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The corneal pocket assay consists of the placement of a stimulus (purified growth factor, cell suspension, tumor tissue) into a corneal pocket in order to induce a vascular outgrowth from the limbal vessels toward the implant. This assay, with respect to the other in vivo assays, such as the chorio-allantoic membrane, has the advantage of measuring only new blood vessels, since the cornea is normally avascular. By using rabbits, additional advantages are gained like the easy manipulation of the animals and monitoring of the neovascular growth and inflammatory reaction over time. Measurement of corneal angiogenesis is useful to quantify the effects of angiogenic stimuli and to evaluate the efficacy of potential inhibitors of neovascularization following local delivery or systemic treatment.

In order to develop angiogenic and antiangiogenic strategies, concerted efforts have been made to provide animal models for more quantitative analysis of in vivo angiogenesis. In vivo techniques consist of the cornea pocket and iris implant in the eye, the rabbit ear chamber, the dorsal skinfold chamber, the cranial window, the hamster cheek pouch window, the sponge implant assay, the fibrin clots, the sodium alginate beads and the Matrigel plugs, the rat mesenteric window, the chick embryo chorioallantoic membrane and the air sac in mice and rats (1). In this chapter we will discuss the avascular cornea assay, and the advantages and disadvantages of using this assay in different species. The cornea assay is based on the placement of an angiogenic inducer (tumor tissue, cell suspension, growth factor) into a corneal pocket in order to evoke vascular outgrowth from the peripherally located limbal vasculature. In comparison to other in vivo assays, this assay has the advantage of measuring only new blood vessels, because the cornea is initially avascular.

Thanks to the advancement of knowledge in the field of angiogenesis, i.e., angiogenic/ angiostatic factors and their receptors as well as the molecular mechanisms activated, it has been possible to design diagnostic tools for new therapeutic strategies for angiogenesisdependent diseases and for assessing the angiogenic potential and malignancy of tumors. It is known that fibroblast growth factor-2 (FGF-2) and vascular endothelial growth factor (VEGF) play a critical role in the acquisition of an angiogenic phenotype. However, the blocking of these specific molecules might not be sufficient to suppress endothelial cell activation. Strategies targeting signal transduction cascades or the invasive phenotype acquired by the endothelium rather than individual factors and/or their receptors may be also proposed. Following this approach, we have targeted the nitric oxide synthase (NOS) pathway, the mitogen activated protein kinase (MAPK) cascade, and the urokinase-type plasminogen activator (uPA)-receptor, with encouraging results. The quantitation of the angiogenic output in tumor specimens or biopsies provides important information for the prognosis and the definition of biological endpoints to direct therapeutic decisions. Here we describe the development of quantitative methodologies for measuring the expression of angiogenic factors and modulators in tumor specimens. Assay methods, with good performance in practicability and analytical quality, when used to measure individual angiogenic factors in tumor specimens, are able to characterize tumor angiogenesis as a prognostic parameter and to select and monitor the efficacy of specific antiangiogenic drugs.

Chemokines, a large family of inflammatory cytokines, have been shown to play a critical role in the regulation of angiogenesis during several pathophysiologic processes, such as tumor growth, wound healing and ischemia. Semiquantitative or quantitative angiogenesis assays are commonly utilized to screen the angiogenic or angiostatic activity of chemokines. These include in vitro endothelial cell activation assays and ex vivo or in vivo models of neovascularization. Chemokines may exert their regulatory activity on angiogenesis directly or as a consequence of leukocyte infiltration and/or the induction of growth factor expression. The effect of chemokines on endothelium can be assessed by performing in vitro assays on purified endothelial cell populations or by in vivo assays. Nevertheless, each model used to evaluate the angiogenic or angiostatic activity of a discrete factor has advantages and limitations. Thus, in order to avoid under- or overestimating the regulatory effect of chemokines on angiogenesis and to evaluate all aspects of the angiogenic process, multiple assays are usually performed. This review summarizes past and recent studies on chemokines as modulators of angiogenesis with particular emphasis on the methods currently used for the assessment of chemokine-mediated angiogenic or angiostatic responses.

Angiogenesis, the development of new capillaries form pre-existing vessels, requires the coordinate activation of endothelial cells, which migrate and proliferate in response to growth factors to form functional vessels. Therapeutic angiogenesis is proposed to restore tissue integrity and function following damage and ischemia, while strategies aimed to block or suppress the neovascular growth are designed as adjuvant therapies for cancer treatment. Different experimental and clinical observations support the existence of a molecular / biochemical link between vasodilation, nitric oxide (NO) production and angiogenesis. NO significantly contributes to the prosurvival / proangiogenic program of capillary endothelium by triggering cell growth and differentiation via endothelial-constitutive NO synthase (ecNOS) activation, and cyclic GMP (cGMP) dependent gene transcription. Re-establishment of a balanced NO production in the cardiovascular system results in a reduction of cell damage during inflammatory and vascular diseases. Elevation of NOS activity in correlation with angiogenesis and tumor growth and aggressiveness has been extensively reported in experimental and human tumors. On these bases, the nitric oxide pathway appears to be a promising target for the development of pro- and anti-angiogenic therapeutic strategies. In particular, the use of NOS inhibitors or NO scavengers seems appropriate to reduce edema, block angiogenesis and facilitate antitumor drug delivery.

Since the initial report of nitric oxide (NO) activity, enormous progress has been made over the last two decades in the field of NO research. Whereas most physiological responses triggered by moderate concentrations of NO are mediated by soluble guanylate cyclase activation and the subsequent production of cyclic GMP as the major signaling messenger, recent studies have provided evidence of alternative signaling pathways triggered by high concentrations of NO. These signals operate in part through redox-sensitive regulation of transcription factors, gene expression, transcription, cellular activation, proliferation, and cell death. Numerous results converge to indicate a role for NO in physiological and pathological angiogenesis. Experimental data indicate that NO synthase, depending on the isoforms, the timing, and the degree of activation, may display contradictory effects, expressed during both physiological and pathological angiogenesis. The dual personality of NO will be reviewed in the context of the angiogenesis process.

Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies. Tumor angiogenesis appears to be governed by alterations of tumor suppressor or oncogenes operant in a broad range of tumors. We have addressed this issue in neuroblastoma, a malignancy characterized by the near-exclusive amplification and overexpression of the N-Myc oncogene. Here, we report that N-Myc overexpression results in down-regulation of interleukin-6 (IL-6) and that IL-6 is an inhibitor of endothelial cell proliferation and VEGF-induced rabbit corneal angiogenesis. STAT3 is instrumental for IL-6 activity as infection with adenoviruses expressing a phosphorylation deficient STAT3 mutant renders endothelial cells insensitive to the antiproliferative action of IL-6. Finally, though IL-6 does not influence neuroblastoma cell growth, IL-6-expressing xenograft tumors in mice exhibit reduced neovascularization and suppressed growth. Our data shed new light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastomas. Oncogene

Publisher Summary Most used in vivo bioassay in laboratory is the rabbit cornea assay. Because the cornea is an avascular tissue, this assay avoids the problems of interpretation inherent in the other bioassays, such as the chick chorion allantoic membrane (CAM). The outgrowth of new vessels into the cornea stroma can be readily identified and their morphology documented. The corneal assay is performed in New Zealand white rabbits. Nitric oxide (NO) directs endothelial cells in each step of angiogenesis and its role is twofold. In vitro administration of NO donor drugs to sparse coronary endothelium increases their mitotic index and favors cell migration and matrix degradation. As a result, in vivo NO donors speed up and potentiate neovascular growth. Thus, NO acts as the true effector of angiogenesis. However, mediators of angiogenesis signal the angiogenic switch in endothelium by releasing free NO and elevating cGMP levels. The development of NO-targeting drugs with higher selectivity for the different isoforms, combined with targeted tissue delivery to minimize side effects, as well as qualitative assessment of NO function from activity, to genomic expression, is a promising strategy for the exploitation of the findings on the role of NO on angiogenesis.

Fibroblast growth factor-2 (FGF2) and vascular endothelial growth factor (VEGF) exert their angiogenic activity by interacting with endothelial cells in a distinct manner. In this study, we investigated the morphological features of endothelial cells of the chick embryo chorioallantoic membrane (CAM) microvasculature after stimulation with FGF2 or VEGF. In order to provide a continuous delivery of the growth factor, we utilized a recently developed gelatin sponge/CAM assay in which a limited number of FGF2- or VEGF-transfected cells were adsorbed onto gelatin sponges and applied on the top of the CAM on day 8 of development. Their angiogenic activity was compared to that exerted by a single bolus of the corresponding growth factor. All the angiogenic stimuli induced a comparable vasoproliferative response, as demonstrated by the appearance of similar numbers of immature blood vessels within the sponge on day 12. No angiogenic response was observed in CAMs implanted with the corresponding parental cell lines or vehicle. Electron microscopy demonstrated that VEGF-overexpressing cells modified the phenotype of the endothelium of the blood vessels at the boundary between the implant and the surrounding CAM mesenchyme. The endothelial lining of 30% of these vessels showed segmental attenuations, was frequently interrupted and became fenestrated, mimicking what is observed in tumor vasculature. In contrast, the vessels consisted of continuous endothelium sealed by tight junctions in all the other experimental conditions. These results indicate that FGF2 and VEGF interact with endothelial cells of the CAM in a distinct manner. Both growth factors induce a potent angiogenic response, but only VEGF delivered in a continuous manner by its transfectants can modify the phenotype of the otherwise quiescent endothelium of CAM blood microvessels. The gelatin sponge/CAM assay may constitute a new model to study the mechanisms leading to endothelial fenestration in tumor growth.

The role of nitric oxide (NO) in the induction of angiogenesis was evaluated in a murine heart endothelioma cell line (H.end.FB) carrying the mT oncogene. Two clonal derivatives of H.end.FB, H80 and H73, exhibiting different NO synthase (NOS) activities were selected and used in the study. The relationship among NOS activity and tumor cell behaviour (growth, and angiogenic capacity) and the molecular control of gene expression were investigated. H.end.FB and H80 on one side and H73 on the other side exhibited the highest and lowest NOS activity, respectively. Cell growth was inversely correlated to the amount of NO produced by the cell lines. Conversely, in the avascular rabbit cornea assay, H.end.FB and H80 cells were strongly angiogenic, while H73 were poorly angiogenic, indicating that the ability of the cells to induce neovascularization was associated with the extent of NO produced. Consistently, systemic administration to rabbits of the NOS inhibitor Nw-nitro- l -arginine methyl ester ( l -NAME) significantly reduced the angiogenicity of H.end.FB cells. RT-PCR evidenced that H.end.FB expressed mRNA for TGF-β1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-β1 expression. These results indicate that the endogenous production of NO in tumor cells can serve as an autocrine/paracrine signalling mechanism of progression, by controlling angiogenic factor/modulator expression.

Angiogenesis, the formation of new blood vessels from existing capillaries, is critical for tumors to grow beyond a few in size. Tumor cells produce one or more angiogenic factors including fibroblast growth factor and vascular endothelial growth factor. Surprisingly, antiangiogenic factors or angiogenesis inhibitors have been isolated from tumors. Some angiogenesis inhibitors, such as angiostatin, are associated with tumors while others, such as platelet-factor 4 and interferon-alpha are not. Endostatin, a C-terminal product of collagen XVIII, is a specific inhibitor of endothelial cell proliferation, migration and angiogenesis. The mechanism by which endostatin inhibits endothelial cell proliferation and migration is unknown. Endostatin was originally expressed in a prokaryotic system and, late, in a yeast system, thanks to which it is possible to obtain a sufficient quantity of the protein in a soluble and refolded form to be used in preclincial and clinical trials.

Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. BK promotes growth of endothelial cells and its mitogenic activity involves c-Fos expression. Only the Bl receptor appears to be responsible for the proliferation induced by BK and suggests that this type of receptor might be implicated in favouring angiogenesis of coronary venules. Phospholipase C (PLC) activation and the endogenous upregulation of nitric oxide synthase (NOS) were monitored in response to Bl and B2 receptor activation. BK-induced IP turnover was triggered by B2 activation, while NOS activation was linked to the Bl receptor. NOS inhibition in CVEC prevented BK/B1 induced cell growth. These results substantiate the protective and trophic role of BK on microvascular endothelium by demonstrating that Bl receptor stimulation on venular endothelium is mandatory for the angiogenic effect of BK through its coupling to NOS activation.

Endostatin, a C-terminal product of collagen XVIII, is a very powerful angiogenesis inhibitor. In vivo experiments in mice indicate that endostatin dramatically reduces tumor mass without causing the onset of any resistance to the treatment. Recently, a 12-aa shorter human endostatin has been purified from plasma, but is ineffective in in vitro angiogenesis assays. Here we report that the full-length human recombinant endostatin has a potent inhibitory activity in in vitro angiogenesis assays. Two powerful angiogenic factors were used to stimulate endothelial cells: FGF-2 and VEGF-165. Endostatin prevented cell growth both in the basal condition and after stimulation with FGF-2 or VEGF-165. Migration of microvascular endothelial cells toward FGF-2 or VEGF-165 was impaired, both when cells were pretreated with the inhibitor and when endostatin was added together with the growth factors. Furthermore, experiments of inhibition of proliferation performed on nonmicroendothelial cells showed that endostatin was ineffective. This study indicates that human endostatin is a potent angiogenesis inhibitor and suggests its use in human anticancer therapy.

Almost any growth of tumors is to some extent associated with an inflammatory reaction which may be anti-tumorigenic by acting directly on tumor cells or protumorigenic cells presumably by inducing tumor-associated angiogenesis. In this study, we have analyzed the angiogenesis-inducing capacity of monocyte chemoattractant protein-1 (MCP-1), a key regulatory molecule of monocyte trafficking to sites of inflammation. MCP-1 was found to be potently angiogenic when implanted into rabbit cornea, exerting potency similar to the specific angiogenic vascular endothelial growth factor (VEGF)-A121. MCP-1-induced angiogenesis in the cornea is associated with prominent recruitment of macrophages, whereas VEGF-A121-induced corneal angiogenesis is devoid of inflammatory cell recruitment. Based on these findings, we studied MCP-1 expression and macrophage recruitment in human invasive ductal mammary carcinomas in comparison with the physiological angiogenic processes in bovine ovarian corpus luteum. Macrophage recruitment was always associated with MCP-1 expression. High macrophage counts in mammary tumors corresponded with poor prognosis. In contrast, physiological ovarian angiogenesis was associated with only minimal inflammatory recruitment of macrophages. Our data show that MCP-1 is an indirect inflammation-associated inducer of angiogenesis and demonstrate distinct qualitative differences between tumor angiogenesis in human mammary tumors and physiological angiogenesis in the ovary. Int. J. Cancer 82:765–770, 1999. © 1999 Wiley-Liss, Inc.

c- fos -induced growth factor/vascular endothelial growth factor D (Figf/Vegf-D) is a secreted factor of the VEGF family that binds to the vessel and lymphatic receptors VEGFR-2 and VEGFR-3. Here we report that Figf/Vegf-D is a potent angiogenic factor in rabbit cornea in vivo in a dose-dependent manner. In vitro Figf/Vegf-D induces tyrosine phosphorylation of VEGFR-2 and VEGFR-3 in primary human umbilical cord vein endothelial cells (HUVECs) and in an immortal cell line derived from Kaposi’s sarcoma lesion (KS-IMM). The treatment of HUVECs with Figf/Vegf-D induces dose-dependent cell growth. Figf/VEGF-D also induces HUVEC elongation and branching to form an extensive network of capillary-like cords in three-dimensional matrix. In KS-IMM cells Figf/Vegf-D treatment results in dose-dependent mitogenic and motogenic activities. Taken together with the previous observations that Figf/Vegf-D expression is under the control of the nuclear oncogene c- fos , our data uncover a link between a nuclear oncogene and angiogenesis, suggesting that Figf/Vegf-D may play a critical role in tumor cell growth and invasion.

The different members of the vascular endothelial growth factor (VEGF) family act as key regulators of endothelial cell function controlling vasculogenesis, angiogenesis, vascular permeability and endothelial cell survival. In this study, we have functionally characterized a novel member of the VEGF family, designated VEGF-E. VEGF-E sequences are encoded by the parapoxvirus Orf virus (OV). They carry the characteristic cysteine knot motif present in all mammalian VEGFs, while forming a microheterogenic group distinct from previously described members of this family. VEGF-E was expressed as the native protein in mammalian cells or as a recombinant protein in Escherichia coli and was shown to act as a heat-stable, secreted dimer. VEGF-E and VEGF-A were found to possess similar bioactivities, i.e. both factors stimulate the release of tissue factor (TF), the proliferation, chemotaxis and sprouting of cultured vascular endothelial cells in vitro and angiogenesis in vivo. Like VEGF-A, VEGF-E was found to bind with high affinity to VEGF receptor-2 (KDR) resulting in receptor autophosphorylation and a biphasic rise in free intracellular Ca2+ concentration, whilst in contrast to VEGF-A, VEGF-E did not bind to VEGF receptor-1 (Flt-1). VEGF-E is thus a potent angiogenic factor selectively binding to VEGF receptor-2. These data strongly indicate that activation of VEGF receptor-2 alone can efficiently stimulate angiogenesis.

We have assessed the role of soluble P-selectin in promoting endothelial cell migration. Human endothelial cells (HUVEC) and bovine microvascular endothelial cells (CVEC) were assessed for migration in the Neuroprobe 48-well microchemotaxis chamber. Soluble P-selectin promoted a dose-dependent (0.1-10 nM) migration of both cell types, with maximal response at 10 nM, producing approximately 60% increment over basal migration. Anti-P-selectin monoclonal antibody (5 microg/ml) selectively blocked P-selectin induced migration. Fibronectin and collagen were essential to disclose the migration induced by P-selectin. It is suggested that at vascular level in the presence of modifications of the extracellular matrix milieu, the production of soluble P-selectin could contribute to angiogenesis by promoting endothelial cell migration.

Angiogenesis, the formation of new microvessels from a pre-existing vascular bed, is stimulated by tissue hypoxia. Adenosine, a key extracellular signal elicited by hypoxia, plays several roles in initiation and/or modulation of angiogenesis. Adenosine governs the angiogenesis process through its actions on endothelial cell proliferation and migration. In addition, adenosine amplifies the mitogenic signal elicited by other angiogenic mediators such as nitric oxide and vascular endothelial growth factor. Through the interplay of tissue oxygen tension, adenosine, nitric oxide and vascular endothelial growth factor, a complex physiological control system ensures the matching of vascularity and metabolic needs in a variety of organs and circumstances.

Basic fibroblast growth factor (bFGF) is expressed in vascular endothelium during tumor neovascularization and angioproliferative diseases. The ultimate significance of this observation is poorly understood. We have investigated the biological consequences of endothelial cell activation by endogeneous bFGF in a mouse aortic endothelial cell line stably transfected with a retroviral expressionvector harboring a human bFGF cDNA. Selected clones expressing 24 kD, 22 kD, and/or 18 kD bFGF isoforms were characterized by a transformed morphology and an increased saturation density. bFGF transfectants showed invasive behavior and sprouting activity in three-dimensional fibrin gels and formed a complex network of branching cord-like structures connecting foci of infiltrating cells when seeded on laminin-rich basement membrane matrix (Matrigel). The invasive and morphogenetic behavior was prevented by anti-bFGF antibody, revealing the autocrine modality of the process. The biological consequences of this autocrine activation were investigated in vivo. bFGF-transfected cells gave rise to highly vascularized lesions resembling Kaposi’s sarcoma when injected in nude mice and induced angiogenesis in avascular rabbit cornea. Injected into the allantoic sac of the chick embryo they caused an increase in vascular density and formation of mangiomas in the choriallantoic membrane. In conclusion, bFGF-overexpressing endothelial cells acquire an angiogeneic phenotype and reclute quiescent endothelium originating angioproliferative lesions in vivo. These findings demonstrated that bFGF overexpression exerts an autocrine role for endothelial cells and support the notion that tumor neovascularization and angioproliferative diseases can be triggered by stimuli that induce vascular endothelium to produce its own autocrine factors(s).

In order to develop angiogenic and antiangiogenic strategies, there are concerted efforts to provide animal model for more quantitative analysis of in vivo angiogenesis. In vivo techniques consist in the rat and rabbit cornea assay, the sponge implant assay, the fibrin clots, sodium alginate beads and matrigel plugs and the chick embryo chorioallantoic membrane. In this chapter we will discuss on the avascular cornea assay and the advantages and divadvantages in the rat and the rabbits, the two animal species most used.

Screening of 84 primary human breast carcinomas for the mRNA expression of seven angiogenic polypeptides showed that the most commonly expressed and/or the most highly expressed when compared to normal breast tissue were vascular endothelial growth factor (VEGF) and platelet-derived endothelial cell growth factor/thymidine Phosphorylase (PDECGF/TP)1. The neurokines midkine (MK) and pleiotrophin were also fairly commonly expressed in tumour but not normal tissue (unpublished data from this group and ref. 1).

bFGF belongs to the family of the heparin-binding growth factors (Basilico and Moscatelli, 1992). The single copy human bFGF gene encodes multiple bFGF isoforms with molecular weights ranging from 24 kD to 18 kD. High molecular weight isoforms (HMW-bFGFs) are colinear NH2-terminal extensions of the better characterized 18 kD protein (Florkiewicz and Sommer, 1989). Both low and high molecular weight bFGFs exert angiogenic activity in vivo and induce cell proliferation, protease production, and Chemotaxis in endothelial cells in vitro (Gualandris, Urbinati, Rusnati, Ziche, and Presta, 1994). Also, bFGF has been shown to stimulate endothelial cells to form capillary-like structures in collagen gels (Montesano, Vassalli, Baird, Guillemin, and Orci, 1989) and to invade the amniotic membrane in vitro (Mignatti, Tauboi, Robbins, and Rifkin, 1989). Moreover, the phenotype induced in vitro by bFGF in endothelial cells includes modulation of integrin expression (Klein, Giancotti, Presta, Albelda, Buck, and Rifkin, 1993), gap-junctional intercellular communication (Pepper and Meda, 1992) and urokinase receptor upregulation (Mignatti, Mazzieri, and Rifkin, 1991a). Studies with neutralizing anti-bFGF antibodies have implicated endogenous bFGF in wound repair (Broadly, Aquino, Woodward, Bickley-Sturrock, Sat, Rifkin, and Davidson, 1989), vascularization of the chorioallantoic membrane during chick embryo development (Ribatti, Urbinati, Nico, Rusnati, Roncali, and Presta, 1995), and tumor growth under defined experimental conditions (Baird, Mormede, and Bohlen, 1986; Gross, Herblin, Dusak, Czerniak, Diamond, Sun, Eidsvoog, Dexter, and Yoayon, 1993).

Systemic administration of the nitric oxide (NO) synthase inhibitor Nomega-nitro--arginine methyl ester (L-NAME) to rabbits bearing a corneal implant blocked vascular endothelial growth factor (VEGF), but not basic fibroblast growth factor (bFGF)-induced angiogenesis. L-NAME completely blocked angiogenesis induced by VEGF-transfected MCF-7 breast carcinoma cells and the cells remained dormant in the cornea. Postcapillary endothelial cell migration and growth induced by VEGF were blocked by both the NO synthase inhibitor Nomega-mono-methyl--arginine and by the guanylate cyclase inhibitor LY 83583. We conclude that NO is a downstream imperative of VEGF-, but not bFGF-induced angiogenesis, and propose that the NO synthase/guanylate cyclase pathway is a potential target for controlling tumor angiogenesis in response to VEGF. Our studies support recent evidence that VEGF and bFGF induce angiogenesis by different mechanistic pathways using the alphavbeta5 and alphavbeta3 integrins, respectively.

The effect of the selective non-peptide antagonist for NK1 receptors (+/-)CP 96,345 on cellular transduction mechanisms elicited by the NK1 selective agonist [Sar9]-substance P-sulfone ([Sar9]-SP) was investigated in a stabilized culture of human skin fibroblasts (HF) and compared to the effects of two peptide antagonists, FK 888 and GR 82, 334. The exposure of the cells to [Sar9]-SP (100 nM) produced an early increase in inositol 1,4,5-trisphosphate (IP3) level, which peaked after 6 s, and a later rise in cellular inositol 1-phosphate (IP1) content which reached the maximum level in 15 min. The cAMP level was not significantly modified. The increase in IP1 was greatly reduced, at approximately the same extent by the 10 min pretreatment with a concentration of (+/-)CP 96,345 (100 nM) 10 times smaller than that of FK 888 and GR 82,334 (1 microM). The cytosolic Ca2+ mobilization in response to the NK1 agonist was monitored both by spectrofluorimetric and single-cell image analysis determinations on adherent cells loaded with the Ca(2+)-sensitive fluorescent indicators Fura-2/AM and Indo-1, respectively. [Sar9]-SP (100 nM) produced a rapid increase in the intracellular Ca2+ level in Fura-2/AM loaded cells. Cytosolic Ca2+ mobilization, measured by single-cell image analysis, indicated a concentration-dependent increase in both the ratio and in the number of cells responding to [Sar9]-SP. Either the non-peptide or the peptide selective NK1 receptor antagonists inhibited the increase in Ca2+ level in both the assays. In the spectrofluorimetric experiments the antagonizing effects of (+/-)CP 96,345 (1-100 nM), FK 888 (10 nM-1 microM) and GR 82,334 (10 nM-1 microM) were concentration-dependent. Moreover, the non-peptide antagonist was more potent than the two peptide antagonists, producing an 82.5% inhibition of Ca2+ mobilization at a concentration (10 nM) at which FK 888 and GR 82,334 decreased the response by only 62.3 and 60%, respectively. Stimulation of phosphatidylinositol turnover and calcium mobilization were also induced by 10 nM bradykinin; these effects were influenced neither by the previous administration of the NK1 receptor agonist nor by the three antagonists tested. These results demonstrate that the cellular transduction mechanisms induced in human skin fibroblasts by NK1 receptor stimulation are specifically and effectively antagonized by (+/-)CP 96,345, and that this non-peptide antagonist is more potent than the two peptide antagonists tested.

We investigated the influence of the Ca(2+)-ATPase inhibitor thapsigargin (TG) on the vasorelaxant response to different endothelium-dependent and endothelium-independent relaxing agents in an isolated thoracic aorta preparation of the rabbit, precontracted by norepinephrine (NE). Pretreatment with 100 microM L-arginine methyl ester (L-NAME) an inhibitor of nitric oxide (NO) synthesis, completely prevented acetylcholine (ACh)-induced relaxation; the inactive stereoisomer D-NAME did not modify the effect of ACh. The exposure of the preparations to 1 microM TG induced a slowly developing slight increase in the basal tension during 30-min contact. The same concentration of TG also slightly reduced the response to the subsequent administration of NE. The antagonist effect of TG on the ACh response was concentration dependent in the range between 0.1 and 10 microM. A 30-min pretreatment with 1 microM TG appeared to be sufficient to induce a consistent antagonism of the ACh (0.01-10 microM) concentration-relaxant effect curve, since an increase to 60 min did not produce a further significant increment in the degree of the antagonist effect. The concentration-dependent relaxation induced by substance P (SP 0.1-3 nM) was also significantly antagonized by 1 microM TG. The effect of the calcium ionophore A23187 (0.01-1 microM) was reduced by the Ca(2+)-ATPase inhibitor only at the higher concentrations tested (0.3-1 microM). However, a 30-min contact time with 1 microM TG was completely ineffective in antagonizing the concentration-relaxant response curves to the two nitrovasodilators sodium nitroprusside (SNP 0.1-100 microM) and nitroglycerin (NTG 1-300 nM) and to the cyclic GMP analogue 8-Bromo-cyclic GMP (3-100 microM). The effects of the beta-adrenoceptor agonist isoprenaline (ISO 0.1-10 microM) and of the direct adenylate cyclase activator forskolin (FK 0.01-10 microM) were also completely unaffected by 1 microM TG. These results demonstrate that TG affects the response to agents that induce an endothelium-dependent relaxation through receptor-dependent calcium mobilization. However, they do not support the hypothesis that sarcoplasmic pump activity is essential for the development of a vasorelaxant response to endothelium-independent agents.

Vascular endothelial growth factor (VEGF) is a secreted protein that is a specific growth factor for endothelial cells. We have recently demonstrated that nitric oxide (NO) donors and vasoactive peptides promoting NO-mediated vasorelaxation induce angiogenesis in vivo as well as endothelial cell growth and motility in vitro; in contrast, inhibitors of NO synthase suppress angiogenesis. In this study we investigated the role of NO in mediating the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary postcapillary venules. VEGF induced a dose-dependent increase in cell proliferation and DNA synthesis. The role of NO was determined by monitoring proliferation or guanosine 3',5'-cyclic monophosphate (cGMP) levels in the presence and absence of NO synthase blockers. The proliferative effect evoked by VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. This effect was potentiated by superoxide dismutase addition and was abolished by NO synthase inhibitors. VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation.

Fibroblast migration is an important component of the tissue response during the repair process, and substance P (SP) has been shown to exert trophic effects. In the present study, cell migration was evaluated as the distance travelled by adherent human skin fibroblasts (HF) at 96 h and by the number of individual cells moving across a filter within 5 h. In control conditions (1% calf serum) adherent fibroblasts moved from the starting line by approximately 700 microns. The addition of SP (10(-11)-10(-7) M) increased HF mobilisation in a concentration-dependent manner, with maximal activity at 10(-8) M (50% increase in migration over control). Migration of individual HF in suspension was also promoted by SP in a concentration-dependent manner, with an EC50 of 2.2 x 10(-9) M. The response produced by the maximally effective concentration of SP was equal to 65 and 90% of the effect elicited by 100 ng/ml Platelet-Derived Growth Factor A/B (PDGF A/B) on adherent and individual cells respectively. The synthetic NK1 receptor agonist [Sar9]SP-sulphone (10(-11)-10(-6) M) reproduced the SP effect. The NK2 and NK3 receptor agonists [beta Ala8]NKA(4-10) and [MePhe7]NKB were devoid of any effect. The effect of SP was antagonised by two selective antagonists of NK1 receptors, namely (+/-) CP 96,345 (10(-10)-10(-8) M) and FK 888 (10(-9)-10(-7) M), while the NK2 receptor antagonist MEN 10627 (10(-8)-10(-7) M) was not effective. Our data indicate that SP is a potent effector of fibroblast migration and the NK1 receptor is responsible for this effect. These observations further support the specific role of the NK1 receptor in mediating the trophic function of SP at the cutaneous level.

Conventional medical knowledge predicates that inflammation and angiogenesis are closely associated phenomena. For decades, angiogenesis, previously described as erythema or redness, has been viewed as one of the cardinal signs of inflammation, the two phenomena being inextricably interwoven. This firmly established notion has been challenged by the discovery of mediators of inflammation endowed with a wide repertoire of angiogenic actions ranging from potent stimulators to suppressors of angiogenesis. The most cogent examples of molecules, which may exert opposing actions on angiogenesis, while invariably contributing to inflammation, are found in the interleukin and cytokine class of mediators (Cozzolino et al., 1990; Szekanecz & Koch 2001; Hatzi et al., 2002). For example, IL-1, IL-6 and IL-10 exert both angiogenic and angiostatic action depending on the experimental conditions (Cervenak et al., 2000; Kohno et al., 2003; Wei et al., 2003; Bar et al., 2004). Thus, the relationship between inflammation and angiogenesis appears less unequivocal than previously thought. Reports on tumor angiogenesis and the prognostic significance of the inflammatory infiltrate are a good example of conflicting evidence (Griffioen et al., 1996; Offersen et al., 2002). Certainly the extrapolation of observations in isolated cell populations to in vivo experimental models designed either for inflammation or angiogenesis contribute to the ambiguity on angiogenic properties of inflammatory mediators (Gullino, 1981; Auerbach et al., 2003). In the current issue of the journal, Ferreira et al. (2004) have revisited the role of endogenous PAF in chronic inflammation, in angiogenesis, and other sequela of inflammation such as cell recruitment and in the production of cytokines. To study endogenous PAF, they used either PAF receptor-deficient mice (PAFR-KO) or wild-type mice orally treated with a PAF receptor antagonist. Chronic inflammation was induced by a sponge implant, a condition that promotes granolomatous tissues closely reproducing that occurring in human pathology. In the study of Ferreira et al. (2004), the absence of the effects of endogenous PAF induced a significant stimulation of angiogenesis, and attenuation of the inflammatory response. This finding sharply contrasts with a wealth of literature data describing the abundance of PAF, rather than its absence, as a potent stimulus for the activation of endothelial cells leading to the formation of new vessels (Brizzi et al., 1999; Montrucchio et al., 2000; Deo et al., 2002). However, the portrayal of PAF as a proangiogenic molecule has been drawn from in vitro experiments on cultured endothelial cells exposed to exogenous PAF (Brizzi et al., 1999), or from in vivo studies in which angiogenesis was inherently primed by matrigel implants (Montrucchio et al., 2000), or a combination of PAF and angiogenic stimulator (Montrucchio et al., 2000). An emerging concept is that the extent of angiogenic response greatly depends on the innate angiogenic tone. Thus, it appears that the activity of PAF, a ubiquitous lipid mediator of inflammation, is strongly influenced by the experimental conditions giving rise to opposing effects. The decreased inflammation observed by Fereira et al. (2004) in PAFR-KO mice was accompanied by a reduced level of inflammatory cell infiltrate and no modification of known angiogenic factors such as VEGF and TNF-a, but unexpectedly by a sustained rise of chemokines CXCL2 (KC) and CCL2 (MCP-1/JE). The enhanced level of these chemokines indicates that loss of PAF's effects does not indiscriminately suppress all inflammatory signals, which perhaps compensates for the absence of an important molecule in maintaining immunogical surveillance. Moreover, the elevation of CCL2 (MCP-1/JE), particularly significant in PAFR-KO mice, may account for the increased vascularization, since this cytokine possesses consistent proangiogenic activity (Goede et al., 1999). In summary, the article by Ferreira et al. (2004) contributes to the better definition of the role of PAF in inflammation and in angiogenesis in conditions that mimic mild chronic inflammation in man. PAF appears to be a physiological mediator of inflammation, which negatively regulates angiogenesis rather than promoting it, a profile that is also shared by other mediators of inflammation. In physiological angiogenesis the interplay among endogenous stimulators and inhibitors, although highly redundant, is tightly regulated to achieve neovascular growth in maintaining tissue integrity. While some molecules have been selectively assigned to either function, others have been shown to possess opposing actions. Whether PAF belongs to the latter class of molecules, as suggested by the report of Ferreira et al. (2004), needs to be substantiated in other models.

The growth and migration of endothelial cells are the prerequisites for vascular remodeling. The effects of endothelin (ET)-1 and -3 (ET-1, ET-3) on the proliferation and migration of endothelial cells isolated from bovine adrenal capillaries (BACE) and human umbilical veins (HUVEC) have been investigated. Cell proliferation (measured as DNA synthesis and as total cell number) and migration were significantly increased by ET-1 and ET-3. Dose-dependent proliferation was produced by ET-1 and ET-3 in both cell lines, with maximal effects at 0.1 nM concentration. ET-1 and ET-3 also stimulated BACE and HUVEC mobilization in a dose-dependent manner. The maximal responses were obtained at 10 nM concentration in both BACE and HUVEC. The full agonist for the ETB receptor, ET-(16--21), was able to reproduce the effects of ET on proliferation and migration of both cell lines. Modification of ET-(16--21) at Leu17 and Ile19 and amidation of the COOH-terminal were accompanied by loss of activity. The ETB-receptor antagonist IRL-1038 blocked the migration induced by ET-3 and ET-(16--21), whereas the ETA-receptor antagonist BQ-123 was not effective. We conclude that ETs, by favoring endothelial cell growth and mobilization, can contribute to neovascularization through an autocrine mechanism that requires ETB-receptor activation.

Calcitonin gene-related peptide (CGRP, 0.1 microM) and forskolin (10 microM) both produced a time-dependent accumulation of cAMP in homogenates of the guinea-pig ureter, while cromakalim (3 microM) was ineffective. Neither agent did increase the cGMP levels. cAMP accumulation induced by CGRP or forskolin was unchanged by glibenclamide (1 microM). In sucrose gap, the application of forskolin (1-10 microM for 15 s) hyperpolarized the smooth muscle membrane and its effect was greatly enhanced when tested in a low-K+ medium (extracellular K+ reduced from 5.9 to 1.2 mM). The hyperpolarization produced by 10 microM forskolin was reduced and abolished by 1 and 10 microM glibenclamide, respectively, in both normal and low-K+ medium. The present findings demonstrate that CGRP determines a selective cAMP accumulation in the guinea-pig ureter and suggest that elevation of cAMP may be involved in the opening of glibenclamide-sensitive K+ channels in the ureter smooth muscle.

The single-copy gene of human basic fibroblast growth factor (bFGF) encodes four co-expressed isoforms, with an apparent molecular weight (Mr) of 24kD, 22.5 kD, 22kD, and 18kD, co-translated from a single mRNA. As a tool for the study of the role exerted by the different bFGF isoforms in the biology of endothelial cells, human recombinant 24-kD bFGF was produced and purified from transformed Escherichia coli cells. To this purpose, the novel CUG start codon present in human bFGF cDNA and responsible for the synthesis of 24-kD bFGF was mutagenized to the classic AUG start codon. Transient expression of the mutagenized cDNA in simian COS-1 cells, followed by immunolocalization and subcellular fractionation, resulted in the synthesis of high levels of 24-kD bFGF, which localizes in the cell nucleus as an intact protein. When the same 24-kD bFGF, cDNA was expressed in E. coli, the recombinant protein was purified to homogeneity by heparin-Sepharose and ion-exchange chromatography. Recombinant 24-kD bFGF was similar to recombinant 18-kD bFGF in receptor-binding activity and in inducing cell proliferation, plasminogen activator production, and chemotactic movement in cultured endothelial cells. In agreement with the in vitro observations, 24-kD bFGF and 18-kD bFGF exerted a similar angiogenic response when assayed in vivo in the rabbit cornea. Experiments performed with the radiolabeled molecule demonstrated that 24-kD bFGF has an intrinsic ability to bind to high-affinity receptors when added to endothelial GM 7373 cell cultures. Receptor-bound 24-kD bFGF is internalized within the cell and associates with the nucleus with kinetics similar to 13-kD bFGF. Internalized 24-kD bFGF is first processed to the 18-kD form via a chloroquine-insensitive pathway and then to smaller fragments into the lysosomal compartment. At variance with the data obtained in transfected COS-1 cells, only limited amounts of exogenous internalized 24-kD bFGF associates with the nucleus in the intact form, mostly of the nuclear-bound molecule being represented by the processed 18-kD protein and by smaller degradation products. In conclusion, human recombinant 24-kD bFGF exerts a biological response in endothelial cells similar to 18-kD bFGF both in vitro and in vivo. Our data point to a different intracellular behavior of the high-molecular-weight bFGF isoform when added exogenously to cultured cells or when produced endogenously in transfected cells. © 1994 Wiley-Liss, Inc.

Under physiological circumstances the ability of a given tissue to produce a neovascular response is under strict control, i.e. the balance between factors and events favoring or interfering with neovascular growth is in equilibrium. Modification of the normal cell/cell and cell/matrix boundaries, which maintain tubular morphology of the endothelium at the postcapillary level, is a prerequisite for capillary endothelial cell mobilization and proliferation to constitute a neovascular response.

Prostaglandins, mostly PGE-2, have assumed an important role in cancer biology. Regarding of PGE-2 production, recent investigations have focused on PGE-2 synthases (mPGEs-1, mPGEs-2 and cPGEs), specifically on mPGEs-1, the only inducible enzyme among those so far identified in cells. Whereas cPGEs and mPGEs-2 are constitutively expressed at relatively low levels, mPGEs-1 is highly inducible by pro-inflammatory stimuli. mPGEs-1 upregulation has been detected in many epithelial tumors, and its silencing has been reported to reduce preneoplastic lesions. Despite the evidence linking mPGEs-1 and tumorigenesis, most studies investigating the inducible nature of the enzyme in cultured cells have been performed using a variety of inflammatory stimuli, while little is known about the behavior of the enzyme in tumor cells challenged with oncogenic stimuli. In this work we investigated mPGES-1 regulation in cultured epithelial tumor cells exposed to epidermal growth factor (EGF), an oncogene product which, through its receptor, EGF-receptor, is responsible for tumorigenisis in a wide array of solid tumors. We used three cell lines, representative of colon (HT-29), epidermoid (A431) and lung (A549) tumors, examining the expression of mPGEs-1 protein following EGF-receptor activation by EGF. We found that cells responded to EGF-receptor stimulation with a transient surge of the transcription factor Egr-1, its translocation from cytosol to necleus, an increase of mPGEs-1 mRNA and protein, as well as, an enhanced PGE-2 formation. These changes were suppressed by silencing Egr-1 and by EGF-receptor blockade or by inhibitors of the EGF-receptor-dependent mediators, such as MAP-kinase, ERK1/2. Moreover, cell activation through EGF-receptor induced a tumorigenic phenotype, detectable by clonogenic assay, which regressed when mPGEs-1 was silenced. We also explored the effect of inhibiting the EGF-receptor in A431 xenografts on mPGEs-1 expression and tumor growth, finding a parallel decline of both. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1130. doi:10.1158/1538-7445.AM2011-1130

To elucidate mechanisms underlying neovascularization that accompanies certain chronic immune/inflammatory disorders, the effects of interferon-alpha (IFN-alpha) and interleukin 2 (IL-2) on endothelial cell (EC) growth in vitro and angiogenesis in vivo were studied. Preincubation of cultured human ECs with IFN-alpha, followed by exposure to IL-2, resulted in effective stimulation of cell growth, whereas either cytokine alone had only a slight effect. The combination of IFN-alpha/IL-2 induced an angiogenic response in the rabbit cornea. IL-2 receptor expression was enhanced on IFN-alpha-treated ECs: p55 was increased and p70 was induced. 125I-IL-2 binding to ECs treated with IFN-alpha was enhanced (Kd from approximately 7 nM to approximately 260 pM with IFN-alpha), and anti-p55 IgG blocked 125I-IL-2/EC interaction as well as IL-2-mediated EC proliferation. Consistent with these findings in cell culture, immunohistologic studies demonstrated p55 and p70 antigen in the vasculature of rheumatoid joints, but not in normal joint tissue. Exposure of cultured ECs to IFN-alpha increased levels of intracellular EC basic fibroblast growth factor (bFGF), and subsequent addition of IL-2 led to bFGF release into the medium. The observation that anti-bFGF IgG largely blocked EC proliferation in response to IFN-alpha/IL-2 suggested that bFGF was a critical agent in this setting. These data suggest a mechanism rendering ECs responsive to IL-2 which may be relevant in immune/inflammatory disorders: IFN-alpha-mediated induction of functional EC receptors for IL-2, which drives cell proliferation by a mechanism dependent on increased synthesis and release of bFGF.

The unrestrained growth and metastatic potential of a tumor is strictly correlated to its capacity to induce an efficient capillary network in the host tissue (Folkman J and Cotran R 1976, Ziehe M and Gullino PM, 1982). Under physiological circumstances the ability of a given tissue to produce a neovascular response is under strict controll, i.e. the balance between factors and events favoring or interfering with neovascular growth is in equilibrium.

The effect of synthetic selective tachykinin receptor antagonists was studied on the growth of cultured human skin fibroblasts (HF). Selective antagonists for the NK 1 receptor ([D-Pro 4 , D-Trp 7,9 , Phe 11 ]-SP(4–11), GR71251 and L 668,169) and the NK 2 receptor (L 659,877) were tested against Substance P (SP), against the selective NK 1 receptor agonist [β-Ala 4 , Sar 9 , Met(O 2 ) 11 ]-SP(4–11) and against basic Fibroblast Growth Factor (bFGF). All the selective NK 1 receptor antagonists, tested at the concentration of 10 −5 M, induced a significant displacement to the right of the dose-response curves induced by SP and by the selective NK 1 receptor agonist. The selective NK 2 receptor antagonist did not modify the proliferative response to the tachykinins used. The growth promoting effect of bFGF was not modified by any of the tachykinin antagonists tested. These results indicate that the newly developed receptor-selective tachykinin antagonists appear to be a useful tool to assess the biological effects of tachykinin in vitro on cultured isolated cells.

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.

The data reported were obtained as an attempt to understand whether the change in total concentration and relative ratios of the 3 major corneal gangliosides (GM3, GM2, GD3) previously observed in corneas stimulated by an angiogenic molecule (Ziche et al., 1989) was a relevant event in the angiogenic response of the tissue. The effect on endothelial cell growth was tested for the 3 corneal gangliosides added singly to the culture medium, and GM3 was found to possess a substantial growth inhibitory effect as compared to GM2 and GD3. The growthlimiting effect of GM3 was counteracted to a different degree by the addition of a second ganglioside to the culture medium. A mixture of GM3 + GM2 + GD3 in the proportion similar to that found in the cornea stimulated by an angiogenic molecule was able to sustain a sharp increment in cell growth and motility when added to cultures of capillary endothelium. On the contrary, when the 3 components of the mixture were in the proportion present in the normal cornea, the increment in growth or motility did not occur. A simple change in the relative proportions of the 3 gangliosides was sufficient to trigger or prevent an increment in growth and motility of the endothelial cells. These data in vitro suggest that the changes in concentration and relative ratios of the 3 major corneal gangliosides observed in vivo when the cornea was stimulated by an angiogenic molecule were an event targeted to favour growth and mobilization of the capillary endothelium located within the limbal vessels at the periphery of the cornea. © 1992 Wiley-Liss, Inc.

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.

The effect of peptidase inhibitors on neuropeptide release from peripheral endings of capsaicin-sensitive sensory neurons was studied in cerebral superior sagittal and transverse sinuses of guinea-pig. Capsaicin (1 microM)-evoked release of substance P-like immunoreactivity (SP-LI) was increased in a concentration-dependent manner by thiorphan (0.1-10 microM). Captopril (10 microM) or a mixture of bestatin (10 microM), leupeptin (10 microM) and bacitracin (10 microM) did not affect the capsaicin-evoked SP-LI release. Thiorphan (10 microM) increased also the capsaicin-evoked release of neurokinin A-like immunoreactivity (TK-LI) and calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) by 228% and 172%, respectively, while captopril (10 microM) was without effect. Thiorphan (10 microM), but not captopril (10 microM), enhanced by 239% CGRP-LI release induced by bradykinin (10 microM). In the cerebral venous vessels neutral endopeptidase (EC 3.4.24.11, NEP)-like activity was 58.8 +/- 6.1 pmol/mg protein/min, while angiotensin converting enzyme-like activity was below the detection limit of the assay. A thiorphan-sensitive mechanism, putatively attributable to NEP, plays a major role in the inactivation of peptides released from or acting on capsaicin-sensitive sensory fibres of cerebral venous sinuses of guinea-pig.

Substance P (SP) has been indicated as a main mediator of neurogenic inflammation, leading to vasodilation, increase in vascular permeability and modulation of immune cell function. Certain vascular effects produced by SP are endothelium mediated. We have studied the effect of SP and of selective NK-1, NK-2 and NK-3 receptor agonists on migration of cultured capillary endothelial cells of bovine origin. Our results indicate that SP (10 −14 –10 −6 M) induces a concentration-dependent migration of endothelial cells with maximal activity at 10 −10 M. This effect was mimicked by the selective NK-1 receptor agonist which showed a similar concentration-dependent curve, while selective NK-2 and NK-3 receptor agonists were ineffective. Our conclusions are that endothelial cells possess specific receptors for SP of the NK-1 type which affect mobilization of capillary endothelial cells.

This study was designed to identify the set of functions activated in cultured endothelial cells by the hematopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage-colony-stimulating factor (GM-CSF), and to compare them with those elicited by prototypic cytokines active on these cells. Moreover, indications as to the in vivo relevance of in vitro effects were obtained. G-CSF and GM-CSF induced endothelial cells to proliferate and migrate. In contrast, unlike appropriate reference cytokines (IL-1 and tumor necrosis factor, IFN-gamma), G-CSF and GM-CSF did not modulate endothelial cell functions related to hemostasis-thrombosis (production of procoagulant activity and of platelet activating factor), inflammation (expression of leukocyte adhesion molecule-1 and production of platelet activating factor), and accessory function (expression of class II antigens of MHC). Other colony-stimulating factors (IL-3 and macrophage-colony-stimulating factor) were inactive on all functions tested. In comparison to basic fibroblast growth factor (bFGF), G-CSF and GM-CSF induced lower maximal proliferation of endothelial cells, whereas migration was of the same order of magnitude. G-CSF and GM-CSF stimulated repair of mechanically wounded endothelial monolayers. Exposure to both cytokines induced shape changes and cytoskeletal reorganization consistent with a migratory phenotype. To explore the in vivo relevance of the in vitro effects of these cytokines on endothelium, we studied the angiogenic activity of human G-CSF in the rabbit cornea. G-CSF, but not the heat-inactivated molecule, had definite angiogenic activity, without any sign of inflammatory reactions. G-CSF was less active than bFGF. However, the combination of a nonangiogenic dose of bFGF with G-CSF resulted in an angiogenic response higher than that elicited by either individual cytokines. Thus, G-CSF and GM-CSF induce endothelial cells to express an activation/differentiation program (including proliferation and migration) related to angiogenesis.

1. The effect of synthetic tachykinin selective receptor agonists was studied on the growth of cultured human skin fibroblasts (HF). 2. Human fibroblasts were grown in serum-free conditions in the presence of natural tachykinins (substance P and neurokinin A) and of three synthetic agonists, [beta-Ala4, Sar9, Met(O2)11]-SP(4-11), [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB selective for NK1-, NK2- and NK3-receptors respectively. Cell proliferation was measured by percentage increase in cell number and by [3H]-thymidine uptake following 48 h exposure to agents compared to baseline condition. 3. Neurokinin A (NKA) and substance P (SP) significantly increased cell proliferation the threshold concentrations being 10(-12) and 10(-11) M, respectively. Addition of thiorphan to culture conditions enhanced the effect of SP but not of NKA. 4. The selective NK1-receptor agonist produced a dose-dependent increase in cell proliferation as judged by total cell number and [3H]-thymidine uptake. No significant effect was observed with NK2- and NK3-receptor agonists. 5. These data indicate that the effect of SP on fibroblast proliferation is mediated by interaction with a NK1-receptor type and local metabolism can interfere with the full expression of this effect of SP on cell proliferation.

The experiments reported were motivated by the observation that in vivo gangli-osides promoted angiogenesis when the dose of the angiogenic factor was too low to be effective (Ziche et al.: Laboratory Investigation 61:629–634, 1989). As an approach to understanding the mechanism of this modulatory effect, we analysed the influence that gangliosides have on survival, growth, and migration of capillary endothelium when and angiogenesis factor like basic fibroblast growth factor (bFGF) was present in the culture medium. Clones of bovine capillary endothelium were cultivated in media unable to sustain survival over a 72 h period. With this experimental approach, cell survival was evaluated after addition of either bFGF or gangliosides or both to the medium. The Boyden chamber procedure was utilized to measure the influence of bFGF or gangliosides on cell mobilization across a micropore filter. Low doses of both molecules, ineffective when added singly to the culture media, improved all three parameters when added in combination. A synergic effect between bFGF and the gangliosides (GM1, GD1b, GT1b) was observed for the improvement of survival or growth and for the acceleration of endothelial cell migration. The removal of sialic acid from the ganglioside molecule prevented any effect on all three parameters. The addition of sialic acid alone to cultures was also totally ineffective. In the aduit organism most angiogenic events occur under conditions of tissue damage. The synergism between gangliosides and bFGF can be interpreted as the initial phase of a process for which endothelial cell survival is the indispensable first step in the formation of a new vascular network.