinase binding in the presence of an unaltered expression of receptor. Transfection of human urokinase plasminogen activator receptor in normal Chinese hamster ovary fibroblasts and in Chinese hamster ovary cells defective for the synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar K(d) values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on urokinase/receptor binding.

Transforming growth factor beta-1 stimulates invasivity of hepatic stellate cells by engagement of the cell-associated fibrinolytic system.

The activation of hepatic stellate cells (HSC) during liver fibrogenesis has been shown to be mediated by paracrine and autocrine loops involving transforming growth factor-beta1 (TGF-beta1) as the main fibrogenic mediator secreted by activated macrophages, endothelial cells and liberated by disintegrated platelets. The cell-associated plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We have studied whether TGF-beta1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-beta1 on HSC. Urokinase plasminogen activator receptors (u-PAR), u-PA and plasminogen activator inhibitor type 1 (PAI-1) were determined by immunoassay and RNase protection assay. Cell migration, evaluated either as chemotaxis or as chemoinvasion, was studied in Boyden chambers after addition of TGF-beta1, and inhibition with anti-u-PA and anti-u-PAR antagonists [antibodies against u-PA and u-PAR and antisense oligonucleotides (aODN) against u-PAR mRNA]. We have shown that TGF-beta1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-beta1 up-regulates the synthesis and expression of PAI-1, as well as u-PAR expression and exposure at the cell membrane, while it does not affect u-PA levels. TGF-beta1-dependent chemoinvasion of reconstituted basement membrane exploits the cell-associated plasminogen activation system, since it is blocked by monoclonal antibodies against u-PA and against various u-PAR domains, as well as by anti-u-PAR aODN. We have also observed a cumulative effect of TGF-beta1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-beta1 the chemoinvasive activity of PDGF and bFGF is dramatically increased. Also this cooperation requires u-PAR and is inhibited by monoclonal antibodies against u-PAR domains I, II and III.

The urokinase-type plasminogen activator system and inflammatory joint diseases.

Much evidence indicates that the urokinase plasminogen activator (u-PA), the urokinase receptor (u-PAR) and the serpin inhibitors are critical in cell invasion processes. The balance between u-PAR-bound u-PA and inhibitors modulate a pericellular proteolytic activity able to give "stop and go" signals to invading cells. The plasminogen activation system operates both directly and in concert with the matrix-metalloproteinase system. Direct interactions of u-PAR with vitronectin and integrins further regulate cell invasion. Another line of evidence suggests that u-PA-u-PAR interaction elicits chemotaxis, chemoinvasion and cell multiplication, events that do not require plasmin generation and therefore are referred to as "plasminogen-independent". Following the description of the main molecular and functional characteristics of the cell-surface-associated plasminogen activation system, we discuss here the observations indicating a role of this system in many aspects of the rheumatic diseases, ranging from the infiltration of inflammatory cells into the affected joint, infiltration of synovial cells into the underlying cartilage, and remodeling of the cartilage itself. Evidence of the intraarticular cytokine- and growth factor-dependent regulation of the components of the plasminogen activation system are presented in terms of the paracrine and autocrine regulation of receptor-associated fibrinolysis. The roles of plasminogen-dependent and plasminogen-independent u-PAR-associated events in various phases of joint inflammation are also discussed. A knowledge of these processes is required for the therapeutic utilization of antagonists of the u-PA/u-PAR system able to control the activity of proliferating and invading cells in inflammatory joint diseases.

Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor.

During liver fibrogenesis, hepatic stellate cells (HSC) proliferate and migrate under the influence of growth factors, including platelet-derived growth factor (PDGF) and basic-fibroblast growth factor (b-FGF). The plasminogen activation system regulates extracellular matrix (ECM) catabolism and cell movement. We evaluated the expression and biological functions of the plasminogen activation system in human HSC and its interaction with PDGF and b-FGF. Urokinase-plasminogen activator receptors (u-PAR) were measured by radioligand binding, cell cross-linking, immunoassay, and RNAse protection assay. u-PA and plasminogen activator inhibitors (PAIs) expression and activities were analyzed by zymography, immunoassay, and RNase protection assay. Cell migration and proliferation, studied in Boyden chambers and by microscopic counting, were evaluated after the addition of PDGF, b-FGF, and blockade with anti-u-PA, anti-u-PAR antibodies, and antisense oligodeoxynucleotides (aODN) against u-PAR mRNA. We have shown that HSC produce u-PAR, u-PA, and PAI-1. PDGF and b-FGF up-regulate u-PA and u-PAR, but not PAI-1, and exogenous addition of u-PA stimulates HSC proliferation, chemotaxis, and chemoinvasion. Inhibition of u-PA/u-PAR with antibodies against u-PA or u-PAR and with u-PAR aODN inhibit the proliferative, chemotactic, and chemoinvasive activity of PDGF and b-FGF. These findings indicate that u-PA and u-PAR are required for the mitogenic and chemoinvasive activity of PDGF and b-FGF on HSC.

Urokinase-dependent angiogenesis in vitro and diacylglycerol production are blocked by antisense oligonucleotides against the urokinase receptor.

The plasminogen activator system is known to play a crucial role in the angiogenesis process by modulating the adhesive properties of endothelial cells to the extracellular matrix and cell-cell interaction. In the present study, we demonstrated that the urokinase-type plasminogen activator (u-PA) induced neovascular growth in the avascular rabbit cornea and dose-dependently promoted growth, chemotaxis, and matrix invasion of cultured endothelial cells. Interaction between u-PA and its receptor appears to be mandatory for the angiogenic effect of u-PA because monoclonal antibodies anti-u-PA and anti-u-PA receptor (u-PAR) blocked the proangiogenic effects of u-PA at the endothelial cell level. We then assessed the signaling pathway activated in endothelial cells by u-PA. u-PAR activation by u-PA produced de novo synthesis of diacylglycerol (DAG) from glucose by a cytochalasin B-inhibitable mechanism, indicating the involvement of a specific glucose transporter (GLUT). Endothelial cells expressed GLUT2, whose activation was tyrosine kinase-dependent and protein kinase C (PKC)-independent. The increase of glucose uptake led to DAG production, which resulted in PKC activation/translocation. Impairment of u-PAR availability by monoclonal antibodies and by antisense oligonucleotides (aODN) against u-PAR mRNA inhibited glucose uptake, DAG neosynthesis, and PKC activation, resulting in the blockade of endothelial cell proliferation, chemotaxis, and chemoinvasion. These data suggest that u-PAR activation consequent to the binding of u-PA can be regarded as an "angiogenic switch" and disclose the possibility that an anti-u-PAR aODN strategy may efficiently target endothelial cell function to control angiogenesis in vivo.

Antisense targeting of the urokinase receptor blocks urokinase-dependent proliferation, chemoinvasion, and chemotaxis of human synovial cells and chondrocytes in vitro.

Proliferation and invasion of synovial pannus in rheumatoid arthritis and cartilage remodeling in osteoarthritis are key events in development of disability of arthritic joints. The mechanisms that trigger these events are still poorly understood. The production of urokinase-type plasminogen activator (u-PA) by synovial cells and chondrocytes and the subsequent interaction of u-PA with its membrane receptor (u-PAR) is under the control of a variety of growth factors and cytokines released within the inflamed joints. Here we show that u-PA, on interaction with the specific receptor, regulates movement and invasion as well as proliferation of human synovial cells and chondrocytes. Targeting the urokinase receptor with an antisense oligonucleotide blocks the u-PA-dependent synoviocyte and chondrocyte proliferation and chemoinvasion, suggesting a possible use for this new class of drugs in the progression of the disease in rheumatoid arthritis and osteoarthritis.