A “cure” for preeclampsia: Improving neonatal outcomes by overcoming excess fetal placental vascular resistance

Accepted Manuscript A “cure” for preeclampsia; improving neonatal outcomes by overcoming excess fetal placental vascular resistance T.J. Byrne PII: DOI: Reference:

S0306-9877(15)00220-0 http://dx.doi.org/10.1016/j.mehy.2015.06.001 YMEHY 7943

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Medical Hypotheses

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23 April 2014 2 June 2015

Please cite this article as: T.J. Byrne, A “cure” for preeclampsia; improving neonatal outcomes by overcoming excess fetal placental vascular resistance, Medical Hypotheses (2015), doi: http://dx.doi.org/10.1016/j.mehy. 2015.06.001

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A “CURE” FOR PREECLAMPSIA; IMPROVING NEONATAL OUTCOMES BY OVERCOMING EXCESS FETAL PLACENTAL VASCULAR RESISTANCE TJ Byrne M.D. Director of Maternal Fetal Medicine Harlem Hospital 506 Lenox Avenue New York, New York, USA 10037 Thomas J. Byrne M.D. 4761 Broadway Apt 3 Z NewYork,New York, USA 10034 phone +1-405-397-5500 fax +1 -212-939-4501 [email protected] No supporting grants

A “CURE” FOR PREECLAMPSIA; IMPROVING NEONATAL OUTCOMES BY OVERCOMING EXCESS FETAL PLACENTAL VASCULAR RESISTANCE 1

ABSTRACT From a broad perspective there are only three arterial systems that respond to relative hypoxia with vasoconstriction. They are the placental, the pulmonic and the renal vascular beds. The renal system's adaptation to hypoxia is markedly different from the other two circulatory beds and will not be further considered here. Regional vasoconstriction is adaptive in the placenta and lung because it redirects red blood cells from areas of relative hypoxia to more oxygenated areas thereby maximizing oxygen uptake for a given cardiac output. The fetal placental and pulmonary vascular systems are unique because their smooth muscle cells have a unique and possibly identical potassium channel that responds to hypoxia by closing, thereby depolarizing the cell membrane allowing calcium ion influx and muscle contraction. It may be that a variety of initial causes of temporary or local placental hypoxia initiate a cascade of first fetal placental then maternal vasoconstriction and endothelial activation leading to the clinical syndrome we call preeclampsia. The response cascades seen in preeclampsia, which for purposes of this article I will abbreviate as (PECL), after development of widespread vasoconstriction, will also be seen to be identical or at least parallel in pulmonary hypertension (PAH). This means that some or all of the pharmacotherapies presently used, tested or considered in early PAH may also have a therapeutic effect in PECL by reducing fetal placental arterial resistance thereby increasing fetal placental flow. This would allow increased oxygen and other nutrient uptake and possibly increased fetal cardiac output in the face of reduced fetal cardiac work. This may allow a delay in delivery in which fetuses grow and are better oxygenated in preterm PECL, improving neonatal outcomes.

SUMMARY The etiology of preeclampsia has remained an enigma for the entire history of medicine. Treatment of preeclampsia has remained unchanged and has not advanced for two generations. The pharmacotherapy of this disease process has focused on minimizing the maternal effect of it's pathophysiology largely through control of maternal blood pressure with beta blockers. Beta blocker cause growth restriction in the fetus but do protect the mother from more serious morbidity. It may be that we have not developed more effective treatment of this disease process because we have not considered the likely origin of at least part of this disease process, the over reactive constriction of fetal placental resistance vessels in reaction to relative reduced local oxygen availability, leading to eventual fetal heart failure manifested by the well documented sequence of Doppler changes seen in fetuses with this disease process. There has been a dearth of effective treatment for preeclampsia because although there are multiple animal models ,there are none are in our primate line because of expense. The non-primate models probably do not completely mimic all aspects of PECL because of a 60 to 90 million year hereditary separation. For instance there was a gene expansion in Luteinizing Hormone (LH) subunits and probably other undiscovered changes in pregnancy biology in our primate lineage. Also the animal models do not walk upright with all the changes in blood pressure control this requires, do not have primarily singleton pregnancies and are born vastly more physiologically mature than humans. All of these apparent and probably some non-apparent changes limit their applicability in humans. There is however a similar disease that has been well studied and whose pathophysiology on a molecular basis has been increasingly well understood in both animal models and patients. It is my proposal that the fetal placental circulation in preeclampsia (PECL) and the arterial circulation in early pulmonary hypertension (PAH) have parallel or identical signaling and further that because of this identical or parallel response there should 2

be fetal improvement in PECL, at least temporarily, in response to some of the plethora of drugs being successfully used to treat PAH. Temporary improvement, even say two or three weeks of continued growth, in all early preeclamptics and even a week in later pregnancy would markedly improve neonatal outcomes.

HYPOTHESIS A large part of the pathophysiology of preeclampsia, which up to this point has been ignored or which clinicians are presently unaware of, involves excess fetal placental arterial resistance leading to eventual fetal heart failure. A close or identical model of this excess resistance is the resistance arterial vessel in early pulmonary hypertension. There are potassium channel signaling systems present in both pulmonary and fetal placental resistance arteries which respond identically to hypoxia with vasoconstriction. This is the opposite of the response in all other vascular beds. After the primary identical response to local hypoxia, the continued response cascades in the two disease processes remain identical or parallel. Treatment of this excess resistance in preeclampsia with medications, presently used for early pulmonary hypertension, that reduce placental resistance, will improve fetal outcome in preeclampsia by increasing fetal placental blood flow, reducing fetal cardiac work and will maximize oxygen and other nutrient uptake in the fetus.

RESPONSE TO HYPOXIA IN THE PLACENTAL AND PULMONARY VASCULATURE There has been great progress in understanding the pathophysiology of pulmonary hypertension. It is known that human pulmonary endothelial cells rapidly increase their intracellular Ca++ levels in response to hypoxia.1 This same response has been noted in human umbilical vein endothelial cells. 2 The mechanism of increased vascular resistance in the face of hypoxia in the pulmonary vascular tree has been discovered to be oxygen sensing potassium channels in smooth muscle cells of pulmonary artery resistance vessels. 3 4 5 After these potassium channels are blocked depolarization occurs and then Ca++ is allowed to ingress into cells and probably is also released from sarcoplasmic reticulum. There is a recently discovered parallel or identical potassium channel response system in placental resistance 1

2 3 4 5

Arnould,T,Michiels,C,Alexandre,I,Remacle,J. . Effect of hypoxia upon intracellular calcium concentration of human endothelial cells. J. Cell. Physiol. (1991)152( 1 ):215-221. Aono,Y,Ariyoshi,H,Sakon,M,Ueda,A,Tsuji,Y,Kawasaki,T,Monden,M. (2000). Human umbilical vein endothelial cells (HUVECS) show Ca(2+) mobilization as well as Ca(2+) influx upon hypoxia. J. Cell Biochem. (2000) 78(3): 458-464 Beech,DJ:. Ion channel switching and activation in smooth-muscle cells of occlusive vascular diseases. Biochem. Soc. Trans. (2007) 35(5)890-894. Smirnov,SV,Beck,R,Tammaro,P,Ishii,T,Aaronson,PI. Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries. Jour. Physiol.(2002) 538(3): 867-878. Shimoda, LA,Polak,J. Hypoxia.4.Hypoxia and ion channel function. Am. J. Physiol Cell Physiol.(2010) 300(5):C951C967.

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vessels.6 This in normal circumstances maximizes the transfer of oxygen across the placental and pulmonary capillary beds by directing blood from relative hypoxic areas to better oxygenated areas. In preeclampsia and pulmonary hypertension this signaling system is overactive leading to increased total vascular resistance in both capillary beds and to reduced blood flow and increased cardiac work through increased total pulmonic and placental vascular resistance. Exactly how hypoxia blocks these channels leading to membrane depolarization, increased calcium ion influx and increased release of calcium from the endoplasmic reticulum is not completely understood in either system but the increased intracellular free calcium ions lead to muscle contraction in vascular smooth muscles. The K+ and Ca++ channels may both be amenable to alteration from pharmacotherapy. There is a lot of animal experimental and human clinical molecular research in pulmonary hypertension but, since there is not an animal model that completely parallels preeclampsia, evidence is fragmentary and animal models usually only invoke some of the responses seen in human preeclampsia. Human trials mostly involve examining peripheral blood changes seen during the disease process or pathologic examination after the disease process. There is a relatively newly described animal model of PECL which may lend itself to use of therapeutic agents presently used with success in PAH.7 The complete mechanism of the pathophysiology of either process is not known but there is now evidence of PAH and PECL evolving in parallel fashion when comparing known animal models of PAH with evidence of the same process in humans with PAH and PECL. It is well established in multiple studies that there is an increased incidence of PECL in patients who live at high altitude. 8 There is also an increased incidence of PAH in patients who live at high altitude.9 So a decrease in ambient alveolar oxygen in PAH and a small decrease in the venous dissolved oxygen in PECL both predispose to development of the two respective diseases. There are multiple animal models of PAH. All the models but one rely on two stages of stress to induce pulmonary hypertension. In one model there is injection of a vascular irritant, such as monocratline, a toxic plant alkaloid that causes pulmonary endothelial inflammation, and then after placement in a relatively hypoxic environment most animals quickly develop PAH and die of heart failure if untreated.10 Another model is removal of one lung and then treatment with SU 4516, a blocker of the vascular endothelial growth factor (VEGF) receptor, and then placement in a hypoxic environment.11,12 Another interesting model to believers in immune system malfunction in preeclampsia is the treatment of rats already genetically deficient in T cell type 2 cells (T2) with SU4516.13 In this model rats with a genetic 6 Brereton,MF,Wareing,M,Jones,RL,Greenwood,SL. Characterization of K+ channels in human fetoplacental vascular smooth muscle cells. PLOSONE.ORG (2013) 8(2)1-10. 7 LI,J,LaMarca,B,Reckelhoff,JF. A model of preeclampsia in rats: the reduced perfusion pressure (RUPP) model. Am. J. Physiol. Heart Circ. Physiol.(2012) 303( 1 ): H1-H8. 8 Palmer,SK,Moore,LG,Young,D,Cregger,B,Berman,JC,Zamudio,S. Altered blood pressure course during normal pregnancy and increased preeclampsia at high altitude (3100 meters) in Colorado. Am. J. Obstet. Gynecol.(1999) 180(5): 1161-1168. 9 Penaloza,D Effects of high-altitude exposure on the pulmonary circulation. Cardiovascular.2012) 65(12): 1075-1078. 10 Saltier,RJ,Olson,JW,Gillespie,MN. Altered pulmonary vascular smooth muscle responsiveness in momocratlineinduced pulmonary hypertension. J Pharmacol.Exp.Ther. (1986)236(2):390-395. 11 Fong,TA,Shawver,LK,Sun,L,et.al.SU4516 is a potent and selective inhibitor of the vascular endothelial growth fact receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis,tumor vascularization ,and growth of multiple tumor types. Cancer Res.(1999)59( 1 ):99-106 12 Sakao,S,Tatsumi,K The effects of antiangiogenetic compound SU 4516 in a rat model of pulmonary arterial hypertension. Resp.(2011) 81(3)::253-261. 13 Taraseviciene-Stewart,L,Nicolis,MR,Kraskauskas,D,et.al. Absence of T cells confers increased pulmonary arterial

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lack of T2 cells develop pulmonary hypertension after exposure to SU4516 without a required exposure to hypoxia. In this same model, reconstitution of the deficient T2 cells leads to prevention of PAH.14 B cell depletion in this same model also prevented PAH.15 There are several pathways in these models that become abnormal or that have been altered to study the effect in these models 16 The following are synopses of the study of the primary vascular signalers and responses to these signals in animal models and human patients with pulmonary hypertension and a comparison to the same signals in preeclampsia. Because of our unique nature: hemochorial placenta, usually singular pregnancy, upright posture with it's effect on our blood pressure controlling system both during and after gestation, relatively large fetal/maternal carcass ratio and our relative immaturity at birth, among other possible and undefined factors, it has been difficult to design an animal model that completely mimics human preeclampsia, we are left with comparison of humans with or being screened for PECL with relatively well defined animal models of PAH and patients with PAH. We now will look at several facets of PAH and PECL that show a parallel response cascades in the two disease processes. First we will look at effectors of angiogenesis, then vascular signaling peptides, then vascular signaling cascades and a final miscellaneous category. Although I have separated these response cascades into some categories it is appropriate to say here that they all interract in ways outside of their categories and more importantly they all must interact on the intracellular level. The effectors of angiogenesis are; Transforming growth factor - beta (TGF-b), Bone morphogenetic protein (BMP), endoglin, vascular endothelial growth factor (VEGF), soluble FMS-like tyrosine kinase -1(s Flt1) and placental growth factor (PlGF). The vascular signaling peptides are the endothelins and the natriuretic peptides. The endothelial response modulators are NO, thromboxane and prostacyclin. The vascular control cascades are Rho- associated protein kinase (ROCK), cyclic GMP phosphodiesterases and renin-angiotensis-aldosterone system (RAAS). Finally there is a short discussion about uric acid. EFFECTORS OF ANGIOGENESIS These factors work mostly locally although there are some distal effects Transforming growth factor beta (TGF-b) This molecule is from a large super family of signaling molecules that have homologous structures, share some of the same receptors and have differing affinities for each others receptors. Family memberscan have the opposite effect on vascular growth;such as BMP causing the invasion and development of new vessels and TGF-b stopping this formation. Both work through signaling pathways that have some common denominators but which lead to alternate transcription effects at the nuclear level. TGF-b blockade prevents development of hypertension in animal PAH models.17 18Blockade of TGF-b by antibodies or blockade of it's receptor prevents or ameliorates PAH in animal models. hypertension and vascular remodeling. Am J. Respir. Crit. Care Med. (2007)175(12):1280-1289. 14 Tamosiuniene,R,Tian,W,Dhillon,G,et.al. Regulatory T cells limit vascular endothelial injury and prevent pulmonary hypertension. Circ. Res.(2011) 109(8):867-879. 15 Mizuno,S,Farkas,L,Al Husseini,A,et.al. Severe pulmonary hypertension induced by SU 4516 and ovalbumin immunization. Am. J. Respir. Cell Mol. Biol.(2012) 47(5):679-687. 16 Nicolls, MR,Mizuno,S,Taraseviciene-Stewart,L, et.al. New models of pulmonary hypertension based on VEGF receptor blockade-induced endothelial cell apotosis. Pulm. Circ.(2012) 2(4): 434-442. 17 Megalou,AJ, Glave,C, Vilaeti,AD,et.al. Transforming growth factor-b inhibition and endothelin receptor blockade in rats with monocratline-induced pulmonary hypertension. Pulm. Circ. (2012)2(4):461-469. 18 Goumans,MJ,Liu,Z,ten Dijke,P. TGF-b signaling in vascular biology and dysfunction. Cell. Res. (2009)19( 1 ):116127.

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Increased signaling in the TGF-b pathway leading to more effect is associated with higher pulmonary pressures.19 In normal pregnancy there is increased expression of TGF-b3 early in pregnancy which drops around the 9th week when trophoblastic invasion starts in earnest. Blockade of TGF-b3 expression by antisense ribonucleic acids (RNA)s leads to increased growth of trophoblastic tissue in culture.20 TGF-b is produced by the cytotrophoblast. 21 In human fetuses from patients with PECL the TGF-b levels are markedly higher than in fetuses in normal pregnancy especially if the affected fetus has growth restriction. 22 Higher levels of TGF-b are found after the first trimester in women with PECL and in those destined to develop it later.23 24 There is a definite correlation with higher maternal serum TGF-b levels and HELLP syndrome.25 This has been demonstrated in high-risk cohorts.26 Bone morphogenetic protein (BMP) BMPs are members of the same signaling family as the transforming growth factor betas but they have the opposite biologic effects. BMP 2 and 4 cause growth of new blood vessels. The function of BMPs in mammalian reproduction has been recently reviewed.27 Levels of some BMPs are decreased in both animal models and patients with clinical PAH.28 29 30 BMP levels have not been reported in PECL. The BMP s are a large family of signals, some with opposing effects and all with incompletely undefined effects in PECL. Further work is needed on this interesting signal family.

Endoglin Endoglin is a receptor for BMP.31 There is an increased level of the soluble form of endoglin present in 19 Graham,BB,Chabon,J,Gebreab,L,et.al.. Transforming growth factor-b signaling promotes pulmonary hypertension caused by schistosoma mansoni. Circ.(2013) 128(12):1354-1364. 20 Caniggia,I,Grisaru-Gravnosky,S,Kuliszewsky,.,Post,M,Lye,SJ. Inhibition of TGF-b3 restores the invasive capability of extravillous trophoblasts in preeclamptic pregnancies. J.Clin.Invest.1999) 103(2): 1641-1650. 21 Graham,CH,Lysiak,JJ,McCrae,KR,Lala,PK. Localization of transforming growth factor -beta at the human fetalmaternal interface: role in trophoblast growth and differentiation..Biol. Reprod.(1991) 46(4): 561-572. 22 Briana,DD,LLiosi,S, Gourgiotis,D,et.al. Fetal concentration of the growth factors TGF-b and TGF-b1 in relation to normal and restricted fetal growth at term. Cytokine (2012)60( 1 ): 157-161. 23 Peracoli,MT,Menegon,FT,Borges,VT,de Araujo Costa,RA,Thomazi-Santos,IA, Peracoli,JC. Platelet aggregation and TGF-beta(1) plasma levels in pregnant women with preeclampsia. J. Reprod. Immunol. (2008)79 ( 1 ): 79-84. 24 Muy-Rivera,MM,Sanchez,SE,Vadachkoria,S,Qiu,C,Bazul,V,Williams,MA. Transforming growth factor-beta (TGF b-1) in plasma is associated with preeclampsia risk in Peruvian women with systemic inflammation. Am. J. Hyper.(2002) 17(4):334-338. 25 Emanueli,M,Giannubilo,SR,Landi,B,et.al. Placental over expression of transforming growth factor-b3 in the HELLP syndrome. Gynecol. Obstet. Invest. (2008)65( 1 ): 1-5 26 Maynard,SE,Moore Simas,TA,Bur,L,Crawford,SL,Slitro,MJ,Meyer,BA. Soluble endoglin for the prediction of preeclampsia in a high risk cohort. Hypertens. Pregnancy.(2010) 29(3): 330-341. 27 Shiamsaki,S,Moore,RK,Otsuka,F,Erickson,GF. The bone morphogenetic protein system in mammalian reproduction. Endocrin. Rev. (2004)25( 1 ): 2003-2007. 28 Long,L,Crosby,A,Yang, X,Southwood,M,Upton,PD,Kim,DK,Morell,NW. Altered bone morphogenetic protein and transforming growth factor-beta signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circ. (2009)119(4):566-576. 29 Loyd,JE,Phillips,JA. Heritable pulmonary arterial hypertension. Gene Reviews (Internet)(2002) seen April 2014 30 Morell,NW,Yang,XY,Upton,PD,et.al. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor b-1 and bone morphogenetic proteins. Circ.(2001) 104(5): 790-795. 31 Barbara,NP,Wrana,JL,Letarte,M. Endoglin is an accessory protein that interacts with the signaling receptor complex

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serum from preeclamptics. The soluble endoglin effectively reduces the cellular effect of circulating BMP.32 This has been verified on multiple occasions and is being considered as a screening test for PECL.3334 So in animal models and human clinical cases elevated TGF-b and decreased effective BMP are associated with both PECL and PAH. The primary source of TGF-b is the cytotrophoblast. There is little information on BMPs or endoglin levels in fetuses. Vascular endothelial growth factor (VEGF) VEGF is a secreted signal which increases vascular endothelial mitosis and new blood vessel growth.35 There are now animal models of PAH involving the blockade of VEGF to start the cascade of PAH after first performing removal of one lung. In another model, treatment of rats with extra VEGF, delivered by a viral vector prevented the development of PAH. 36 Treatment of newborn rats with SU 4516, a blocker of the VEGF receptor, even without hypoxia causes the development of PAH.37 Once pulmonary hypertension develops in humans levels of VEGF are elevated. 3839 This is a physiological response to try to develop more pulmonary vessels and thus reduce the pressure in each one. In human studies of PECL patients have increasing levels of VEGF as they develop blood pressure elevation compared to normal cohorts.40 Soluble fms like tyrosine kinase 1 (s Flt-1) There are many reports now of increased VEGF concentrations in preeclampsia but less cellular effect probably because of high levels of a soluble VEGF receptor sFlt-1. 4142 At a given blood pressure in mid-gestation the higher the sFlt-1 level the greater the risk of developing PECL by term. 43 Placental of multiple members of the transforming growth factor-beta super family. J.Biol.Chem.1999) 274(@): 584-594. 32 Castonguay,R,Werenr,ED, Matthews,RG,et.al. Soluble endoglin specifically binds bone morphogenetic proteins 9 and 10 via its orphan domain,inhibits blood vessel formation,and suppresses tumor growth. J. Biol. Chem.(2011) 286(34): 30034-30046. 33 Chen,Y. Novel angiogenic factors for predicting preeclampsia: sFlt1,PIGF, and soluble endoglin. Open Clin. Chem. Jour.(2009) 2:1-6. 34 Benian,A,Madazi,R,Aksu,F,Uzun,H,Aydin,S. Plasma and placental levels of interleukin-10,transforming growth factro-b1, and epithelial-cadherin in preeclmapsia. Obstet. Gynecol. (2002)100(2): 327-331. 35 Leung,DW,Cachianes,G,Kuang,WJ,Goeddel,DV,Ferrara,N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science (1989)246(4935): 1306-1309. 36 Farkas,L,Farkas,D,Ask,K,et.al.. VEGF ameliorates pulmonary hypertension through inhibition of endothelial apotosis in experimental lung fibrosis in rats. J. Clin. Invest. (2009)119(5): 1298-1311.. 37 LeCras,TD,Markham,NE,Tuder,RM,Voelkel,NF,Abman,SH. Treatment of rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure. Am.J.Physiol.Lung Cell. Mol. Physiol.(2002)28(3): L555-L562. 38 Papaioannou,AI,Zakynthinos,E,Kostikas,K,et.al. Serum VEGF levels are related to the presence of pulmonary arterial hypertension in systemic sclerosis. BMC Pulm. Med.(2009) 9(18): doi 10.1186/1471-2466-9-18. 39 Shehata,SMK,Mooi,Wj Okasaki,T, El-Banna,I, Sharma,HS,Tibboel,D. Enhanced expression of vascular endothelial growth factor in lungs of newborn infants with congenital diaphragmatic hernia and pulmonary hypertension. Thorax (1999)54(5):427-431. 40 Hunter,A,Aitkenhead,M,Caldwell,C,McCracken,G,Wilson,D,McClure,N. Serum levels of vascular endothelial growth factor in preeclamptic and normotensive pregnancy.. Hyper. (2000) 36(6): 965-969. 41 Maynard,SE,Min,JY,Merchan,J,et.al. Excess placental soluble fms- like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension,and proteinuria in preeclampsia. J. Clin. Invest. (2003) 111(5): 649-658. 42 Levine,RJ, Maynard,SE,Qian,C,et.al. Circulating angiogenic factors and the risk of preeclampsia. New Eng. J. Med. (2004)350(7): 672-683. 43 Levine,RJ,Qian,C,Maynard,SE,YU,KF,Epstein,FH,Karumanchi,SA. Serum s Flt-1 concentration during preeclampsia and mid trimester blood pressure in healthy nulliparous women. Am. J. Obstet. Gynecol.(2006) 194($): 1034-1041.

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expression of messenger RNA for VEGF is markedly increased in preeclamptics probably because the effect of VEGF is lessened due to the soluble receptor preventing it's effect upon cells.44 There is a gradient present in the circulation with respect to the maternal and fetal levels of free VEGF. The maternal level of total VEGF is more but there is more sFlt-1 so the free level of VEGF is higher in the fetus.45 The absolute levels in both mothers and their neonates corresponded to their altitude, with higher altitudes associated with higher VEGF levels. So there is a parallel between animal models of PAH in which VEGF is blocked experimentally and preeclampsia in which a soluble receptor for VEGF effectively blocks it's tissue effect before the development of clinical disease.

Placental derived growth factor (PlGF) PlGF is a placental derived growth factor in the VEGF superfamily. It is produced by placentas but also by other tissues. It is an important signaling molecule involved in producing new vascular growth and is produced in large amounts by syncytiotrophoblasts in human pregnancy.46 Patients with preeclampsia have lower levels of PlGF lower than normal controls. 47 This has been repeated in multiple studies.48 49 Lower levels of PlGF presumably lead to lower production of new decidual bed blood vessels. In hypoxic pulmonary hypertension PlGF causes new blood vessel growth.50 Knockout mice with absent PlGF develop pulmonary hypertension in response to a relatively small reduction in ambient 02.51 After patients with sickle cell disease develop pulmonary hypertension there is general agreement that PlGF levels are elevated, presumably in an attempt to try to develop new blood vessels and bypass obstructed ones. This parallels the elevation of VEGF in PAH after the development of disease. So in both PECL and PAH parallel developments occur in relation to PlGF and clinical disease. Low levels of PlGF are seen in the onset of both disease processes.

TABLE 1 SUMMARY OF EFFECTORS OF ANGIOGENESIS IN PECL AND PAH

VASCULAR SIGNALING PEPTIDES

44 Zhou,Q,Liu,H,Qiao,F,Wu,Y,Xu,J.. VEGF deficit is involved in endothelium dysfunction in preeclampsia. J. Huazhong Univ. Sci. Tech. (2010)30(3): 370-374. 45 Ali,KZ,Burton,GJ,Al-Binali,et.al.. Concentration of free vascular endothelial growth factor and its soluble receptor,s Flt-1in the maternal and fetal circulations of normal term pregnancies at high and low altitudes. J. Matern. Fetal Neonatal. Med. (2012)25(10): 2066-2070.. 46 Khaliq,A,Li,XF,Shams,M,Sisi,P,Acevedo,CA,Whittle,MJ,Weich,H,Ahmed,A. Localization of placental growth factor (PlGF) in human term placenta. Growth Factors (1996)13(3-4): 243-250. 47 Levine,RJ, Thadhani,R, Qian,c,et.al. Urinary placental growth factor and risk of preeclampsia. JAMA (2005)293( 1 ):77-85. 48 Aggarawal,PK,Jain,V,Sakhuja,V,KKarumanchi,SA,Jha,V. Low urinary placental growth factor is a marker of preeclampsia. Kid. Inter.(2006) 69(5):621-624. 49 Akolekar,R,Zaragoza,E,Poon,CY,Pepes,S,Nicolaides,KH. Maternal serum placental growth factor at 11+ 0 to 13 + 6 weeks of gestation in the prediction of pre-eclampsia. Ultrasound Obstet. Gynecol.(2002) 32 (6 ): 732-739. 50 Ma,C,Wang,Y, Shen,T,et.al. Placental growth factor mediates angiogenesis in hypoxic pulmonary hypertension. Prost.Leuktr.Ess 51 Colfert,E,Costello,C,DeFalco,S,McLoughlin,P,Howell,K. Placental growth factor knockout mice demonstrate elevated pulmonary hypertension and increased vessel leak. Proc. Physiol. Soc.(2012) 27:C71 oral communication.

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These peptides are produced and released locally but also are transferred in the vascular compartment and have distal effects. Endothelins (ETs ) Enbdothelins are the most potent vasoconstrictors known. There are three of them known at present, (1,2,3), all produced from similar genes by vascular endothelium. They differ by substitution of several non-critical amino acids.52 They are produced and probably mostly act locally. They are produced as proendothelins then stored and then released by cleavage into an active segment and an inactive segment. They have multiple receptors, four known at present (A,B1,B2,and C). As in most systems we are discussing, some receptors for endothelin act as constrictors, ET-A, and some as vasodilators, ET-B. ET-A is present in endothelium and vascular smooth muscle cells. ET-B is present only in vascular smooth muscle cells. It has been known for 10 years that endothelins are increased in both patients with and animal models of pulmonary hypertension.53 It has been demonstrated that blockade of the endothelin system with blockers of just ET-A, or B or A and B blockers or a blocker of the activation of endothelin by cleavage, all work equally well to prevent PAH in an air embolism animal model.54 The previously mentioned monocratline animal models also show elevated ET-1 in animals developing PAH and that ET-A blockade prevents hypertension but ET-B blockade does not.55 Messenger RNA for both endothelin and endothelin receptors are increased by hypoxia in animal models of PAH.56 This has been shown in humans with PAH from chronic pulmonary thrombosis.57 Serum levels of preeclamptic patients have also been shown to have elevated endothelin levels.58 59 It has been calculated to be responsible for a large amount of blood pressure elevation in PECL.60 A recent review wonders whether endothelin-1 is the key factor in preeclampsia.61 In fetuses with severe growth restriction and increased placental resistance there is increased endothelin in the fetal serum compared to normal controls. 62 So in summary PAH and the placental vessels in PECL have identical endothelin responses during and after disease development and in animal models blockade of ET-1 prevents PAH.

52 Inoue,A,Yanagishawa,M,Kimura,S,et.al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc.Nat.Acad.Sci.1989) 86(8 ): 2863-2867. 53 Gailie,N,Manes,A,Branzi,A. The endothelin system in pulmonary arterial hypertension. Cardiovasc. Res.(2004) 61(2):227-237. 54 Battasini,B,Verreault,M,Ayach,B,et.al.(2004). Role of the endothelin system in secondary pulmonary hypertension related to air embolism: lessons learned from testing four classes of endothelin blockers in a rat model. .J. Cardiovasc. Pharmacol. 55 Frasch,HF,Marshall,C,Marshall,BE.(1999). Endothelin-1 is elevated in monocratline pulmonary hypertension. Am. J. Physiol.Feb, 276(2Pt 1): L304-310. 56 Li,H,Chen,SJ,Chen,YJ,Meng,QC,Durand,J,Aparil,S,Elton,TS. (1985). Enhanced endothelin-1 and endothelin receptor gene expression in chronic hypoxia. J.Appl.Physiol.77(3): 1451-1459. 57 Bauer,m,Wilkins,H,Langer,F,et.al.(2002). Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension. Circ.105(9):1034-1036. 58 Taylor,RN,Varma,M,Teng,NN,Roberts,JM.(1990). Women with preeclampsia have higher plasma endothelin level s than women with normal pregnancies. J.Clin.Endocrinol.Metab.71(^): 1675-1677. 59 Baksu,B,Davas,I,Baksu,A,Akyol,A,Gulbaba,G. (2005).Plasma nitric oxide,endothelin-1 and urinary nitric oxide and cyclic guanosine monophosphate levels in hypertensive pregnant women. Int. J. Gynaecol. Obstet. 90(2): 112-117. 60 George,EM,Granger,JP. (2011). Endothelin: a key mediator of hypertension in preeclampsia. Am.J.Hypertens. 24(9): 964-969. 61 Jain,A.(2012). Endothelin-1: a key pathological factor in pre-eclampsia?. Reprod. Bio Med. 25(4): 443-449. 62 McQeen,J,Kingdom,JC,Connell,JM,Whittle,MJ. (1993). Fetal endothelin levels and placental vascular endothelin receptors in intrauterine growth retardation. Obstet. Gynecol. 82(6):992-998.

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Natriuretic peptides atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) These are a family of small peptides which share a conserved peptidal ring of 17 amino acids. They are stored as a large protein which is cleaved prior to release of the active peptide. There are five known members of this family to date, each produced from a different gene.63 We will focus mostly on ANP and BNP because the most is known about them and there are standardized clinical tests for them available. They also share a family of related receptors but have markedly stronger affinities for there preferred receptors by ten or more fold. In animal models, disruption of the ANP gene and subsequent placement even in a normal oxygen environment is enough for the development of PAH and very mild reduction in oxygen exacerbates this.64 In rat models of PAH, rats genetically disposed to produce less ANP had more severe PAH. 65 BNP in this same lab has been found to be involved in the same way as ANP.66 Ctype natriuretic peptide (CNP) is produced in high amounts in rats who respond to hypoxia by adaptation instead of developing pulmonary hypertension. 67It is important to note that low levels of these peptides are involved in initiation of pathology in both animal models and human patients with PAH. After the development of PAH the cardiac strain causes the increased production of both of these peptides.68 69 Maternal serum ANP and BNP are elevated in diabetic pregnancies at nine weeks gestation compared to normal pregnancies.70 There are no other studies of the natriuretic peptides in early pregnancy. There are no serial measurements of the level of ANP in normal pregnancy or preeclampsia. There is some research on corin, this is the enzyme that cleaves ANP and BNP to their active forms before release. It is produced from the heart.71 There are high levels of corin in the uterus of humans and it is especially involved in the trophpoblastic invasion of maternal vessels. 72 There are genetic variations of this gene that render it relatively ineffective in activating ANP and BNP. In these patients with inactive corin there is less invasion by trophoblasts histologically and a higher incidence of preeclampsia.73 Knockout mice lacking ANP and mice lacking corin have similar courses of hypertension development and markedly increased blood pressure during pregnancy.74 Humans with ineffective corin from mutations have a very high risk of developing hypertension and PAH, these corin variants are

63 Cea,lb. (2005). Natriuretic peptide family: new aspects. Curr.Med.Chem.Cardiovasc.Hematol.Agents 3(2): 87-98 64 Klinger,JR,WArburton,RR,Pietras,LA,Smithies,O,Swift,R,Hill,NS. (1999). Genetic disruption of atrial natriuretic peptide causes pulmonary hypertension in normoxic and hypoxic mice. Am.J.Physiol. 276(5 PT1 ):L868-L874. 65 Klinger,JR,Wrenn,DS,Warburton,RR,Pietras,L,Ou,LC,Hill,NS. (1997). Atrial natriuretic peptide expression in rats with different pulmonary hypertensive response to hypoxia. Am.J.Physiol. 273( 1 PT 2): H411-H417. 66 Klinger,JR,Warburton,RR,Pietras,L,Hill,NS. (1985). Brain natriuretic peptide inhibits hypoxic pulmonary hypertension in rats. J. Appl. Phys.84(5): 1646-1652. 67 Klinger,JR,Siddiq,FM,Swift,RA,et.al.(1998). C-type natriuretic peptide expression and pulmonary vasodilation in hypoxia-adapted rats. Am.J.Physiol. 275(PT 1 ):L645-L652. 68 Casserly,B,Klinger,JR. (2009). Brain natriuretic peptide in pulmonary arterial hypertension:biomarker and potential therapeutic agent. Drug Des, Develop.Ther. 3: 269-287. 69 Leuche,HH,Holzapfel,M,BaumgartnerRA,et.al.(2004). Clinical significance of brain natriuretic peptide in primary pulmonary hypertension. J.Am.Coll.Cardiol. 43(3): 764. 70 Ringholm,L,Pederson-Bjergaard,U,Thorsteinsson,B,et.al. (2011). Atrial natriuretic peptide (ANP) in early pregnancy is associated with development of preeclampsia in type1 diabetics. Diab.REs. Clin. Prac. 93(3): e106-e109. 71 Yan,W,Sheng,N,Seto,M,Morser,J,Wu,Q.(1999). Corinn, a mosaic transmembrane serine protease encoded by a novel cDNA from human heart.J.Biol.Chem. 274(21): 14926-14935. 72 Cui,Y,Wang,W,Dong,N,et.al. (2010). Role of corin in trophoblast invasion and uterine spiral artery remodeling in pregnancy. Nature 484(4): 246-250. 73 Zhou,Y,Wu,Q.(2013). Role of corin and atrial natriuretic peptide in preeclampsia. Placenta. 34(2): 89-94. 74 John,SW,Krege,JH,Oliver,PM,Hagaman,JR,Hodgin,JB,PengJ.(1995). Genetic decreases in atrial natriuretic peptide and salt-sensitive hypertension. Science 267(5198):679-681.

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common in north American blacks.75 So, in another parallel, low apparent levels of ANP or BNP from animal models and humans leads to the development of both PAH and PECL. Levels of ANP were higher in fetuses with severe placental dysfunction than in there mothers. 76 After the onset of clinical disease in humans levels of these two peptides are elevated probably in part from strain on the heart and from the placenta.77 This is probably true in both the fetal and maternal circulation. ENDOTHELIAL RESPONSE MODULATORS These are local response products of the endothelium and also act locally. Thromboxane and Prostacyclin There are many active signaling compounds that are produced from arachidonic acid split from the cell wall. There are also a whole family of related receptors for these compounds. A thorough review of this complex system is beyond this article. 78 As with BMP and TGF-b these are closely related compounds which have an opposite effect on our vascular systems, platelets and endothelia. It gets more complicated than that. Both molecules can act as agonists on each others receptors. In fact it has been shown at pharmacologic doses all prostaglandins and thromboxanes interact as agonists with all of each others receptors.79 When in animal models you completely blockade production of thromboxane the enhanced prostacyclin production also interacts with thromboxane receptors so you still get some of thromboxane's effect. This family of compounds and their receptor families are very old, conserved and closely related genetically. Nonetheless there are many studies examining the levels of these compounds in PECL and looking at their effects in animal models and human clinical cases of pulmonary hypertension and preeclampsia. Thromboxane levels are increased in animal models of PAH.80 They are also increased in patients with PAH. 81 Blockade of synthesis of thromboxane in a newborn infection model prevents development of PAH in a hypoxic environment.82 In newborn rat models of PAH a lot of the pressure increase in the pulmonary resistance vessels has been calculated to be mediated by Thromboxane A2 receptors. 83 A

75 Dries,DL,Victro,RG,Rama,JE,et.al. (2005). Corin gene minor allele defined by 2 missense mutations is common in blacks and associated with blood pressure and hypertension. Circ. 112(16):2403-2410. 76 Furahashi,N,Tsujiei,M,Kimura,H,Yajima,A,Nagae,H,Kimura. Maternal and fetal atrial natriuretic peptide levels, maternal plasma renin activity,angiotensin II,prostacyclin and thromboxane A2 levels in normal and preeclamptic pregnancies. Tohoku, J. .Exp. Med. (1991) 165(2): 79-86. 77 Furuhashi,N,Kimura,H,Nagae,H,Yajima,A,Kimura,C,Saito,T. Brain natriuretic peptide and atrial natriuretic peptide levels in normal pregnancy and preeclampsia. Gyncol. Obstet. Invest. (1994)38( 1 ): 73-77. 78 Bos,CL,Richel,DJ,Ritsema,T,Peppelenbosch,Versteeg,HH. Prostanoids and prostanoid receptors in signal transduction. Int. J. Biochem. Cell Biol. (2004) 36(7 ): 1187-1205 79 Tsuboi,K,Sugimoto,Y,Idcikawa,A..Prostanoid receptor subtypes. Molecular biology of the arachidonate cascade: Prostaglandins and other lipid mediators. (2002) 68-69: 535-556. 80 Jankow,RP,Belcstro,R,Ovcina,E,Lee,J,Massaeli,H,Lye,SJ,Tanswell,AK. Thromboxane A2 receptors mediate pulmonary hypertension in 60% oxygen-exposed newborn rats by a cyclooxygenase- independent mechanism. Am. J. Resp. Crit. Care Med.(2002) 166(3): 208-214. 81 Christman, BW,McPherson,CD,Newman,JH,et.al. An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. NEJM.(1992) 327(2): 70-75. 82 Li,XJ,Gray,BM,Oliver,JR,Lu,CY,Philips,JB.Delayed thromboxane synthesis inhibition but not cholinergic blockade, reverses group B streptococcus-induced pulmonary hypertension. Dev.Pharmacol.Ther.1992) 19( 1 ): 40-49. 83 Jankov,RP,Belcastro,R,Ovcina,E,et.al. Thromboxane A2 receptors mediate pulmonary hypertension in 60% oxygenexposed newborn rats by a cyclooxygenase- independent mechanism. Resp. Crit. Care Med. (2002)166(2): 208-214.

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porcine model of PAH demonstrates a marked diminution in prostacyclin production and levels.84 In humans with primary PAH there has been noted a decrease in prostacyclin synthase expression. Urinary levels of prostacyclin metabolites are decreased in PAH patients. Prostacyclin therapy improves blood flow in animal models of PAH.85 In culture, prostacyclin stimulates human lung endothelial cells to produce more VEGF. 86It is well established that there is an identical imbalance between thromboxane and prostacyclin in preeclampsia. This is seen even very early in pregnancy. 87 88 89After development of clinical disease in PECL some authors claim there is an increased production of prostacyclin but not if the production of thromboxane is compared to it. 90 There are also increased levels of thromboxane in the circulation of fetuses with maternal preeclampsia. The fetal levels are much higher than levels in the maternal circulation in each patient pair.91 So in PAH and PECL the placental and maternal vasculature have parallel responses in arachidonic acid metabolism. Nitric oxide (NO) Nitric oxide , carbon monoxide and possibly hydrogen sulfide are the gases known to be involved in cell to cell signaling. Nitric oxide, first named endothelial derived relaxation factor, has been the best and longest described gas produced in signaling. The effect of the other two is basically unknown in PAH and PECL. Nitric oxide is produced in vascular endothelium by denitrification of arginine by a membrane bound nitric oxide synthase and then diffuses through to the underlying smooth muscle cells on the outside of the endothelial vascular tube. In smooth muscle cells it stimulates soluble guanosine cyclase and inhibits membrane bound adenyl cyclase causing an increased production of cyclic GMP and a decreased production of cyclic AMP which, through amplification cascades, cause smooth muscle cell relaxation.92 It thus does not act in the cell where it is produced but diffuses locally. The effects of various nitrate compounds used in medicine for over a hundred years were thus discovered to be due to local donation of nitric oxide from these compounds. Nitric oxide levels are decreased in animal models of PAH.93 Transgenetic models with increased production of nitric oxide are associated with prevention of PAH.9495 In patients with pulmonary hypertension from chronic hemolytic anemia it has been found 84 Soukka,H.,Vinikka,L,Kaapa,P. Involvement of thromboxane A2 and prostacyclin in the early pulmonary hypertension after porcine meconium aspiration. Pediatr.Res. (1998) 44(6): 838-842. 85 Van Albada,ME,Berger,RM,Niggebrugge,M,van Veghel,R,Cromme-Dijkhuis,AH,Schoemaker,RG. Prostacyclin therapy increases right ventricular capillarization in a model for flow-associated pulmonary hypertension. Eur.J.Pharmacol. (2006)549(1-):107-116. 86 Kamio,K,Sato,T,Liu,X,et.al. Prostacyclin analogs stimulate VEGF production from human lung fibroblasts in culture. Am. J. Physiol. (2008) 294 (6):L1226-!1232 87 Chavarria,ME,Lara-Gonzalez,L,Gonzalez-Gleason,A,et.al. Prostacyclin /thromboxane early changes in pregnancies that are complicated by preeclampsia. Am.J.Obstet.Gyn.2003) 188(4):986-992. 88 Eiland,E,Nzerue,c,Faulkner,M.(2012). Preeclampsia . J. Preg.:(2012) :586578. 89 Lewis,DF,Canzoner,BJ,Gu,Y,Zhao,S,Wang,Y. Maternal levels of prostacyclin,thromboxane,ICAM,VCAM in normal and preclamptic pregnancies. Am. J. Reprod. Immun.(2010) 64(6): 376-383. 90 Walsh,SW,Behgr,MJ,Allen,NH. Placental prostacyclin production in normal and toxemic pregnancies. AM. J. Obstet. Gynecol. (1985) 151( 1 ):110-115. 91 Liu,HS,Chu,TY,Yu,MH,Chang,YK,Ko,CS,Chao,CF. Thromboxane and prostacyclin in maternal and fetal circulation in pre-eclampsia. Int. J. Gynaecol. Obstet. (1998) 63( 1 ): 1-6 92 Francis,SH,Busch,JL,Cor bin,JD. cGMP-dependent protein kinase and cGMP phosphodiesterases in nitric oxide and cGMP action. Pharmacol. Rev. (2010) 62(3): 525-563. 93 Perrella,MA,Deell.ES,Krowka,MJ,Cortese,DA,Burnett,JC,jr. Endothelium derived relaxation factor in pulmonary and renal circulations during hypoxia. Am. J. Physiol.(1992) 263(Pt 2): R45-R50. 94 Firth,AL,Mandel,J,YuanJX. Idiopathic pulmonary arterial hypertension. Dis. Mod. Mech.(2010) 3(5-6):268-273. 95 West,J,Hemnes,A. Experimental and transgenic models of pulmonary hypertension. Compr. Physiol.(2011) 1(2): 769-

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that dysregulation of the arginine synthase system leading to a decreased nitric oxide production is present in those developing pulmonary arterial hypertension compared to those with the same disease process but normal pulmonary blood pressure.96 After the development of clinically evident PAH , there is reduced production of nitric oxide by the endothelium of patients with PAH.97 In pregnancy, nitric oxide generation by human trophoblasts enhances decidual invasion and new blood vessel formation. There is a markedly reduced concentration of nitric oxide synthase in the umbilical endothelium of rat fetuses born after hypertension was present during pregnancy. 98 In a cellular model of human umbilical endothelial cells, hypoxia causes a reduced production of nitric oxide partially from altered arginine metabolism. In humans during pregnancy there is a large increase in nitric oxide production. This is exacerbated with the onset of clinical PECL in both mothers and fetuses.99 This is probably a compensatory mechanism and the results have been reproduced frequently.100101 INTRACELLULAR SIGNALING CASCADES These systems respond to cellular, usually surface, signals and amplify or reduce intracellular responses to these signals.

Cyclic GMP phosphodiesterase 5 (PDE 5) Cyclic nucleotide phosphodiesterases are a family of enzymes involved in breaking the phosphodiester ring in the cyclic nucleotides, cyclic GMP and cyclic AMP. There are currently eleven known phosphodiesterases with various expressions in different tissues. Some have specificity for AMP or GMP and some cleave both. Cyclic GMP phosphodiesterase 5 (PDE5) is an intracellular enzyme that breaks the ring of cyclic GMP rendering it GMP, an inactive compound in signaling. It is an important regulator of vascular tone, causing vasoconstriction.102 Animal models previously described for PAH have shown increased mRNAs for cGMP specific phosphodiesterases. 103 Patients who develop high altitude PAH have been shown to have increased levels of PDE 5 in their lung arterioles. 104 In adults with PAH the 782. 96 Morris,Cr,Gladwin,MT,Kato,GJ. Nitric oxide and arginine dysregulation: a novel pathway to pulmonary hypertension in hemolytic disorders. Curr.Mol.Med.2008) 8(7): 620-632. 97 Giaid,A,Saleh,D. Reduced expression of endothelial nitric oxide synthase in the lung of patients with pulmonary hypertension. NEJM (1995) 333(4): 214-221. 98 Villamor,E,lLeCras,TD,Horan,MP,Halblower,AC,Tuder,RM,Abman,SH. Chronic intrauterine hypertension impairs endothelial nitric oxide synthase in the ovine fetus. Am J. Physiol.(1997) 272(5 PT 1 ): L1013-L1020. 99 Shaamash,AH,Elsnosy,ED,Maklouf,AM,Zakhari,MM,Ibrahim,OA,El-dien,HM. Maternal and fetal serum nitric oxide (NO) concentrations in normal pregnancy, preeclamppsia and eclampsia. . Int.J.Gynaecol.Obstet. (2000) 68(3): 207214. 100 Von Mandach,U,Lauth,D,Huch,R. Maternal and fetal nitric oxide production in normal and abnormal pregnancy. J. Mat. Fet. Neonat. Med. (2003) 13( 1 ):22-27. 101 Smarasom,AK,Allman,KG,Young,D,Redman,CW. Elevated levels of serum nitrate,a stable end product of nitric oxide, in women with pre-eclampsia. Br. J. Obstet. Gynaecol. (1997)104(5): 538-543. 102 Rybalkin,SD,Yan,C,Bornfeldt,KE,Beavo,JA. Cyclic GMP phosphodiesterases and regulation of smooth muscle function. Circ. Res. (2013) 93( 4): 280-291 103 Murray,F,MacLean,MR,Pyne,NJ. Increased expression of the cGMP -inhibited cAMP- specific(PDE3) and cGMP binding cGMP- specific (PDE5) phosphodiesterases in models of pulmonary hypertension. Br. J. Pharmacol.(2002) 137(8 ): 1187-1194 104 Aldashev,AA, Kojonazarov,BK, Amatov,TA,et.al.. Phosphodiesterase type 5 and high altitude pulmonary hypertension. Thorax (2005)60( 8): 683-687.

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increased PDE 5, which is specific for cGMP, has been accepted as being overactive and is a target for therapy at present. 105 In patients with PECL there is a marked increase in PDE 5 activity.106 The uterus shows a marked elevation of PDE 5 activity in preeclamptic as opposed to normal pregnant uterine vessels. 107 In fetuses with chronic pulmonary hypertension levels of cGMP phosphodiesterase are increased.108 I have been unable to find measurements of PDE 5 in fetuses from normal or preeclamptic pregnancies.

RhoA/Rho kinase (ROCK) RhoA/Rhokinase is a secondary intracellular messenger system stimulated by a variety of G protein associated receptors which acts to increase vascular tone through phosphorylation of other proteins involved in modulation of actin/myosin interaction in smooth muscle cells. Activators of this system include: endothelin-1(ET-1), Angiotensin II, Serotonin and other vasoconstrictors. There are a variety of stimulators of these G protein associated receptors. In animal models previously described for PAH there is enhanced activation of the RhoA-Rho kinase system in PAH before the development of disease. 109 110 It has been postulated that inhibition of this system through specific inhibitors can reduce pulmonary blood pressure in PAH.111 There are animal models of PAH that develop simply by being raised in Denver at 5,000 feet above sea level instead of at sea level. Blockade of the RhoA/rho-kinase system, in these altitude driven models, prevents the natural development of PAH.112 In human pulmonary hypertension, overactivity of the same pathway, possibly from excess Serotonin stimulation has been found. 113 In fact some authors believe that this signaling pathway is the key in the development of PAH in humans.114 In normal mammalian pregnancy there is a reduction in activity of the RhoA/ROCK pathways downstream of surface ignaling.115 In humans with PECL compared to normal there is a large increase in placental RhoA/ROCK expression from RNA libraries.116 In human 105 Kass,DA,Champion,HC,Beavo,JA. Phosphodiesterase type 5 : expanding roles in cardiovascular regulation. Circ. Res. 101( 11 ): 1084-1095. 106 Da Costa,BEP,Scocco,C,Poli de figueiredo,CE, Guimaraes,JA. (2006)Increased serum phosphodiesterase activity in women with preeclampsia.. Br.J.Obstet. Gynaecol. 113 (5): 577-579. 107 Wareing,M,Myers,JE, O'Hara,M,et.al. (2003). Effects of a phosphodiesterase-5 (PDE5) inhibitor on endothelium dependent relaxation of myometrial small arteries. Am. J. Obstet. Gynecol. 190(5): 1283-1290 108 Hanson,KS,Ziegler,JW,Rybalkin,SD,Miller,JW,Abman,SH,Clarke,WR. (1998). Chronic pulmonary hypertension increases fetal ling cGMP phosphodiesterase activity. Am. J.Physiol. 275(Pt 1): L931-L941. 109 Shimokawa,H,Takeshita,A Rho- kinase is an important therapeutic target in cardiovascular medicine. Arterioscler.Thromb.Vasc.Biol. (2005)25(9):1767-1775. 110 Satoh,K,Fukumoto,Y,Shimokawa,H. Rho- kinase : important new therapeutic target in cardiovascular disease. Am. J.Physiol.Heart Circ. Physiol. (2011)301(2): H287-H298. 111 Noosaman,BD,Kadowitz,PJ.The role of the RhoA/rho- kinase pathway in pulmonary hypertension.Curr. Drug Discov.Technol.(2009) 6( 1 ): 59-71. 112 Nagaoka,T,Gebb,S,Karoor,V,Homma,N,Morris,KG,McMurtry,Oka,M. Involvement of the RhoA/rho- kinase signaling in pulmonary hypertension of the fawn-hooded rat. J.Appl.Physiol.(2006) 100(3): 996-1002. 113 Guilluy,C,Eddahibi,S,Agard,C,et.al. RhoA and rho kinase activation in human pulmonary hypertension. Am. J. Resp.Crit.Care Med. (2009)179(6): 1151-1158. 114 Connolly,MJ,Aaronson,PI. Key role of the RhoA/Rho kinase system in pulmonary hypertension. Pulm.Pharm.Ther. (2011)24( 1 ): 1-14. 115 Goulopoulou,S,Hannan,JL,Matsumopto,T,Webb,RC. Pregnancy reduces RhoA/Rho kinase and protein kinase C signaling pathways downstream of thromboxane receptor activation in the rat uterine artery..Am. J.Physiol.Heart Circ.Physiol.2012) 302(12): H2477-H2488. 116 Ark,M,Yilmaz,N,Yazici,G,Kubat,H,Aktas,S. Rho-associated kinase II (rock II) expression in normal and preeclamptic human placentas. Placenta (2005) 26( 1 ): 81-84.

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umbilical cells from PECL pregnancies there is a markedly enhanced Rho A/ ROCK expression compared to human umbilical cells from normal pregnancy. 117In preeclampsia placental tissue overexpresses RhoA compared to normal term pregnancy placentae.118 In human omental blood vessels obtained from preeclamptics at caesarean section most of the vasoconstriction and secondary reactive oxygen species production was blocked by blocking the ROCK pathway.119 There is enhanced ROCK expression in placental resistance vessels in preeclamptic placentas.120 In a sign of how all these signaling systems are interrelated it has been found that RhoA/ROCK pathway stimulation blocks endothelial production of nitric oxide.121 So the Rho A/Rock signaling system in pulmonary vasculature in PAH and the maternal and placental beds in preeclampsia respond in an identical or parallel fashion. VASCULAR CONTROL SYSTEM The system described here requires an intact vascular system to transport and complete this signal system. Renin Angiotensin system (RAAS) The RAAS system is activated in early stage of pulmonary hypertension before the development of clinical disease in animal models ,but it has not been well studied in these models.122 In patients with PAH it has been found that there is an increased incidence of genetically overactive Angiotensin converting enzyme, (ACE).123 There is a dysregulated RAAS system with increased activity in patients with progressive PAH.124 It was discovered that normal pregnant patients were resistant to the effect of Angiotensin II and that preeclamptic patients were sensitive to it's vasoconstrictive effects.125 More recently it has been found that the RAAS system is more active in the placental vascular beds of preeclamptics compared to normal patients. There are no receptors for Angiotensin II in the decidual or placental bed.126 There is a very active RAAS system in the fetus and this becomes more active with Angiotensin converting enzyme (ACE) blockade.127 Angiotensin II from the mother is probably required 117 Friel,AM,Sexton,DJ, O'reilly,MW,Smith,TJ,Morrison,JJ.. RhoA/Rho kniase : human umbilical artery m RNS expression in normal and pre eclamptic pregnancies and functional role in isoprostane- induced vasoconstriction. Reprod. (2006)132( 1 ): 169-176. 118 Zhou,L,Qiao,F. Expression of RhoA in placenta of preeclampsia. J. Huazhong.Univ. Sci.Tschnol.Med.Sci.2002) 26(6): 744-746. 119 Mishra,N,Nugent,WH,HMahadavi,S,Walsh,SW. Mechanisms of enhanced vascular reactivity in preeclampsia. Hypertens.(2011) 58(3): 867-873. 120 Orozco-Hernandez,EG,Clemente-Guerrero,M, Saavedra-Molina,A,Manzo-Avalos,S. Determination of RhoA GTPase effector proteins in preeclamptic placentas. FASEB J.(2013) 27(meeting abstract supplement):598.6 121 Takemoto,M,Sun,J,Hiroki,J,Shimokawa,H,Liao,JK. Rho- kinase mediates hypoxia-induced down regulation of endothelial nitric oxide synthase. Circulation (2002) 106( 1 ): 57-62. 122 Mikami,O,Nagaoka,I,Hasunuma,K,Takahishi,H. Angiotensin II receptors in a rat model of hypobaric hypoxic hypertension. Nihon Kyobu Shikkan Gakkai Zasshi.(1996)34(2):186-193. This is true 123 Abraham,WT,Raynolds,MV,Gottschll,B.et.al. Importance of angiotensin- converting enzyme in pulmonary hypertension. Cardiol. 124 De Man,FS,Tu,L, Handoko,ML,et.al. Dysregulated renin-angiotensin-aldosterone system contributes to pulmonary arterial hypertension. Amer. J. Resp. Crit. Care Med (2012) 186(8): 780-789. 125 Gant,N,Worley,R. Hypertension in pregnancy concepts and management. (1980)New York,NY.Appleton Century Crofts first edition. 126 Anton,L,Merrill,DC,Liomar,AA,et.al. The uterine placental bed renin-angiotensin system in normal and preeclamptic pregnancy. Endocrinol.(2009) 150(9):4316-4325. 127 Forhead,AJ,Broughton-Pipkin,F,Sutherland,MF,Fowden,AL. Changes in the maternal and fetal renin-angiotnsin systems in response to angiotensin II type 1 receptor blockade and angiotensin- converting enzyme inhibition in pregnant sheep during late gestation. Exp. Physiol. (1997) 82(4): 761-776.

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for trophoblast invasion of the endometrium in mammals.128 In patients followed prospectively there was an association between higher Angiotensin 1-7, which is a split by a different peptidase from the common precursor Angiotensinogen and, has the opposite effect of Angiotensin II and the later development of PECL. 129 A recent review of the RAAS system in normal pregnancy and preeclampsia confirms these presented findings. 130 So as in all of the presented signaling systems in which there is hypoactivity in normal early pregnancy , there is hyperactivity after the development of clinical disease. TABLE 2 SUMMARY OF CHANGES IN SYSTEM RESPONSES IN PECL AND PAH Miscellaneous It is unknown whether uric acid is a signal or a response or a bystander in vascular biology. Uric acid Uric acid is a metabolic breakdown product of purine metabolism. It has been known for decades that elevated levels of this compound were uniquely present in preeclamptic patients as opposed to pregnant patients with hypertension. 131 It has been postulated in the past that this was a unique marker in pregnancy for PECL but was found later to lack specificity for PECL. It has been recently been discovered that serum uric acid elevations in patients with PAH correlate strongly with patient mortality.132 This has been verified in multiple causes of pulmonary hypertension. It has been suggested that reduction of uric acid with diet and medication may reduce mortality in PAH. 133 In neonates with pulmonary hypertension there is also an elevation in uric acid. 134 It has been suggested that this elevation interferes with production of nitric oxide by arginine synthase. So it has been suggested that pharmacologically reducing uric acid may improve endothelial production of nitric oxide by endothelium which might be helpful in both PECL and PAH. Conflict of interest: none SUMMARY There are probably other primary signaling factors involved in the pathogenesis of both PECL and PAH but these discussed factors are the most well-studied known and agreed upon. The striking parallel signaling seen in PECL and PAH leads me to believe that there are identical signals at the start of both 128 Pijacka,W,Hunter,MG,Broughton-Pipkin,F,Luck,MR. Expression of renin-angiotensin system components in the early bovine embryo. Endocrin. Conn.(2013) 1 ( 1 ): 22-30. 129 Sykes,SD,Pringle,KG,Zhou,A,Dekker,GA,Roberts,CT,Lumbers,ER. Fetal sex and the circulating reninangiotensin system during early gestation in women who later develop preeclampsia or gestational hypertension. J.Hum.Hypertens.2013) 28( 2): 133-139. 130 Irani,R,Xia,Y. The functional role of the renin-angiotensin system in pregnancy and preeclampsia. Placenta.(2008) 29(9):763-771. 131 Chesley,L.(1978). Hypertensive disorders in pregnancy. New York,New York, Appleton-Century-Crofts.first edition. 132 Nagaya,N, Uematsu,M,Satoh,T,et.al. Serum uric acid levels correlate with the severity and the mortality of primary pulmonary hypertension. Am. J. Respir Crit. Care Med. (1999)160(2): 487-492. 133 Zharikov,SI, Swenson,ER, Lanaspa,M,Block,ER, Patel,JM,Johnson,RJ. Could uric acid be a modifiable risk factor in subjects with pulmonary hypertension. Med. Hypotheses (2010)74(6): 1069-1074. 134 Pearson,DL,Dawling,S,Walsh,WF,et.al. Neonatal pulmonary hypertension-urea cycle intermediates, nitric oxide production, and carbamoyl- phosphate synthetase function. NEJM. (2001)344(24): 1832-1828.

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pathophysiologies because the two vascular beds have the same endpoint, maximizing the oxygen uptake by their respective capillaries. They also have the same original signaling process, blockade of an oxygen sensitive potassium channel leading to resistance vessel constriction. As a short summary positive effectors of new vessel growth, vasodilators and endothelial relaxants are inhibited and negative effectors of vessel growth, vasoconstrictors and endothelial activators are enhanced in both preecclampsia (PECL) and pulmonary hypertension (PAH). TREATMENT OF PECL WITH DRUGS USED IN PAH The usage of beta blockers for hypertension has been our only treatment for PECL for more than twenty years. Beta blockers are really a class D drug if you would consider fetal growth restriction, in my humble opinion. In the face of obvious fetal heart failure in severe preeclampsia and growth restriction it seems illogical to use beta blockers which reduce fetal cardiac output and RAAS activity. The fetal circulation has to expand daily to keep up with growing carcass size and inhibitors of this process like beta blockers and ACE inhibitors have to have negative effects on fetal physiology. Recent national opinion by ACOG did nothing but condone their continued usage.135 From an adaptive perspective and from the primary and secondary molecular signaling involved in pulmonary hypertension and the placental circulation in preeclampsia, it is apparent that they evolve in a parallel or identical fashion. It is apparent from the known facts that this is especially true when looking at the molecular signaling changes present in the placental and pulmonary arterial vascular systems in the pathophysiology of PAH and PECL both during the onset and the development of disease. They both end with cardiac failure, the fetus and sometimes the mother in PECL and the patient in PAH. The fetal maladaptation of PECL should be amenable to the same pharmacotherapies that have improved patient outcomes in pulmonary hypertension. There are at present at least eleven whole classes of drugs being presently used to treat pulmonary hypertension. There are: calcium channel blockers, nitrates, PDE5 inhibitors, endothelin receptor blockers, prostacyclin agonists, leukotriene receptor blockers, thromboxane receptor antagonists, Rho A/rhokinase inhibitors, soluble guanylate cyclase activators and uric acid oxidase inhibitors. We presently use just one of these drug classes in pregnancy; calcium channel blockers and most physicians use it as a last resort in hypertension. All these classes of drugs should theoretically reduce fetal placental arterial resistance in PECL as they all reduce pulmonic arterial resistance in PAH. There have been some small studies of the use of nitrates and arginine supplementation in pregnancy to prevent or treat PECL. 136 137 The nitrate donors were found to acutely increase fetal cardiac output. This has to be from reduction in placental resistance. Supplementation with L-arginine was found to reduce the occurrence of PECL. Both consistent with the same response in PAH. A review of the other drugs on the above list show all are B or C class drugs and therefore most are probably safe in pregnancy, especially after eighteen weeks of gestation. It is almost sure that some are not appropriate in PECL ,such as the inhaled prostacyclin agonists, because they will not reach the effective target, placental resistance arteries. Some will probably have too great a side effect profile in pregnant women. But some may work and may improve fetal placental blood flow and reduce the 135 ACOG taskforce on hypertension in pregnancy. ACOG November 2013. 136 Luzi,G,Caserta,G,Iammarino,G,Clerici,G,Di Renzo,GC. Nitric oxide donors in pregnancy: fetomaternal hemodynamic effects induced in mild pre-eclampsia and threatened labor. Ultras. Obstet.Gynecol.1999) 14(2):101-109. 137 Vadillo-Ortega,F,Perichart-Perera,O,Espino,S,et.al.. Effect of supplementation during pregnancy with L-arginine and antioxidant vitamins in medical food on pre-eclampsia in high risk population: randomized controlled trial. Brit.Med.J. May 11, 2011 342:d2901.

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vascular resistance that causes fetal heart failure in PECL and secondarily reduce the placental derived maternal endothelial overactivation in PECL. These will not cure PECL. If we introduce a therapy effective in increasing fetal uptake of oxygen and other nutrients it will cause the fetus to once again outgrow the increased supply and be in the same situation several or more weeks down the road. This seems self defeating but if every preterm baby with PECL could grow normally for just two or three weeks imagine the effect upon nenonatal outcomes , not to mention neonatal costs.

CONCLUSION We have stood still for more than twenty years. Now is the time to start clinical trials of some or all of the pregnancy class B and C drug classes used in pulmonary hypertension. Trials should start with small numbers and with close ultrasound supervision of each pregnancy. But we should start, staying where we are has not helped babies for the last two decades. It is scary for obstetricians, we have been taught to be uncomfortable with pharmacology. Imagine whole new classes of drugs to learn and develop comfort using. It is daunting but we, or some of us, have to move forward, otherwise we are leaving our small patients behind.

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TABLE 1 Summary of effectors of angiogenesis in PIH and PAH Peptide Effector TGF-b

Change in PIH INCREASED ACTIVITY

Change in PAH INCREASED ACTIVITY

BMP s

DECREASED ACTIVITY

DECREASED ACTIVITY

ENDOGLIN

INCREASED ACTIVITY

INCREASED ACTIVITY

VEGF

DECREASED ACTIVITY

DECREASED ACTIVITY

sFlt-1

INCREASED ACTIVITY

INCREASED ACTIVITY

PlGF

DECREASED ACTIVITY

DECREASED ACTIVITY

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TABLE 2 SUMMARY OF CHANGES IN RESPONSE SYSTEMS IN PIH AND PAH Response System RENIN-ANGIOTENSINALDOSTERONE SYSTEM

Change in PIH INCREASED ACTIVITY

Change in PAH INCREASED ACTIVITY

ENDOTHELINs

INCREASED ACTIVITY

INCREASED ACTIVITY

NATRIURETIC PEPTIDES

DECREASED ACTIVITY

DECREASED ACTIVITY

THROMBOXANE

INCREASED ACTIVITY

INCREASED ACTIVITY

PROSTACYCLIN

DECREASED ACTIVITY

DECREASED ACTIVITY

NITRIC OXIDE

DECREASED ACTIVITY

DECREASED ACTIVITY

CYCLIC GMP PHOSPHODIESTERASE

INCREASED ACTIVITY

INCREASED ACTIVITY

RHO A /RHO KINASE

INCREASED ACTIVITY

INCREASED ACTIVITY

ENDOTHELIAL RESPONSE MODULATORS

INTRACELLULAR RESPONSE CASCADES

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CONFLICT OF INTEREST STATEMENT I have had no financial or other involvements in any organization which in any way could have ant interest in this article. Thomas J. Byrne M.D.

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