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Biomedical subjects

A Sachinidis

Publications and source records attributed to A Sachinidis.

At least 73 records · Page 4Linked to original sources
PubMed1996

Carbocyclic thromboxane A2 enhances the angiotensin II-induced DNA synthesis in smooth muscle cells.

The present study describes the influence of carbocyclic thromboxane A2 on the proliferative effects of angiotensin II on vascular smooth muscle cells. Angiotensin II (10(-7) M) and carbocyclic thromboxane A2 (10(-6) M) per se caused an increase in [3H]thymidine incorporation and cell number. The exposure of cells to both agonists resulted in a 2.5-fold elevation of the angiotensin II dependent effect on DNA synthesis and a 1.6-fold increase in cell number. 2-Ethoxy-1-[[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylic acid (CV-11974), the active metabolite of the specific non-peptide angiotensin AT1 receptor antagonist (+/-)-1-(cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1 H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1 H-benzimidazole-7-carboxylate (TCV-116, Candesartan) suppressed the effect of angiotensin II on cell growth as well as reduced the synergistic effect of carbocyclic thromboxane A2. Simultaneous cell stimulation with carbocyclic thromboxane A2 and angiotensin II for 30 min resulted in a 26 +/- 9% elevation of the angiotensin II-induced increase of c-fos mRNA (100%).

Angiotensin II↗
PubMed1996

Regulation of angiotensin AT1 receptor gene expression during cell growth of vascular smooth muscle cells.

Cell proliferation influences the expression of numerous tissue-specific genes. The angiotensin AT1 receptor is highly expressed on vascular smooth muscle cells where it mediates cell contraction upon activation with angiotensin II. Since vascular smooth muscle cell de-differentiation leads to differential expression of several genes, we investigated the effects of cell growth on angiotensin AT1 receptor gene expression in vascular smooth muscle cells in culture. Northern hybridization analysis revealed a decrease of angiotensin AT1 receptor mRNA levels to approximately 20% in proliferating cells in comparison to growth-arrested cells. There is a correlative loss of membrane-associated angiotensin AT1 receptor protein in growing cells versus non-growing cells, as assessed by saturation radioligand binding assays. In addition, the BB-isoform of platelet-derived growth factor (PDGF-BB), which induces proliferation of quiescent vascular smooth muscle cells, causes a marked down-regulation of angiotensin AT1 receptor mRNA. These data suggest that proliferation of vascular smooth muscle cells leads to reduced angiotensin AT1 receptor gene expression. The mechanisms underlying this process and its physiological implications remain to be defined.

Animals↗
PubMed1996

Competitive reverse transcription/polymerase chain reaction for the quantification of p53 and mdm2 mRNA expression.

Wild-type p53 (wtp53) is a tumour suppressor gene involved in cell cycle regulation. The mdm2 protein can complex with the p53 protein and influence its function as a regulator of cell growth. To detect and quantify wtp53 and mdm2 mRNA expression, we established the competitive reverse transcription/polymerase chain reaction for these genes and for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The target RNA differed from the competitor cRNA by having 183 bp, 205 bp and 173 bp deletions for p53, mdm2 and GAPDH, respectively. Target RNA and known concentrations of competitor cRNA were co-reverse transcribed and co-amplified with the same primers. Target cDNA and the corresponding competitor cDNA were amplified at the same efficiency.

Endothelium, Vascular↗
PubMed1996

Effect of the Na+/H+ antiport inhibitor Hoe 694 on the angiotensin II-induced vascular smooth muscle cell growth.

1. Hoe 694 (3-methylsulphonyl-4-piperidinobenzoyl)guanidine methanesulphonate) was characterized as a new, potent, non-amiloride inhibitor of the Na+/H+ exchanger. In order to elucidate the role of the Na+/H+ exchanger isoform 1 (NHE-1) in the regulation of vascular smooth muscle cell growth, we investigated the effects of different amiloride analogues and of Hoe 694 on angiotensin II-induced cell growth. Since intracellular pH, the intracellular free Ca2+ concentration and the expression of the transcription factor c-fos seem to be involved in the regulation of cell growth, the effects of the amiloride analogues and Hoe 694 on the angiotensin II-induced changes in these three parameters were examined. 2. Measurement of cytosolic Ca2+ and pH in cell monolayers was performed using fura-2/AM and BCECF/AM, respectively. The effect of angiotensin II on cell growth was examined using (1) [3H]-thymidine incorporation, (2) the bromo-2-deoxyuridine (BrdU) immunfluorescence assay, (3) the colorimetric determination of cell mitochondrial dehydrogenase activity and (4) determination of cell number. Total RNA was extracted from cells by the guanidinium isothiocyanate/CsCl procedure. The expression of c-fos was quantitated by Northern blotting. 3. Various amiloride analogues inhibited the angiotensin II-induced stimulation of the Na+/H+ exchanger, the increase in cytosolic Ca2+ and cell growth but not the induction of c-fos mRNA. Hoe 694 (1-25 microM) dose-dependently inhibited the angiotensin II-induced stimulation of the Na+/H+ exchanger but had no significant effects on cytosolic Ca2+, c-fos mRNA levels or cell growth. 4. Our findings support the concept that activation of the Na+/H+ exchanger is not essential for angiotensin II-induced vascular smooth muscle cell growth.

Angiotensin II↗
PubMed1996

Gangliosides GM1, GM2 and GM3 inhibit the platelet-derived growth factor-induced signalling transduction pathway in vascular smooth muscle cells by different mechanisms.

Gangliosides appear to regulate proliferation of different cell types. In the present study, we investigated the effects of gangliosides GM1, GM2 and GM3 on platelet-derived growth factor (PDGF)-induced vascular smooth muscle cell (VSMC) growth. In addition, we examined the effects of gangliosides on the PDGF-BB-dependent signalling transduction pathway in rat aortic VSMC. GM2 and GM1 inhibit the PDGF-BB-dependent receptor tyrosine autophosphorylation, stimulation of the PLC-gamma 1, increase of inositol-1,4,5-trisphosphate (InsP3), elevation in cytosolic free Ca2+ ([Ca2+]i), expression of the immediate early growth response gene c-fos and cell proliferation with the following rank order of potency GM2 > GM1. Although GM3 did not influence the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activation, it effectively inhibited the PDGF-BB-dependent InsP3 formation, [Ca2+]i and cell growth. Binding studies with 125I-PDGF-BB on VSMC in the presence and absence of 10 to 50 microM of each ganglioside revealed that GM1 and GM2 effectively inhibited the specific binding of PDGF-BB with an IC50 value of 20 microM for GM2 and 30 microM for GM1. GM3 had no significant effect on the specific 125I-PDGF-BB binding. These observations suggest that GM1 and GM2 may interact with PDGF-BB or its receptor resulting in a prevention of its binding. GM3 was able to suppress the PDGF-BB-dependent increase of InsP3 and [Ca2+]i downstream of the PDGF-BB-dependent receptor autophosphorylation and PLC-gamma 1 activity.

Animals↗
PubMed1995

Effects of TCV-116 and CV-11974 on angiotensin II-induced responses in vascular smooth muscle cells.

(+/-)-1-(Cyclohexyloxycarbonyloxy)ethyl 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylate (TCV-116, Candesartan) and its active metabolite 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]-1H- benzimidazole-7-carboxylic acid (CV-11974) are specific nonpeptide angiotensin AT1 receptor antagonists. In the present study, the inhibitory potency of these two antagonists on the angiotensin II-induced responses in aortic vascular smooth muscle cells from Wystar Kyoto rats was investigated. The specific binding of 125I-angiotensin II to cells was inhibited by CV-11974 and TCV-116 with a half-maximal inhibitory concentration (IC50) of 3 x 10(-11) M and 1 x 10(-9) M, respectively. CV-11974 and TCV-116 inhibited the angiotensin II-induced increase in [3H]thymidine incorporation with an IC50 of 3 x 10(-10) and 5 x 10(-9) M, respectively. Both CV-11974 and TCV-116 (10(-7) M) completely blocked the angiotensin II-induced increase in c-fos mRNA. The inhibitory potency of the metabolite CV-11974 was about 30-100-fold higher than that of the prodrug TCV-116.

Angiotensin II↗
PubMed1995

Potentiation of PDGF-induced growth responses in coronary artery smooth muscle cells by thromboxane.

Mitogenic effects of TXA2 in vascular smooth muscle cells are discussed to be dependent on the age of the donor organism. The present study investigates the contribution of TXA2 on PDGF-induced proliferation of bovine coronary artery smooth muscle cells (BCA-SMC) isolated from adult animals. Radioligand binding studies revealed high affinity TXA2 binding sites (Kd = 1.6 nM) in these cells. TXA2-mimetics alone showed no proliferative effect in BCA-SMC, assessed by [3H]thymidine incorporation. However, PDGF-stimulated proliferation was potentiated two-fold receptor-dependently by TXA2-mimetics. Thus, vasoconstrictory eicosanoids released from activated platelets might aggravate proliferation of vascular smooth muscle cells at sites of vessel injury in the adult organism.

Animals↗
PubMed1995

Reverse transcriptase-polymerase chain reaction (RT-PCR): a sensitive method to examine basic fibroblast growth factor-induced expression of the early growth response gene-1 (egr-1) in human umbilical arterial endothelial cells.

Immediate-early genes are expressed upon growth and differentiation in a large variety of cells and species. In the present study we investigated the effect of basic fibroblast growth factor (bFGF) on early growth response gene-1 (egr-1)-mRNA expression in human umbilical arterial endothelial cells (HUAEC). The detection of this gene in HUAEC was performed by Northern blotting and by reverse transcriptase-polymerase chain reaction (RT-PCR). For RT-PCR specific primers for egr-1 and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) were constructed and PCR conditions were optimized. bFGF induced a time- and concentration-dependent increase of egr-1 expression. Maximal expression occurred within 30 min of stimulation with bFGF at a concentration of 50-100 ng ml-1. RT-PCR gave highly reproducible and specific results. The comparison of both methods showed comparable results but a higher sensitivity for RT-PCR in detecting the egr-1 mRNA. RT-PCR is an excellent method for detecting the expression of egr-1 mRNA in HUAEC.

Base Sequence↗
PubMed1995

Kinetics of Na+/H+ exchange in vascular smooth muscle cells from WKY and SHR: effects of phorbol ester.

The kinetic properties of Na+/H+ exchange were investigated in vascular smooth muscle cells (VSMC) in culture from normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). Antiport activity was measured in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein-loaded cells after nigericin-induced cytosolic acidification. Studies were performed without (control) and with pretreatment of the cells with phorbol 12-myristate 13-acetate (PMA; 200 nM). Na+/H+ exchange markedly differed between the two strains with lower Hill coefficients [1.56 +/- 0.17 (SE) vs. 2.62 +/- 0.36] and higher maximal activity (Vmax) values (55.85 +/- 5.24 vs. 31.11 +/- 2.38 mmol H+.l-1.min-1) in SHR compared with WKY cell lines. PMA markedly altered the antiport kinetics in WKY VSMC with a decrease in the Hill coefficient (1.75 +/- 0.14) without affecting Vmax (31.88 +/- 1.55 mmol H+.l-1.min-1). In VSMC from SHR, PMA had no effect on the kinetic variables investigated. Thus two kinetic abnormalities are present with respect to Na+/H+ antiport activity in VSMC from SHR compared with WKY, i.e., increased Vmax and decreased Hill coefficient. The observation that PMA does not affect the kinetics of the Na+/H+ antiport in VSMC from SHR suggests a marked degree of antiporter prestimulation in this animal model of genetic hypertension.

Animals↗
PubMed1995

Thromboxane A2 and vascular smooth muscle cell proliferation.

In the present study we describe the intracellular pathways for the transmission of growth signals by the potent vasoconstricting eicosanoids prostaglandin H2 and thromboxane A2 in smooth muscle cells from rat aorta. Carbocyclic thromboxane A2 and U46619 are stable thromboxane A2 mimetics acting at the common thromboxane A2/prostaglandin H2 receptor. Carbocyclic thromboxane A2 (10(-6) mol/L) induced an approximately 2.5-fold increase in [Ca2+]i above the basal value at 25 seconds. Maximal stimulation of the 42-kD mitogen-activated protein kinase isoform by both thromboxane A2 mimetics occurred at 5 minutes. Both thromboxane A2 mimetics at a concentration of 10(-6) mol/L induced the expression of c-fos and early growth response gene-1 (egr-1) mRNA, with a maximum at 30 minutes. Carbocyclic thromboxane A2 (10(-6) mol/L) induced a 3.3-fold increase in [3H]thymidine incorporation into cell DNA above the basal value and produced a 3.5-fold elevation of platelet-derived growth factor-BB-dependent [3H]thymidine incorporation into cell DNA. Similar effects of U46619 (10(-6) to 10(-5) mol/L) alone did in combination with platelet-derived growth factor-BB on cell DNA synthesis were obtained. The thromboxane A2/prostaglandin H2 receptor antagonist SQ29548 (10(-6) mol/L) completely suppressed the mitogenic effect of both thromboxane A2 mimetics (10(-6) mol/L). Pertussis toxin (10 to 100 ng/mL) did not influence the mitogenic effects of the thromboxane A2 mimetics. Carbocyclic thromboxane A2 (10(-6) mol/L) and platelet-derived growth factor-BB (20 ng/mL) per ser caused a 44% and 100% increase in cell number, respectively. In the presence of carbocyclic thromboxane A2 (10(-6) mol/L), platelet-derived growth factor-BB induced a 152% increase in cell number. Similar results were obtained with U46619 alone or in combination with platelet-derived growth factor-BB.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1995

Native low-density lipoprotein (LDL) induces the expression of the early growth response gene-1 in human umbilical arterial endothelial cells.

Low-density lipoprotein (LDL) is thought to be involved in the growth of various cell types including human endothelial cells. Nevertheless, little is known about the signal transduction mechanisms underlying the growth-promoting effects of LDL in endothelial cells. Furthermore, the question whether native LDL participates in the described effects remains unanswered. Here, we show that native LDL induces a dose-dependent elevation in free intracellular Ca(2+)-concentration ([Ca2+]i) as well as a rapid and prolonged increase in intracellular pH (pHi) in human umbilical arterial endothelial cells (HUAEC). Native LDL induces a dose-dependent increase of early growth response gene-1 (egr-1) mRNA expression. The effect is maximal 30 min after addition of LDL to the culture medium. Moreover, native LDL causes an increase in DNA-synthesis and cell proliferation. In addition, the effect of acidic fibroblast growth factor (aFGF) on HUAEC proliferation was enhanced by native LDL.

Calcium↗
PubMed1994

[Bone pain, polydipsia, polyuria].

This 20 year old man suffered increasingly from multifocal bone- and back pain over the last 6 months. Painful weakness of the left leg with dysesthesia of the 4th and 5th toe, a weight loss of 15 kg and polydipsia and pollakiuria had developed. The clinical workup disclosed hard tumors in the right mandible and tibia, a waddling gait with bilateral sign of Trendelenburg, reduced muscular force in the left leg with missing achilles tendon reflex and a loss of sensibility in the distal S1 segment, epigastric tenderness on pressure and hypertension with a value of 160/100 mmHg. X-rays revealed multiple cystic bone lesions at all sites. Hypercalcemia and massively elevated parathyroid hormone were measured. Since the parathyroids were enlarged on sonography, primary hyperthyroidism with fibrosing osteitis v. Recklinghausen was diagnosed.

Adenoma↗
PubMed1994

Differential activation by platelet-derived growth factor-BB of mitogen activated protein kinases in starved or nonstarved AKR-2B fibroblasts.

More than 90% of serum-deprived (starved) AKR-2B mouse fibroblasts are stimulated to divided by the addition of platelet-derived growth factor (PDGF)-BB. In density-arrested (nonstarved) cells, PDGF-BB affords protection from cell death without stimulation of cell division. In both cultivation conditions the cells express similar amounts of PDGF beta-receptors and the receptor kinase activity was identical as judged by its autophosphorylation capacity. Three signaling pathways were studied in detail: 1) Phospholipase C-gamma (PLC-gamma) and [Ca2+]i increase, 2) activation of the phosphatidylinositol-3 kinase (PI-3 kinase), and 3) activation of mitogen activated kinases I and II (MAP kinases I and II). There was no difference in starved or nonstarved cells regarding PLC-gamma activation, increase of [Ca2+]i, and stimulation of PI-3 kinase activity. But most remarkably the activation of MAP-I was largely suppressed in nonstarved cells. The implications of these signaling pathways in cell protection or cell division are discussed.

Animals↗
PubMed1994

The differential activation of phosphatidylinositol-3 kinase and mitogen-activated protein kinases by PDGF-AA and IGF-I might explain the synergistic effect of the two growth factors on the proliferation of AKR-2B fibroblasts.

AKR-2B mouse fibroblasts express similar numbers of alpha- or beta-type PDGF receptors on their surface. Previous studies showed that PDGF-AA alone was unable to stimulate cell proliferation. Simultaneous addition together with insulin-like growth factor I (IGF-I), itself also inactive, led to a significant proliferation of the cells. In an effort to explain this synergism of the two growth factors we describe here the effects of the isoforms PDGF-AA or -BB and insulin-like growth factor I on three distinct signaling pathways, i.e., polyphosphoinositol turnover and [Ca2+]i increase, phosphatidylinositol-3 kinase, and mitogen-activated protein kinases. Whereas PDGF-BB effectively stimulated all three events, PDGF-AA failed to stimulate the phosphatidylinositol-3 kinase activity, but stimulated the mitogen-activated protein kinase to a maximum extent. In contrast IGF-I had no effect on mitogen-activated kinase but strongly stimulated phosphatidylinositol-3 kinase activity.

Animals↗
PubMed1994

Plasma lipids and lipoproteins and essential hypertension.

In recent years there have been many studies demonstrating a correlation between increased arterial blood pressure and altered lipid profiles, and there has been an especially positive correlation between high cholesterol levels and blood pressure. There are differences between the various reports that are important. In our study the lipid distribution in 105 hypertensive patients with mild or moderate arterial hypertension according to WHO criteria without clinically or ultrasonographically apparent atherosclerosis was compared to the lipid distribution in 65 age-matched healthy persons. On the epidemiological level a significant, positive association was found between LDL serum levels (P < or = 0.001), Apo B serum levels (P < or = 0.001), serum triglyceride levels (P < or = 0.05) and VLDL serum levels (P < or = 0.01) and arterial hypertension. However, in contrast to recent reports, no significant difference was found between total serum cholesterol levels in normotensives and hypertensives, and there was no difference in HDL serum levels. No evidence could be found for a significant increase in lipoprotein (a) serum levels in hypertensives.

Adult↗
PubMed1994

Oxymetazoline enhances epidermal- and platelet-derived growth factor-induced DNA synthesis.

In the present study, the effect of 10(-9) to 10(-6) M epinephrine (alpha- and beta-agonist), norepinephrine (alpha- and beta 1-antagonist) isoproterenol (beta-agonist) salbutamol (beta 2-agonist), phenylephrine (alpha 1-agonist) and oxymetazoline (mainly alpha 2-agonist) on DNA synthesis in vascular smooth muscle cells (VSMCs) from rat aorta has been investigated. Our results show that only oxymetazoline induced a moderate dose-dependent elevation of [3H]thymidine incorporation into cell DNA (10(-6) M, 100-300%). Epidermal growth factor (EGF) (50 ng/ml) and platelet-derived growth factor (PDGF)-BB induced an elevation of the [3H]thymidine incorporation into cell DNA from 154 +/- 7 (basal value) to 1270 +/- 95 and 1552 +/- 178 cpm/microgram protein (mean +/- S.D., n = 3). Oxymetazoline (10(-6) M) and phenylephrine induced an increase of [3H]thymidine incorporation to 368 +/- 53 and 205 +/- 27 cpm/microgram protein, respectively. In contrast to phenylephrine, oxymetazoline caused an elevation of the PDGF-BB- and EGF-induced [3H]thymidine incorporation to 1561 +/- 143 and 2086 +/- 235 (means S.D., n = 3), respectively. In addition, EGF (1 to 50 ng/ml) induced a dose-dependent increase of [3H]thymidine incorporation from 154 +/- 7 (basal value) to 486 +/- 35 (1 ng/ml), 912 +/- 74 (5 ng/ml), 1019 +/- 40 (25 ng/ml) and 1270 +/- 95 (50 ng/ml) cpm/microgram protein (mean +/- S.D.). In the presence of 10(-6) M oxymetazoline, 1, 5, 25 and 50 ng/ml EGF caused an increase of [3H]thymidine incorporation to 633 +/- 101, 1124 +/- 87, 1231 +/- 101, and 1561 +/- 89 cpm/microgram protein (mean +/- S.D.).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic Agonists↗
PubMed1993

Low density lipoprotein subfractions increase thromboxane formation in endothelial cells.

Correlations between dyslipidemia and high blood pressure have been shown. It is unclear whether there is a causal relationship but there are indications that blood lipids might have a direct effect on mechanisms regulating the vascular tone. As secretory products of endothelial cells (EC) have been suggested to be involved in blood pressure regulation, we studied the influence of low density lipoprotein subfractions on thromboxane (T) synthesis in human umbilical vein endothelial cells (HUVEC). LDL can be separated into at least three major subfractions. In the present study, subfractions LDL1 (d = 1.030-1.033 g/ml), LDL 2 (d = 1.033-1.040 g/ml), and LDL 3 (d = 1.040-1.045 g/ml) were obtained by density gradient ultracentrifugation. T was measured by a radioimmuno-assay. LDL subfractions induced a dose dependent (10-100 micrograms/ml) increase in T synthesis. Control T concentration was 96 +/- 12 pg/ml. LDL 3 (205 +/- 17 pg/ml, p < 0.01) and LDL 2 (194 +/- 13 pg/ml, p < 0.05) caused a significantly higher T concentration compared to the LDL 1 subfraction (152 +/- 11 pg/ml). These results indicate that LDL 2 and 3- the subfractions with the higher density-might have a more pronounced effect on T synthesis by EC than the other LDL subfractions.

Cells, Cultured↗