MPST but not CSE is the primary regulator of hydrogen sulfide production and function in the coronary artery.

Hydrogen sulfide (H2S) has emerged as an important gasotransmitter in the vasculature. In this study, we tested the hypothesis that H2S contributes to coronary vasoregulation and evaluated the physiological relevance of two sources of H2S, namely, cystathionine-γ-lyase (CSE) and 3-mercaptypyruvate sulfertransferase (MPST). MPST was detected in human coronary artery endothelial cells as well as rat and mouse coronary artery; CSE was not detected in the coronary vasculature. Rat coronary artery homogenates produced H2S through the MPST pathway but not the CSE pathway in vitro. In vivo coronary vasorelaxation response was similar in CSE knockout mice, wild-type mice (WT), and WT mice treated with the CSE inhibitor propargylglycine, suggesting that CSE-produced H2S does not have a significant role in coronary vasoregulation in vivo. Ex vivo, the MPST substrate 3-mercaptopyruvate (3-MP) and H2S donor sodium hydrosulfide (NaHS) elicited similar coronary vasoreactivity responses. Pyruvate did not have any effects on vasoreactivity. The vasoactive effect of H2S appeared to be nitric oxide (NO) dependent: H2S induced coronary vasoconstriction in the presence of NO and vasorelaxation in its absence. Maximal endothelial-dependent relaxation was intact after 3-MP and NaHS induced an increase in preconstriction tone, suggesting that endothelial NO synthase activity was not significantly inhibited. In vitro, H2S reacted with NO, which may, in part explain the vasoconstrictive effects of 3-MP and NaHS. Taken together, these data show that MPST rather than CSE generates H2S in coronary artery, mediating its effects through direct modulation of NO. This has important implications for H2S-based therapy in healthy and diseased coronary arteries.

Vasopressin-mediated enhancement of adrenergic vasoconstriction involves both the tyrosine kinase and the protein kinase C pathways.

BACKGROUND: Vasopressin is frequently used to treat catecholamine-resistant vasodilatory shock. It enhances the vasoconstrictor effects of catecholamines at concentrations of vasopressin that have none or only minimal intrinsic pressor effects. However, the vascular mechanisms underlying this combined pharmacological approach have not been fully elucidated. METHODS: We used isometric tension measurements in vascular rings to investigate potential cellular mechanisms. Vascular rings (0.2 mm diameter) were harvested from the superior mesenteric artery of Wistar rats (2 to 4 months of age). Dose-response relationships were derived for vasopressin (VP) and norepinephrine (NE), in the absence and presence of a subpressor dose of VP (10(-9) M). The contribution of tyrosine kinase (TK), the TK pathway proteins SRC and PYK2, as well as protein kinase C (PKC) were determined by measuring the modulating influence of specific inhibitors on the pressor response to NE (10(-5) M) alone and the augmented pressor response to VP (10(-9) M). RESULTS: VP (10(-9) M) had only minimal pressor effect alone (10% of maximal response), but significantly increased the E(max) response to NE (587.8 ± 40.7 vs 331.2 ± 10.4 mg). TK inhibition completely abolished the pressor response to NE (100% vs 1.0% 0.5%) and the augmented VP response alone (100% vs 2.0% ± 1.01%). Both responses were significantly, but equally, decreased by SRC inhibition (63% ± 4.0% and 69% 1.0%). In contrast, inhibition of the TK molecule PYK2 with salicylate had differential inhibitory effects on the vasoconstrictor responses. Salicylate caused a greater inhibition of VP-induced augmented NE response in comparison with NE alone (62.1% ± 7% and 15% ± 2%). Inhibition of either the µ or γ PKC isoform significantly decreased both responses, but the magnitude of the inhibition was significantly different for each isoform. Inhibition of the γ PKC isoform significantly decreased the vasoconstriction responses to both NE and VP plus NE (82.9 ± 3.9 vs 32.8 ± 3.8). Inhibition of the µ PKC isoform significantly decreased both responses and completely abolished the VP-augmented response to NE. CONCLUSION: These data are consistent with a central role for TK in mediating both the NE response and the VP-augmented response. Moreover, PYK2 and the µ and γ isoforms of PKC seem to play a preferential role in mediating the augmented VP response. The apparent divergent roles of these pathways in mediating NE- versus VP-augmented pressor responses could potentially lead to new targeted therapies in catecholamine-resistant shock.

Upregulation of arginase-II contributes to decreased age-related myocardial contractile reserve.

Arginase-II (Arg-II) reciprocally regulates nitric oxide synthase (NOS) and offsets basal myocardial contractility. Furthermore, decreased or absent myocardial NOS activity is associated with a depression in myocardial contractile reserve. We therefore hypothesized that upregulation of Arg-II might in part be responsible for depressed myocardial contractility associated with age. We studied arginase activity/expression, NOS expression, NO production in the presence and absence of the arginase inhibitor S-(2-boronoethyl)-L: -cysteine (BEC) in old (22 months) and young (3 months) rat hearts and myocytes. The spatial confinement of Arg-II and NOS was determined with immuno-electron-miocrographic (IEM) and immuno-histochemical studies. We tested the effect of BEC on the force frequency response (FFR) in myocytes, as well as NOS abundance and activity. Arginase activity and Arg-II expression was increased in old hearts (2.27 ± 0.542 vs. 0.439 ± 0.058 nmol urea/mg protein, p = 0.02). This was associated with a decrease in NO production, which was restored with BEC (4.54 ± 0.582 vs. 12.88 ± 0.432 µmol/mg, p < 0.01). IEM illustrates increased mitochondrial density in old myocytes (51.7 ± 1.8 vs. 69 ± 2.2 × 10(6)/cm(2), p < 0.01), potentially contributing to increased Arg-II abundance and activity. Immunohistochemistry revealed an organized pattern of mitochondria and Arg-II that appears disrupted in old myocytes. The FFR was significantly depressed in old myocytes (61.42 ± 16.04 vs. -5.15 ± 5.65%), while inhibition of Arg-II restored the FFR (-5.15 ± 5.65 vs. 70.98 ± 6.10%). NOS-2 is upregulated sixfold in old hearts contributing to increased production of reactive oxygen species which is attenuated with NOS-2 inhibition by 1400 W (4,735 ± 427 vs. 4,014 ± 314 RFU/min/mg protein, p = 0.005). Arg-II upregulation in aging rat hearts contributes to age-related decreased contractile function.

ß2-adrenergic receptor-coupled phosphoinositide 3-kinase constrains cAMP-dependent increases in cardiac inotropy through phosphodiesterase 4 activation.

BACKGROUND: Emerging evidence suggests that phosphoinositide 3-kinase (PI3K) may modulate cardiac inotropy; however, the underlying mechanism remains elusive. We hypothesized that ß(2)-adrenergic receptor (AR)-coupled PI3K constrains increases in cardiac inotropy through cyclic adenosine monophosphate (cAMP)-dependent phosphodiesterase (PDE) activation. METHODS: We tested the effects of PI3K and PDE4 inhibition on myocardial contractility by using isolated murine cardiac myocytes to study physiologic functions (sarcomere shortening [SS] and intracellular Ca(+) transients), as well as cAMP and PDE activity. RESULTS: PI3K inhibition with the reversible inhibitor LY294002 (LY) resulted in a significant increase in SS and Ca(2+) handling, indicating enhanced contractility. This response depended on G(iα) protein activity, because incubation with pertussis toxin (an irreversible G(iα) inhibitor) abolished the LY-induced hypercontractility. In addition, PI3K inhibition had no greater effect on SS than both a PDE3,4 inhibitor (milrinone) and LY combined. Furthermore, LY decreased PDE4 activity in a concentration-dependent manner (58.0% of PDE4 activity at LY concentrations of 10 µM). Notably, PI3K(γ) coimmunoprecipitated with PDE4D. The ß(2)-AR inverse agonist, ICI 118,551 (ICI), abolished induced increases in contractility. CONCLUSIONS: PI3K modulates myocardial contractility by a cAMP-dependent mechanism through the regulation of the catalytic activity of PDE4. Furthermore, basal agonist-independent activity of the ß(2)-AR and its resultant cAMP production and enhancement of the catalytic activity of PDE4 through PI3K represents an example of integrative cellular signaling, which controls cAMP dynamics and thereby contractility in the cardiac myocyte. These results help to explain the mechanism by which milrinone is able to increase myocardial contractility in the absence of direct ß-adrenergic stimulation and why it can further augment contractility in the presence of maximal ß-adrenergic stimulation.

Early changes in vasoreactivity after simulated microgravity are due to an upregulation of the endothelium-dependent nitric oxide/cGMP pathway.

Emerging evidence suggests that nitric oxide (NO) plays a pivotal role in the mechanism of vascular hyporesponsiveness contributing to microgravity-induced orthostatic intolerance. The cellular and enzymatic source of the NO, however, remains controversial. In addition, the time course of the endothelial-dependent contribution remains unstudied. We tested the hypotheses that the change in vasoresponsiveness seen in acute (3-day) hindlimb unweighted (HLU) animals is due to an endothelium-dependent mechanism and that endothelial-dependent attenuation in vasoreactivity is due to endothelial nitric oxide synthase (NOS-3) dependent activation. Vasoreactivity was investigated in rat aortic rings following acute HLU treatment. Dose responsiveness to norepinepherine (NE) was depressed after 3-day HLU [1,338 +/- 54 vs. 2,325 +/- 58 mg at max (NE), HLU vs. C, P < 0.001]. However, removal of the endothelium restored the vascular contractility to that of C. In addition, 1H-oxadiazole quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor, restored the reduced vasoconstrictor responses to phenylephrine (PE) seen in 3-day HLU rings (1.30 +/- 0.10 vs. 0.53 +/- 0.07 g, HLU + ODQ vs. HLU, P = 0.0001). Ca(+) dependent nitric oxide synthase (NOS) activity was increased, as was vascular NO products as a result of HLU. While NOS-3 expression was not increased in HLU rats, phosphorylation of NOS-3 at serine-1177 (an activator of NOS-3) was increased while phosphorylation of serine-495 (an inactivator of NOS-3) was decreased. These findings demonstrate that changes in vasoresponsiveness in the acute HLU model of microgravity are due to an upregulation of the endothelial-dependent NO/cGMP pathway through NOS phosphorylation.

Single exposure to radiation produces early anti-angiogenic effects in mouse aorta.

Radiation exposure can increase the risk for many non-malignant physiological complications, including cardiovascular disease. We have previously demonstrated that ionizing radiation can induce endothelial dysfunction, which contributes to increased vascular stiffness. In this study, we demonstrate that gamma radiation exposure reduced endothelial cell viability or proliferative capacity using an in vitro aortic angiogenesis assay. Segments of mouse aorta were embedded in a Matrigel-media matrix 1 day after mice received whole-body gamma irradiation between 0 and 20 Gy. Using three-dimensional phase contrast microscopy, we quantified cellular outgrowth from the aorta. Through fluorescent imaging of embedded aortas from Tie2GFP transgenic mice, we determined that the cellular outgrowth is primarily of endothelial cell origin. Significantly less endothelial cell outgrowth was observed in aortas of mice receiving radiation of 5, 10, and 20 Gy radiation, suggesting radiation-induced endothelial injury. Following 0.5 and 1 Gy doses of whole-body irradiation, reduced outgrowth was still detected. Furthermore, outgrowth was not affected by the location of the aortic segments excised along the descending aorta. In conclusion, a single exposure to gamma radiation significantly reduces endothelial cell outgrowth in a dose-dependent manner. Consequently, radiation exposure may inhibit re-endothelialization or angiogenesis after a vascular injury, which would impede vascular recovery.

Simulated microgravity-induced aortic remodeling.

We have previously shown that microgravity and simulated microgravity induce an increase in human and rat aortic stiffness. We attempted to elucidate the mechanism(s) responsible for this increase in stiffness. We hypothesize that an alteration in vessel wall collagen or elastin content or in extracellular matrix (ECM) cross-linking either individually or in a combination is responsible for the increased vessel stiffness. Rats underwent hindlimb unweighting (HLU) for a period of 7 days to simulate microgravity. The contribution of ECM cross-linking to the vessel wall stiffness was evaluated by measuring aortic pulse wave velocity following inhibition of the cross-linking enzymes lysyl oxidase (LOX) and transglutaminase (tTG) and the nonenzymatic advanced glycation end product cross-linking pathway during HLU. Aortic collagen and elastin content was quantified using established colorimetric assays. Collagen subtype composition was determined via immunofluorescent staining. The increase in aortic pulse wave velocity after HLU was significantly attenuated in the LOX and tTG inhibition groups compared with saline (1.13 +/- 0.11 vs. 3.00 +/- 0.15 m/s, LOX vs. saline, P < 0.001; 1.16 +/- 0.25 vs. 3.00 +/- 0.15 m/s, tTG vs. saline, P < 0.001). Hydroxyproline content, a measure of collagen content, was increased in all groups after HLU (2.01 +/- 0.62 vs. 3.69 +/- 0.68% dry weight, non-HLU vs. HLU, P = 0.009). Collagen subtype composition and aortic elastin content were not altered by HLU. Together, these data indicate that HLU-induced increases in aortic stiffness are due to both increased aortic collagen content and enzyme cross-linking activity.

Selective fluorescent labeling of S-nitrosothiols (S-FLOS): a novel method for studying S-nitrosation.

Protein S-nitrosation is a reversible post-translation modification critical for redox-sensitive cell signaling that is typically studied using the Biotin Switch method. This method and subsequent modifications usually require avidin binding or Western blot analysis to detect biotin labeled proteins. We describe here a modification of the Biotin Switch assay that eliminates the need for Western blot or avidin enrichment protocols and allows direct comparison of the S-nitrosation state proteins from two different samples in the same gel lane or on the same 2D gel. This S-FLOS method offers detection, identification and quantification of S-nitrosated proteins, with the potential for site-specific identification of nitrosation events.

Microgravity-induced changes in aortic stiffness and their role in orthostatic intolerance.

Microgravity (microG)-induced orthostatic intolerance (OI) in astronauts is characterized by a marked decrease in cardiac output (CO) in response to an orthostatic stress. Since CO is highly dependent on venous return, alterations in the resistance to venous return (RVR) may be important in contributing to OI. The RVR is directly dependent on arterial compliance (C(a)), where aortic compliance (C(ao)) contributes up to 60% of C(a). We tested the hypothesis that microG-induced changes in C(a) may represent a protective mechanism against OI. A retrospective analysis on hemodynamic data collected from astronauts after 5- to 18-day spaceflight missions revealed that orthostatically tolerant (OT) astronauts showed a significant decrease in C(a) after spaceflight, while OI astronauts showed a slight increase in C(a). A ground-based animal model simulating microG, hindlimb-unweighted rats, was used to explore this phenomenon. Two independent assessments of C(ao), in vivo pulse wave velocity (PWV) of the thoracic aorta and in vitro pressure-diameter squared relationship (PDSR) measurements of the excised thoracic aorta, were determined. PWV showed a significant increase in aortic stiffness compared with control, despite unchanged blood pressures. This increase in aortic stiffness was confirmed by the PDSR analysis. Thus both actual microG in humans and simulated microG in rats induces changes in C(ao). The difference in C(a) in OT and OI astronaut suggests that the microG-induced decrease in C(a) is a protective adaptation to spaceflight that reduces the RVR and allows for the maintenance of adequate CO in response to an orthostatic stress.

Impaired shear stress-induced nitric oxide production through decreased NOS phosphorylation contributes to age-related vascular stiffness.

Endothelial dysfunction and increased arterial stiffness contribute to multiple vascular diseases and are hallmarks of cardiovascular aging. To investigate the effects of aging on shear stress-induced endothelial nitric oxide (NO) signaling and aortic stiffness, we studied young (3-4 mo) and old (22-24 mo) rats in vivo and in vitro. Old rat aorta demonstrated impaired vasorelaxation to acetylcholine and sphingosine 1-phosphate, while responses to sodium nitroprusside were similar to those in young aorta. In a customized flow chamber, aortic sections preincubated with the NO-sensitive dye, 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate, were subjected to steady-state flow with shear stress increase from 0.4 to 6.4 dyn/cm(2). In young aorta, this shear step amplified 4-amino-5-methylamino-2',7'-difluorofluorescein fluorescence rate by 70.6 +/- 13.9%, while the old aorta response was significantly attenuated (23.6 +/- 11.3%, P < 0.05). Endothelial NO synthase (eNOS) inhibition, by N(G)-monomethyl-l-arginine, abolished any fluorescence rate increase. Furthermore, impaired NO production was associated with a significant reduction of the phosphorylated-Akt-to-total-Akt ratio in aged aorta (P < 0.05). Correspondingly, the phosphorylated-to-total-eNOS ratio in aged aortic endothelium was markedly lower than in young endothelium (P < 0.001). Lastly, pulse wave velocity, an in vivo measure of vascular stiffness, in old rats (5.99 +/- 0.191 m/s) and in N(omega)-nitro-l-arginine methyl ester-treated rats (4.96 +/- 0.118 m/s) was significantly greater than that in young rats (3.64 +/- 0.068 m/s, P < 0.001). Similarly, eNOS-knockout mice demonstrated higher pulse wave velocity than wild-type mice (P < 0.001). Thus impaired Akt-dependent NO synthase activation is a potential mechanism for decreased NO bioavailability and endothelial dysfunction, which likely contributes to age-associated vascular stiffness.

Nitric oxide regulation of myocardial contractility and calcium cycling: independent impact of neuronal and endothelial nitric oxide synthases.

The mechanisms by which nitric oxide (NO) influences myocardial Ca2+ cycling remain controversial. Because NO synthases (NOS) have specific spatial localization in cardiac myocytes, we hypothesized that neuronal NOS (NOS1) found in cardiac sarcoplasmic reticulum (SR) preferentially regulates SR Ca2+ release and reuptake resulting in potentiation of the cardiac force-frequency response (FFR). Transesophageal pacing (660 to 840 bpm) in intact C57Bl/6 mice (WT) stimulated both contractility (dP/dtmax normalized to end-diastolic volume; dP/dt-EDV) by 51+/-5% (P<0.001) and lusitropy (tau; tau) by 20.3+/-2.0% (P<0.05). These responses were markedly attenuated in mice lacking NOS1 (NOS1-/-) (15+/-2% increase in dP/dt-EDV; P<0.001 versus WT; and no change in tau; P<0.01 versus WT). Isolated myocytes from NOS1-/- (approximately 2 months of age) also exhibited suppressed frequency-dependent sarcomere shortening and Ca2+ transients ([Ca2+]i) compared with WT. SR Ca2+ stores, a primary determinant of the FFR, increased at higher frequencies in WT (caffeine-induced [Ca2+]i at 4 Hz increased 107+/-23% above 1 Hz response) but not in NOS1-/- (13+/-26%; P<0.01 versus WT). In contrast, mice lacking NOS3 (NOS3-/-) had preserved FFR in vivo, as well as in isolated myocytes with parallel increases in sarcomere shortening, [Ca2+]i, and SR Ca2+ stores. NOS1-/- had increased SR Ca2+ ATPase and decreased phospholamban protein abundance, suggesting compensatory increases in SR reuptake mechanisms. Together these data demonstrate that NOS1 selectively regulates the cardiac FFR via influences over SR Ca2+ cycling. Thus, there is NOS isoform-specific regulation of different facets of rate-dependent excitation-contraction coupling; inactivation of NOS1 has the potential to contribute to the pathophysiology of states characterized by diminished frequency-dependent inotropic responses.

Arginase reciprocally regulates nitric oxide synthase activity and contributes to endothelial dysfunction in aging blood vessels.

BACKGROUND: Although abnormal L-arginine NO signaling contributes to endothelial dysfunction in the aging cardiovascular system, the biochemical mechanisms remain controversial. L-arginine, the NO synthase (NOS) precursor, is also a substrate for arginase. We tested the hypotheses that arginase reciprocally regulates NOS by modulating L-arginine bioavailability and that arginase is upregulated in aging vasculature, contributing to depressed endothelial function. METHODS AND RESULTS: Inhibition of arginase with (S)-(2-boronoethyl)-L-cysteine, HCl (BEC) produced vasodilation in aortic rings from young (Y) adult rats (maximum effect, 46.4+/-9.4% at 10(-5) mol/L, P<0.01). Similar vasorelaxation was elicited with the additional arginase inhibitors N-hydroxy-nor-L-arginine (nor-NOHA) and difluoromethylornithine (DFMO). This effect required intact endothelium and was prevented by 1H-oxadiazole quinoxalin-1-one (P<0.05 and P<0.001, respectively), a soluble guanylyl cyclase inhibitor. DFMO-elicited vasodilation was greater in old (O) compared with Y rat aortic rings (60+/-6% versus 39+/-6%, P<0.05). In addition, BEC restored depressed L-arginine (10(-4) mol/L)-dependent vasorelaxant responses in O rings to those of Y. Arginase activity and expression were increased in O rings, whereas NOS activity and cyclic GMP levels were decreased. BEC and DFMO suppressed arginase activity and restored NOS activity and cyclic GMP levels in O vessels to those of Y. CONCLUSIONS: These findings demonstrate that arginase modulates NOS activity, likely by regulating intracellular L-arginine availability. Arginase upregulation contributes to endothelial dysfunction of aging and may therefore be a therapeutic target.

Estrogen has opposing effects on vascular reactivity in obese, insulin-resistant male Zucker rats.

We hypothesized that estradiol treatment would improve vascular dysfunction commonly associated with obesity, hyperlipidemia, and insulin resistance. A sham operation or 17beta-estradiol pellet implantation was performed in male lean and obese Zucker rats. Maximal vasoconstriction (VC) to phenylephrine (PE) and potassium chloride was exaggerated in control obese rats compared with lean rats, but estradiol significantly attenuated VC in the obese rats. Estradiol reduced the PE EC50 in all groups. This effect was cyclooxygenase independent, because preincubation with indomethacin reduced VC response to PE similarly in a subset of control and estrogen-treated lean rats. Endothelium-independent vasodilation (VD) to sodium nitroprusside was similar among groups, but endothelium-dependent VD to ACh was significantly impaired in obese compared with lean rats. Estradiol improved VD in lean and obese rats by decreasing EC50 but impaired function by decreasing maximal VD. The shift in EC50 corresponded to an upregulation in nitric oxide synthase III protein expression in the aorta of the estrogen-treated obese rats. In summary, estrogen treatment improves vascular function in male insulin-resistant, obese rats, partially via an upregulation of nitric oxide synthase III protein expression. These effects are counteracted by adverse factors, such as hyperlipidemia and, potentially, a release of an endothelium-derived contractile agent.

Measuring ascending aortic stiffness in vivo in mice using ultrasound.

We present a protocol for measuring in vivo aortic stiffness in mice using high-resolution ultrasound imaging. Aortic diameter is measured by ultrasound and aortic blood pressure is measured invasively with a solid-state pressure catheter. Blood pressure is raised then lowered incrementally by intravenous infusion of vasoactive drugs phenylephrine and sodium nitroprusside. Aortic diameter is measured for each pressure step to characterize the pressure-diameter relationship of the ascending aorta. Stiffness indices derived from the pressure-diameter relationship can be calculated from the data collected. Calculation of arterial compliance is described in this protocol. This technique can be used to investigate mechanisms underlying increased aortic stiffness associated with cardiovascular disease and aging. The technique produces a physiologically relevant measure of stiffness compared to ex vivo approaches because physiological influences on aortic stiffness are incorporated in the measurement. The primary limitation of this technique is the measurement error introduced from the movement of the aorta during the cardiac cycle. This motion can be compensated by adjusting the location of the probe with the aortic movement as well as making multiple measurements of the aortic pressure-diameter relationship and expanding the experimental group size.

Alagebrium in combination with exercise ameliorates age-associated ventricular and vascular stiffness.

Advanced glycation end-products (AGEs) initiate cellular inflammation and contribute to cardiovascular disease in the elderly. AGE can be inhibited by Alagebrium (ALT), an AGE cross-link breaker. Moreover, the beneficial effects of exercise on aging are well recognized. Thus, we investigated the effects of ALT and exercise (Ex) on cardiovascular function in a rat aging model. Compared to young (Y) rats, in sedentary old (O) rats, end-systolic elastance (Ees) decreased (0.9±0.2 vs 1.7±0.4mmHg/µL, P<0.05), dP/dt(max) was attenuated (6054±685 vs 9540±939mmHg/s, P<0.05), ventricular compliance (end-diastolic pressure-volume relationship (EDPVR)) was impaired (1.4±0.2 vs 0.5±0.4mmHg/µL, P<0.05) and diastolic relaxation time (tau) was prolonged (21±3 vs 14±2ms, P<0.05). In old rats, combined ALT+Ex (4weeks) increased dP/dt(max) and Ees (8945±665 vs 6054±685mmHg/s, and 1.5±0.2 vs 0.9±0.2 respectively, O with ALT+Ex vs O, P<0.05 for both). Diastolic function (exponential power of EDPVR and tau) was also substantially improved by treatment with Alt+Ex in old rats (0.4±0.1 vs 0.9±0.2 and 16±2 vs 21±3ms, respectively, O with ALT+EX vs O, P<0.05 for both). Pulse wave velocity (PWV) was increased in old rats (7.0±0.7 vs 3.8±0.3ms, O vs Y, P<0.01). Both ALT and Ex alone decreased PWV in old rats but the combination decreased PWV to levels observed in young (4.6±0.5 vs 3.8±0.3ms, O with ALT+Ex vs Y, NS). These results suggest that prevention of the formation of new AGEs (with exercise) and breakdown of already formed AGEs (with ALT) may represent a therapeutic strategy for age-related ventricular and vascular stiffness.

HZE 56Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase.

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.

Dietary inhibition of xanthine oxidase attenuates radiation-induced endothelial dysfunction in rat aorta.

Radiation exposure is associated with the development of various cardiovascular diseases. Although irradiation is known to cause elevated oxidant stress and chronic inflammation, both of which are detrimental to vascular function, the molecular mechanisms remain incompletely understood. We previously demonstrated that radiation causes endothelial dysfunction and increased vascular stiffness by xanthine oxidase (XO) activation. In this study, we investigated whether dietary inhibition of XO protects against radiation-induced vascular injury. We exposed 4-mo-old rats to a single dose of 0 or 5 Gy gamma radiation. These rats received normal drinking water or water containing 1 mM oxypurinol, an XO inhibitor. We measured XO activity and superoxide production in rat aorta and demonstrated that both were significantly elevated 2 wk after radiation exposure. However, oxypurinol treatment in irradiated rats prevented aortic XO activation and superoxide elevation. We next investigated endothelial function through fluorescent measurement of nitric oxide (NO) and vascular tension dose responses. Radiation reduced endothelium-dependent NO production in rat aorta. Similarly, endothelium-dependent vasorelaxation in the aorta of irradiated rats was significantly attenuated compared with the control group. Dietary XO inhibition maintained NO production at control levels and prevented the development of endothelial dysfunction. Furthermore, pulse wave velocity, a measure of vascular stiffness, increased by 1 day postirradiation and remained elevated 2 wk after irradiation, despite unchanged blood pressures. In oxypurinol-treated rats, pulse wave velocities remained unchanged from baseline throughout the experiment, signifying preserved vascular health. These findings demonstrate that XO inhibition can offer protection from radiation-induced endothelial dysfunction and cardiovascular complications.

Cyclohexanone contamination from extracorporeal circuits impairs cardiovascular function.

Extracorporeal circulation provides critical life support in the face of cardiopulmonary or renal failure, but it also introduces a host of unique morbidities characterized by edema formation, cardiac insufficiency, autonomic dysfunction, and altered vasomotor function. We tested the hypothesis that cyclohexanone (CHX), a solvent used in production of extracorporeal circuits and intravenous (IV) bags, leaches into the contained fluids and can replicate these clinical morbidities. Crystalloid fluid samples from circuits and IV bags were analyzed by gas chromatography-mass spectrometry to provide a range of clinical CHX exposure levels, revealing CHX contamination of sampled fluids (9.63-3,694 microg/l). In vivo rat studies were conducted (n = 49) to investigate the effects of a bolus IV infusion of CHX vs. saline alone on cardiovascular function, baroreflex responsiveness, and edema formation. Cardiovascular function was evaluated by cardiac output, heart rate, stroke volume, vascular resistance, arterial pressure, and ventricular contractility. Baroreflex function was assessed by mean femoral arterial pressure responses to bilateral carotid occlusion. Edema formation was assessed by the ratio of wet to dry organ weights for lungs, liver, kidneys, and skin. CHX infusion led to systemic hypotension; pulmonary hypertension; depressed contractility, heart rate, stroke volume, and cardiac output; and elevated vascular resistance (P < 0.05). Mean arterial pressure responsiveness to carotid occlusion was dampened after CHX infusion (from +17.25 +/- 1.8 to +5.61 +/- 3.2 mmHg; P < 0.05). CHX infusion led to significantly higher wet-to-dry weight ratios vs. saline only (3.8 +/- 0.06 vs. 3.5 +/- 0.05; P < 0.05). CHX can reproduce clinical cardiovascular, neurological, and edema morbidities associated with extracorporeal circulatory treatment.

Arginase inhibition restores NOS coupling and reverses endothelial dysfunction and vascular stiffness in old rats.

There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2(-)) production than young. Acute inhibition of both NOS, with N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-amino- 6-boronohexanoic acid (ABH), significantly reduced O2(-) production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692-702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2(-) production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.