Rat Model of Menopausal/Andropausal Hypertension with Different Sensitivities to Non-Genomic Antihypertensive Responses of Female and Male Sex Steroids

Blood pressure (BP) higher than the normal range (hypertension) is a medical condition and the leading risk factor for the development of cardiovascular disease (CVD) and mortality [1, 2]. Hypertension is prevalent in older women and men; however, although BP rates increase with age, the progression and severity of hypertension are unclear between sexes and a gap has been observed in identifying the differences between older women and men and its control by gonadal steroids.

Female sex steroids such as 17β-estradiol (E2) were considered cardioprotectors in women to regulate CVD including hypertension, directly through non-genomic effects, and indirectly through genomic actions [3‒5]. Decreased estrogen production during and after menopause is associated with hypertension in women [6] by genomic mechanisms [7]. Regarding the action of androgens on BP, the obsolete view of the adverse effects of androgens on CVD has been documented [8]. Various studies have considered testosterone (T) as pro-hypertensive [5, 9, 10]. Some evidence has demonstrated the prominent non-genomic mechanisms of androgens to produce vascular relaxation, revised in [11‒14]. Consequently, antihypertensive responses are supported by recent studies in hypertensive rat models. We reported that some T metabolites could block vasopressor responses to noradrenaline or a calcium channel agonist in vagosympathectomized, pithed male Wistar rats [15]. Subsequently, T and its 5β-reduced metabolite, 5β-dihydrotestosterone (5β-DHT), have been reported to induce a marked reduction in BP in both conscious, normotensive Sprague-Dawley, and testicular feminized male rats (androgen receptor-deficient), this BP reduction induced by androgens in testicular feminized male rats, was confirmed as a non-genomic action, i.e., androgen receptor-independent [16]. Androgens can also elicit a significant antihypertensive response in conscious, spontaneously hypertensive male rats, and normotensive Kyoto rats that were deprived of androgen by orchidectomy to induce hypertension [17]. A relevant study in a rat model of preeclampsia has indicated that T, its precursor dehydroepiandrosterone (DHEA), or its 5-reduced metabolites 5α-dihydrotestosterone (5α-DHT) and 5β-DHT are also antihypertensives in females [18]. Likewise, androgen administration induces antihypertensive responses in hypertensive rats ([19], revised by [14]). In addition, we recently observed that hyperandrogenism in non-obese rats and women with polycystic ovary syndrome reduces BP [20].

Few studies have reported that androgens are more potent than estrogens in inducing vasorelaxation [21‒23]. Female steroids also induce relaxation of airway smooth muscle, although with lower relaxant potency than androgens [24]. These studies may be the only published reports comparing the efficacy and potency of androgens vs. estrogens. Some studies have suggested that estrogen and androgen deficiency in women and men, respectively, may contribute to the development of CVD [25‒31]. Notoriously, androgen deficiency in men is accepted as a primary risk factor for several disorders, including CVD [26, 32‒39]. However, its association with hypertension has been poorly studied.

We hypothesized that hypertension in males is more severe and rapid than in females because males are unprotected by early androgen deficiency. The present study aimed to (i) explore the sex differences in the progression of hypertension by gonadal steroid deprivation using an accepted rat model of menopausal or andropausal hypertension; (ii) investigate the potential prevention of hypertension by treatment of E2 in gonadectomized females and T in males; (iii) study the impact of E2 in males and T in females on BP increment by opposite treatment; and (iv) analyze the potential antihypertensive responses in conscious hypertensive rats caused by gonadectomy with a special emphasis on determining the different hypotensive potency and efficacy of various sex steroid hormones with different molecular structures (various vasoactive androgens vs. E2).

This study was conducted in the Department of Cell Biology and Physiology, Institute for Biomedical Research, National Autonomous University of Mexico. The animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (NIH publication 86-23, revised 2014). The Institutional Animal Care and Use Committee (IACUC; ID 6307) reviewed and approved all protocols at the National Autonomous University of Mexico, Institute for Biomedical Research.

Eight-week-old female and male Wistar rats (body weight: 180–200 g for females and 200–220 g for males) were obtained from the Animal Center of the Institute for Biomedical Research. All the rats were housed in standard experimental laboratory rodent housing for 2 weeks before experiment initiation. The rats were housed in cages (3–5 animals per cage). The room temperature was maintained at 20–22°C with a 12-h light-dark cycle. Food (Teklad Global Diets 2018S) and water were provided ad libitum. The rats were used at 12 weeks old and randomly divided into eight experimental groups (with the exception of group 3 (n = 30) and 4 (n = 30), the remaining groups were six rats per group) as follows:

• 1.

  Group 1, n = 6; intact female rats (female control group).

• 2.

  Group 2, n = 6; intact male rats (male control group).

• 3.

  Group 3, n = 30; orchiectomized (ORX) male rats.

• 4.

  Group 4, n = 30; ovariectomized (OVX) female rats.

• 5.

  Group 5, n = 6; OVX rats + estradiol benzoate (E2B).

• 6.

  Group 6, n = 6; ORX rats + testosterone propionate (TP).

• 7.

  Group 7, n = 6; OVX rats + TP.

• 8.

  Group 8, n = 6; ORX rats + E2B.

Both male and female rats (groups 3-8) were gonadectomized, resulting in the loss of gonadal production of sex steroids. Briefly, the rats underwent standard bilateral ORX or OVX surgery. The rats were anesthetized with 80/10 mg/kg of ketamine and xylazine administered intraperitoneally. The ovaries of females and testes of males were excised. In females, a single small abdominal incision was made from the area caudal to the 2nd pair of mammary papillae, and the ovaries were removed, after which the uterine horns were returned to the cavity. In males, a small bilateral incision was made at the posterior end of the scrotum, and the spermatic cord was ligated. In both cases, the cuts were closed using silk sutures. This experimental model is conventionally accepted to mimic human menopause or andropause. Sex hormonal levels dramatically decrease a few hours after surgery and are reestablished with hormone replacement therapy [40‒42]. After surgery, the rats were housed individually for 1 week to allow recovery.

In all the groups, systolic/diastolic BP (SBP/DBP; mm Hg of BP) was measured preoperative (time 0) and every week during the next 15 weeks with an indirect method, by the same researcher, in conscious and slightly restrained rats by tail-cuff plethysmography using a small animal tail-cuff BP system (LE 5002 storage pressure; Panlab Harvard Apparatus, Spain). Details are described in a previous study [19].

Immediately following the recovery period (1 week), female and male gonadectomized rats (groups 5 and 8) were provided estrogen replacement therapy using E2B (estradiol benzoate, 0.1 mg/Kg, dissolved in extra virgin olive oil) twice weekly via subcutaneous injection for the next 15 weeks. This physiological dose of E2B had been previously demonstrated to induce complete estrogenization [42‒44]. Female and male gonadectomized rats (groups 6 and 7) were provided androgen replacement therapy using TP (testosterone propionate, 8.75 mg/kg; dissolved in extra virgin olive oil), once per week by subcutaneous injection over the next 15 weeks; this dose of TP was selected on our previous experience [17]. These groups were compared with their respective controls.

In other experiments, the opposite treatment was also determined, i.e., female rats with replacement therapy with TP (groups 7: OVX rats + TP) and male rats with replacement therapy with E2B (group 8; ORX rats + E2B), considering the same application scheme mentioned above for each sex steroid hormone replacement during the next 15 weeks, and was compared with their controls.

The BP of non-gonadectomized intact rats was recorded (control groups 1 and 2). In addition, in other experiments, oil-treated rats (control experiments with the vehicle used) were examined to compare the response of E2B and TP and their vehicle (extra virgin olive oil), and separated vehicle controls were run for OVX and ORX rats with subcutaneous bolus injection of the same volume of vehicle, extra virgin olive oil (0.1 mL in the dose of E2B, and 0.8 mL in each dose of TP; n = 6) at identical time intervals as hormone replacement therapy.

The gonadectomized rats were anesthetized with a combination of ketamine (80 mg/kg) and xylazine (10 mg/kg) administered intraperitoneally, and chronic indwelling catheters were inserted into the left carotid artery and right jugular vein, using polyurethane tubing (internal diameter 0.36 mm, external diameter 0.84 mm, “Microrenathane,” Braintree Scientific, Inc., USA). The catheters were passed subcutaneously to the dorsal surface of the neck and exteriorized. The rats were then fitted with polyester cloth vests with Velcro closures that surrounded the chest and neck to protect the catheters.

After the gonadectomized rats had completely recovered from anesthesia and catheter implant surgery (24–48 h), they were placed in clear Plexiglas rodent restrainers, allowing acclimatization to the experimental conditions for 90 min. The carotid arterial catheter was connected to a pressure transducer (Grass P23 XL) adapted to an MP150 Research System (BIOPAC Systems, Inc.). The output was combined with the AcqKnowledge software for data acquisition and analysis. When hemodynamic variables were stable for at least 60 min, baseline values of BP, i.e., SBP, and DBP (SBP/DBP; mm Hg of BP) were monitored continuously during the experiment and calculated using the AcqKnowledge software. In all the groups, the recording continued for at least 90 min after the highest dose of androgen was administered.

Based on our previous report the most potent antihypertensive androgens in different rat models of hypertension [16‒19] are T, its precursor DHEA, and its immediate 5β-reduced metabolite, 5β-DHT. This study determined their potency and efficacy compared with the female sex steroid, E2. After a 60–90-min stabilization period (baseline values), dose-response curves were obtained from OVX or ORX rats for each steroid hormone (DHEA, E2, T, or 5β-DHT; only a single androgen was tested in each animal) and compared with E2. These steroid hormones were separately administered intravenously by bolus injection (through the jugular vein catheter) cumulatively at log doses of 1 × 10⁻⁷, 1 × 10⁻⁶, 1 × 10⁻⁵, and 1 × 10⁻⁴M kg⁻¹ min⁻¹ which were administered over 1 min employing a digital programmable single syringe pump (KD Scientific, MA; Kds 100) with an interval of ∼20 min between each dose, according to our previous reports [17‒19]. The hemodynamic values after each dose were compared with the baseline values to report (a) the difference in mm Hg of BP (mm Hg) and (b) the changes in heart rate (HR; BPM). Cumulative dose-response curves for each androgen in gonadectomized rats were determined independently. The potency of each steroid was evaluated by calculating its effective dose 50 (ED₅₀). The antihypertensive efficacy of each androgen was evaluated as its maximal effect (E_max). The ED₅₀ and E_max values of each steroid hormone were statistically compared.

Data are expressed as means ± standard deviation (n = 6, number of animals). In the text and figures, changes in BP in vivo are expressed as changes from the baseline (mm Hg). The progression of BP over 15 weeks was compared between gonadectomized and intact male and female rats.

The antihypertensive potency of each androgen in vivo was evaluated by calculating the effective dose 50 (ED₅₀ = dose of androgen required to reduce BP by 50% of the maximal response compared to the control BP). The ED₅₀ was calculated using linear regression from the cumulative dose-response curve obtained from each animal. The antihypertensive efficacy of each androgen was evaluated by calculation of the maximal response (E_max; antihypertensive response at 1 × 10⁻⁴M kg⁻¹ min⁻¹). Non-paired Student’s t tests were used to compare responses between the two groups. A two-way analysis of variance followed by Tukey’s test was used to compare E_max or ED₅₀ values in BP studies. Significance was set at p < 0.05.

With the exception of ketamine (Pisa Farmaceutica, Mexico), xylazine (Bayer, Germany), and 5β-dihydrotestosterone (5β-DHT; 17β-hydroxy-5β-androstan-3-one) from Steraloids (Newport, RI., USA), the remaining compounds were purchased from Sigma (St. Louis, MO., USA), and included 1,3,5(10)-estratriene-3,17β-diol (17β-estradiol; E2), estradiol benzoate (E2B; 1,3,5(10)-estratriene-3-benzoate,17β-diol), dehydroepiandrosterone (DHEA; 3β-hydroxy-5-androsten-17-one), testosterone (T; 17β-hydroxy-4-androsten-3-one), and testosterone propionate (TP; 3-oxoandrosten-4-en-17β-hydroxyl-2-methylpropanoate).

Figure 1 presents arterial BP (SBP/DBP mm Hg). Rats, before gonadectomy indicated as time “0” were normotensives as follows: females 120 ± 2.4/85 ± 2.5 mm Hg, body weight 204±4 g and males 121 ± 1.9/84 ± 2.0 mm Hg, body weight 225 ± 10 g (n = 48). In intact rats, females 121 ± 2/86 ± 3 mm Hg and males 121 ± 1/85 ± 1 mm Hg were also normotensives during the experiments (15 weeks). By contrast, the BP of gonadectomized rats displayed a progressive increase (time-dependent) in SBP/DBP at 15 weeks after gonadectomy reaching hypertensive values at the end of the experiment (15 weeks) in OVX (138 ± 2/95 ± 2 mm Hg, body weight 240 ± 14 g) and ORX (156 ± 4/105 ± 2 mm Hg, body weight 403 ± 15 g) rats, indicating that the BP of ORX males was significantly higher than that of OVX females (p < 0.001). The BP of ORX male rats was significantly higher (p < 0.05) at week 5 than in previous weeks. Thus, ORX males displayed hypertension values at week 7, and in the subsequent weeks, the increment of BP was more rapid than that observed in OVX female rats who showed hypertension values until week 14 (Fig. 2). Regarding the body weight, we observed as expected, that all rats increased their body weight at the end of the experiment (15 weeks). We also noticed a difference in the grams of body weight gain in gonadectomized males versus females during the experiment where the males had greater weight gain than females; likewise, the percent of the increase in body weight was 18% in females versus 79% in males. A correlation in gonadectomized rats without treatment was observed, i.e., at high body weight major BP. In the case of females treated with TP showed masculinization with the same body weight as the males but not the BP.

Figure 1 also demonstrates the BP in both gonadectomized females and males with sex steroid hormone replacement. The results revealed that the replacement of E2B in OVX females and TP in ORX males completely prevented an increase in BP. Similarly, the replacement of TP in OVX females also completely prevented the increase in BP. This is the consequence when the deficiency of steroids is reestablished after gonadectomy. Meanwhile, the replacement of E2B did not completely prevent the increase in BP in ORX males (85% prevention).

All sex steroid hormones caused an immediate (∼2 min) decrease in BP in conscious female and male hypertensive rats. HR was unchanged in all doses, although a minimal difference of 18.25 ± 3.1% less BPM was observed only at the highest doses. The dose of each steroid was measured when the BP stopped falling and expressed as a drop of BP in mm Hg from the baseline values, and then the next dose was administered. The total dose-response cumulative curve is represented in Figure 3 which summarizes the response to i.v. Bolus injection of each steroid hormone tested at cumulative doses on the hypertensive response induced by gonadectomy in conscious female and male rats which produced a dose-dependent reduction in BP (SBP/DBP mm Hg) until the values were normotensive to T. Meanwhile, in males (Fig. 3), BP continued to decline until attaining hypotensive values after administering the highest dose of DHEA (92 ± 5/55 ± 5 mm Hg) and 5β-DHT (93 ± 4/56 ± 4 mm Hg). In females (Fig. 3), the highest dose also induced hypotensive values after the last dose of DHEA (96 ± 4/62 ± 5 mm Hg), and 5β-DHT (99 ± 3/69 ± 5 mm Hg). The vehicle control did not significantly alter BP; however, the response induced by each steroid was significantly higher than that of the vehicle control at all doses tested (p < 0.01). Figure 4 displays the immediate drop in BP caused by each dose of steroid administration (5β-DHT), 15 min after the last dose, we observed a partial recovery of BP which was maintained at least for 90 min.

The analysis of the curves (Table 1) indicates that no significant differences were observed in the antihypertensive potency, i.e., ED₅₀ of androgens; DHEA, T, and 5β-DHT only in females. By contrast, in males, the most potent were 5β-DHT and DHEA, although T was two times less potent (ED₅₀) and with less efficacy (E_max) than them, whereas E2 was 4 times less potent than them. In conclusion, 5β-DHT and DHEA were more effective than T in males, while in females, the potency and efficacy of the three androgens were similar. Remarkably, E2 exhibited the lowest antihypertensive potency and efficacy in both females and males. Thus, the rank order of antihypertensive potency was DHEA = 5β-DHT = T >> E2 in females and 5β-DHT = DHEA >> T >> E2 in males. Likewise, the antihypertensive efficacy (E_max) was 5β-DHT = DHEA = T >> E2 in females and 5β-DHT = DHEA >> T >> E2 in males.

In the present study we observed that estrogens are effective in preventing hypertension only in females and that androgens are beneficial for both females and males to control high BP. Androgens may be more strongly involved than estrogens in the regulation of BP in both females and males. On the other hand, androgens and E2 are capable of reducing high BP (hypertension) such as in other rat models of hypertension [17‒19]. This decrease of BP to steroids tested was structure-dependent and non-genomically mediated. The present findings suggest the potential therapeutic use for arterial hypertension of the 5β-reduced metabolite of T (5β-DHT).

As expected, hypertension that developed after gonadectomy was completely prevented by E2B replacement in OVX females and TP replacement in ORX males. No significant differences were observed in the BP of rats treated with hormone replacement therapy when compared to the BP of intact rats. This evidence suggests that hormone replacement therapy reduces the risk of hypertension in postmenopausal and andropausal periods. Therefore, hypertension is accelerated by gonadectomy and attenuated by androgen or estrogen replacement. These results are in line with previous studies [41, 45]. By contrast, the male sex steroid, TP, is also effective in preventing high BP in OVX female rats. Moreover, E2B replacement in ORX male rats did not completely prevent hypertension.

Despite the biomedical relevance of sex differences in the development of hypertension in older adults, little consideration has been given to them. In this study, ORX male rats demonstrate a greater predisposition to hypertension, more rapid and severe, than OVX female rats. The change in BP was significantly greater in males versus females from week 7 onward. Since the severity of the increase in BP was more substantial in males (156 ± 4/105 ± 2 mm Hg, 18/10 mm Hg in SBP/DBP more than in females 138 ± 2/95 ± 2 mm Hg at the end of experiment, where males and females had similar BP at the beginning of the study; a mean increase in SBP of 29% in males vs. 15% in females, and a mean increase in DBP of 25% in males and 12% in females between weeks 0 and 15). We also observed that the BP was more rapidly established in male ORX rats than in female OVX rats. This finding suggests that androgen deficiency, particularly in aging men, increases the risk of hypertension in postmenopausal women. Consistent with these results, numerous male studies have associated low circulating testosterone levels with cardiovascular dysfunction, including hypertension [46‒50]. A decrease in estrogen production during and after menopause has been reported to be associated with hypertension in women [6] although the cause of hypertension has not been established between older women and men.

Although the autonomic nervous system plays an important role in BP regulation and contributes to modulating CVD [51], the rapid non-genomic membrane regulation of steroids in the cardiovascular system should be considered to prevent hypertension was demonstrated here by the rapid (∼2 min) antihypertensive response induced by sex steroids tested in conscious hypertensive rats caused by orchidectomy such as in other rat models of hypertension [17‒19]. It is well known that both E2 and T have rapid non-genomic vasodilating effects, these actions are short-term and very fast and the hormone might be administered intravenously as a bolus and could be considered a first-line therapy for acute hypertensive emergency to treat severe hypertension.

The doses of androgen used in this study are seemingly high compared to physiological plasma levels of T in the rats; however, since androgens, and other steroids, can accumulate at much higher local concentrations in target tissues than in plasma, published plasma levels of endogenous androgens likely do not reflect the true biologically relevant levels of androgens. In addition, the doses of androgens used in the present study are in the same range as those used in our previous reports [16‒19].

This study using an animal model excluded demographics or comorbidities or medication presented in humans in particular. The rat model of menopausal/andropausal used in the present work was very advantageous for our aims. However, subtle differences in hypertension were observed between menopausal and andropausal conditions that have not been well studied. Our findings corroborated that gonadectomy causes hypertension, which supports the fact that when the ovaries and testes are dysfunctional with aging, the control of BP is affected, and hypertension is triggered presumably by the lack of male or female gonadal sex steroids. This highlights the importance of gonadal sex steroid status and age.

Different molecular structures of each gonadal steroid tested to induce hypotension were analyzed. Our data indicate that androgens (C19-steroids) are more potent than estrogens (C18-steroids) in reducing BP. Regarding the potency and efficacy of androgens, 5β-DHT, and DHEA displayed the highest potency and T exhibited a moderate potency. Conformation analysis, i.e., the molecular structure of these three androgens, revealed that the Ring A (A/B cis conformation) of 5β-DHT is folded under the plane of Δ4-3keto configuration of T, and Δ5,3β-hydroxyl structure in DHEA which also has the inclusion of 17-keto group, a difference that may be relevant to increase its potency and efficacy. The structural change of the 5β configuration (when C₅ hydrogen is β/cis oriented) in 5β-DHT is critical for enhancing hypotensive potency and efficacy. These results agree with our previous evidence regarding the rank order of both potency and efficacy to induce vasorelaxing and antihypertensive responses characterized as a non-genomic effect whose mechanism of action has been previously associated with inactivation of L-type voltage-operated calcium channels and/or activation of potassium channels, revised in detail in [14]. By contrast, a possible reason for the low E2 potency may be that this estrogen has an aromatic A-ring with a C₃ hydroxyl and lacks a C19 methyl group. Therefore, the outstanding antihypertensive responses produced by all the androgens tested suggest that male sex steroids may strongly modulate BP which may open a window of opportunity for therapeutic use. Our findings also suggest that replacement therapy in aging women with 5β-DHT, a nonaromatizable androgen without androgen receptor binding and consequently without androgenic action, may be useful. This has special value since it has been reported that arterial hypertension affects more than a quarter of the adult female population worldwide [52], with the prevalence increasing markedly in postmenopausal women [6].

Consistent with this view, the 5β-DHT property of not bioconverting to estrogens, i.e., without estrogenic effects as well as without androgenic action, i.e., this 5-reduced metabolite has no affinity for androgen receptor should be considered. Moreover, coupled with its outstanding non-genomic (rapid) vasorelaxing and antihypertensive action (revised in [14]), this androgen has been suggested as an excellent therapeutic candidate for controlling hypertension in females and males.

Our results provide new information on sex differences in the development of BP in old age. This study demonstrates that both estrogen deficiency in women and androgen deficiency in men might contribute to the development of hypertension, underlining that androgen deficiency in females and males is the key point for the development of hypertension with special emphasis on the structure-function relationship of each androgen to induce hypotension as well as the potential therapeutic use for arterial hypertension of the 5β-reduced metabolite of T (5β-DHT). However, further studies are required to validate these findings in humans. Remarkably, the non-genomic action of sex steroids, is, however, still ignored by physicians, even by geriatricians, in the prescription of drugs and hormones to elder people.

To fill the gap between hypertension and older women and men, obesity is an important risk factor for hypertension should be discussed. Obesity is well known to play an important role in the pathogenesis of hypertension. Excessive visceral adipose cells that increase the release of inflammatory cytokines and decrease adiponectin production, thereby promoting endothelial dysfunction and hypertension [53], may be the main cause of hypertension. In this context, polycystic ovary syndrome is associated with hyperandrogenism in women, and most women with polycystic ovary syndrome have obesity and exhibit hypertension. Thus, androgen excess in women causing hypertension is plausible [54]. Nonetheless, our recent study revealed that hyperandrogenism in non-obese rats and women with polycystic ovary syndrome can regulate BP [20] and that obesity is responsible for hypertension, a situation that can also be synergized with sex steroid deficiency to induce hypertension.

Sex steroids can affect several mechanisms and many interactions of T or E2 with females or males have been proposed. For example, it has been indicated that estrogen treatment decreases BP in females [55], while T therapy increases BP in females [54, 56, 57] which can be attributed to the use of the wrong androgen, and/or a genomic action that can be associated with decreased estrogen receptor [57]. The present study showed that androgens have non-genomic antihypertensive properties. Finally, it is important to keep in mind that the regulation of BP by sex steroids occurs through synergistic action between genomic and non-genomic mechanisms.

This work shows two different aspects of gonadal steroids: (1) the development of hypertension by gonadectomy prevented with hormone replacement therapy in females and males which is a long-term genomic action, and (2) the fast non-genomic decrease in BP of hypertensive rat model by different androgens and estrogen. Significant evidence demonstrates that both female and male sex steroids play important roles in the development and control of hypertension in old age when the risk of this disease is remarkable. Gonadal steroid deficiency, particularly androgen deficiency is a key factor in the development of hypertension; in addition, BP increase was more severe in males versus females. Moreover, androgen 5β-DHT without estrogenic and androgenic properties may have potential therapeutic use as an antihypertensive in women and men.

The authors would like to thank David Rico Malfavón for his help with software execution.

The Institutional Animal Care and Use Committee (IACUC; ID 6307) reviewed and approved all protocols at the National Autonomous University of Mexico, Institute for Biomedical Research.

The authors have no conflicts of interest to declare. The authors declare no conflicts of interest that could be perceived as prejudicing the impartiality of the reported research.

This study was supported by M. Perusquía’s research budget from the Institute of Biomedical Research, Universidad Nacional Autónoma de México.

M.P. designed and directed the project. N.H. performed the experiments. All authors critically revised the report, commented on drafts of the manuscript, and approved the final report.

This manuscript contains all of the data generated or analyzed during this research. Further questions should be submitted to the corresponding author.