M. Del Rosso

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.

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. Copyright

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 β1, 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. Copyright

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-β1 (TGF-β1) 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-β1 could modulate the plasminogen activation system in human HSC and the role of such protease system in the activity of TGF-β1 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-β1, 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-β1 is not mitogenic for HSC, while it is a powerful motogen either in chemotaxis or chemoinvasion assays. TGF-β1 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-β1-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-β1, b-FGF and PDGF in the invasion assay of HSC: in the presence of low amounts of TGF-β1 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.

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.

The β-Subunit of Cholera Toxin has a High Affinity for Ganglioside GM1 Embedded into Solid Supported Lipid Membranes with a Lipid Raft-Like Composition

In this communication, we report on the fabrication of GM1-rich solid-supported bilayer lipid membranes (ssBLM) made of sphingomyelin and cholesterol, the main components of lipid rafts, which are the physiological hosting microenvironment of GM1 on the cell membrane. The functionality of the ganglioside has been checked by measuring the apparent dissociation constant KD of the complex formed by the β-subunit of the cholera toxin and GM1. The value found deviates less than one order of magnitude from that measured for in vivo cells, indicating the potential of these ssBLM as optimized in vitro biomimetic platforms.

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 µg/mL were evaluated, with/without interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNFα) 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‐1β and TNFα 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.

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 μM and 1 μM 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 μM and 1 μM 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 μg/106 cells, respectively; p < 0.01), lower levels of u-PA (1.04 ± 0.05 versus 3.1 ± 0.4 ng/106 cells, respectively; p < 0.001), and lower levels of u-PAR (11.28 ± 0.22 versus 23.6 ± 0.1 ng/106 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.

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

BackgroundMultiple 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.MethodsCAF 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.ResultsWe 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.ConclusionsOur 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.