**4. RAS and gestational pathophysiological conditions**

Since the RAS has a wide array of important functions in the body, any dysfunction in this system may lead to complications [41]. Studies have shown that the RAS is involved in repro‐ ductive conditions such as preeclampsia, polycystic ovary (PCOS) [76]. Moreover, it has a role in tumour progression in gynaecological cancers, highlighting the implication of the RAS in on tumour cell proliferation, vascular function and angiogenesis [54]. The following sections will describe the implication of RAS in the development of gestational pathologies, with the main emphasis being put on preeclampsia.

### **4.1. Polycystic ovary syndrome**

**2.6. RAS and the uterus/endometrium**

90 Renin-Angiotensin System - Past, Present and Future

**3. Pregnancy and RAS**

for the growing placenta and foetus [64].

Most components of the RAS can be found in both myometrium and endometrium of the uterus. However, the role of the RAS in the non‐pregnant uterus is still unknown [55]. Elevated expression and secretion of prorenin in stromal cells have been associated with decidualisation of the endometrium in early to mid‐proliferative phase [56]. Activation of the (P)RR by prorenin has been shown to promote vascular endothelial growth factor (VEGF) expression and could thus increase vascularity of the decidua to ensure an adequate blood flow to the placenta [56]. In addition, Ang II as well as AT1R and AT2R show a cyclical pattern of expression depending on the phase of the uterine cycle. First, AT2R is expressed at higher levels compared to AT1R, although both receptors show a similar expression pattern. Their expression gradually increases during the proliferative phase, reaching a maximum in late proliferative and early secretory phases, followed by a gradual decrease in expression through the rest of the secretory phase [57]. In comparison, plasma Ang II levels gradually increase through the menstrual cycle, reach‐ ing a peak in the late secretory phase [58]. Moreover, in the early to mid‐proliferative phase, endometrial Ang II levels and ATRs expression are mostly localized to the glandular and stro‐ mal cells of the endometrium, which could highlight a role for the RAS in modulating decidu‐ alisation and neovascularisation of the endometrium. Alternatively, in late secretory phase, they are localized mostly around blood vessels, where Ang II could contribute to the vasoconstriction of spiral arterioles which is necessary for the induction of menstruation [57]. In addition, angio‐ tensin‐(1‐7) (Ang‐(1‐7), a heptapeptide generated from Ang II cleavage by the enzyme ACE 2) and its receptor MAS (MAS‐R) have been shown to be expressed in the endometrium. While MAS‐R expression is localized to the epithelial and stromal cells and does not change through‐ out the menstrual cycle, Ang‐(1‐7) concentrations are highest in the glandular epithelium and in the stroma of the endometrium in mid‐ to late‐secretory phase [59]. Although the function of the Ang‐(1‐7)—Mas‐R axis is not well understood in the endometrium, by its vasodilatory, antiangiogenic and antimitotic properties, Ang‐(1‐7) could counterbalance Ang II actions and,

possibly regulate endometrial regenerating processes according to homeostatic needs.

Pregnancy is characterized by an elevation in the levels of maternal circulating estrogen. Consequently, maternal circulating prorenin and renin are also increased during pregnancy. Prorenin reaches a peak within 20 days after conception and remains high until parturition while plasma‐renin activity rises during the first few weeks of pregnancy [60]. ACE is the only RAS component that decreases during pregnancy [61] while plasma Agt and Ang II levels are particularly elevated during the last trimester of normal gestation [62]. The elevated Ang II levels could be attributed in part to the stimulatory effect of estrogen on Agt expression but also to the elevated renin levels [63]. In addition, increased urinary and plasma aldosterone levels are observed during pregnancy which produces the increased plasma volume required

The increase in RAS in pregnant women should normally be associated with an increase in blood pressure. However, elevated blood pressure in not typically observed during normal Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility in women of reproductive age. Evidence of enhanced systemic RAS activity (increased plasma renin, Ang II and aldosterone) has been demonstrated to be responsible for the development of this disease [76, 77]. In PCOS patients, the maturation and oocyte quality are both affected by the increased intra‐follicular renin level [54]. Moreover, there is evidence indicating that a polymorphism in ACE gene is associated to insulin resistance (IR) in women with PCOS [76, 78, 77]. Thus, treatment with ACE inhibitors aiming at increasing insulin sensitivity could result in an increased fertility in PCOS patients, but since RAS inhibitors are known to be teratogenic, further studies and much care would be needed to validate this therapeutic approach.

### **4.2. Ovarian cancer**

Ovarian cancer is the most lethal gynaecological malignancy in women worldwide [79]. Ovarian cancer cells express Ang II and AT1R [80]. Elevated AT1R levels have been mea‐ sured in borderline lesions and in invasive epithelial ovarian cancers [81]. Moreover, prog‐ nosis is worse for patients with tumours expressing high AT1R levels compared to patients with AT1R‐negative tumours. The Ang II—AT1R pathway stimulates cell proliferation while the simultaneous increase in VEGF expression and Ang II levels promotes angiogenesis [54]. Therefore, targeting the Ang II‐AT1R pathway could be part of a future treatment strategy for invasive epithelial ovarian cancer.

### **4.3. Endometrial cancer**

Endometrial cancer (EC) is the most common gynaecological malignancy. Moreover, since obesity is a major risk factor, its incidence could increase in the future in parallel with the growing metabolic syndrome pandemic [82]. The endometrial RAS, like other tissue RASs, has been implicated in angiogenesis, neovascularisation and cell proliferation, which are processes involved in tumour growth and metastasis. Increased expression of Ang II, AT1R, AT2R, VEGF and estrogen receptor alpha (NR3A1) has been identified in EC tissues [83]. Moreover, a strong positive correlation has been detected between the levels of Ang II and AT1R/AT2R expression in endometrial tumours with advancing stage of the tumour [54, 83]. Overactivation of the RAS can often be attributed to single nucleotide polymorphisms (SNPs) in a RAS gene [84]. In a study by Freitas‐Silva et al., an ACE polymorphism was described to be associated with early onset of EC. In summary, high activity of the local RAS in endome‐ trial cancer is associated with higher incidence, earlier onset and increased rates of angiogen‐ esis [54].

### **4.4. Preeclampsia**

### *4.4.1. Definition of the pathology*

Preeclampsia is a gestational complication that affects 2–5% of women in North America [85]. Preeclampsia risk factors include primiparity, multiparity as well as pre‐existing conditions such as type 2 diabetes mellitus, obesity, hypertension and thrombophilia [86]. Moreover, women with preeclampsia are more likely to develop cardiovascular diseases later in life [87]. Clinical diagnostic is determined by the presence of new onset of hypertension (systolic pres‐ sure ≥140 mmHg or diastolic pressure ≥90 mmHg) and proteinuria (≥300 mg in 24h) after 20 weeks of gestation. Other potential clinical manifestations are placental alterations, cerebral ischemia, liver abnormalities, cardiac hypertrophy and impaired vascular reactivity, although they are not seen in all preeclamptic women [88]. Patients with severe preeclampsia can also develop pulmonary oedema, haemolysis, elevated liver enzymes and low platelets syndrome, severe central nervous system symptoms, renal failure and intrauterine growth restriction [89].

Several factors have been involved in the development of preeclampsia, such as placental abnormalities, oxidative stress, endothelial dysfunction, inflammation and immunity, but none have been clearly proven [86]. Preventive therapies such as antioxidants have not demonstrated any beneficial effects while calcium supplementation only helps patients with calcium deple‐ tion [90, 91]. Therefore, physicians usually try to control the progression of the disease using antihypertensive therapies, such as methyldopa (an α‐adrenergic agonist), labetalol (an α‐ and β‐blocker) and nifedipine (a calcium channel antagonist), which are considered relatively safe for the foetus. On the contrary, other drugs, such as RAS inhibitors, which are teratogenic and diuretic, are not compatible with regards to the hypovolemic state associated with preeclamp‐ sia. As such, they are not recommended for the treatment of this disease [92]. Ultimately, pre‐ mature delivery of the foetus is the only effective treatment available, which can be problematic if the development of the foetus, has not sufficiently progressed.

### *4.4.2. Preeclampsia and RAS*

**4.2. Ovarian cancer**

invasive epithelial ovarian cancer.

92 Renin-Angiotensin System - Past, Present and Future

**4.3. Endometrial cancer**

esis [54].

**4.4. Preeclampsia**

*4.4.1. Definition of the pathology*

Ovarian cancer is the most lethal gynaecological malignancy in women worldwide [79]. Ovarian cancer cells express Ang II and AT1R [80]. Elevated AT1R levels have been mea‐ sured in borderline lesions and in invasive epithelial ovarian cancers [81]. Moreover, prog‐ nosis is worse for patients with tumours expressing high AT1R levels compared to patients with AT1R‐negative tumours. The Ang II—AT1R pathway stimulates cell proliferation while the simultaneous increase in VEGF expression and Ang II levels promotes angiogenesis [54]. Therefore, targeting the Ang II‐AT1R pathway could be part of a future treatment strategy for

Endometrial cancer (EC) is the most common gynaecological malignancy. Moreover, since obesity is a major risk factor, its incidence could increase in the future in parallel with the growing metabolic syndrome pandemic [82]. The endometrial RAS, like other tissue RASs, has been implicated in angiogenesis, neovascularisation and cell proliferation, which are processes involved in tumour growth and metastasis. Increased expression of Ang II, AT1R, AT2R, VEGF and estrogen receptor alpha (NR3A1) has been identified in EC tissues [83]. Moreover, a strong positive correlation has been detected between the levels of Ang II and AT1R/AT2R expression in endometrial tumours with advancing stage of the tumour [54, 83]. Overactivation of the RAS can often be attributed to single nucleotide polymorphisms (SNPs) in a RAS gene [84]. In a study by Freitas‐Silva et al., an ACE polymorphism was described to be associated with early onset of EC. In summary, high activity of the local RAS in endome‐ trial cancer is associated with higher incidence, earlier onset and increased rates of angiogen‐

Preeclampsia is a gestational complication that affects 2–5% of women in North America [85]. Preeclampsia risk factors include primiparity, multiparity as well as pre‐existing conditions such as type 2 diabetes mellitus, obesity, hypertension and thrombophilia [86]. Moreover, women with preeclampsia are more likely to develop cardiovascular diseases later in life [87]. Clinical diagnostic is determined by the presence of new onset of hypertension (systolic pres‐ sure ≥140 mmHg or diastolic pressure ≥90 mmHg) and proteinuria (≥300 mg in 24h) after 20 weeks of gestation. Other potential clinical manifestations are placental alterations, cerebral ischemia, liver abnormalities, cardiac hypertrophy and impaired vascular reactivity, although they are not seen in all preeclamptic women [88]. Patients with severe preeclampsia can also develop pulmonary oedema, haemolysis, elevated liver enzymes and low platelets syndrome, severe central nervous system symptoms, renal failure and intrauterine growth restriction [89]. Several factors have been involved in the development of preeclampsia, such as placental abnormalities, oxidative stress, endothelial dysfunction, inflammation and immunity, but none Dysregulation of the RAS has been observed in preeclampsia compared to women with healthy pregnancies [6, 93, 94]. In particular, contrarily to normal pregnancy, preeclamptic women suffer from a hypovolemic hypertension (as mentioned above) characterized by a reduction in plasma renin, Ang I, and Ang II levels [70]. However, PE is characterized by a heightened sensitivity to vasoconstrictors when compared to normal pregnancy [6] partly due to an upregulation of the Ang II type 1 receptors [93], which would contribute to the increased blood pressure associated with this condition. Moreover, recent human studies revealed that both plasma Ang‐(1‐7) and Ang II are increased in normal pregnancy but decreased in pre‐ eclampsia [70]. However, the analysis of the Ang‐(1‐7)/Ang II ratio demonstrates that there is a greater decrease in Ang‐(1‐7) relatively to Ang II levels in preeclamptic [70], tipping the vasopressive balance towards increased vasoconstriction in pathological pregnancies. In addi‐ tion, many epidemiological studies have suggested a relation between alleles of the RAS and PE [95]. For instance, women carrying specific polymorphisms of ACE [96] or Ang [97–99] genes have been reported to have an increased PE risk. Interestingly, these alleles are associ‐ ated with an increase in systemic RAS [100].

In contrast, patients with preeclampsia have also been reported to have an increased Ang II content and AT1R expression in maternal decidua and in the placenta itself. Brosnihan's group also found in placental chorionic villi from human preeclamptic pregnancies an increase in Ang II and AT1R while Ang‐(1‐7) was not elevated and the Mas‐R was significantly decreased [44]. They proposed that this increased Ang II effect in the chorionic villi could produce a decrease in foetal blood flow, and thus contribute to a reduction in foetal oxygen and nutrients as well as to the development of the intra‐uterine growth restriction observed in these pregnancies. The same group showed that the placental increase in Ang‐(1‐7) content observed during normal pregnancy was reduced in a rat model of PE (the reduced uterine perfusion pressure model), although this was not accompanied by a concomitant decrease in ACE2 [101]. Moreover, we have demonstrated that R+ A+ mice, an animal model of preeclamp‐ sia, have increased AT1Rand decreased Mas‐R protein in both placenta and aorta, a condition expected to decrease angiotensin‐(1‐7) effects in favour of angiotensin II effects [102]. The importance of different RAS components in the development of preeclampsia will be further discussed below.

### *4.4.3. Prorenin and prorenin receptor ((P)RR) and preeclampsia*

Expression of the (P)RR has been shown to be localized to the syncytiotrophoblasts both in normotensive and preeclamptic pregnant women [103]. Placental prorenin and (P)RR levels as well as the circulating soluble form of (P)RR (s(P)RR) were shown to be significantly higher in preeclamptic compared to normotensive pregnant women [104]. Moreover, placental (P)RR expression positively correlates with systolic blood pressure only in preeclamptic women. The concomitant modulations of prorenin and (P)RR in preeclamptic women reinforce the idea that an increase in RAS local activation could promote the elevation of blood pressure in this pathol‐ ogy. However, the implication of an increase in s(P)RR in the development of preeclampsia is still misunderstood.

### *4.4.4. AT1 receptors autoantibodies in preeclampsia*

In recent years, a wealth of evidence has emerged supporting a role for AT1R autoantibodies (AT1‐AA) in the development of preeclampsia. Studies have shown that these autoantibod‐ ies are elevated in patients with preeclampsia compared to normal pregnancies and have been shown to specifically stimulate Ang II type 1 receptors, suggesting that these autoan‐ tibodies may be involved in the development of preeclampsia [93, 105]. Studies in animal models of preeclampsia have shown that the hypoxia used to induce the disease (caused by the reduction in placental perfusion in pregnant rats) strongly stimulated AT1‐AA produc‐ tion [106]. Moreover, infusion of AT1‐AA from preeclamptic patients in normal pregnant animal was able to trigger hypertension through an increase in endothelin‐1 expression, a potent vasoconstrictor [107]. *In vitro* and *in vivo* studies have demonstrated the binding of those autoantibodies to AT1R on different cell types [108]. In particular, AT1‐AA binding at the surface of human trophoblastic cells cause an activation of NADPH oxidase, contribut‐ ing to the rise in oxidative stress putatively involved in the development of preeclampsia [109]. In addition, activation of AT1R in this cell‐type stimulates the release of PAI‐1, result‐ ing in decreased trophoblastic invasiveness causing a defect in placentation [110]. It was also observed that AT1‐AA stimulates the release of sFlt‐1 and s‐Eng by the placenta which stimulates endothelial dysfunction [111, 112]. Overall, these results indicate that the vaso‐ constrictor angiotensin receptor signalling is a key pathway involved in the development of PE.

### *4.4.5. RAS and angiogenic factors in preeclampsia*

A molecular hallmark of preeclampsia is a decrease in plasmatic angiogenic markers, free VEGF and placental growth factor (PlGF), along with an increase in the circulating levels of anti‐angiogenic markers, soluble fms‐like tyrosine‐1 (sFlt‐1, a soluble variant of the VEGF receptor) and soluble endoglin (sEng), compared to normal pregnancies [113–115]. The decrease in VEGF and PlGF would lead to the improper spiral artery remodelling which is associated with preeclampsia [116]. Moreover, hypoxia, through an increased expression of hypoxia‐inducible factor 1 α (HIF‐1α), stimulates the expression of sFlt‐1, and therefore amplifies the hypoxic placental microenvironment [117, 118]. HIF‐1α has also been shown to upregulate the expression of both endothelin‐1 and endoglin, a membrane‐bound precur‐ sor of sEng [119, 120]. In addition, increased secretion of sEng has been measured from both chorionic villi from preeclamptic placenta and hypoxic trophoblastic cells [121]. The increase in endothelin‐1 would promote the increase in blood pressure associated with preeclampsia, while the increase in sEng levels would prevent trophoblastic differentiation and invasion.

### *4.4.6. Beneficial effects of exercise training on preeclampsia could be through modulation of the RAS*

While exercise training is well known for its health benefits in the general population, it has also been shown to improve pregnancy outcome during normal human gestation [122]. Moreover, there are data demonstrating that it can also reduce the prevalence of human pregnancy disorders such as gestational diabetes. There is also a significant body of evi‐ dence supporting the exercise training‐induced reduction in risk of developing PE by 35% to 78% [123]. We have recently demonstrated that exercise training (mouse voluntary wheel running) before and during gestation significantly prevents the development of preeclamp‐ sia superimposed on chronic hypertension phenotypes in our mouse model of that disease [102]. We noted that the pregnant mice naturally reduce the duration and intensity of their exercise training throughout pregnancy and cease exercising 2–3 days prior to delivery, a phenomenon we call the graded intensity or GI‐exercise training program. Indeed, this GI‐exercise training program normalized the mouse preeclampsia phenotypes, and: (1) pre‐ vented the increase in blood pressure; (2) reduced the development of the proteinuria; (3) abolished the increase in placental mRNA and circulating levels of sFlt‐1; and (4) prevented the development of the placental pathology characteristic of preeclampsia, and thus also prevented the associated foetal intra‐uterine growth restriction phenotype. In support of this beneficial effect of the GI‐exercise training program, we also observed similar benefits in a mouse model of preeclampsia (*hAGT\*hREN model*; normotensive female mice which overexpress human angiotensinogen, bred with males that overexpress human renin) [124]. Interestingly, we found that these beneficial effects of exercise training in R<sup>+</sup> A+ mice were associated to a normalisation of AT1R and MasR in the placenta as well as an increase Mas receptor content in the aorta [102]. Hence, this could contribute to the prevention of the increase in blood pressure and the normalisation of placental development observed in this animal model.

### **5. Conclusion**

*4.4.3. Prorenin and prorenin receptor ((P)RR) and preeclampsia*

94 Renin-Angiotensin System - Past, Present and Future

*4.4.4. AT1 receptors autoantibodies in preeclampsia*

*4.4.5. RAS and angiogenic factors in preeclampsia*

still misunderstood.

of PE.

Expression of the (P)RR has been shown to be localized to the syncytiotrophoblasts both in normotensive and preeclamptic pregnant women [103]. Placental prorenin and (P)RR levels as well as the circulating soluble form of (P)RR (s(P)RR) were shown to be significantly higher in preeclamptic compared to normotensive pregnant women [104]. Moreover, placental (P)RR expression positively correlates with systolic blood pressure only in preeclamptic women. The concomitant modulations of prorenin and (P)RR in preeclamptic women reinforce the idea that an increase in RAS local activation could promote the elevation of blood pressure in this pathol‐ ogy. However, the implication of an increase in s(P)RR in the development of preeclampsia is

In recent years, a wealth of evidence has emerged supporting a role for AT1R autoantibodies (AT1‐AA) in the development of preeclampsia. Studies have shown that these autoantibod‐ ies are elevated in patients with preeclampsia compared to normal pregnancies and have been shown to specifically stimulate Ang II type 1 receptors, suggesting that these autoan‐ tibodies may be involved in the development of preeclampsia [93, 105]. Studies in animal models of preeclampsia have shown that the hypoxia used to induce the disease (caused by the reduction in placental perfusion in pregnant rats) strongly stimulated AT1‐AA produc‐ tion [106]. Moreover, infusion of AT1‐AA from preeclamptic patients in normal pregnant animal was able to trigger hypertension through an increase in endothelin‐1 expression, a potent vasoconstrictor [107]. *In vitro* and *in vivo* studies have demonstrated the binding of those autoantibodies to AT1R on different cell types [108]. In particular, AT1‐AA binding at the surface of human trophoblastic cells cause an activation of NADPH oxidase, contribut‐ ing to the rise in oxidative stress putatively involved in the development of preeclampsia [109]. In addition, activation of AT1R in this cell‐type stimulates the release of PAI‐1, result‐ ing in decreased trophoblastic invasiveness causing a defect in placentation [110]. It was also observed that AT1‐AA stimulates the release of sFlt‐1 and s‐Eng by the placenta which stimulates endothelial dysfunction [111, 112]. Overall, these results indicate that the vaso‐ constrictor angiotensin receptor signalling is a key pathway involved in the development

A molecular hallmark of preeclampsia is a decrease in plasmatic angiogenic markers, free VEGF and placental growth factor (PlGF), along with an increase in the circulating levels of anti‐angiogenic markers, soluble fms‐like tyrosine‐1 (sFlt‐1, a soluble variant of the VEGF receptor) and soluble endoglin (sEng), compared to normal pregnancies [113–115]. The decrease in VEGF and PlGF would lead to the improper spiral artery remodelling which is associated with preeclampsia [116]. Moreover, hypoxia, through an increased expression of hypoxia‐inducible factor 1 α (HIF‐1α), stimulates the expression of sFlt‐1, and therefore amplifies the hypoxic placental microenvironment [117, 118]. HIF‐1α has also been shown

In conclusion, the reproductive system's local RAS has been clearly shown to be implicated in fertility, reproduction and pregnancy. Moreover, dysregulation of the RAS has been asso‐ ciated with gestational pathologies, although more work is needed to clearly identify the molecular mechanisms involved. As such, the development of new therapies aiming at ampli‐ fying the vasodilating arm of the RAS could help in improving both maternal and foetal out‐ comes although caution needs to be taken given that RAS inhibitors have been shown to be teratogenic.
