De-endothelialized Segments Research Articles

Therapeutic Benefits of Pomegranate Flower Extract: A Novel Effect That Reduces Oxidative Stress and Significantly Improves Diastolic Relaxation in Hyperglycemic In Vitro in Rats.

The pomegranate flower is an ancient herb in traditional Chinese medicine with multiple properties. Recent studies have shown that pomegranate flower extract is beneficial, especially for hyperglycemia. In this experiment, we investigated the diastolic effect of pomegranate flower polyphenol (PFP) extract on the isolated thoracic aorta of rats in both the absence and presence of high glucose levels. Isotonic contractile forces were recorded from aortic rings (about 3 mm in length) from rats using the BL-420F Biological Function Test System. Tissues were precontracted with 60 mM KCl to obtain maximum tension under 1.0 g load for 1 hour before the balance was achieved, and the fluid was changed every 15 minutes. PFP (700 mg/L–900 mg/L) showed a concentration-dependent relaxant effect on the aortic rings; vasodilation in the endothelium-intact was significantly higher than that in the de-endothelialized segments (P < 0.01). The endothelium-dependent vasorelaxant effect of PFP was partially attenuated by K+ channel blockers, tetraethylammonium (TEA), glibenclamide (Glib), and BaCl2, as well as L-NAME (eNOS inhibitor) on the denuded endothelium artery ring. Concentration-dependent inhibition of PFP on releasing intracellular Ca2+ in the Ca2+-free solution and vasoconstriction of CaCl2 in Ca2+-free buffer plus K+ (60 mM) was observed. In addition, PFP (0.1–10 mg/L) showed significant inhibition of acetylcholine-induced endothelial-dependent relaxation in the aorta of rats in the presence of high glucose (44 mmol/L). Nevertheless, the vasodilating effect of PFP was inhibited by atropine and L-NAME. The results indicated that PFP-induced vasodilation was most likely related to the antioxidant effects through enhanced NO synthesis, as well as the blocking of K+ channels and inhibition of extracellular Ca2+ entry. In conclusion, these observations showed that PFP ameliorates vasodilation in hyperglycemic rats. Hence, our results suggest that PFP supplementation may be beneficial for hypertensive patients with diabetes.

Angiotensin II increases neurogenic nitric oxide metabolism in mesenteric arteries from hypertensive rats

We investigated, in mesenteric arteries from hypertensive rats (SHR), the possible changes in neurogenic nitric oxide (NO) release produced by angiotensin II (AII), and the possible mechanisms involved in this process. In deendothelialized segments the NO synthase inhibitor N G-nitro-L-arginine (L-NAME, 10 μM) increased the contractions caused by electrical field stimulation (EFS, 200 mA, 0.3 ms, 1–16 Hz, for 30 s). AII (0.1 nM) enhanced the response to EFS, which was unmodified by the subsequent addition of L-NAME. The AII antagonist receptor saralasine (0.1 μM) prevented the effect of AII, and the subsequent addition of L-NAME restored the contractile response. SOD (25 u/ml) decreased the reponse to EFS and the subsequent addition of L-NAME increased this response. AII did not modify the decrease in EFS response induced by SOD, and the addition of L-NAME increased the response. None of these drugs altered the response to exogenous noradrenaline (NA) or basal tone except SOD, which increased the basal tone, an effect blocked by phentolamine (1 μM). In arteries preincubated with [ 3H]-NA, AII did not modify the tritium efflux evoked by EFS, which was diminished by SOD. AII did not alter basal tritium efflux while SOD significantly increased it. These results suggest that EFS of SHR mesenteric arteries releases neurogenic NO, the metabolism of which is increased in the presence of AII by the generation of superoxide anions.

Characterization of the contractile and relaxant action of the endothelin-1 precursor, big endothelin-1, in the isolated rat basilar artery

The presence of functional endothelin converting enzyme (ECE) activity in basilar artery ring segments was investigated by measuring the contractile and relaxant effects of big endothelin (ET)-1. Under resting tension conditions cumulative application of big ET1-1 elicited a concentration-related contraction with the concentration-effect curve (CEC) shifted to the right against ET-1 by a factor of 31 and 29 in segments with the endothelium intact or mechanically removed, respectively. Preincubation with the ET(A) receptor antagonist, BQ123, induced an apparently parallel rightwards shift without affecting the maximum contraction. This shift was more pronounced for ET-1 than for big ET-1. With the putative ECE inhibitor phosphoramidon (10 −3 M) in the bath a small rightwards shift of the CEC for big ET-1 was observed in control segments and a more marked one in de-endothelialized segments. In segments precontracted with prostaglandin (PG) F 2α big ET-1 induced a significant although transient relaxation whereas ET-1 did not. However, in the presence of BQ123 both ET-1 and big ET-1 elicited concentration-related relaxation with a significantly higher maximum effect obtained with big ET-1. The potency was 13 fold higher for ET-1, which is markedly less than that found for contraction. The results, therefore, suggest 1) the presence of functional ECE-activity in the rat basilar artery wall, and 2) differences in the functional ECE activity located in the endothelium and media.

Evidence for functional endothelin-converting enzyme activity in isolated rat basilar artery: effect of inhibitors.

Endothelin-1 (ET-1) may be involved in the upregulation of cerebroarterial resistance under pathologic conditions, most notably in the development of vasospasm after subarachnoid hemorrhage. Therefore, blocking the contractile action of ET-1 by receptor antagonists may prove to be a new and worthwhile approach. However, decreasing synthesis and release of ET-1 by blocking the endothelin-converting enzyme (ECE) activity may also prove worthwhile. In this study we have therefore investigated the effect of several putative ECE inhibitors in isolated rat basilar artery by measuring isometric contraction after application of big ET-1, the precursor peptide which is not vasoactive in itself. In the presence of phosphoramidon (10(-4) M in segments with an intact endothelium or 5 x 10(-4) M in de-endothelialized segments), there was only a small shift to the right of the concentration-effect curve for big ET-1. Similarly, 10(-3) M thiorphan (a selective inhibitor of the neutral endopeptidase) did not affect big ET-1-induced contraction, both alone and in combination with phosphoramidon (10(-3) M). When the big ET-1 analogue [22Phe]big ET-1[19-37] was applied, an increase in resting tension occurred irrespective of whether or not the endothelium was present. Furthermore, in the presence of 10(-5) M [22Phe]big ET-1[19-37], contraction induced by big ET-1 was not affected in de-endothelialized segments but rather was enhanced in endothelium-intact segments. These results suggest the presence of functional ECE activity in the rat basilar artery wall. However, such activity could not be markedly inhibited with different putative enzyme inhibitors. Therefore, the chemical nature of the cerebroarterial ECE activity must be further elucidated before rational development of efficient ECE inhibitors for treatment of cerebral vasospasm becomes possible.

914-74 Estradiol 17βProtects Against Homocysteine-induced Vascular Injury in Rat Thoracic Aorta

Hyperhomocysteinemia produces atherosclerotic lesions with characteristic endothelial desquamation and intimal smooth muscle cell (VSMC) proliferation. In vitro , homocysteine is cytotoxic to endothelial cells and recent observations suggest that it may directly stimulate VSMC proliferation. Estrogen, on the other hand, inhibits VSMC proliferation both in vivo and in vitro . We evaluated the effect of estradiol 17 β (E 2 β ) on homocysteine-induced VSMC proliferation in arterial segments from the rat thoracic aorta. Segments were placed overnight in Dulbecco's Minimum Essential Medium, supplemented with gentamycin (25 μg/ml), glutamine (2 mM) and 0.4% fatal bovine serum, before incubation for 30 hours with DL-homocysteine thiolactone (0.1–5.0 mM). Radiolabelled thymidine uptake was assessed in intact and deendothelialized arterial segments, in presence of E 2 β (30 nM) or vehicle (1% ethanol). In deendothelialized segments homocysteine elicited a concentration-dependent increase in 3 H-thymidine uptake, expressed as cpm/mg protein. Thymidine uptake increased from a basal value of 8694 ± 1465 to 36338 ± 2025, at 5 mM homocysteine concentration (p &lt; 0.01). Intact arterial segments showed a significantly attenuated response to homocysteine stimulation. On the other hand, incubation of deendothelialized segments with E 2 β (30 nM) caused a significant inhibition of homocysteinestimulated response, without affecting basal 3 H-thymidine incorporation. These results provide evidence for a direct stimulatory effect of homocysteine on VSMC proliferation in rat aortic segments. Further, our data show estradiol 17 β protects against homocysteine-induced vascular injury, possibly via a direct effect on VSMC.

13,14-dihydro-prostaglandin E 1 decreases low-density lipoprotein influx into rabbit aorta

The effect of 4-week daily administration of 13,14-dihydro-prostaglandin E 1 (13,14-DH-PGE 1; 2 μg/kg) on LDL influx into the rabbit aortic wall was examined versus the effect of the same dose of PGE 1 and sham-treatment in 108 male animals fed an 1% cholesterol supplemented diet. Treatment was started after de-endothelialization of the abdominal aorta with a Fogarty catheter. After the 1-month treatment period the animals were injected with 10 μCi 125I-low-density lipoprotein (LDL; 0.5 mg protein/ml). Uptake of the radiolabelled LDL was measured in morphologically verified endothelialized, re-endothelialized and de-endothelialized abdominal aortic segments. The LDL influx into the aorta was significantly (P < 0.001) lower in 13,14-DH-PGE 1- and PGE 1-treated rabbits than in the controls. This reduction was most pronounced in re- and de-endothelialized segments. No significant difference between effect of PGE 1 and of its biologically active derivative, 13,14-DH-PGE 1, on arterial LDL entry was found. These data demonstrate a comparable beneficial effect of 13,14-DH-PGE 1 and PGE 1 on vascular wall lipid metabolism by decreasing LDL entry into the aortic wall in vivo.

Low density lipoprotein labelling characterizes experimentally induced atherosclerotic lesions in rabbits in vivo as to presence of foam cells and endothelial coverage

The entry of autologous iodine-125 low density lipoprotein (125I-LDL) into the aortic wall in rabbits was measured. After abdominal endothelium abrasion with a Fogarthy catheter the animals were fed a 1% cholesterol-supplemented diet for 4 weeks. The animals were killed 1-48 h after administration of 25 muCi 125I-LDL. Local entry of radiolabelled LDL was estimated and correlated to endothelial surface lining and foam cell content, both controlled morphologically. Endothelialized segments showed the lowest entry of 125I-LDL, the maximum uptake was reached at around 8 h. In de-endothelialized segments the entry was higher and the peak later (12 h), while in re-endothelialized segments a continuous increase in 125I-LDL entry up to 48 h was measured. Number and extent of foam cells correlated with the entry of LDL. The data indicate the usefulness of LDL radiolabelling for qualitative in vivo information on surface lining and foam cell content.

Diabetes-Induced Enhancement of Prostanoid-Stimulated Contraction in Mesenteric Veins of Mice

We investigated the influence of the diabetic state on the contractile response of longitudinal segments of isolated mesenteric vein to prostanoids and leukotriene (LT), and the contribution of the vascular endothelium to modulation of the contractile response was determined. The normal mesenteric vein and de-endothelialized veins of normal (ddY), diabetic KK-CAy and streptozotocin ddY mice (150 mg/kg, i.v., 6 weeks) were used. In the diabetic state, the contractions produced by noradrendline (60 μM), high K+ solution (143.4 mM), and the thromboxane A2 analogue U-46619 (29 nM–29 mM) were not affected, and LTD4 (0.1 nM-1 μM)-induced contraction was suppressed. Contractions induced by prostaglandin (PG) E2 (0.2 μM-2 mM), PGF2α (0.3 μM-0.3 mM) and the prostacyclin derivatives PGI2-Na (10–100 μM) and TRK-100 (0.2 μM–2 mM) were significantly enhanced in the presence of an intact vascular endothelium, but not in de-endothelialized segments. The increase in PGF2α (0.28 mM) contractions was dependent on age (correlation coefficient r=0.36, significant difference, P< 0.05) and blood glucose (r=0.88, significant difference, P<0.01), but was independent of obesity. The contractile response to PGD2 (0.3–0.9 mM) was enhanced in both intact and de-endothelialized segments. These results indicate that the diabetic state affects prostanoid responses in an endothelium-dependent manner, except for the PGD2 response, which is independent of the endothelium.

The Basement Membrane Underlying the Vascular Endothelium Is Not Thrombogenic: In Vivo and In Vitro Studies with Rabbit and Human Tissue

Studies examining the interaction of platelets with exposed subendothelium in vivo have reported conflicting results. To examine possible explanations for the apparently discrepant findings, we measured the platelet reactivity of subendothelium prepared by a number of methods both in vivo and in vitro. In addition, we examined the possibility that 13-hydroxyoctadecadinoic acid (13-HODE), an endothelial cell-derived chemorepellant, modulates the reactivity of the subendothelium to platelets. In vivo, the subendothelium of segments of rabbit carotid arteries was exposed by removing the endothelial cells by air perfusion or by balloon catheter stripping. Platelet accumulation onto the de-endothelialized segments was assessed by 3H-radioactivity uptake, using 3H-adenine-labelled platelets, and by scanning electron microscopy. In vitro, 3H-adenine-labelled platelet adhesion was measured onto plain plastic discs and onto plastic discs coated with the following purified basement membrane components: collagens type I, III, IV, V, laminin, or fibronectin. In addition, 3H-adenine-labelled platelet adhesion was measured onto plastic discs covered with human endothelial cells or onto the basement membrane underlying the endothelial cells. In vivo, there was marked 3H-platelet accumulation onto the balloon catheter carotid arteries one hour after injury. In contrast, there was no platelet accumulation onto the subendothelium of carotid arteries de-endothelialized by air perfusion. These differences were confirmed by scanning electron microscopy. Transmission electron microscopic examination demonstrated that the extracellular matrix was intact following the air perfusion injury whereas the majority of it was removed by the balloon catheter injury.(ABSTRACT TRUNCATED AT 250 WORDS)