According to the American Heart Association, men are more likely than women to have hypertension between the ages of 20 and 65. Nonetheless, beyond that age range, women have a higher prevalence of hypertension [16]. Furthermore, hypertension is more common in women aged 45–54 and 55–64, the ages at which the majority of women enter menopause. This is consistent with previous findings that estrogen protects against hypertension and CVD in premenopausal women versus men [17]. On the other hand, hypertension is more common in RA patients, with studies estimating that 53–71% of RA patients are hypertensive [18]. It has also been observed that hypertensive RA patients receiving antihypertensive medication still had significantly higher blood pressure compared to non-hypertensive RA patients [19]. Furthermore, RA patients are more likely to develop CVD than healthy people [20]. Since it is known that RA is more prevalent in women around menopausal age, in this study we investigated the changes of blood pressure in ovariectomized rats (Ovx), which mimic postmenopausal state, and RA-induced ovariectomized rats (Ovx + RA), which represent postmenopausal RA state. We then evaluated the effects of oestrogen, a commonly used HT in postmenopausal women, on the blood pressure in RA induced ovariectomised rats (Ovx + RA + Oestrogen). Additionally, we also evaluated blood pressure changes in RA induced ovariectomised rats treated with baricitinib (Ovx + RA + Baricitinib), a JAKi used in RA treatment, and losartan (Ovx + RA + Losartan), an ARB used to treat hypertension, as comparison drugs to oestrogen treatment.
Our findings show that ovariectomized (Ovx) rats have significantly higher SBP, DBP, and MAP than sham rats, which is consistent with a previous study showing that postmenopausal women have higher SBP and DBP than premenopausal women [21]. Furthermore, when compared to the Ovx rat’s, the Ovx + RA rat’s had significantly higher SBP and partially elevated MAP, lending credence to the notion that RA contributes to hypertension. However, when compared to Ovx + RA rats, estrogen treatment significantly reduced SBP, MAP, and DBP, indicating that estrogen is protective in blood pressure regulation. Previous research has shown that estrogen therapy lowers blood pressure in postmenopausal women [22, 23]. Baricitinib treatment also significantly reduced SBP and partially decreased DBP and MAP in Ovx + RA rats. This could be attributed to baricitinib's beneficial effects on RA disease activity and the associated reduction in hypertension [24]. Furthermore, among the three treatment groups, losartan treatment resulted in the most significant decrease in blood pressure. This finding was in line with the current knowledge and the wide clinical use of losartan as an anti-hypertensive medication.
This study examined the histological changes associated with blood pressure in ovariectomized collagen-induced arthritis rats. Our H&E results revealed that the tunica adventitia layer of the aorta thickened in the Ovx and Ovx + RA rats, and oestrogen treatment reversed these changes. The Picro-sirius staining demonstrated increased collagen deposition in the aorta in the Ovx and Ovx + RA rats, and oestrogen treatment reduced this collagen deposition. The changes observed in the tunica adventitia thickness in our study may be associated with the changes in collagen deposition. Various studies have shown that hypertension leads to increased collagen synthesis and hypertensive aortas have elevated collagen levels [25]. These findings are supported by our study where we demonstrated similar patterns of changes between the blood pressure and collagen deposition in the aorta. The aorta's tunica adventitia layer was thickened in the Ovx and Ovx + RA groups, consistent with previous research reporting the collagen deposition and aortic stiffening in RA patients [26, 27]. The Ovx and Ovx + RA groups had more collagen deposition in the aorta, whereas estrogen, baricitinib, and losartan treatments all reduced this collagen deposition. These findings are consistent with previous studies that show estrogen benefits aortic collagen deposition and arterial stiffness [28, 29]. Our study also observed that baricitinib and losartan treatments reduced the thickness of the tunica adventitia layer and slightly reduced collagen deposition in the aorta, which may be related to the less severe RA caused by these medications. These results were supported by a study which showed that angiotensin Ⅱ promotes collagen production through the AT1 receptors in the aortic adventitial fibroblast, and losartan inhibits this collagen production by antagonising Angiotensin (AT1) receptor [30].
In this study, we investigated the mechanism underlying blood pressure changes in rats by examining various oxidative, inflammatory, growth, fibrosis, and apoptosis markers in the aorta tissues through immunohistochemistry, immunofluorescence, and qPCR. According to our findings, eNOS mRNA level, iNOS, Nox2, and Nox4 protein expressions increased in the aorta of Ovx + RA rats and the expressions decreased with oestrogen treatment. The oxidative stress has been attributed to upregulated Nox and NOS activity [31]. Previous research conducted on RA rat model have showed that eNOS mRNA expression and protein level were significantly elevated, while iNOS mRNA expression and protein level were not changed in the aorta. However, the eNOS dimer to monomer ratio was decreased suggesting eNOS uncoupling [31]. This suggests that despite the elevated levels of eNOS mRNA expression observed in our study, the eNOS enzyme present in our Ovx + RA rats may be highly uncoupled. Similar to our iNOS findings, another study demonstrated upregulated iNOS activity in RA patients [32].
It is known that eNOS uncoupling is associated with diminished NO production, elevated ROS production and oxidative stress [33]. Additionally, although iNOS produces NO which is crucial for vasodilation, it is known that overexpression of iNOS may lead to production of reactive nitrogen species which may contribute to inflammatory and oxidative stress seen in RA [34]. Additionally, Nox4 is widely known to produce ROS suggesting that the elevated Nox4 observed in our Ovx + RA rats also contribute to the oxidative stress.
Nox2 has also been widely studied in RA and is known to produce ROS contributing to oxidative stress, and these ROS are also known to regulate the chronic inflammation observed in RA through various immune mechanisms [34]. Although Nox4 expression has not been previously studied in the cardiovascular system in RA, Nox4 is widely known to produce ROS suggesting that the elevated Nox4 observed in our Ovx + RA rats also contribute to the oxidative stress.
Studies have shown that estrogen reduces oxidative stress in the vascular system and myocardium by regulating NOS and NOX, resulting in lower ROS production and increased NO synthesis [35, 36]. Furthermore, estrogen increases the availability of Tetrahydrobiopterin (BH4), which prevents eNOS uncoupling and ROS production [36]. Studies have also shown that oestrogen related eNOS inhibition may occur through oestrogen receptors ER-α, ER-β and GPR30 [37], while oestrogen related Nox4 inhibition may occur through GPR30 [38]. In our study, the reversal of oxidative markers suggests that the estrogen treatment reduced oxidative stress in Ovx + RA + Oestrogen rats. Additionally, we also showed that eNOS mRNA expressions and Nox4 protein expressions were reduced with baricitinib and losartan treatment. These findings suggest that baricitinib may also exert some anti-oxidative effects through the inhibition of JAK signalling. The anti-oxidative effects observed with losartan treatment in this study are also supported by previous findings that have demonstrated protective effects of losartan against oxidative stress [39].
In addition to oxidative stress, we looked at the expression of inflammatory markers linked to RA, such as TLR4, Phospho IĸBα/β, NF-ĸB and TNF-α. Protein expressions of TLR4, TNF-α, Phospho-IKKα/β, and NF- ĸB were found to be higher in the aorta of Ovx + RA rats and lower when estrogen was administered. TLR4 signaling has been linked to an increase in the occurrence and severity of RA [40], and our findings indicate that estrogen therapy reduces inflammation by inhibiting the TLR4- NF-ĸB signaling pathway. Furthermore, treatment with baricitinib and losartan decreased TLR4, TNF-α and NF-ĸB protein expression, suggesting they may exert anti-inflammatory effects via the TLR4-NF-ĸB pathway. Previous research has also shown that losartan treatment reduces upregulated TLR4 expression in the hypertensive aorta [41], implying that TLR4-NF-ĸB-associated inflammation may play a role in hypertension. Additionally, we observed upregulated NF-ĸB expressions in the aorta of our Ovx + RA rats and it is known that prolonged activation of NF-ĸB in the cardiovascular is detrimental due to the corresponding chronic inflammatory changes [42] suggesting that this may be one of the contributing factors of increased CVD risk in RA patients. It is known that NF-ĸB can be activated by various factors such as TNF, IL-1, IL-17, IL-18, LPS and H2O2 [43]. Activation by TNF leads to the serine phosphorylation, ubiquitination, and degradation of the inhibitory IĸBα protein. The degradation of the inhibitor protein allows the p65 subunit phosphorylation and translocation of NF-ĸB into the nucleus, where it binds to DNA allowing gene transcription [43]. Since it is known that in RA both TNF and ROS are elevated, the NF-ĸB activation observed in our Ovx + RA rats may have occurred through both these pathways as well as other IL associated pathways. The changes in expression of Phospho IĸBα/β and NF-ĸB noted in our study can be explained by the IĸBα degradation associated with TNF-induced NF-ĸB activation.
Chronic RA inflammation has been linked to increased synovial angiogenesis and vascular reorganization [44]. Inflammation plays a vital role in angiogenesis [45]. While angiogenic growth factors like VEGF, TGFβ1 and FGF2 have been studied in the joint development of RA [46, 47], their expression and effects on the cardiovascular system in RA are unknown. Thus, we further evaluated the expression of growth factors and their signaling and fibrosis factors such as TGFβ-1, VEGF, FGF2, αSMA, Smad2 and Fibronectin in the aorta.
We observed that VEGF protein expression and FGF2 mRNA level were increased in the aorta of Ovx + RA rats and the expressions were decreased with oestrogen treatment. In line with our findings, studies on other diseases such as retinopathy [48] and breast cancer [49] had previously demonstrated that oestrogen may decrease VEGF expression. Our findings support previous research that serum VEGF levels are significantly higher in RA patients [50] and that IL-6, a critical inflammatory marker in RA, is linked to increased VEGF expression [51]. Since we have demonstrated elevated levels of NF-ĸB expression in the aorta of Ovx + RA rats, the downstream elevation of IL-6 may have contributed to the upregulated VEGF expression observed in our study. However, our findings contradict previous research, which found that estrogen protects the cardiovascular system after injury by increasing VEGF expression and angiogenesis [52]. This suggests that the regulatory effect of estrogen on VEGF expression in the cardiovascular system may differ depending on the tissues and underlying disease.
In addition to VEGF, our experiment observed that FGF2 mRNA expression was upregulated in the aorta of Ovx + RA rats and that estrogen treatment decreased this expression. FGF2 has been shown to promote fibroblast proliferation and fibrosis in the myocardium [53], and its expression in the endothelium is upregulated in response to inflammation [54]. Our findings suggest that increased FGF2 expression in our Ovx + RA rats contributed to collagen deposition and fibrotic changes in the aorta, which were mitigated by estrogen treatment. Another study on the effects of estradiol on endothelial cells demonstrated that 17 β-oestradiol upregulated the high molecular weight FGF2 protein expression however the FGF2 mRNA expressions were reduced in FGF2 knock out mice Fgf2−/− model [55]. The study highlighted that the Fibroblast growth factor-2 (FGF2) is expressed in multiple isoforms: the low molecular weight 18 kDa protein (FGF2lmw) is secreted which activates FGFRs, while the high molecular weight (21 and 22 kDa) isoforms (FGF2hmw) stay intranuclear, although their function is largely unclear [55]. The study continues to examine how different FGF2 isoforms contribute to the effects of estradiol and generated models of mice deficient only in the FGF2lmw. Similar to Fgf2+/+ mice, estradiol induced in vitro migration and in vivo angiogenesis in endothelial cells from Fgf2lmw−/−. In endothelial cell cultures from Fgf2+/+ and Fgf2lmw−/− mice, estradiol increased the quantity of FGF2hmw protein [55]. These contrasting findings were suggested to possibly occur due to FGF2 regulation at the translational level [55]. This research partly supports the reduced aortic FGF2 mRNA expression observed with oestrogen treatment in our study. Although the mechanism behind this differential regulation of oestrogen on FGF2 mRNA expression in different tissues are unclear, it is suggestive that oestrogen may regulate FGF2 in different tissues via different mechanism and the alterations in the FGF2 mRNA expression may not directly represent the alterations in the FGF2 protein expressions. Treatment with baricitinib and losartan also reduced VEGF protein and FGF2 mRNA expression in the aortas of Ovx + RA rats, implying that these drugs may have anti-fibrotic properties. This is consistent with previous animal studies on inflammatory arthritis, showing that losartan treatment significantly reduces inflammation and serum VEGF levels [56]. Our findings suggest that the increased VEGF and FGF2 expression seen in the aorta of Ovx + RA rats was caused by inflammation, which also contributed to the collagen deposition and fibrotic changes observed. Furthermore, our findings suggest that estrogen, baricitinib and losartan may have anti-fibrotic effects in the Ovx + RA aorta.
In our study we also observed that oestrogen treatment resulted in significantly increased mRNA fold changes for TGFβ1 in the aorta of experimental group. TGFβ1 mRNA fold changes were increased in Ovx rats as compared to normal and were reduced in Ovx + RA rats as compared to Ovx group in aorta tissues. However, after oestrogen treatment, TGFβ1 levels were significantly increased in the aorta tissues. Controversial data exist in many findings related to TGFβ1 expression and signaling in cardiovascular system [57]. TGFβ1 family mediates its action through heteromeric complexes of type I and type II receptors and activates members of the Smad family of signal transducers [58]. Context-dependence is the primary characteristic of the TGF-β signaling pathway [58]. A study showed that TGF-β signaling prevents both abdominal and thoracic aneurysmal disease but does so by distinct mechanisms. Smooth muscle extrinsic signaling protects the abdominal aorta and smooth muscle intrinsic signaling protects the thoracic aorta [59]. Another study showed that TGF-β expression was significantly increased in the abdominal aortic smooth muscle cells (SMCs) of ovariectomised female mice incubated with 17β estradiol. Since TGF-β promotes wound healing, the same study performed a wound healing assay on abdominal aortic SMCs in the absence and presence of estradiol. In this assay, the area unoccupied by cells 24 hours after introduction of a wound was decreased significantly by estradiol [60]. This study also quantified estradiol effects on αSMA expression on abdominal aortic SMCs. The results showed that Ovx females have increased smooth muscle α-actin expression on dose dependent administration of estradiol [60]. However, in our study the smooth muscle α-actin expression was highly increased in Ovx and Ovx + RA group and the expression was reduced with estradiol treatment. α-SMA is located primarily in the microfilament bundles of vascular SMCs and exerts contractile functions, activation of myofibroblast and arterial tones [61]. An immunohistochemical analysis showed α-SMA positivity was more intense in patients with aortic dissection than in those with aortic aneurysm [62]. α-SMA is associated with TGF-β pathway and overexpression may lead to fibrosis [63]. However, in our study TGFβ1 was found to be reduced in Ovx + RA group, we suggest that upregulated expression of α-SMA could be due to inflammatory changes associated with RA, affecting arterial tone and consequent increase in arterial blood pressure. In contrast to α-SMA expression, our study found that fibronectin expression was significantly reduced in Ox + RA group. Studies have shown that Fibronectin synthesis is regulated by transforming growth factor-β (TGF-β) and in diabetic smooth muscle cells increased expression was associated in response to TGF-β1 [64]. Fibronectin influences diverse processes including cell growth, adhesion, migration, and wound repair and reduced expression may cause loss of these functions affecting normal aortic structure in RA. Additionally, ongoing inflammation in RA may affect TGF-β-fibronectin pathway in aorta, as the expression of both are found to be decreased in Ovx + RA group in our study.
In our study, we also sought Samd 2 expression as prior research has demonstrated that TGF-β can trigger cellular signaling by phosphorylating Smad2 or Smad3 [65]. We analysed the Smad2 protein expressions in the aorta of ovariectomized rats and found that expressions were decreased in the aorta of Ovx and Ovx + RA rats, however, the expressions were increased in all treated groups. This shows the TGFβ1 might have been involved in the signal transmissions to smad2 and modulate transcription of cell proliferation and apoptosis. This is further validated in our study by analysing apoptotic markers.
Apoptosis has been widely studied in RA joints, and despite the loss of apoptotic activity it has been identified that apoptosis markers such as caspase-3 and Bax are upregulated in the RA joints. These contrasting effects are explained by the simultaneous upregulation of anti-apoptotic markers in RA joints [66–67]. However, more research must be conducted on apoptosis and apoptosis markers in RA patients' cardiovascular systems. To address this knowledge gap, we investigated the expression of apoptosis markers such as caspase-3 and Bax in the aortas of rats with RA with ovariectomy (Ovx + RA) and how estrogen treatment affected these markers.
Caspase-3 protein expression was shown to be more significant in the aortas of Ovx + RA rats, and it was reduced by estrogen treatment. In contrast, Bax mRNA expression in the aorta decreased in Ovx + RA rats and increased following estrogen treatment. Our data suggest that apoptotic marker increase in RA is not restricted to the joints and may occur systemically. Moreover, a previous study has shown that caspase-3 mRNA is raised in the serum of RA patients [68]. Consequently, the higher caspase-3 expression seen in our study might be linked to elevated TNF- levels. This inflammatory marker binds to TNF receptor 1 and activates caspase-3, resulting in apoptosis and caspase-3 activation [69]. Thus, elevated caspase-3 levels observed in our study may have occurred due to the elevated TNF-α levels secondary to CIA induction in our rats. Additionally, the simultaneous downregulation of the Bax mRNA and upregulation of the caspase-3 protein expression observed in the aorta of the Ovx + RA rats in our study further suggest that caspase-3 in the cardiovascular system in RA may have been upregulated by other mechanisms not involving Bax. However, it remains unclear why RA has opposing effects on the Bax mRNA expression in the aorta. Moreover, treatment with 17 β estradiol resulted in significantly elevated Bax levels in the aorta of ovaroectomised RA rats. We propose that this mediation could occur due to estrogen upregulation of TGFβ1 via smad pathway.