Maura Camozzi

Role of the soluble pattern recognition receptor PTX3 in vascular biology

•  Introduction •  PTX3 gene and expression •  PTX3 protein structure •  PTX3 ligands •  PTX3 in vascular pathology ‐  PTX3 as a marker of vascular damage ‐  Atherosclerosis ‐  Angiogenesis ‐  Restenosis •  Concluding remarks

Heparin derivatives as angiogenesis inhibitors

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot growth as a mass above few mm(3) unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.

Identification of an Antiangiogenic FGF2-binding Site in the N Terminus of the Soluble Pattern Recognition Receptor PTX3

Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 comprises a pentraxin-like C-terminal domain involved in complement activation via C1q interaction and an N-terminal extension with unknown functions. PTX3 binds fibroblast growth factor-2 (FGF2), inhibiting its pro-angiogenic and pro-restenotic activity. Here, retroviral transduced endothelial cells (ECs) overexpressing the N-terminal fragment PTX3-(1–178) showed reduced mitogenic activity in response to FGF2. Accordingly, purified recombinant PTX3-(1–178) binds FGF2, prevents PTX3/FGF2 interaction, and inhibits FGF2 mitogenic activity in ECs. Also, the monoclonal antibody mAb-MNB4, which recognizes the PTX3-(87–99) epitope, prevents FGF2/PTX3 interaction and abolishes the FGF2 antagonist activity of PTX3. Consistently, the synthetic peptides PTX3-(82–110) and PTX3-(97–110) bind FGF2 and inhibit the interaction of FGF2 with PTX3 immobilized to a BIAcore sensor chip, FGF2-dependent EC proliferation, and angiogenesis in vivo. Thus, the data identify a FGF2-binding domain in the N-terminal extension of PTX3 spanning the PTX3-(97–110) region, pointing to a novel function for the N-terminal extension of PTX3 and underlining the complexity of the PTX3 molecule for modular humoral pattern recognition.

Pentraxin 3 Inhibits Fibroblast Growth Factor 2–Dependent Activation of Smooth Muscle Cells In Vitro and Neointima Formation In Vivo

Objective—The fibroblast growth factor (FGF)/FGF receptor system plays an important role in smooth muscle cell (SMC) activation. Long-pentraxin 3 (PTX3) is a soluble pattern recognition receptor with non-redundant functions in inflammation and innate immunity. PTX3 is produced by different cell types of the vessel wall, including SMCs. PTX3 binds FGF2 and inhibits its angiogenic activity on endothelial cells. We investigated the capacity of PTX3 to affect FGF2-dependent SMC activation in vitro and in vivo. Methods and Results—When added to human coronary artery SMCs, human PTX3 inhibits cell proliferation driven by endogenous FGF2 and the mitogenic and chemotactic activity exerted by exogenous recombinant FGF2. Accordingly, PTX3 prevents 125I-FGF2 interaction with FGF receptors on the same cells. Also, PTX3 overexpression after recombinant adeno-associated virus-PTX3 gene transfer inhibits human coronary artery SMC proliferation and survival promoted by FGF2 in vitro. Consistently, a single local endovascular injection of recombinant adeno-associated virus-PTX3 gene inhibits intimal thickening after balloon injury in rat carotid arteries. Conclusions—PTX3 is a potent inhibitor of the autocrine and paracrine stimulation exerted by FGF2 on SMCs. Local PTX3 upregulation may modulate SMC activation after arterial injury.

Anti-FGF2 approaches as a strategy to compensate resistance to anti-VEGF therapy: long-pentraxin 3 as a novel antiangiogenic FGF2-antagonist

Angiogenesis, the formation of new blood vessels from the endothelium of the existing vasculature, plays a pivotal role in tumor growth, progression and metastasis. Over the last 30 years, numerous pro- and antiangiogenic molecules, their ligands, and intracellular signaling pathways have been identified, and significant efforts have been undertaken to develop antiangiogenic strategies for cancer therapy. Agents that selectively target vascular endothelial growth factor (VEGF) and its receptors have shown promising activity in clinical trials and have been approved for use in selected cancer indications. However, patients may ultimately develop resistance to these drugs. One proposed mechanism of tumor escape from anti-VEGF therapy is the up-regulation of fibroblast growth factor-2 (FGF2). FGF2 is a pleiotropic, angiogenesis inducer belonging to the family of the heparin-binding FGF growth factors. FGF2 is expressed by numerous tumor types and exerts its proangiogenic activity by interacting with tyrosine kinase receptors, heparan-sulfate proteoglycans, and integrins expressed on the endothelial cell surface. Experimental evidence suggests that targeting FGF2, in addition to VEGF, might provide synergistic effects in the treatment of angiogenesis-related diseases, including cancer. Several FGF2 inhibitors, with different chemical structure and mechanism of action, have been identified. Recent observations have shown the ability of the soluble pattern recognition receptor long-pentraxin-3 (PTX3) to bind FGF2, thus acting as a FGF2 antagonist. PTX3 binds FGF2 with high affinity and specificity. This interaction prevents the binding of FGF2 to its cognate tyrosine kinase receptors, leading to inhibition of the angiogenic activity of the growth factor. Further, preliminary observations support the hypothesis that PTX3 may inhibit FGF2-mediated tumor angiogenesis and growth. The identification of the FGF2-binding domain in the unique N-terminal extension of PTX3 has allowed the design of PTX3-derived synthetic peptides endowed with significant antiangiogenic activity in vitro and in vivo. These findings may provide the basis for the development of novel antiangiogenic FGF2 antagonists, with potential implications for cancer therapy.

A pro-inflammatory signature mediates FGF2-induced angiogenesis

Fibroblast growth factor‐2 (FGF2) is a potent angiogenic growth factor. Here, gene expression profiling of FGF2‐stimulated microvascular endothelial cells revealed, together with a prominent pro‐angiogenic profile, a pro‐inflammatory signature characterized by the up‐regulation of pro‐inflammatory cytokine/chemokines and their receptors, endothelial cell adhesion molecules and members of the eicosanoid pathway. Real‐time quantitative PCR demonstrated early induction of most of the FGF2‐induced, inflammation‐related genes. Accordingly, chick embryo chorioallantoic membrane (CAM) and murine Matrigel plug angiogenesis assays demonstrated a significant monocyte/macrophage infiltrate in the areas of FGF2‐driven neovascularization. Similar results were obtained when the conditioned medium (CM) of FGF2‐stimulated endothelial cells was delivered onto the CAM, suggesting that FGF2‐upregulated chemoattractants mediate the inflammatory response. Importantly, FGF2‐triggered new blood vessel formation was significantly reduced in phosphatidylinositol 3‐kinase‐γ null mice exhibiting defective leucocyte migration or in clodronate liposome‐treated, macrophage‐depleted mice. Furthermore, the viral pan‐chemokine antagonist M3 inhibited the angiogenic and inflammatory responses induced by the CM of FGF2‐stimulated endothelial cells and impaired FGF2‐driven neovascularization in the CAM assay. These findings point to inflammatory chemokines as early mediators of FGF2‐driven angiogenesis and indicate a non‐redundant role for inflammatory cells in the neovascularization process elicited by the growth factor.

Antiangiogenic Activity of Semisynthetic Biotechnological Heparins. Low-Molecular-Weight-Sulfated Escherichia coli K5 Polysaccharide Derivatives as Fibroblast Growth Factor Antagonists

Objective— Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. Methods and Results— Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and αvβ3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/αvβ3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. Conclusions— LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/αvβ3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.

Heparin derivatives and semisynthetic biotechnological heparins as angiogenesis inhibitors

Angiogenesis is the process of generating new capillary blood vessels. Uncontrolled endothelial cell proliferation is observed in tumor neovascularization and in angioproliferative diseases. Tumors cannot grow as a mass above few mm unless a new blood supply is induced. It derives that the control of the neovascularization process may affect tumor growth and may represent a novel approach to tumor therapy. Angiogenesis is controlled by a balance between proangiogenic and antiangiogenic factors. The angiogenic switch represents the net result of the activity of angiogenic stimulators and inhibitors, suggesting that counteracting even a single major angiogenic factor could shift the balance towards inhibition. Heparan sulfate proteoglycans are involved in the modulation of the neovascularization that takes place in different physiological and pathological conditions. This modulation occurs through the interaction with angiogenic growth factors or with negative regulators of angiogenesis. Thus, the study of the biochemical bases of this interaction may help to design glycosaminoglycan analogs endowed with angiostatic properties. The purpose of this review is to provide an overview of the structure/function of heparan sulfate proteoglycans in endothelial cells and to summarize the angiostatic properties of synthetic heparin-like compounds, chemically modified heparins, and biotechnological heparins.