Increased vascular contractility and oxidative stress in beta(2)-adrenoceptor knockout mice: the role of NADPH oxidase

Background/Aims: beta(2)-adrenoceptor (beta(2)-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal beta(2)-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional beta(2)-AR (beta 2KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from beta 2KO and wild-type mice (WT) were studied. beta 2KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between beta 2KO and WT mice. Basal NO availability was reduced in aortas from beta 2KO mice. Incubation of beta 2KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. beta 2KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47(phox) expression was enhanced in beta 2KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of beta 2KO mice. This study extends the knowledge of the relevance of the endogenous activity of beta(2)-AR to the maintenance of the vascular physiology. Copyright (C) 2012 S. Karger AG, Basel

Interaction between Advanced Glycation End Products Formation and Vascular Responses in Femoral and Coronary Arteries from Exercised Diabetic Rats

Background: The majority of studies have investigated the effect of exercise training (TR) on vascular responses in diabetic animals (DB), but none evaluated nitric oxide (NO) and advanced glycation end products (AGEs) formation associated with oxidant and antioxidant activities in femoral and coronary arteries from trained diabetic rats. Our hypothesis was that 8-week TR would alter AGEs levels in type 1 diabetic rats ameliorating vascular responsiveness. Methodology/Principal Findings: Male Wistar rats were divided into control sedentary (C/SD), sedentary diabetic (SD/DB), and trained diabetic (TR/DB). DB was induced by streptozotocin (i.p.: 60 mg/kg). TR was performed for 60 min per day, 5 days/week, during 8 weeks. Concentration-response curves to acetylcholine (ACh), sodium nitroprusside (SNP), phenylephrine (PHE) and tromboxane analog (U46619) were obtained. The protein expressions of eNOS, receptor for AGEs (RAGE), Cu/Zn-SOD and Mn-SOD were analyzed. Tissues NO production and reactive oxygen species (ROS) generation were evaluated. Plasma nitrate/nitrite (NOx-), superoxide dismutase (SOD), catalase (CAT), thiobarbituric acid reactive substances (TBARS) and N-epsilon-(carboxymethyl) lysine (CML, AGE biomarker). A rightward shift in the concentration-response curves to ACh was observed in femoral and coronary arteries from SD/DB that was accompanied by an increase in TBARS and CML levels. Decreased in the eNOS expression, tissues NO production and NOx- levels were associated with increased ROS generation. A positive interaction between the beneficial effect of TR on the relaxing responses to ACh and the reduction in TBARS and CML levels were observed without changing in antioxidant activities. The eNOS protein expression, tissues NO production and ROS generation were fully re-established in TR/DB, but plasma NOx- levels were partially restored. Conclusion: Shear stress induced by TR fully restores the eNOS/NO pathway in both preparations from non-treated diabetic rats, however, a massive production of AGEs still affecting relaxing responses possibly involving other endothelium-dependent vasodilator agents, mainly in coronary artery.

Red wine and equivalent oral pharmacological doses of resveratrol delay vascular aging but do not extend life span in rats

Objective: To investigate, in male Wistar rats, the effects of long-term moderate red wine (RW) consumption (equivalent to similar to 0.15 mg% resveratrol RS), or RS in low (L, 0.15 mg%) or high (H, 400 mg%) doses in chow. Background: Both RW and RS exhibit cardioprotection. RS extends lifespan in obese rats. It is unclear whether RW consumption or low-dose RS delay vascular aging and prolong life span in the absence of overt risk factors. Methods: Endpoints were aerobic performance, exercise capacity, aging biomarkers (p53,p16,p21, telomere length and telomerase activity in aortic homogenates), vascular reactivity. Data were compared with controls (C) given regular chow. Results: Expressions of p53 decreased similar to 50% similar to with RW and LRS (p < 0.05 vs. C), p16 by similar to 29% with RW (p < 0.05 vs. C) and p21 was unaltered. RW and LRS increased telomere length >6.5-fold vs. C, and telomerase activity increased with LRS and HRS. All treatments increased aerobic capacity (C 32.5 +/- 1.2, RW 38.7 + 1.7, LRS 38.5 + 1.6, HRS 38.3 + 1.8 mlO2 min(-1) kg(-1)), and RW or LRS also improved time of exercise tolerance vs. C (p < 0.05). Endothelium-dependent relaxation improved with all treatments vs. C. Life span, however, was unaltered with each treatment vs. C = 673 +/- 30 days, p = NS. Conclusions: RW and LRS can preserve vascular function indexes in normal rats, although not extending life span. These effects were translated into better aerobic performance and exercise capacity. (c) 2012 Elsevier Ireland Ltd. All rights reserved.

Alterations in vasoconstrictor responses to the endothelium-derived contracting factor uridine adenosine tetraphosphate are region specific in DOCA-salt hypertensive rats

Uridine adenosine tetraphosphate (Up(4)A) has been recently identified as a novel and potent endothelium-derived contracting factor and contains both purine and pyrimidine moieties, which activate purinergic P2X and P2Y receptors. The present study was designed to compare contractile responses to Up(4)A and other nucleotides such as ATP (P2X/P2Y agonist), UTP (P2Y(2)/P2Y(4) agonist), UDP (P2Y(6) agonist), and alpha,beta-methylene ATP (P2X(1) agonist) in different vascular regions [thoracic aorta, basilar, small mesenteric, and femoral arteries] from deoxycorticosterone acetate-salt (DOCA-salt) and control rats. In DOCA-salt rats [vs. control uninephrectomized (Uni) rats]: (1) in thoracic aorta, Up(4)A-, ATP-, and UP-induced contractions were unchanged; (2) in basilar artery, Up(4)A-, ATP-, UTP- and UDP-induced contractions were increased, and expression for P2X(1), but not P2Y(2) or P2Y(6) was decreased; (3) in small mesenteric artery, Up(4)A-induced contraction was decreased and UDP-induced contraction was increased; expression of P2Y(2) and P2X(1) was decreased whereas P2Y(6) expression was increased; (4) in femoral artery, Up(4)A-. UTP-, and UDP-induced contractions were increased, but expression of P2Y(2), P2Y(6) and P2X(1) was unchanged. The alpha,beta-methylene ATP-induced contraction was bell-shaped and the maximal contraction was reached at a lower concentration in basilar and mesenteric arteries from Uni rats, compared to arteries from DOCA-salt rats. These results suggest that Up(4)A-induced contraction is heterogenously affected among various vascular beds in arterial hypertension. P2Y receptor activation may contribute to enhancement of Up(4)A-induced contraction in basilar and femoral arteries. These changes in vascular reactivity to Up(4)A may be adaptive to the vascular alterations produced by hypertension. (C) 2011 Elsevier Ltd. All rights reserved.

Emerging role of angiotensin type 2 receptor (AT2R)/Akt/NO pathway in vascular smooth muscle cell in the hyperthyroidism

Hyperthyroidism is characterized by increased vascular relaxation and decreased vascular contraction and is associated with augmented levels of triiodothyronine (T3) that contribute to the diminished systemic vascular resistance found in this condition. T3 leads to augmented NO production via PI3K/Akt signaling pathway, which in turn causes vascular smooth muscle cell (VSMC) relaxation; however, the underlying mechanisms involved remain largely unknown. Evidence from human and animal studies demonstrates that the renin-angiotensin system (RAS) plays a crucial role in vascular function and also mediates some of cardiovascular effects found during hyperthyroidism. Thus, in this study, we hypothesized that type 2 angiotensin II receptor (AT2R), a key component of RAS vasodilatory actions, mediates T3 induced-decreased vascular contraction. Marked induction of AT2R expression was observed in aortas from T3-induced hyperthyroid rats (Hyper). These vessels showed decreased protein levels of the contractile apparatus: α-actin, calponin and phosphorylated myosin light chain (p-MLC). Vascular reactivity studies showed that denuded aortic rings from Hyper rats exhibited decreased maximal contractile response to angiotensin II (AngII), which was attenuated in aortic rings pre-incubated with an AT2R blocker. Further study showed that cultured VSMC stimulated with T3 (0.1 µmol/L) for 24 hours had increased AT2R gene and protein expression. Augmented NO levels and decreased p-MLC levels were found in VSMC stimulated with T3, both of which were reversed by a PI3K/Akt inhibitor and AT2R blocker. These findings indicate for the first time that the AT2R/Akt/NO pathway contributes to decreased contractile responses in rat aorta, promoted by T3, and this mechanism is independent from the endothelium.

Reduced endothelium-dependent relaxation to anandamide in mesenteric arteries from young obese Zucker rats

Impaired vascular function, manifested by an altered ability of the endothelium to release endothelium-derived relaxing factors and endothelium-derived contracting factors, is consistently reported in obesity. Considering that the endothelium plays a major role in the relaxant response to the cannabinoid agonist anandamide, the present study tested the hypothesis that vascular relaxation to anandamide is decreased in obese rats. Mechanisms contributing to decreased anandamide-induced vasodilation were determined. Resistance mesenteric arteries from young obese Zucker rats (OZRs) and their lean counterparts (LZRs) were used. Vascular reactivity was evaluated in a myograph for isometric tension recording. Protein expression and localization were analyzed by Western blotting and immunofluorescence, respectively. Vasorelaxation to anandamide, acetylcholine, and sodium nitroprusside, as well as to CB1, CB2, and TRPV1 agonists was decreased in endothelium-intact mesenteric arteries from OZRs. Incubation with an AMP-dependent protein kinase (AMPK) activator or a fatty acid amide hydrolase inhibitor restored anandamide-induced vascular relaxation in OZRs. CB1 and CB2 receptors protein expression was decreased in arteries from OZRs. Incubation of mesenteric arteries with anandamide evoked endothelial nitric oxide synthase (eNOS), AMPK and acetyl CoA carboxylase phosphorylation in LZRs, whereas it decreased phosphorylation of these proteins in OZRs. In conclusion, obesity decreases anandamide-induced relaxation in resistance arteries. Decreased cannabinoid receptors expression, increased anandamide degradation, decreased AMPK/eNOS activity as well as impairment of the response mediated by TRPV1 activation seem to contribute to reduce responses to cannabinoid agonists in obesity.

Untersuchung der zellulären Antwort von Tumor und Endothelium auf radioaktive Bestrahlung in vitro und in vivo

Tumore des Kopf-Hals Bereiches sprechen aufgrund schneller Resistenzbildung häufig schlecht auf die derzeit praktizierten Bestrahlungstherapien an. Der Erfolg dieser Behandlung wird dabei maßgeblich durch die Strahlenresistenz des malignen Gewebes limitiert. Das Verständnis der zugrunde liegenden zellulären und molekularen Mechanismen ist diesbezüglich unvollständig. Die Resistenzzunahme während der klinischen Behandlung könnte durch die Selektion strahlenresistenter Einzelzellen verursacht werden oder durch die Aktivierung von Resistenzmechanismen. Im Rahmen dieser Arbeit wurde die bestrahlungsvermittelte Freisetzung möglicherweise protektiv wirkender Faktoren durch Tumorzelllinien des Kopf-Hals Bereiches untersucht. Durch Bestrahlung erfolgte eine Induktion von VEGF (vascular endothelial growth factor) und FGF-2 (fibroblast growth factor 2), IL-8 (Interleukin-8) und PGE2 (Prostaglandin E2). Die Untersuchung von VEGF und FGF-2 zeigte weiterhin ein zytoprotektives Potential dieser Faktoren, d.h. die T

Airway Basal Cell Vascular Endothelial Growth Factor-mediated Cross-Talk Regulates Endothelial Cell Dependent Growth Support of Human Airway Basal Cells

The human airway epithelium is a pseudostratified heterogenous layer comprised of cili-ated, secretory, intermediate and basal cells. As the stem/progenitor population of the airway epi-thelium, airway basal cells differentiate into ciliated and secretory cells to replenish the airway epithelium during physiological turnover and repair. Transcriptome analysis of airway basal cells revealed high expression of vascular endothelial growth factor A (VEGFA), a gene not typically associated with the function of this cell type. Using cultures of primary human airway basal cells, we demonstrate that basal cells express all of the 3 major isoforms of VEGFA (121, 165 and 189) but lack functional expression of the classical VEGFA receptors VEGFR1 and VEGFR2. The VEGFA is actively secreted by basal cells and while it appears to have no direct autocrine function on basal cell growth and proliferation, it functions in a paracrine manner to activate MAPK signaling cascades in endothelium via VEGFR2 dependent signaling pathways. Using a cytokine- and serum-free co-culture system of primary human airway basal cells and human endothelial cells revealed that basal cell secreted VEGFA activated endothelium to ex-press mediators that, in turn, stimulate and support basal cell proliferation and growth. These data demonstrate novel VEGFA mediated cross-talk between airway basal cells and endothe-lium, the purpose of which is to modulate endothelial activation and in turn stimulate and sustain basal cell growth.

Thermal model for optimization of vascular laser tissue soldering

Laser tissue soldering (LTS) is a promising technique for tissue fusion based on a heat-denaturation process of proteins. Thermal damage of the fused tissue during the laser procedure has always been an important and challenging problem. Particularly in LTS of arterial blood vessels strong heating of the endothelium should be avoided to minimize the risk of thrombosis. A precise knowledge of the temperature distribution within the vessel wall during laser irradiation is inevitable. The authors developed a finite element model (FEM) to simulate the temperature distribution within blood vessels during LTS. Temperature measurements were used to verify and calibrate the model. Different parameters such as laser power, solder absorption coefficient, thickness of the solder layer, cooling of the vessel and continuous vs. pulsed energy deposition were tested to elucidate their impact on the temperature distribution within the soldering joint in order to reduce the amount of further animal experiments. A pulsed irradiation with high laser power and high absorbing solder yields the best results.

Antibodies recognising sulfated carbohydrates are prevalent in systemic sclerosis and associated with pulmonary vascular disease

Glycosylation represents an important modification that regulates biological processes in tissues relevant for disease pathogenesis in systemic sclerosis (SSc), including the endothelium and extracellular matrix. Whether patients with SSc develop antibodies to carbohydrates is not known.

Concomitant vascular GRP-receptor and VEGF-receptor expression in human tumors: molecular basis for dual targeting of tumoral vasculature

Gastrin-releasing peptide (GRP) and GRP receptors (GRPR) play a role in tumor angiogenesis. Recently, GRPR were found to be frequently expressed in the vasculature of a large variety of human cancers. Here, we characterize these GRPR by comparing the vascular GRPR expression and localization in a selection of human cancers with that of an established biological marker of neoangiogenesis, the vascular endothelial growth factor (VEGF) receptor. In vitro quantitative receptor autoradiography was performed in parallel for GRPR and VEGF receptors (VEGFR) in 32 human tumors of various origins, using ¹²⁵I-Tyr-bombesin and ¹²⁵I-VEGF₁₆₅ as radioligands, respectively. Moreover, VEGFR-2 was evaluated immunohistochemically. All tumors expressed GRPR and VEGFR in their vascular system. VEGFR were expressed in the endothelium in the majority of the vessels. GRPR were expressed in a subpopulation of vessels, preferably in their muscular coat. The vessels expressing GRPR were all VEGFR-positive whereas the VEGFR-expressing vessels were not all GRPR-positive. GRPR expressing vessels were found immunohistochemically to co-express VEGFR-2. Remarkably, the density of vascular GRPR was much higher than that of VEGFR. The concomitant expression of GRPR with VEGFR appears to be a frequent phenomenon in many human cancers. The GRPR, localized and expressed in extremely high density in a subgroup of vessels, may function as target for antiangiogenic tumor therapy or angiodestructive targeted radiotherapy with radiolabeled bombesin analogs alone, or preferably together with VEGFR targeted therapy.

Vascular endothelial cell-specific phosphotyrosine phosphatase (VE-PTP) activity is required for blood vessel development

VE-PTP, a receptor-type phosphotyrosine phosphatase, associates with the tyrosine kinase receptor Tie-2 and VE-cadherin and enhances the adhesive function of the latter. Here, VE-PTP was found to be restricted to endothelial cells, with a preference for arterial endothelium. Mutant mice expressing a truncated, secreted form of VE-PTP lacking the cytoplasmic and transmembrane domains and the most membrane-proximal extracellular fibronectin type III repeat, showed severe vascular malformations causing lethality at 10 days of gestation. Although blood vessels were initially formed, the intraembryonic vascular system soon deteriorated. Blood vessels in the yolk sac developed into dramatically enlarged cavities. In explant cultures of mutant allantoides, endothelial cells were found next to vessel structures growing as cell layers. No signs for enhanced endothelial apoptosis or proliferation were observed. Thus, the activity of VE-PTP is not required for the initial formation of blood vessels, yet it is essential for their maintenance and remodeling.

Impaired vascular function in normoglycemic mice prone to autoimmune diabetes: role of nitric oxide

Type 1 diabetes is an immuno-inflammatory condition which increases the risk of cardiovascular disease, particularly in young adults. This study investigated whether vascular function is altered in mice prone to autoimmune diabetes and whether the nitric oxide (NO)-cyclic GMP axis is involved. Aortic rings suspended in organ chambers and precontracted with phenylephrine were exposed to cumulative concentrations of acetylcholine. To investigate the role of NO, some experiments were performed in the presence of either 1400W (N-(3-aminomethyl)benzyl-acetamidine hydrochloride), a selective inhibitor of the iNOS-isoform, L-NAME (N(G)-nitro-L-arginine methyl ester hydrochloride), an inhibitor of all three NOS-isoforms, or ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a selective inhibitor of guanylate cyclase. Moreover, contractility to phenylephrine, big endothelin-1, and endothelin-1 was assessed and histological analysis and iNOS immunohistochemistry were performed. Endothelium-dependent relaxation was reduced in prediabetic NOD mice (78+/-4 vs. 88+/-2%, respectively, P<0.05 vs. control) despite normal plasma glucose levels (n.s. vs. control). Preincubation with 1400W further attenuated responses in prediabetic (P<0.05 vs. untreated) but not in diabetic or in control mice. In contrast, basal NO bioactivity remained unaffected until the onset of diabetes in NOD mice. Contractile responses to big endothelin-1 and endothelin-1 were reduced in prediabetic animals (P<0.05 vs. control), whereas in diabetic mice only responses to big endothelin-1 were decreased (P<0.05 vs. control). These data demonstrate that endothelium-dependent and -independent vascular function in NOD mice is abnormal already in prediabetes in the absence of structural injury. Early proinflammatory activation due to iNOS in diabetes-prone NOD mice appears to be one of the mechanisms contributing to impaired vasoreactivity.

Vascular adaptation of the internal thoracic artery graft early and late after bypass surgery

OBJECTIVE: Flow mismatch between the supplying artery and the myocardial perfusion region has been observed in patients with internal thoracic artery grafts. Thus coronary flow changes of arterial (internal thoracic artery grafts) and saphenous (saphenous vein grafts) bypass grafts were studied early and late after coronary artery bypass grafting. METHODS: Thirty patients undergoing elective bypass surgery (internal thoracic artery and saphenous vein grafts) were studied intraoperatively and (17 patients) 3 to 10 months postoperatively. Coronary flow was measured intraoperatively with the transit-time Doppler scanning technique. Postoperatively, flow velocity and coronary flow reserve were determined with the Doppler flow wire technique. Quantitative angiographic analysis was used to determine vessel size for calculation of absolute flow. RESULTS: Intraoperatively, internal thoracic artery graft flow was significantly lower than saphenous vein graft flow (31 +/- 8 vs 58 +/- 29 mL/min, P < .01). Postoperatively, internal thoracic artery graft flow increased significantly to 42 +/- 24 mL/min at 3 months and to 56 +/- 30 mL/min (P < .02 vs intraoperative value) at 10 months, respectively. However, saphenous vein graft flow remained unchanged over time (58 +/- 29 to 50 +/- 27 mL/min at 3 months and 46 +/- 27 mL/min at 10 months). Coronary flow reserve was abnormally low intraoperatively in the internal thoracic artery (1.3 +/- 0.3) and saphenous vein (1.6 +/- 0.5) grafts but increased significantly to normal values in both types of graft at follow-up. CONCLUSIONS: Bypass flow of the internal thoracic artery graft is significantly reduced intraoperatively when compared with that of the saphenous vein graft. However, 3 and 10 months after the operation, flow of the internal thoracic artery graft increases significantly and is similar to saphenous vein graft flow. This finding can be explained by an early flow mismatch of the native internal thoracic artery in the presence of a large perfusion territory. During follow-up, there is vascular remodeling of the internal thoracic artery, probably because of endothelium-mediated mechanisms.

The role of purinergic signaling in the liver and in transplantation: effects of extracellular nucleotides on hepatic graft vascular injury, rejection and metabolism

Extracellular nucleotides (e.g. ATP, UTP, ADP) are released by activated endothelium, leukocytes and platelets within the injured vasculature and bind specific cell-surface type-2 purinergic (P2) receptors. This process drives vascular inflammation and thrombosis within grafted organs. Importantly, there are also vascular ectonucleotidases i.e. ectoenzymes that hydrolyze extracellular nucleotides in the blood to generate nucleosides (viz. adenosine). Endothelial cell NTPDase1/CD39 has been shown to critically modulate levels of circulating nucleotides. This process tends to limit the activation of platelet and leukocyte expressed P2 receptors and also generates adenosine to reverse inflammatory events. This vascular protective CD39 activity is rapidly inhibited by oxidative reactions, such as is observed with liver ischemia reperfusion injury. In this review, we chiefly address the impact of these signaling cascades following liver transplantation. Interestingly, the hepatic vasculature, hepatocytes and all non-parenchymal cell types express several components co-ordinating the purinergic signaling response. With hepatic and vascular dysfunction, we note heightened P2- expression and alterations in ectonucleotidase expression and function that may predispose to progression of disease. In addition to documented impacts upon the vasculature during engraftment, extracellular nucleotides also have direct influences upon liver function and bile flow (both under physiological and pathological states). We have recently shown that alterations in purinergic signaling mediated by altered CD39 expression have major impacts upon hepatic metabolism, repair mechanisms, regeneration and associated immune responses. Future clinical applications in transplantation might involve new therapeutic modalities using soluble recombinant forms of CD39, altering expression of this ectonucleotidase by drugs and/or using small molecules to inhibit deleterious P2-mediated signaling while augmenting beneficial adenosine-mediated effects within the transplanted liver.