Nitric Oxide Signaling and Atherothrombosis Redux: Evidence From Experiments of Nature and Implications for Therapy

HomeCirculationVol. 137, No. 3Nitric Oxide Signaling and Atherothrombosis Redux Free AccessEditorialPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessEditorialPDF/EPUBNitric Oxide Signaling and Atherothrombosis ReduxEvidence From Experiments of Nature and Implications for Therapy Joseph Loscalzo, MD, PhD Joseph LoscalzoJoseph Loscalzo Brigham and Women’s Hospital, Harvard Medical School, Boston, MA. Originally published16 Jan 2018https://doi.org/10.1161/CIRCULATIONAHA.117.032901Circulation. 2018;137:233–236Article, see p 222Don’t reinvent the wheel, just realign it.1—Anthony J. D’AngeloThe relationship of nitric oxide (NO) to vascular disease has a long and storied history. Even before the identification of the endothelium-derived relaxing factor as NO, abnormalities in endothelium-dependent vasorelaxation were recognized in human atherosclerotic coronary arteries.2 Soon thereafter, Bossaller and colleagues3 showed that this impairment in endothelium-dependent relaxation in response to muscarinic agonists is associated with attenuated cyclic guanosine 5′-monophosphate (cGMP) accumulation, consistent with a key signaling mechanism for NO. Vita and colleagues4 subsequently demonstrated that impaired endothelium-dependent vasodilation of angiographically smooth coronary arteries correlates with risk factor burden, ushering in the notion that endothelial dysfunction and associated NO insufficiency is a precursor to overt coronary atherosclerosis.5 From the thrombosis perspective, platelet-derived NO was shown to be critical in modulating hemostatic potential,6 largely by impairing recruitment of platelets to the growing platelet thrombus.7 In keeping with this key biological role, a deficiency of platelet-derived NO was shown to predict the presence of an acute coronary syndrome8 and to contribute to the overall propensity to arterial thrombosis.9 Taken together, these observations show that altered NO bioavailability and signaling functionally link NO to the pathogenesis of atherothrombosis. Furthermore, early evidence suggested that adequate bioavailable NO can promote the regression of established atherosclerosis, in part, by improving endothelial function,10 and by inducing monocyte/macrophage apoptosis, as well.11Functional polymorphisms in the endothelial NO synthase gene (NOS3) that attenuate NO production are experiments of nature that confirm the adverse consequences of inadequate generation of NO by the endothelium. Casas and colleagues12 reported a meta-analysis of 26 studies involving >23 000 subjects and showed that homozygosity for the loss-of-function Glu298Asp variant and for the intron-4a (27-bp insertion/deletion variable number tandem repeats) polymorphism(s) are associated with a significantly higher risk of ischemic heart disease. Similarly, a risk variant (rs7692387) in the α3-subunit of the soluble guanylyl cyclase-1 gene (GUCY1A3), which lowers gene expression in whole blood and decreases inhibition of platelet aggregation by NO donors, has been associated with an increase in coronary artery disease risk linked to chromosomal locus 4q32.1.13 These and other (lower frequency) variants in these genes have also been associated with altered coronary artery disease risk in select populations.In this issue of Circulation, Emdin and colleagues14 examine rare variants in NOS3 and GUCY1A3 that are associated with gain of function. They explore individual-level data from >335 000 participants in the UK Biobank, and summary association analysis from 7 large-scale genome-wide association studies, as well, that include nearly 1.5 million subjects. Their results indicate that variants that increase NO and NO-mediated cGMP synthesis are associated with decreased mean arterial pressure and reduced risk of coronary heart disease, peripheral arterial disease, and stroke. It is important to note that mediation analysis showed that the benefit of these variants with respect to coronary heart disease risk was not accounted for solely by their blood pressure–lowering effect. In a complementary analysis, the authors also demonstrated that rare loss-of-function variants in NOS3 and GUCY1A3 are associated with a higher mean arterial pressure and a significantly increased risk of coronary heart disease.This study is instructive for two important reasons. First, it confirms the functional studies and phenotype association studies performed >25 years earlier, and does so by using the rare variant approach in large populations. Second, it emphasizes the importance of the nonlipid determinants of atherothrombosis. The attributable risk of coronary heart disease has not yet been fully ascertained, and genetic variants in lipid metabolism, despite the expectation that they alone are necessary and sufficient to explain the disease, simply do not do so. To vascular biologists and clinical cardiologists alike, this is hardly surprising given the extensive prior evidence for endothelial dysfunction as a risk factor for coronary heart disease even in the absence of dyslipidemias. What is unique in this report, however, is that, in contrast to the earlier work in which endothelial dysfunction was felt to be an integrative intermediate phenotype produced by well-established risk factors, the authors now convincingly show that naturally occurring, intrinsic (germline) variation in genes that govern the endothelial NO-cGMP pathway can alone modulate the risk of atherothrombotic vascular disease, presumably by affecting endothelial function and its consequences for blood pressure, thrombosis, and inflammation. In effect, this study demonstrates that the causal relationship between endothelial dysfunction and the endophenotypic determinants of atherothrombosis is bidirectional. These observations led the authors to conclude that identifying intrinsic, heritable abnormalities in a well-studied signaling pathway in the vasculature offers the possibility of novel therapeutic strategies by which to enhance NO signaling for the prevention and treatment of cardiovascular disease.But is this conclusion truly novel, and if so, would simple enhancement of NO signaling be an effective way to prevent, treat, or even cure the disease? Very soon after the discovery of endothelial NO, investigators began to explore the potential therapeutic effects of NO donors in preventing or reversing atherothrombosis.15–17 The mechanistic argument underlying this approach was that the oxidant stress accompanying inflammation in atherothrombosis generates reactive oxygen species, such as superoxide anion, that can react with and inactivate NO (in forming peroxynitrite, for example).18 This impairment in NO bioavailability was addressed in early studies by the use of small-molecule antioxidants, such as α-tocopherol,19 and met with variable success because of the potential for pro-oxidant effects that depend on concentrations and related redox couples.20 Other strategies that have been used include increasing tetrahydrobiopterin, pharmacologically21 or by upregulating GTP-cyclohydrolase 1,22 to prevent biochemical uncoupling of endothelial NO synthase and its generation of superoxide anion21; supplemental folic acid to reverse endothelial NO synthase dysfunction via its effects on tetrahydrobiopterin metabolism23; and supplemental l-arginine to increase the generation of endothelial NO by endothelial NO synthase. Supplemental l-arginine, in particular, seems a straightforward and relatively safe approach to restoring endothelial NO production. In fact, another experiment of nature, lysinuric protein intolerance caused by a mutation in the cationic amino acid transporter SLC7A7 gene, provides additional insight in this regard. Mutations in this gene cause a deficiency of l-arginine and NO, and lead to impaired coronary perfusion with exercise and enhanced platelet activation,24 effects that can be corrected with supplemental l-arginine. Although l-arginine can increase NO production in the healthy vasculature, reported benefits in the atherosclerotic vasculature have been inconsistent, likely because it also serves as a substrate for inducible NO synthase.25 In this setting, the adverse effect is likely caused, in part, by enhanced peroxynitrite formation in the setting of oxidant stress and the creation of a methylation stress state.26An alternative approach to improving the bioactivity and bioavailability of NO involves enhancing NO production via the enterosalivary pathway.27,28 This pathway involves the oral microbiome, which reduces dietary nitrate to nitrite. This oral nitrite pool is then reduced in the acidified stomach to nitrous acid, which is ultimately converted to nitrogen oxides via N2O3, including NO itself. As a source of NO, this pathway represents a significant fraction of the total circulating pool of NO, is modifiable by dietary nitrate, and can improve endothelial function in the setting of hypercholesterolemia, in part, by increasing the bacterial taxa producing nitrate reductase.29Yet another strategy for improving NO-cGMP pathway signaling in vascular disease involves enhancing cGMP synthesis or inhibiting its degradation. The former can be achieved by activating the oxidatively inactivated NO receptor guanylyl cyclase,30 whereas the latter can be realized by inhibiting phosphodiesterase-5 in the endothelium.31 Activation of guanylyl cyclase has been shown not only to improve endothelial dysfunction, but also to prevent atherosclerosis by impairing foam cell formation.32Ideally, of course, rational combinations of therapies that optimize NO-cGMP pathway activity would be best. With the use of modern combinatorial systems pharmacology approaches, this pathway can be kinetically dissected to determine the key steps that can be inhibited or activated to maximize cGMP production over time, as we have recently reported.33 Such an approach takes the guesswork out of the typically semiempirical approach to polypharmaceutical dosing, maximizing efficacy while minimizing side effects.Clearly, we have come a long way from the early history of the pharmacology of NO donors.34 From the first demonstration of the vascular effects of nitroglycerin and related nitrovasodilators,35 to the recognition that NO is a naturally occurring substance with multiple cardiovascular actions, and now to the identification of naturally occurring genetic variants that modulate NO signaling and cGMP production, we are poised to begin to make rational choices about optimal therapeutic strategies for prevention. The work of Emdin and colleagues14 puts the importance of this key pathway in cardiovascular biology once again in perspective, and highlights its potential as a therapeutic target. This is clearly a potential therapeutic pathway that is well worth revisiting.AcknowledgmentsThe author thanks Rose Cannistraro for expert technical assistance.DisclosuresNone.FootnotesThe opinions expressed in this article are not necessarily those of the editors or of the American Heart Association.Circulation is available at http://circ.ahajournals.org.Correspondence to: Joseph Loscalzo, MD, PhD, Department of Medicine, Brigham and Women’s Hospital, 75 Francis Street, Boston, MA 02115. E-mail [email protected]References1. Anthony J. D’Angelo quotes.BrainyQuote.com, Xplore Inc; 2017. https://www.brainyquote.com/quotes/anthony_j_dangelo_377829. Accessed December 8, 2017.Google Scholar2. Ludmer PL, Selwyn AP, Shook TL, Wayne RR, Mudge GH, Alexander RW, Ganz P. Paradoxical vasoconstriction induced by acetylcholine in atherosclerotic coronary arteries.N Engl J Med. 1986; 315:1046–1051. doi: 10.1056/NEJM198610233151702.CrossrefMedlineGoogle Scholar3. Bossaller C, Habib GB, Yamamoto H, Williams C, Wells S, Henry PD. Impaired muscarinic endothelium-dependent relaxation and cyclic guanosine 5’-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta.J Clin Invest. 1987; 79:170–174. doi: 10.1172/JCI112779.CrossrefMedlineGoogle Scholar4. Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD, Yeung AC, Vekshtein VI, Selwyn AP, Ganz P. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease.N Engl J Med. 1990; 81:491–497.Google Scholar5. Cohen RA. The role of nitric oxide and other endothelium-derived vasoactive substances in vascular disease.Prog Cardiovasc Dis. 1995; 38:105–128.CrossrefMedlineGoogle Scholar6. Freedman JE, Sauter R, Battinelli EM, Ault K, Knowles C, Huang PL, Loscalzo J. Deficient platelet-derived nitric oxide and enhanced hemostasis in mice lacking the NOSIII gene.Circ Res. 1999; 84:1416–1421.LinkGoogle Scholar7. Freedman JE, Loscalzo J, Barnard MR, Alpert C, Keaney JF, Michelson AD. Nitric oxide released from activated platelets inhibits platelet recruitment.J Clin Invest. 1997; 100:350–356. doi: 10.1172/JCI119540.CrossrefMedlineGoogle Scholar8. Freedman JE, Ting B, Hankin B, Loscalzo J, Keaney JF, Vita JA. Impaired platelet production of nitric oxide predicts presence of acute coronary syndromes.Circulation. 1998; 98:1481–1486.LinkGoogle Scholar9. Loscalzo J. Nitric oxide insufficiency, platelet activation, and arterial thrombosis.Circ Res. 2001; 88:756–762.LinkGoogle Scholar10. Hayashi T, Sumi D, Juliet PA, Matsui-Hirai H, Asai-Tanaka Y, Kano H, Fukatsu A, Tsunekawa T, Miyazaki A, Iguchi A, Ignarro LJ. Gene transfer of endothelial NO synthase, but not eNOS plus inducible NOS, regressed atherosclerosis in rabbits.Cardiovasc Res. 2004; 61:339–351.CrossrefMedlineGoogle Scholar11. Wang BY, Ho HK, Lin PS, Schwarzacher SP, Pollman MJ, Gibbons GH, Tsao PS, Cooke JP. Regression of atherosclerosis: role of nitric oxide and apoptosis.Circulation. 1999; 99:1236–1241.LinkGoogle Scholar12. Casas JP, Bautista LE, Humphries SE, Hingorani AD. Endothelial nitric oxide synthase genotype and ischemic heart disease: meta-analysis of 26 studies involving 23028 subjects.Circulation. 2004; 109:1359–1365. doi: 10.1161/01.CIR.0000121357.76910.A3.LinkGoogle Scholar13. Kessler T, Wobst J, Wolf B, Eckhold J, Vilne B, Hollstein R, von Ameln S, Dang TA, Sager HB, Moritz Rumpf P, Aherrahrou R, Kastrati A, Björkegren JLM, Erdmann J, Lusis AJ, Civelek M, Kaiser FJ, Schunkert H. Functional characterization of the GUCY1A3 coronary artery disease risk locus.Circulation. 2017; 136:476–489. doi: 10.1161/CIRCULATIONAHA.116.024152.LinkGoogle Scholar14. Emdin CA, Khera AV, Klarin D, Natarajan P, Zekavat SM, Nomura A, Haas M, Aragam K, Ardissino D, Wilson JG, Schunkert H, McPherson R, Watkins H, Elosua R, Bown MJ, Samani NJ, Baber U, Erdmann J, Gormley P, Palotie A, Stitziel NO, Gupta N, Danesh J, Saleheen D, Gabriel S, Kathiersan S. Phenotypic consequences of a genetic predisposition to enhanced nitric oxide signaling.Circulation. 2018; 137:222–232. doi: 10.1161/CIRCULATIONAHA.117.028021.LinkGoogle Scholar15. Kojda G, Stein D, Kottenberg E, Schnaith EM, Noack E. In vivo effects of pentaerythrityl-tetranitrate and isosorbide-5-mononitrate on the development of atherosclerosis and endothelial dysfunction in cholesterol-fed rabbits.J Cardiovasc Pharmacol. 1995; 25:763–773.CrossrefMedlineGoogle Scholar16. Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview.Circ Res. 2002; 90:21–28.LinkGoogle Scholar17. Herman AG, Moncada S. Therapeutic potential of nitric oxide donors in the prevention and treatment of atherosclerosis.Eur Heart J. 2005; 26:1945–1955. doi: 10.1093/eurheartj/ehi333.CrossrefMedlineGoogle Scholar18. Forstermann U, Xia N, Li H. Roles of oxidative stress and nitric oxide in the pathogenesis of atherosclerosis.Circ Res. 2017; 120:713–735.LinkGoogle Scholar19. Keaney JF, Gaziano JM, Xu A, Frei B, Curran-Celentano J, Shwaery GT, Loscalzo J, Vita JA. Low-dose alpha-tocopherol improves and high-dose alpha-tocopherol worsens endothelial vasodilator function in cholesterol-fed rabbits.J Clin Invest. 1994; 93:844–851. doi: 10.1172/JCI117039.CrossrefMedlineGoogle Scholar20. Handy DE, Loscalzo J. Responses to reductive stress in the cardiovascular system.Free Radic Biol Med. 2017; 109:114–124. doi: 10.1016/j.freeradbiomed.2016.12.006.CrossrefMedlineGoogle Scholar21. Moens AL, Kass DA. Therapeutic potential of tetrahydrobiopterin for treating vascular and cardiac disease.J Cardiovasc Pharmacol. 2007; 50:238–246. doi: 10.1097/FJC.0b013e318123f854.CrossrefMedlineGoogle Scholar22. Antoniades C, Cunnington C, Antonopoulos A, Neville M, Margaritis M, Demosthenous M, Bendall J, Hale A, Cerrato R, Tousoulis D, Bakogiannis C, Marinou K, Toutouza M, Vlachopoulos C, Leeson P, Stefanadis C, Karpe F, Channon KM. Induction of vascular GTP-cyclohydrolase I and endogenous tetrahydrobiopterin synthesis protect against inflammation-induced endothelial dysfunction in human atherosclerosis.Circulation. 2011; 124:1860–1870. doi: 10.1161/CIRCULATIONAHA.111.029272.LinkGoogle Scholar23. Stroes ES, van Faassen EE, Yo M, Martasek P, Boer P, Govers R, Rabelink TJ. Folic acid reverts dysfunction of endothelial nitric oxide synthase.Circ Res. 2000; 86:1129–1134.LinkGoogle Scholar24. Kamada Y, Nagaretani H, Tamura S, Ohama T, Maruyama T, Hiraoka H, Yamashita S, Yamada A, Kiso S, Inui Y, Ito N, Kayanoki Y, Kawata S, Matsuzawa Y. Vascular endothelial dysfunction resulting from L-arginine deficiency in a patient with lysinuric protein intolerance.J Clin Invest. 2001; 108:717–724. doi: 10.1172/JCI11260.CrossrefMedlineGoogle Scholar25. Detmers PA, Hernandez M, Mudgett J, Hassing H, Burton C, Mundt S, Chun S, Fletcher D, Card DJ, Lisnock J, Weikel R, Bergstrom JD, Shevell DE, Hermanowski-Vosatka A, Sparrow CP, Chao YS, Rader DJ, Wright SD, Puré E. Deficiency in inducible nitric oxide synthase results in reduced atherosclerosis in apolipoprotein E-deficient mice.J Immunol. 2000; 165:3430–3435.CrossrefMedlineGoogle Scholar26. Loscalzo J. Adverse effects of supplemental L-arginine in atherosclerosis: consequences of methylation stress in a complex catabolism?Arterioscler Thromb Vasc Biol. 2003; 23:3–5.LinkGoogle Scholar27. Buckwold VE. Stomach synthesis of NO.Nature. 1994; 368:502.CrossrefMedlineGoogle Scholar28. Lundberg JO, Weitzberg E, Cole JA, Benjamin N. Nitrate, bacteria and human health.Nat Rev Microbiol. 2004; 2:593–602. doi: 10.1038/nrmicro929.CrossrefMedlineGoogle Scholar29. Velmurugan S, Gan JM, Rathod KS, Khambata RS, Ghosh SM, Hartley A, Van Eijl S, Sagi-Kiss V, Chowdhury TA, Curtis M, Kuhnle GG, Wade WG, Ahluwalia A. Dietary nitrate improves vascular function in patients with hypercholesterolemia: a randomized, double-blind, placebo-controlled study.Am J Clin Nutr. 2016; 103:25–38. doi: 10.3945/ajcn.115.116244.CrossrefMedlineGoogle Scholar30. Stasch JP, Schmidt PM, Nedvetsky PI, Nedvetskaya TY, H S AK, Meurer S, Deile M, Taye A, Knorr A, Lapp H, Müller H, Turgay Y, Rothkegel C, Tersteegen A, Kemp-Harper B, Müller-Esterl W, Schmidt HH. Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels.J Clin Invest. 2006; 116:2552–2561. doi: 10.1172/JCI28371.CrossrefMedlineGoogle Scholar31. Korkmaz S, Radovits T, Barnucz E, Neugebauer P, Arif R, Hirschberg K, Loganathan S, Seidel B, Karck M, Szabó G. Dose-dependent effects of a selective phosphodiesterase-5-inhibitor on endothelial dysfunction induced by peroxynitrite in rat aorta.Eur J Pharmacol. 2009; 615:155–162. doi: 10.1016/j.ejphar.2009.05.020.CrossrefMedlineGoogle Scholar32. Tsou CY, Chen CY, Zhao JF, Su KH, Lee HT, Lin SJ, Shyue SK, Hsiao SH, Lee TS. Activation of soluble guanylyl cyclase prevents foam cell formation and atherosclerosis.Acta Physiol (Oxf). 2014; 210:799–810. doi: 10.1111/apha.12210.CrossrefMedlineGoogle Scholar33. Garmaroudi FS, Handy DE, Liu YY, Loscalzo J. Systems pharmacology and rational polypharmacy: nitric oxide-cyclic GMP signaling pathway as an illustrative example and derivation of the general case.PLoS Comput Biol. 2016; 12:e1004822. doi: 10.1371/journal.pcbi.1004822.CrossrefMedlineGoogle Scholar34. Steinhorn BS, Loscalzo J, Michel T. Nitroglycerin and nitric oxide–a rondo of themes in cardiovascular therapeutics.N Engl J Med. 2015; 373:277–280. doi: 10.1056/NEJMsr1503311.CrossrefMedlineGoogle Scholar35. Divakaran S, Loscalzo J. The role of nitroglycerin and other nitrogen oxides in cardiovascular therapeutics.J Am Coll Cardiol. 2017; 70:2393–2410. doi: 10.1016/j.jacc.2017.09.1064.CrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Zhang Q, Liu J, Duan H, Li R, Peng W and Wu C (2021) Activation of Nrf2/HO-1 signaling: An important molecular mechanism of herbal medicine in the treatment of atherosclerosis via the protection of vascular endothelial cells from oxidative stress, Journal of Advanced Research, 10.1016/j.jare.2021.06.023, 34, (43-63), Online publication date: 1-Dec-2021. Lee L, Oldham W, He H, Wang R, Mulhern R, Handy D and Loscalzo J (2021) Interferon-γ Impairs Human Coronary Artery Endothelial Glucose Metabolism by Tryptophan Catabolism and Activates Fatty Acid Oxidation, Circulation, 144:20, (1612-1628), Online publication date: 16-Nov-2021. Schirra C, Xia N, Schüffler A, Heck A, Hasselwander S, Förstermann U and Li H (2020) Phosphorylation and activation of endothelial nitric oxide synthase by red fruit (Pandanus conoideus Lam) oil and its fractions, Journal of Ethnopharmacology, 10.1016/j.jep.2019.112534, 251, (112534), Online publication date: 1-Apr-2020. Nagy Z and Senis Y (2019) Platelet Inhibitory Receptors Platelets, 10.1016/B978-0-12-813456-6.00015-1, (279-293), . January 16, 2018Vol 137, Issue 3 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.117.032901PMID: 29335284 Originally publishedJanuary 16, 2018 Keywordsendotheliumcyclic GMPcardiovascular diseasesEditorialsvasodilationnitric oxidePDF download Advertisement SubjectsCardiovascular DiseaseEndothelium/Vascular Type/Nitric Oxide