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The Food and Drug Administration recently approved the new drug avacopan for a relatively rare disease, anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. Avacopan is an antagonist of receptor-1 for anaphylatoxin C5a (C5aR

Aryl chloroethyl ureas (CEUs) are new protein alkylating agents exhibiting anticancer activity both in vitro and in vivo. We report herein that14C-labeled CEU derivatives, designated CEU-025 and CEU-027, covalently bind to thioredoxin-1 (TRX1). Covalent binding of these molecules slightly decreases the disulfide-reducing activity of recombinant TRX1, when compared with the effect of strong thioalkylating agents such as N-ethylmaleimide. Moreover, site-directed mutagenesis and diamide competition assays demonstrated that TRX1 cysteinyl residues are not the prime targets of CEUs. CEU-025 abrogates the nuclear translocation of TRX1 in human cancer cells. In addition, we show that CEU-025 can block TRX1 nuclear translocation induced by cisplatin. Unexpectedly, pretreatment with sublethal CEU-025 concentrations that block TRX1 nuclear translocation protected the cells against cisplatin cytotoxicity. Overexpression of TRX1 in HT1080 fibrosarcoma cells attenuated CEU-025 cytotoxicity, while its suppression using TRX1-specific siRNA increased the effects of CEU-025, suggesting that loss of function of TRX1 is involved, at least in part, in the cytotoxic activity of CEU-025. These results suggest that CEU-025 and CEU-027 exhibit anticancer activity through a novel, unique mechanism of action. The importance of TRX1 and the dependence of the cytotoxicity of CEU-025 and CEU-027 on TRX1 intracellular localization are also discussed.

The blood vessels are one of the important target tissues for the mediators of inflammation and allergy; further cytokines affect them in a number of ways. We review the use of the isolated blood vessel mounted in organ baths as an important source of pharmacological information. While its use in the bioassay of vasoactive substances tends to be replaced with modern analytical techniques, contractility assays are effective to evaluate novel synthetic drugs, generating robust potency and selectivity data about agonists, partial agonists and competitive or insurmountable antagonists. For instance, the human umbilical vein has been used extensively to characterize ligands of the bradykinin B(2) receptors. Isolated vascular segments are live tissues that are intensely reactive, notably with the regulated expression of gene products relevant for inflammation (e.g., the kinin B(1) receptor and inducible nitric oxide synthase). Further, isolated vessels can be adapted as assays of unconventional proteins (cytokines such as interleukin-1, proteases of physiopathological importance, complement-derived anaphylatoxins and recombinant hemoglobin) and to the gene knockout technology. The well known cross-talks between different cell types, e.g., endothelium-muscle and nerve terminal-muscle, can be extended (smooth muscle cell interaction with resident or infiltrating leukocytes and tumor cells). Drug metabolism and distribution problems can be modeled in a useful manner using the organ bath technology, which, for all these reasons, opens a window on an intermediate level of complexity relative to cellular and molecular pharmacology on one hand, and in vivo studies on the other.

Restenosis, the re-occlusion of a diseased vessel following a surgical intervention, is a major cause of failure of angioplasty, stenting, and bypass grafting with natural and synthetic vessels. In healthy vessels, the endothelium exerts a control over smooth muscle cell (SMC) proliferation and migration. Unfortunately, revascularization procedures damage the endothelium of natural vessels and bypass vessels are completely devoid of endothelial cells. Many strategies have been developed to inhibit SMC proliferation and reduce intimal hyperplasia, yet most of the drugs tested thus far simultaneously inhibit endothelialization and do not selectively target SMCs. The ideal biological agent should have anti-proliferative effects on SMCs while preserving vascular healing and endothelialization so as to prevent late thrombosis. Imatinib mesylate is a specific inhibitor of three tyrosine kinase receptors, two of which, PDGF-R and c-Kit, are implicated in the pathogenesis of intimal hyperplasia. In this study, we investigated in vitro the potential of imatinib mesylate to inhibit SMCs and its effect on ECs. Our findings indicate that low doses of imatinib mesylate successfully inhibit SMC proliferation. Furthermore, at these concentrations, the drug was not only harmless to ECs, but also enhanced their proliferation. In light of these in vitro results, imatinib mesylate shows potential as a good candidate to inhibit intimal hyperplasia without delaying neo-endothelialization.

Recently, a subset of N-phenyl-N'-(2-chloroethyl)ureas (CEU) was found abrogating the nuclear translocation of thioredoxin-1 and arresting the cell cycle in G(0)/G(1) phase. Several derivatives were prepared to assess their effect on cell cycle progression and on the intracellular location of Trx-1. Compounds 1-20, 21-40, and 41-60 exhibited GI(50) between 1 and 80 microM. Immunocytochemistry analysis showed compounds 4, 6, 8, 10, 11, 23, 24, 26-31, 34, 37, 41, 44, 46-51, 53, 56, and 57 inhibiting the nuclear translocation of Trx-1. Our results suggest that increasing the electrophilic character of these molecules might enhance the antiproliferative activity at the expense of the selectivity toward thioredoxin-1 and the G(0)/G(1) phase arrest.

This study presents two-step and multistep reactions for modifying the surface of plasma-functionalized poly(tetrafluoroethylene) (PTFE) surfaces for subsequent conjugation of biologically relevant molecules. First, PTFE films were treated by a radiofrequency glow discharge (RFGD) ammonia plasma to introduce amino groups on the fluoropolymer surface. This plasma treatment is well optimized and allows the incorporation of a relative surface concentration of approximately 2-3.5% of amino groups, as assessed by chemical derivatization followed by X-ray photoelectron spectroscopy (XPS). In a second step, these amino groups were further reacted with various chemical reagents to provide the surface with chemical functionalities such as maleimides, carboxylic acids, acetals, aldehydes, and thiols, that could be used later on to conjugate a wide variety of biologically relevant molecules such as proteins, DNA, drugs, etc. In the present study, glutaric and cis-aconitic anhydrides were evaluated for their capability to provide carboxylic functions to the PTFE plasma-treated surface. Bromoacetaldehyde diethylacetal was reacted with the aminated PTFE surface, providing a diethylacetal function, which is a latent form of aldehyde functionality. Reactions with cross-linkers such as sulfo-succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB) were evaluated to provide a highly reactive maleimide function suitable for further chemical reactions with thiolated molecules. Traut reagent (2-iminothiolane) was also conjugated to introduce a thiol group onto the fluoropolymer surface. PTFE-modified surfaces were analyzed by XPS with a particular attention to quantify the extent of the reactions that occurred on the polymer. Finally, surface immobilization of fibronectin performed using either glutaric anhydride or sulfo-SMPB activators demonstrated the importance of selecting the appropriate conjugation strategy to retain the protein biological activity.

The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to β-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

Little is known about the interaction of tumor cells with host vascular smooth muscle cells. In reconstitution experiments, tumorigenic cell lines (including the rat hepatocarcinoma Morris 7777 and human melanoma M-21) were cultured for 17 hr in the presence of rat aortic rings, subsequently evaluated in contractility assays (response to phenylephrine and KCl). An agonist-independent loss of contractility was observed in rings pre-incubated with either tumorigenic cell lines or their conditioned medium (CM). The depressing effect of Morris cells depends largely on the expression of inducible nitric oxide synthase (iNOS) in smooth muscle cells and was reversed by an inhibitor of this enzyme; iNOS immunoreactivity was verified in some muscular vessels at the periphery of tumors formed by the Morris cell line in rats. The M-21 melanoma produces cytotoxicity in rat aortic rings (presence of single stranded DNA, cleavage of PARP, in differentiated smooth muscle only), accounting for the irreversible loss of contractility. The cytotoxicity produced by M-21 CM is not dependent on NO. Gel filtration of CM suggests that both the iNOS- and cytotoxicity-inducing substances from Morris hepatoma cells or M-21 cells, respectively, are mainly of low molecular weight (1 kDa or less). Other cell lines derived from rat or human tumors produce minimal effects on the rat aorta smooth muscle (H4-II-E-C3) or an irreversible anergy (RBL, MDA-MB-231, HEP-3B, HEP G2). The results emphasize that inhibition of vascular smooth muscle is relevant to tumor biology both by modulation of tumoral hemodynamics and by influencing the state of vessel maturation. © 2004 Wiley-Liss, Inc.

Two types of receptors (B1R, B2R) for kinins are defined in mammalian species. Comparative experiments involving recombinant fusion proteins consisting of rabbit B1R or B2R fused to GFP-related proteins are exploited to study the regulation of the response to kinins at the receptor level. The following points will be briefly reviewed and supported by some novel data. (1) The constitutive B2Rs are internalized upon agonist stimulation, but completely recycled to the cell surface; however, B2R destruction can be achieved following limited proteolysis (extracellular trypsin, neutrophil proteases), a plausible down-regulation mechanism in pathology. (2) The inducible B1Rs, stimulated by des-Arg9-kinins, are not phosphorylated nor internalized upon agonist stimulation, but rather undergo a reversible redistribution to caveolae-related rafts. B2Rs are also subjected to this translocation, but only transiently (before endocytosis). (3) Based on the analysis of rabbit aortic smooth muscle cells, B1R induction by cytokines is dependent on nuclear factor KB in rabbit vascular tissue, but exogenous kinins acting on either receptor type do not induce B1R expression.

Menstrual cycle activity predisposes to ovarian epithelial tumors based on numerous epidemiological studies. We showed that the hormones involved in menstrual cycle regulation modulate cell cycle activity in these tumors in an accompanying paper. We investigated whether such hormones could also influence angiogenesis, an important determinant of tumor progression, in the same tumors. Treatment with progesterone (P4) stimulated VEGF protein secretion in 4 of 5 ovarian carcinoma cell lines examined. Northern blot analyses performed in MCV50 cells showed that this effect was accompanied by increased VEGF mRNA levels. P4 also stimulated VEGF promoter activity in these cells. Estradiol (E2) showed a similar, but substantially smaller effect on VEGF secretion which was additive to that of P4. Conditioned medium from P4-treated cells strongly stimulated angiogenesis on chicken chorio-allantoic membranes. Incubating the conditioned medium with a neutralizing anti-VEGF antibody, but not with non-specific immunoglobulins abolished this effect. Angiogenic activity was not altered by treatment of the membranes with P4 directly. We conclude that P4 can stimulate angiogenic activity via induction of VEGF secretion in some ovarian epithelial tumors. Therapeutic use of progestins may be most effective when administered in combination with an anti-angiogenic agent, at least against a subset of ovarian carcinomas.

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.

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.

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 α2(IV), α3(IV), and α6(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 αv and β1integrin-dependent mechanisms. Systemic administration of recombinant α2(IV), α3(IV), and α6(IV) NC1 domains potently inhibit angiogenesis and tumor growth, whereas α1(IV), α4(IV), and α5(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.

Forty one 4-(2-oxoimidazolidin-1-yl)benzenesulfonamide derivatives (I) are synthesized and screened for their biological activity.

Sixty-one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) and 13 of their tetrahydro-2-oxopyrimidin-1(2H)-yl analogues (PPB-SOs) were prepared and biologically evaluated. The antiproliferative activities of PIB-SOs on 16 cancer cell lines are in the nanomolar range and unaffected in cancer cells resistant to colchicine, paclitaxel, and vinblastine or overexpressing the P-glycoprotein. None of the PPB-SOs exhibit significant antiproliferative activity. PIB-SOs block the cell cycle progression in the G(2)/M phase and bind to the colchicine-binding site on β-tubulin leading to cytoskeleton disruption and cell death. Chick chorioallantoic membrane tumor assays show that compounds 36, 44, and 45 efficiently block angiogenesis and tumor growth at least at similar levels as combretastatin A-4 (CA-4) and exhibit low to very low toxicity on the chick embryos. PIB-SOs were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships.

The importance of the bridge linking the two phenyl moieties of substituted phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonates (PIB-SOs) was assessed using a sulfonamide group, which is a bioisostere of sulfonate and ethenyl groups. Forty one phenyl 4-(2-oxoimidazolidin-1-yl)benzenesulfonamide (PIB-SA) derivatives were prepared and biologically evaluated. PIB-SAs exhibit antiproliferative activities at the nanomolar level against sixteen cancer cell lines, block the cell cycle progression in G 2 /M phase, leading to cytoskeleton disruption and anoikis. These results were subjected to CoMFA and CoMSIA analyses to establish quantitative structure-activity relationships. These results evidence that the sulfonate and sulfonamide moieties are reciprocal bioisosteres and that phenylimidazolidin-2-one could mimic the trimethoxyphenyl moiety found in the structure of numerous potent antimicrotubule agents. Finally, compounds 16 and 17 exhibited potent antitumor and antiangiogenic activities on HT-1080 fibrosarcoma cells grafted onto chick chorioallantoic membrane similar to CA-4 without significant toxicity for the chick embryos, making this class of compounds a promising class of anticancer agents.

Seven subsets of aromatic urea and amide analogues of N-phenyl-N'-(2-chloroethyl)ureas (CEU) have been synthesized by nucleophilic addition of 3-chloropropylisocyanate, 2-chloroacetylisocyanate, ethylisocyanate, 2-chloroacetyl chloride, 3-chloropropanoyl chloride, 4-chlorobutanoyl chloride, and acryloyl chloride, respectively, to selected anilines or benzylamines to afford 3-chloropropylureas (1, CPU), 2-chloroacetylureas (2, CAU), ethylureas (3, EU), 2-chloroacetamides (4, CA), 3-chloropropionamides (5, CPA), 4-chlorobutyramides (6, CBA) and acrylamides (7, Acr). The molecular structure of these compounds has been confirmed by IR, (1)H and (13)C NMR, and MS spectra and their purity also confirmed by HPLC. The CEU analogues were evaluated for their antiproliferative activity against three human tumor cell lines, namely human colon carcinoma HT-29, human skin melanoma M21, and human breast carcinoma MCF-7. CAU (2c to 2g), CA (4a to 4d, 4f and 4 g), CPA (5a) and Acr (7a and 7b) had IC(50) ranging from 1.4 to 25 microM. CAU, CA, CPA and Acr exhibited interesting antiproliferative activity through mechanism(s) of action unrelated to the acylation of glutamic acid at position 198 on beta-tubulin that is characterizing CEU.

Development of antimitotic binding to the colchicine-binding site for the treatment of cancer is rapidly expanding. Numerous antimicrotubule agents are prepared every year, and the determination of their binding affinity to tubulin requires the use of purified tubulins and radiolabeled ligands. Such a procedure is costly and time-consuming and therefore is limited to the most promising candidates. Here, we report a quick and inexpensive method that requires only usual laboratory resources to assess the binding of antimicrotubules to colchicine-binding site. The method is based on the ability of N,N'-ethylene-bis(iodoacetamide) (EBI) to crosslink in living cells the cysteine residues at position 239 and 354 of β-tubulin, residues which are involved in the colchicine-binding site. The β-tubulin adduct formed by EBI is easily detectable by Western blot as a second immunoreacting band of β-tubulin that migrates faster than β-tubulin. The occupancy of colchicine-binding site by pertinent antimitotics inhibits the formation of the EBI: β-tubulin adduct, resulting in an assay that allows the screening of new molecules targeting this binding site.

The majority of contrast agents used in magnetic resonance imaging (MRI) is based on the rare-earth element gadolinium. Gadolinium-based nanoparticles could find promising applications in pre-clinical diagnostic procedures of certain types of cancer, such as glioblastoma multiforme. This is one of the most malignant, lethal and poorly accessible forms of cancer. Recent advances in colloidal nanocrystal synthesis have led to the development of ultra-small crystals of gadolinium oxide (US-Gd(2)O(3), 2-3 nm diameter). As of today, this is the smallest and the densest of all Gd-containing nanoparticles. Cancer cells labeled with a sufficient quantity of this compound appear bright in T(1)-weighted MRI images. Here we demonstrate that US-Gd(2)O(3) can be used to label GL-261 glioblastoma multiforme cells, followed by localization and visualization in vivo using MRI. Very high amounts of Gd are efficiently internalized and retained in cells, as confirmed with TEM and ICP-MS. Labeled cells were visualized in vivo at 1.5 T using the chicken embryo model. This is one more step toward the development of "positively contrasted" cell tracking procedures with MRI.

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.

The expression of tissue factor (TF) in tumors reportedly exacerbates the agressiveness of several types of cancers. The shedding of TF-containing membrane particles is believed to influence the ability of tumors to expand and metastasize, and these microparticles may also be harmful in the onset of disseminated intravascular coagulation in specific cancers. Futhermore, the intracellular signaling that is elicited after the formation of the TF / coagulation factor VIIa complex at the cell membane modulates the activity of adhesion molecules and mitogen-activated protein (MAP) kinases. To evaluate whether TF overexpression in tumor cells modulates its shedding and neighboring stromal cells by its catalytic or intracellular activity, TF-GFP (green fluorescent protein) and a tailless form (TFΔC-GFP) were stably expressed in the rat Morris hepatoma and human HT1080 fibrosarcoma cell lines. Both TF proteins were efficiently produced by tumor cells and functionally active, and their clotting activity could be blocked by the active site-inhibited factor VIIa (ASIS). TF-expressing tumorigenic cells produced a soluble factor that increased the migration of arterial smooth muscle cells in vitro. This effect was abrogated by ASIS and the PAR-1 receptor antagonist ATAP-2, showing that it is dependent on the proteolytic activity of the TF ligand factor VIIa and the thrombin-activated cell membrane receptor. We propose that TF-containing microparticles that are released in the culture medium by tumor cells influence the migratory behavior of neighboring stromal cells, thus aiding the cancer cell’s tumorigenic potential.

Computational tools such as CoMSIA and CoMFA models reported in a recent study revealed the structure-activity relationships ruling the interactions occurring between hydrophobic N-phenyl-N'-(2-chloroethyl)ureas (CEU) and the colchicine-binding site (C-BS) on beta(II)-tubulin. Here, we describe the mechanisms involved in the covalent binding of three subsets of CEU derivatives to the C-BS. The FlexiDock experiments confirmed that the interaction of non-covalent portions of the CEU auxophore moiety of CEU is involved in the binding of the drug to the C-BS facilitate the nucleophilic attack of Glu-beta198 rather than Cys-beta239. In addition, these studies suggest that Cys-beta239 together with Asn-alpha99, Ser-alpha176, Thr-alpha177, Leu-beta246, Asn-beta247, Ala-beta248, Lys-beta252 and Asn-beta256 are implicated in the stabilization of a C-BS-CEU complex prior to the acylation of Glu-beta198 by CEU. Our molecular models propose the formation of a stabilized C-BS-CEU complex before the completion of the Glu-beta198 acylation; acylation triggering conformational changes of beta-tubulin, microtubule depolymerization and anoikis. The computational models presented here might be useful to the design of selective and more potent C-BS inhibitors. Of interest, in vivo acylation of acidic amino acid residues by xenobiotics is an unusual reaction and may open new approaches for the design of irreversible protein inhibitors such as tubulin.

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.

Prolonged in vitro incubation of rabbit aortic rings allows recording contractile responses mediated by the inducible bradykinin B(1) receptors; addition of interleukin (IL)-1 or epidermal growth factor (EGF) to the bathing fluid increases the rate of sensitization, a process partially inhibited by the nonspecific Tyr-kinase inhibitor genistein. The recent development of specific inhibitors for receptor associated Tyr-kinase activities (tyrphostin AG 1478 for EGF receptor, sunitinib for VEGF receptor and others) allows assessing the role of such signaling molecules in this process. AG 1478 reduced the potentiating effects of exogenous EGF, and also the spontaneous sensitization to the agonist des-Arg(9)-bradykinin. Sunitinib or GM 6001, a wide spectrum inhibitor of metalloproteinases, had no effect on these responses. In rabbit aortic smooth muscle cells, the cytokines IL-1beta and EGF increased the density of binding sites for [(3)H]Lys-des-Arg(9)-bradykinin in 4 h; AG 1478 reduced only the effect of exogenous EGF. IL-1 receptor antagonist decreased both the effect of IL-1beta and of EGF in rabbit smooth muscle cells. EGF was weakly and slowly coupled to nuclear factor-kappaB nuclear translocation in these cells, as compared to the effect of IL-1beta. EGF-induced EGF receptor autophosphorylation and ERK1/2 phosphorylation was selectively inhibited by AG 1478 in smooth muscle cells; Lys-des-Arg(9)-bradykinin did not transactivate EGF receptor in these cells. While the Tyr-kinase activity sensitive to AG 1478 is recruited by tissue damage and exogenous EGF to upregulate bradykinin B(1) receptors in freshly isolated aortas, this signaling system interacts with others (e.g., IL-1) for the optimal expression of B(1) receptors.

Aryl chloroethylureas (CEUs) are potent anti-neoplastic agents alkylating specific intracellular proteins such as beta(II)-tubulin. Recently we have identified a new subset of CEU derived from compound 36 that alkylates thioredoxin isoform 1 (Trx-1), inhibits the nuclear translocation of Trx-1, and favors the accumulation of cells in G(0)/G(1) phase. We have evaluated the effects of various substituents and their position on the aromatic ring of a series of derivatives of 36 on (i) the anti-proliferative activity, (ii) the cell cycle progression, (iii) the nuclear translocation of Trx-1, and (iv) their covalent binding to beta-tubulin. The same experiments were performed on representative CEU derivatives where the 2-chloroethyl amino moiety is replaced by either an ethyl, a 2-aminooxazolinyl or a 2-chloroacetyl group. On one hand, our results suggest that CEUs substituted on the phenyl ring at position 3 or 4 by cycloalkyl and substituted cycloalkyl or cycloalkoxy groups inhibit the nuclear translocation of Trx-1 and arrest the cell cycle progression in G(0)/G(1). On the other hand, CEUs substituted by a fused aromatic ring, an aliphatic chain, or a fused aliphatic ring are alkylating beta(II)-tubulin but not Trx-1. Beside the expected inactivity of the ethylurea derivatives, none of the modification to the electrophilic moiety led to cross-selectivity of the drugs toward beta-tubulin but increased the anti-proliferative activity and resulted in mitigated effects on Trx-1 translocation.

1-(2-Chloroethyl)-3-(4-cyclohexylphenyl)urea (cHCEU) has been shown to abrogate the presence of thioredoxin-1 into the nucleus through its selective covalent alkylation. In the present letter we have evaluated the structure-activity relationships of the substituents at positions 3 and 4 of the phenyl ring of cHCEU derivatives on cell cycle progression and thioredoxin-1 nuclear translocation. Active CEU derivatives exhibited GI(50) ranging from 1.9 to 49muM on breast carcinoma MCF-7, skin melanoma M21, and colon carcinoma HT-29 cells. On one hand, compounds 1, 2, 9c, 10c, 13, and 14 arrested the cell cycle in G(2)/M phase while CEUs 3, 4, 5c, 6c, 11c, and 12c blocked the cell division in G(0)/G(1) phase. On the other hand, CEUs 2-4, 5c, 7c, 8c, 11c, and 12c abrogated the translocation of thioredoxin-1 while the other CEU derivatives were inactive in that respect. Our results suggest that CEU substituted on the phenyl ring at position 3 or 4 by lower cycloalkyl or cycloalkoxy groups arrest cell progression in G(0)/G(1) phase through mechanism of action different from their antimicrotubule counterparts, presumably via thioredoxin-1 alkylation and modulation of its activity. The mechanism of action of these new molecules is still undetermined. However, the significant accumulation of cells in G(0)/G(1) phase suggests that these molecules may act similarly to known chemopreventive agents against cancers. In addition, the inhibition of Trx-1 nuclear localization also suggests the abrogation of an important chemoresistance mechanism towards a variety of chemotherapeutic agents.

Surface coating of synthetic materials is often considered to improve biomedical devices biocompatibility. In this study, we covalently bound fibronectin (FN) onto ammonia plasma-treated PTFE via two crosslinkers, namely glutaric anhydride (GA) and sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-SMPB). With respect to clean PTFE, cell adhesion increased markedly on both FN grafted surfaces, although it was twice higher on PTFE-GA-FN than on PTFE-SMPB-FN. ELISA experiments performed with a polyclonal antibody revealed that the amount of FN is identical on both surfaces while monoclonal antibody specific to the RGD binding site clearly demonstrated a greater availability when FN is surface grafted through GA. These results provide evidence of a variation in protein conformation correlated with the surface conjugation strategy.

Chloroethylureas (CEU) are soft alkylating agents that covalently bind to β-tubulin (βTAC) and affect microtubule polymerization dynamics. Herein, we report the identification of a CEU subset and its corresponding oxazolines, which induce cell growth inhibition, apoptosis, and microtubule disruption without alkylating β-tubulin (N-βTAC). Both βTAC and N-βTAC trigger the collapse of mitochondrial potential (ΔΨm) and modulate reactive oxygen species levels, following activation of intrinsic caspase-8 and caspase-9. Experiments using human fibrosarcoma HT1080 respiratory-deficient cells (ρ0) and uncoupler of the mitochondrial respiratory chain (MRC) showed that βTAC and N-βTAC impaired the MRC. ρ0 cells displayed an increased sensitivity toward N-βTAC as compared with ρ+ cells but, in contrast, were resistant to βTAC or classic chemotherapeutics, such as paclitaxel. Oxazoline-195 (OXA-195), an N-βTAC derivative, triggered massive swelling of isolated mitochondria. This effect was insensitive to cyclosporin A and to Bcl-2 addition. In contrast, adenine nucleotide translocator (ANT) antagonists, bongkrekic acid or atractyloside, diminished swelling induced by OXA-195. The antiproliferative activities of the N-βTACs CEU-025 and OXA-152 were markedly decreased in the presence of atractyloside. Conversely, pretreatment with cyclosporin A enhanced growth inhibition induced by βTAC and N-βTAC. One of the proteins alkylated by N-βTAC was identified as the voltage-dependent anion channel isoform-1, an ANT partner. Our results suggest that βTAC and N-βTAC, despite their common ability to affect the microtubule network, trigger different cytotoxic mechanisms in cancer cells. The role of mitochondria in these mechanisms and the potential of N-βTAC as a new therapeutic approach for targeting hypoxia-resistant cells are discussed. [Cancer Res 2007;67(5):2306–16]

A number of N -phenyl- N ′-(2-chloroethyl)ureas ( CEUs ) have been shown to be potent antimitotics through their covalent binding to the colchicine-binding site on intracellular β-tubulin. The present communication aimed to evaluate the role of the electrophilic 2-chloroethyl amino moiety of CEU on cell growth inhibition and the specificity of the drugs as irreversible antagonists of the colchicine-binding site. To that end, several N -phenyl- N ′-(2-ethyl)urea ( EU ), N -phenyl- N ′-(2-chloroethyl)urea ( CEU ), N -aryl amino-2-oxazoline ( OXA ), and N -phenyl- N ′-(2-chloroacetyl)urea ( CAU ) derivatives were prepared and tested for their antiproliferative activity, their effect on the cell cycle, and their irreversible binding to β-tubulin. EU derivatives were devoid of antiproliferative activity. CEUs ( 2h – 2i , 2k , 2l , OXA 3e , 3h , 3i , 3k , 3l , tBCEU, and ICEU), OXA ( 3h , 3i , 3k , 3l , tBOXA, and IOXA), and CAU ( 4a – 4m , tBCAU, and ICAU) had GI 50 between 1.7 and 10 μM on three tumor cell lines. Cytotoxic CEU and OXA arrested the cell cycle in G 2 /M phase, while the corresponding CAU were not phase specific. Finally, Western blot analysis clearly showed that only CEUs 2h , 2k , 2l , tBCEU, ICEU and OXA 3h , 3i , 3k , 3l , tBOXA ,and IOXA were able to bind irreversibly to the colchicine-binding site. Our results suggest that increasing the potency of the electrophilic moiety of the aromatic ureas enhances their antiproliferative activity but decreases significantly their capacity to covalently bind to the colchicine-binding site.

Microtubule disruption provokes cytoskeleton and cell adhesion changes whose importance for apoptosis induction remains unclear. The present study focuses on the functional and the molecular adhesion kinetics that are induced by microtubule disruption-mediated apoptosis. We showed that antimicrotubules induce a biphasic sequence of adhesion response that precedes the onset of apoptosis and focal adhesion kinase hydrolysis. Antimicrotubules first induced an increase of the cellular adhesion paralleled by the raise of focal adhesion sites and actin contractility, which was followed by a sharp decrease of cell adhesion and disorganization of focal adhesion and actin stress fibers. The latter sequence of events ends by cell rounding, detachment from the extracellular matrix, and cell death. Microtubule-disrupting agents induced a sustained paxillin phosphorylation, before the activation of apoptosis, that requires the prior activation of extracellular signal-regulated kinase and p38 but not c-Jun NH 2 -terminal kinase. Interestingly, integrin-linked kinase overexpression rescued the antimicrotubule-mediated loss of cell viability. Altogether, these results propound that antimicrotubule agents induce anoikis through the loss of focal adhesion structure integrity.

MMPs are recognized to play a major role in tumor progression and metastasis of many forms of cancers. The purpose of this study was to compare the expression and activity of MMP-2 in uveal melanoma cell lines grown either in vitro on plastic culture plates or in vivo as tumors produced in chick embryos.The chick chorioallantoic membrane (CAM) model was used to evaluate the tumorigenic potential of uveal melanoma cell lines derived either from the primary uveal melanoma tumor isolated from three different patients (cell lines SP6.5, SP8.0, and TP31) or from a metastatic lesion derived from the liver of a patient diagnosed with uveal melanoma (cell line H79). The presence of MMP-2 in the vicinity of the tumor cells was determined by immunofluorescence analyses. Gelatin zymography was used for the detection of latent and activated forms of MMP-2 in uveal melanoma cell lines when grown in vitro on plastic, or in the solid tumors these cell lines produced in vivo on the CAM of the chick embryo. The gelatinase activity was quantified by densitometric analyses and the active/(active+pro-form) ratio was calculated as the MMP-2 activation ratio. Western blot analyses were performed to confirm the zymographic profile.Only the inactive form of MMP-2 was expressed and secreted in vitro by all uveal melanoma cell lines, higher levels being found for the liver-derived H79 cell line whereas SP8.0 only expressed MMP-2 to a very low level. On the other hand, all solid tumors produced in the CAM from these cell lines expressed and secreted, although to varying levels (SP6.5 and SP8.0, TP31 and H79), primarily the active form of MMP-2. Gelatinolytic activities of active MMP-2 were significantly higher in uveal melanoma tissues than in the non-neoplastic CAM, as revealed by the measurement of the activation ratio. The immunolocalization of MMP-2 revealed that all cell lines were MMP-2-positive although a reduced and more diffuse staining was observed for H79 and SP6.5 than in SP8.0 and TP31 cells.These results suggest the activation of proMMP-2 as an important event in the process of uveal melanoma progression. An elevated active to inactive MMP-2 ratio in the tumor environment of uveal melanoma suggests that a potential MMP-2 activity could be related to the progression of this type of cancer.

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.

Uveal melanoma is the most frequent primary intraocular tumor in the adult population. This malignancy has a high mortality rate and responds poorly to existing chemotherapy. Recently, the tumor environment has been found to exert a profound influence on drug response through cell interaction with components from the extracellular matrix (ECM). In the present study, we investigated whether individual components from the ECM may affect cell survival and/or cell death induced by the cytotoxic agent cisplatin on the SP6.5 uveal melanoma cell line. Tumor cells were shown by immunofluorescence analyses to be surrounded by the ECM proteins fibronectin (FN), type IV collagen (CIV) and laminin (LM), both at the primary and metastatic sites. Binding of SP6.5 cells to FN, LM and CIV is primarily dictated by the expression of membrane bound integrins from the beta1 family as revealed by cell adhesion assays conducted on ECM-coated culture plates. Analysis of cell death by flow cytometry demonstrated that culturing SP6.5 cells in the presence of FN, CIV and LM, substantially reduced the percentage of cells undergoing apoptosis after cisplatin treatment when compared with those seeded on a non-permissive matrix. These results suggest that adhesion of the SP6.5 uveal melanoma cells to the ECM proteins FN, CIV and LM might therefore confer resistance to the chemotherapeutic agent cisplatin. The cellular resistance induced by the ECM proteins toward cisplatin could explain in part the local recurrence of metastasis derived from uveal melanoma often observed clinically after chemotherapy.

The development of new anticancer agents with lower toxicity, higher therapeutic index, and weaker tendency to induce resistant phenotypes in tumor cells is a continuous challenge for the scientific community. Toward that end, we showed previously that a new class of soft alkylating agents designed as phenyl-3-(2-chloroethyl)ureas (CEUs) inhibits tumor cell growth in vitro and that their efficiency is not altered by clinically relevant mechanisms of resistance such as overexpression of multidrug resistance proteins, increase in intracellular concentration of glutathione and/or glutathione S-transferase activity, alteration of topoisomerase II, and increased DNA repair. Mechanistic studies have showed recently that the cytotoxic activity of several CEUs was mainly related to the disruption of microtubules. Here, we present results supporting our assumption that 4-tert-butyl-[3-(2-chloroethyl)ureido]phenyl (tBCEU) (and its bioisosteric derivative 4-iodo-[3-(2-chloroethyl)ureido]phenyl (ICEU) are potent antimicrotubule agents both in vitro and in vivo. They covalently bind to beta-tubulin, leading to a microtubule depolymerization phenotype, consequently disrupting the actin cytoskeleton and altering the nuclear morphology. Accordingly, tBCEU and ICEU also inhibited the migration and proliferation of endothelial and tumor cells in vitro in a dose-dependent manner. It is noteworthy that ICEU efficiently blocked angiogenesis and tumor growth in three distinct animal models: (a) the Matrigel plug angiogenesis assay; (b) the CT-26 tumor growth assay in mice; and (c) the chick chorioallantoic membrane tumor assay. In addition, we present evidence that CEU cytotoxicity is unaffected by additional resistance mechanisms impeding tumor response to DNA alkylating agents such as cisplatin, namely the cell adhesion mediated-drug resistance mechanism, which failed to influence the cytocidal activity of CEUs. On the basis of the apparent innocuousness of CEUs, on their ability to circumvent many classical and recently described tumor cell resistance mechanisms, and on their specific biodistribution to organs of the gastrointestinal tract, our results suggest that CEUs represent a promising new class of anticancer agents.

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.

Angiogenesis, the formation of new blood vessels from preexisting vessels, is an essential component of many normal biological processes such as embryonic development, wound healing, and endometrial maturation in premenopausal women (1–3). This process is similar to, but not identical with vasculogenesis, which is associated with the development of blood vessels from precursor cells termed angioblasts (4,5). Under normal physiological conditions the complex cellular events controlling vascular development are tightly regulated. However, when the molecular and biochemical mechanisms controlling angiogenesis are disrupted, uncontrolled neovascularization can contribute to a number of pathologies. In fact, several clinically important human diseases are characterized by abnormal vascular development including solid tumor growth, rheumatoid arthritis, diabetic retinopathy, and psoriasis (1–3,6–8). Thus, the pathological consequences of abnormal neovascularization impacts a large segment of the population and clearly demonstrates the need for an in depth understanding of the molecular mediators involved in the regulation of angiogenesis. To this end, an expanding body of work has identified a wide variety of molecules as potential targets for antiangiogenic strategies including a complex network of cytokines, cell adhesion receptors, proteolytic enzymes, and extracellular matrix components (9–11). Interestingly, many of these important discoveries were first identified by the use of in vitro and in vivo angiogenesis models.

Plasminogen activator inhibitor-1 (PAI-1) is the principal inhibitor of urokinase type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), and as such is thought to play an important role in the regulation of extracellular matrix remodeling. In blood, PAI-1 is bound to the adhesion protein vitronectin and is associated with vitronectin in fibrin clots and the provisional matrix. Elevated levels of PAI-1 are associated with atherosclerosis and an increased thrombotic tendency, while PAI-1 deficiency leads to increased fibrinolysis and bleeding. PAI-1 is also elevated in many solid tumors and is associated with a poor prognosis in cancer. PAI-1 has been shown to be a potent regulator of both vascular cell migration in vitro and of angiogenesis and tumor growth in vivo. PAI-1 can both promote and inhibit tumor growth and angiogenesis. Low concentrations of PAI-1 can stimulate tumor angiogenesis while treatment of animals with high doses of PAI-1 inhibits angiogenesis and tumor growth. Hence, PAI-1 appears to have a multifunctional role in regulating the migratory and fibrinolytic activity of vascular cells, and this, in turn, may help to explain the many varied actions of PAI-1.

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 B 1 type and interleukin-1 amplifies this upregulation process. Tissues continuosly treated with the protein synthesis inhibitor cycloheximide (71 μM) or with the protein trafficking inhibitor, brefeldin A (18 μM), failed to develop a contractile response to the bradykinin B 1 receptor agonist, des-Arg 9 -bradykinin (1.7 μM) (72−100% inhibition of kinin response recorded at 3 or 6 h), whether or not they were exposed to interleukin-1β (290 pM). The protein glycosylation inhibitor tunicamycin exerted a selective and significant, but partial (50−76%), inhibition of des-Arg 9 -bradykinin- induced responses. The biochemical effect of the metabolic inhibitors on the tissue has been validated in assays involving incorporation of [ 3 H]leucine and of [ 3 H]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 B 1 receptors is the molecular basis of both the spontaneous and the interleukin-1-stimulated upregulation phenomenon.

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.

PURPOSE. We investigated the involvement of MAPK signaling in the cell death mechanisms of classical microtubule interfering agents (MIA) and aryl-3-(2-chloroethyl)ureas (CEU) acting as antimitotics, along with CEU that don’t affect directly microtubules (non-MIA CEU). METHODS. To ascertain the activated signaling pathway profile of MIA and non-MIA CEU, Western blot, immunoprecipitation and transfection experiments were performed. RESULTS. Non-MIA CEU do not activate p38, as opposed to MIA, and the extent of ERK and JNK activation is lower than in response to MIA. The effect of MIA and non-MIA CEU on focal adhesion associated protein was also studied; MIA were shown to induce focal adhesion dismantlement associated with a sustained increase in paxillin phosphorylation and FAK cleavage, as opposed to non-MIA CEU. In addition, bcl-2 phosphorylation and AKT cleavage, induced by all MIA tested, was not observed in response to non-MIA CEU further emphasizing the differential cell death mechanisms induced by MIA and non-MIA CEU. Pharmacologic and genetic approaches emphasize that the ASK1-p38 pathway activation contributes to the cytotoxic mechanism of MIA, in contrast to non-MIA CEU. ASK1-p38 is important for increased paxillin phosphorylation and FAK cleavage, suggesting that ASK-1-p38 is an upstream event of FA structure dismantlement induced by MIA. Moreover, the endogen inhibitor of ASK-1, thioredoxin, is released from ASK-1 in response to MIA as opposed to non-MIA CEU. CONCLUSION. Our study supports that ASK1-p38 activation is an important signaling event, induced by MIA, which impairs focal adhesion structure and induces anchorage-dependent apoptosis or anoikis.

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.

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.

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.

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.