Correspondence Acknowledgements
Response to Letter Regarding Article, “Dysregulation of Hydrogen Sulfide (H2S) Producing Enzyme Cystathionine γ-lyase (CSE) Contributes to Maternal Hypertension and Placental Abnormalities in Preeclampsia”
We thank Dr Colin Murdoch for his helpful suggestions.
Sources of Funding This work was supported by program grants from the British Heart Foundation (RG/09/001/25940) and Medical Research Council (G0700288). Generous financial support from the High Sheriff for West Midlands Dr Tim Watts (Pertemps) to Aston Medical School’s research program is gratefully appreciated.
We thank Drs Tsikas and Cooper for their interest in our recent publication on plasma H2S reduction in preeclampsia (Figure 1A1). They questioned the merit of the widely used methylene blue assay to measure plasma H2S levels. We agree that this assay may overestimate the amount of free H2S present in plasma.2 However, the experimental design in our study investigated the relative change in plasma H2S levels between normal pregnancy and preeclampsia. Furthermore, the reduction in plasma H2S in preeclampsia compared with normal pregnancy was accompanied by decreased CSE mRNA and protein expression (Figure 11). Therefore, 3 independent observations (RNA, protein, activity) provide compelling evidence of a net loss of H2S activity in preeclampsia. The cytoprotective pathway of heme oxygenase-1 and cystathionine γ-lyase (CSE), which generate carbon monoxide (CO) and hydrogen sulfide (H2S), respectively, hold promise for preeclampsia therapy because of their unique ability to suppress the antiangiogenic state, systemic inflammation, and oxidative stress.1,3 We respectfully disagree with the suggestion that because an overall decrease in total reactive sulfur maybe modest, this is inconsistent with the H2S/CSE role in preeclampsia. The changes in H2S levels between preeclampsia and normal pregnancy were significant (Figure 1A).1 More importantly, a reduction of H2S in pregnant mice administered DL-propargylglycine to inhibit CSE activity, resulted in hypertension, liver damage, and abnormal labyrinth vascularization in the placenta, leading to fetal growth restriction and preeclampsia-like features.1 As is true with other labile gases, the focus needs to remain on the biological relevance. In agreement with our results, Shen and colleagues demonstrated that free H2S levels and acid-labile sulfide in CSE deficient (CSE−/−) mice were reduced compared with wildtype animals (see Figure 52). The acid labile pool was in the low micromolar range and likely liberated and detected by the methylene blue method along with free H2S as was the case in our study. The change, whether modest or large, is associated with the CSE−/− mouse exhibiting abnormal vascular phenotype.4 Consistent with our notion of loss of CSE as a pathogenic mechanism, supplementation with H2S reverses abnormalities in the mouse, suggesting therapeutic potential.1 The importance of H2S in preventing damage is further supported by a recent study showing that H2S attenuates adenovirusmediated overexpression of soluble Flt-1–induced hypertension and renal damage in nonpregnant Sprague-Dawley rats.5 Further transgenic studies are needed to delineate the relative importance of placental- and endothelial-specific knockdown of CSE. A more precise determination of the distribution of H2S in its various biochemical forms using advanced analytic methods2 is underway on well-defined longitudinal clinical samples in collaboration with a leading H2S analytic scientist. Absolute values are important in studying the physiology of endogenous H2S and in determining the accurate dose of H2S donors for biological systems. Any future pharmacological safety studies on H2S donors in pregnant women will have to precisely measure H2S levels before the Food and Drug Administration could approve a clinical trial. There is therefore a need to develop high-throughput methods to accurately measure the labile gases in biological fluids. In conclusion, our studies show that the restoration of effective endogenous cytoprotective pathway1,3 now offers a new paradigm to resolve preeclampsia and protect mother and baby.
Disclosures None. Keqing Wang, MD, PhD Shakil Ahmad, PhD Meng Cai, PhD Vascular Medicine Unit, Aston Medical School Aston University Birmingham, UK Jillian Rennie, BSc Takeshi Fujisawa, PhD Gustav Born Centre for Vascular Biology and BHF Centre for Cardiovascular Sciences University of Edinburgh Edinburgh, UK Fatima Crispi, MD, PhD Department of Maternal-Fetal Medicine University of Barcelona Barcelona, Spain James Baily, MD Mark R. Miller, PhD Melissa Cudmore, PhD Patrick W. F. Hadoke, PhD Gustav Born Centre for Vascular Biology and BHF Centre for Cardiovascular Sciences University of Edinburgh Edinburgh, UK Rui Wang, MD Lakehead University Thunder Bay, Ontario, Canada Eduard Gratacós, MD Department of Maternal-Fetal Medicine University of Barcelona Barcelona, Spain Irina A. Buhimschi, MD Catalin S. Buhimschi, MD Department of Obstetrics, Gynecology and Reproductive Science Yale School of Medicine, Yale University New Haven, Connecticut Asif Ahmed, PhD Vascular Medicine Unit, Aston Medical School Aston University Birmingham, UK
(Circulation. 2014;129:e517-e518.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.114.010273
e517
e518 Circulation June 3, 2014
References 1. Wang K, Ahmad S, Cai M, Rennie J, Fujisawa T, Crispi F, Baily J, Miller MR, Cudmore M, Hadoke PW, Wang R, Gratacós E, Buhimschi IA, Buhimschi CS, Ahmed A. Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia. Circulation. 2013;127:2514–2522. 2. Shen X, Peter EA, Bir S, Wang R, Kevil CG. Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radic Biol Med. 2012;52:2276–2283. 3. Cudmore M, Ahmad S, Al-Ani B, Fujisawa T, Coxall H, Chudasama K, Devey LR, Wigmore SJ, Abbas A, Hewett PW, Ahmed A. Negative
regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation. 2007;115:1789–1797. 4. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, Snyder SH, Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science. 2008;322:587–590. 5. Holwerda KM, Burke SD, Faas MM, Zsengeller Z, Stillman IE, Kang PM, van Goor H, McCurley A, Jaffe IZ, Karumanchi SA, Lely AT. Hydrogen sulfide attenuates sFlt1-induced hypertension and renal damage by upregulating vascular endothelial growth factor. J Am Soc Nephrol. 2014;25:717–725.
Response to Letter Regarding Article, “Dysregulation of Hydrogen Sulfide (H2S) Producing Enzyme Cystathionine γ-lyase (CSE) Contributes to Maternal Hypertension and Placental Abnormalities in Preeclampsia”
We thank Dr Colin Murdoch for his helpful suggestions.
Sources of Funding This work was supported by program grants from the British Heart Foundation (RG/09/001/25940) and Medical Research Council (G0700288). Generous financial support from the High Sheriff for West Midlands Dr Tim Watts (Pertemps) to Aston Medical School’s research program is gratefully appreciated.
We thank Drs Tsikas and Cooper for their interest in our recent publication on plasma H2S reduction in preeclampsia (Figure 1A1). They questioned the merit of the widely used methylene blue assay to measure plasma H2S levels. We agree that this assay may overestimate the amount of free H2S present in plasma.2 However, the experimental design in our study investigated the relative change in plasma H2S levels between normal pregnancy and preeclampsia. Furthermore, the reduction in plasma H2S in preeclampsia compared with normal pregnancy was accompanied by decreased CSE mRNA and protein expression (Figure 11). Therefore, 3 independent observations (RNA, protein, activity) provide compelling evidence of a net loss of H2S activity in preeclampsia. The cytoprotective pathway of heme oxygenase-1 and cystathionine γ-lyase (CSE), which generate carbon monoxide (CO) and hydrogen sulfide (H2S), respectively, hold promise for preeclampsia therapy because of their unique ability to suppress the antiangiogenic state, systemic inflammation, and oxidative stress.1,3 We respectfully disagree with the suggestion that because an overall decrease in total reactive sulfur maybe modest, this is inconsistent with the H2S/CSE role in preeclampsia. The changes in H2S levels between preeclampsia and normal pregnancy were significant (Figure 1A).1 More importantly, a reduction of H2S in pregnant mice administered DL-propargylglycine to inhibit CSE activity, resulted in hypertension, liver damage, and abnormal labyrinth vascularization in the placenta, leading to fetal growth restriction and preeclampsia-like features.1 As is true with other labile gases, the focus needs to remain on the biological relevance. In agreement with our results, Shen and colleagues demonstrated that free H2S levels and acid-labile sulfide in CSE deficient (CSE−/−) mice were reduced compared with wildtype animals (see Figure 52). The acid labile pool was in the low micromolar range and likely liberated and detected by the methylene blue method along with free H2S as was the case in our study. The change, whether modest or large, is associated with the CSE−/− mouse exhibiting abnormal vascular phenotype.4 Consistent with our notion of loss of CSE as a pathogenic mechanism, supplementation with H2S reverses abnormalities in the mouse, suggesting therapeutic potential.1 The importance of H2S in preventing damage is further supported by a recent study showing that H2S attenuates adenovirusmediated overexpression of soluble Flt-1–induced hypertension and renal damage in nonpregnant Sprague-Dawley rats.5 Further transgenic studies are needed to delineate the relative importance of placental- and endothelial-specific knockdown of CSE. A more precise determination of the distribution of H2S in its various biochemical forms using advanced analytic methods2 is underway on well-defined longitudinal clinical samples in collaboration with a leading H2S analytic scientist. Absolute values are important in studying the physiology of endogenous H2S and in determining the accurate dose of H2S donors for biological systems. Any future pharmacological safety studies on H2S donors in pregnant women will have to precisely measure H2S levels before the Food and Drug Administration could approve a clinical trial. There is therefore a need to develop high-throughput methods to accurately measure the labile gases in biological fluids. In conclusion, our studies show that the restoration of effective endogenous cytoprotective pathway1,3 now offers a new paradigm to resolve preeclampsia and protect mother and baby.
Disclosures None. Keqing Wang, MD, PhD Shakil Ahmad, PhD Meng Cai, PhD Vascular Medicine Unit, Aston Medical School Aston University Birmingham, UK Jillian Rennie, BSc Takeshi Fujisawa, PhD Gustav Born Centre for Vascular Biology and BHF Centre for Cardiovascular Sciences University of Edinburgh Edinburgh, UK Fatima Crispi, MD, PhD Department of Maternal-Fetal Medicine University of Barcelona Barcelona, Spain James Baily, MD Mark R. Miller, PhD Melissa Cudmore, PhD Patrick W. F. Hadoke, PhD Gustav Born Centre for Vascular Biology and BHF Centre for Cardiovascular Sciences University of Edinburgh Edinburgh, UK Rui Wang, MD Lakehead University Thunder Bay, Ontario, Canada Eduard Gratacós, MD Department of Maternal-Fetal Medicine University of Barcelona Barcelona, Spain Irina A. Buhimschi, MD Catalin S. Buhimschi, MD Department of Obstetrics, Gynecology and Reproductive Science Yale School of Medicine, Yale University New Haven, Connecticut Asif Ahmed, PhD Vascular Medicine Unit, Aston Medical School Aston University Birmingham, UK
(Circulation. 2014;129:e517-e518.) © 2014 American Heart Association, Inc. Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.114.010273
e517
e518 Circulation June 3, 2014
References 1. Wang K, Ahmad S, Cai M, Rennie J, Fujisawa T, Crispi F, Baily J, Miller MR, Cudmore M, Hadoke PW, Wang R, Gratacós E, Buhimschi IA, Buhimschi CS, Ahmed A. Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia. Circulation. 2013;127:2514–2522. 2. Shen X, Peter EA, Bir S, Wang R, Kevil CG. Analytical measurement of discrete hydrogen sulfide pools in biological specimens. Free Radic Biol Med. 2012;52:2276–2283. 3. Cudmore M, Ahmad S, Al-Ani B, Fujisawa T, Coxall H, Chudasama K, Devey LR, Wigmore SJ, Abbas A, Hewett PW, Ahmed A. Negative
regulation of soluble Flt-1 and soluble endoglin release by heme oxygenase-1. Circulation. 2007;115:1789–1797. 4. Yang G, Wu L, Jiang B, Yang W, Qi J, Cao K, Meng Q, Mustafa AK, Mu W, Zhang S, Snyder SH, Wang R. H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase. Science. 2008;322:587–590. 5. Holwerda KM, Burke SD, Faas MM, Zsengeller Z, Stillman IE, Kang PM, van Goor H, McCurley A, Jaffe IZ, Karumanchi SA, Lely AT. Hydrogen sulfide attenuates sFlt1-induced hypertension and renal damage by upregulating vascular endothelial growth factor. J Am Soc Nephrol. 2014;25:717–725.
