Carvacrol Improves Vascular Function in Hypertensive Animals by Modulating Endothelial Progenitor Cells

Carvacrol, a phenolic monoterpene, has diverse biological activities, highlighting its antioxidant and antihypertensive capacity. However, there is little evidence demonstrating its influence on vascular regeneration. Therefore, we evaluated the modulation of carvacrol on endothelial repair induced by endothelial progenitor cells (EPC) in hypertension. Twelve-week-old spontaneously hypertensive rats (SHR) were treated with a vehicle, carvacrol (50 or 100 mg/kg/day), or resveratrol (10 mg/kg/day) orally for four weeks. Wistar Kyoto (WKY) rats were used as the normotensive controls. Their systolic blood pressure (SBP) was measured weekly through the tail cuff. The EPCs were isolated from the bone marrow and peripherical circulation and were quantified by flow cytometry. The functionality of the EPC was evaluated after cultivation through the quantification of colony-forming units (CFU), evaluation of eNOS, intracellular detection of reactive oxygen species (ROS), and evaluation of senescence. The superior mesenteric artery was isolated to evaluate the quantification of ROS, CD34, and CD31. Treatment with carvacrol induced EPC migration, increased CFU formation and eNOS expression and activity, and reduced ROS and senescence. In addition, carvacrol reduced vascular ROS and increased CD31 and CD34 expression. This study showed that treatment with carvacrol improved the functionality of EPC, contributing to the reduction of endothelial dysfunction.


Introduction
Endothelial progenitor cells (EPC), precursors of mature endothelial cells, are capable of exerting functional effects on arteries, capillaries, and veins, which include maintenance of the endothelial layer and vascular homeostasis, as well as participation in vascular regeneration and neovascularization [1].
Under normal conditions, most EPC remains in the bone marrow, relatively inactivated, in contact with stromal cells. However, in response to an injury through chemotactic factors, EPCs are released from the bone marrow and mobilized to the injured tissue directly into the damaged vessel structures, preventing blood vessel dysfunction [2,3]. In contrast, when the percentage of EPC decreases, or its function is impaired, the angiogenic capacity is weakened, reducing its regeneration capacity and potentiating vascular injury [4].
A series of experimental and clinical studies have been conducted in the context of hypertension that demonstrate reductions in functionality and proliferative capacity [5] with lower numbers of circulating EPCs [6][7][8]. The pathological mechanisms involving Then, the cells were centrifuged at 600× g for 10 min at 4 • C. Next, the supernatant was discarded, and the pellet was resuspended in 10 mL of medium 199 (Sigma-Aldrich ® , St. Louis, MO, USA). Finally, the blood was collected, and bone marrow (BM) washing was added to the Ficoll ® -Paque PLUS density gradient media (Cytiva Life Sciences, Uppsala, Sweden) for cell separation. Thus, the mononuclear cell layer was used for flow cytometry to quantify, characterize, and follow seven-day cell cultures.

Functional Evaluation of Endothelial Progenitor Cells (EPCs) by Colony-Forming Units
Following carvacrol treatment, the EPC clonogenicity properties were evaluated. Thus, to determine the number of colony-forming units (CFU), the cells were visually inspected after seven days through an inverted microscope with 40× magnification (NIKON Eclipse TS100, Tokyo, Japan). The results were expressed as a percentage of the average number of colonies formed by 10 6 plated cells. Two different observers manually calculated the average number of colonies from ten wells under a light microscope.

Evaluation of EPC-eNOS Expression after Carvacrol Treatment
The expression of total endothelial nitric oxide synthase (eNOS) and phosphorylated endothelial nitric oxide synthase (ps1177) are performed by the flow cytometry technique as previously described [30]. In brief, after seven days of cell culture, EPC was fixed with 4% paraformaldehyde for 20 min at 37 • C, following permeabilization with 0.1% Triton X-100 for 10 min at 37 • C. Then, a blocking solution consisting of PBS and 5% BSA was added for 2 h at room temperature. Subsequently, the EPCs were resuspended and incubated with primary anti-eNOS (1:50; Bd Biosciences, Franklin Lakes, NJ, USA) or anti-eNOS ps1177 (1:100; Bd Biosciences, Franklin Lakes, NJ, USA) antibodies for 1 h at room temperature. Finally, the samples were washed with PBS and incubated with PE-conjugated anti-mouse IgG (Invitrogen™, Waltham, MA, USA) for 1 h in the dark. The samples were analyzed using a FACS Canto-II cytometer (BD, Santa Monica, CA, USA).

Evaluation of the Effect of Carvacrol Treatment on ROS Production
Superoxide anions (ROS) were measured by a dihydroethidium (DHE) probe (Sigma-Aldrich, St. Louis, MO, USA). DHE, an oxidative fluorescent dye, was oxidized to ethidium bromide and intercalated into DNA in the presence of superoxide anions. Briefly, cultured EPC or superior mesenteric artery sections (10 µm) from different groups were loaded with 10 µM DHE for 40 min at 37 • C in the dark. Subsequently, the samples were washed with PBS and analyzed by fluorescence microscopy (NIKON Eclipse TS100, Tokyo, Japan). The analysis of ROS production in EPC was performed by two different analyzers, quantifying the fluorescence intensity using NIS elemental ® software version 4.2 through the observations of 10 other fields. Data were normalized by the control group.

Evaluation of Cellular Senescence by Flow Cytometry
To measure SA-β-gal activity by flow cytometry, the fluorogenic substrate C12FDG (5-dodecanoylaminofluorescein Di-β-D-galactopyranoside, Invitrogen, Life Technologies SAS; Waltham, MA, USA) was used as previously described [31]. Briefly, the EPC was pretreated for 1 h with chloroquine (300 µM) to raise the pH of the lysosomes (pH = 6.0), plus an additional 1 h of C12FDG (33 µM) in the dark. Finally, EPCs were washed with ice-cold PBS, trypsinized, and immediately analyzed with a FACS Canto-II (BD, Santa Monica, CA, USA).

Immunofluorescence
Initially, superior mesenteric artery segments of the treated animals were included in Tissue Tek Compound (OCT), frozen in liquid nitrogen, and kept at −80 • C until the experimental protocols. Subsequently, superior mesenteric artery sections (10 µm) from different groups were fixed with 4% paraformaldehyde for 30 min at 37 • C. Next, the vessels were blocked with PBS with 5% BSA for 30 min at room temperature. Finally, monoclonal antibodies were incubated: FITC-conjugated anti-PECAM antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) and PE-conjugated anti-CD34 antibody (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 • C. Subsequently, the sections were washed with PBS and mounted with DAPI mounting medium (Fluoroshield™, Sigma Aldrich, St. Loius, MO, USA) for nuclear identification [32]. Data were acquired using fluorescence microscopy (NIKON Eclipse TS100, Tokyo, Japan) and analyzed with ImageJ ® software version 1.50i.

Statistical Analysis
All data were expressed as the mean plus or minus the standard error of the mean (e.p.m). The significant difference between the tested groups was tested by one-way or twoway analysis of variance (ANOVA), followed by Tukey's post-test. Data were considered significant when p < 0.05. All analyses performed were calculated using the statistical program Graph Pad Prism version 7.0 ® .

Carvacrol Reduces SBP in SHR Animals
Changes in the SBP of WKY and SHR animals from the different experimental groups after four weeks of treatment are shown in Table 1. At the beginning of the treatment, the baseline SBP of the WKY-CTL group was significantly lower than the SHR-CTL (Table 1), SHR-C50, SHR C100, and SHR-RE10 groups (p > 0.05). The pressure levels of the WKY-CTL group remained lower than the SHR-CTL group (p > 0.05) at the end of the treatment. The SHR-C50, SHR-C100, and SHR-RE10 groups significantly reduced SBP compared to the SHR-CTL group at the end of the treatment (p < 0.05). Similarly, the SHR-C50, SHR-C100, and SHR-RE10 groups significantly reduced SBP compared to the respective groups at the beginning of the treatment. Thus, carvacrol effectively reduced SBP in hypertensive animals.

Characterization of EPC after Cell Culture
After 7 days of cell culture, EPC was characterized in the bone marrow and peripheral circulation. The results obtained after evaluating the expression of the CD34 and VEGFR-2 surface antigens demonstrated the achievement of EPC in all the groups under study, with no statistically significant differences between the treated groups (Figure 2a,b).
indicated that carvacrol reduced the entrapment of EPC in the bone marrow, favoring its migration to the peripheral circulation.

Characterization of EPC after Cell Culture
After 7 days of cell culture, EPC was characterized in the bone marrow and peripheral circulation. The results obtained after evaluating the expression of the CD34 and VEGFR-2 surface antigens demonstrated the achievement of EPC in all the groups under study, with no statistically significant differences between the treated groups (Figure 2a,b).

Carvacrol Treatment Increases eNOS Activity and Expression
The depletion in NO levels is linked to the low repair capacity mediated by EPC. In contrast, NO signaling mediates the induction of proliferation, angiogenesis, and migration of EPC to the peripheral circulation. Thus, we evaluated the expression and activity of eNOS, the enzyme that synthesizes NO. In EPC isolated from bone marrow, the different experimental groups did not show significant changes in eNOS expression

Carvacrol Reduced Oxidative Stress and Cellular Senescence in EPCs
Elevated ROS levels, such as oxidative stress conditions, lead to reduced cell migration due to, at least in part, an increase in senescence and cell apoptosis, in addition to reducing the mobilization, migration, and adhesion factors. Therefore, we evaluated the fluorescence intensity by quantifying superoxide anions in EPC after 7 days of cultivation using the DHE probe.
(d) Figure 3. Evaluation of the total eNOS and phosphorylated eNOS in EPC cultured 7 days after treatment. Evaluation of the total eNOS (a) and p-eNOS (b) expression in cultured EPC from bone marrow. Quantification of the total eNOS (c) and p-eNOS (d) expression in EPC isolated from the peripheral circulation. Results are expressed as the mean ± e.p.m. The ANOVA test was used for the statistical analysis, followed by Tukey's post-test. * p < 0.05 vs. WKY-CTL; # p < 0.05 vs. SHR-CTL.

Carvacrol Reduced Oxidative Stress and Cellular Senescence in EPCs
Elevated ROS levels, such as oxidative stress conditions, lead to reduced cell migration due to, at least in part, an increase in senescence and cell apoptosis, in addition to reducing the mobilization, migration, and adhesion factors. Therefore, we evaluated the fluorescence intensity by quantifying superoxide anions in EPC after 7 days of cultivation using the DHE probe.

Carvacrol Reduces Vascular Oxidative Stress and Induces EPC-Mediated Reendothelialization
Vascular oxidative stress is related to a lower recovery of the endothelial layer, as it can damage adjacent endothelial cells and reduce the cell adhesion capacity. In our study, the SHR-CTL animals (112.9 ± 1.5; n = 6) showed an increase in the intensity of DHE fluorescence compared to the WKY-CTL group (100 ± 0.6; n = 6) (Figure 5a,d). Interestingly, the treatments of the SHR-C50 (101.9 ± 1.9; n = 6), SHR-C100, and SHR-RE10 groups were able to reduce the fluorescence intensity observed in the SHR-CTL group (p < 0.05). These results show that carvacrol inhibited the production of ROS characteristic of hypertension, reducing vascular damage.

Carvacrol Reduces Vascular Oxidative Stress and Induces EPC-Mediated Reendothelialization
Vascular oxidative stress is related to a lower recovery of the endothelial layer, as it can damage adjacent endothelial cells and reduce the cell adhesion capacity. In our study, the SHR-CTL animals (112.9 ± 1.5; n = 6) showed an increase in the intensity of DHE fluorescence compared to the WKY-CTL group (100 ± 0.6; n = 6) (Figure 5a,d). Interestingly, the treatments of the SHR-C50 (101.9 ± 1.9; n = 6), SHR-C100, and SHR-RE10 groups were able to reduce the fluorescence intensity observed in the SHR-CTL group (p < 0.05). These results show that carvacrol inhibited the production of ROS characteristic of hypertension, reducing vascular damage. The improvement in EPC functionality was directly related to the increase in regenerative capacity. Thus, we evaluated the expressions of CD34 and CD31 in the blood vessels of treated animals. SHR-CTL animals showed a reduction in CD34 (Figure 5b,e) and CD31 (Figure 5c,f) fluorescence intensity compared to WKY-CTL animals. However, this effect was reversed by treating animals SHR-C50, SHR-C100, and SHR-RE10. Therefore, these results indicate that carvacrol improved endothelial dysfunction in the treated hypertensive animals. The improvement in EPC functionality was directly related to the increase in regenerative capacity. Thus, we evaluated the expressions of CD34 and CD31 in the blood vessels of treated animals. SHR-CTL animals showed a reduction in CD34 (Figure 5b,e) and CD31 (Figure 5c,f) fluorescence intensity compared to WKY-CTL animals. However, this effect was reversed by treating animals SHR-C50, SHR-C100, and SHR-RE10. Therefore, these results indicate that carvacrol improved endothelial dysfunction in the treated hypertensive animals.

Discussion
A critical component of the Mediterranean diet includes the herb oregano, which is commonly added to salads, spaghetti, soups, and sauces and is also used in the preparation of meats, sausages, and canned foods and to preserve cucumbers, mushrooms, and tomatoes [12]. Essential oils are responsible for the main pharmacological properties attributed to oregano, with carvacrol as the main constituent [34]. This monoterpene has been extensively studied and has been shown to present several benefits, including antioxidant [35], anti-inflammatory [36], and cardioprotective [17,22,37] properties, especially antihypertensive action [18,38], as demonstrated by our results.
Furthermore, in the present study, carvacrol prevents endothelial dysfunction in hypertensive animals by upregulating EPC mobilization, increasing the proliferative and functional capacity, and reducing the oxidative stress and senescence of cells isolated from bone marrow and peripheral blood, which contribute to EPC-mediated reendothelialization.
Multiple data indicate that EPC has diverse surface markers, which depend on the EPC maturation levels. Furthermore, EPC is a heterogeneous group of cells characterized by expressing on their surface CD133 and CD34 as hematopoietic progenitor cell markers and VEGFR2 as an endothelial lineage marker, characterizing the most reliable cell marking [39]. Thus, to characterize the EPC population, we adopted the concomitant labeling of CD133 + , CD34 + , and VEGRF-2 + .
When quantifying the number of EPCs, we verified that carvacrol reduced the entrapment of EPCs in the bone marrow, making them more available to perform endothelial repair in the peripheral circulation. Studies have reported that increased EPC mobilization in the circulation aims to preserve the endothelial integrity in response to vascular damage [8]. The increase in EPC migration is associated with functional maintenance, a necessary condition for their repair capacity [40]. Thus, carvacrol efficiently increased the mobilization of EPC to the peripheral circulation, improving cell function. Similar results were observed by treatment with resveratrol, as demonstrated by Huang, who reported an increase in the biological process of EPC [41].
The clonogenic capacity of EPC has been used as an index of functional analyses, being correlated as a cardiovascular risk factor when it presents a low formation of CFU [9]. In hypertension, the decline in the functionality of the EPC occurs primarily in the reduction of circulating EPC, and this factor is accentuated as hypertension progresses [42]. The low functionality of these cells has been reported in patients with hypertension that is difficult to control and with damage to the target organs [43][44][45]. In our study, we also demonstrated that hypertensive animals have a deficiency in repair mediated by these cells by reducing the proliferation of these cells, corroborating with studies in SHR animals [46,47] and hypertensive patients [48][49][50].
Furthermore, treatment with carvacrol increased the proliferative capacity of EPC similarly to animals treated with resveratrol and the normotensive group. Studies have demonstrated that consuming resveratrol can enhance the proliferative capacity of EPCs through increasing nitric oxide (NO) signaling [41].
NO is an essential mediator for angiogenesis by inducing EPC mobilization and improving the migratory and proliferative capacity [4]. This fact was evidenced through experiments with knockout eNOS-/-mice, which failed in the mobilization of EPC to the peripheral circulation [51]. The malfunction in NO bioavailability is a characteristic of endothelial dysfunction, which depends on the balance between eNOS production and inactivation triggered by ROS. Interestingly, carvacrol treatment not only repaired the inactivation of the PI3K/AKT-eNOS-NO pathway but also attenuated ROS production in SHR EPCs. The NO-mediated pathway can promote the growth and mobilization of EPC from the bone marrow into the peripheral circulation [52,53]. Thus, we suggest that carvacrol increases the EPC migratory capacity by reversing the damage to the NO signaling pathway in hypertensive animals.
Experimental studies have indicated that hypertensive patients have high rates of ROS formation associated with increased cellular senescence [54,55]. Thus, when produced in the bone marrow, due to the deficiency of chemotactic factors and the accelerated state of senescence, the mobilization of these cells does not occur properly, becoming trapped in the bone marrow and intensifying the endothelial dysfunction [56]. Therefore, we demon-strated that carvacrol reduced cellular senescence, causing an increase in the migratory capacity of EPC and making them available to perform the repair of damaged organs.
The antioxidant capacity of carvacrol [20] acts beneficially on the cardiovascular system, restoring vascular function [18,38] and reducing the activity of the NADPH oxidase enzyme, the main enzyme involved in the formation of ROS [21]. Recently, carvacrol has been shown to increase SOD activity in human mesenchymal stem cells [24]. Thus, we can suggest that the increase in performance observed in EPC isolated from hypertensive animals treated with carvacrol is associated with its antioxidant capacity.
Marketou et al. reported that EPC plays an important role in arterial stiffness and remodeling in hypertensive patients, where reduced mobilization reflects inefficient repair [57]. Therefore, we investigated how the increase in EPC mobilization induced by carvacrol altered vascular remodeling. In SHR animals, treatment with carvacrol reversed vascular oxidative stress, which may contribute to the increase in EPC-mediated reendothelialization, as demonstrated by the increase in CD31-and CD34-labeled cells in the vessels of treated hypertensive animals. Similar results were observed with SHR-RE10, corroborating previous studies demonstrating that resveratrol accelerates the reendothelialization of balloon-injured arterial segments [58].
Furthermore, Matluobi et al. showed through in vitro experiments that carvacrol increased the migration, angiogenesis, and transdifferentiation of mesenchymal cells to endothelial cells. This effect was mediated by an increase in the expression of VEGF, a critical chemotactic factor involved in EPC-mediated reendothelialization [24]. However, in our study, the expression of chemotactic factors was not quantified, which is the main limitation. Therefore, additional studies are needed to prove this hypothesis and better elucidate the mechanisms by which carvacrol modulates EPC function and the reendothelization process.

Conclusions
This study elucidated the effects of carvacrol on EPC-mediated endothelial repair, improving mobilization, proliferation, and cell function; increasing eNOS expression; and reducing oxidative stress and cell senescence. However, additional studies to evaluate the intracellular signaling mechanisms involved in the regenerative potential of carvacrol are necessary to consolidate its protective action in the cardiovascular system.  Institutional Review Board Statement: The animal study protocol was approved by the Ethics Committee on Animal Use of the Federal University of Paraiba (protocol code 2171120320 and approved on 29 May 2020).

Data Availability Statement:
The data presented in this study are available on request from the corresponding authors.