Improving arterial prosthesis neo-endothelialization: application of a proactive VEGF construct onto PTFE surfaces.

The formation of a confluent endothelium on expanded polytetrafluoroethylene (PTFE) vascular prostheses has never been observed. This lack of endothelialization is known to be one of the main reasons leading to the development of thromboses and/or intimal hyperplasia. In this context, several efforts were put forward to promote endothelial cell coverage on the internal surface of synthetic vascular prostheses. The goal of the present study was to immobilize the vascular endothelial growth factor (VEGF) onto Teflon PTFE surfaces to generate a proactive polymer construct favoring interaction with endothelial cells. An ammonia plasma treatment was first used to graft amino groups on PTFE films. Subsequent reactions were performed to covalently bind human serum albumin (HSA) on the polymer surface and to load this protein with negative charges, which allows adsorbtion of VEGF onto HSA via strong electrostatic interactions. X-ray photoelectron spectroscopy (XPS) experiments along with surface derivatization strategies were performed between each synthesis step to ascertain the occurrence of the various molecules surface immobilization. Finally, the electrostatic binding of VEGF to the negatively charged HSA matrix was performed and validated by ELISA. Endothelial cell adhesion and migration experiments were carried out to validate the potential of this VEGF-containing biological construct to act as a proactive media toward the development of endothelial cells.

Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis.

Elevated expression of plasminogen activator inhibitor-1 (PAI-1) in tumors is associated with a poor prognosis in many cancers. Reduced tumor growth and angiogenesis have also been reported in mice deficient in PAI-1. These results suggest that PAI-1 may be required for efficient angiogenesis and tumor growth. In the present study, we demonstrate that PAI-1 can both enhance and inhibit the growth of M21 human melanoma tumors in nude mice and that this appears to be due to PAI-1 regulation of angiogenesis. Quantitative analysis of angiogenesis in a Matrigel implant assay indicated that in PAI-1 null mice angiogenesis was reduced approximately 60% compared with wild-type mice, while in mice overexpressing PAI-1, angiogenesis was increased nearly 3-fold. Furthermore, addition of PAI-1 to implants in wild-type mice enhanced angiogenesis up to 3-fold at low concentrations but inhibited angiogenesis nearly completely at high concentrations. Together, these data demonstrate that PAI-1 is a potent regulator of angiogenesis and hence of tumor growth and suggest that understanding the mechanism of this activity may lead to the development of important new therapeutic agents for controlling pathologic angiogenesis.

Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1.

The process of angiogenesis is important in both normal and pathologic physiology. However, the mechanisms whereby factors such as basic fibroblast growth factor promote the formation of new blood vessels are not known. In the present study, we demonstrate that exogenously added plasminogen activator inhibitor-1 (PAI-1) at therapeutic concentrations is a potent inhibitor of basic fibroblast growth factor-induced angiogenesis in the chicken chorioallantoic membrane. By using specific PAI-1 mutants with either their vitronectin binding or proteinase inhibitor activities ablated, we show that the inhibition of angiogenesis appears to occur via two distinct but apparently overlapping pathways. The first is dependent on PAI-1 inhibition of proteinase activity, most likely chicken plasmin, while the second is independent of PAI-1's anti-proteinase activity and instead appears to act through PAI-1 binding to vitronectin. Together, these data suggest that PAI-1 may be an important factor regulating angiogenesis in vivo.

The chick embryo metastasis model.

The growth and dissemination of malignant tumors continues to have a devastating impact on people throughout the United States and the rest of the world. In fact, it is estimated that well over a half a million new cases of cancer will be diagnosed per year (1). The most commonly used clinical approaches to treat cancer include surgical removal of the primary tumor, chemotherapy, and radiation, all of which have varying degrees of success. Importantly, a major obstacle contributing to the failure of treatment in many cases involves the metastatic dissemination of tumor cells from the primary tumor mass to distant sites. While some progress has been achieved in understanding the complex biochemical and molecular mechanisms regulating tumor invasion, much remains to be learned.

New functions for non-collagenous domains of human collagen type IV. Novel integrin ligands inhibiting angiogenesis and tumor growth in vivo.

Collagen type IV is a major component of the basal lamina of blood vessels. Six genetically distinct collagen type IV chains have been identified and are distributed in a tissue-specific manner. Here we define a novel function for soluble non-collagenous (NC1) domains of the alpha2(IV), alpha3(IV), and alpha6(IV) chains of human collagen type IV in the regulation of angiogenesis and tumor growth. These NC1 domains were shown to regulate endothelial cell adhesion and migration by distinct alpha(v) and beta(1) integrin-dependent mechanisms. Systemic administration of recombinant alpha2(IV), alpha3(IV), and alpha6(IV) NC1 domains potently inhibit angiogenesis and tumor growth, whereas alpha1(IV), alpha4(IV), and alpha5(IV) showed little if any effect. These findings suggest that specific NC1 domains of collagen type IV may represent an important new class of angiogenesis inhibitors.

Integrin alpha(v)beta3 promotes M21 melanoma growth in human skin by regulating tumor cell survival.

Growth and dissemination of malignant melanoma has a profound impact on our population, and little is known concerning the mechanisms controlling this disease in humans. Evidence is provided that integrin alpha(v)beta3 plays a critical role in M21 melanoma tumor survival within human skin by a mechanism independent of its known role in angiogenesis. Antagonists of alpha(v)beta3 blocked melanoma growth by inducing tumor apoptosis. Moreover, M21 melanoma cell interactions with denatured collagen, a known ligand for alpha(v)beta3, caused a 5-fold increase in the relative Bcl-2:Bax ratio, an event thought to promote cell survival. Importantly, denatured collagen colocalized with alpha(v)beta3-expressing melanoma cells in human tumor biopsies, suggesting that alpha(v)beta3 interaction with denatured collagen may play a critical role in melanoma tumor survival in vivo.

Mechanisms of action of antimalarials in inflammation: induction of apoptosis in human endothelial cells.

Antimalarials are beneficial therapeutic agents in systemic lupus and rheumatoid arthritis. These autoimmune diseases have abnormally low apoptosis of inflammatory cells. Both disorders have an abnormal angiogenesis. In the present report, antimalarials were demonstrated to selectively increase apoptosis of HUVECs in vitro. A 24-h exposure to 50 or 150 microM of the drugs was associated with a significant loss of substrate-adherent cells. Chloroquine exhibited an inhibitory effect on HUVEC proliferation over 7 days. Programmed cell death in HUVECs rendered nonadherent by chloroquine was confirmed by the induction of DNA fragmentation in floating cells. Northern blot analysis revealed a rapidly increased expression of the bcl-x(s) gene without any change in the expression of the bcl-2 gene, indicating that HUVECs under chloroquine were undergoing apoptosis. The onset of the apoptotic cascade in HUVECs appeared shortly after the addition of chloroquine. The effect of chloroquine on apoptosis was distinct from acute cell lysis and was restricted to HUVECs. Antimalarials also induced IL-1alpha production. In parallel, chloroquine alone did not increase the expression of IL-6. Anti-IL-1alpha Ab or IL-1Ra only marginally reversed chloroquine-induced depression of proliferation for the low drug concentration, but not the massive cell death effect at and above 50 microM. Taken together, these data may indicate that antimalarials repress angiogenesis. The autocrine mechanism involving IL-1alpha accounts only for a minor fraction of the full antiendothelial effect of chloroquine, which is mainly dependent on apoptosis.

Pathologic leukocyte infiltration of the rabbit aorta confers a vasomotor effect to chemotactic peptides through cyclooxygenase-derived metabolites.

We determined the effect of chemotactic peptides FMLP and C5a, postulated to be relatively selective activators of phagocytes, on the thoracic aorta of rabbits subjected to experimental pathologies that allowed infiltration by leukocytes, i.e., dietary atherosclerosis and serum sickness. Aortic ring tissues isolated from hypercholesterolemic rabbits, precontracted or not by phenylephrine, exhibited a rapid and relatively sustained (10 to 20 min) contractile response when challenged by FMLP (10 nM and above); precontracted tissues also responded to C5a (2.5 nM and above). Aortic rings from rabbits with serum sickness (13 days post-BSA injection) exhibited brief contractions that were often followed by a relaxation in phenylephrine-precontracted tissues. In both models, tissues from normal weight-matched animals were not consistently responsive to these peptides. The cyclooxygenase inhibitor indomethacin extensively reduced the contractile effect of either peptide on precontracted aortic rings in both models. Chemotactic peptide-induced increased prostanoid secretion was evident only in the fluid bathing atherosclerotic aortic rings. Morphologic correlations included the demonstration of cells positive for the RAM-11 macrophage marker and the C5a receptors in tissues from rabbits with hypercholesterolemia (numerous clusters of cells) or serum sickness (modest infiltration). Control aortic rings responded to FMLP by a significant contraction if cultured for 2 h in the presence of resident peritoneal cells (84% macrophages), but not in the presence of a high density of PBL (less than 0.5% monocytes). Infiltrating or adherent macrophages in the blood vessel wall confer to some phagocyte activating peptides the role of eicosanoid-dependent vasoconstrictor agents.

Further analysis of the upregulation of bradykinin B1 receptors in isolated rabbit aorta by using metabolic inhibitors.

Kinins exert a contractile effect that develops as a function of the in vitro incubation time with isolated rabbit aorta. This response is mediated via receptors of the bradykinin B1 type and interleukin-1 amplifies this upregulation process. Tissues continuously treated with the protein synthesis inhibitor cycloheximide (71 microM) or with the protein trafficking inhibitor, brefeldin A (18 microM), failed to develop a contractile response to the bradykinin B1 receptor agonist, des-Arg9-bradykinin (1.7 microM) (72-100% inhibition of kinin response recorded at 3 or 6 h), whether or not they were exposed to interleukin-1 beta (290 pM). The protein glycosylation inhibitor tunicamycin exerted a selective and significant, but partial (50-76%), inhibition of des-Arg9-bradykinin-induced responses. The biochemical effect of the metabolic inhibitors on the tissue has been validated in assays involving incorporation of [3H]leucine and of [3H]mannose into protein or glycoprotein fractions, respectively. The modulatory effects of metabolic inhibitors on the responses to kinins of the isolated rabbit aorta support the idea that a de novo formation of membrane bradykinin B1 receptors is the molecular basis of both the spontaneous and the interleukin-1-stimulated upregulation phenomenon.

Recombinant human hemoglobin inhibits both constitutive and cytokine-induced nitric oxide-mediated relaxation of rabbit isolated aortic rings.

A genetically engineered recombinant human hemoglobin (rHb1.1) was recently developed for use as a blood substitute (Nature 1992;356:258-60). Like other mammalian hemoglobin (Hb) molecules, it might bind and antagonize the actions of nitric oxide (NO). We used an isolated rabbit aortic ring preparation to examine the ability of rHb1.1 to inhibit acetylcholine (ACh)- and interleukin-1 beta (IL-1 beta)-induced reductions of vasoconstrictor responses to the alpha-adrenoceptor agonist phenylephrine (PE). rHb1.1 (0.04-4.4 microM) rapidly and reversibly inhibited, in a concentration-dependent manner, both ACh- and IL-1 beta-induced decreases in PE contractile responses. These inhibitory effects of rHb1.1 were non-competitive and were equipotent to those of purified, cell-free human Hb (p.hHb). These two forms of soluble Hb were at least 10 times more potent than Hb in erythrocytes (red blood cells: RBC-Hb). Both NG-nitro-L-arginine (10 microM) a NO synthase inhibitor, and LY-83583 (10 microM), a guanylyl cyclase inhibitor, mimicked the effects of rHb1.1. The inhibitory effects of rHb1.1 were not shared by either human serum albumin (HSA 44 microM), which combines with but does not deactivate NO, or cytochrome C (44 microM), a heme-containing protein that does not bind NO; neither were they reversed by L-arginine (L-ARG) (1 mM), the presumed NO precursor. These and other results suggest that the chemical antagonism of NO is likely to be the mechanism by which rHb1.1 and other Hbs inhibit ACh- and IL-1 beta-induced decreases in the response to PE in rabbit aortic rings.

Epidermal growth factor-induced rapid relaxation of the isolated rabbit mesenteric artery.

Epidermal growth factor (EGF) (10-100 ng ml-1) induced a rapidly developing relaxation of precontracted rabbit mesenteric artery rings within 30 min of exposure. Indomethacin or protein synthesis inhibitors prevented or acutely reversed the effect of EGF on the preparation and an erbstatin analogue significantly reduced it. It is concluded that the EGF-mediated relaxation may be induced by the rapid production of prostanoids via a cascade of biochemical events initiated by the tyrosine-kinase activity of the receptors for EGF. One or more proteins rapidly produced and rapidly depleted appear to be involved in the activation of arachidonate metabolism.

Rapid protein synthesis and turnover is involved in interleukin-1-induced relaxation of the rabbit isolated mesenteric artery. Analysis of the arachidonate cascade.

The rabbit isolated superior mesenteric artery, when precontracted with phenylephrine, relaxes in a sustained manner in less than 30 min when exposed to recombinant interleukin-1 (IL-1), and this is a prostaglandin (PG)-dependent, endothelium- and 5-lipoxygenase-independent process. We have studied the dependency of IL-1-induced relaxation on protein synthesis and trafficking using isolated rings of rabbit mesenteric arteries. Three chemically unrelated protein synthesis inhibitors (PSIs) (cycloheximide, anisomycin and puromycin) completely prevented the response to IL-1. Moreover, the PSIs reversed IL-1-induced relaxations within 15 to 30 min of application. The amplitude and/or the duration of the relaxation induced by arachidonic acid and iloprost (a PGI2 mimetic) were partially inhibited by cycloheximide treatment, but not those induced by nitroglycerin or by cromakalim. However, tissues initially relaxed by IL-1 and then recontracted by a PSI are still able to relax when challenged with either arachidonic acid or iloprost, suggesting that cyclooxygenase is not depleted and that the responsiveness to PGs is intact under these conditions. Manoalide, thioether amide glycerophosphocholine (type II phospholipase A2 inhibitors) or brefeldin A (an inhibitor of intracellular protein trafficking) did not inhibit IL-1-induced relaxation. The data suggest the involvement of newly synthesized protein(s) with a very rapid turnover in the vascular response to IL-1. PSIs probably act at several levels to inhibit the relaxant response to IL-1, but depletion of tissue cyclooxygenase does not appear to be the limiting mechanism for the PSI effect on IL-1-induced relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of interleukin-1 receptor antagonist on three types of responses to interleukin-1 in rabbit isolated blood vessels.

Interleukin-1 receptor antagonist (IRA) is a secretory product of human monocytes or related cell lines that acts as a pure interleukin-1 (IL-1) antagonist in several bioassays. IRA administration was reportedly a life-saving intervention in rabbits injected with lethal doses of bacterial lipopolysaccharide (LPS). We report the inhibitory effect of IRA on three distinct types of vascular responses to IL-1 in rabbit isolated blood vessels. The rabbit isolated superior mesenteric artery, when precontracted with phenylephrine, relaxed in a sustained manner in less than 30 min following application of recombinant interleukin-1 beta (12-290 pM), and this was a prostaglandin (PG)-dependent and endothelium-independent process. IRA (human recombinant sequence; 0.9-15 nM) behaved as an antagonist of IL-1 alpha or IL-1 beta, based on the surmountability and the concentration dependence, but could only prevent the effect of IL-1, not reverse it. IRA had no direct effect on the preparation and did not influence the acute relaxing effect elicited by substance P or iloprost, a PGI2 mimetic. Exposure to IL-1 beta depressed the response to noradrenaline (NA) in several hours in rabbit aorta rings. The inhibitory effect of IL-1 beta was endothelium and prostaglandin independent, but was prevented by a treatment with NG-nitro-L-arginine (a nitric oxide synthesis inhibitor), cycloheximide, dexamethasone, or IRA. Using the residual NA-induced contraction as a quantification of the IL-1 agonist effect, IRA was a very potent antagonist of IL-1 beta but was not totally surmountable.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation of the antimicrobial activity of bacitracin USP from its renovascular effects.

Bacitracin is a nephrotoxic antibiotic that has recently been shown to induce contractile effects in aortas isolated from rabbits by stimulating receptors for 5-hydroxytryptamine (5-HT). The possible renovascular actions of this antibiotic were investigated. Bacitracin USP increased the vascular resistance in a concentration-dependent manner (9 to 175 micrograms/ml) in rat kidneys perfused with a constant flow of Krebs solution. This was significantly inhibited by 5-HT antagonists, but only partially at the higher bacitracin concentration. An antagonist of the chemotactic peptide fMet-Leu-Phe failed to influence the pressor effect of bacitracin in rat kidneys. Indomethacin modestly reduced the effect of all potent pressor agents in the rat organ. Bacitracin USP was separated in several fractions by using C18 reverse-phase chromatography. Two distinct fractions were vasoconstrictive when infused in rat kidneys; both fractions were 5-HT mimetics. These peaks were different from the major antibiotic peak, bacitracin A, which was identified by using analytical high-pressure liquid chromatography, mass spectrometry, and inhibition of Micrococcus luteus growth. The less polar vasoactive peak corresponded to at least two minor peptides of the bacitracin family. The most abundant of these vasoactive peptides had no direct contractile effect on an aorta isolated from a rabbit, but a preliminary metabolic study in rat kidneys suggests that it is apparently transformed into a potent 5-HT agonist that is active on the aorta preparation. Bacitracin A, the major constituent of bacitracin with antimicrobial activity, had no vasoconstrictor effect in the test systems that we used; however, we did rule out the possibility that the renovascular stimulants found in the bacitracin mixture do not derive spontaneously or by biotransformation from the antibacterial forms of bacitracin.

Synergism between the contractile effect of epidermal growth factor and that of des-Arg9-bradykinin or of alpha-thrombin in rabbit aortic rings.

1. Rabbit aortic rings were used to test the possible contractile effects of growth factors and their interaction with other stimuli. A rapid potentiation of kinin-induced contraction by epidermal growth factor (EGF) has been previously observed in this preparation. 2. EGF (5-1500 ng ml-1) and the isoform BB of platelet-derived growth factor (PDGF-BB; 1-126 ng ml-1) exerted modest but sustained contractile effects in rabbit aortic rings. 3. EGF pretreatment (100 ng ml-1) potentiated the contractile responses to des-Arg9-bradykinin (des-Arg9-BK), an agonist of the B1 receptors for kinin found in this preparation, and to human alpha-thrombin but not to several other contractile stimuli. The interaction appeared also relatively selective for the growth factor, because PDGF-BB pretreatment potentiated neither des-Arg9-BK nor alpha-thrombin-induced contraction. 4. EGF, applied on a contraction plateau induced by des-Arg9-BK or alpha-thrombin, exerted a synergistic contractile effect, with a time course and a half-maximal concentration for EGF-induced contraction similar to the ones recorded in resting tissues (between 67 and 220 ng ml-1, depending on the series of experiments). 5. The direct or synergistic contractile effects of EGF were not modified by the removal of the endothelium or by treatment with indomethacin. However, the tyrosine kinase inhibitors, erbstatin or genistein, inhibited the synergistic effect of EGF with des-Arg9-BK. The small direct contractile effect of EGF was significantly reduced by genistein. The synergistic effect of EGF with alpha-thrombin was comparatively more resistant to the tested tyrosine kinase inhibitors.6. An inhibitor of the catalytic activity of alpha-thrombin, D-Phe-Pro-Arg-CH2Cl, prevented the contractile effect of x-thrombin in the aortic rings. In this system, a tetradecapeptide derived from a recently cloned alpha-thrombin receptor was a contractile stimulus at and above 10 microM. Consistent with the hypothesis that this peptide could behave as an alpha-thrombin receptor agonist, its contractile effect was potentiated by EGF pretreatment. Pharmacological evidence was provided to show that the receptors for alpha-thrombin were distinct from the B, receptors for kinins. Together, these findings suggest that a model of a cleavable receptor recently elaborated to account for alpha-thrombin effects on human platelets is valid in blood-free vascular smooth muscle preparations such as the rabbit isolated aorta.7. The synergism between EGF and kinin- or alpha-thrombin-induced contractions constitutes a novel mode of myotropic action for growth factors. The synergism is probably dependent on the tyrosine kinase activity of receptors for EGF. These combinations of stimuli could occur in various types of vascular disease and account for abnormal vascular reactivity often associated with atheroma lesions or vascular wound healing.

Human interleukin-1 induces a rapid relaxation of the rabbit isolated mesenteric artery.

1. Strips of rabbit superior mesenteric artery, precontracted with phenylephrine, relaxed when exposed to human recombinant interleukin-1 (IL-1) of the alpha or beta types. The effect was observed within 10 min, was optimal 32 min after the application of the cytokines and concentration-dependent (12-290 pM). 2. IL-1 alpha and IL-1 beta were equipotent in relaxing the rabbit mesenteric artery. A synthetic fragment corresponding to IL-1 beta 163-171 was approximately one million fold less active than IL-1 beta. The tripeptide Lys-D-Pro-Thr, an analogue of IL-1 beta 193-195, was inactive as an antagonist of IL-1 beta on the preparation. 3. Indomethacin (2.8 microM) prevented or acutely reversed IL-1-induced relaxations in the rabbit mesenteric artery. Purified haemoglobin (10 microM) or the removal of endothelium had no effect on relaxations elicited by IL-1 beta. 4. The preparation exhibited some selectivity for IL-1 as recombinant human tumour necrosis factor-alpha (TNF-alpha), IL-2 or IL-6 failed to influence it. TNF-alpha was not synergistic with a subthreshold concentration of IL-1 beta. 5. Immunoreactive 6-keto-prostaglandin F1 alpha and prostaglandin E2 were increased in the bathing fluid of isolated mesenteric arteries exposed to IL-1 beta as compared to controls. 6. A supernatant of lipopolysaccharide-stimulated human monocytes produced a relaxation of the preparation with a profile similar to that produced with IL-1s and there was a good quantitative agreement between the extent of the relaxation and the enzyme immunoassay measurements of IL-1 alpha and IL-1 beta in the supernatant.Furthermore the relaxation of crude monocyte IL-i was prevented by preincubating with antibodies to IL-l alpha and IL-1 beta. This experiment illustrates the possible use of the preparation for bioassay of IL-1. 7. It is concluded that either form of IL-I relaxes the precontracted rabbit mesenteric artery by a prostaglandin-dependent, nitric oxide-independent mechanism. The model is also useful for distinguishing the mechanism of IL-1-induced hypotension in vivo in rabbits.

Relaxant effect of chemotactic peptides on rabbit vascular strips: evidence for nitric oxide release from a nonendothelial source.

Two distinct peptides, C5a and f-Met-Leu-Phe (FMLP), that are chemotactic for phagocytic leukocytes affect profoundly the circulation in various in vivo systems. These peptides are known to relax strips of rabbit isolated blood vessels, the portal vein and pulmonary artery. In the present study, the effect of recombinant human C5a (2.5-25 nM) was examined and found to be qualitatively similar to that previously reported for FMLP. Indomethacin completely or partially inhibited the vasorelaxations induced by either peptide in the pulmonary artery and the portal vein, respectively. The relaxation induced by C5a was not abolished by removing the endothelial lining of the model vessels. The C5a or FMLP effects were more tachyphylactic in tissues continuously exposed to cycloheximide; this phenomenon was particularly pronounced for FMLP. A series of experiments were focused on the indomethacin-resistant component of the relaxation induced by either peptide on the portal vein. This component was not inhibited by capsaicin pretreatment or by endothelium removal, but was suppressed by treatment with NG-nitro-L-arginine or reduced by LY-83583. These findings suggest that the chemotactic peptides elicit their mechanical effect on rabbit vascular smooth muscle through the release of secondary mediators not related to the endothelium; the mediators are tentatively identified as prostaglandins and nitric oxide. It is the coordinated combinations of the metabolic pathways that are involved in the final responses, with inherent differences between vessel sources and the agonists used.

Contractile effect of the chemotactic factors f-Met-Leu-Phe and C5a on the human isolated umbilical artery. Role of cyclooxygenase products and tissue macrophages.

Factors that are chemotactic for phagocytic leukocytes are known to elicit important acute circulatory changes, and the role of circulating leukocytes in these models is controversial. To evaluate the role of the blood vessel wall in the absence of circulating cells, spiral strips of human umbilical artery were exposed in vitro to the chemotactic peptides f-Met-Leu-Phe (1-100 nM) or C5a (2.5-25 nM). Contractile responses were observed for both peptides. Certain agonist analogues and a selective antagonist of the chemotactic action of f-Met-Leu-Phe behaved correspondingly as agonists and antagonist of the contractile effect on umbilical artery. The anaphylatoxin C3a also exerted a contractile effect on the tissue (25 nM and above), but this effect was highly tachyphylactic. Inhibitory drugs were used to examine the contributions of secondary mediators in eliciting the effects of C5a and f-Met-Leu-Phe. The contractile effect of both peptides was massively inhibited either by indomethacin or the thromboxane A2/prostaglandin H2 antagonist SQ 29548. Dazmegrel, a thromboxane A2 synthetase inhibitor, had partial inhibitory effects on contractions induced by either peptide. The contractile effect of C3a was prevented by indomethacin pretreatment. Vascular strips did not release measurable histamine in the bathing fluid after challenge with C5a or f-Met-Leu-Phe. The tissue apparently contains neither histamine nor mast cells. Autoradiography of 125I-labeled C5a or f-Met-Leu-Phe analogue showed specific binding of the peptides to cells dispersed in the vessel wall, but more frequently at the periphery. Cells stained positively for alpha-naphthyl acetate esterase showed a similar distribution. Pure cultures of smooth muscle cells derived from the umbilical artery failed to release prostanoids when exposed to f-Met-Leu-Phe or C5a, whereas fresh strips of this artery released more thromboxane B2 than the baseline in response to these peptides. We conclude that macrophagelike cells, present in the vessel wall, are the likely target cells for the chemotactic peptides. These cells trigger a contractile effect of the smooth muscle by generating cyclooxygenase products.