**1. Introduction**

With a global prevalence of about 40% in the adult population [1], arterial hypertension is the main risk factor for morbidity and mortality of cardiovascular diseases and is considered to be the leading cause of death in industrialized countries [2–4]. In the overwhelming majority of patients, hypertension manifests in adulthood. The number of hypertensive adults aged 30–79 years in the world is more than 1.2 billion people at present [5]. The prevalence of hypertension increases with advancing age to 60–70% in people aged over 60–65 years [3,6]. From all hypertensive patients, only about 50% are diagnosed, and in about 40–50% of diagnosed patients, treatment is lacking or insufficient to stabilize blood pressure (BP) at values below 140/90 mmHg [5,7–9]. Untreated or insufficiently treated arterial hypertension leads to constant pressure load of the left ventricle (LV), which first results in cardiac hypertrophy. With long maintenance of hypertension, it may progress to cardiac remodeling, fibrosis, dilatation, and finally heart failure. One of the main causes of lacking or insufficient treatment of diagnosed hypertension is poor adherence or even non-adherence to treatment [3]. The term adherence denotes the extent to which the patient complies with the therapy recommendation. Non-adherence to antihypertensive therapy affects 10–80% of all hypertensive patients, preferably elderly patients [9,10]. Poor adherence to antihypertensive treatment is associated with elevated BP values, an increased cardiovascular risk, and a poor prognosis in these patients [3,9].

**Citation:** Rassler, B.; Hawlitschek, C.; Brendel, J.; Zimmer, H.-G. How Do Young and Old Spontaneously Hypertensive Rats Respond to Antihypertensive Therapy? Comparative Studies on the Effects of Combined Captopril and Nifedipine Treatment. *Biomedicines* **2022**, *10*, 3059. https://doi.org/10.3390/ biomedicines10123059

Academic Editors: Josef Zicha and Ivana Vanˇeˇcková

Received: 20 October 2022 Accepted: 21 November 2022 Published: 28 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Although the prevalence of hypertension is significantly higher in persons above 60 years, about 13% of younger adults between 20 and 40 years of age worldwide are hypertensive with an average BP above 140/90 mmHg [11]. In a study on young adults, only 11% of the hypertensive persons received antihypertensive medication at the time of enrolment [12]. Hypertension is less frequently diagnosed in young than in old adults [11]. Younger adults may be in their prehypertensive state, which may remain undetected or underestimated for a longer period of time. However, four out of five prehypertensive people aged 40–49 years would become hypertensive in the next 10 years [13]. Prehypertensive and even hypertensive young people are often not aware of their BP [14]. However, several studies have demonstrated that elevated BP at youth correlates with higher rate of LV hypertrophy and cardiovascular mortality at an older age [15–17]. Therefore, the ESC/ESH recommends that all adults ≥18 years should have their office BP measured and recorded, and be aware of their BP [3].

Several years ago, the SPRINT study was the first randomized trial that clearly demonstrated the benefits of a systolic BP (SBP) reduction below previous target values to 120 mmHg [18,19]. A change in BP of 20/10 mmHg is associated with a 50% difference in cardiovascular risk [9]. In patients who were at increased risk of cardiovascular disease, antihypertensive therapy targeting SBP below 120 mmHg resulted in lower rates of major adverse cardiovascular events and mortality compared with target SBP of less than 140 mmHg [20]. Consequently, the BP targets have been revised in the recent European, U.S., and WHO guidelines [3,21,22]. Early initiation of therapy and faster achievement of the recommended BP targets (within 6 months, ideally even within 3 months) are associated with a significant reduction in heart failure and other major cardiovascular events [23]. For initiation of antihypertensive therapy, a combination treatment of a blocker of the renin-angiotensin system (RAS; either an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin receptor blocker) with a calcium channel blocker (CCB) or diuretic is the preferred recommendation for most hypertensive patients. Combination therapy, preferably as single-pill combination, has been proven to be more effective in fast achievement of the recommended BP targets and to be associated with a better adherence of patients to treatment [3,23].

#### *Purpose of the Present Study*

To prevent the development of cardiovascular complications, adequate therapy should start as early as possible. In animal models, efficient antihypertensive therapy started in their prehypertensive state proved to be more efficient than the same therapy started in adulthood when hypertension was manifest [24,25]. In humans, there is still much controversy about the treatment of younger adults with low-risk grade 1 hypertension and about the efficacy of antihypertensive treatment in young hypertensives compared with old patients. Conventional clinical outcome studies are difficult to perform in people before age 40, hence there is a lack of randomized clinical trials for young hypertensive patients [3,11]. Consequently, it is not clear whether the effects of antihypertensive treatments in old and young people are comparable. Even in animal models, there are only very few studies comparing treatment effects in young and old animals. Previously, we had performed studies on antihypertensive treatments in young (aged 7–10 weeks) [26] and old (aged 60–82 weeks) spontaneously hypertensive rats (SHRs) [27]. The present study was designed to directly compare the treatment effects achieved in young and old SHRs. The relative reduction of SBP, parameters of LV hypertrophy and LV remodeling and fibrosis induced by a combination of the ACE inhibitor captopril and the CCB nifedipine was compared using a two-way ANOVA with the factors treatment and age. The purpose of this analysis is to show how effectively the reduction in SBP and attenuation of cardiac hypertrophy and remodeling can be achieved with early-onset compared with late-onset therapy.

#### **2. Materials and Methods**

We re-evaluated data from two previously published studies on male SHRs [26,27]. The raw data were obtained from these studies and were re-analyzed using a new statistical approach allowing a direct comparison between the data from both animal studies. All animal protocols were approved by the state agency (Landesdirektion Sachsen, number and date of approval: TVV 36/08; 13 May 2009) in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health and with the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes".

In brief, we analyzed data from 12 young SHRs aged 7–10 weeks (ySHRs) and fourteen old SHRs aged 60–82 weeks (oSHRs). The study on ySHRs [26] was designed as a pilot study to identify the most appropriate treatments to be applied to oSHRs. The ySHRs were intended to be in their adolescence throughout the experiment. For this reason, the experimental period was defined from 7 to 10 weeks of age. In contrast, the old SHRs should be treated over a long period of time, which was set to 22 weeks [27].

All animals were cultured at Charles River, Sulzfeld, Germany. Both groups were subdivided at random into two subgroups each, one remaining untreated and serving as control (yCtrl, n = 6; oCtrl, n = 7) and the other one being treated with a combination of captopril (60 mg kg−<sup>1</sup> d−1, Axxora, Lörrach, Germany) and nifedipine (10 mg kg−<sup>1</sup> d−1, Sigma-Aldrich Chemie, Steinheim, Germany). The treated groups were labeled as yC+N (n=6) and oC+N (n = 7). ySHRs were 7 weeks of age at the beginning of the treatment period, which lasted 3 weeks. In oSHRs, the treatment period started at 60 weeks of age and lasted 22 weeks. The medication (or placebo in Ctrl animals) was administered as tablets for oral uptake given into the cages along with chow once daily between 9:00 a.m. and 10:00 a.m.

Before and during the experimental period, we regularly measured systolic blood pressure (SBP) using the tail-cuff-method (TSE Blood Pressure Monitor, Series 209002, TSE Systems GmbH, Bad Homburg, Germany; for more details, see [26,27]). Values from the final SBP measurement performed in the last experimental week were taken for the present re-evaluation.

At the end of the experiments, the animals were sacrificed and their hearts were extracted. We determined heart weight (HW) as a measure of cardiac hypertrophy. We analyzed the absolute HW as well as HW normalized to final body weight (HW/BW). Pieces of the left ventricle (LV) were obtained to perform ribonuclease protection assays to determine several markers of LV hypertrophy and remodeling. mRNA expressions of atrial natriuretic peptide (ANP) and collagen types I (Coll 1) and III (Coll 3) were included into the present analysis. Histological preparations were made from the cardiac apex. A score ranging from 0 (= no fibrosis) to 3 (= fibrosis of the entire heart) was applied to assess the histological degree of fibrosis (for more details, see [26,27]). The degree of fibrosis was also included in the present analysis.

Statistical analyses were carried out with the software package SigmaPlot Version 14.0 (Systat Software GmbH, Erkrath, Germany) for Windows. First, we directly compared final SBP, HW, HW/BW, ANP mRNA, Coll 1 mRNA, Coll 3 mRNA, and degree of fibrosis among all four groups (yCtrl, yC+N, oCtrl, oC+N) using a two-way ANOVA with the factors A = treatment (Ctrl or C+N) and B = age (y or o). As a post-hoc test, an all pairwise multiple comparison procedure according to the Holm–Sidak method was applied.

In addition, we assessed the treatment effect for each parameter and compared the treatment effects in young versus old SHRs. The treatment effect was calculated for each treated animal as the difference between the average value of the respective Ctrl group (y or o) and the individual value of the treated animal. The difference was normalized to the average yCtrl or oCtrl value (given in %). A positive value of the treatment effect marks an improvement (i.e., reduction) in the parameter. For the comparison of young versus old, we first performed a Shapiro–Wilk test for normality. In the case of normal distribution, we

performed a t-test, otherwise a Mann–Whitney rank sum test was applied. *p*-values < 0.05 were considered significant.

#### **3. Results**

#### *3.1. Treatment Effects on SBP*

SBP of young SHRs at baseline (age 7 weeks) was 173.8 ± 3.1 and 157.5 ± 3.7 mmHg for Ctrl and C+N animals, respectively (Table 1). This difference was not significant. The original experiment [26] included a total of 42 ySHRs with an average SBP at baseline of 171.2 ± 3.5 mmHg. In untreated ySHRs, SBP increased continuously over the 3-week period of observation to 201.7 ± 6.5 mmHg at 10 weeks of age. This was significantly higher than SBP of age-matched SHRs after 3 weeks of C+N treatment. In the first two weeks of treatment, SBP decreased to 115.8 ± 2.4 mmHg. SBP of age-matched Wistar–Kyoto rats (WKY) was with 123.1 ± 0.9 mmHg in a similar range [26]. However, in the final treatment week, SBP re-increased to 152.5 ± 2.8 mmHg. The difference to untreated ySHRs was with 49.2 ± 2.8 mmHg significant (*p* < 0.001) corresponding to a treatment effect of 24.4 ± 1.4% of untreated Ctrls.

**Table 1.** SBP, HR, and BW of young and old SHRs.


Data are given as means ± SEM. Baseline values were obtained at 7 weeks of age in young SHRs and at 60 weeks of age in old SHRs. Final values were obtained at 10 weeks of age in young SHRs and at 82 weeks of age in old SHRs. Treatment groups: y/oCtrl: untreated animals, y/oC+N: animals treated with captopril + nifedipine. \* significant vs. corresponding Ctrl group (*p* < 0.05); ◦ significant vs. baseline value (*p* < 0.01); ◦ ◦ significant vs. baseline value (*p* < 0.001).

Age had a significant effect on SBP (*p* < 0.001). In untreated oSHRs aged 60 weeks, SBP was 231.0 ± 8.7 mmHg and hardly rose during the following 22 weeks to 239.0 ± 5.1 mmHg at week 82 of age (*p* < 0.001 compared with untreated ySHRs). Treatment with C+N over 22 weeks significantly reduced SBP by 45.4 ± 4.8 mmHg to 193.6 ± 4.8 mmHg (*p* < 0.001; Table 1); this is, however significantly higher than in yC+N rats (*p* < 0.001; Figure 1A). The treatment effect is 19.0 ± 2.0% of the untreated oCtrls. However, despite a much longer period of therapy in oSHRs, this treatment effect is lower than in ySHRs (*p* = 0.059; Figure 1B).
