FGF-2 overexpression opposes the beta amyloid toxic injuries to the vascular endothelium.

Recent evidences suggest that Abeta peptides modulate endothelial cell (EC) functions. At low concentrations, Abeta1-40 enhances the pro-angiogenic activity of FGF-2, whereas deposition of excess Abeta causes EC dysfunction and cerebral amyloid angiopathy (CAA). We investigated whether FGF-2 attenuates EC dysfunction caused by pathological Abeta levels. We studied Abeta1-40 on EC survival, as well as on signals responsible of their angiogenic phenotype. At 5-50 microM Abeta1-40 reduced EC population, caused apoptosis, downregulated FGF-2 production, inhibited FGF-2 binding to heparin, and FGFR1 phosphorylation. Toxic effects were owing to lack of FGF-2 stimulation, as EC overexpressing FGF-2 displayed extraordinary resistance to Abeta1-40 injuries. The FGF-2 mechanism responsible for reversing damages, involves the downstream enhancement of Akt, a pathway independent of eNOS activation. In conclusion, we demonstrate that FGF-2 protects EC from the effects of excess Abeta1-40, suggesting that it may attenuate the consequences of Abeta deposition in pathologies as CAA.

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

Interferon-alpha and interleukin 2 synergistically enhance basic fibroblast growth factor synthesis and induce release, promoting endothelial cell growth.

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.

Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis.

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.

In vitro and in vivo activation of endothelial cells by colony-stimulating factors.

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.

Soluble N-cadherin fragment promotes angiogenesis.

Endothelial cells express two dependent intercellular adhesion molecules: vascular endothelial (VE)-cadherin, specific for endothelial cells, and N-cadherin, also present in neuronal, lens, skeletal and heart muscle cells, osteoblasts, pericytes and fibroblasts. While there exists a vast amount of evidence that VE-cadherin promotes angiogenesis, the role of N-cadherin still remains to be elucidated. We found that a soluble 90-kDa fragment N-cadherin promotes angiogenesis in the rabbit cornea assay and in the chorioallantoic assay when cleaved enzymatically from the extracellular domain of N-cadherin. Soluble N-cadherin stimulates migration of endothelial cells in the wound healing assay and stimulates phosphorylation of extracellular regulated kinase. In vitro experiments with PD173074 and knock-down of N-cadherin and fibroblast growth factor (FGF)-receptor, showed that the pro-angiogenic effect of soluble N-cadherin is N-cadherin- and FGF-receptor-dependent. Our results suggest that soluble N-cadherin stimulates migration of endothelial cells through the FGF-receptor.

Role of nitric oxide in angiogenesis and tumor progression in head and neck cancer.

BACKGROUND: Angiogenesis (formation of new blood vessels) is associated with tumor growth and metastasis in patients with solid tumors, including those of the head and neck. Nitric oxide (NO) production may contribute to these processes. We assessed the role of the NO pathway in angiogenesis and tumor progression in patients with head and neck cancer. METHODS: Biochemical assays were used to measure NO synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) levels in specimens of tumor and normal mucosa obtained from 27 patients. Microvessels in tumor specimens were identified by CD-31-specific immunohistochemical staining. Associations between microvessel densities, levels of NOS, and cGMP were examined by use of two-sided statistical tests. Tumor specimens and human squamous carcinoma A-431 cells were grown as explants on the corneas of rabbits, and the effect of the NOS inhibitor N(omega)-nitro-L-arginine-methyl ester (L-NAME) was tested. RESULTS: Levels of total NOS, inducible NOS, and cGMP were higher in tumor specimens than in specimens of normal mucosa (all P<.0001). Tumor specimens from patients with lymph node metastases presented a higher total NOS activity (P = .005) and were markedly more vascularized than tumor specimens from patients with no lymph node involvement (P = .0002). Microvessel density at the tumor edge was an independent predictor of metastasis for this series of patients (odds ratio = 1.19; 95% confidence interval = 1.07-2.89; P = .04). A-431 cells and tumor specimens exhibiting high levels of NOS activity induced angiogenesis in the rabbit cornea assay; when NO production was blocked, tumor angiogenesis and growth were repressed. CONCLUSIONS: The NO pathway appears to play a key role in tumor angiogenesis and spread in patients with head and neck cancer.

Purine analogue 6-methylmercaptopurine riboside inhibits early and late phases of the angiogenesis process.

Angiogenesis has been identified as an important target for antineoplastic therapy. The use of purine analogue antimetabolites in combination chemotherapy of solid tumors has been proposed. To assess the possibility that selected purine analogues may affect tumor neovascularization, 6-methylmercaptopurine riboside (6-MMPR), 6-methylmercaptopurine, 2-aminopurine, and adenosine were evaluated for the capacity to inhibit angiogenesis in vitro and in vivo. 6-MMPR inhibited fibroblast growth factor-2 (FGF2)-induced proliferation and delayed the repair of mechanically wounded monolayer in endothelial GM 7373 cell cultures. 6-MMPR also inhibited the formation of solid sprouts within fibrin gel by FGF2-treated murine brain microvascular endothelial cells and the formation of capillary-like structures on Matrigel by murine aortic endothelial cells transfected with FGF2 cDNA. 6-MMPR affected FGF2-induced intracellular signaling in murine aortic endothelial cells by inhibiting the phosphorylation of extracellular signal-regulated kinase-2. The other molecules were ineffective in all of the assays. In vivo, 6-MMPR inhibited vascularization in the chick embryo chorioallantoic membrane and prevented blood vessel formation induced by human endometrial adenocarcinoma specimens grafted onto the chorioallantoic membrane. Also, topical administration of 6-MMPR caused the regression of newly formed blood vessels in the rabbit cornea. Thus, 6-MMPR specifically inhibits both the early and the late phases of the angiogenesis process in vitro and exerts a potent anti-angiogenic activity in vivo. These results provide a new rationale for the use of selected purine analogues in combination therapy of solid cancer.

Characterization of zofenoprilat as an inducer of functional angiogenesis through increased H2 S availability.

BACKGROUND AND PURPOSE: Hydrogen sulfide (H2 S), an endogenous volatile mediator with pleiotropic functions, promotes vasorelaxation, exerts anti-inflammatory actions and regulates angiogenesis. Previously, the SH-containing angiotensin-converting enzyme inhibitor (ACEI), zofenopril, was identified as being effective in preserving endothelial function and inducing angiogenesis among ACEIs. Based on the H2 S donor property of its active metabolite zofenoprilat, the objective of this study was to evaluate whether zofenoprilat-induced angiogenesis was due to increased H2 S availability. EXPERIMENTAL APPROACH: HUVECs were used for in vitro studies of angiogenesis, whereas the Matrigel plug assay was used for in vivo assessments. KEY RESULTS: Zofenoprilat-treated HUVECs showed an increase in all functional features of the angiogenic process in vitro. As zofenoprilat induced the expression of CSE (cystathionine-γ-lyase) and the continuous production of H2 S, CSE inhibition or silencing blocked the ability of zofenoprilat to induce angiogenesis, both in vitro and in vivo. The molecular mechanisms underlying H2 S/zofenoprilat-induced angiogenesis were dependent on Akt, eNOS and ERK1/2 cascades. ATP-sensitive potassium (KATP ) channels, the molecular target that mediates part of the vascular functions of H2 S, were shown to be involved in the upstream activation of Akt and ERK1/2. Moreover, the up-regulation of fibroblast growth factor-2 was dependent on CSE-derived H2 S response to H2 S and KATP activation. CONCLUSIONS AND IMPLICATIONS: Zofenoprilat induced a constant production of H2 S that stimulated the angiogenic process through a KATP channel/Akt/eNOS/ERK1/2 pathway. Thus, zofenopril can be considered as a pro-angiogenic drug acting through H2 S release and production, useful in cardiovascular pathologies where vascular functions need to be re-established and functional angiogenesis induced.

Nitric oxide modulates the angiogenic phenotype of middle-T transformed endothelial cells.

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 N(w)-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-beta1 and all VEGF isoforms, VEGF165 being predominantly expressed. NOS inhibition reduced the basal expression of VEGF isoforms, while it markedly potentiated TGF-beta1 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.

Distinct capillary density and progression promoted by vascular endothelial growth factor-A homodimers and heterodimers.

The aim of this study was to characterize the capillary density, progression and persistence of new capillaries induced by different isoforms of vascular endothelial growth factor (VEGF)-A. They were produced and purified using the same protocol and assessed in the same experimental model, the rabbit cornea assay. Monogenic homodimers for VEGF121 and VEGF165 together with the heterodimer VEGF121/165 were tested as slow release polymer pellets implanted into the avascular rabbit cornea and examined up to 18 days post-implantation. The implants consistently stimulated angiogenesis in the absence of inflammation. The VEGF121 isoform produced the strongest increase of new capillary vessels which rapidly and persistently progressed into the corneal stroma. VEGF165 promoted the growth of a smaller number of capillaries which ten-ded to regress over time. Heterodimers of VEGF121/165 produced intermediate in vivo activities between the two homodimers. In vitro endothelial cell proliferation, mobilization and adhesion were promoted by all VEGF isoforms under serum-free or serum-reduced conditions with the same order of potency. Anti-soluble KDR (sKDR) antibody completely inhibited the effects of all the isoforms. These results indicate that monogenic homodimer preparations of VEGF121 and VEGF165 can display distinct biological effects which are functionally retained after the heterodimeric assembly of VEGF121 and VEGF165. The observed different biological behavior of the VEGF isoforms reveals the possibility that in vivo the assembly of dimers derived from splicing of a single gene may yield molecules with either different matrix or receptor interaction, stability or diffusion rate according to specific needs.

The effect of linomide on the migration and the proliferation of capillary endothelial cells elicited by vascular endothelial growth factor.

In order to assess the mechanism of action of the quinoline-3-carboxyamide linomide as an antiangiogenic drug, the effect of linomide was studied in vitro on postcapillary endothelial cells exposed to vascular endothelial growth factor (VEGF). Linomide did not block the spontaneous replication of endothelial cells, but significantly suppressed endothelial cell growth and migration elicited by VEGF. It is concluded that linomide appears to be an effective tool to inhibit VEGF-dependent angiogenesis.

NK1-receptors mediate the proliferative response of human fibroblasts to tachykinins.

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.

B1 receptor involvement in the effect of bradykinin on venular endothelial cell proliferation and potentiation of FGF-2 effects.

1. Bradykinin (BK) contributes to the inflammatory response inducing vasodilation of postcapillary venules and has been demonstrated to induce neovascular growth in subcutaneous rat sponges. 2. In this study the ability of BK to stimulate cell growth and migration in cultured endothelium from coronary postcapillary venules (CVEC) has been investigated. 3. [3H]-thymidine incorporation in subconfluent and synchronised CVEC was used to monitor DNA synthesis over 24 h. BK promoted a concentration-dependent increase of DNA synthesis with maximal activity at 100 nM. At this concentration BK also induced 18 fold accumulation of c-Fos protein immunoreactivity in the nucleus within 1 h from peptide exposure. 4. The total number of cells recovered after 48 h exposure to BK was increased in a concentration-dependent manner. Maximal effect was produced by 100 nM concentration of the peptide which produced 50% increase in cell number. The selective B1 receptor agonist Des-Arg9-BK mimicked the proliferative effect of BK, while the B2 receptor agonist kallidin was devoid of any activity. The proliferation induced by BK was abolished in a concentration-dependent manner by the addition of the B1 selective antagonist Des-Arg9-Leu8-BK, while the selective B2 receptor antagonist HOE140 did not modify BK-induced growth. 5. DNA synthesis and growth promoted by a threshold concentration of fibroblast growth factor-2 (FGF-2) (0.25 nM) were potentiated by increasing concentrations of BK and Des-Arg9-BK. 6. Endothelial cell migration assessed by the Boyden Chamber procedure was not promoted by BK or the selective B1 and B2 receptor agonists. 7. These data are the first demonstration that BK promotes growth of endothelial cells from postcapillary venules. The mitogenic activity of BK involves c-Fos expression and potentiates the growth promoting effect of FGF-2. Only the B1 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.

Distinct chemotactic and angiogenic activities of peptides derived from Kaposi's sarcoma virus encoded chemokines.

The vMIPs are chemokine-like proteins expressed by the Kaposi's sarcoma-associated herpesvirus (KSHV/HHV8) during the lytic phase of viral infection. vMIP-I activates CCR8, a chemokine receptor expressed by Th2 lymphocytes and cultured monocytes. vMIP-II is an agonist for CCR3, a receptor expressed by eosinophils, and an antagonist for several other chemokine receptors. Both are highly angiogenic in the chick chorio-allantoic membrane. We designed and tested three 26-mer peptides, derived from vMIP-I (pK-I), from vMIP-II (pK-II) and from the control MIP-1alpha (pM), spanning key residues of chemokines. pK-I, pK-II and pM all were able to activate a strong chemotactic response in monocytes, higher than parental vMIP-I and II. This corresponded to induction of calcium fluxes in these cells, typical of chemokines. Interestingly, pK-II and pM were also active on PMN neutrophils. In vivo studies (matrigel sponge and rabbit cornea models) showed that pK-I retains the strong angiogenic potential exerted by vMIP-I, while pK-II and pM induced an inflammatory response, probably mediated by PMN recruitment. Our observations indicate that chemokine-derived peptides can show biological activity at pharmacological concentrations. pK-I, in particular, displays the angiogenic activity of full-length vMIP-I, while all peptides appear to have acquired additional properties, stimulating new cellular targets.

Effect of selective tachykinin receptor antagonists on the growth of human skin fibroblasts.

The effect of synthetic selective tachykinin receptor antagonists was studied on the growth of cultured human skin fibroblasts (HF). Selective antagonists for the NK1 receptor ([D-Pro4, D-Trp7,9,Phe11]-SP(4-11), GR71251 and L 668,169) and the NK2 receptor (L 659,877) were tested against Substance P (SP), against the selective NK1 receptor agonist [beta-Ala4,Sar9, Met(O2)11]-SP(4-11) and against basic Fibroblast Growth Factor (bFGF). All the selective NK1 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 NK1 receptor agonist. The selective NK2 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.

Calcitonin gene-related peptide selectively increases cAMP levels in the guinea-pig ureter.

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.

Endothelial cell migration is induced by soluble P-selectin.

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.

Substance P induces migration of capillary endothelial cells: a novel NK-1 selective receptor mediated activity.

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.

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial 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.