W. Robert Taylor

Role of NADH/NADPH Oxidase–Derived H2O2 in Angiotensin II–Induced Vascular Hypertrophy

Recent evidence suggests that oxidative mechanisms may be involved in vascular smooth muscle cell (VSMC) hypertrophy. We previously showed that angiotensin II (Ang II) increases superoxide production by activating an NADH/NADPH oxidase, which contributes to hypertrophy. In this study, we determined whether Ang II stimulation of this oxidase results in H2O2 production by studying the effects of Ang II on intracellular H2O2 generation, intracellular superoxide dismutase and catalase activity, and hypertrophy. Ang II (100 nmol/L) significantly increased intracellular H2O2 levels at 4 hours. Neither superoxide dismutase activity nor catalase activity was affected by Ang II; the SOD present in VSMCs is sufficient to metabolize Ang II-stimulated superoxide to H2O2, which accumulates more rapidly than it is degraded by catalase. This increase in H2O2 was inhibited by extracellular catalase, diphenylene iodonium, an inhibitor of the NADH/NADPH oxidase, and the AT1 receptor blocker losartan. In VSMCs stably transfected with antisense p22phox, a critical component of the NADH/NADPH oxidase in which oxidase activity was markedly reduced, Ang II-induced production of H2O2 was almost completely inhibited, confirming that the source of Ang II-induced H2O2 was the NADH/NADPH oxidase. Using a novel cell line that stably overexpresses catalase, we showed that this increased H2O2 is a critical step in VSMC hypertrophy, a hallmark of many vascular diseases. Inhibition of intracellular superoxide dismutase by diethylthiocarbamate (1 mmol/L) also resulted in attenuation of Ang II-induced hypertrophy (62+/-2% inhibition). These data indicate that AT1 receptor-mediated production of superoxide generated by the NADH/NADPH oxidase is followed by an increase in intracellular H2O2, suggesting a specific role for these oxygen species and scavenging systems in modifying the intracellular redox state in vascular growth.

p22phox mRNA Expression and NADPH Oxidase Activity Are Increased in Aortas From Hypertensive Rats

Recent studies suggest that superoxide production by the NADPH/NADH oxidase may be involved in smooth muscle cell growth and the pathogenesis of hypertension. We previously showed that angiotensin II (Ang II) activates a p22phoxbased NADPH/NADH oxidase in cultured rat vascular smooth muscle cells and in animals made hypertensive by infusion of Ang II. To investigate the mechanism responsible for this increased oxidase activity, we examined p22phox mRNA expression in rats made hypertensive by implanting an osmotic minipump that delivered Ang II (0.7 mg/kg per day). Blood pressure began to increase 3 days after the start of Ang II infusion and remained elevated for up to 14 days. Expression of p22phox mRNA in aorta was also increased after 3 days and reached a maximum increase of 338 +/- 41% by 5 days after pump implantation compared with the value after sham operation. This increase in mRNA expression was accompanied by an increase in the content of the corresponding cytochrome (twofold) and NADPH oxidase activity (179 +/- 11% of that in sham-operated rats 5 days after pump implantation). Treatment with the antihypertensive agents losartan (25 mg/kg per day) or hydralazine (15 mg/kg per day) inhibited this upregulation of mRNA levels and activity. Furthermore, infusion of recombinant heparin-binding superoxide dismutase decreased both blood pressure and p22phox mRNA expression. In situ hybridization of aortic tissue showed that p22phox mRNA was expressed in medial smooth muscle as well as in the adventitia. These findings suggest that Ang II-induced hypertension activates the NADPH/NADH oxidase system by upregulating mRNA levels of one or several components of this oxidase system, including the p22phox, and that the NADPH/NADH oxidase system is associated with the pathology of hypertension in vivo.

Nox1 Overexpression Potentiates Angiotensin II-Induced Hypertension and Vascular Smooth Muscle Hypertrophy in Transgenic Mice

Background— Reactive oxygen species (ROS) have been implicated in the development of cardiovascular pathologies. NAD(P)H oxidases (Noxes) are major sources of reactive oxygen species in the vessel wall, but the importance of individual Nox homologues in specific layers of the vascular wall is unclear. Nox1 upregulation has been implicated in cardiovascular pathologies such as hypertension and restenosis. Methods and Results— To investigate the pathological role of Nox1 upregulation in vascular smooth muscle, transgenic mice overexpressing Nox1 in smooth muscle cells (TgSMCnox1) were created, and the impact of Nox1 upregulation on the medial hypertrophic response during angiotensin II (Ang II)–induced hypertension was studied. These mice have increased expression of Nox1 protein in the vasculature, which is accompanied by increased superoxide production. Infusion of Ang II (0.7 mg/kg per day) into these mice for 2 weeks led to a potentiation of superoxide production compared with similarly treated negative littermate controls. Systolic blood pressure and aortic hypertrophy were also markedly greater in TgSMCnox1 mice than in their littermate controls. To confirm that this potentiation of vascular hypertrophy and hypertension was due to increased ROS formation, additional groups of mice were coinfused with the antioxidant Tempol. Tempol decreased the level of Ang II-induced aortic superoxide production and partially reversed the hypertrophic and hypertensive responses in these animals. Conclusions— These data indicate that smooth muscle-specific Nox1 overexpression augments the oxidative, pressor, and hypertrophic responses to Ang II, supporting the concept that medial Nox1 participates in the development of cardiovascular pathologies.

Angiotensin II–Induced Hypertension Accelerates the Development of Atherosclerosis in ApoE-Deficient Mice

BackgroundAngiotensin II may contribute to the development and progression of atherosclerotic lesions because of its growth and proinflammatory effects. We sought to determine whether angiotensin II–induced hypertension would augment and accelerate the development of atherosclerotic lesions in apoE-deficient mice. Methods and ResultsAngiotensin II (0.7 mg · kg−1 · d−1 SC) was administered to apoE-deficient mice via osmotic minipumps. The animals were placed on either standard chow or an atherogenic diet. After 8 weeks, the mean atherosclerotic lesion area in the descending thoracic and abdominal aortas of animals on a standard chow diet was 0.4±0.1% compared with 5.2±1.2% in those animals maintained on an atherogenic diet (P <0.0001). In angiotensin II–treated animals on standard chow, the mean lesion area was increased to 11.0±2.3%, which was further increased to 69.9±9.4% (P <0.0001) in angiotensin II–treated animals on an atherogenic diet. Similar findings were obtained when tissues from the ascending aorta were analyzed. At 8 weeks in mice receiving a standard chow diet, angiotensin II dramatically increased the atherosclerotic lesion area by 840±83 &mgr;m2 (P <0.0001). Animals on a high-fat diet had a similar marked increase in lesion area in response to angiotensin II (217±19 &mgr;m2, P <0.0001). In contrast, when hypertension was induced with norepinephrine, only a modest effect on the atherosclerotic lesion area was observed. ConclusionsAngiotensin II–induced hypertension specifically increased the development of atherosclerosis in apoE knockout mice. This response was seen in animals receiving either standard chow or an atherogenic diet. These studies demonstrate the profound effect of angiotensin II on the development of atherosclerosis.

Hydrocyanines: A Class of Fluorescent Sensors That Can Image Reactive Oxygen Species in Cell Culture, Tissue, and In Vivo

The development of fluorescent probes for superoxide and the hydroxyl radical is a central problem in the field of chemical biology. Superoxide and the hydroxyl radical play a significant role in a variety of inflammatory diseases, and probes that can detect these reactive oxygen species (ROS) have tremendous potential as medical diagnostics and research tools. Fluorescent sensors for superoxide and the hydroxyl radical, such as dihydroethidium (DHE), have been developed; however, they have had limited applicability because of their spontaneous autoxidation, rapid photobleaching, low emission wavelengths, and multiple reaction products with ROS. New chemical probes for superoxide and the hydroxyl radical are therefore greatly needed. Herein, we present a new family of fluorescent ROS sensors, termed the hydrocyanines, which can be synthesized in one step from the commercially available cyanine dyes and can detect superoxide and the hydroxyl radical in living cells, tissue samples, and for the first time in vivo. We anticipate widespread interest in the hydrocyanines given their physical/ chemical characteristics and ease of synthesis. The synthesis of and mechanism by which hydrocyanines image ROS are shown in Figure 1a. The hydrocyanines are synthesized by reducing the iminium cations of the cyanine dyes with NaBH4. Hydrocyanines detect ROS through fluorescent imaging; they are weakly fluorescent because of their disrupted p conjugation; however, oxidation with either superoxide or the hydroxyl radical dramatically increases their fluorescence by regenerating their extended p conjugation. Figure 1b demonstrates the proof of principle of this methodology. Hydro-IR-676 has minimal fluorescence; however, oxidation with superoxide causes a 100-fold increase in its fluorescence intensity. The cyanine dyes comprise a family of approximately 40 dyes, and the reduction methodology presented in Figure 1a was used to synthesize several new fluorescent ROS sensors, which have the physical and chemical properties needed to detect intracellular and extracellular ROS, both in vitro and in vivo. For example, five new ROS sensors, termed hydro-Cy3, hydro-Cy5, hydroCy7, hydro-IR-783, and hydro-ICG, were synthesized by reduction with NaBH4 (Table 1). These molecules have negligible fluorescence, as a result of reduction of their iminium cations; however, after reaction with either superoxide or the hydroxyl radical, they fluoresce at 560, 660, 760, 800, and 830 nm, respectively (see Table 1 and the Supporting Information for details). Hydro-Cy3, hydro-Cy5, hydro-IR676, and hydro-Cy7 have the physical properties needed to detect intracellular ROS, in that they are initially membranepermeable molecules; however, oxidation with intracellular ROS converts them into charged and membrane-impermeable molecules, which should accumulate within cells that are overproducing ROS. In contrast, hydro-ICG and hydro-IRFigure 1. a) Synthesis of hydrocyanines by a one-step reduction with NaBH4. Reaction with superoxide or the hydroxyl radical oxidizes the hydrocyanines to produce the fluorescent cyanine dyes. b) Hydro-IR676 has negligible fluorescence emission; however, oxidation with superoxide causes a 100-fold increase in fluorescence (lex = 675 nm, lem = 693 nm).

Monocyte Chemoattractant Protein-1 Expression in Aortic Tissues of Hypertensive Rats

Monocyte chemoattractant protein-1 (MCP-1), a potent monocyte chemoattractant synthesized by vascular cells and monocytes, has been proposed to be an important mediator of inflammatory responses in the arterial vasculature. It was recently demonstrated that hypertension is associated with an inflammatory response in the arterial wall. To determine the effect of hypertension on arterial MCP-1 expression, we induced hypertension in Sprague-Dawley rats by infusing angiotensin II (0.75 mg x kg[-1] x d[-1] SC) for 7 days. Using Northern blot analysis, we detected a 3.6-fold increase in MCP-1 mRNA in the aortas of hypertensive rats. When we normalized blood pressure in angiotensin II-treated rats through oral administration of the nonspecific vasodilator hydralazine (15 mg x kg[-1] x d[-1]), aortic MCP-1 mRNA expression was significantly reduced. Similar results were obtained with a norepinephrine model of hypertension. Taken together, these data suggest that mechanical factors may be responsible in part for the upregulation of expression. Consistent with this interpretation, we found that cultured rat aortic vascular smooth muscle cells exposed to mechanical strain (20% peak deformation at 1 Hz) exhibited a marked increase in MCP-1 expression, suggesting the hemodynamic strain imparted onto arterial cells in hypertension is an important stimulus underlying this phenomenon. These results provide important insights into the in vivo regulation of MCP-1 and have potential implications for understanding the influence of hypertension on atherosclerosis.

Bioartificial matrices for therapeutic vascularization.

Therapeutic vascularization remains a significant challenge in regenerative medicine applications. Whether the goal is to induce vascular growth in ischemic tissue or scale up tissue-engineered constructs, the ability to induce the growth of patent, stable vasculature is a critical obstacle. We engineered polyethylene glycol–based bioartificial hydrogel matrices presenting protease-degradable sites, cell-adhesion motifs, and growth factors to induce the growth of vasculature in vivo. Compared to injection of soluble VEGF, these matrices delivered sustained in vivo levels of VEGF over 2 weeks as the matrix degraded. When implanted subcutaneously in rats, degradable constructs containing VEGF and arginine-glycine-aspartic acid tripeptide induced a significant number of vessels to grow into the implant at 2 weeks with increasing vessel density at 4 weeks. The mechanism of enhanced vascularization is likely cell-demanded release of VEGF, as the hydrogels may degrade substantially within 2 weeks. In a mouse model of hind-limb ischemia, delivery of these matrices resulted in significantly increased rate of reperfusion. These results support the application of engineered bioartificial matrices to promote vascularization for directed regenerative therapies.

Hemodynamic Shear Stresses in Mouse Aortas: Implications for Atherogenesis

Objective—The hemodynamic environment is a determinant of susceptibility to atherosclerosis in the vasculature. Although mouse models are commonly used in atherosclerosis studies, little is known about local variations in wall shear stress (WSS) in the mouse and whether the levels of WSS are comparable to those in humans. The objective of this study was to determine WSS values in the mouse aorta and to relate these to expression of gene products associated with atherosclerosis. Methods and Results—Using micro-CT and ultrasound methodologies we developed a computational fluid dynamics model of the mouse aorta and found values of WSS to be much larger than those for humans. We also used a quantum dot-based approach to study vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 expression on the aortic intima and demonstrated that increased expression for these molecules occurs where WSS was relatively low for the mouse. Conclusions—Despite large differences in WSS in the two species, the spatial distributions of atherogenic molecules in the mouse aorta are similar to atherosclerotic plaque localization found in human aortas. These results suggest that relative differences in WSS or in the direction of WSS, as opposed to the absolute magnitude, may be relevant determinants of flow-mediated inflammatory responses.

The Study of the Influence of Flow on Vascular Endothelial Biology

It is now recognized that the mechanical environment of a cell has an influence on its structure and function. For the vascular endothelial cell that resides at the interface of the flowing blood and the underlying vessel wall, there is mounting evidence of the importance of flow and the associated wall shear stress in the regulation of endothelial biology. Not only is it a sensitive regulator of endothelial structure and function, but different flow environments will influence endothelial cell biology differently. Furthermore, there may be an interaction between the chemical environment of a cell and its mechanical environment. This is illustrated by the inhibition by steady laminar shear stress of the cytokine induction of VCAM-1. Results also are presented in which flow studies have been conducted using a co-culture model of the vessel wall. These experiments provide evidence of a quiescent endothelium; however, much more needs to be done to engineer the cell culture environment to make it more physiologic.

Angiotensin II and atherosclerosis.

Numerous clinical and laboratory data are now available supporting the hypothesis that the renin-angiotensin system is mechanistically relevant in the pathogenesis of atherosclerosis. The traditional role of the renin-angiotensin system in the context of blood pressure regulation has been modified to incorporate the concept that angiotensin II (Ang II) is a potent proinflammatory agent. In vascular cells, Ang II is a potent stimulus for the generation of reactive oxygen species. As a result, Ang II upregulates the expression of many redox-sensitive cytokines, chemokines, and growth factors that have been implicated in the pathogenesis of atherosclerosis. Extensive data now confirm that inhibition of the renin-angiotensin system inhibits atherosclerosis in animal models as well as in humans. These studies provide mechanistic insights into the precise role of Ang II in atherosclerosis and suggest that pharmacologic interventions involving the renin-angiotensin system may be of fundamental importance in the treatment and prevention of atherosclerosis.