Francesca Margheri

Endothelial progenitor cell–dependent angiogenesis requires localization of the full-length form of uPAR in caveolae

Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

Antisense oligodeoxynucleotides for urokinase-plasminogen activator receptor have anti-invasive and anti-proliferative effects in vitro and inhibit spontaneous metastases of human melanoma in mice

We have targeted the urokinase‐type plasminogen activator receptor (uPAR) with phosphorothioate antisense oligonucleotides (aODN) in vitro to evaluate the anti‐invasive and anti‐proliferative effects of uPAR down‐regulation, as well as in vivo to evaluate anti‐tumor and anti‐metastatic activity. aODN‐dependent uPAR downregulation in vitro was induced in cells of human melanoma, mammary carcinoma, ovarian carcinoma and SV‐40‐transformed embryonic lung fibroblasts. uPAR was determined by an antibody‐based assay and by semiquantitative polymerase chain reaction. Cell invasion was evaluated by Matrigel invasion assay and cell proliferation by direct cell counting. aODN reduced uPAR, invasion and proliferation in all the treated cell lines. Following aODN treatment, human melanoma cells exhibited a strong decrease of uPAR‐dependent ERK1/2 activation and were used in vivo to control metastasis in CD‐1 male nude (nu/nu) mice by uPAR aODN injection. 60 mice were injected in the hind leg muscles with a suspension of 106 melanoma cells. After 4 days, when a tumor mass of about 350 mg was evident in all the mice injected, 20 mice were treated i.v. with aODN and 20 with dODN at 0.5 mg/day for 5 consecutive days. Twenty control mice were not treated. A second and third cycle of treatment was administered at 2‐day intervals. Treatment with aODN resulted into a 78% reduction of lung metastases and 45% reduction of the primary tumor mass with no loss of body weight. Our results suggest to evaluate the utility of uPAR aODN in controlling the metastatic spreading of human melanoma.

The Urokinase Receptor System, A Key Regulator at the Intersection between Inflammation, Immunity, and Coagulation

The urokinase plasminogen activator (uPA) and its receptor (uPAR) provide a cell surface integrated multimolecular complex that exerts pleiotropic functions influencing the development of inflammatory, immune, coagulation and fibrinolytic responses. Here we review the evidences indicating a role of the uPA/uPAR system in the regulation of the innate immune system in the inflammation process, of the adaptive immune response, as well as the role of fibrin and fibrin degradation products at the cross-road between coagulation and inflammation. Comparative studies have clearly highlighted the notion that coagulation and immunity are co-regulated and intertwined. The implication is that the vertebrate blood clotting system is evolutionarily by product of the innate immune system, where the blood clotting proteases have diverged from those comprising the complement system. Differences have emerged gradually, as shown by the acquisition of unique protein structures, such as kringle domains and gla (glutammic acid) domains, in order to comply with the increasingly complex vertebrate systems and to defend higher organisms against a range of infections and injuries. Plasminogen activation also controls the formation of complement anaphylotoxins (responsibe for vasodilatation, increase of venular permeability and leukocyte chemotaxis) and of bradykinin (which accounts for vasodilatation, increase of venular permeability and pain) by regulating the plasma contact system. The urokinase plasminogen activator and its cellular receptor, expressed on the surface of human leukocytes, provide a functional unit that, by regulating interaction of leukocytes with extracellular matrix, as well as its degradation, is critical for the migration of leukocytes and for their movement in the damaged tissues.

Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain-loss of function of pentraxin 3 and matrix metalloproteinase 12.

OBJECTIVE Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. METHODS Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. RESULTS Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. CONCLUSION Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge.

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

Mesenchymal stem cells are recruited and activated into carcinoma-associated fibroblasts by prostate cancer microenvironment-derived TGF-β1.

Tumor stromal cells can supply appropriate signals that may develop aggressive phenotypes of carcinoma cells and establish a complex scenario which culminates in metastasis. Recent works proposed that bone marrow‐derived mesenchymal stem cells (MSC) are recruited to primary tumors. However, the exact functions of these cells in the tumor microenvironment are not well characterized, as it is reported that MSC can either promote or inhibit tumor progression. In the present study, we aim at investigating the signaling molecules which regulate the interplay between MSC, prostate carcinoma (PCa) cells and two important cellular types constituting the tumor‐associated stroma, macrophages and fibroblasts, during their progression toward malignancy. We identified TGF‐β1 as a crucial molecule able to attract MSC recruitment both to PCa cells as well as to tumor stroma components. Moreover, PCa‐ and tumor stroma‐secreted TGF‐β1 is important to induce MSC transdifferentiation into carcinoma‐associated fibroblast (CAF)‐like cells. Consequently, the CAF‐like phenotype acquired by MSC is central to promote tumor progression related effects. Thus, tumor‐educated MSC enhance PCa invasiveness compared to nonactivated MSC. Additionally, differing from normal MSC, CAF‐like MSC perform vascular mimicry and recruit monocytes, which can be further polarized to M2 macrophages within the PCa environment. Our findings indicate a prominent role for TGF‐β1 in MSC mobilization and activation strengthened by the fact that the blockade of TGF‐β1 signaling impairs MSC promotion of PCa progression. Stem Cells 2016;34:2536–2547

Overexpression of the transmembrane carbonic anhydrase isoforms IX and XII in the inflamed synovium

Abstract Juvenile idiopathic arthritis (JIA) is the most common form of chronic rheumatic disease affecting children worldwide, with some features similar to adult rheumatoid arthritis (RA). In the present study, we aim at investigating novel markers that will allow in the future for tailored, more personalized treatment strategies. Hence, taking notice of several reports proving the role of local acidosis as a causal link between inflammatory diseases and related pain, and the involvement of several carbonic anhydrases (CA, EC 4.2.1.1) isoforms in articular diseases, we evaluated in JIA patients the expression of these metalloenzymes. We identified that JIA patients show high levels of active CA IX and XII isoforms. Our results represent the first evidence of the identification of these enzymes as potential therapeutic targets and development of novel innovative therapies for arthritis, also considering that the two isoforms are validated antitumor targets.

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

TGFβ1 antagonistic peptides inhibit TGFβ1-dependent angiogenesis

The role of transforming growth factor beta (TGFbeta) in tumor promotion and in angiogenesis is context-dependent. While TGFbeta prevents tumor growth and angiogenesis in early phases of tumor development, evidence is accumulating about its pro-angiogenic and tumor promotion activities in late-stages of tumor progression. Here we have studied, in an experimental context previously reported to disclose the pro-angiogenic effects of TGFbeta, the blocking activity of TGFbeta antagonist peptides. In agreement with previous results, we have observed that TGFbeta exerts a powerful pro-angiogenic activity on human normal dermal microvascular endothelial cells (MVEC), by promoting invasion and capillary morphogenesis in Matrigel. No apoptotic activity of TGFbeta was observed. By RT-PCR we have shown that TGFbeta up-regulates expression not only of plasminogen activator inhibitor type-1 (PAI-1), but also of the urokinase-type plasminogen activator receptor (uPAR), whose inhibition by specific antibodies blunted the TGFbeta angiogenic response in vitro. The SMAD2/3 and FAK signaling pathways were activated by TGFbeta in MVEC, as an early and late response, respectively. The use of two different TGFbeta1 antagonist peptides, derived from TGFbeta type III receptor sequence and 15-mer phage display technology, inhibited the signaling and pro-angiogenic response in vitro, as well as uPAR and PAI-1 up-regulation of MVEC following TGFbeta challenge. The anti-angiogenic properties of both inhibitors were evident also in the in vivo TGFbeta Matrigel Sponge Assay. These results may be relevant to develop a potentially fruitful strategy for the therapy of late-stage-associated tumor angiogenesis.

TGFbeta1 antagonistic peptides inhibit TGFbeta1-dependent angiogenesis.

The role of transforming growth factor beta (TGFbeta) in tumor promotion and in angiogenesis is context-dependent. While TGFbeta prevents tumor growth and angiogenesis in early phases of tumor development, evidence is accumulating about its pro-angiogenic and tumor promotion activities in late-stages of tumor progression. Here we have studied, in an experimental context previously reported to disclose the pro-angiogenic effects of TGFbeta, the blocking activity of TGFbeta antagonist peptides. In agreement with previous results, we have observed that TGFbeta exerts a powerful pro-angiogenic activity on human normal dermal microvascular endothelial cells (MVEC), by promoting invasion and capillary morphogenesis in Matrigel. No apoptotic activity of TGFbeta was observed. By RT-PCR we have shown that TGFbeta up-regulates expression not only of plasminogen activator inhibitor type-1 (PAI-1), but also of the urokinase-type plasminogen activator receptor (uPAR), whose inhibition by specific antibodies blunted the TGFbeta angiogenic response in vitro. The SMAD2/3 and FAK signaling pathways were activated by TGFbeta in MVEC, as an early and late response, respectively. The use of two different TGFbeta1 antagonist peptides, derived from TGFbeta type III receptor sequence and 15-mer phage display technology, inhibited the signaling and pro-angiogenic response in vitro, as well as uPAR and PAI-1 up-regulation of MVEC following TGFbeta challenge. The anti-angiogenic properties of both inhibitors were evident also in the in vivo TGFbeta Matrigel Sponge Assay. These results may be relevant to develop a potentially fruitful strategy for the therapy of late-stage-associated tumor angiogenesis.