WO2024126977A1 - Hydrogen sulphide (h2s) generating compounds for use in the treatment of preeclampsia and related disorders in obese pregnant women - Google Patents

Abstract

The application provides Hydrogen Sulphide (H2S), or a (H2S) generating compound or compound capable of stimulating H2S production in an overweight or obese pregnant subject for use in the treatment or prevention of pre-eclampsia (PE)or fetal growth restriction or preterm birth. Also provided are methods of treating or preventing pre-eclampsia (PE) or fetal growth restriction comprising administering to an overweight or obese pregnant subject, a pharmaceutically effective amount of H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject. H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject for use in the treatment pre-term labour and described, as are a method of treating pre-term labour comprising administering to a pregnant subject, a pharmaceutically effective amount of H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject. Methods of monitoring treatment of pre-eclampsia or impaired fetal growth in overweight or obese pregnant subject, comprising measuring the amount of H2S in a sample of blood, serum or plasma in a subject prior to treatment and comparing it to the amount in a sample taken after treatment are also described.

Description

HYDROGEN SULPHIDE (H2S) GENERATING COMPOUNDS FOR USE IN THE TREATMENT OF PREECLAMPSIA AND RELATED DISORDERS IN OBESE PREGNANT WOMEN

The invention relates to the prevention or treatment of overweight and obese women in pregnancy using hydrogen sulphide (H2S) generating compounds.

Introduction

Obesity (Body Mass Index (BMI) >30kg/m²) is a major health concern globally. The World Health Organisation (WHO) estimates approximately 1.9 billion adults are overweight and of this population about 650 million adults are classified as obese¹. The leading cause for increased BMI and metabolic disorders is a 'Western lifestyle'. This is a combination of high fat/sugar and low nutrient diets paired with low physical activity². High BMI is a major risk factor for chronic diseases such as cardiovascular disease and diabetes but in pregnancy it dramatically exacerbates the risk of pregnancy-related complications, such as preeclampsia³' ⁴.

Preeclampsia is a hypertensive disorder that affects 1 in 12 women during pregnancy. Interestingly, 4 in 10 women who are overweight before or during pregnancy (BMI 25<29.9 kg/m²) develop preeclampsia³' ⁵. Critically, preeclampsia manifests as high blood pressure after 20 weeks of gestation, caused by widespread endothelial dysfunction^6-8. Preeclampsia also increases life-long risk of major adverse cardiovascular events after pregnancy⁹. The exact aetiology of preeclampsia is unknown but excessive inflammation^10-14, imbalance of angiogenic factors¹⁵ and the defect in the protective pathways of heme oxygenase-1 (HO-1)¹⁶' ¹⁷ and Cystathionine y-Lyase (CSE)¹⁸' ¹⁹ are strongly associated with preeclampsia development. It is unlikely that inflammation alone is the cause of preeclampsia as a systemic inflammatory response during pregnancy can occur in the absence of preeclampsia-related symptoms²⁰. On the other hand, anti- angiogenic proteins, such as vascular endothelial growth factor (VEGF) antagonist soluble Fms-like tyrosine kinase receptor-1 (sFlt-1), a splice variant of membrane-bound vascular endothelial growth factor receptor-1 (VEGFR-1)²¹' ²², and soluble endoglin (sEng) induces severe preeclampsia-like signs in rats and are increased dramatically in pregnant women weeks prior to the clinical onset of preeclampsia²³. The defect in HO-1 and CSE lead to an increase in sFlt-1 in the maternal circulation and causes preeclampsia, which is prevented by a hydrogen sulfide-releasing compound ²⁴' ²⁵. This is discussed in WO2014/132083, incorporated herein in its entirety.

Increased adiposity in overweight individuals generates a low-grade chronic inflammatory profile, with increased levels of adipokine secretions and endothelial activation^26-28. This inflammatory state increases the risk of preeclampsia; yet, the underlying mechanism is unclear. The Applicants propose that low-grade chronic inflammation before pregnancy, induced by high fat diet (HFD) consumption, primes the vascular endothelium for activation, and that in the presence of angiogenic imbalance and inflammation during pregnancy, anti-angiogenic factors sensitise the endothelium to an exacerbated inflammatory phenotype, leading to widespread endothelial dysfunction and adverse pregnancy outcomes.

This was unexpected. The effect of maternal obesity on anti-angiogenic factors levels are poorly understood. It has been reported that mature adipocytes produce soluble Flt- l(sFlt-l) and the expression of sFlt-1 and BMI showed inverse correlation (https;//doi.org/10.1161/HYPERTENSIONAHA.111.1171322). Furthermore, sFlt-1 levels are not significantly different in normal and obese women in first gestation. Moreover, sFItl levels in second and third trimester were significantly higher in normal weight women compared to overweight or obese women (https;//doi,org/10.1177/1933719112452472). This suggests the pathway is not involved and teaches away from treating obese women with H2S.

The Applicant did not know that HFD in pregnant individual will lead to decrease in placental CSE expression and increase in sFlt-1. Compared to a normal chow diet (CD), the high fat diet (HFD)-fed mice had significantly increased body weight after eight weeks of consumption. However, compared to CD, HFD consumption had no effects on the mean arterial pressure (MAP) in non-pregnant animals, whereas in pregnant mice, MAP was significantly increased in mice fed with HFD. This itself one would not anticipate. Levels of kidney injury marker, KIM-1, were elevated in HFD-fed pregnant mice but not non- pregnant mice when compared to CD fed animals. Clearly, pregnancy and HFD compounded poor outcome. The resistance index (RI) of uterine arteries from dams maintained on either CD or HFD was significantly decreased at E17.5 compared to EO (Fig. IE), indicating that vascular adaptation had taken place during pregnancy in both groups. Regardless of the status, pregnancy or non-pregnancy, there were no significant differences in RI observed between the HFD and CD groups. The Applicant found decreased expressions of H₂S-producing enzyme, cystathionine-gamma lyase (CSE) in placenta (Fig. 8A, B) and endothelium of mesenteric arteries in HFD-fed dams compared to CD controls. CSE enzyme activity in HFD placentas was also significantly decreased, reducing H2S producing potential. These would not be obvious from previously published work. A high fat intake increased the levels of pro-inflammatory cytokines in both pregnant and non-pregnant animal groups, however, elevated arterial blood pressure and kidney damage were only observed in pregnant mice fed with HFD. We found that high fat intake not only increased the levels of inflammatory cytokines but also circulating levels of sFIt- 1 and sEng in pregnant mice. The Applicant shows for the first time that down-regulation of vascular CSE leads to the HFD-induced endothelial dysfunction and poor outcomes in pregnancy. To pinpoint the defect as HFD induces down regulation of placental CSE, we used CSE deficient individuals.

There was no significant differences in vessel contractility or endothelium-dependent relaxation between HFD and CD groups in CSE knockout (CSE^-/-) pregnant mice. Thus, HFD induced endothelial dysfunction during pregnancy was mediated by CSE dysregulation. Consistent with the above notion, exogenously adding the slow-releasing H2S donor, GYY4137, was able to restore endothelium dependent vessel relaxation in HFD exposed vessels.

The invention provides hydrogen sulphide (H2S), a H2S generating compound or compound capable of stimulating H2S production in an obese pregnant subject, for use in the treatment or prevention of pre-eclampsia (PE) or fetal growth restriction (FGR or intra uterine fetal growth restriction).

The invention also provides a method of treating or preventing pre-eclampsia (PE) or FGR in an overweight or an obese pregnant subject, comprising administering to a pregnant subject a pharmaceutically effective amount of H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject.

The addition of H2S has been shown to encourage, for example, angiogenesis, resulting in the restoration of blood supply to the fetus. The use of H2S, a H2S generating compound or compound capable of stimulating H2S production as a prophylactic in an overweight or anobese pregnant subject to prevent the onset of PE is therefore also provided.

This also means that, for example, pre-term labour may also be treated.

Overweight or obese typically means a BMI (Body Mass Index) or 25 or more. Overweight may be within the BMI range 25.0<30 and obese is typically classified as a BMI of 30.0 or higher Pre-eclampsia, as described above, is a hypertension syndrome and a major contributor to fetal morbidity. Additionally, fetus may have impaired fetal growth due to the pregnancy induced hypertension. This "impaired fetal growth" produces lower birth weight babies and babies with increased risk of complications later.

The subject is typically a mammal, especially a human.

H2S may be administered, for example as a gas or solution, such as in a carrier solvent.

Naturally occurring H2S donating compounds are known. These include allicin from garlic which decomposes to diallyl disulphide and diallyl trisulphide. Sulforaphane is produce by broccoli and erucin is found in rocket (Eruca Sativa). These may be provided orally, such as in the form of tablets or capsules.

A number of synthetic H2S compounds are known. These include GYY4137 (morpholin- 4-ium 4 methoxy phenyl (morpholino) phosphinodithiolate) from Cayman Chemical. This has been previously used in rat studies to study H2S activity by injection intraperitoneally (ip) or intravenously (iv). Lawesson's reagent is another H2S donor.

SG1002 (Sulfa GENIX Inc) is also a H2S producing compound and may be used. See also US 8,361,514 B.

Anethole trithione is also a commonly used H2S donor. Sodium sulphide in buffer (produced by Ikaria as IK-1001) has been used in clinical trails for reperfusion/injury. Other H2S generating compounds are disclosed in Bannenberg G.L. and Viera HLA (Expert Opin. Ther. Patents (2009) 19(5) 663-682).

Other compounds are disclosed in the article by Predmore B.L. et al (Antioxidants and Redox Signalling (2012) 17 (1) 119-140), including compounds ADT-OH, TBZ and 4 hydroxyphenylisothiocyanate.

The compound may be ACS-14, AC583, ACS 84, ACS 85, ACS 86 (Lee M, J. Biol Chem (2010) 285, 17318-17328), DATS (diallyl trisulphide), S-diclofenac, sulfane sulphur, thiocysteine, GSH hydropersulphide, GYY4137, SG1002, a H2S-donating derivative of sildenafil (ACS6-Sparatore A et al Expert Rev, Clin. Pharmacol (2011) 4, 109-121), ADT- OH, TBZ and 4-hydroxyphenyl isothiocyanate, thioglycine, 1-thiolysine, 1-thiovaline, or salts thereof. Alternatively, the production of H2S may be induced in the body for example by inducing CSE production or inducing other enzymes that produce H2S in the body. For example, statins, such as simvastatin or pravastatin have been found to upregulate CSE production.

The compounds may be introduced by any suitable means, including ip, iv, orally, intrauterine for example as a pessary, or intramuscularly. They may be administered together with one or more pharmaceutically acceptable carriers or excipients. Typical doses may be lOmmol/kg to 0.01 mol/kg, typically lOmmol/kg to O.lmmol/kg.

Methods of monitoring treatment of pre-eclampsia or impaired fetal growth, comprising measuring the amount of H2S in a sample of blood, serum or plasma in a subject prior to treatment with H2S, or compound as described above and comparing it to the amount in a sample taken after treatment. Pre-term labour treatment may be similarly monitored.

This allows the amount of H2S in the body to be subject to ensure that optimal levels of H2S are provided. The amount of H2S detected may be detected by techniques generally known in the art, such as the assay method described below.

The subject may have been treated with a compound as described above or alternatively another unrelated anti-PE as anti-impaired fetal growth compound.

The invention will now be described by way of example only with reference to the following Figures:

Figure 1: HFD consumption induced a preeclampsia-like phenotype. (A) Arterial pressure was measured in non-pregnant and pregnant mice fed with HFD or CD using Millar Tip catheter. (B) Marker of kidney damage, KIM-1, was measured in urine from non-pregnant and pregnant mice fed a CD or HFD (n=5-13). (C) Pup weight distribution (dotted line represented 10^th percentile) and (D) placental weight were measured at E17.5 from dams fed a HFD or CD. (n=9). Uterine artery waveforms using colour Doppler were collected from non-pregnant and pregnant mice fed a HFD and CD. (E) Resistance index and (F) Pulsatility index were calculated. Data are expressed as mean ± S.E.M. and analysed by Mann-Whitney test or one-way ANOVA. n = 6-13.

****p<0.0001***p<0.001, **p<0.01, *P<0.05.

Figure 2: HFD increased placental pro-inflammatory cytokines and circulating anti-angiogenic factors. Plasma or placental tissue were harvested at E17.5 from pregnant mice fed a HFD or CD. (A) Placental levels of TNFa and (B) IL-6; (C) plasma levels of sFlt-1 and (D) sEng were measured using ELISA. Data are expressed as mean ± S.E.M. and analysed by Mann-Whitney test. n=6-12 ***p<0.001, **p<0.01, *p<0.05.

Figure 3: HFD induces vascular complications during pregnancy and anti- angiogenic factors promote pro-inflammatory cytokine-induced endothelial dysfunction. Mesenteric arteries were isolated from CD or HFD fed pregnant mice at E17.5. (A) Vessel contractility in response a series of concentrations of phenylephrine, and (B) vessel relaxation in response to acetylcholine (ACh) were measured by wire myography. Human Umbilical Vein Endothelial cells (HUVECs) were transfected with combinations of adenovirus (Ad) encoding soluble Flt-1, soluble Eng or an empty vector (CMV) before treated with different concentrations of Tumour Necrosis Factor Alpha (TNFa) for six hours. Endothelial activation markers, (C) Intracellular Adhesion Molecule- 1 (ICAM-1) and (D) Vascular Cell Adhesion Molecule-1 (VCAM-1) were measured using FACS analysis. Data are expressed as mean ± S.E.M. and analysed by Mann-Whitney test or Two-way ANOVA. n=6-7. *AdCMV + AdsFlt-1 vs AdsFlt-1 + AdsEng corresponding TNFa treatment, ^{^}AdCMV + AdsEng vs AdsFlt-1 + AdsEng corresponding TNFa treatment, #AdCMV vs AdsFlt-1 corresponding TNFa treatment, #AdCMV vs AdsEng corresponding TNFa treatment. ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05; ^{^^^^}p<0.0001, ^{^^^}p<0.001, ^{^^}p<0.01, ^{^}p<0.05; ^####p<0.0001, ^###p<0.001, ^##p<0.01, ^#p<0.05; ****p<0.0001, ***p<0.001, **p<0.01, *p<0.05.

Figure. 4: HFD consumption dysregulates CSE expression in endothelium and H2S rescues HFD induced vascular complication. (A) Representative immunohistochemical staining of CSE (red) and endothelial marker CD31 (green) in mesenteric arteries isolated from CD or HFD fed pregnant mice at E17.5. (B) Trimethylsulfonium, a metabolite of H₂S methylation was measured in urine collected for 24 hours at E16.5. (C) Endothelial dependent vessel relaxation in response to ACh in mesenteric arteries isolated from pregnant cystathionine gamma-lyase wild-type (CSE^+/+) and knockout mice (CSE^-/-), fed a CD or HFD at E17.5, were measured using wire myography. (D) Mesenteric arteries isolated from HFD fed CSE^+/+ mice and were treated with or without H2S donor, GYY4137 compound. Vessel relaxation in response to ACh was measured using myography. Results are representative or expressed as mean ± S.E.M. and analysed by Mann-Whitney test or Two-way ANOVA. n=5-7. **p<0.01, *p<0.05.

Figure 5: HFD increases body weight and induces inflammation. Wild-type mice were fed with either 45% fat diet (HFD) or chow diet (CD) from 4 weeks old. (A) Mice were weighed weekly up to 12 weeks old and the percentage of weight gain was calculated. (B) Levels of TNFa in the livers in non-pregnant mice fed a CD or HFD were measured using ELISA. Data are expressed as mean ± S.E.M. and analysed by Mann- Whitney test. n=6-8. **p<0.01, *p<0.05.

Figure 6: HFD increases maternal systolic and diastolic aterial pressure in pregnant mice. (A) Systolic blood pressure (SBP) and (B) Diastolic blood pressure (DBP) were measured in age matched non-pregnant and pregnant mice fed with CD and HFD. Data are expressed as mean ± S.E.M. and analysed by Mann Whitney test. n=6-7. *p<0.05.

Figure 7: Levels of sFlt-1 and sEng in conditioned media of HUVECs treated with Adenovirus encoding sFlt-1 and sEng. HUVECs were transfected with combinations of adenovirus (Ad) encoding sFlt-1, sEng or an empty vector (CMV). (A) sFlt-1 and (B) sEng release were measured in the conditioned media using ELISA. Data are expressed as mean ± S.E.M. and analysed by Mann Whitney test. n=6-7. *p<0.05.

Figure 8: HFD dysregulates CSE expression in the placenta in pregnant mice. Placenta were collected from mice fed either CD or HFD at E17.5. (A) Placental CSE mRNA expression was determined by real-time PCR. (B) Placental CSE protein expression was confirmed by Western blot analysis and quantified using densitometry analysis. (C) Placental CSE enzyme activity assay was determined using ninhydrin reagent to measure cysteine produced from crude extracts treated with CSE specific substrate cystathionine. Data are expressed as mean ± S.E.M. and analysed by Mann Whitney test. n = 5-6. *p<0.05.

Table 1: Maximum responses (Emax) and pD2 values of phenylephrine (PE) and acetylcholine (ACh) in mesenteric arteries from pregnant mice fed a chow diet (CD) or high fat diet (HFD). Data are expressed as mean ± S.E.M. and analysed by Mann-Whitney test. n = 6-7. **p<0.01; CD vs. HFD.

Table 2: Maximum responses (Emax) and pD2 values of phenylephrine (PE) and acetylcholine (ACh) in mesenteric arteries from pregnant CSE^+/+ and CSE^-/- mice fed a chow diet (CD) or high fat diet (HFD). Data are expressed as mean ± S.E.M. and analysed by Mann-Whitney test. n=6-7. * CSE^+/+ CD vs. CSE^-/- CD, *p<0.05, **p<0.01, ***p<0.001; # ACh vs. ACh+GYY4137 in CSE+/+ HFD, * p<0.05. Methods

Animal Experimental Protocol

Four-week-old female C57BL/6j x 129SvE (B6;129) mice were weighed and littermates were divided randomly to be given either a high fat diet (HFD; 45% fat) or chow diet (CD) ad libitum. Mice remained on their respective diets for the entire experiment. Once mice reached 12 weeks old and the consumption of the HFD led to a weight increase of >20%, mice were time mated with males fed CD. All mice were housed in a controlled 12-hour light-dark cycle environment maintained at 22°C.

The CSE knockout mouse (CSE^-/-) was kindly provided by G. Yang²⁹. The protocol for the generation of the CSE^-/- mouse has been published previously²⁹. Positive cell clones were microinjected into C57BL/6j mice blastocysts and planted back into a surrogate mother. Chimera's were chosen for mating and the generation of Fl. Heterozygous genotypes (CSE^+/_) were then used to generate F2. The resultant experimental mice were generated using B6;129. All experimentation was conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act, 1986 using procedures approved by the University Ethical Review Committee.

Blood Pressure Measurement

Arterial blood pressure was measured at E17.5 by Millar System through carotid artery using microtip catheter as described previously¹⁸. Briefly, mice were anaesthetized using 2% isoflurane. Adequacy of anaesthesia was monitored through the disappearance of the pedal withdrawal reflex. The carotid artery was isolated and cannulated with a Millar 1- French Mikro-Tip pressure catheter connected to a pressure transducer (ADInstruments Ltd. Oxford, UK). After 30 minutes of blood pressure stabilisation, arterial pressure recorded and averaged over a further 10 minute-period. On E17.5 of pregnancy, blood sampling was undertaken, the animals sacrificed via cervical dislocation and organs were collected. The live fetuses and placentas were counted and weighed.

Ultrasonography

Mice were anesthetised with 2% isoflurane. Maternal heart rates were maintained at 450 ± 50 BPM and body temperature was monitored using rectal thermometer. All hair was removed from the abdomen and pre-warmed ultrasound gel was applied for imaging. Mice were imaged at baseline (virgin) and E17.5 of pregnancy. The bladder was first identified as a reference point for finding the uterine artery. Colour Doppler mode was used to visualise uterine artery blood flow in pregnant and non-pregnant mice. Once flow was located, power Doppler mode was applied. The relevant flow was gated in the direction of flow and at an angle greater than 40°. The resistance index and Pulsatility index were calculated using Vevo® Labs software.

Myography

Ice cold Krebs solution was used to collect the mesenteric bed from pregnant mice. The superior mesenteric artery was identified and second order branches were isolated and cleaned of fat and connective tissues. Cleaned segments were placed into myograph chambers (DMT, Denmark) containing fresh Krebs solution bubbled with medical grade oxygen and heated to 37°C. Stainless steel wires (25pm in diameter) were threaded through the lumen consecutively.

Following initial 30-minute incubation, each vessel was normalised in a standardised procedure. Arteries were first challenged with 5mM KCI to test contractile response at baseline. Each vessel was then stimulated as follows; cumulative addition of phenylephrine (Sigma-Aldrich, Dorset, UK): 10^-9, 3xl0^-9, 10^-8, 3xl0^-8, 10^-7, 3xl0^-7, 10^-6, 3xl0^-6, and 10^-5 mol/L, with 5 minutes' incubation per concentration. (2) cumulative response to acetylcholine (Sigma; in mol/L): 10^-9, 3xl0^-9, 10^-8, 3xl0^-8, 10^-7, 3xl0^-7, 10^-6, 3x l0^-6, and 10^-5, to a vessel pre-constricted with optimal dose of phenylephrine (dose which resulted in 70% of maximal contraction).

Subsequently, vessels were then incubated with H₂S-donor; GYY4137 (Sigma-Aldrich, Dorset, UK) for 30 minutes before being subjected to contraction and relaxation protocol again.

Cell culture and flow cytometry

Human umbilical vein endothelial cells (HUVECs) were cultured in EGM-2 medium. Cells were treated with adenovirus (Ad) for sFlt-1, sEng or empty vector (CMV), in reduced serum media (M199 medium containing 5% FBS). After 24 hours, media was changed to fresh reduced serum media containing one of the following concentrations of recombinant TNFo; 0, 0.1, 0.5, 1, 5, 10 ng/mL and incubated for six hours. Following treatment, the cells were stained using appropriate fluorophore conjugated antibodies against vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-Selectin. Isotype controls were used. Cells were incubated for one hour at room temperature with gentle agitation. Immunoreactivity was analysed using Beckman Coulter FC 500 and FlowJo analysis software.

Western Blotting

Proteins were extracted from placental tissues with RIPA buffer. Equal amounts of proteins were separated on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (GE Healthcare, UK). Subsequent blots were blocked with LICOR intercept blocking buffer and incubated with anti-CSE (Proteintech) and anti-pactin (Sigma) at 4°C overnight. Antibody reactions were detected using LICOR secondary antibody system. Densitometry was carried out using image studio software.

Enzyme linked immunosorbent assay

Enzyme-linked immunosorbent assay (ELISA) kits for murine sVEGFRl/Flt-1, sEng, TNFo, IL-6 and kidney injury molecule-1 (KIM-1) were obtained from R&D Systems and performed according to the manufacturer's specifications.

Real-time Polymerase Chain Reaction

Placental RNA was prepared using Qiagen RNeasy mini kit and was reverse-transcribed into cDNA using Evoscript cDNA synthesis kit (Roche). Triplicate cDNA samples and standards were amplified in LightCycler® 480 SYBR Green I Master (Roche Life Science) with primers specific for mouse CSE (forward; 5'-TTC CTG CCT AGT TTC CAG CAT-3, reverse; 5'-GGA AGT CCT GCT TAA ATG TGG TG-3') and (3-actin (forward; 5'- CGTGAAAAGATGACCCAGATCA-3', reverse; 5'-TGGTACGACCAGAGGCATACAG-3') The relative expression of target mRNA levels was quantified relative to that of the control β- actin from the same reaction.

CSE activity assay

Cysteine production was measured using a sensitive colorimetric reaction as previously described³⁰. The standard assay was performed with 35μ I of lysate (2mg) in the presence of 0.5mg/ml BSA, 50pM PLP, ImM DTT and 200mM Bis-Tris Propane buffer pH 8.25 to make a 200pl volume total. The reaction was started with the addition of cystathionine (40mM). This was incubated at 37°C for one hour. Mixing 50pl of the incubated lysate with 50pl ninhydrin reagent and 50|j I glacial acetic acid terminated the reaction. The tubes were boiled for 10 minutes on a heat block and then rapidly cooled in cold tap water. The contents were diluted with 850pl of 95% ethanol. Absorbance of the samples was measured at 560nm.

To correct for cysteine already present in the tissue, another set of the same samples was assayed alongside the originals. Cystathionine was not added to these samples. The cysteine present in these samples at the end of the protocol could be deemed natural and used to normalise the data treated with cystathionine.

Trimethylsulfonium measurement

Urine was collected from pregnant mice fed HFD or CD using a metabolic cage. Urine was vortexed for 30 seconds before being filtered through 0.2pm nylon filters. The samples were diluted 1 : 10 in samples buffer (80% acetonitrile with lOmM ammonium formate).

Commercially available trimethylsulfonium (TMS) iodide and isotopica I ly labelled d9-TMS, were analysed using liquid chromatography on-line coupled to the ESI-QqLIT- MS/MS. TMS and d9-TMS were individually injected to identify optimal fragments based on their abundance for multiple reactions monitoring (MRM) analysis. The precursor ion scans were performed between 40 m/z to 100 m/z mass range with ESI-MS in a positive ion mode. At least three diagnostic product ions were selected for each analyte, and collision energy, depolarisation potential and exit potential was optimised for each transition pair. Urine samples (lOpI) were separated on a cation exchange column using mobile phase, at 25°C. Flow rate was maintained at 250pl/min. Acquired data were processed using Analyst Software (version 1.6.2, AB Sciex).

Statistical analysis

Statistical comparison between two groups was performed using Student t-test, paired t- test or Mann-Whitney U-test where appropriate. Comparisons between three or more groups were performed using one-way ANOVA. Statistical significance was set at P<0.05. Distribution curves were creating by constructing fetal weight histograms and performing non-linear regression (Gaussian distribution). Results

HFD consumption induces a preeclampsia-like phenotype in mice

To study the impact of pre-pregnancy low-grade chronic inflammation on pregnancy outcomes, four-week-old mice were fed a diet consisting of 45% fat (HFD) and monitored for weight gain. Compared to a normal CD, the HFD-fed mice had significantly increased body weight after eight weeks of consumption (Fig.5A). Non-pregnant mice also showed three times more liver TNFo protein levels compared to age-matched CD-fed mice (Fig5B).

To determine the effects of HFD consumption on cardiovascular function, blood pressure was measured in pregnant mice at E17.5 and in age-matched non-pregnant controls. Compared to CD, HFD consumption had no effects on the mean arterial pressure (MAP) in non-pregnant animals, whereas in pregnant mice, MAP was significantly increased in mice fed with HFD (Fig. 1A). This was also seen in both systolic (Fig. 6A) and diastolic (Fig. 6B) blood pressure. Similarly, levels of kidney injury marker, KIM-1, were elevated in HFD-fed pregnant mice but not non-pregnant mice when compared to CD fed animals (Fig. IB).

Fetal weight distribution analysis of offspring from HFD-fed dams showed larger variation compared to CD pregnancy. Fetuses from HFD-fed dams were three times more likely to be of low birth weight with 24.1% of offspring falling below the 10^th percentile (Fig. 1C) compared to 7.2% of fetuses from CD-fed dams. Placental weights were also significantly smaller from the HFD-fed pregnancies when compared to CD placentas (Fig. ID).

In order to provide sufficient blood supply to the placenta, flow resistance in the uterine arteries decreases progressively in normal pregnancy³¹. Failure to reduce resistance to blood flow and sustain placental perfusion has previously been identified in pregnancy complications such as preeclampsia and fetal growth restriction (FGR)³²' ³³. To further investigate the influence of HFD on vascular adaptation during pregnancy, uterine artery flow resistance was examined in mice fed with HFD at two time points, EO (non-pregnant baseline) and E17.5 (termination) by Doppler ultrasound. The resistance index (RI) of uterine arteries from dams maintained on either CD or HFD was significantly decreased at E17.5 compared to EO (Fig. IE), indicating that vascular adaptation had taken place during pregnancy in both groups. Interestingly, regardless of the status, pregnancy or non-pregnancy, there were no significant differences in RI observed between the HFD and CD groups.

Pulsatility index (PI), marker of distal resistance and vascular sufficiency for placental perfusion, was also calculated. We found that PI of the uterine artery was significantly reduced in pregnant mice fed with either HFD or CD (Fig. IF). Interestingly, compared to mice fed with CD, HFD consumption increased the PI in both non-pregnant and pregnant mice. An increase in the uterine artery PI during pregnancy is indicative of impaired placental perfusion and vascular tree development³²' ³⁴. Taken together, these findings confirmed that uterine artery blood flow was impeded by the consumption of HFD in pregnancy, and that HFD increased the risk of developing preeclampsia partly due to insufficient vascular adaptation.

HFD increases the levels of anti-angiogenic factors and pro-inflammatory cytokines during pregnancy

Inflammation and angiogenic imbalance are associated with preeclampsia. We found that placental levels of the key pro-inflammatory cytokines, TNFo and interleukin-6 (IL-6) were significantly increased in HFD-fed dams compared to CD controls (Fig. 2A, B). Interestingly, the levels of anti-angiogenic proteins, sFlt-1 and sEng were also increased in maternal plasma at E17.5 in HFD-fed dams, following normalisation for live litter size (Fig. 2C, D). This is similar to the expression pattern seen in women suffering from preeclampsia.

HFD exacerbates vascular complications during pregnancy and anti-angiogenic factors promote pro-inflammatory cytokine-induced endothelial dysfunction We propose that under excess inflammation and a high anti-angiogenic condition induced by HFD consumption during pregnancy, maternal endothelial function is compromised. To determine the impact of HFD on maternal peripheral vascular function during pregnancy, mesenteric artery reactivity was measured at E17.5. Vessel contractility in response to phenylephrine was significantly increased in HFD-fed dams compared to CD-fed controls in a dose-dependent manner (Fig. 3A). Endothelium-dependent relaxation induced by acetylcholine (ACh) was impaired in HFD-fed pregnant dams (Fig. 3B). In addition, vessels from HFD-fed dams were consistently unable to relax to the same degree as control vessels with an Emax of 59.1% for HFD vessels and 84.0% for the CD group (Table 1). The dose of ACh needed to result in at least 50% of the total response (pD₂) was also higher in HFD vessels (Table 1), indicative of a systemic vascular dysfunction.

To further investigate the specific relationship between angiogenic imbalance and inflammation on endothelial function, HUVECs were transfected with adenovirus (Ad) encoding sFlt-1 or sEng alone, or AdsFlt-1 and AdsEng together. An empty vector (AdCMV) was used as a control. Overexpression of sFlt-1 and sEng was confirmed in the conditioned media (Fig. 7A, B). AdsFlt-1 and/or AdsEng treatment in the absence of TNFo, did not induce changes in the cell surface expression of endothelial cell activation markers, VCAM- 1 or ICAM-1 (Fig. 3C, D). However, overexpression of sFlt-1 or sEng significantly increased TNFo-induced up-regulation of ICAM-1 on the cell surface at higher doses (5-10ng/ml). Furthermore, the combination of AdsFlt-1 and AdsEng significantly augmented TNFo- induced endothelial cell activation as evidenced by the increased expression of VCAM-1 or ICAM-1 as compared to cells pre-treated with AdsFlt-1 or AdsEng alone, suggesting that sFlt-1 and sEng act synergistically to enhance TNFo mediated endothelial activation.

HFD consumption dysregulates CSE expression in endothelium and H2S rescues HFD induced vascular complication

H2S is an important vasodilator²⁹. H₂S-producing enzyme, cystathionine-y lyase (CSE), plays a crucial role in pregnancy as inhibition of CSE activity induces preeclampsia-like signs and up-regulates sFlt-1 and sEng production¹⁸. We found that expressions of CSE in placenta (Fig. 8A, B) and endothelium of mesenteric arteries (Fig. 4A) were reduced in HFD-fed dams compared to CD controls. Furthermore, CSE enzyme activity in HFD placentas was also significantly decreased reducing H2S producing potential (Fig. 8B).

To further determine the impact of HFD on CSE/H2S pathway, trimethylsulfonium (TMS), a methylated metabolite of H2S, was measured in urine as a marker of H2S metabolism by mass spectroscopy³⁵' ³⁶. The expelled levels of TMS were severely decreased in HFD fed pregnant dams compared to pregnant controls (Fig. 4B). This decrease was not due to dietary sources of methionine and cysteine, which are upstream of TMS production³⁵ as both methionine (CD 0.37%, HFD 0.53%) and cysteine (CD 0.35%, HFD 0.42%) are higher in the HFD composition.

To confirm that CSE down-regulation under inflammatory and high anti-angiogenic conditions is responsible for HFD induced endothelial dysfunction, vessel activities were measured in mesenteric arteries isolated from pregnant CSE knockout (CSE^-/-) mice fed with HFD. It was noted that the CSE^-/-HFD animals did not undergo the same increases in weight and body fat as the wild-type mice (CSE^+/+) from the same genetic background (data not shown). We found that unlike in wild-type mice, there were no significant differences in vessel contractility (Table 2) or endothelium-dependent relaxation (Fig. 4C, Table 2) between HFD and CD groups in CSE^-/- pregnant mice. In addition, maximum relaxant effect in response to ACh in CSE^-/- mice fed with CD was similar to that of CSE^+/+ mice fed with HFD suggesting that HFD induced endothelial dysfunction during pregnancy was mediated by CSE dysregulation (Table 2).

Consistent with the above notion, exogenously adding the slow-releasing H2S donor, GYY4137, was able to restore endothelium dependent vessel relaxation in HFD exposed vessels (Fig. 4D).

Discussion

In this study, we investigated the impact of chronic inflammation on maternal endothelial function during pregnancy. We found that HFD consumption induced elevated levels of maternal pro-inflammatory cytokines and anti-angiogenic factors, accompanied by impaired endothelial relaxation and adverse pregnancy outcomes, importantly, we identified the novel mechanism by which HFD causes endothelium dysfunction, which is through down-regulation of CSE, the key H2S producing enzyme in the vasculature.

High fat food consumption is the leading cause for obesity throughout the world³⁷. It has been increasingly recognised that obesity is an inflammatory disease, and that high fat consumption is the major contributor of the observed chronic inflammation³⁸. More importantly, chronic inflammatory conditions such as obesity and diabetes, increase the overall risk of preeclampsia by approximately 2 to 3-fold³' ⁵' ²⁸, albeit the underlying mechanisms are not fully understood. Widespread endothelial dysfunction is a common phenomenon in preeclampsia and chronic inflammatory conditions such as obesity³⁹ contributing to both the pathophysiology of preeclampsia and obesity-related cardiovascular diseases. This suggests that there may be overlapping mechanisms between these two conditions. Although it is evident that inflammation is associated with preeclampsia, it is unlikely that excess inflammation is the cause⁴⁰. Indeed, we found that high fat intake increased the levels of pro-inflammatory cytokines in both pregnant and non-pregnant animal groups, however, elevated arterial blood pressure and kidney damage were only observed in pregnant mice fed with HFD. This shows that inflammation per se is not the cause but pregnancy itself is a related risk factor contributing to the impaired cardiovascular and kidney function.

As imbalance of angiogenic factors are key features of preeclampsia. The loss of VEGF activity due to increase in sFlt-1²¹ and the loss of TGF-β activity as a consequence of elevated sEng⁴¹' ⁴², appear to be responsible for the clinical presentation of preeclampsia. In the present study, we found that high fat intake not only increased the levels of inflammatory cytokines but also circulating levels of sFlt-1 and sEng in pregnant mice. Furthermore, endothelium-dependent vasodilation was compromised in these animals. It is likely that chronic inflammation works in concert with anti-angiogenic factors during pregnancy, to promote endothelial dysfunction. Indeed, consistent with a study by Cindrova-Davies and collegues⁴³, we found that loss of VEGF activity by overexpression of sFlt-1 sensitises endothelial cells to inflammatory cytokine stimulation. Additionally, we found that compared to sFlt-1 or sEng alone, the combination of overexpression of sFlt-1 and sEng in endothelial cells further exacerbated TNF-o-induced endothelial activation. This supports our premise that under chronic inflammatory conditions, the rise in anti- angiogenic factors acts to disrupt normal vascular homeostasis.

Increased production of nitric oxide (NO)⁴⁴ and H2S⁴⁵ during pregnancy indicates that these factors play important roles in maintaining vascular homeostasis in pregnancy. Vascular H2S production is largely regulated by CSE²⁹ and the loss of CSE in mice leads to increased blood pressure and impaired vessel relaxation¹⁸' ²⁹. Previously, we have discovered that compared to normotensive pregnancy, women with preeclampsia had much lower levels of circulating H2S¹⁸ and this was associated with reduced placental expression of CSE. These discoveries are further supported by a more recent study that found reduced H2S levels in the blood serum of women with severe preeclampsia⁴⁶. Our present study shows for the first time that down-regulation of vascular CSE leads to the HFD-induced endothelial dysfunction and poor outcomes in pregnancy. Interestingly, HFD has been reported to up-regulate the expression of CSE in liver and epididymal fat⁴⁷' ⁴⁸. It is likely the increase of CSE expression is to protect the organ against the excessive inflammation as H2S has anti-inflammatory properties⁴⁹' ⁵⁰. However, during pregnancy, this chronic inflammation is accompanied with progressively increased level of anti- angiogenic factors such as sFlt-1⁵¹. More importantly, CSE and H2S negatively regulate the production of anti-angiogenic factors such as sFlt-1 and sEng in pregnancy¹⁸' ²⁵' ⁵². As discussed earlier, the excess of anti-angiogenic factors under the chronic inflammatory condition results in exacerbated widespread endothelium dysfunction.

Overall, our data strongly support the idea that HFD increases the risk of preeclampsia due to the combined insults of inflammation and anti-angiogenic factors on the endothelium through compromising CSE/H2S protective pathway. Our findings provide a potential therapeutic intervention for the obese individuals who are more likely to develop pregnancy complications.

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Table 1 . Maximum responses (Emax) and pD2 values of phenylephrine (PE) and Asyinmesenteric arteries from pregnant mice fed a chow diet (CD) or high fat diet (HFD).

Table 2. Maximum responses (Emax) and pD2 values of phenylephrine (PE) and acetylcholine (ACh) in mesenteric arteries from pregnant CSE+/+ and CSE-/- mice fed a chow diet (CD) or high fat diet (HFD).

Claims

Claims

1. Hydrogen Sulphide (H2S), or a (H2S) generating compound or compound capable of stimulating H2S production in an overweight or obese pregnant subject for use in the treatment or prevention of pre-eclampsia (PE)or fetal growth restriction or preterm birth

2. A method of treating or preventing pre-eclampsia (PE) or fetai growth restriction comprising administering to an overweight or obese pregnant subject, a pharmaceutically effective amount of H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject.

3. H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject for use in the treatment pre-term labour.

4. A method of treating pre-term labour comprising administering to a pregnant subject, a pharmaceutically effective amount of H2S, an H2S generating compound or a compound capable of inducing H2S production in the subject.

5. A use or method according to claims 1 to 4 wherein the compound is H2S, ACS 14, AC583, ACS 84, ACS 85, ACS 86, DATS (diallyl trisulphide), S-diclofenac, sulfane sulphur, thiocysteine, GSH hydropersulphide, GYY4137, SG1002, a H2S-donating derivative of sildenafil, ADT-OH, TBZ and 4-hydroxyphenyl isothiocyanate, thioglycine, 1-thiolysine, 1- thiovaline, or salts thereof.

6. A method of monitoring treatment of pre-eclampsia or impaired fetal growth in overweight or obese pregnant subject, comprising measuring the amount of H2S is a sample of blood, serum or plasma in a subject prior to treatment and comparing it to the amount in a sample taken after treatment.

7. A method according to claim 6, wherein the subject has been treated with H2S, a H2S generating compound or compound of stimulating H2S production in a pregnant subject.