Angiotensin II acts through the angiotensin 1a receptor to upregulate pendrin

Pendrin is an anion exchanger expressed in the apical regions of B and non-A, non-B intercalated cells. Since angiotensin II increases pendrin-mediated Cl(-) absorption in vitro, we asked whether angiotensin II increases pendrin expression in vivo and whether angiotensin-induced hypertension is pendrin dependent. While blood pressure was similar in pendrin null and wild-type mice under basal conditions, following 2 wk of angiotensin II administration blood pressure was 31 mmHg lower in pendrin null than in wild-type mice. Thus pendrin null mice have a blunted pressor response to angiotensin II. Further experiments explored the effect of angiotensin on pendrin expression. Angiotensin II administration shifted pendrin label from the subapical space to the apical plasma membrane, independent of aldosterone. To explore the role of the angiotensin receptors in this response, pendrin abundance and subcellular distribution were examined in wild-type, angiotensin type 1a (Agtr1a) and type 2 receptor (Agtr2) null mice given 7 days of a NaCl-restricted diet (< 0.02% NaCl). Some mice received an Agtr1 inhibitor (candesartan) or vehicle. Both Agtr1a gene ablation and Agtr1 inhibitors shifted pendrin label from the apical plasma membrane to the subapical space, independent of the Agtr2 or nitric oxide (NO). However, Agtr1 ablation reduced pendrin protein abundance through the Agtr2 and NO. Thus angiotensin II-induced hypertension is pendrin dependent. Angiotensin II acts through the Agtr1a to shift pendrin from the subapical space to the apical plasma membrane. This Agtr1 action may be blunted by the Agtr2, which acts through NO to reduce pendrin protein abundance.     

Sexual dimorphism in prostanoid-potentiated vascular contraction: roles of endothelium and ovarian steroids

The effects of constrictor prostanoid (CP) pathway inhibitors on vascular reactivity to vasopressin (VP) and phenylephrine (PE) were examined in thoracic aortas of male, female, and ovariectomized (OVX) female Sprague-Dawley rats. Maximal contractile response of control (Cont) aortas to VP was markedly higher in females (3,885 +/- 332 mg/mg ring wt) than in males (810 +/- 148 mg). Indomethacin (Indo; 10 microM) attenuated maximal response to VP in females (3,043 +/- 277 mg) but not in males. SQ-29,548 (SQ; 1 microM) attenuated maximal response to VP in females (3,042 +/- 290 mg) to a similar extent as Indo. Dazoxiben (Daz; 10 microM) alone had no effect, but Daz + SQ attenuated maximal contractile response to VP to a similar extent as SQ alone. Removal of the endothelium in female aortas attenuated contractile responses to VP in Cont aortas. OVX attenuated maximal contractile response to VP in Cont aortas (2,093 +/- 329 mg) and abolished the attenuating effects of Indo. Indo, SQ, and Daz exerted identical effects on contractile responses of male, female, and OVX female aortas to PE. These findings establish the following in the rat aorta: 1) CP, probably thromboxane and/or endoperoxide, is responsible for approximately 25-30% of contractile responses of females, but not males, to VP and PE; 2) CP production by the female aorta is primarily endothelial in origin; and 3) ovarian steroids modulate production and/or actions of CP in female aortas.     

Effects of streptozotocin-induced hyperglycemia on agonist-stimulated phosphatidylinositol turnover in rat aorta

Altered vascular sensitivity and responsiveness has been previously described in various stages of experimental diabetes mellitus. Increases in membrane bound Ca2+ and intracellular calcium in diabetic aorta have been postulated to explain excitation-coupling dysfunction in diabetic vascular smooth muscle (VSM). Receptor-mediated phosphatidylinositol (PI) hydrolysis is known to activate VSM contraction; thus contractile changes in diabetic aorta could be functionally linked to abnormal PI turnover. To evaluate this possibility, parallel experiments were undertaken to study contractility and receptor-stimulated PI turnover with norepinephrine (NE), phenylephrine (PE), and serotonin (5-HT). At 7 and 28 days following injection of streptozotocin with production of the hyperglycemic state, aortas were harvested for contractile and PI turnover experiments. No differences in the contractile cumulative dose responses or receptor-mediated PI turnover were measured in the 7 day group. At 28 days, vascular supersensitivity and increased responsiveness were observed. PI hydrolysis in basal and agonist-stimulated aorta was, however, markedly decreased at 28 days. These findings suggest that mobilization and utilization of Ca2+ during contraction occur independently of receptor-stimulated PI hydrolysis in aorta from hyperglycemic rats. The duration of hyperglycemia also significantly effects contractility and PI turnover in rat aorta.     

The anion exchanger pendrin (SLC26A4) and renal acid-base homeostasis

The anion exchanger pendrin (Pds, SLC26A4) transports various anions including bicarbonate, chloride and iodide. In the kidney, pendrin is exclusively expressed on the luminal pole of bicarbonate-secretory type B intercalated cells. Genetic ablation of pendrin in mice abolishes luminal chloride-bicarbonate exchanger activity from type B intercalated cells suggesting that pendrin is the apical bicarbonate extruding pathway. The renal expression of pendrin is developmentally adapted and pendrin positive cells originate from both the uretric bud and mesenchyme. In adult kidney, pendrin expression and activity is regulated by systemic acid-base status, dietary electrolyte intake (mostly chloride), and hormones such as angiotensin II and aldosterone which can affect subcellular localization, the relative number of pendrin expressing cells, and the overall abundance consistent with a role of pendrin in maintaining normal acid-base homeostasis. This review summarizes recent findings on the role and regulation of pendrin in the context of the kidneys role in acid-base homeostasis in health and disease.     

Remodeling of Aorta Extracellular Matrix as a Result of Transient High Oxygen Exposure in Newborn Rats: Implication for Arterial Rigidity and Hypertension Risk

Neonatal high-oxygen exposure leads to elevated blood pressure, microvascular rarefaction, vascular dysfunction and arterial (aorta) rigidity in adult rats. Whether structural changes are present in the matrix of aorta wall is unknown. Considering that elastin synthesis peaks in late fetal life in humans, and early postnatal life in rodents, we postulated that transient neonatal high-oxygen exposure can trigger premature vascular remodelling. Sprague Dawley rat pups were exposed from days 3 to 10 after birth to 80% oxygen (vs. room air control) and were studied at 4 weeks. Blood pressure and vasomotor response of the aorta to angiotensin II and to the acetylcholine analogue carbachol were not different between groups. Vascular superoxide anion production was similar between groups. There was no difference between groups in aortic cross sectional area, smooth muscle cell number or media/lumen ratio. In oxygen-exposed rats, aorta elastin/collagen content ratio was significantly decreased, the expression of elastinolytic cathepsin S was increased whereas collagenolytic cathepsin K was decreased. By immunofluorescence we observed an increase in MMP-2 and TIMP-1 staining in aortas of oxygen-exposed rats whereas TIMP-2 staining was reduced, indicating a shift in the balance towards degradation of the extra-cellular matrix and increased deposition of collagen. There was no significant difference in MMP-2 activity between groups as determined by gelatin zymography. Overall, these findings indicate that transient neonatal high oxygen exposure leads to vascular wall alterations (decreased elastin/collagen ratio and a shift in the balance towards increased deposition of collagen) which are associated with increased rigidity. Importantly, these changes are present prior to the elevation of blood pressure and vascular dysfunction in this model, and may therefore be contributory.     

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.     

Cathepsin K knockout alleviates aging‐induced cardiac dysfunction

Aging is a major risk factor for cardiovascular disease. It has previously been shown that protein levels of cathepsin K, a lysosomal cysteine protease, are elevated in the failing heart and that genetic ablation of cathepsin K protects against pressure overload‐induced cardiac hypertrophy and contractile dysfunction. Here we test the hypothesis that cathepsin K knockout alleviates age‐dependent decline in cardiac function. Cardiac geometry, contractile function, intracellular Ca²⁺ properties, and cardiomyocyte apoptosis were evaluated using echocardiography, fura‐2 technique, immunohistochemistry, Western blot and TUNEL staining, respectively. Aged (24‐month‐old) mice exhibited significant cardiac remodeling (enlarged chamber size, wall thickness, myocyte cross‐sectional area, and fibrosis), decreased cardiac contractility, prolonged relengthening along with compromised intracellular Ca²⁺ release compared to young (6‐month‐old) mice, which were attenuated in the cathepsin K knockout mice. Cellular markers of senescence, including cardiac lipofuscin, p21 and p16, were lower in the aged‐cathepsin K knockout mice compared to their wild‐type counterpart. Mechanistically, cathepsin K knockout mice attenuated an age‐induced increase in cardiomyocyte apoptosis and nuclear translocation of mitochondrial apoptosis‐inducing factor (AIF). In cultured H9c2 cells, doxorubicin stimulated premature senescence and apoptosis. Silencing of cathepsin K blocked the doxorubicin‐induced translocation of AIF from the mitochondria to the nuclei. Collectively, these results suggest that cathepsin K knockout attenuates age‐related decline in cardiac function via suppressing caspase‐dependent and caspase‐independent apoptosis.     

Ablation of cardiac myosin–binding protein-C accelerates contractile kinetics in engineered cardiac tissue

Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin–binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C–null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca²⁺ transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C–null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca²⁺ transient kinetics. Strikingly, the ability of cMyBP-C–null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca²⁺ transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.     

Effect of hindlimb suspension on the functional properties of slow and fast soleus fibers from three strains of mice.

Cross-sectional area (CSA), peak Ca2+-activated force (Po), fiber specific force (Po/CSA), and unloaded shortening velocity (Vo) were measured in slow-twitch [containing type I myosin heavy chain (MHC)] and fast-twitch (containing type II MHC) chemically skinned soleus muscle fiber segments obtained from three strains of weight-bearing and 7-day hindlimb-suspended (HS) mice. HS reduced soleus slow MHC content (from approximately 50 to approximately 33%) in CBA/J and ICR strains without affecting slow MHC content in C57BL/6 mice ( approximately 20% of total MHC). Two-way ANOVA revealed HS-induced reductions in CSA, Po, and Po/CSA of slow and fast fibers from all strains. Fiber Vo was elevated post-HS, but not consistently across strains. No MHC x HS treatment interactions were observed for any variable for C57BL/6 and CBA/J mice, and the two significant interactions found for the ICR strain (CSA, Po) appeared related to inherent pre-HS differences in slow vs. fast fiber CSA. In the mouse HS models studied here, fiber atrophy and contractile dysfunction were partially dependent on animal strain and generally independent of fiber MHC isoform content.     

Changes in muscle fiber populations and muscle enzyme activities in the primiparous lactating sow

An experiment involving 12 primiparous Large White sows was conducted to investigate changes in contractile and metabolic characteristics of skeletal muscle during the first 3 weeks of lactation. The sows lost 19.7 +/- 6.6 kg of body weight. No change in DNA concentration was observed in the longissimus dorsi (LD), a fast-twitch glycolytic muscle, and the trapezius (T), a mainly slow-twitch oxidative muscle during lactation. The percentage of type I fibers increased (P less than 0.05) in LD, but not in T. The muscle fiber cross sectional area (CSA) of IIB fibers, which represents about 78% of the total number of LD fibers, decreased by 18% (P less than 0.01) by lactation; the CSAs of I and IIA fibers were not significantly affected. Marker enzyme activities for oxidative and glycolytic metabolisms decreased in both muscles during lactation. The decrease in oxidative enzyme activities was particularly dramatic in T (P less than 0.001). No significant relationship was observed between sow weight loss and changes in muscle fiber CSA or enzyme activities. The extent to which the results could be related to a negative nutritional balance or to changes in hormonal status is discussed.     

Effects of hindlimb unweighting on the mechanical and structure properties of the rat abdominal aorta.

Previous studies have shown that hindlimb unweighting of rats, a model of microgravity, reduces evoked contractile tension of peripheral conduit arteries. It has been hypothesized that this diminished contractile tension is the result of alterations in the mechanical properties of these arteries (e.g., active and passive mechanics). Therefore, the purpose of this study was to determine whether the reduced contractile force of the abdominal aorta from 2-wk hindlimb-unweighted (HU) rats results from a mechanical function deficit resulting from structural vascular alterations or material property changes. Aortas were isolated from control (C) and HU rats, and vasoconstrictor responses to norepinephrine (10(-9)-10(-4) M) and AVP (10(-9)-10(-5) M) were tested in vitro. In a second series of tests, the active and passive Cauchy stress-stretch relations were determined by incrementally increasing the uniaxial displacement of the aortic rings. Maximal Cauchy stress in response to norepinephrine and AVP were less in aortic rings from HU rats. The active Cauchy stress-stretch response indicated that, although maximum stress was lower in aortas from HU rats (C, 8.1 +/- 0.2 kPa; HU, 7.0 +/- 0.4 kPa), it was achieved at a similar hoop stretch. There were also no differences in the passive Cauchy stress-stretch response or the gross vascular morphology (e.g., medial cross-sectional area: C, 0.30 +/- 0.02 mm(2); HU, 0.32 +/- 0.01 mm(2)) between groups and no differences in resting or basal vascular tone at the displacement that elicits peak developed tension between groups (resting tension: C, 1.71 +/- 0.06 g; HU, 1.78 +/- 0.14 g). These results indicate that HU does not alter the functional mechanical properties of conduit arteries. However, the significantly lower active Cauchy stress of aortas from HU rats demonstrates a true contractile deficit in these arteries.     

Altered clock gene expression and vascular smooth muscle diurnal contractile variations in type 2 diabetic db/db mice

This study was designed to determine whether the 24-h rhythms of clock gene expression and vascular smooth muscle (VSM) contractile responses are altered in type 2 diabetic db/db mice. Control and db/db mice were euthanized at 6-h intervals throughout the day. The aorta, mesenteric arteries, heart, kidney, and brain were isolated. Clock and target gene mRNA levels were determined by either real-time PCR or in situ hybridization. Isometric contractions were measured in isolated aortic helical strips, and pressor responses to an intravenous injection of vasoconstrictors were determined in vivo using radiotelemetry. We found that the 24-h mRNA rhythms of the following genes were suppressed in db/db mice compared with control mice: the clock genes period homolog 1/2 (Per1/2) and cryptochrome 1/2 (Cry1/2) and their target genes D site albumin promoter-binding protein (Dbp) and peroxisome proliferator-activated receptor-γ (Pparg) in the aorta and mesenteric arteries; Dbp in the heart; Per1, nuclear receptor subfamily 1, group D, member 1 (Rev-erba), and Dbp in the kidney; and Per1 in the suprachiasmatic nucleus. The 24-h contractile variations in response to phenylephrine (α(1)-agonist), ANG II, and high K(+) were significantly altered in the aortas from db/db mice compared with control mice. The diurnal variations of the in vivo pressor responses to phenylephrine and ANG II were lost in db/db mice. Moreover, the 24-h mRNA rhythms of the contraction-related proteins Rho kinase 1/2, PKC-potentiated phosphatase inhibitory protein of 17 kDa, calponin-3, tropomyosin-1/2, and smooth muscle protein 22-α were suppressed in db/db mice compared with control mice. Together, our data demonstrated that the 24-h rhythms of clock gene mRNA, mRNA levels of several contraction-related proteins, and VSM contraction were disrupted in db/db mice, which may contribute to the disruption of their blood pressure circadian rhythm.     

Deletion of angiotensin II type 1 receptor gene or scavenge of superoxide prevents chronic alcohol-induced aortic damage and remodelling

To investigate whether chronic alcohol consumption induces vascular injury via angiotensin II (Ang II) type 1 (AT1) receptor‐dependent superoxide generation, male transgenic mice with knockout of AT1 gene (AT1‐KO) and age‐matched wild‐type (WT) C57BL/6 mice were pair‐fed a modified Lieber‐DeCarli alcohol or isocaloric maltose dextrin control liquid diet for 2 months. Ethanol content (%, W/V) in the diet was 4.8 (34% of total calories) at initiation, and gradually increased up to 5.4 (38% of total calories). For some WT mice with and without alcohol treatment, superoxide dismutase mimetic (MnTMPyP) was given simultaneously by intraperitoneal injection at 5 mg/kg body weight daily for 2 months. At the end of studies, aortas were harvested for histopathological and immunohistochemical examination. Significant increases in the wall thickness and structural disarrangement of aorta were found in alcohol group, along with significant increases in aortic oxidative and/or nitrosative damage, expressions of NADPH oxidases (NOXs), inflammatory response, cell death and proliferation, and remodelling (fibrosis). However, these pathological changes were completely attenuated in alcohol‐treated AT1‐KO mice or in alcohol‐treated WT mice that were also simultaneously treated with MnTMPyP for 2 months. These results suggest that chronic alcohol consumption may activate NOX via Ang II/AT1 receptor, to generate superoxide and associated peroxynitrite that in turn causes aortic nitrosative damage, inflammation, cell death and proliferation, and remodelling. Therefore, blocking Ang II/AT1 system or scavenging superoxide may become a potential preventive and/therapeutic approach to alcoholic vascular damage.     

Development and characterisation of a large diameter decellularised vascular allograft

The aims of this study were to develop a biological large diameter vascular graft by decellularisation of native human aorta to remove the immunogenic cells whilst retaining the essential biomechanical, and biochemical properties for the ultimate benefit of patients with infected synthetic grafts. Donor aortas (n = 6) were subjected to an adaptation of a propriety decellularisation process to remove the cells and acellularity assessed by histological analysis and extraction and quantification of total DNA. The biocompatibility of the acellular aortas was determined using standard contact cytotoxicity tests. Collagen and denatured collagen content of aortas was determined and immunohistochemistry was used to determine the presence of specific extracellular matrix proteins. Donor aortas (n = 6) were divided into two, with one half subject to decellularisation and the other half retained as native tissue. The native and decellularised aorta sections were then subject to uniaxial tensile testing to failure [axial and circumferential directions] and suture retention testing. The data was compared using a paired t-test. Histological evaluation showed an absence of cells in the treated aortas and retention of histoarchitecture including elastin content. The decellularised aortas had less than 15 ng mg^?1 total DNA per dry weight (mean 94% reduction) and were biocompatible as determined by in vitro contact cytotoxicity tests. There were no gross changes in the histoarchitecture [elastin and collagen matrix] of the acellular aortas compared to native controls. The decellularisation process also reduced calcium deposits within the tissue. The uniaxial tensile and suture retention testing revealed no significant differences in the material properties (p > 0.05) of decellularised aorta. The decellularisation procedure resulted in minimal changes to the biological and biomechanical properties of the donor aortas. Acellular donor aorta has excellent potential for use as a large diameter vascular graft.     

Akita Spontaneously Type 1 Diabetic Mice Exhibit Elevated Vascular Arginase and Impaired Vascular Endothelial and Nitrergic Function

Background

Elevated arginase (Arg) activity is reported to be involved in diabetes-induced vascular endothelial dysfunction. It can reduce L-arginine availability to nitric oxide (NO) synthase (NOS) and NO production. Akita mice, a genetic non-obese type 1 diabetes model, recapitulate human diabetes. We determined the role of Arg in a time-course of diabetes-associated endothelial dysfunction in aorta and corpora cavernosa (CC) from Akita mice.

Methods and Results

Endothelium-dependent relaxation, Arg and NOS activity, and protein expression levels of Arg and constitutive NOS were assessed in aortas and CC from Akita and non-diabetic wild type (WT) mice at 4, 12 and 24 wks of age. Systolic blood pressure (SBP) was assessed by tail cuff. In aorta and CC, Akita mice exhibited a progressive impairment of vascular endothelial and nitrergic function increased Arg activity and expression (Arg1 in aorta and both Arg1 and Arg2 in CC) compared with that of age-matched WT mice. Treatment of aorta and CC from Akita mice with an Arg inhibitor (BEC or ABH) reduced diabetes-induced elevation of Arg activity and restored endothelial and nitrergic function. Reduced levels of phospho-eNOS at Ser¹¹⁷⁷ (in aorta and CC) and nNOS expression (in CC) were observed in Akita mice at 12 and 24 wks. Akita mice also had decreased NOS activity in aorta and CC at 12 and 24 wks that was restored by BEC treatment. Further, Akita mice exhibited moderately increased SBP at 24 wks and increased sensitivity to PE-induced contractions in aorta and sympathetic nerve stimulation in CC at 12 and 24 wks.

Conclusions

Over 24 wks of diabetes in Akita mice, both aortic and cavernosal tissues exhibited increased Arg activity/expression, contributing to impaired endothelial and nitrergic function and reduced NO production. Our findings demonstrate involvement of Arg activity in diabetes-induced impairment of vascular function in Akita mouse.     

Baicalein protects against the development of angiotensin II-induced abdominal aortic aneurysms by blocking JNK and p38 MAPK signaling

An abdominal aortic aneurysm(AAA) is a permanent, localized dilatation of the abdominal aorta. In western countries, the morbidity of AAA is approximately 8%. Currently, pharmacotherapies for AAA are limited. Here, we demonstrate that baicalein(BAI), the main component of the Chinese traditional drug "Huang Qin", attenuates the incidence and severity of AAA in Apoe儃/儃 mice infused with angiotensin II(AngII). Mechanically, BAI treatment decreases AngII-induced reactive oxygen species(ROS) production in the aortic wall. Moreover, BAI inhibits inflammatory cell accumulation in the aortas of mice infused with AngII. It also inhibits AngII-induced activation of matrix metalloproteinase 2(MMP-2) and MMP-9 to maintain elastin content in vivo. In addition, it blocks AngII cascade by downregulating angiotensin type 1 receptor(AT1R) and inhibiting mitogen-activated protein kinases(MAPKs). Taken together, our findings show that BAI is an effective agent for AAA prevention.     

Functional and structural pattern of arterial responses in hereditary hypertriglyceridemic and spontaneously hypertensive rats in early stage of experimental hypertension

It has been shown that endothelium-derived nitric oxide (NO) plays an important role in regulation of vascular tone in the prenatal and early postnatal period. The aim of this paper was to determine the reactivity and accompanying structural changes in thoracic aorta from 4-week-old spontaneously hypertensive rats (SHR) and rats with hereditary hypertriglyceridemia (hHTG) in comparison with age-matched normotensive controls. For functional studies thoracic aorta was excised, cut into rings and mounted in organ baths for measurement of isometric contractile force. For morphological studies cardiovascular system of rats was perfused with glutaraldehyde fixative (at 100 mm Hg) via cannula placed in the left ventricle. Morphological changes of thoracic aorta were measured using light microscopy. Systolic blood pressure (SBP) in SHR (98+/-1 mm Hg) did not significantly differ from that of age-matched control rats (95+/-4 mm Hg), but was slightly increased in hHTG rats (110+/-2 mm Hg, P<0.05). Heart weight/body weight ratio was higher in SHR and hHTG rats than in control group indicating the hypertrophy of the heart in both models of hypertension. Endothelium-dependent relaxation of aorta induced by acetylcholine was preserved in all groups and did not differ from that in control normotensive rats. The maximal isometric contraction of thoracic aorta to noradrenaline (NA) was reduced in hypertensive groups and the concentration-response curves to NA were shifted to the right indicating increased sensitivity of smooth muscle to NA. The values of wall thickness and cross sectional area as well as inner diameter of thoracic aorta in SHR and hHTG rats were significantly decreased in comparison to control groups. Endothelial dysfunction seems to be absent in all young rats before development of hypertension. In conclusion, our observations indicate that in early stage of experimental hypertension NO-dependent relaxation is preserved so that putative impairment of this function provides no significant pathogenic contribution to the onset of hypertension in these two experimental models.     

Inflammatory Monocytes Determine Endothelial Nitric-oxide Synthase Uncoupling and Nitro-oxidative Stress Induced by Angiotensin II

Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. Angiotensin II (ATII, 1 mg/kg/day for 7 days) increased Ly6C^high and CD11b⁺/iNOS^high leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C57BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM⁺) monocytes in ATII-infused LysM^iDTR transgenic mice prevented eNOS glutathionylation and eNOS-derived N^ω-nitro-l-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysM^iDTR mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM⁺ monocytes.