Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached t... more Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing ou...
Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transforme... more Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug d...
Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable a... more Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. ...
Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to stand... more Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to standard therapeutic approaches that combine surgery, radiotherapy, and chemotherapy. As opposed to healthy tissues, EphA2 has been found highly expressed in specimens of glioblastoma, and increased expression of EphA2 has been shown to correlate with poor survival rates. Accordingly, agents blocking Eph receptor activity could represent a new therapeutic approach. Herein, we demonstrate that UniPR1331, a pan Eph receptor antagonist, possesses significant anti-angiogenic and anti-vasculogenic properties which lead to a significant anti-tumor activity in xenograft and orthotopic models of GBM. UniPR1331 halved the final volume of tumors when tested in xenografts (p<0.01) and enhanced the disease-free survival of treated animals in the orthotopic models of GBM both by using U87MG cells (40 vs 24 days of control, p<0.05) or TPC8 cells (52 vs 16 days, p<0.01). Further, the association of...
Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and ora... more Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and orally bioavailable compounds is impairing the development of the field. Since 2009 our research group has been devoted to the discovery and development of small molecules targeting Eph/ephrin system and our research culminated with the synthesis of UniPR129, a potent but problematic Eph/ephrin antagonist. Herein, we describe the in vitro pharmacological properties of two derivatives (UniPR139 and UniPR502) stemmed from structure of UniPR129. These two compounds acted as competitive and reversible antagonists of all Eph receptors reducing both ephrin-A1 and -B1 binding to EphAs and EphBs receptors in the low micromolar range. The compounds acted as antagonists inhibiting ephrin-A1-dependent EphA2 activation and UniPR139 exerted an anti-angiogenic effect, inhibiting HUVEC tube formation in vitro and VEGF-induced vessel formation in the chick chorioallantoic membrane assay. Finally, the oral ...
Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has ... more Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na(+),K(+)-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.
Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and pare... more Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and parenchymal tumor cells. Even though FGFs have been firstly characterized as angiogenic factors, they exert autocrine and paracrine functions not only on endothelial cells but also on tumor cells and other stromal components. Thus, the FGF/FGF receptor (FGFR) pathway may represent a key player in tumor growth by regulating the complex cross-talk between stromal and tumor compartments. The ligand dependent or independent activation of the FGF/FGFR system by gene upregulation, oncogenic mutation or amplification occurs in a variety of human tumors and is implicated in various key steps of tumor growth and progression. In addition, FGF/FGFR activation has been described as a mechanism of tumor escape in response to antiangiogenic/anti-VEGF therapies. Experimental and clinical evidences provide a compelling biologic rationale for the development of anti-FGF/FGFR targeting agents in cancer therapy. However, the development of drugs specifically targeting the FGF/FGFR pathway proved to be difficult, also due to the high redundancy and pleiotropic effects of FGF and FGFR family members. On the other hand, the possibility to develop "two-compartment" targeting agents endowed with both antiangiogenic and antitumor activities remains promising. Here we will review the preclinical and clinical approaches and potential therapeutics currently available to block the FGF/FGFR system in human cancer.
Angiogenesis plays a key role in various physiological and pathological processes, including infl... more Angiogenesis plays a key role in various physiological and pathological processes, including inflammation and tumor growth. Numerous angiogenic growth factors (AGFs) have been identified. Usually, the angiogenic process is assumed to represent the outcome of a straightforward interaction of AGFs with specific signalling receptors of the endothelial cell (EC) surface. Actually, the mechanisms by which AGFs induce neovascularization are much more complex. Indeed, angiogenesis is the result of the simultaneous actions of various AGFs and angiogenesis modulators; multiple EC surface receptors with different structure and biological properties are engaged by AGFs to exert a full angiogenic response; AGFs bind a variety of free and immobilized proteins, polysaccharides, and complex lipids of the extracellular milieu that affect AGF integrity, stability, and bioavailability; some of the AGF-binding molecules interact also with AGF receptors. In this review the authors summarize literature data and discuss the current knowledge about the extracellular molecules able to interact with AGFs, thus representing possible key regulators of the angiogenesis process and targets/templates for the development of novel antiangiogenic drugs. This work represents an attempt to highlight common theme in the AGF interactome that occurs at the extracellular level during neovascularization.
Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied... more Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected both in normal and neoplastic tissues. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, and its capacity to induce plasminogen activator production in cultured endothelial GM 7373 cells. The levels of immunoreactive and biologically active bFGF appeared to be significantly higher in the extracts of adrenal pheochromocytoma and chemodectoma than in the extracts of normal adrenal medulla. bFGF immunostaining was detectable in the nuclei of chief (Type-I) cells and of endothelial cells both in normal adrenal medulla and in pheochromocytoma. Cytoplasmic bFGF positivity of endothelial cells was also observed in pheochromocytoma but not in normal tissue. The data are in keeping with the hypothesis that bFGF may exert autocrine and paracrine functions in the growth and neovascularization of human pheochromocytoma.
International Journal of Clinical Laboratory Research, Feb 1, 1996
Basic fibroblast growth factor is an angiogenic molecule involved in several physiological and pa... more Basic fibroblast growth factor is an angiogenic molecule involved in several physiological and pathological processes, including wound repair, embryonic development, and tumor growth. In vitro, basic fibroblast growth factor induces an "angiogenic phenotype" in endothelial cells, which includes chemotaxis, mitogenesis, protease production, fl-integrin expression, and tube formation in three-dimensional gels. It acts by binding to specific tryosine kinase receptors and to cell-associated heparan sulfate proteoglycans. The physiological significance of the interaction with cell-associated and soluble heparan sulfate proteoglycans is manyfold. Heparan sulfate proteoglycans protect basic fibroblast growth factor from inactivation in the extracellular environment and modulate its bioavailability. At the cell surface, soluble and cell-associated heparan sulfate proteoglycans may play different roles in modulating the dimerization of the growth factor and its interaction with tyrosine kinase receptors. Finally, they affect the internalization and the intracellular fate of basic fibroblast growth factor, suggesting that growth factor slash proteoglycan complexes are involved in intracellular delivery. The bioavailability and the biological activity of basic fibroblast growth factor on endothelial cells strictly depend on the glycosaminoglycan milieu of the extracellular environment. Hence the angiogenic activity of the growth factor in vivo might be modulated by using exogenous glycosaminoglycans. The capacity of glycosaminoglycans to bind to and to influence the biological activity of basic fibroblast growth factor depends on size, degree of sulfation, and disaccharide composition. In the present paper we discuss the physiological significance and the biochemical bases of the interaction of the growth
The extracellular matrix (ECM) plays a crucial role in the process of endothelial cell migration,... more The extracellular matrix (ECM) plays a crucial role in the process of endothelial cell migration, proliferation, and differentiation during angiogenesis [1]. Several ECM proteins, like fibronectin (FN), affect cell behavior by binding to the various members of the integrin family of receptors, molecules that recognize the cell adhesion signal sequence Arg-Gly-Asp (RGD) [2].
The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhib... more The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylaminosubstituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors. Deregulation of FGFs (fibroblast growth factors) and their tyrosine kinase receptors (FGFRs) have a fundamental role in a variety of human cancers. Aberrant activation of this pathway, caused by gene amplification, chromosomal translocation, mutations, autocrine activation and impaired receptor down-regulation 1,2 contributes to cancer progression by inducing tumor angiogenesis 3-5. Moreover, FGFs directly promote tumor cell mitogenesis, survival, motility, invasion, epithelial-mesenchymal transition, and metastasis, and exert pleiotropic effects on the surrounding stroma 6. In addition, FGFs/FGFRs have been reported to mediate tumor escape/resistance to VEGF-targeted therapies 7 as well as resistance to targeted therapies such as Imatinib 8. The FGFs/FGFRs system is therefore a recognized actionable target to simultaneously affect angiogenesis, tumor cells and the stroma compartment. A rapidly expanding number of therapeutic compounds is being developed to target FGFs, their receptors, or downstream signaling, including tyrosine kinase receptor inhibitors, monoclonal antibodies, FGF traps, and ligands of the growth factors or their receptors 6,9-11 .
Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor... more Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.
The development of topical microbicides is a valid approach to protect the genital mucosa from se... more The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections.
Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached t... more Glycosaminoglycans (GAGs) are linear polysaccharides. In proteoglycans (PGs), they are attached to a core protein. GAGs and PGs can be found as free molecules, associated with the extracellular matrix or expressed on the cell membrane. They play a role in the regulation of a wide array of physiological and pathological processes by binding to different proteins, thus modulating their structure and function, and their concentration and availability in the microenvironment. Unfortunately, the enormous structural diversity of GAGs/PGs has hampered the development of dedicated analytical technologies and experimental models. Similarly, computational approaches (in particular, molecular modeling, docking and dynamics simulations) have not been fully exploited in glycobiology, despite their potential to demystify the complexity of GAGs/PGs at a structural and functional level. Here, we review the state-of-the art of computational approaches to studying GAGs/PGs with the aim of pointing ou...
Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transforme... more Cystic fibrosis transmembrane conductance regulator (CFTR)-rescuing drugs have already transformed cystic fibrosis (CF) from a fatal disease to a treatable chronic condition. However, new-generation drugs able to bind CFTR with higher specificity/affinity and to exert stronger therapeutic benefits and fewer side effects are still awaited. Computational methods and biosensors have become indispensable tools in the process of drug discovery for many important human pathologies. Instead, they have been used only piecemeal in CF so far, calling for their appropriate integration with well-tried CF biochemical and cell-based models to speed up the discovery of new CFTR-rescuing drugs. This review will give an overview of the available structures and computational models of CFTR and of the biosensors, biochemical and cell-based assays already used in CF-oriented studies. It will also give the reader some insights about how to integrate these tools as to improve the efficiency of the drug d...
Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable a... more Thrombospondin (TSP)-1 and TSP-2 share similar structures and functions, including a remarkable antiangiogenic activity. We have previously demonstrated that a mechanism of the antiangiogenic activity of TSP-1 is the interaction of its type III repeats domain with fibroblast growth factor-2 (FGF2), affecting the growth factor bioavailability and angiogenic activity. Since the type III repeats domain is conserved in TSP-2, this study aimed at investigating whether also TSP-2 retained the ability to interact with FGF2. The FGF2 binding properties of TSP-1 and TSP-2 and their recombinant domains were analyzed by solid-phase binding and surface plasmon resonance assays. TSP-2 bound FGF2 with high affinity (Kd = 1.3 nM). TSP-2/FGF2 binding was inhibited by calcium and heparin. The FGF2-binding domain of TSP-2 was located in the type III repeats and the minimal interacting sequence was identified as the GVTDEKD peptide in repeat 3C, corresponding to KIPDDRD, the active sequence of TSP-1. ...
Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to stand... more Glioblastoma multiforme (GBM) is the most malignant brain tumor, showing high resistance to standard therapeutic approaches that combine surgery, radiotherapy, and chemotherapy. As opposed to healthy tissues, EphA2 has been found highly expressed in specimens of glioblastoma, and increased expression of EphA2 has been shown to correlate with poor survival rates. Accordingly, agents blocking Eph receptor activity could represent a new therapeutic approach. Herein, we demonstrate that UniPR1331, a pan Eph receptor antagonist, possesses significant anti-angiogenic and anti-vasculogenic properties which lead to a significant anti-tumor activity in xenograft and orthotopic models of GBM. UniPR1331 halved the final volume of tumors when tested in xenografts (p<0.01) and enhanced the disease-free survival of treated animals in the orthotopic models of GBM both by using U87MG cells (40 vs 24 days of control, p<0.05) or TPC8 cells (52 vs 16 days, p<0.01). Further, the association of...
Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and ora... more Eph/ephrin system is an emerging target for cancer therapy but the lack of potent, stable and orally bioavailable compounds is impairing the development of the field. Since 2009 our research group has been devoted to the discovery and development of small molecules targeting Eph/ephrin system and our research culminated with the synthesis of UniPR129, a potent but problematic Eph/ephrin antagonist. Herein, we describe the in vitro pharmacological properties of two derivatives (UniPR139 and UniPR502) stemmed from structure of UniPR129. These two compounds acted as competitive and reversible antagonists of all Eph receptors reducing both ephrin-A1 and -B1 binding to EphAs and EphBs receptors in the low micromolar range. The compounds acted as antagonists inhibiting ephrin-A1-dependent EphA2 activation and UniPR139 exerted an anti-angiogenic effect, inhibiting HUVEC tube formation in vitro and VEGF-induced vessel formation in the chick chorioallantoic membrane assay. Finally, the oral ...
Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has ... more Besides its essential role in the activation of HIV-1 gene expression, the viral Tat protein has the unusual property of trafficking in and out of cells. In contrast to Tat internalization, the mechanism involved in extracellular Tat release has so far remained elusive. Here we show that Tat secretion occurs through a Golgi-independent pathway requiring binding of Tat with three short, non-consecutive intracytoplasmic loops at the C-terminus of the cellular Na(+),K(+)-ATPase pump alpha subunit. Ouabain, a pump inhibitor, blocked this interaction and prevented Tat secretion; virions produced in the presence of this drug were less infectious, consistent the capacity of virion-associated Tat to increase HIV-1 infectivity. Treatment of CD4+ T-cells with short peptides corresponding to the Tat-binding regions of the pump alpha subunit impaired extracellular Tat release and blocked HIV-1 replication. Thus, non canonical, extracellular Tat secretion is essential for viral infectivity.
Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and pare... more Fibroblast growth factors (FGFs) are a family of pleiotropic factors produced by stromal and parenchymal tumor cells. Even though FGFs have been firstly characterized as angiogenic factors, they exert autocrine and paracrine functions not only on endothelial cells but also on tumor cells and other stromal components. Thus, the FGF/FGF receptor (FGFR) pathway may represent a key player in tumor growth by regulating the complex cross-talk between stromal and tumor compartments. The ligand dependent or independent activation of the FGF/FGFR system by gene upregulation, oncogenic mutation or amplification occurs in a variety of human tumors and is implicated in various key steps of tumor growth and progression. In addition, FGF/FGFR activation has been described as a mechanism of tumor escape in response to antiangiogenic/anti-VEGF therapies. Experimental and clinical evidences provide a compelling biologic rationale for the development of anti-FGF/FGFR targeting agents in cancer therapy. However, the development of drugs specifically targeting the FGF/FGFR pathway proved to be difficult, also due to the high redundancy and pleiotropic effects of FGF and FGFR family members. On the other hand, the possibility to develop "two-compartment" targeting agents endowed with both antiangiogenic and antitumor activities remains promising. Here we will review the preclinical and clinical approaches and potential therapeutics currently available to block the FGF/FGFR system in human cancer.
Angiogenesis plays a key role in various physiological and pathological processes, including infl... more Angiogenesis plays a key role in various physiological and pathological processes, including inflammation and tumor growth. Numerous angiogenic growth factors (AGFs) have been identified. Usually, the angiogenic process is assumed to represent the outcome of a straightforward interaction of AGFs with specific signalling receptors of the endothelial cell (EC) surface. Actually, the mechanisms by which AGFs induce neovascularization are much more complex. Indeed, angiogenesis is the result of the simultaneous actions of various AGFs and angiogenesis modulators; multiple EC surface receptors with different structure and biological properties are engaged by AGFs to exert a full angiogenic response; AGFs bind a variety of free and immobilized proteins, polysaccharides, and complex lipids of the extracellular milieu that affect AGF integrity, stability, and bioavailability; some of the AGF-binding molecules interact also with AGF receptors. In this review the authors summarize literature data and discuss the current knowledge about the extracellular molecules able to interact with AGFs, thus representing possible key regulators of the angiogenesis process and targets/templates for the development of novel antiangiogenic drugs. This work represents an attempt to highlight common theme in the AGF interactome that occurs at the extracellular level during neovascularization.
Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied... more Biopsies of human normal adrenal medulla, adrenal pheochromocytoma, and chemodectoma were studied for the presence of basic fibroblast growth factor (bFGF). An immunoreactive Mr 18,000 bFGF-like molecule was detected both in normal and neoplastic tissues. This molecule was identified as bFGF on the basis of its molecular weight, its affinity for heparin, and its capacity to induce plasminogen activator production in cultured endothelial GM 7373 cells. The levels of immunoreactive and biologically active bFGF appeared to be significantly higher in the extracts of adrenal pheochromocytoma and chemodectoma than in the extracts of normal adrenal medulla. bFGF immunostaining was detectable in the nuclei of chief (Type-I) cells and of endothelial cells both in normal adrenal medulla and in pheochromocytoma. Cytoplasmic bFGF positivity of endothelial cells was also observed in pheochromocytoma but not in normal tissue. The data are in keeping with the hypothesis that bFGF may exert autocrine and paracrine functions in the growth and neovascularization of human pheochromocytoma.
International Journal of Clinical Laboratory Research, Feb 1, 1996
Basic fibroblast growth factor is an angiogenic molecule involved in several physiological and pa... more Basic fibroblast growth factor is an angiogenic molecule involved in several physiological and pathological processes, including wound repair, embryonic development, and tumor growth. In vitro, basic fibroblast growth factor induces an "angiogenic phenotype" in endothelial cells, which includes chemotaxis, mitogenesis, protease production, fl-integrin expression, and tube formation in three-dimensional gels. It acts by binding to specific tryosine kinase receptors and to cell-associated heparan sulfate proteoglycans. The physiological significance of the interaction with cell-associated and soluble heparan sulfate proteoglycans is manyfold. Heparan sulfate proteoglycans protect basic fibroblast growth factor from inactivation in the extracellular environment and modulate its bioavailability. At the cell surface, soluble and cell-associated heparan sulfate proteoglycans may play different roles in modulating the dimerization of the growth factor and its interaction with tyrosine kinase receptors. Finally, they affect the internalization and the intracellular fate of basic fibroblast growth factor, suggesting that growth factor slash proteoglycan complexes are involved in intracellular delivery. The bioavailability and the biological activity of basic fibroblast growth factor on endothelial cells strictly depend on the glycosaminoglycan milieu of the extracellular environment. Hence the angiogenic activity of the growth factor in vivo might be modulated by using exogenous glycosaminoglycans. The capacity of glycosaminoglycans to bind to and to influence the biological activity of basic fibroblast growth factor depends on size, degree of sulfation, and disaccharide composition. In the present paper we discuss the physiological significance and the biochemical bases of the interaction of the growth
The extracellular matrix (ECM) plays a crucial role in the process of endothelial cell migration,... more The extracellular matrix (ECM) plays a crucial role in the process of endothelial cell migration, proliferation, and differentiation during angiogenesis [1]. Several ECM proteins, like fibronectin (FN), affect cell behavior by binding to the various members of the integrin family of receptors, molecules that recognize the cell adhesion signal sequence Arg-Gly-Asp (RGD) [2].
The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhib... more The FGFs/FGFRs system is a recognized actionable target for therapeutic approaches aimed at inhibiting tumor growth, angiogenesis, metastasis, and resistance to therapy. We previously identified a non-peptidic compound (SM27) that retains the structural and functional properties of the FGF2binding sequence of thrombospondin-1 (TSP-1), a major endogenous inhibitor of angiogenesis. Here we identified new small molecule inhibitors of FGF2 based on the initial lead. A similarity-based screening of small molecule libraries, followed by docking calculations and experimental studies, allowed selecting 7 bi-naphthalenic compounds that bound FGF2 inhibiting its binding to both heparan sulfate proteoglycans and FGFR-1. The compounds inhibit FGF2 activity in in vitro and ex vivo models of angiogenesis, with improved potency over SM27. Comparative analysis of the selected hits, complemented by NMR and biochemical analysis of 4 newly synthesized functionalized phenylaminosubstituted naphthalenes, allowed identifying the minimal stereochemical requirements to improve the design of naphthalene sulfonates as FGF2 inhibitors. Deregulation of FGFs (fibroblast growth factors) and their tyrosine kinase receptors (FGFRs) have a fundamental role in a variety of human cancers. Aberrant activation of this pathway, caused by gene amplification, chromosomal translocation, mutations, autocrine activation and impaired receptor down-regulation 1,2 contributes to cancer progression by inducing tumor angiogenesis 3-5. Moreover, FGFs directly promote tumor cell mitogenesis, survival, motility, invasion, epithelial-mesenchymal transition, and metastasis, and exert pleiotropic effects on the surrounding stroma 6. In addition, FGFs/FGFRs have been reported to mediate tumor escape/resistance to VEGF-targeted therapies 7 as well as resistance to targeted therapies such as Imatinib 8. The FGFs/FGFRs system is therefore a recognized actionable target to simultaneously affect angiogenesis, tumor cells and the stroma compartment. A rapidly expanding number of therapeutic compounds is being developed to target FGFs, their receptors, or downstream signaling, including tyrosine kinase receptor inhibitors, monoclonal antibodies, FGF traps, and ligands of the growth factors or their receptors 6,9-11 .
Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor... more Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation. To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA. The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity. Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner. Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF. However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells. The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium. In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF. However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.
The development of topical microbicides is a valid approach to protect the genital mucosa from se... more The development of topical microbicides is a valid approach to protect the genital mucosa from sexually transmitted infections that cannot be contained with effective vaccination, like HSV and HIV infections. A suitable target of microbicides is the interaction between viral proteins and cell surface heparan sulfate proteoglycans (HSPGs). AGMA1 is a prevailingly cationic agmatine-containing polyamidoamine polymer previously shown to inhibit HSPGs dependent viruses, including HSV-1, HSV-2, and HPV-16. The aim of this study was to elucidate the mechanism of action of AGMA1 against HSV infection and assess its antiviral efficacy and biocompatibility in preclinical models. The results show AGMA1 to be a non-toxic inhibitor of HSV infectivity in cell cultures and human cervicovaginal histocultures. Moreover, it significantly reduced the burden of infection of HSV-2 genital infection in mice. The investigation of the mechanism of action revealed that AGMA1 reduces cells susceptibility to virus infection by binding to cell surface HSPGs thereby preventing HSV attachment. This study indicates that AGMA1 is a promising candidate for the development of a topical microbicide to prevent sexually transmitted HSV infections.
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Papers by Marco Rusnati