Arterial stiffness has been recognized as an independent predictor of all-cause mortality, not only in population with diseases like hypertension, diabetes, and renal disease (Hamilton et al., 2007; Sakuragi and Abhayaratna, 2010; Vlachopoulos et al., 2010), but also in overall population (Mitchell, 2014; Nilsson Wadstrom et al., 2019; Scuteri et al., 2020). It is well established that hypertension, associated or not with aging, leads to increased arterial stiffness (Morgan et al., 2014; Phillips et al., 2015; Lindesay et al., 2016, 2018; Lacolley et al., 2017; Rode et al., 2020), assessed by pulse wave velocity (PWV), even though some authors have shown that the development of arterial stiffness may be prior to hypertension (Celik et al., 2006; Kaess et al., 2012), which may cause increases in afterload and left ventricular remodeling (Zieman et al., 2005; Ohyama et al., 2016).

Assessment of PWV has been performed in humans using ultrasound, Doppler, magnetic resonance imaging, and applanation tonometry techniques (Mitchell et al., 2010; Phillips et al., 2015; Ohyama et al., 2016; Obeid et al., 2017a, b; Nilsson Wadstrom et al., 2019; Rode et al., 2020; Scuteri et al., 2020); although human studies precisely determine the compliance and arterial stiffness via the dynamic properties of the arterial wall, they are limited in advancing our knowledge on conditioning mechanisms. The possibility of having an experimental model with the measurement of both blood pressure (BP) and PWV, simultaneously with direct access to the arteries for gene, protein, histological studies, and other assays, represents an important advancement for better understanding the mechanisms involved in arterial stiffness changes. In this regard, our group recently standardized a new non-invasive device for assessment of arterial stiffness in rats (Fabricio et al., 2020) and showed that it is able to detect changes in arterial stiffness that are conditioned by age- and pressure-related arterial remodeling.

Aortic stiffening is associated with either a remodeling or disorganization of the vascular wall, which derives from an increased collagenous material, fibrotic components, the presence of elastin fiber fracture, arterial elasticity, or vascular smooth muscle cell (VSMC) hypertrophy (Morgan et al., 2014; Lindesay et al., 2016, 2018; Fabricio et al., 2020; Steppan et al., 2020). Among several causes of aortic stiffness, the central role of the renin–angiotensin system (RAS) is well known, and therefore, some studies have shown the effects of RAS inhibition on arterial stiffness (Marque et al., 2002; Gonzalez et al., 2018) and, consequently, on BP, but the exact molecular mechanisms induced by RAS on aortic stiffness are not completely understood.

Since either hypertension may induce arterial stiffness or arterial stiffness may induce hypertension, understanding the mechanisms involved in aortic stiffness would be an emerging target for therapeutic intervention to prevent and/or treat hypertension. Thus, the aim of this study was to evaluate the effects of perindopril treatment on PWV and to identify the differentially expressed proteins in the aorta of spontaneously hypertensive rats (SHR), using a proteomic approach.

Twenty-two SHR (250–300 g, 3 months) and ten Wistar rats (similar age) were obtained from the Animal Facility of Institute of Biomedical Sciences, University of São Paulo, (USP) and São Paulo State University (UNESP), campus of Botucatu, SP, Brazil, respectively. All rats were housed at the animal facility maintenance at School of Sciences, São Paulo State University—UNESP, campus of Bauru. All rats received water and food (Biobase, Águas Frias, SC, Brazil) ad libitum and were maintained in a dark–light cycle (12–12 h) in a controlled temperature room (22 ± 2°C). All methods used were approved by the Committee for Ethical Use of Animals at School of Sciences, UNESP (#778/2017 vol. 1).

The main results observed in the present study were that perindopril treatment significantly reduced blood pressure and PWV in SHR. Also, plasma nitrite concentration was negatively correlated with PWV. The proteomic approach identified some differentially expressed proteins induced by hypertension and perindopril treatment, which may contribute to identify possible targets for the management of arterial stiffness.

Even though it is not clear whether arterial stiffness precedes hypertension or it is a consequence (Kaess et al., 2012; Mitchell, 2014; Celik et al., 2017; Rode et al., 2020), several studies have shown a significant correlation between PWV and BP (Laurent et al., 2009; Phillips et al., 2015; Steppan et al., 2020), including the present study, and the growing incidence of cardiovascular events in patients with high PWV values is eminent (Niiranen et al., 2017; Nilsson Wadstrom et al., 2019; Scuteri et al., 2020). Therefore, it is important to determine the mechanisms involved in vascular stiffening.

It is well known that angiotensin II (Ang II) plays a central role in hypertension due to its potent contractile action, and drugs that inhibit Ang II signaling are widely used to treat hypertension (Schmidt-Ott et al., 2000; Marque et al., 2002; Varagic et al., 2010; Gonzalez et al., 2018). While there are several classes of therapeutic agents to control pressure (Chobanian, 2017; Cuspidi et al., 2018; Unger et al., 2020), ACE inhibitors are highly recommended because of their cardio- and vascular protective effects (Ferrari et al., 2005; Janic et al., 2014; Chobanian, 2017). This study confirmed the participation of RAS in hypertension, as shown elsewhere (Marque et al., 2002; Safar, 2010; Hong et al., 2013; Natalin et al., 2016; Mancini et al., 2017; Steppan et al., 2020), since 8 weeks of perindopril treatment significantly reduced BP (−11%), as shown by the time course of SBP. We did not measure PWV at the beginning of the treatment, but since we have shown that PWV positively correlates with BP in SHR (Fabricio et al., 2020), we may assume that PWV was also high at the beginning of the protocol. Because RAS has also been implicated in vessel remodeling and aortic stiffening, ACE inhibitors play a crucial role in the restoration of the balance between plasma (and tissue) angiotensin II and bradykinin levels (Ferrari et al., 2005), which improves arteriolar structure, independent of their ability to reduce BP (Mancini et al., 2017). Ong et al. (2011) demonstrated in their meta-analysis that antihypertensive treatment can improve aortic stiffness beyond BP reduction in essential hypertensive patients and that the decrease in arterial stiffness was less under calcium antagonist treatment than under ACEi in a short-term trial, whereas all classes were equivalent in long-term trials.

Several clinical trials show interesting results, even though they are still inconclusive, but it is already known that other classes of anti-hypertensive drugs like diuretics and beta-blockers have few effects on PWV (Ong et al., 2011; Janic et al., 2014). Therefore, we chose to use perindopril, which has been shown to be a promising anti-hypertensive drug, capable to control/reverse artery stiffening, mainly because it causes changes on the mechanisms responsible to increase stiffness and it can be independent of BP reduction (Mahmud and Feely, 2002; Nakamura et al., 2005). In addition, perindopril is a prodrug ester that has a strong affinity for ACE and it inhibits 50% of ACE activity at a lower concentration than enalapril (Louis et al., 1992). Likewise, it was shown that lisinopril had about one-tenth the potency of perindopril with respect to its effects on plasma angiotensin peptide levels (Campbell et al., 1994). In addition, perindopril has high lipid solubility, it crosses the blood–brain barrier, and, because of that, it decreases brain ACE activity by 50%, different from enalapril and imidapril. For review, see Perini et al. (2020), which is a recent review that shows the pharmacological data of several ACEi. Also, it has been shown that perindopril reduces oxidative stress, which is another mechanism responsible to increase arterial stiffness (Ulu et al., 2014). Although there are several clinical trials investigating and demonstrating the effectiveness of perindopril, regular use in the clinical management of hypertension is not common yet (specially in public health units), mainly because of its elevated costs, when compared with other ACEi.

At the end of the experimental protocol, this study revealed that PWV and SBP were similar between Wistar and perindopril-treated SHR, but different from untreated SHR. We cannot avoid the possibility that PWV in SHR-treated rats could also be due to the hypotensive effect of perindopril. The use of another antihypertensive drug could help to solve this dilemma; however, it has been shown that clonidine as well as calcium blocker diltiazem reduced AP in parallel with a reduction of β-stiffness index and PWV (Vayssettes-Courchay et al., 2011; Lindesay et al., 2016, 2018), at least in young SHR. So, it seems that there is a lack of hypertension-independent arterial stiffening in young SHR, as suggested by Lindesay et al. (2016). On the other hand, it is important to note that the decrease on BP per se does not always reduce stiffness; for instance, Lindesay et al. (2016) demonstrated that when hypertension-induced vessel remodeling is already present, like in aged hypertensive rat, vessel distensibility remains, even after a pharmacological reduction of pressure (clonidine administration). In agreement, Mahmud and Feely (2002) have shown that PWV reduces independent of BP in hypertensive humans treated with Valsartan (AT1 antagonist) and captopril (ACEi). Recently, Steppan et al. (2020) showed that the restoration of normal BP in hypertensive mice (recovery period after stopping Ang II infusion) results in a partial recovery of overall in vivo stiffness (PWV). These authors suggest that restoration of BP improves the viscoelastic nature of blood vessels and partially recover the matrix mechanics, but the stiffness, which is irreversible, results from endothelial dysfunction and molecular changes to the vascular matrix, which contribute to VSMC dysregulation and are the major contributor to the overall in vivo vascular stiffness in essential hypertension. Therefore, further studies are still necessary to better understand the effects of perindopril treatment on PWV of SHR.

In addition, Ang II is a powerful mitogen that causes structural alterations in the vessel wall by stimulating the hypertrophy and hyperplasia of VSMCs, causing increases in the intralamellar distance on the vessel wall, increase of collagen, reduction of the elastin, and stiffness of intact and decellularized segments, among others (Wagenseil and Mecham, 2012; Mancini et al., 2017; Steppan et al., 2020). However, how exactly Ang II may modulate PWV is not completely known. Actually, there are several components that contribute to the arterial stiffening, like extracellular matrix (ECM) proteins that support the mechanical load, such as collagen and elastin (Lacolley et al., 2017). Lacerda et al. (2015) have suggested that PWV may be correlated with some components of the ECM in the vessel wall, like metalloproteinase biomarkers (MMP-9 and MMP-2) and collagen in patients with hypertensive heart disease; however, these biomarkers were evaluated in blood plasma. In addition, alterations of VSMCs, which regulate actomyosin interactions for contraction and cell–ECM interactions and depend on the architecture of cytoskeletal proteins and focal adhesion, are also contributing to regulate arterial stiffness; see Lacolley and colleagues for review (Lacolley et al., 2017). In agreement, phosphorylation or dephosphorylation of contractile proteins in VSMCs contributes to determine the dynamic modifications of the vessel diameter (Touyz et al., 2018). In fact, functional, structural, and biochemical alterations in the vessel wall have been investigated in different types of hypertension, but the molecular mechanisms involved remain unclear and most of these studies were performed in vitro. For this reason, in this study, we performed a proteomic analysis of the aorta tissue, excised from normotensive and hypertensive rats, treated or not with perindopril and compared SHR_C × Wistar to comprehend the effects of hypertension and SHR_P × SHR_C to identify the differentially expressed proteins after 8 weeks of perindopril treatment. As far as we know, this is the first work that aimed to identify the proteins related to vascular stiffening in aorta of SHR treated with perindopril.

Only few studies have analyzed the protein expression profile of the aorta during hypertension (Lee et al., 2004, 2006, 2009; Moriki et al., 2004; Bian et al., 2008; Feng et al., 2015; Lyck Hansen et al., 2015), and the results are fairly different. In the present study, when the functional classification according to the cellular component was performed, 30 different components were changed by hypertension (SHR_C × Wistar), and most of them were related to the mechanical integration of the various components of the cytoskeleton and responsible for physical support for cellular constituents, as shown in Figure 2. From 42 differentially expressed proteins in the aortic wall from SHR_C × Wistar rats (see Supplementary Table 1), 21 were upregulated and most of them were associated with cytoskeleton organization, stabilization of the aorta, and apoptosis like Cofilin-1, Tubulin β-5 chain, and Tropomyosin alpha-3 chain, among others (see Supplementary Table 1). In agreement, Lyck Hansen et al. (2015) have shown that several proteins related to smooth muscle cell function and organization of actin cytoskeleton, like tubulin β-2A, tropomyosin α-4, and α-actinin 4, among others, were significantly upregulated in patients with high PWV compared with those patients with normal PWV. In particular, these authors (Lyck Hansen et al., 2015) found that Tropomyosin α-4 chain was a significant predictor of PWV. In the present study, Tropomyosin α-4 chain was expressed in SHR_C’s aorta but did not reach statistical significance; however, Tropomyosin α-3 chain was upregulated. Using a two-dimensional electrophoresis system (2-DE), Bian et al. (2008) performed a proteomic analysis of the aorta from SHR and found that only two upregulated proteins were related to vessel stiffness: GDP dissociation inhibitor protein (RhoGDIa) and Non-muscle myosin alkali light chain.

It has been shown that Ang II levels in VSMC of hypertensive rats are higher than those in normotensive ones (Moriki et al., 2004) and Ang II is one of the main activators of the small GTPase family member (Moriki et al., 2004; Guan et al., 2013) and its downstream effector Rho-associated protein kinase (ROCK) (Zhou et al., 2017). In turn, activation on the RhoA/Rho-kinase pathway reduces the activity of myosin light-chain phosphatase (MLCP) through phosphorylation of its myosin targeting subunit (MYPT1) (Brozovich et al., 2016). This process sustains vasoconstriction, since myosin light chain (MLC) is not dephosphorylated by MLCP. In agreement, Zhou et al. (2017) demonstrated that VMSC from SHR’s aorta presents high activity of ROCK and high MYPT1 protein level. Similarly, Han et al. (2008) have shown that aorta of SHR had higher expression of myosin light chain kinase (MLCK) and myosin light chain phosphorylation (MLC-P), which in turn induces contraction (Guan et al., 2013).

In addition, ROCK is also an activator of LIM kinase-LIMK (Lacolley et al., 2017), an enzyme that phosphorylates and inactivates Cofilin-1 (Morales-Quinones et al., 2020). Since the main activity of Cofilin-1 is to sever F-actin cytoskeletal stress fibers, inactivation of Cofilin-1 reduces actin depolymerization (Lacolley et al., 2017; Sousa-Squiavinato et al., 2019) and induces arterial stiffening (Lam et al., 2007; Williams et al., 2019; Morales-Quinones et al., 2020). Although PWV of SHR was higher than that of Wistar, the present study showed that Cofilin-1 was upregulated in stiffened aorta from SHR_C compared with Wistar rats. We may speculate that this increased cofilin-1 expression could be a compensatory mechanism against aortic stiffening in SHRc, induced by high activity of the RhoA/Rho-kinase pathway, observed in hypertension (Moriki et al., 2004; Ying et al., 2004; Wynne et al., 2009; Walsh, 2011; Zhou et al., 2017). Furthermore, this cofilin-1 could be dephosphorylated, as demonstrated by Lee et al. (2006). These authors have shown that Cofilin-1 protein level was upregulated by hydrogen peroxide in rat aortic smooth muscle, but further analysis revealed that this cofilin-1 was dephosphorylated, which may lead to an inhibition of actin polymerization. Due to the nature of the proteomic analysis performed in the present study, this confirmation could not be performed, and future studies are necessary to confirm the activation state of cofilin-1 in SHR’s aorta.

It is well known that reactive oxygen species (ROS) decreases nitric oxide (NO) bioavailability and induces hypertension and arterial stiffness (Landmesser et al., 2003; Bellien et al., 2010; Eleuterio-Silva et al., 2013; Roque et al., 2013; Wu et al., 2014). In agreement, using a 2D gel electrophoresis, Lee et al. (2009) identified seven proteins in the aorta artery that were differentially expressed between SHR and Wistar rats, including downregulation of the dihydropteridine reductase (DHPR), which is associated with the regeneration of tetra-hydrobiopterin (BH₄) (Bendall et al., 2014). It has been shown that decreases in BH₄ increases the generation of superoxide anion, which reduces NO bioavailability (Bellien et al., 2010). Reduction of NO availability has also been found after RhoA/ROCK pathway activation, which negatively regulates eNOS phosphorylation and eNOS expression (Ming et al., 2002).

In our study, we did not observe altered expression of DHPR or BH4 in aorta of SHR, but if we consider that hypertension and arterial stiffness are associated with reduction of NO bioavailability (induced by RhoA/Rho-kinase pathway activation or increases of ROS), we may hypothesize that increases in NO, due to perindopril treatment (indicated by the plasma nitrite concentration), could be involved in the PWV reduction observed in the present study in treated SHR (SHR_P). Actually, the present study revealed that perindopril increased the nitrite concentration by 83% in SHR, and it was negatively correlated with PWV. In agreement with our results, other studies have shown that perindopril treatment increases plasma concentrations of nitrite/nitrate, indirectly indicating plasma NO contents (Kedziora-Kornatowska et al., 2006; Ceconi et al., 2007). Ceconi et al. (2007) have also observed an increase on eNOS protein expression and activity in coronary artery disease patients treated with perindopril. It has been shown that NO may relieve vascular stiffness by inactivation of RhoA/Rho-kinase pathway through a cGMP-dependent protein kinase activation (Krepinsky et al., 2003; Sauzeau et al., 2003). In fact, NO has been considered the most powerful physiological endothelial relaxing factor that negatively regulates RhoA/Rho-kinase activation in the vasculature. For review, see Nunes and Webb (2020).

In addition, we have identified an upregulation of GDP dissociation inhibitor protein (GDIs) in aorta of perindopril-treated SHR (SHR_P), which is an internal regulator of RhoA activation. Actually, the activity of RhoA is normally controlled by three regulatory proteins, such as guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and GDP dissociation inhibitors (GDIs) (Guan et al., 2013), the last one being an inhibitory protein, which is involved in the suppression of the transformation between Rho-GDP and Rho-GTP forms (Guan et al., 2013) and may contribute to a decrease in the RhoA/ROCK/LIMK/Cofilin-1 pathway. Recently, Morales-Quinones et al. (2020) have shown that inhibition of LIMK reduces p-Cofilin/Cofilin and reduces arterial stiffness, which suggests the involvement of RhoA/ROCK/LIMK/Cofilin-1 on vascular stiffening. Similarly, losartan (an Ang II receptor type 1 antagonist) inhibits RhoA/Rho-kinase pathway activity in hypertensive rats (Moriki et al., 2004; Wynne et al., 2009). Likewise, inhibition of this RhoA/Rho-kinase pathway by Y-27632 (an inhibitor of ROCK) has been associated with reduction of BP (Seko et al., 2003; Moriki et al., 2004; Zhou et al., 2017) and vascular stiffness (Wehrwein et al., 2004). In agreement, Masumoto et al. (2001) have shown that Fasudil, an inhibitor of Rho-kinase, increased forearm blood flow in humans.

Moreover, the proteins Long-chain specific acyl-CoA dehydrogenase mitochondrial and Heat shock protein beta-1, interacting with Desmin in the network of SHR_P × SHR_C comparison, were downregulated in aorta of SHR_C and became upregulated after perindopril treatment. Long-chain specific acyl-CoA dehydrogenase mitochondrial is involved with the energy production and Heat shock protein beta-1 is involved in protein folding. Interesting, Feng et al. (2015) identified the same proteins after physical exercise in aorta of SHR, which means that perindopril treatment may be contributing to restart the normal function of the vessel, as well as exercise training does.

In summary, the results of the present study revealed that treatment with perindopril reduced arterial pressure and PWV in SHR. In addition, the proteomic analysis in aorta suggested for the first time that RhoA/Rho-kinase/LIMK/Cofilin-1 pathway may be inhibited by perindopril-induced upregulation of GDIs or increases in NO bioavailability in SHR. Therefore, we may propose that activation of GDIs or inhibition of RhoA/Rho-kinase pathway could be a possible strategy to treat arterial stiffness.

This study has some limitations. First, although we have shown a positive correlation between PWV and SBP, we cannot say that perindopril treatment improved PWV, since we did not measure PWV at the beginning of the experimental protocol; however, we may say that SHR-treated rats showed lower PWV compared with non-treated SHR. Second, since perindopril reduced pressure and PWV, we cannot avoid the possibility that PWV response was dependent of the BP, even though some studies have shown that sometimes arterial stiffness reduction may be independent of BP reduction; third, due to the limitation of the technique, PWV was measured in anesthetized rats. Finally, due to the nature of the proteomic analysis performed in the present study, we cannot confirm if the upregulation of the cofilin-1 in SHR’s aorta is a compensatory mechanism or if this protein is dephosphorylated. Future studies, using more specific techniques, are necessary to further confirm the results observed in this study. Besides that, we do believe that this kind of study is important, mainly because the currently available studies using proteomics looking for a better management of hypertension and cardiovascular diseases are relatively small, not standardized, and difficult to compare with each other (Delles et al., 2018).

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

The animal study was reviewed and approved by Committee for Ethical Use of Animals at School of Sciences, UNESP (#778/2017 vol.1).

SA and DM designed the study and wrote the manuscript. DM, AD, and AJ conducted the experiments, acquired the data, and analyzed the data. AZ supervised nitrite concentration experiments. SA and MB supervised the study. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor declared a shared affiliation with several of the authors, AD and MB, at the time of review.