bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of L-Type Calcium Channels in Hypoxic Relaxation of Vascular Smooth Muscle
Authors: Herrera Gerald M. , Walker Benjimen R.
Published: April 2003

In the systemic vasculature, hypoxia elicits a local vasodilator response that may be partially mediated by ionic channels on vascular smooth muscle, such as adenosine triphosphate sensitive K+ channels. Recent electrophysiological studies suggest that hypoxia may also inhibit voltage-dependent Ca2+ channels (L type) on peripheral vascular smooth muscle cells. We hypothesized that hypoxia elicits relaxation of vascular smooth muscle by inhibiting L-type Ca2+ channels. In endothelium-denuded rat thoracic aortic rings contracted with phenylephrine, mild and moderate hypoxia (PO2 35 and 20 mm Hg, respectively) elicited significant relaxation. Pretreatment with the L-type Ca2+ channel antagonist nifedipine completely inhibited mild hypoxic relaxation and diminished relaxation under moderate hypoxia, whereas glibenclamide, a blocker of adenosine triphosphate sensitive potassium channels, only attenuated the response to moderate hypoxia. In rings contracted with the L-type Ca2+ channel agonist (–)BAY K 8644 both mild and moderate hypoxia elicited almost complete relaxation. Furthermore, in rings contracted with hyperkalemic solutions (85 mM K+ or 120 mM K), mild and moderate hypoxia elicited significant relaxations. Thus, we conclude that hypoxia acts directly on vascular smooth muscle to cause relaxation in part by inhibiting L-type Ca2+ channels.
Role of Smooth Muscle Activation in Conduction of Vasodilation along Isolated Hamster Feed Arteries
Authors: Walker Bryan D. , Segal Steven S.
Published: April 2003

Vasodilation originating within a muscle is conducted into feed arteries, but the determinants of conduction along feed arteries are undefined. We have investigated the effect of vasomotor tone on conducted vasodilation in feed arteries of the hamster retractor muscle. Unbranched segments (length, 2–3 mm) were isolated, cannulated, and studied in vitro at 37°C while pressurized to 25 mm Hg, 75 mm Hg (in vivo pressure), or 120 mm Hg in the absence and presence of norepinephrine (NE, 10–7 M + 10–6 M propanolol). Internal diameter decreased (p < 0.05) as pressure increased (85 ± 7, 73 ± 4, 66 ± 6 µm, respectively), confirming myogenic reactivity. Acetylcholine (ACh, 0.1 M) was delivered (1 µA, 500 ms) from a micropipette; vasomotor responses were recorded at the site of release (‘local’) and at distances up to 1,500 µm (‘conducted’) along the vessel. There were no responses to ACh at 25 mm Hg. However, dilations (diameter change, µm) were pronounced at 75 mm Hg (local: 22 ± 3; conducted: 18 ± 2, respectively), without further change at 120 mm Hg. NE constricted vessels (p < 0.05) at each pressure (to 65 ± 9, 57 ± 6, and 56 ± 8 µm, respectively); this enabled both local (14 ± 2 µm) and conducted (10 ± 1 µm) vasodilation at 25 mm Hg but was without effect on conduction at 75 or 120 mm Hg. Our findings indicate that conducted responses plateau at a physiological level of myogenic tone. Whereas NE had no effect on conduction under normal or hypertensive conditions, it enabled conduction during hypotension. We conclude that pressure and NE influence the ability of conducted vasodilation to govern muscle blood flow.
Heparin Fails to Inhibit the Proliferation of Human Vascular Smooth Muscle Cells in the Presence of Human Serum
Authors: Underwood P.A. , Mitchell S.M. , Whitelock J.M.
Published: April 2003

The proliferation of vascular smooth muscle cells (VSMC) plays a significant part in both the developing atherosclerotic lesion and in restenosis. Heparin has been widely reported to inhibit the growth of VSMC in culture and intimal VSMC in some animal models of vascular hyperplasia. Clinical trials with heparin, however, have failed to inhibit restenosis following angioplasty. Bovine serum is normally used as a growth supplement in in vitro VSMC growth assays. We have compared the effects of human serum with those of bovine serum on the cellular response to heparin in human VSMC culture. While heparin inhibited the proliferation of human VSMC in the presence of bovine serum, it was totally ineffective in the presence of human serum. These observations were consistent over a wide range of serum and VSMC samples. Experiments utilizing neutralizing antibodies to a number of growth factors showed that cells in either serum were similarly dependent on platelet-derived growth factor for proliferation. In contrast, proliferation in the presence of bovine serum was shown to be dependent on extracellular basic fibroblast growth factor, whereas that in human serum was not. Direct binding of [3H]-heparin to VSMC was significantly reduced in the presence of human serum compared with bovine serum, and the former contained twice the concentration of heparin-binding factors of the latter. Removal of heparin-binding factors from either serum type significantly reduced the proliferation potential. Fractionation of heparin-binding factors from human serum showed that the major growth-promoting activity, together with heparin resistance, was contained within a fraction excluded by a 100,000 molecular weight membrane. We conclude that the mechanism of resistance to heparin in human serum is likely to be due to a combination of differential growth factor binding and interference with heparin interaction with cellular receptors by a high molecular weight heparin-binding factor. This phenomenon may significantly contribute to the lack of success of heparin as an antirestenotic agent in clinical trials.
Binding of Gold-Protein Ligand Complexes to Neurokinin Receptors
Authors: Cathieni MariaM. , Taban CharlesH. , Sors Patrick , Mastrangelo Dominique
Published: April 2003

Active neurokinin (NK) receptors were visualized with 5-nm colloidal gold-protein-substance P (GPSP) and -senktide (GPSenk) complexes on vascular tissues. Electron micrographs of pig coronary strips incubated with GPSP showed gold particles either bound to the plasmalemma or inside intracytoplasmic vesicles of endothelial cells. Preincubation with SP or the NK1 receptor antagonist L-703606 prevented GPSP marking. No gold particles were seen after incubation with GPSenk. On coronary strips in vitro, which had been precontracted with U46619, GPSP induced relaxations similar to those produced by equimolar concentrations of SP, both relaxations being inhibited by L-703606. Analogous to senktide, GPSenk was totally inactive on arterial strips. Incubation of rat portal veins with GPSenk showed gold particles bound to the plasmalemma or inside intracytoplasmic vesicles of smooth muscle cells. Preincubation with senktide or the NK3 receptor antagonist R-820 prevented GPSenk marking. On portal veins in vitro GPSenk induced contractions similar to those induced by equimolar concentrations of senktide or NKB; these effects were inhibited by R-820. Our results show that colloidal gold-protein complexes present biological activity and selectivity similar to those of their respective native ligand and detect the presence of active receptors; in addition, they suggest the presence of NK-receptor-mediated endocytosis in endothelial cells of coronary artery and in smooth muscle cells of the portal vein.
Stimulation of Tissue Factor Expression in Human Microvascular and Macrovascular Endothelial Cells by Cultured Vascular Smooth Muscle Cells in vitro
Authors: Zhang Jing-Chuan , Ruan Qiu-Rong , Paucz Ljubomir , Fabry Astrid , Binder Bernd R. , Wojta Johann
Published: April 2003

The effect of conditioned media obtained from different smooth muscle cells (SMC) on tissue factor (TF) expression in endothelial cells (EC) in vitro was investigated. We could show that conditioned media from cultured human aortic SMC, human umbilical artery SMC or human umbilical vein SMC all resembling the synthetic phenotype of SMC induced TF activity in human umbilical vein EC and human skin microvascular EC in a dose- and time-dependent fashion. This induction was also seen at the level of specific TF mRNA as evidenced by Northern blotting. The TF inducing activity was heat-labile and acid-stable and had an approximate molecular mass of 38 kD. This activity was found to be distinct from known inducers of TF expression in EC such as interleukin-1, tumor necrosis factor-α, bacterial lipopolysaccharide or vascular endothelial growth factor. Such as factor, if released by SMC in vivo, could contribute to the activation of EC under conditions such as when EC are in close contact with SMC of the synthetic (nondifferentiated) phenotype seen in processes like vessel development or neo-intima formation.
Assessment of Local Differences in Intima-Media Thickness in the Human Common Carotid Artery
Authors: Willekes Christine , Brands Peter J. , Willigers Jean M. , Hoeks Arnold P.G. , Reneman Robert S.
Published: April 2003

Intimal thickening may be focal in nature and is especially found in areas with low shear rate. To be able to study the relation between intima-media thickness (IMT) and wall shear rate appropriately, a method to assess IMT locally is required. It was the aim of the present study to investigate the ability of a recently developed automated method to assess local differences in IMT, if any, in relatively short arterial segments. Therefore, intrasession interlocation differences in IMT were assessed at the posterior wall of the common carotid artery close to the bulb (0 mm) and 10 and 20 mm more upstream in terms of mean difference ± 2 standard deviations. Prior to this study we investigated the ability of the system to reproducibly assess IMT locally (intersession intralocation) in terms of repeatability coefficient (= 2 standard deviations). The measurements were performed in the common carotid artery 20 mm proximal to the bulb. The study was performed on young and older subjects presumed to be healthy. The intersession intralocation repeatability coefficient was 0.07 mm in the young group and 0.11 mm in the older group. The IMT close to the bulb (0 mm) was significantly larger (±0.050–0.065 mm) than that at the other locations in both age groups. We conclude that local IMT can be assessed reproducibly and local differences in wall morphology in short arterial segments can be studied reliably.
Leukocyte Margination in Alveolar Capillaries: Interrelationship with Functional Capillary Geometry and Microhemodynamics
Authors: Kuebler W.M. , Kuhnle G.E.H. , Goetz A.E.
Published: April 2003

The pulmonary capillary microvasculature harbors a large pool of intravascularly marginated leukocytes. In this study, we investigated the interrelationship of leukocyte margination with characteristics of functional capillary geometry and microhemodynamics in alveolar capillary networks. In 22 anesthetized rabbits we assessed functional capillary density, average capillary length, red blood cell velocity and leukocyte kinetics in alveolar capillary networks in vivo by intravital fluorescence microscopy. In alveolar wall areas of 12,800 ± 1,800 μm2, we detected 3.6 ± 0.5 sticking leukocytes and 21.0 ± 1.9 functional capillary segments with an average capillary length of 35.7 ± 2.1 μm. We calculated that approximately 15% of functional capillary segments are blocked by marginated leukocytes. Leukocyte margination was predominantly observed in capillary networks characterized by a high functional capillary density, short capillary segments and low red blood cell velocities. The multitude of interconnected capillary channels in these networks may allow alveolar blood flow to bypass marginated leukocytes. Hence, this interrelationship may be relevant for maintenance of adequate alveolar perfusion and low capillary network resistance despite excessive leukocyte margination in the pulmonary microvasculature. Local microhemodynamic factors may play a regulatory role in the spatial distribution of leukocyte margination.
Moderate Hypoxia Increases Heat Shock Protein 90 Expression in Excised Rat Aorta
Authors: Almgren C.M. , Olson L.E.
Published: April 2003

We hypothesized that heat shock protein 90 (HSP90) expression would be increased in vascular tissue exposed to a hypoxic stress that resulted in altered contractile function. We tested this hypothesis by subjecting excised rat aortic rings to a hypoxic stress that has been shown to reduce contractile force induced by arginine vasopressin (PO2 ≈50 mm Hg for 1 h) and determining the effect on HSP90 expression. Concentration-response curves were determined for control and hypoxic excised rat aortic rings exposed to norepinephrine (n = 8) or KCl (n = 8). Hypoxia reduced the force generated in response to the highest concentration of each agonist. HSP90 expression was evaluated by immunoblotting (n = 6) and immunohistochemistry (n = 7). Both methods documented increased expression of HSP90 in hypoxic aortae as compared to controls. HSP90 expression was increased within the cytoplasm and in conjunction with the nucleus of vascular smooth muscle cells in the tunica media and also within vascular myointimal cells. We conclude that a hypoxic stress sufficient to induce contractile dysfunction increases HSP90 expression in rat aortic smooth muscle cells.
Effects of NO Synthase Inhibitors on the Synovial Microcirculation in the Mouse Knee Joint
Authors: Veihelmann A. , Krombach F. , Refior H.J. , Messmer K.
Published: April 2003

Production of nitric oxide by the inducible NO synthase (iNOS) is known to be enhanced in chronic joint inflammation and osteoarthritis as well as aseptic loosening of joint prostheses. Initial studies yielded promising results after inhibition of the nitric oxide synthase (NOS). However, the effect of NOS inhibition has not been studied at the site of the primary function of NO, the microcirculation of the synovium in vivo. Using our recently developed model for the in vivo study of synovial microcirculation in the mouse knee joint, the effects of selective versus nonselective inhibition of iNOS were investigated by means of intravital fluorescence microscopy. After resection of the patella tendon, the synovial fatty tissue was exposed for intravital microscopy. Diameter of arterioles, functional capillary density (FCD), diameter of venules, venular red blood cell velocity and leukocyte-endothelial cell interaction were quantitatively analyzed before, and 10 and 60 min after intravenous injection of NOS inhibitors [selective iNOS inhibitor N-iminoethyl-L-lysine (L-NIL), and nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME)]. Our results demonstrate that L-NAME causes a significant decrease in the arteriolar diameter and FCD associated with an increase in the leukocyte accumulation in the synovium in vivo. In contrast, L-NIL neither altered the microhemodynamics nor the leukocyte-endothelial cell interaction in the synovium, indicating its potential use for selective inhibition of iNOS in joint inflammation. Using our method, further studies will provide new insights into the unknown effect of NOS inhibition on the synovial microvasculature in inflammatory joint disease in vivo.
Impaired Endothelium-Mediated Relaxation in Coronary Arteries from Insulin-Resistant Rats1
Authors: Miller Allison W. , Katakam Prasad V.G. , Ujhelyi Michael R.
Published: April 2003

Objective: The insulin resistance syndrome is associated with atherosclerosis and cardiovascular events; however, the underlying mechanism of vascular dysfunction is unknown. The purpose of the current study was to assess endothelium- and smooth-muscle-mediated vasodilation in isolated coronary arteries from insulin-resistant rats and to determine whether insulin resistance alters the activity of the specific endothelium-derived relaxing factors. Methods: Male Sprague-Dawley rats were randomized to insulin resistance or control. Insulin resistance was induced by a fructose-rich diet. After 4 weeks of diet, coronary arteries were removed and vascular function was assessed in vitro using videomicroscopy. Acetylcholine (10–9–3 × 10–5 M)- or sodium-nitroprusside (10–9–3 × 10–4 M)-induced relaxations were determined. To evaluate the role of the specific endothelium-derived relaxing factors, several inhibitors were used, including N-nitro-L-arginine (LNNA), charybdotoxin/apamin (CTX/apamin), and indomethacin. Results: Studies with nitroprusside showed that smooth-muscle-dependent relaxation did not differ between insulin resistance and control groups. In contrast, maximal relaxation (Emax) to acetylcholine was decreased in the insulin resistance group (56 ± 7%) versus control (93 ± 3%). LNNA pretreatment further impaired Emax in the IR group from 56 ± 7 to 17 ± 2% (p < 0.01). In control, Emax was only slightly impaired by LNNA (93 ± 3 to 63 ± 6%; p < 0.05). The addition of CTX/apamin also decreased relaxation in the control group (93 ± 3 to 47 ± 7%; p < 0.05), whereas relaxation in insulin-resistant rats was not affected (45 ± 5% with CTX/apamin vs. 56 ± 7% with acetylcholine alone, NS). Pretreatment with indomethacin did not affect relaxation in either group, while pretreatment with the combination of LNNA and CTX/ apamin completely abolished relaxation in both groups. Conclusions: Endothelium-dependent relaxation is impaired in small coronary arteries from insulin-resistant rats. The mechanism of this defect is related to a decrease in an endothelium-dependent, nitric oxide/prostanoid-independent relaxing factor or endothelium-derived hyperpolarizing factor.
L-Arginine Availability Is Not Limiting for Nitric Oxide Generation from Recombinant Endothelial Nitric Oxide Synthase
Authors: Mohacsi Tibor , Mozes Geza , Sato Jun’ichi , Gloviczki Peter , Katusic Zvonimir , O’Brien Timothy
Published: April 2003

L-Arginine (L-Arg) may be limiting for inducible nitric oxide synthase (NOS) activity and under certain circumstances, such as increased concentrations of a NOS inhibitor, may also be limiting for endothelial NOS activity. It is unknown if L-Arg is limiting for recombinant eNOS activity in the vascular wall after adenoviral mediated gene transfer. Our aim was to examine, if L-Arg is limiting for recombinant eNOS activity in the normal or atherosclerotic vessel wall. Rings of rabbit aorta from chow or cholesterol fed animals were transduced with adenovirus vector encoding eNOS (AdeNOS) or β-galactosidase (AdβGal). After 24 h, transgene expression was confirmed and vasomotor studies were performed in the absence or presence of L-Arg. During maximal contractions to phenylephrine (10–5 M), L-Arg (3 mM) was added to the organ chamber for 30 min. Subsequently, relaxations to acetylcholine during half-maximal contractions were obtained. In the chow- and cholesterol-fed animals, relaxations were significantly enhanced in the NOS and NOS + L-Arg groups compared to the βGal and βGal + L-Arg groups. There was no difference between NOS and NOS + L-Arg or βGal and βGal + L-Arg rings from chow- or cholesterol-fed animals. While gene transfer of eNOS enhances endothelium-dependent vasorelaxation in the normal and atherosclerotic vessel wall, L-arginine is not limiting for recombinant eNOS activity.
Increased (Na+K+Cl) Cotransport in Rat Arterial Smooth Muscle in Deoxycorticosterone (DOCA)/Salt-Induced Hypertension
Authors: Brown R.A. , Chipperfield A.R. , Davis J.P.L. , Harper A.A.
Published: April 2003

The inhibition by loop diuretics of K efflux (tracer 86Rb) from the rat femoral arterial smooth muscle was measured in normotension and in DOCA-salt hypertension. The sensitivity sequence (bumetanide > piretanide > furosemide) was the characteristic pharmacological profile of (Na+K+Cl) cotransport. In hypertension, cotransport activity was 46% greater than in normotension and the sensitivity to loop diuretics was threefold less. Intracellular [K] and the Na, K and Cl permeability ratios and electrogenic Na pump activity were assessed electrophysiologically in normotension and hypertension. [K]i was lower in hypertension (173 mM) than normotension (198 mM) but the other parameters (PNa/Cl = 0.14, PCl/PK = 0.19 and electrogenic pump = –8.3 mV in normotension) were not significantly different. Ionic permeabilities to Na, K and Cl were significantly lower in hypertension than normotension. Plasma [Na], but not [K], was higher in hypertension than normotension. The conclusion is that increased activation of (Na+K+Cl) cotransport in hypertension plays a major role in the elevation of [Cl]i and depolarisation of the membrane potential in vascular smooth muscle in DOCA-salt hypertension. The role of (Na+K+Cl) cotransport in vascular smooth muscle in this model of hypertension is discussed in relation to [Cl]i, depolarisation of the membrane potential and contraction and in relation to cell growth.
Effect of TGF-β1 Antisense S-Oligonucleotide on Synthesis and Accumulation of Matrix Proteoglycans in Balloon Catheter-Injured Neointima of Rabbit Carotid Arteries
Authors: Merrilees Mervyn , Beaumont Brent , Scott Lesley , Hermanutz Valentin , Fennessy Paul
Published: April 2003

Arterial matrix proteoglycans (PG) are necessary for the maintenance of viscoelastic properties of the vessel wall, but excess levels, particularly of versican and biglycan in primary and restenotic intimal thickenings, are correlated with increased tissue volume and with atherogenicity. There is good evidence that the primary stimulus to increased PG synthesis, including versican and biglycan, is transforming growth factor-β1 (TGF-β1). The aim of this study was to determine the effects of reducing endogenous TGF-β1 on rates and patterns of PG synthesis and on versican, biglycan and decorin accumulation in vivo. Rabbit common carotid arteries subjected to balloon catheter injury were treated with a TGF-β1 antisense phosphorothioate oligonucleotide applied in a pluronic gel to the adventitia. Control animals received a nonsense oligonucleotide or gel alone. TGF-β1 antisense (1) significantly (p < 0.005) inhibited, at day 2, the balloon catheter-induced increase in TGF-β1 mRNA relative to β-actin mRNA; (2) inhibited intimal thickening at 23 days by ∼40% (p < 0.05); (3) inhibited (p < 0.05) PG synthesis, measured by autoradiographic detection of [3H]glucosamine, in the media of day 2 ballooned carotids and in the subendothelial zone of day 23 neointima, and (4) decreased immunostaining intensity for versican (p < 0.03) and TGF-β1 (p < 0.001) in the neointima. Biglycan was reduced to a lesser extent but not significantly and decorin was not affected. Proliferating cell nuclear antigen indices were variable and not significantly changed. These findings confirm a role for TGF-β1 in developing neointima and demonstrate a specific effect on the synthesis, distribution, and accumulation of matrix PG, particularly versican.
Effect of Venous and Lymphatic Congestion on Lymph Capillary Pressure of the Skin in Healthy Volunteers and Patients with Lymph Edema
Authors: Gretener Silvia B. , Läuchli Severin , Leu Anders J. , Koppensteiner Renate , Franzeck Ulrich K.
Published: April 2003

The aim of the present study was to assess the influence of venous and lymphatic congestion on lymph capillary pressure (LCP) in the skin of the foot dorsum of healthy volunteers and of patients with lymph edema. LCP was measured at the foot dorsum of 12 patients with lymph edema and 18 healthy volunteers using the servo-nulling technique. Glass micropipettes (7–9 μm) were inserted under microscopic control into lymphatic microvessels visualized by fluorescence microlymphography before and during venous congestion. Venous and lymphatic congestion was attained by cuff compression (50 mm Hg) at the thigh level. Simultaneously, the capillary filtration rate was measured using strain gauge plethysmography. The mean LCP in patients with lymph edema increased significantly (p < 0.05) during congestion (15.7 ± 8.8 mm Hg) compared to the control value (12.2 ± 8.9 mm Hg). The corresponding values of LCP in healthy volunteers were 4.3 ± 2.6 mm Hg during congestion and 2.6 ± 2.8 mm Hg during control conditions (p < 0.01). The mean increase in LCP in patients with lymph edema was 3.4 ± 4.1 mm Hg, and 1.7 ± 2.0 mm Hg in healthy volunteers (NS). The maximum spread of the lymph capillary network in patients increased from 13.9 ± 6.8 mm before congestion to 18.8 ± 8.2 mm during thigh compression (p < 0.05). No increase could be observed in healthy subjects. In summary, venous and lymphatic congestion by cuff compression at the thigh level results in a significant increase in LCP in healthy volunteers as well as in patients with lymph edema. The increased spread of the contrast medium in the superficial microlymphatics in lymph edema patients indicates a compensatory mechanism for lymphatic drainage during congestion of the veins and lymph collectors of the leg.
Hirulog-1 Reduces Expression of Platelet-Derived Growth Factor in Neointima of Rat Carotid ArteryInduced by Balloon Catheter Injury
Authors: Xue Mengzhou , Fenton II John W. , Shen Garry X.
Published: April 2003

Vascular restenosis is one of the major concerns for the treatment of atherosclerotic cardiovascular diseases using therapeutic vascular procedures. Hirulog-1, a synthetic thrombin inhibitor, effectively reduced ischemic events in coronary heart disease patients and caused less hemorrhagic complications compared to heparin. Thrombin stimulated the expression of platelet-derived growth factor (PDGF) in vascular cells. PDGF receptor blockers reduced angioplasty-induced restenosis in the swine model. The present study examined the effects of hirulog-1 on vascular stenosis, platelet deposition and the expression of PDGF in rat carotid arteries injured by balloon catheter. Multiple intravenous infusions of hirulog-1 (1 mg/kg/h for 4 h for 6 times), but not bolus injection or 1–2 times of infusion, reduced neointima/media ratio by 50% in balloon-injured carotid arteries compared to injured animals receiving saline alone. Activated partial thromboplastin time in hirulog-1-treated rats was significantly prolonged compared to saline controls but shorter than that in animals receiving heparin (50 U/kg/h). One of heparin-treated rat, but none of hirulog-1-treated, died from bleeding complication. Hirulog-1 injection transiently reduced platelet deposition on denuded intima visualized by scanning electron microscopy. Abundance of PDGF in neointima of injured carotid arteries detected by immunohistochemistry was significantly decreased following infusions of hirulog-1. The results suggest that balloon catheter injury induced neointima formation and the overexpression of PDGF in the neointima of rat carotid artery may be effectively suppressed by infusions with hirulog-1, a thrombin-specific inhibitor.
Prevention of Increases in Blood Pressure and Left Ventricular Mass and Remodeling of Resistance Arteries in Young New Zealand Genetically Hypertensive Rats: The Effects of Chronic Treatment with Valsartan, Enalapril and Felodipine
Authors: Ledingham Janet M. , Phelan Edward L. , Cross Marie A. , Laverty Richard
Published: April 2003

The relative efficacy of three antihypertensive drugs in the prevention of further elevation of blood pressure (BP) and cardiovascular structural remodeling in 4-week-old genetically hypertensive (GH) rats was studied by means of two complementary methods, stereology and myography. Four to 10-week-old GH rats were treated with valsartan (10 mg/kg/day), enalapril (10 mg/kg/day) or felodipine (30 mg/kg/day). Untreated GH and normotensive control rats of Wistar origin served as controls. Tail-cuff systolic SBP was measured weekly and left ventricular (LV) mass determined at the end of the experiment. Mesenteric resistance arteries (MRA) were either fixed by perfusion, embedded in Technovit and sections stained for stereological analysis, or mounted on a wire myograph for structural and functional measurements. BP and LV mass were significantly reduced by all drugs; decreases in BP and LV mass were smaller after felodipine treatment. Valsartan and enalapril caused a decrease in BP to normotensive control values. Felodipine kept BP at the 4-week level and prevented further rise with age. Valsartan caused hypotrophic outward remodeling of MRA, enalapril eutrophic outward remodeling and felodipine hypotrophic remodeling. Myograph measurements showed remodeling of the same order. While all drugs lowered the media/lumen ratio in GH to normal, the outward remodeling after valsartan and enalapril indicates that valsartan and enalapril might be more effective in reversing the inward remodeling of resistance arteries found in essential hypertension.
Toxicity, Uptake Kinetics and Efficacy of New Transfection Reagents: Increase of Oligonucleotide Uptake
Authors: Axel Dorothea I. , Spyridopoulos Ioakim , Riessen Reimer , Runge Heike , Viebahn Richard , Karsch Karl R.
Published: April 2003

Human arterial smooth muscle cell (haSMC) proliferation is stimulated by platelet-derived growth factor (PDGF) release of human arterial endothelial cells (haEC) whereas transforming growth factor-β1 (TGF-β1) secretion by haSMC promotes extracellular matrix formation. Inhibitory concepts with antisense oligonucleotides (ASO) against those growth factors might be promising, requiring, however, sufficient transfection efficacy. Thus, toxicity and efficacy of new transfection reagents were examined. MTT tests showed that high doses >1.6 μg/ml of the liposome Cytofectin GSV® (CF) and the dendrimer SuperFect® (SF) reduced mitochondrial activity of haEC after ≥4 h transfection whereas viability of haSMC was not influenced. DAC-30® showed significant toxic effects on haEC and haSMC at each dose after ≥4 h and Lipofectin® (LF) caused complete detachment of haEC and haSMC in medium containing 10% serum. Uptake studies demonstrated that ‘naked’ ASO were not incorporated intracellularly whereas transfection within CF or SF resulted in a strong cytoplasmic and nuclear labeling after 2–5 h. With DAC-30®, only a slight cytoplasmic fluorescence was found. SF caused an unexpected stimulation of endothelial PDGF-AB synthesis. Thus, CF was favored for inhibition studies. ELISA, Western and Northern blotting showed a significant inhibition of endothelial PDGF-B and smooth muscle TGF-β1 mRNA expression and synthesis after transfection for 3–5 h using 0.1–1.0 μM ASO versus control oligonucleotides. We conclude that Cytofectin GSV® is superior to the other transfection reagents, predominantly at haEC, showing an improved efficacy and less toxicity than the classical liposome Lipofectin®. Cytofectin GSV® might offer a promising tool for antisense strategies in the treatment of vascular disorders.
The Fractal Nature of Myocardial Blood Flow Emerges from a Whole-Organ Model of Arterial Network
Authors: Beard Daniel A. , Bassingthwaighte James B.
Published: April 2003

Mammalian hearts exhibit a heterogeneous spatial distribution of blood flows, but flows in near-neighbor regions correlate strongly. Also, tracer 15O-water washout after injection into the inflow shows a straight log-log relationship between outflow concentration and time. To uncover the role of the arterial network in governing these phenomena, morphometric data were used to construct a mathematical model of the coronary arterial network of the pig heart. The model arterial network, built in a simplified three-dimensional representation of tissue geometry, satisfies the statistical morphometric data on segment lengths, diameters and connectivities reported for real arterial networks. The model uses an avoidance algorithm to position successive vascular segments in the network. Assuming flows through the network to be steady, the calculated regional flow distributions showed (1) the degree of heterogeneity observed in normal hearts; (2) spatial self-similarity in local flows; (3) fractal spatial correlations, all with the same fractal dimension found in animal studies; (4) pressure distributions along the model arterial network comparable to those observed in nature, with maximal resistances in small vessels. In addition, the washout of intravascular tracer showed tails with power law slopes that fitted h(t) = at–α–1 with the exponents α = 2 for the reconstructed networks compared with those from experimental outflow concentration-time curves with α = 2.1 ± 0.3. Thus, we concluded that the fractal nature of spatial flow distribution in the heart, and of temporal intravascular washout, are explicable in terms of the morphometry of the coronary network.
Ambulatory Infusion of Noradrenaline for Long-Term Treatment of Shy-Drager Syndrome
Authors: Kribben Andreas , Bremer Christoph , Fritschka Emanuel , Koeppen Susanne , Ahrens Oliver , Philipp Thomas
Published: April 2003

A 70-year-old female patient with advanced Shy-Drager syndrome exhibited severe orthostatic hypotension, low serum catecholamine levels, and autonomic dysfunction. She was bedridden despite oral medication with fludrocortisone, etilefrin, dihydroergotamine, L-dopa, yohimbine, and amezinium methyl sulfate. Only intravenous application of noradrenaline (30 ng/kg/min) provided complete mobilization. After implantation of a port-a-cath system, intravenous noradrenaline treatment could be continued on an outpatient basis. Over the following 5 years, the patient was throughout sufficiently mobile and did not show any significant side effects of this treatment. However, during the 5th year she suffered from nonhemorrhagic brain stem infarction due to cerebral hypoperfusion after orthostatic stress in the absence of noradrenaline infusion. We conclude that ambulatory noradrenaline infusion is a new valuable tool for long-term treatment of advanced Shy-Drager syndrome.
Osmotically Induced Conductance and Capacitance Changes in in vitro Perfused Rectal Gland Tubules of Squalus acanthias
Authors: Thiele I. , Warth R. , Bleich M. , Waldegger S. , Lang F. , Greger R.
Published: April 2003

The rectal gland of Squalus acanthias is critically involved in the homeostasis of NaCl and water metabolism and hence in overall osmoregulation. In the present study, we have examined the acute responses of rectal gland slices and in vitro perfused rectal gland tubule (RGT) cells to the exposure to dilute and hypertonic peritubule solutions. Five series were performed. (i) With changes in osmolality, Western blots to monitor tyrosine, threonine and serine phosphorylation in rectal gland slices did not reveal clear–cut changes in phosphorylation patterns. All other series were performed in in vitro perfused RGT. (ii) Relative cell volume was estimated by fura–2 fluorescence using the emission at the isosbestic excitation wavelength of 360 nm. Hypotonic solution (–100 mmol/l NaCl) reduced fura–2 fluorescence by 16% and hypertonic solution (+100 mmol/l NaCl) had the opposite effect (+12%). (iii) Transepithelial resistance was increased markedly by hypotonic solution, probably by cell swelling, and the opposite was seen with hypertonic solutions. (iv) Whole–cell patch clamp experiments indicated that hypotonic solution hyperpolarized the cells, and increased membrane conductance and membrane capacitance. The latter two changes correlated significantly with each other. Hypertonic solution had the opposite effect. (v) Measurements of the fura–2 fluorescence ratio (340/380 nm) revealed that hypotonic solution (–NaCl) increased cytosolic Ca2+ activtiy ([Ca2+]i). Hypertonic solution had no detectable effect on [Ca2+]i. These data indicate that RGT cells are swollen by removal of NaCl from the bath solution. This causes an increase in [Ca2+]i and a predominant increase in K+ conductance and hyperpolarization. Urea apparently permeates these cells quite well and its addition (+U) or its removal (–U) had only moderate osmotic effects. The removal of urea and replacement by mannitol produced effects similar to those seen with hypertonic NaCl solution.
Analysis of the Role of Apoptosis and Cell Proliferation in Renal Cystic Disorders
Authors: Kovács Judit , Gomba Szabolcs
Published: April 2003

Cell proliferation and apoptosis were studied in 8 patients with inherited polycystic kidney disease and in 34 patients with acquired cystic kidney conditions including solitary and multilocular cysts and segmental tubular dilation. Intact renal tissue of 20 surgically removed tumorous kidneys served as control. Cell proliferation and apoptosis were demonstrated by immunohistochemical and in situ end–labeling methods. The percentage of positively stained nuclei was calculated and statistically analyzed. Both apoptosis and cell proliferation were significantly higher (p<0.001) in polycystic kidney disease. The percentage of positively stained nuclei in the whole kidney tissue with acquired cysts did not differ from controls although cell proliferation was significantly higher (p<0.001) in cells lining the cysts. Apoptotic cells were rarely found in the cystic epithelium or were even absent in these cases. Our data indicate that while polycystic kindneys seem to be characterized by abnormal cell survival, acquired renal cysts have different behavior in which so far unknown intracellular changes are more likely to cause tubular distension probably through induced cell proliferation.
Protopanaxatriol Ginsenosides Inhibit Glucose Uptake in Primary Cultured Rabbit Renal Proximal Tubular Cells by Arachidonic Acid Release
Authors: Han Ho-Jae , Park Soo-Hyun , Koh Hyun-Ju , Nah Seung-Yeol , Shin Dong-Ho , Choi Han-Sun
Published: April 2003

Ginsenosides are involved in protective action against renal dysfunction and the regulation of renal functions. However, the effects of ginsenosides on glucose reabsorption are not yet known in renal proximal tubular cells. The aim of this study was to examine the effects of ginsenosides, protopanaxadiol (PD) saponin and protopanaxatriol (PT) saponin, on α–methyl–D–glucopyranoside (α–MG) uptake and its mechanism of action in primary cultured rabbit renal proximal tubular cells (PTCs). The α–MG uptake was inhibited by 90% by 0.5mM phloridizin and by removal of Na+ in the PTCs. These are typical characteristics described for the proximal tubule. To determine the time– and dose–dependent effects of PD and PT saponins on α–MG uptake, PTCs were incubated with different concentrations of PD and PT saponins (10–100 μg/ml) and for different time periods (from 10 min to 24 h). PT saponin (≥50 μg/ml) from 30 min inhibited α–MG uptake; however, PD saponin did not alter the α–MG uptake at any doses and time periods. In the kinetic analysis of α–MG uptake, PT saponin produced a significant decrease in Vmax. The PT saponin induced inhibition of α–MG uptake was blocked by mepacrine, a phospholipase A2 inhibitor. In addition, PT saponin increased [3H] arachidonic acid release by 218% of that of control, and this effect was also completely blocked by mepacrine. In conclusion, PT saponin inhibited, in part, α–MG uptake through the phospholipase A2 signal pathway in primary cultured rabbit renal PTCs.
Nitric Oxide–Dependent Renal Vasodilatation Is Not Altered in Rat with rHuEpo–Induced Hypertension
Authors: Migliori M. , Taccola D. , Panichi V. , De Pietro S. , Andreini B. , Di Benedetto A. , Filippi C. , Palla R. , Giovannini L.
Published: April 2003

Background: Recombinant human erythropoietin (rHuEpo) is the treatment of choice in anemia associated with end–stage renal disease. Its major side effect is hypertension, which occurs in 8–30% of uremic patients. The exact mechanism of rHuEpo–induced hypertension has not been fully elucidated, and several possibilities have been proposed, such as a direct vascular effect of the drug with a shift in the balance of constrictor and relaxing endothelial factors (endothelins and nitric oxide (NO)). Recent papers suggested an enhanced rather than reduced activity of endogenous NO system in rats with normal renal function and rHuEpo–induced hypertension. Our study was designed to verify whether, in spite of enhanced activity of the renal NO system, rHuEpo may affect endothelium–dependent (acetylcholine–induced) and/or endothelium–independent (sodium nitroprusside–induced) vasorelaxation and to evaluate basal NO release by the infusion of NG–nitro–L–arginine methyl ester (L–NAME) in an isolated and perfused rat kidney model. Methods: To investigate this hypothesis, we have determined systemic and renal NO activity in Wistar rats treated with a hypertensive dose of rHuEpo (150 IU/kg b.w. every other day for 2 weeks) by measuring stable NO metabolites (NO2+NO3) in the urine and have also evaluated variations in renal vascular resistance after the injection of Ach, SNP and the infusion of L–NAME. Results: Hematocrit, hemoglobin concentration and arterial blood pressure were significantly increased in the treated group as compared with the controls. Urinary excretion of NO2+NO3 was significantly higher in treated than in the controls (438±66 vs. 294±36 nM/ml/min, p<0.01, respectively). There were no significant differences in the dose–response curves to Ach and SNP between the two groups. The renal vasoconstriction following the infusion of L–NAME was also similar in the two groups. Conclusions: The analysis of our results seems to indicate that the endogenous NO system activity was enhanced in rHuEpo–induced hypertension in rats with normal renal function and a resistance to NO was not developed in renal circulation. Further studies seem to be necessary to better clarify the exact mechanisms underlying the development of rHuEpo–induced hypertension.
Indirect and Direct Simultaneous, Comparative Blood Pressure Measurements with the Bosotron 2® Device
Authors: Weber F. , Lindemann M. , Erbel R. , Philipp T.
Published: April 2003

Testing the accuracy of the automatic blood pressure measuring device Bosotron2®, we performed simultaneous, same–arm, comparative blood pressure measurements with three instruments (A, B, and C) of the device and by auscultation in the antecubital fossa by a trained observer in 24 volunteers each. The volunteers were evenly matched for age, sex, and blood pressure level. The mean systolic differences and their standard deviations (24 subjects each, n = 288 single measurements) were –1.32±3.8 mm Hg for instrument A, –0.99±3.9 mm Hg for instrument B, and –1.12±5.0 mm Hg for instrument C. The corresponding values for the diastolic differences were –2.71±4.4, –3.46±4.3, and –2.72±3.9 mm Hg, respectively. Combining the results for the three instruments, the comparison yielded a good accuracy (mean difference) and sufficient repeatability of the differences (standard deviation) for the systolic and diastolic blood pressures (systolic –1.14±4.3 mm Hg; diastolic –2.96±4.2 mm Hg). In addition to the British Hypertension Society protocol, in 33 patients with coronary artery disease the measurements using the three Bosotron 2 devices were compared with measurements within the aortic arch. The mean systolic/diastolic differences (±SD) were +1.45±4.3 and +7.27±4.7 mm Hg for instrument A, +4.70±12.8 and +1.73±7.4 mm Hg for instrument B, and –3.74±8.0 and +5.61±2.6 mm Hg for instrument C. Combining the results of the three instruments, the blood pressure was determined to be only slightly higher (systolic +1.74±9.6 mm Hg; diastolic +4.87±5.6 mm Hg) by the Bosotron 2® device as compared with the aortic arch pressure. The Bosotron 2® device seems to be suitable for clinical use and for monitoring blood pressure during clinical–pharmacological studies.
Chronic Treatment with Angiotensin II Type 1 Receptor Antagonist Suppresses Glomerular Activator Protein–1 Activity in Salt–Sensitive Hypertensive Rats
Authors: Otsuka Fumio , Ogura Toshio , Yamauchi Takayoshi , Sato Minoru , Kataoka Hideo , Kageyama Jingo , Makino Hirofumi
Published: April 2003

We examined the effects of angiotensin–converting enzyme inhibitor (ACEI) and angiotensin II type 1 receptor antagonist (AT1a) on the action of protooncogene c–fos in salt–sensitive hypertensive rats. Seven–week old Dahl salt–sensitive rats fed a high (8%)–salt diet were treated with ACEI, cilazapril (10 mg/kg) or AT1a, TCV–116 (1mg/ kg) every day for 6 weeks. The control animals were fed a low (0.3%)–salt diet. Systolic blood pressure gradually increased in high–salt–loaded rats and was higher than low–salt–treated rats throughout the study. However, both medications had no significant antihypertensive effect. After 6 weeks of therapy, glomerular mRNA and nuclear protein were extracted from the resected kidneys. Competitive reverse transcription–polymerase chain reaction showed a high level of glomerular c–fos mRNA in high–salt–loaded rats and that ACEI or AT1a treatment did not significantly change its level. Electrophoretic mobility shift assay demonstrated that treatment with AT1a significantly decreased the activator protein–1 (AP–1) binding activity in the glomerular nuclear extract compared to ACEI. Our findings suggest that, compared with ACEI treatment, long–term treatment with AT1a may contribute to attenuation of the glomerular injury in salt–sensitive hypertension by inhibiting AP–1 transcription activity independent of its antihypertensive effect.
Hypertension in Unilaterally Nephrectomized Rats Induced by Single–Kidney Transfection with Angiotensinogen cDNA
Authors: Marley Wanda Sue , Bowen Richard , Gotshall Robert W. , Wilke Lee
Published: April 2003

Plasmid expression vectors containing angiotensinogen (ATG) cDNA were complexed to cationic liposomes and injected into the renal artery of unilaterally nephrectomized rats to evaluate the effect of intrarenal ATG cDNA on arterial blood pressure and the renin–angiotensin system. Systolic blood pressures measured by tail cuff on days 12, 16, and 18 after transfection were significantly higher in rats that received ATG cDNA than in control rats that received the lac Z reporter gene. Plasma renin activity and plasma ATG concentration were unchanged. These results provide direct evidence that the availability of intrarenal ATG may be instrumental in the development of systemic hypertension.
Influence of Mycophenolic Acid and FK–506 on Human Platelet Activation in vitro
Authors: Klein Barbara C. , Bach Dieter , Rehfeld Irene , Kirchhoff Elisabeth , Hohlfeld Thomas , Schrör Karsten , Scharf Rüdiger , Grabensee Bernd
Published: April 2003

Background/Aim: FK–506 (FK) and mycophenolic acid (MPA) are immunosuppressive agents used in kidney transplant recipients. Their effect on posttransplant thromboembolic complications is either controversial (FK) or has not been described (MPA). Thromboembolic events are among the consequences of platelet hyperaggregability which can be identified by measuring platelet aggregation. The aim of this study was to evaluate the in vitro effects of MPA and FK upon platelet activation in healthy subjects. Methods: Platelet–rich plasma from healthy volunteers (n = 18) was incubated with FK (70 ng/ml), FK vehicle, and MPA (30 μg/ml) before platelet aggregation was induced by the platelet agonists adenosine diphosphate (2 and 5 μM) and collagen 0.5 and 1.0 μg/ml). Aggregation was measured by recording the optical density. Results: MPA resulted in a significant decrease in the platelet response to collagen (1.0 μg/ml) in platelet–rich plasma, whereas FK significantly increased platelet aggregation in response to collagen (0.5 μg/ml). The vehicle of FK had no influence on platelet aggregation with either agonist. Conclusions: The decreased platelet–activating response following preincubation with MPA may favor its use in kidney transplant recipients to reduce thromboembolic events. The FK–induced enhancement of platelet aggregation shown in vitro may lead to thromboembolic complications in transplant recipients.