doi: 10.1161/01.CIR.0000094403.78467.C3
(Circulation. 2003;108:2172.)
© 2003 American Heart Association, Inc.
Special Review |
Physiology and Pathophysiology of Vascular Signaling Controlled by Cyclic Guanosine 3',5'-Cyclic Monophosphate–Dependent Protein Kinase
From the Division of Cardiology, University Hospital Eppendorf, Hamburg, Germany (T.M.); Institut für Pharmakologie und Toxikologie, Technische Universität, München, Germany (R.F., F.H.); the Institute of Cardiovascular Physiology, J.W. Goethe–University Clinic, Frankfurt/Main, Germany (A.M.); and the Institute of Clinical Biochemistry and Pathobiochemistry, University of Würzburg, Würzburg, Germany (S.M.L., U.W.).
Correspondence to Prof Thomas Münzel, MD, Professor of Internal Medicine, Division of Cardiology, University Hospital Eppendorf, Martinistr 52, 20246 Hamburg, Germany. E-mail muenzel{at}uke.uni-hamburg.de
Key Words: nitric oxide • natriuretic peptides • endothelium • cardiovascular diseases • free radical
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Introduction |
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 Top Introduction cGMP SynthesisReferences |
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Despite impressive medical advances1 that have led to diminished cardiovascular death rates in some countries over the past 20 years, cardiovascular disease remains the leading cause of death in developed countries such as the United States. This promises to worsen as a result of aging populations; the incipient obesity and type II diabetes epidemic; sedentary lifestyle; and continued abuse of tobacco, alcohol, and other substances. Cardiovascular disease is clearly multifactorial, and the approach to its prevention necessarily likewise. Candidates for prevention include cyclic guanosine 3',5'-cyclic monophosphate (cGMP)–dependent signaling networks initiated by natriuretic peptides (NPs) and nitric oxide (NO), which demonstrate characteristics deemed worthy of diagnostic and therapeutic exploitation. cGMP signaling contributes to the function and interaction of several vascular cell types, and its dysfunction could be involved in major destructive processes such as atherosclerosis, hypertension, diabetic complications, (re)stenosis, and tissue infarction, as well as the undermining of clinical therapy like in the case of nitrate tolerance. This review takes a focused look at key elements of the cGMP signaling cascade in vascular tissue, particularly recent advances in our knowledge of cGMP-dependent protein kinase (cGK, also known as PKG) function. Finally, we discuss the potential of clinical monitoring of cGK activity for assessing the functional status of cGMP signaling and for guiding the design of therapeutic strategies to improve vascular function.
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cGMP Synthesis |
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TopIntroductioncGMP SynthesisReferences |
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One of the 2 major synthetic pathways for cGMP generation from guanosine 5'-triphosphate (GTP) is directed by NPs (Figure 1), consisting of atrial (ANP), B-type (BNP), and C-type (CNP) natriuretic peptides, which act via the membrane receptor guanylate cyclases GC-A (highest affinity for ANP, BNP) and GC-B (highest affinity for CNP). ANP and BNP, released from the heart by mechanical stretch in response to increased atrial pressure/volume, and CNP, released from the endothelium, can all cause smooth muscle (SM) relaxation and vasodilation.2,3 Particularly, ANP has multiple effects, which lead to vascular smooth muscle (VSM) relaxation, increased vascular permeability and glomerular filtration rate, inhibition of the sympathetic and renin-angiotensin-aldosterone systems, and natriuresis/diuresis.3 Germline deletion of ANP or its GC-A receptor in mice resulted in varying degrees of hypertension, cardiomyopathy, and heart failure (reviewed in reference 2), but also cardiac hypertrophy that is independent of hypertension.4,5 ANP effects on VSM itself were analyzed in transgenic mice engineered to lack GC-A selectively in smooth muscle. These mice displayed resistance to vasodilation by acute ANP treatment or vascular volume expansion, but normal resting blood pressure,6 suggesting that the function of GC-A in VSM can be compensated for in chronic blood pressure regulation.
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The second major pathway for cGMP synthesis (Figure 1) entails stimulation of nitric oxide synthases (NOS) to produce NO, which activates soluble guanylate cyclases (sGC).2 NO inhibits VSM constriction, proliferation, and migration, and reduces platelet adhesion and activation as well as vascular inflammation.7 Transgenic overexpression and deletion of endothelial NOS (eNOS, NOSIII) and inducible NOS (iNOS, NOSII) have substantiated the role of NO in reducing blood pressure and the consequences of vascular injury.2,7 These results with experimental models have led to the suggestion of members of the NO pathway as promising candidates for gene therapy, and a phase I NOS gene transfer study has been announced.7 Recommended potential clinical applications of NOS gene transfer include inhibition of vascular proliferation, relevant, for example, for atherosclerosis and restenosis, and inhibition of pulmonary hypertension, vasospasm, vasculitis, oxidative and reperfusion injury, thrombosis, and (through antisense NOS) septicemia.7 Already idiopathic pulmonary hypertension has been shown to respond to inhaled NO in some patients with apparent defects in their endogenous NO system.8 Promising as this sounds, incipient signals generated by NO or NPs need to traverse long, divergent, and interacting chains of multiple mediators before reaching effector molecules, which trigger final effects. Also, defects, desensitization, or other aberrations in the pathways distal to NO/NPs must be recognized as capable of derailing the desired procession of events. The ensuing discussion illustrates that understanding the complex array of interactions beyond NO/NPs is a serious challenge and an integral aspect of achieving insights into successful strategies for opposing cardiovascular disease.
cGMP Effectors
The cGMP signal is diversified through cell type–specific collections of intracellular cGMP receptors. In the cardiovascular system, the major cGMP effectors are cGMP-regulated phosphodiesterases (PDEs)9,10 and cGKs (see subsequently in this article).11–14 Cyclic nucleotide PDEs hydrolyze cAMP and/or cGMP and thus terminate their action. They are presently classified as PDE families 1 through 11, encoded by 20 genes, generating more than 50 PDE isozymes.14a cGMP-hydrolyzing PDEs include ones that specifically (PDE 5, 6, and 9) or preferentially (PDE 1A1) hydrolyze cGMP,15 as well as ones that hydrolyze both cGMP and cAMP (PDE 1 to 3 and 10 to 11).9,10,16 cGMP may also be transported out of cells by the multidrug-resistant protein 5.17 The activity of certain PDEs can be stimulated (PDE 2) or inhibited (PDE 3) by cGMP. Also, cGKI phosphorylates and activates PDE 5 to rapidly terminate cGMP action.18,19 Platelets contain at least PDE 2, 3, and 519,20; arterial VSM cells at least PDE 1 to 5; and venous VSM cells at least PDE 1, 4, and 5.21
There is limited information on other cGMP effectors, which include cyclic nucleotide-gated channels (CNG) and cAMP-dependent protein kinase (cAK). Endothelial cells can express CNG22; however, the functional significance of these channels is unclear, because no cardiovascular phenotype has been reported for various CNG-knockout mice.23,24 cGMP signals can be mediated by cAMP-dependent protein kinase25 through either of 2 different mechanisms. cGMP can inhibit PDE 3 (also called cGI-PDE) at concentrations similar to those that activate cGK,26,26a,26b and thus increase cAMP and its activation of cAK. In contrast to the native cGMP increased by physiological activators, membrane-permeable cGMP analogs inhibit PDE 3 with different efficiencies, 100 µmol/L 8-Br-cGMP26b apparently reaching intracellular concentrations sufficient to inhibit PDE 3, whereas 100 µmol/L 8-pCPT-cGMP does not.26a,27 Alternatively, cGMP has been proposed to directly activate cAK under certain circumstances. However, compared with cGK, much higher levels of cGMP are required to directly activate cAK.26 Studies of SM and platelets from cGK I–deficient transgenic mice,28,29 or cGK I-deficient platelets from chronic myelocytic leukemia patients,30 suggest that cAK is not involved in NO/cGMP signaling under physiological conditions, although cAK could come into play in pathophysiological settings such as inflammatory conditions30a and septic shock when high NO/cGMP levels are produced.31
cGMP-Dependent Protein Kinases
The mammalian cGK family consists of cGKI
and Iß, splice forms derived from 1 gene, and cGKII, encoded by a second gene.11,13 Several cardiovascular cells, and other cell types which regulate the cardiovascular system, contain cGKs. These include fibroblasts and certain types of endothelial cells (cGKI), but not HUVECs from human umbilical veins,32,33 SM cells (cGKI and Iß),34,35 platelets and T lymphocytes (cGKIß),36,37 renin-secreting juxtaglomerular cells (cGKI and cGKII),38 other renal cells (cGKI and cGKII),39 and cardiac myocytes (cGKI).40 It is important to note that cGKs are lost in many primary cell types on passaging in cell culture and are undetectable in many cell lines.32,37,41 This and the use of selective but not totally specific agonists and inhibitors of cGK has compounded the problems associated with establishing cGK functions. A notable example is the cGKI inhibitor KT5823, which can stimulate cGKI in certain cell types.42
cGKs can achieve specificity of function partly through their selective binding to and phosphorylation of various substrates. Both cGKI and Iß have been shown to associate with troponin T and phosphorylate troponin I in the heart43; however, only cGKI interacted with and phosphorylated the myosin-binding subunit of myosin light chain (MLC) phosphatase in VSM.44 Only cGKIß occurs in a complex with and phosphorylates the IP3 receptor-associated cGKI substrate (IRAG) in VSM.45
Insight into cGK functions has been obtained both from analyses of substrates phosphorylated by cGKs and from cGK knockout mice. cGKI substrates and functions (Table) have been reviewed11,14,46,47 and are briefly discussed here. Except for CFTR,48 there are almost no other well-studied substrates of cGKII. In conventional cGKI knockout mice, the major phenotype observed was decreased lifespan28; impaired relaxation of vascular,28 visceral,28,49 and penile50 SM; and disturbed platelet activation.29 The phenotype of cGKII knockout mice included normal lifespan,51 decreased longitudinal bone growth,51 decreased intestinal chloride secretion,51,52 and altered renin secretion.53 Results obtained with knockout mice discussed here support an important role of cGKI in the cardiovascular system, particularly in VSM cells and platelets, as well as potential cGKII influences on hemodynamic parameters through regulation of renin release and ion transport in the kidney.
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cGKI—Endothelial Cells
Two lines of evidence exist for activation of endothelial NOSIII by cGKI. cGKI54 as well as several other protein kinases (cAK, Akt kinase, AMP kinase, protein kinase C [PKC], and calmodulin-dependent kinase II)54–57 regulate (eg, cGKI stimulates) the phosphorylation and function of NOSIII, and in some cases (including cGKI) reduce its Ca2+ dependence. Furthermore, cGKI and cGKII phosphorylate and activate 6-pyruvoyltetrahydropterin synthase to produce tetrahydrobiopterin (BH4) needed by NOS for NO synthesis.58
cGKI—Vascular Smooth Muscle Cells
NO/ANP/cGMP interfere with VSMC signaling initiated by vasoconstrictor receptors and G-proteins,59 which elevate Ca2+ to promote activation of MLC kinase, phosphorylation of MLCs, interaction of myosin/actin filaments, and contraction (Figure 1). Evidence supports a dual role of cGKI in inhibition of both intracellular Ca2+ concentrations and Ca2+ sensitization to evoke SM relaxation.14,46,60 Primary cells cultured from cGKI knockout mice have also been important in demonstrating differences in cGKI isoform involvement in SMC Ca2+ regulation. NO/cGMP inhibited noradrenaline-induced Ca2+-transients in wild-type VSMCs containing endogenous cGKI and cGKIß, but not in cGKI-deficient cells.28,61 Interestingly, the defective Ca2+ regulation in cGKI-deficient VSMCs was rescued by transfection of only cGKI, not cGKIß,61 suggesting that cGKI relaxes VSM by decreasing the cytosolic Ca2+ level. The role of cGKIß is unclear at present, but it is perhaps more relevant to cGKI effects on SM proliferation, differentiation, and gene expression.14,46
Contributing to reduction of intracellular Ca2+ concentrations could be a spectrum of SM cGKI substrates, including the IP3 receptor and IRAG14,45 (Figure 1). cGKI also phosphorylates a SM BKCa channel, which leads to K+ efflux from the cell, hyperpolarization, inhibition of Ca2+ entry through voltage-dependent ion channels, and relaxation.14,46 cGKI phosphorylation of the sarcoplasmic reticulum (SR) protein phospholamban (PLB) was suggested to deinhibit the SR Ca2+-ATPase, thus increasing Ca2+ loading of SR for later release and activation of BKCa.46 However, PLB deletion in mice has only a minor effect on cGMP-mediated VSM relaxation.62
At constant Ca2+ concentration, SM relaxation can be promoted by cGKI phosphorylation of Rho A to inhibit Rho kinase and thus activate MLC phosphatase to dephosphorylate MLC (Figure 1).63 cGKI binds to and phosphorylates the myosin-binding subunit of MLC phosphatase, which could be important for localization of cGKI near the enzyme it regulates.44 Furthermore, cGKI inhibition of Rho reinforces insulin signaling and its stimulation of myosin-bound phosphatase activity.64 The importance of the Rho pathway is illustrated by the fact that treatment of rats with the Rho kinase inhibitor Y-27632 corrected hypertension in several hypertensive rat models65 and also suppressed neointimal formation in balloon-injured carotid arteries.66
In agreement with a role of cGKI in SM relaxation, juvenile (4 to 5 week old) cGKI knockout mice had elevated blood pressure.28 These and in vitro studies with small arteries from cGKI-deficient mice31 suggested that cGKI mediates at least part of the antihypertensive effects of NO and ANP. The release of renin from the kidney and subsequent generation of angiotensin II also affect blood pressure. Inactivation of the cGKII gene, but not the cGKI gene, abolished cGMP inhibition of renin secretion in vitro and in vivo.53 Thus, both cGKI and cGKII could be involved in the regulation of blood pressure, although through different mechanisms and cell types.
Interestingly, 7-week-old and older cGKI knockout mice have normal, or only slightly elevated, blood pressure,28 indicating that lack of cGKI can be bypassed in older animals. Similarly, the cardiac phenotype of young NOSIII null mutants67 is not observed in older NOSIII knockout mice.68,69 These results suggest that mice with defects in the NO/cGMP pathway develop mechanisms to compensate for lost gene functions or, alternatively, that the physiological role of components of this signaling pathway is age-dependent. However, cGKI null mutants develop multiple defects with increasing age, including infections and inflammation, which are known to induce massive NO synthesis. Therefore, it is tempting to speculate that the apparent 
normalization
of blood pressure in older cGKI mutants could be the result of cross-activation of cAK by the high NO/cGMP levels potentially generated in these diseased mice.
Although conventional cGKI knockout mice have yielded important insights into cGKI functions in the cardiovascular system, the analysis of these mice has several limitations. These mice have low life expectancy (approximately 50% of these mice die before 5 to 6 weeks of age), precluding analysis of adult mice and performance of long-term experiments like telemetric blood pressure measurements. Furthermore, the interpretation of phenotypes of conventional null mutants (in which the cGKI gene is inactivated in the germline and thus in every cell) is complicated by the lack of temporal and regional specificity of gene disruption. cGKI null mutants show not only a cardiovascular phenotype, but also develop severe defects of the gastrointestinal28 and immune systems (C. Werner and F. Hofmann, unpublished results, 2003). It is also difficult to distinguish whether the age-related hypertension of these mice reflects a function of cGKI in VSMCs, endothelial cells, or other cell types. To overcome these limitations, a mouse line has been generated that allows tissue-specific inactivation of the cGKI gene using the Cre/lox site-specific recombination system.70 In the future, the combined use of conventional and conditional cGKI knockout mice and of cGKI-deficient primary cells will further advance our understanding of cGKI and cGKIß function in the vessel wall in health and disease, and provide insight needed to develop new strategies for the treatment of cardiovascular disorders.
Reductions in cGMP/cGKI signaling have also been associated with detrimental conditions such as nitrate tolerance (discussed in a later section) and neointimal formation. In some studies,46 but not others,71 cGKI has been shown to be reduced in the synthetic or proliferative SMC phenotype found in neointima. Adenovirus-mediated gene transfer of cGKI increased the sensitivity of cultured VSMC to the antiproliferative and proapoptotic effects of NO and cGMP,72 and cGKI inhibited VSMC migration.46,73,74 Adenovirus-mediated gene transfer of constitutively active cGKI reduced neointima formation after balloon injury in rat carotid arteries and reduced in-stent restenosis in a porcine model.74
cGKI—Platelets
Antiplatelet therapy is presently standard in the secondary prevention of cardiovascular events.75 Therapeutically used platelet inhibitors are aspirin (a cyclooxygenase inhibitor), glycoprotein IIb/IIIa inhibitors, ADP receptor P2Y12 inhibitors such as Clopidogrel, and adenosine uptake/PDE 5 inhibitors such as dipyridamole, which increase cGMP.20,75,76 cGKI can mediate inhibitory effects on platelets through several of these same targets. Experiments with both cGK-deficient human and murine platelets demonstrated that cGKI mediates many aspects of NO/cGMP inhibition of platelet activation29,30 (Figure 2). Importantly, a prominent role of cGKI in the inhibition of platelet adhesion/activation in vivo during ischemia/reperfusion of the microcirculation was conclusively demonstrated by intravital video microscopy analyses comparing cGKI-deficient versus wild-type murine platelets perfused back into mice.29 These experiments clearly showed that platelet cGKI, but not endothelial or SM cGKI, is essential to prevent intravascular adhesion and aggregation of platelets after ischemia. Furthermore, NO/cGMP/cGKI causes phosphorylation of platelet VASP,77 which closely correlates with inhibition of platelet activation both in vitro20 and in vivo,78 with fibrinogen receptor (integrin GPIIb/IIIa) inhibition, and with inhibition of both VASP binding to F-actin and VASP localization to focal adhesions/integrins.20,77 Other platelet substrates phosphorylated by cGKI include the IP3 receptor, heat shock protein 27,20 the LIM and SH3 protein LASP,79 the small GTPase Rap 1b,80 and PDE 5.18,19
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NO/cGMP signaling through cGKI is also known to inhibit platelet Gq/Gi-coupled receptor responses,20 and in particular, the platelet ADP receptor P2Y12.81 Thus, one consequence of the impaired NO/cGMP signaling found in cardiovascular diseases characterized by endothelial dysfunction could be enhanced signaling through purinergic Gq/Gi-coupled receptors (P2Y1 and P2Y12), leading to enhanced platelet activation. cGKI also contributes to negative feedback or to cycling in signaling systems it transduces. Recent results indicated that cGKI phosphorylation of PDE 5 and activation of cGMP degradation could also contribute to desensitization of the platelet NO/cGMP response and to the contraction–relaxation cycle in SM.18,19 Furthermore, evidence indicates that cGKI mediates effects of dipyridamole, which, in combination with low-dose aspirin, is very effective in preventing recurrent stroke; dipyridamole inhibits cGMP hydrolysis by PDE 5 and enhances antiplatelet effects of NO, but not those of cAMP.76 There is still lack of in vivo evidence in volunteers and/or patients of direct platelet inhibition by NO-generating drugs such as NTG, which activate sGC. However, recent preclinical studies with NO-independent sGC activators suggest that elevation of platelet cGMP in vivo is in fact associated with platelet inhibition.82,83
cGKI—Fibroblasts
NO, ANP, and cGMP inhibit proliferative effects of norepinephrine on cardiac myocytes and fibroblasts.84 Cardiac fibrosis, a hallmark of cardiac failure, is a major consequence of genetic deletion of BNP in mice.85 Some data suggests that cGKI can mediate inhibitory effects on fibrosis. In NIH 3T3 fibroblasts, cGKI phosphorylates MAP/ERK kinase kinase (MEKK1), causing activation of JNK kinase, an inducer of apoptosis.86 In adventitial fibroblasts, cGKI increased expression of p21 (Waf1/Cip1), a cyclin-dependent kinase inhibitor that can arrest cell proliferation during the cell cycle by preventing DNA replication in S-phase.87 In 293T fibroblasts, cGKI stimulated p38 kinase phosphorylation and ATF-2-dependent gene expression.88
cGKI—Cardiac Myocytes
In the heart, cGKI inhibits L-type Ca2+ channels,40 has antihypertrophic effects,89 and mediates the negative inotropic effect of cGMP.70 Ca2+ signaling has been implicated in cardiac hypertrophy,90–92 and cGKI inhibition of Ca2+ influx resulted in inhibition of the Ca2+-dependent calcineurin-NFAT signaling pathway and cell hypertrophy.91 cGKI also phosphorylates PLB,93 releasing PLB inhibition of Ca2+-ATPase and enhancing Ca2+ reuptake into the SR, which would presumably enhance cardiac relaxation, availability of Ca2+ for contraction, and the maximal rate of contraction.94,95 In a mouse model of dilated cardiomyopathy, ablation of PLB rescued phenotypes that resemble human heart failure,96 suggesting that cGKI phosphorylation and inactivation of PLB might have a similar inhibitory effect with regard to heart failure.
Mice with cardiomyocyte-specific cGKI deletion are fully viable and can be studied throughout adulthood. The combined analysis of conventional and cardiomyocyte-specific cGKI knockout mice demonstrated that cGKI mediates the negative inotropic effect of cGMP in juvenile as well as adult murine heart, whereas the NO/cGMP/cGKI pathway seems not to be involved in the negative inotropic action of acetylcholine.68–70 Potential mechanisms involved in the negative inotropic effects of cGKI might include cGKI inhibition of L-type Ca2+ channels,40 and cGKI interaction with troponin T and phosphorylation of troponin I.43
Endothelial Dysfunction, Oxidative Stress, and Defective NO/cGMP Signaling
The preceding sections provide considerable evidence that the NO/cGMP/cGK signaling axis is important in the control of normal cardiovascular function and should have important implications for our understanding and treatment of vascular disease. Endothelial dysfunction, manifested as thrombosis, inflammation, and vasoconstriction, is a common feature of atherosclerosis, hypercholesterolemia, hypertension, and nitrate tolerance. Quite often, endothelial dysfunction is characterized by impairment of endothelium-dependent, NO-mediated relaxation, in contrast to essentially normal vascular relaxation to endothelium-independent nitrovasodilators such as sodium nitroprusside (SNP) or NTG. In endothelial dysfunction, defects have been described in the coupling of stimuli to activation of NOSIII, in the activity and/or expression of NOSIII, and in NO bioavailability as a result of increased production of reactive oxygen species (ROS=oxidative stress). Impaired VSM relaxation can result from not only endothelial dysfunction, but also from defective activity and/or expression of components of the NO downstream signaling pathway (sGC, cGKI, PDEs).
Antioxidants are effective in combating the vascular oxidative stress, which reduces NO production or bioactivity. Acute administration of the antioxidant vitamin C improved endothelial dysfunction in patients with hypercholesterolemia, overt atherosclerosis, diabetes mellitus, hypertension, smoking, ischemia/reperfusion, and NTG therapy (reviewed in reference 97) The major extent to which oxidative stress contributes to endothelial dysfunction is demonstrated by the fact that the degree to which vitamin C improves endothelial dysfunction is a strong and independent predictor of subsequent cardiovascular events.98
In mammalian cells, potential enzymatic sources of reactive oxygen species (ROS) include mitochondrial respiration enzymes, lipoxygenases, cyclooxygenases, cytochrome P450 enzymes, NAD(P)H oxidases, xanthine oxidase, and the ceramide-activated protein kinase (reviewed in reference 99). Another source of vascular ROS production that has received substantial attention is NOSIII itself. Circumstances in which NOSIII generates superoxide include inadequate intracellular concentrations of L-arginine (the NOSIII substrate) and BH4 (a NOSIII cofactor), or interactions of ROS with the Zn thiolate complex of NOSIII itself.100 Diminished intracellular L-arginine bioavailability can occur by diminished L-arginine recycling, increased L-arginine metabolism, or increased formation of asymmetric dimethyl-arginines, which inhibit NOSIII.101 BH4 as well as the Zn thiolate complex are susceptible to oxidation by peroxynitrite (ONOO-), the reaction product of NO and superoxide (Figure 3). Consistent with this, recent animal102 and human studies103,104 of endothelial dysfunction indicate that administration of either BH4, or folic acid (which can restore endothelial BH4 concentrations) improves endothelial dysfunction and simultaneously reduces oxidative stress.
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NO/cGMP Signaling Pathway as Monitor of Endothelial Dysfunction
In general, assessment of any individual component (NOSIII, sGC, PDE) of the NO/cGMP pathway has not been uniformly reliable as a predictor of endothelial dysfunction. However, because cGKI occupies a very distal position in this signaling chain, its activity could theoretically be used to reflect the sum of changes in activity and/or expression of upstream components as well as cGKI itself. Assessment of cGKI activity is facilitated by immunoblotting using antibodies that detect phosphorylation of the cGKI substrate, VASP, at Ser239 (P-VASP) (Figure 3). This assay has proven to reliably reflect NO/cGMP signaling activity in platelets, cultured endothelial and SM cells,33,105 and even intact vascular tissues.106 Immunoblot detection of P-VASP is even sufficiently sensitive to allow the assessment of basal NO release in endothelium-intact arteries. Indeed, either endothelial denudation or incubation with NOS inhibitors significantly attenuates the proportion of arterial P-VASP.106 Conversely, exogenous NO donors such as SNP and NTG markedly increase P-VASP in vessels regardless of the presence or absence of intact endothelium.106 P-VASP also proved to be a suitable monitor for reduced NO bioactivity as a result of oxidative stress in vascular tissue. For example, inhibition of Cu/Zn superoxide dismutase (SOD) increased vascular superoxide, reduced endothelial NO bioactivity, and resulted in marked reduction in the proportion of P-VASP.106
Hypercholesterolemia
Hypercholesterolemia impairs endothelium-dependent vasodilation, and also arterial relaxation to authentic NO, but has little effect on responses to nitrovasodilators such as SNP and NTG. This hypercholesterolemia-dependent defect in NO bioactivity appears linked to oxidative stress, because animals rendered hyperlipidemic by cholesterol feeding,107 LDL receptor deficiency,106 or ApoE deletion102 all exhibit increased steady-state superoxide flux compared with control animals. Sources of superoxide that have been implicated in hypercholesterolemia include xanthine oxidase,107 NAD(P)H oxidase,108 and NOSIII.102,106
The effects of hypercholesterolemia on sGC expression are inconsistent. Cholesterol feeding is associated with a 3- to 4-fold increase in the vascular expression of sGC subunits 1 and ß1 (mainly within neointimal lesions), whereas no change was observed in vessels from Watanabe heritable hyperlipidemic rabbits (WHHL).109 The catalytic activity of sGC is unchanged but the specific activity reduced, perhaps as a result of increased smooth muscle-derived ROS production.110
The expression of cGKI appears to be normal, whereas cGK activity, assessed by the P-VASP/VASP ratio, is strongly decreased compared with controls, suggestive of a defect in the NO/cGMP pathway upstream of cGK.106 Incubation of vessels from controls, as well as WHHL animals, with high concentrations of the exogenous NO donor, SNP, resulted in comparable maximal P-VASP levels, demonstrating that the cGMP signaling pathway downstream of sGC was, per se, not impaired.106 Chronic treatment of WHHL with AT1 receptor blockers reduced oxidative stress within vascular tissue and improved endothelial function, most likely by inhibiting NADPH-oxidase and NOSIII-mediated superoxide production.106,111 The improvement of endothelial function was associated with increased vascular NO concentrations detected with electron paramagnetic resonance, and a quite marked enhancement in vascular P-VASP levels, again demonstrating the usefulness of P-VASP as a reliable biomonitor of NO function in vascular tissue.
Hypertension
Endothelial dysfunction in different animal models of hypertension has been shown to be associated with increased expression of NOSIII112,113 but decreased vascular NO bioavailability, as revealed by P-VASP analysis.112 Uncoupled NOSIII112 and NAD(P)H oxidase114 were identified as significant superoxide sources based on observations that (1) NOSIII inhibition decreased vascular superoxide production in hypertensive animals,112 and (2) these animals exhibited increased NAD(P)H oxidase subunit (nox1, p22phox, p67phox, and gp91phox) expression and enzymatic activity.112,115 Increased superoxide production involved all vessel layers112,116 and was causally linked to both endothelial dysfunction and hypertension, because treatment with either antioxidants or SOD not only improved endothelial function, but also markedly reduced blood pressure.117
Blood vessels from hypertensive animals demonstrate a phenotype that is distinct from that observed with hypercholesterolemia. In particular, vessels from hypertensive animals displayed reduced vasodilation to both endothelium-dependent vasodilators and the endothelium-independent exogenous nitrovasodilators SNP and NTG.114 This finding can be explained in part by reduced expression of sGC because different hypertensive animal models typically demonstrate reduced levels of 1 or both sGC subunits (1 and ß1) as well as reduced NO-dependent sGC activity.112,118,119 The specific mechanism(s) for reduced sGC expression in hypertension are not entirely clear. Changes in sGC expression seem not to be exclusively linked to increased superoxide formation. Reducing blood pressure normalizes or even enhances sGC expression,112,113,120 but does not consistently alter vascular superoxide production. In the angiotensin II-infusion model, inhibition of PKC in vivo reduces both blood pressure and vascular superoxide formation112 concomitant with normalization of sGC levels (M. Oelze, unpublished observation, 2003). In contrast, hydralazine treatment significantly lowers blood pressure but fails to normalize vascular superoxide formation in chronically, NOS-inhibited rats.119
In angiotensin II-induced hypertension, cGKI activity assessed by P-VASP was markedly reduced, indicative of a signaling defect upstream of cGKI.112 Oxidative stress could be involved in this defect, because PKC inhibition with chelerythrine reduced oxidative stress, increased VASP phosphorylation, and improved endothelial dysfunction (M. Oelze, unpublished observation, 2003). Interpretation of these observations is not straightforward, however, because in vitro VSM cell experiments indicate that angiotensin II also increases the activity and expression of the cGMP-specific PDE1A1 isoform (Figure 3).15 Thus, increased cGMP hydrolysis could also contribute to the endothelial dysfunction and decreased cGKI activity observed in angiotensin II-induced hypertension.
Nitrate Tolerance
NTG produces potent vasodilatory and antiischemic effects that are rapidly lost on chronic treatment as a result of a phenomenon known as nitrate tolerance. Like in the case of hypertension and hypercholesterolemia, nitrate tolerance is associated with endothelial dysfunction (=cross-tolerance); increased expression of a dysfunctional, uncoupled NOSIII121; and activation of NAD(P)H oxidase.122 Treatment with antioxidants such as vitamin E123 or C124 ameliorates nitrate tolerance. Moreover, other substances that reduce oxidative stress such as ACE inhibitors and AT1 receptor blockers (both of which can reduce NADPH oxidase activation) or BH4125 and folic acid (which can prevent NOSIII uncoupling)126 also reverse, in part, nitrate tolerance as well as cross-tolerance to endothelium-dependent vasodilators.
Baseline cGMP levels, and cGMP responses to acute NTG challenges, are lower in tolerant than control vessels.127 Mechanisms involved can include decreased vascular NO bioavailability,121 increased production of SM endothelin-1,128 and inhibition of NO-dependent sGC activity by NTG-stimulated ROS production. Posttranslational modifications of sGC such as heme oxidation can also be a cause of impaired sGC-specific activity in the setting of nitrate tolerance.129 Changes in sGC expression do not explain the impaired cGMP response, because both mRNA and protein expression of sGC subunits 1 and ß1 appear increased rather than decreased in aortas from tolerant rats and rabbits.130 cGKI expression does not appear to be changed in arterial tissue from either NTG-treated animals or from humans treated with therapeutic doses of NTG.130,131 Only higher, clinically nonrelevant doses of ISDN and NTG appear to decrease cGKI expression by approximately 50%.130–132 However, in vivo treatment with NTG consistently decreased the cGKI activity assessed by P-VASP in aortas from rats and rabbits,130 and in mammary arteries from patients with coronary artery disease.131 Incubation of arterial tissue from NTG-treated animals with the antioxidant vitamin C restored P-VASP, improved tolerance, and reduced vascular superoxide levels, indicating a crucial role of ROS in inhibiting cGKI activity in the setting of nitrate tolerance.130
In summary, in experimental and clinical examples of hypercholesterolemia, hypertension, and nitrate tolerance, ROS were shown to be a major cause of inhibition of the NO/cGMP/cGKI signaling pathway and induction of endothelial dysfunction. Analysis of distal pathway cGKI activity was a more reliable indicator of endothelial function/dysfunction than were the levels of individual components of the NO/cGMP/cGKI pathway. Monitoring of cGKI activity through P-VASP levels not only serves as an effective predictor of endothelial function, but also potentially represents a much needed biochemical assay for early recognition of disease states and for evaluation of therapeutic strategies.
Diagnostic and Therapeutic Potential of cGKI-Mediated Vascular Signaling
cGKI is clearly an important mediator of vasorelaxant, antithrombotic, and other cardiovascular protective effects of NO and NPs, and new avenues of diagnostic and therapeutic exploitation of NO/NP/cGMP/cGK pathways are emerging. Recently, measurement of plasma BNP has been demonstrated to be useful in the diagnosis of congestive heart failure.133,134 Analysis of VASP phosphorylation as a reporter of cGKI activity in vessels is a reliable monitor of endothelial integrity in model systems of atherosclerosis, hypertension, and nitrate tolerance. Importantly, P-VASP analysis in vessels was found applicable for monitoring nitrate tolerance in humans. Analysis of P-VASP has proven useful for monitoring platelet inhibition by NO-independent sGC activators,83,135 by PDE 5 inhibitors such as dipyridamole,76 and by the ADP P2Y12-receptor inhibitor Clopidogrel.20 Present studies focus on analyzing P-VASP in whole blood to expedite clinical monitoring of the antithrombotic/antiplatelet capacity of the endothelium as an indicator of cardiovascular disease status and the success of therapeutic regimens.
Components of the NO/NP/cGMP/cGK pathways also constitute promising targets for therapy of chronic vascular diseases. The large number of PDEs with different regulators and cellular distributions make them interesting candidate targets for specific intervention into cGMP signaling. The success of Sildenafil, a PDE 5 inhibitor for increasing cGMP and SM relaxation, used in clinical treatment of erectile dysfunction, is well known.136 Other results have shown that PDE1C (hydrolyzes cAMP and cGMP with equal efficiency) is expressed only in proliferating aortic SMCs, and that PDE1C inhibition suppresses proliferation of SMC from normal and atherosclerotic human tissue, potentially useful in prevention of atherosclerotic lesions or restenosis.137 Recently, short-term use of Nesiritide (human recombinant BNP) was shown to be beneficial in the management of decompensated congestive heart failure.138 The longstanding clinical use of NO donors for the treatment of angina pectoris and coronary artery disease is of proven value, however, with the detraction that chronic treatment leads to tolerance and endothelial dysfunction. A promising strategy designed to circumvent this would be use of ROS inhibitors, NO-independent activators of sGC that do not invoke tolerance,82,83 selective PDE 5 inhibitors, direct activators of cGKI, and combinations thereof. Currently, non-NO-based activators of sGC used in in vivo animal studies have caused long-term reduction in blood pressure and thrombosis,83 as well as cardiorenal effects, which would be beneficial for treatment of diseases such as congestive heart failure.139 By definition, these activators would also avoid any undesirable cGMP-independent effects of NO. Further downstream, activators of cGKI or cGKII would have even more focused effects by excluding cGMP actions on other mediators.
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