Control Rat Aorta Research Articles

Acute Elution of TGFβ2 Affects the Smooth Muscle Cells in a Compliance-Matched Vascular Graft.

Transforming growth factor beta 2 (TGFβ2) is a pleiotropic growth factor that plays a vital role in smooth muscle cell (SMC) function. Our prior in vitro work has shown that SMC response can be modulated with TGFβ2 stimulation in a dose dependent manner. In particular, we have shown that increasing concentrations of TGFβ2 shift SMCs from a migratory to a synthetic behavior. In this work, electrospun compliance-matched and hypocompliant TGFβ2-eluting tissue engineered vascular grafts (TEVGs) were implanted into Sprague Dawley rats for 5 days to observe SMC population and collagen production. TEVGs were fabricated using a combined computational and experimental approach that varied the ratio of gelatin:polycaprolactone to be either compliance matched or twice as stiff as rat aorta (hypocompliant). TGFβ2 concentrations of 0, 10, 100 ng/mg were added to both graft types (n = 3 in each group) and imaged in vivo using ultrasound. Histological markers (SMC, macrophage, collagen, and elastin) were evaluated following explanation at 5 days. In vivo ultrasound showed that compliance-matched TEVGs became stiffer as TGFβ2 increased (100 ng/mg TEVGs compared to rat aorta, p < 0.01), while all hypocompliant grafts remained stiffer than control rat aorta. In vivo velocity and diameter were also not significantly different than control vessels. The compliance-matched 10 ng/mg group had an elevated SMC signal (myosin heavy chain) compared to the 0 and 100 ng/mg grafts (p = 0.0009 and 0.0006). Compliance-matched TEVGs containing 100 ng/mg TGFβ2 had an increase in collagen production (p < 0.01), general immune response (p < 0.05), and a decrease in SMC population to the 0 and 10 ng/mg groups. All hypocompliant groups were found to be similar, suggesting a lower rate of TGFβ2 release in these TEVGs. Our results suggest that TGFβ2 can modulate in vivo SMC phenotype over an acute implantation period, which is consistent with our prior in vitro work. To the author's knowledge, this is the first in vivo rat study that evaluates a TGFβ2-eluting TEVG. Impact statement TGFβ2 affects the SMCs in a vascular graft.

Abstract P524: Vasoprotective Effect of Hesperidin in the Onset of Arterial Hypertension Induced by L-NAME

Hesperidin has been used to manage venous disease and has been shown to have vasodilator, antiinflammatory and antioxidant properties. The aim of this work was to evaluate the efects of acute treatment with hesperidin (HD) on the vascular reactivity of rats in the onset of arterial hypertension induced by L-NAME. Male Wistar rats were divided into 3 groups as follow: 1- Control (CTL); 2- L-NAME (LN); 3- L-NAME + Hesperidin (LNHD). LN was given by gavage at the dose of 75mg/Kg, concomitantly with HD at the dose of 0.5 mmol/Kg, 24 hours previously to the sacrifice. After sacrifice, the blood was collected and the aorta removed and mounted in a wire myograph to assess concentration response curves to acetylcholine (ACh) and sodium nitroprusside (SNP). ACh elicited concentration dependent relaxation in aorta of CTL rats, reaching a maximum relaxation of 89 ± 3%, which was significantly reduced in LN group (37 ± 7%). Treatment with HD partially prevented this decrease, reaching the maximum relaxation of 72 ± 2%. On the other hand, SNP induced relaxation was more potent on the aorta of LN (pEC 50 9.67 ± 0.11) when compared to CTL (pEC 50 8.51 ± 0.13) and LNHD rats (pEC 50 8.90 ± 0.09). The oxidative status was not changed by any treatment since SOD activity was 11 ± 1, 9 ± 2 and 12 ± 0.5 U/mL, for CTL, LN and LNHD, respectively and lipid peroxidation measured by TBARS assay showed a MDA concentration of 29 ± 8, 35 ± 7 and 23 ± 3 μM for CTL, LN and LNHD, respectively. Altogether, these data suggest that that Hesperidin prevents the impairment of endothelium-dependent vascular relaxation, through a mechanism not related to its antioxidant property.

Effect of angiotensin II infusion on rhythmic clock gene expression and local renin-angiotensin system in the aorta of Wistar rats.

Endogenous daily rhythms in physiology are regulated by the circadian system consisting of the central and peripheral components. The renin-angiotensin system, involved predominantly in water balance and blood pressure control, exerts 24 h rhythmicity in many of its parameters. The present study is aimed to study possible interactions between these two control systems. We analyzed effects induced by angiotensin II administration on clock gene expression in the aorta of rat and an ability of angiotensin II to influence the local tissue renin-angiotensin system. Angiotensin II was infused in a dose of 100 ng/kg/min by subcutaneously implanted osmotic minipumps for 28 days to male Wistar rats. Gene expression was measured by real time PCR. Angiotensin II administration resulted in an increase in blood pressure, heart weight/body weight index, and water intake in comparison with controls. We observed a significant phase advance in per2 and npas2 mRNA rhythms and decreased mesor of npas2 rhythmic expression in the aorta of angiotensin II-treated rats compared to control. Angiotensin II administration did not influence daily pattern and level of at1 mRNA expression. The ratio ace/ace2 showed a rhythmic pattern in the aorta of control rats with peak levels in the dark period. Angiotensin II infusion influenced clock gene expression and diminished a daily rhythm in ace/ace2 mRNA ratio indicating modulatory effect of angiotensin II on tissue renin-angiotensin system in the aorta.

Selective inhibition of NADPH oxidase reverses the over contraction of diabetic rat aorta

Abnormal vascular responsiveness in diabetes has been attributed to a number of changes in contractile pathways, affected in part by the overproduction of reactive oxygen species (ROS). It has been reported that NADPH oxidase (NOX) is increased in diabetic (streptozotocin treated; STZ) rat arteries; however the pharmacological agents used to inhibit NOX activity are known to be unsuitable for in vitro studies and have a high level of non-selectivity. Here we have used the highly selective NOX inhibitor VAS2870 in diabetic rat aorta and compared its effects with apocynin, SOD, and allopurinol on phenylephrine and U46619 induced contraction.Male Wistar rats were injected intraperitoneally with 65mg/kg STZ and development of diabetes was confirmed by testing blood glucose levels. Rats were killed by CO2 asphyxiation, and the thoracic aorta removed and mounted in an organ bath under a tension of 1g. Diabetic rat aortas exhibit a greatly increased response to phenylephrine, which was reduced to a level consistent with control rat aorta by 10−5M VAS2870 and 150U/ml SOD. Incubation with VAS2870 led to an increase in normal rat aorta contraction, but led to a significant reduction in phenylephrine and U46619 induced tone in diabetic rat aorta, which indicates that ROS in diabetic rats directly contributes to these contractile responses. Apocynin and allopurinol had no effect on contraction in diabetic or normal rat aorta. This data is the first to show that selective inhibition of NOX reduces diabetic arterial contraction in direct comparison with inhibition of other known contributors of ROS.

Abstract 353: Diabetes Induces Increased Expression of Matricellular Osteoblast Factor Periostin and Inhibits the Calcification Inhibitor Matrix Gla Protein (mgp) in Rat Aorta

It is increasingly recognized that diabetes is associated with increased risk of cardiovascular events and vascular calcification. To understand the mechanism of vascular calcification in diabetes we used a rat model of type 2 diabetes. Rats were fed ad libitum with diet containing 45 kcal % fat (soybean oil and lard with 0.95 mg cholesterol /gram of lard), 35 kcal % carbohydrates and 20% kcal protein (HF diet) for eight weeks, followed by a single low dose of stz (30mg/kg intra peritoneally). Rats were then allowed to developed diabetes for sixteen weeks. Average blood glucose in diabetic HF rats 517.6mg/dl vs 87.8mg/dl in HF rats. The expression and immunohistochemical changes were analyzed in aorta of these rats after sixteen weeks of diabetes. Conformational specific gamma carboxylated (Gla) and undercarboxylated (Glu) MGP antibodies were used to assess the expression of Gla and Glu MGP in control and diabetic HF rat aorta. Western blot analysis show significantly reduced expression of Gla MGP in diabetic HF rat aorta and higher Glu MGP expression compared to control rat aorta suggesting that diabetic conditions inhibit the gamma carboxylation of MGP in aorta, gamma carboxylation modification is necessary for the MGP to work as physiological calcification inhibitor. Similarly, immunohistochemical analysis of diabetic HF rat and control rat aorta show increased staining of Glu MGP than Gla MGP compared to control rat aorta further confirm our Western data that modification of Gla MGP is inhibited in diabetic HF rats. Furthermore, immuno-histochemical data also show significantly increased in diabetic HF rat aorta expression of Periostin a matricellular osteoblast specific factor expression compared to control rat aorta. Our findings suggest that active diabetes impair the physiological properties of calcification inhibitor Gla MGP in aorta and increases the osteogenic factors in diabetic HF rats. Increased expression of osteoblastic factor and inhibition of Gla MGP suggests that diabetic HF rat aorta is under active transformation to become more bone like, precursor of arterial calcification.

Comparison of Combination Therapy of Olmesartan plus Azelnidipine or Hydrochlorothiazide on Renal and Vascular Damage in SHR/NDmcr-cp Rats

Background: Although the recommended target blood pressure for patients with chronic kidney disease is <130/80 mm Hg, this is difficult to achieve by treatment with an angiotensin receptor blocker alone. Addition of either a calcium channel blocker or a diuretic is suggested as second-line medication; however, which combination is most beneficial for target-organ protection remains unknown. Methods: SHR/NDmcr-cp rats were administered no medications (control) or low-dose olmesartan for 2 weeks and then either olmesartan at an increased dose, azelnidipine, or the hydrochlorothiazide for 3 weeks. We assessed oxidative stress in the kidney and aorta, and endothelial function. Results: Urinary protein excretion was lower in all treated rats than in control rats. Oxidative stress caused by activation of NAD(P)H oxidase was observed in the glomeruli and aorta of control rats and was significantly suppressed in the olmesartan/azelnidipine (Olm/Azl) groups. Combination therapy with olmesartan and hydrochlorothiazide (Olm/HCTZ) however failed to suppress oxidative stress. The Olm/Azl groups maintained the endothelial surface layer in the glomeruli and protected endothelial function in the aorta. Conclusion: In an animal model of metabolic syndrome, a combination of Olm/Azl is superior to a combination of Olm/HCTZ in terms of prevention of glomerular and vascular injuries.

Depression of cyclooxygenase-2 induction in aortas of rats with type 1 and type 2 diabetes mellitus

The purpose of this study was to determine whether cyclooxygenase expression in arteries is affected by diabetes. Streptozotocin-injected rats and Goto-Kakizaki rats were used as animal models for type 1 and type 2 diabetes, respectively. Cyclooxygenase-2 expression was induced by lipopolysaccharide. Lipopolysaccharide-induced cyclooxygenase-2 expression was significantly lower in aortas isolated from streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats, while expression level of cyclooxygenase-1 was not affected by lipopolysaccharide and was not different in aortas of the three groups of rats. The level of 6-keto-prostaglandin F 1α that accumulated in the presence of lipopolysaccharide as well as the basal accumulation level in the absence of lipopolysaccharide was significantly lower in aortas of streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats. The net increase in 6-keto-prostaglandin F 1α level in response to stimulation with lipopolysaccharide, which was calculated by subtracting the basal accumulation level from the total accumulation level, was also significantly lower in aortas of streptozotocin-injected rats and Goto-Kakizaki rats than in aortas of control rats. There were no significant differences in the accumulated 6-keto-prostaglandin F 1α levels in the absence or presence of lipopolysaccharide and the levels of basal and lipopolysaccharide-induced cyclooxygenase-2 expression in control or Goto-Kakizaki rat aortas under the conditions of different glucose concentrations in the medium. These results suggest that lipopolysaccharide-induced cyclooxygenase-2 expression and subsequent prostacyclin production are decreased in aortas isolated from both type 1 and type 2 diabetes rats.

Insulin-induced impairment via peroxynitrite production of endothelium-dependent relaxation and sarco/endoplasmic reticulum Ca 2+-ATPase function in aortas from diabetic rats

We designed this study to determine whether a high insulin level and a diabetic state need to exist together to cause an impairment of endothelium-dependent relaxation. In diabetic rat aortas organ-cultured with insulin [vs both control rat aortas cultured with insulin and diabetic rat aortas cultured in serum-free medium]: (1) the relaxation responses to both acetylcholine (endothelium-dependent relaxation) and Angeli’s salt (nitric oxide donor) were significantly weaker, (2) acetylcholine-stimulated nitric oxide production was significantly smaller, (3) superoxide and nitric oxide production into the culture medium was greater, and (4) the levels of both nitrotyrosine and tyrosine-nitrated sarco/endoplasmic reticulum calcium ATPase (SERCA) protein were greater. The insulin-induced effects were prevented by cotreatment with either a superoxide scavenger or a peroxynitrite scavenger. After preincubation with an irreversible SERCA inhibitor, the relaxation induced by the nitric oxide donor was significantly impaired in control aortas cultured with or without insulin and in diabetic aortas cultured without insulin, but not in diabetic aortas cultured with insulin. These results suggest that the coexistence of a high insulin level and an established diabetic state may lead to an excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in SERCA function.

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.

IGF-I-induced enhancement of contractile response in organ-cultured aortae from diabetic rats is mediated by sustained thromboxane A2 release from endothelial cells

We have investigated the mechanisms underlying the changes in vascular contractile responsiveness induced by insulin and IGF-I in established streptozotocin-induced diabetic rats. The contractile response to noradrenaline (NA) in organ-cultured diabetic rat aortae cultured with insulin or IGF-I was significantly greater than the corresponding responses in (a) diabetic rat aortae cultured in serum-free medium and (b) control rat aortae cultured with insulin or IGF-I. In aortae from which the endothelium was removed after organ culture the contractile response to NA was greater in those cultured with insulin or IGF-I than in those cultured in serum-free medium. This was not true of aortae endothelium denuded before organ culture. The IGF-I-induced enhancement was prevented by treatment with indomethacin (cyclo-oxygenase inhibitor), SQ29548 (thromboxane (TX) A2 receptor antagonist) or fregrelate (TXA2 synthase inhibitor). IGF-I-induced production of TXB2, a metabolite of TXA2, was greater in diabetic than in control aortae and was attenuated by endothelium denudation, indomethacin or AG1024 (IGF-I receptor inhibitor). The expression of the protein and mRNA for the IGF-I receptor (as assessed by RT-PCR and immunohistochemistry) was markedly increased within endothelial cells in diabetic aortae but only slightly increased within smooth muscle cells (versus control rat aortae). Thus, the NA-induced contractile response in aortae from diabetic rats was enhanced by both insulin and IGF-I and this enhancement may be mediated by sustained cyclo-oxygenase-dependent TXA2 production from endothelial cells. The observed enhancement of IGF-I receptor expression within endothelial cells may be causally related to the potentiation of vascular contractility and the increase in TXA2 production.

Pentose phosphate pathway, glutathione-dependent enzymes and antioxidant defense during oxidative stress in diabetic rodent brain and peripheral organs: effects of stobadine and vitamin E.

The aim of the present study was to investigate the effects of treatment with antioxidant stobadine (ST) on the activities of enzymes related with pentose phosphate pathway and glutathione-dependent metabolism and the other markers of oxidative stress in brain and peripheral organs of diabetic rats, and to compare the effects of ST treatment alone with the effects of treatments with another antioxidant vitamin E and ST plus vitamin E. Rats were made diabetic by the injection of streptozotocin (STZ; 55 mg/kg IP), and, 2 days later, some control and diabetic rats were left untreated or treated with ST (24.7 mg/kg/day, orally), vitamin E (400-500 U/kg/day, orally), or both substances together. In the brain, although 6-phosphogluconate dehydrogenase activity (6-PGD) did not change, glucose-6-phosphate dehydrogenase activity (G-6PD) was markedly increased in diabetic rats compared with controls; only combined treatment with ST and vitamin E produced a partial prevention on this alteration. The aorta G-6PD and 6-PGD of diabetic rats were 52% and 36% of control values, respectively. Neither single treatments with each antioxidant nor their combination altered the G-6PD and 6-PGD in aorta of diabetic rats. Glutathione peroxidase (GSHPx) activity was increased by STZ-diabetes in brain, heart, and kidney. In diabetic brain, vitamin E alone or combination with ST kept GSHPx at normal levels. Diabetes-induced stimulation in GSHPx did not decrease in response to the treatment with vitamin E in heart and kidney, but was greatly prevented by ST alone. The activity of glutathione reductase (GR) was decreased in brain and heart of diabetic rats. The treatment with each antioxidant or with a combination of both agents completely prevented this deficiency and resulted in further activation of GR in diabetic tissues. Glutathione S-transferase (GST) activity did not significantly change in diabetic brain and aorta. GST was stimulated by all treatment protocols in the brain of diabetic rats and was depressed in aorta of control rats. Catalase (CAT) was activated in diabetic heart but depressed in diabetic kidney. Diabetes-induced abnormalities in CAT activity did not respond to vitamin E alone in heart, was moderately ameliorated by the treatment with this vitamin in kidney, and was completely prevented by ST alone in both tissues. Superoxide dismutase (SOD) activity of brain and heart was unchanged by the diabetes but inhibited in diabetic kidney after the treatment ST alone or ST plus vitamin E. The lipid peroxidation (MDA) was increased in diabetic brain and heart. ST or vitamin E alone partly prevented diabetes-induced increase in MDA in brain and heart; however, antioxidant combination achieved a completely amelioration in MDA of these tissues of diabetic rats. Kidney MDA levels were similar in control and untreated diabetic animals. ST and vitamin E treatments, when applied separately or together, significantly reduced kidney MDA in both control and diabetic rats; and the combined effect of antioxidants was greater than that of each alone. These results are consistent with the degenerative role of hyperglycemia on cellular reducing equivalent homeostasis and antioxidant defense, and provide further evidence that pharmacological intervention of different antioxidants may have significant implications in the prevention of the prooxidant feature of diabetes and protects redox status of the cells.

An angiotensin-converting enzyme inhibitor, not an angiotensin II type-1 receptor blocker, prevents β-aminopropionitrile monofumarate-induced aortic dissection in rats

Cystic medial degeneration (CMD) is a histologic abnormality that is common in aortic diseases such as aortic dilation, aneurysm, or dissection. Although little is known about the mechanism underlying CMD, we have previously demonstrated that angiotensin II signaling via angiotensin II type 2 receptor (AT2R) plays a central role in apoptosis of vascular smooth muscle cells (VSMCs) occurring in CMD associated with Marfan syndrome. The aim of this study is to elucidate the role of angiotensin II signaling in THE pathogenesis of aortic diseases associated with CMD. We investigated the effects of angiotensin-converting enzyme inhibitor (ACEI), temocapril (n = 15), angiotensin II receptor type-1 (AT1R) blocker, CS-866 (n = 15), and vehicle control (n = 17) on 0.25% beta-aminopropionitrile monofumarate (BAPN)-induced aortic dissection and histopathologic findings in a rat model. Temocapril significantly prevented aortic dissection (P <.05), CMD (P <.01), and VSMC apoptosis (P <.01) compared with vehicle control in BAPN-fed rats. However, CS-866 did not show any preventive effect. Reversed transcriptase-polymerase chain reaction demonstrated that expression of both AT1R and AT2R was detected in control rat aortas, and that AT2R expression was significantly upregulated in the aortas of BAPN-fed rats (P <.01). Blood pressure was significantly and equally lowered in both temocapril and CS-866 groups compared with control. Differential expression of angiotensin II receptors and AT2R signaling are involved in the pathogenesis of CMD and aortic dissection in BAPN-fed rats. ACEIs might be of clinical value for the prevention and treatment of aortic diseases related to CMD.

Chronic inhibition of nitric oxide synthesis in rats increases aortic superoxide anion production via the action of angiotensin II.

Chronic inhibition of nitric oxide (NO) synthesis by Nomega-nitro-L-arginine methyl ester (L-NAME) increases vascular tissue angiotensin II activity and oxidative stress in animals by incompletely understood mechanisms. In a rat model, we investigated the role of local angiotensin II activity in the pathogenesis of increased oxidative stress. We studied the aortas of control rats and others receiving L-NAME or L-NAME plus an angiotensin II type 1 receptor antagonist (CS-866). Administration of L-NAME for 7 days significantly increased superoxide anion (O2-) and both immunoreactivity and electrophoretically demonstrable activity of redox-sensitive transcription factors (NF-kappaB and AP-1). Treatment with the angiotensin II type 1 receptor antagonist prevented all of the above changes. The observed effects of the type 1 receptor antagonist was independent of the L-NAME-induced arterial hypertension. These findings suggest that chronic inhibition of NO synthesis may increase vascular oxidative stress and oxidative stress-sensitive signals via the action of angiotensin II mediated via type 1 receptors.

Metformin treatment corrects vascular insulin resistance in hypertension

In states of insulin resistance, the vasorelaxant actions of insulin are blunted, which may contribute towards the development of increased vascular tone/hypertension and reduced glucose uptake. To examine whether treating insulin resistance in hypertension restores the vascular actions of insulin, we studied the long-term effects of metformin on the contractile responses of isolated aortas from control and insulin-resistant, hyperinsulinaemic fructose-hypertensive rats in the presence and absence of insulin. Sprague Dawley rats were divided into control, control metformin-treated, fructose and fructose metformin-treated groups (n = 8 per group). The treated groups received metformin (500 mg/kg per day for 6 weeks), following which isometric responses to noradrenaline (NA) and angiotensin II (A-II) were examined in thoracic aortas in the presence and absence of insulin (100 mU/ml for 2 h) using isolated organ-bath apparatus. In addition, endothelium-dependent and independent vascular responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were also studied. Metformin treatment prevented the development of fructose-induced insulin resistance, hyperinsulinaemia and hypertension. Insulin attenuated the contractile responses to NA and A-II in control rat aortas; however, blood vessels from untreated fructose rats were refractory to insulin-induced vasodilation. Strikingly, long-term metformin treatment restored the vasodepressor actions of insulin in fructose rats. Metformin did not affect the contractile responses to NA or A-II in either control or fructose rats. In addition, metformin treatment restored ACh-induced endothelium-dependent vasorelaxation in aortas from fructose rats without affecting SNP-induced relaxation. These data show, for the first time, that long-term metformin treatment corrects vascular insulin resistance and improves endothelium-dependent vasorelaxation in hypertension. These effects appear to be secondary to metformin-induced improvements in metabolic derangements (versus a direct vascular action of metformin). Improving the vascular effects of insulin may serve to decrease peripheral tone, attenuate blood pressure and improve insulin sensitivity.

Increased hyaluronan and hyaluronidase production and hyaluronan degradation in injured aorta of insulin-resistant rats.

Diabetic patients have a greater incidence of restenosis, which has been shown to be related to exaggerated intimal hyperplasia. Hyaluronan (HA) has been shown to be closely involved in arterial smooth muscle cell proliferation and migration, which provoke intimal hyperplasia after balloon catheter injury. Our aim was to determine the effect of fructose feeding, which produces certain characteristics of non-insulin-dependent diabetes (ie, insulin resistance, hyperinsulinemia, and hypertriglyceridemia), on production of HA and hyaluronidase and degradation of HA in rat aorta. Treated rats received fructose (25% in tap water) 12 weeks before balloon catheter injury and 14 days afterward. Fructose-fed rats had hyperinsulinemia and hypertriglyceridemia. Injury increased intima-media wet weight (7.5%) and DNA content (20%) in control rats. This increase was significantly greater in fructose-fed rats (22% for wet weight and 34% for DNA content) and was associated with greater HA and hyaluronidase production (123% and 41%, respectively) than in control rats (49% and 7%, respectively). Determination of HA molecular mass showed that balloon catheter injury increased the number of HA fragments in the aorta of control rats. Normal aorta of fructose-fed rats contained more HA fragments than that of control rats. Injury to the aorta of fructose-fed rats increased HA fragments and induced the appearance of a very-high-molecular-mass (>2000 kDa) HA. In conclusion, fructose treatment, which induced hyperinsulinemia and hypertriglyceridemia, increased HA and hyaluronidase production and HA degradation in injured aorta. This finding suggests that HA, which has been shown to play a crucial role in proliferation and migration of arterial smooth muscle cells, may be involved in the promotional effect of long-term fructose feeding on arterial wall reaction to injury.

Effect of Long-Term Ouabain Treatment on Contractile Responses of Rat Aortae

Male Sprague-Dawley rats were infused with 50 microg/kg/day of ouabain for 4 weeks to address the question whether prolonged exposure to the drug affects blood pressure, the in vitro contractile responses to agonists and high K+ of their aortae, and the influence of endothelium on these responses. Systolic blood pressure was not affected by ouabain treatment. The responsiveness of endothelium-intact aortae from ouabain-treated rats to endothelin-1 increased, that to phenylephrine decreased, and that to high K+ was unchanged, as compared with control. The responses of endothelium-free aortae to endothelin-1, phenylephrine, and high K+ were lower in ouabain-treated than in control rats. The removal of endothelium increased the response to phenylephrine and decreased that to high K+ in either control or ouabain-treated rat aortae, whereas it did not affect the response to endothelin-1 in control rat aortae and decreased it in ouabain-treated rat aortae. The response to caffeine was unaffected by either ouabain treatment or endothelium removal. Thus rat ouabain long-term treatment induces opposing effects on the responsiveness of their intact aortae to an alpha-adrenergic agonist and endothelin-1. If these effects observed in the ex vivo experiments occur also in vivo on rat microvasculature, they could balance out and contribute to the lack of effect on systolic blood pressure of prolonged ouabain treatment.

Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction.

Nitric oxide release from the endothelium plays an important role in regulation of vascular tone, inhibition of both platelet and leukocyte aggregation and adhesion, and inhibition of cell proliferation. These properties suggest that the level of NO production by the endothelium may play a pivotal role in the regulation of vascular disease. Analysis using mass spectrometry has revealed that NO is produced by NOS from the terminal guanidino nitrogen of the precursor amino acid L-arginine. Thus, utilization of L-arginine and conversion to NO may establish a regulatory site in the development of endothelial dysfunction. Endothelial dysfunction is characterized by defective endothelium-dependent relaxation, and some reviews regarding endothelial dysfunction in diabetes have been published. These reviews have focused on factors that might contribute to defective relaxation, some of which will not be addressed in detail in this review. The purpose of the present review is to summarize evidence that specifically supports either decreased NO production by diabetic vascular endothelium and/or impaired NO-mediated endothelium-dependent relaxation. Second, this review provides reasonable alternatives to explain some of the controversies in this research area. Third, since there is growing evidence that arginine appears to have some benefits for diabetes-associated abnormalities, this review summarizes the current state of knowledge of effects of acute and chronic administration of L-arginine on diabetesinduced endothelial dysfunction and discusses potential NOdependent and -independent mechanisms whereby therapeutic intervention with L-arginine might benefit the diabetic endothelium.

Protein kinase C alterations in aortic vascular smooth muscle cells from rats with cirrhosis

Background/Aims: Alterations in signal transduction in vascular smooth muscle cells may contribute to vascular hyporeactivity in cirrhosis. Protein kinase C plays a role in vascular cell contraction by modifying contractile proteins and intracellulawr [Ca 2+] homeostasis. The aim of this study was to examine the vascular reactivity and expression of protein kinase Cα in aortae from rats with cirrhosis. Methods: The contractile response to phorbol ester, a protein kinase C activator, was evaluated in endothelium-denuded aortic rings from normal and cirrhotic rats. Protein kinase Cα expression was determined by Western blot analysis. Results: Maximal contraction was significantly less marked in cirrhotic (1.24±0.24 g) than in control (3.43±0.27 g) aortae. Phorbol myristate-acetate-induced contraction was dependent on extracellular [Ca 2+] concentrations, as shown by a reduction in maximal contraction when control and cirrhotic aortic rings were exposed to a Ca 2+-free medium. Increasing the intracellular [Ca 2+], by incubation with a Ca 2+ ionophore, significantly increased the maximal contraction induced by phorbol myristate-acetate in cirrhotic but not in control rat aortae. Protein kinase Cα expression was significantly lower in aortae in cirrhotic than in control rats. Conclusion: these results confirm alterations in protein kinase C in aortae from cirrhotic rats.

Lack of Endothelin ETBReceptor Binding and Function in the Rat with a Mutant ETBReceptor Gene

Congenital aganglionosis rat is a mutant with an autosomal recessive gene (sl). Recent studies have revealed that the endothelin ETBreceptor gene ofsl/slrat has a deletion of 301-bp region spanning exon 1 and intron 1 corresponding to the first and the second transmembrane domains of the receptor. In the present experiments, we examined the functions of ETBreceptors in thesl/slrats. In the membranes of cerebellum, heart, and lung of control (+/+ andsl/+) rats, ET-1 induced a monophasic, competitive displacement of [125I]ET-1 binding, whereas ET-3, IRL 1620, and BQ-123 showed biphasic displacement. In the membranes ofsl/slrats, in contrast, ET-1, BQ-123, ET-3, and IRL 1620 showed only monophasic displacement. Scatchard analysis revealed a single [125I]ET-3 binding site in the membrane of control heart but not in thesl/slrat heart, and the specific binding sites for [125I]ET-1 in both control andsl/slrat hearts. In the control rat aorta but not in thesl/slrat aorta, ET-3 induced endothelium-dependent relaxation. These results suggest thatsl/slrats do not have functional ETBreceptors.

Chronic renal failure and human growth hormone treatment do not modify endothelium-dependent reactions in the rat aorta in vitro.

1. Growth hormone (GH) increases glomerular filtration rate and renal plasma flow and decreases renal vascular resistance. Sustained GH-induced hyperfiltration might be undesirable in children with chronic renal failure (CRF) who are receiving recombinant human GH (rhGH) therapy. 2. In order to determine the effect of CRF on vascular reactivity and the modifications induced by rhGH administration, two endothelium-dependent effects, acetylcholine relaxation and decrease of contractile response to noradrenaline, were studied in aorta segments of various groups of male Sprague-Dawley rats: CRF rats (CRF, n = 8) with serum urea nitrogen (SUN) 68 +/- 16 mg dl-1 (mean +/- SEM), CRF rats treated with intraperitoneal rhGH at 10 IU kg-1 day-1 for 13 days (CRFGH, n = 6, SUN = 88 +/- 15 mg dl-1), sham operated rats (SHAM, n = 8, SUN: 21 +/- 1 mg dl-1) and control rats (CONTROL, n = 8, SUN 20 +/- 1 mg dl-1), housed in identical conditions but without undergoing surgical intervention or manipulation. CRF was induced by 5/6 two stage nephrectomy. 3. Rats were sacrificed and a segment of thoracic aorta was immediately removed, cut into spirals, and suspended in organ baths according to standard procedures. First, dose-response curves to noradrenaline and acetylcholine relaxation, in strips previously exposed to noradrenaline, were determined. Then, the endothelium was removed and both dose-response curves were repeated. Acetylcholine induced a greater relaxation, P < 0.05, in the aorta of CONTROL rats (82.6 +/- 6.1%) as compared with SHAM (60.3 +/- 4.7%), CRF (60.0 +/- 6.8%) and CRFGH (54.8 +/- 8.2%) rats. 4. Endothelium removal only caused a greater contractile response to noradrenaline (10(-9) and 3 x 10(-9)M) in the CONTROL group, P < 0.05. 5. No differences to acetylcholine and noradrenaline responses were found among the SHAM, CRF and CRFGH groups. 6. These results suggest that the endothelium-dependent vascular reactivity was modified by the experimental protocol to induce chronic renal failure but no further changes resulted from uraemia and rhGH treatment.