Vaccinia virus-mediated expression of human erythropoietin in tumors enhances virotherapy and alleviates cancer-related anemia in mice.

Recombinant human erythropoietin (rhEPO), a glycoprotein hormone regulating red blood cell (RBC) formation, is used for the treatment of cancer-related anemia. The effect of rhEPO on tumor growth, however, remains controversial. Here, we report the construction and characterization of the recombinant vaccinia virus (VACV) GLV-1h210, expressing hEPO. GLV-1h210 was shown to replicate in and kill A549 lung cancer cells in culture efficiently. In mice bearing A549 lung cancer xenografts, treatment with a single intravenous dose of GLV-1h210 resulted in tumor-specific production and secretion of functional hEPO, which exerted an effect on RBC progenitors and precursors in the mouse bone marrow, leading to a significant increase in the number of RBCs and in the level of hemoglobin. Furthermore, virally expressed hEPO, but not exogenously added rhEPO, enhanced virus-mediated green fluorescent protein (GFP) expression in tumors and subsequently accelerated tumor regression when compared with the treatment with the parental virus GLV-1h68 or GLV-1h209 that expressed a nonfunctional hEPO protein. Moreover, intratumorally expressed hEPO caused enlarged tumoral microvessels, likely facilitating virus spreading. Taken together, VACV-mediated intratumorally expressed hEPO not only enhanced oncolytic virotherapy but also simultaneously alleviated cancer-related anemia.

Vasorelaxation induced by L-glutamate in porcine coronary arteries.

Isolated porcine coronary arteries (PCA) contracted by depolarization with high K0 or by histamine (10 microM) were relaxed concentration-dependently by glutamic acid, aspartic acid, N-methyl-D-aspartate (NMDA) and, gamma-aminobutyric acid (GABA). In the PCA preparations contracted by high K0 or histamine the effects were monophasic, but the histamine-induced effects were more sustained and of larger amplitude. The ED50 values of cumulative concentration-response (CCR) curves obtained for the relaxation induced by L-glutamate in histamine-stimulated PCA preparations were shifted from 0.8 mM to 0.25 microM in presence of 1 mM glycine, a co-agonist required for the activation of NMDA receptors. The relaxations resulting from low-affinity binding of L-glutamic were dependent on Ca0 as evidenced by the shift of CCR curves to the right in the presence of 5-100 mM K0. In contrast, CCR curves obtained for contractions induced by NaF (1.5-12 mM), were significantly shifted to the left (from 6.3 to 3.1 mM). A depression of the maximum effect observed at higher F- concentrations was reversed by addition of 5 mM Mg0. Data show that glutamate induces a vasorelaxation that may be associated with symptoms seen in Chinese restaurant syndrome.

Effects of manipulations of cytoplasmic pH on the mechanical responses of isolated porcine coronary arteries.

Isolated porcine coronary arteries contracted transiently when exposed to 20 mM Na salts of organic acids (acetate, propionate, butyrate and lactate) at constant external pH (pH0 = 7.4). The peak of these phasic contractions, completely inhibited by 1 mM amiloride, amounted to about 10% of that elicited by depolarization with 50 mM K0. The slope of the relaxing branch of the phasic contractions induced by 20 mM acetate in HEPES buffered physiological salt solution decreased from 1.05 +/- 0.12 to 0.49 +/- 0.07 mN/min (S.E.M.; n = 14), when Na0 was reduced from 137 to 20 mM. In Na free HEPES-buffered PSS only sustained contractions were obtained. The EC50 values for the concentration-response curves for contractions induced by K-propionate (2-20 mM) in Tris-PSS were 8.2 +/- 0.5 mM, n = 6. After total isoosmotic replacement of external Na+ by sucrose in bicarbonate-buffered PSS, contractions induced by 20 mM K-propionate had the same height as those induced with 50 mM K0+. Longlasting exposure to 0 Ca0 PSS did not affect significantly contractions induced by 20 mM propionate. During an exposure to procaine (10(-4) M) acetate-induced contractions were transiently and significantly enhanced, when induced in bicarbonate buffered PSS, but not in HEPES buffered PSS. In normally polarized PCA preparations 20 mM NH4+ induced both sustained relaxations and polyphasic responses; in depolarized PCA preparations only polyphasic responses were evoked. The time course of the polyphasic responses to application and removal of weak bases (NH(4+)-pulse technique) or to removal and reintroduction of CO2/HCO3- paralleled exactly the expected pHi perturbations, as they have been described in a great variety of cells and tissues, alkalinization leading however to relaxation, acidification to contraction. In presence of NH4+, a transient relaxation (phase 1) was immediately followed by an overshooting contraction (phase 2); at removal of NH4+ a rebound contraction (phase 3) was observed, reaching a peak and changing thereafter to a relaxation (phase 4), the slope of which was sensitive to variation of the external Na0 concentration. These mechanical effects were abolished by 1 hr exposure to 0 Ca PSS. Phase 2 was enhanced 2-5 fold in presence of TEA, Ba ions, Na3 VO4 or beta-methyldigoxin. Contractile responses of PCA preparations activated by various agonists (acetylcholine, histamine and serotonin) to pHi manipulations were similar and generally amplified when compared to unactivated preparations.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of vasoactive drugs on fluoride-induced contractions of vascular smooth muscle in calcium-free solution.

Cumulative dose-response curves were obtained for contractions induced by fluoride in bovine facial veins and arteries in the presence and absence of external Ca (Cao). In 1.4 mmol/l Cao the threshold for contractions was lower for veins than for the arteries, i.e. at 1 mmol/l vs. 2 mmol/l F-. After Ca withdrawal from the external medium, the ED50 values for veins and arteries were shifted from 2.2 to 10 and from 4.5 and 8.5 mmol/l F-, respectively. Several vasodilators (order of potency to relax veins: caffeine less than verapamil less than sulmazole less than sodium nitroprusside less than papaverine) were investigated for their ability to inhibit contractions induced by F- in Ca2+-free solution. None of them was able to counteract F- induced contractions in Ca2+-free solutions. Furosemide and beta-methyldigoxine were also ineffective. However, nitroprusside, sulmazole and papaverine were able to relax strips pretreated with Ca2+-free solutions. By contrast, contractions of depolarized veins induced by F- in Ca2+-free solutions were significantly, though transiently, inhibited by 10 mmol/l ammonium ions. The vasodilators tested in this study seem thus to act by inhibiting Ca influx at some level of the membrane, while F- may act by mobilizing Ca ions from some unidentified, cellular pools not accessible to the drugs tested in this study.

Relaxant effect of trans-resveratrol on isolated porcine coronary arteries.

Recent studies provided evidence that trans-resveratrol (3,4',5-trihydroxystilbene, found in high concentrations in some red wines, may possibly decrease the risk of coronary heart disease mortality. The aim of this study, performed with large epicardial porcine coronary arteries (PCA) strips, was to investigate the relaxant effect of trans-resveratrol on these main conductance vessels, which have been described to be pathologically prone for vasospastic contractions. The data show that the tonic component of the biphasic contractions induced by histamine, as well as the contractions induced by F- ions (10 mmol/l), which activate G proteins downstream of the receptors, could dose-dependently be inhibited by trans-resveratrol (0.1-100 mumol/l). The EC50 values of the dose-response curves established for the inhibition of the sustained component of histamine-induced contractions were very similar to those obtained for the relaxations of fluoride-induced contractions: 0.45 +/- 0.08 and 0.29 +/- 0.05 mumol/l, resp. (n = 6). Ouabain (10 mumol/l)-induced contractions and rhythmic contractions elicited by tetraethylammonium (12 mmol/l) were also strongly inhibited by trans-resveratrol (20 mumol/l). It may be inferred from the results obtained in this study, that the relaxation of the coronary conductance vessels induced by trans-resveratrol is possibly based on a nongenomic interaction with steroid-like receptors located on the cell membrane.

Cyclic AMP and the potassium-induced vasodilation.

The cAMP concentration of bovine facial arteries (helical strips) increased from 0.84 +/- 0.14 to 1.51 +/- 0.24 nmole/g wet weight, when the strips were relaxed by exposure to a physiological salt solution containing 10 instead of 2.7 mM KCl. A similar result (increase from 1.13 +/- 0.15 to 2.07 +/- 0.29 nmole cAMP/g wet weight) was obtained in strips pretreated with 10(-7) g/ml beta-methyldigoxin. These strips reacted to 10 mM KCl with a constriction. Addition of the phosphodiesterase activator N-methyl-imidazole (3 - 10(-3)M) did not affect the mechanical response to 10 mM KCl. It is concluded that for the potassium-induced vasodilation cAMP does not have a mediating function comparable to that in beta-adrenergic relaxation of vascular smooth muscle.