Simona Serratì

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.

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

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.

Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor β–dependent mesenchymal-to-mesenchymal transition

OBJECTIVE Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation. METHODS Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor β (TGFβ) antagonist peptide p17. RESULTS SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFβ and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFβ antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition. CONCLUSION SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.

Reduction of in vitro invasion and in vivo cartilage degradation in a SCID mouse model by loss of function of the fibrinolytic system of rheumatoid arthritis synovial fibroblasts.

OBJECTIVE Urokinase plasminogen activator (uPA), uPA receptor (uPAR), and PA inhibitor 1 (PAI-1) have pivotal roles in the proliferation and invasion of several cell types, including synovial fibroblasts (SFs). The aim of this study was to investigate the possibility of controlling the invasion of rheumatoid arthritis (RA) SFs in vitro and in vivo by inhibiting uPA and uPAR. METHODS Normal SFs, SFs from patients with RA, and SFs from patients with psoriatic arthritis (PsA) were used. The levels of uPA, uPAR, and PAI-1 were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction analysis of messenger RNA. The activity of uPA was studied by zymography. Proliferation was measured by cell counting, and cell invasion was measured with a Boyden chamber assembled with Matrigel-coated porous filters. Human cartilage and RA SF implantation in the SCID mouse model of RA were used to study cartilage invasion in vivo. RESULTS RA SFs and PsA SFs overexpressed uPAR and as a result were more active than their normal counterparts in terms of both Matrigel invasion and proliferation. This effect was counteracted by a specific inhibitor of uPA enzymatic activity (WX-340) and by uPAR antisense treatment. The use of both WX-340 and uPAR antisense treatment in vitro showed cooperative effects in RA SFs that were more intense than the effects of either treatment alone. Significant inhibition of cartilage invasion was obtained in vivo with uPAR antisense treatment, while uPA inhibition was inefficient, either alone or in combination with antisense treatment. CONCLUSION The decrease in uPAR expression in RA SFs reduced invasion of human cartilage in vitro and in the SCID mouse model.

Challenges and Opportunities of MicroRNAs in Lymphomas

MicroRNAs (miRNAs) are small non-coding RNAs that control the expression of many target messenger RNAs (mRNAs) involved in normal cell functions (differentiation, proliferation and apoptosis). Consequently their aberrant expression and/or functions are related to pathogenesis of many human diseases including cancers. Haematopoiesis is a highly regulated process controlled by a complex network of molecular mechanisms that simultaneously regulate commitment, differentiation, proliferation, and apoptosis of hematopoietic stem cells (HSC). Alterations on this network could affect the normal haematopoiesis, leading to the development of haematological malignancies such as lymphomas. The incidence of lymphomas is rising and a significant proportion of patients are refractory to standard therapies. Accurate diagnosis, prognosis and therapy still require additional markers to be used for diagnostic and prognostic purpose and evaluation of clinical outcome. The dysregulated expression or function of miRNAs in various types of lymphomas has been associated with lymphoma pathogenesis. Indeed, many recent findings suggest that almost all lymphomas seem to have a distinct and specific miRNA profile and some miRNAs are related to therapy resistance or have a distinct kinetics during therapy. MiRNAs are easily detectable in fresh or paraffin-embedded diagnostic tissue and serum where they are highly stable and quantifiable within the diagnostic laboratory at each consultation. Accordingly they could be specific biomarkers for lymphoma diagnosis, as well as useful for evaluating prognosis or disease response to the therapy, especially for evaluation of early relapse detection and for greatly assisting clinical decisions making. Here we summarize the current knowledge on the role of miRNAs in normal and aberrant lymphopoiesis in order to highlight their clinical value as specific diagnosis and prognosis markers of lymphoid malignancies or for prediction of therapy response. Finally, we discuss their controversial therapeutic role and future applications in therapy by modulating miRNA.