Folic acid enhances the cardiovascular protective effect of amlodipine in renal hypertensive rats with elevated homocysteine

ABSTRACT

Objectives

To investigate the actions of amlodipine-folic acid (amlodipine-FA) preparation on hypertension and cardiovascular in renal hypertensive rats with hyperhomocysteinemia (HHcy), so as to provide experimental basis for clinical research of amlodipine folic acid tablets.

Methods

Rats model of renal hypertension with HHcy were established. The rats were randomly divided into groups of model, amlodipine, folic acid (FA) and amlodipine-FA of various dosages. Normal rats were used as normal control group. Blood pressure, Hcy as well as plasma NO, ET-1 and hemodynamics were assayed. Histological alterations of heart and abdominal aorta were also examined.

Results

Compared with the normal group, blood pressure, plasma Hcy, and NO of the rats in model group were significantly increased, while the plasma ET-1 was decreased. Compared with the normal group, the animals in the model group had reduced cardiac function, thickened wall of the aorta and narrowed lumen. In FA group and amlodipine group, the rat plasma NO was increased while ET-1 was decreased, the protective effect of amlodipine-FA group on endothelial cells was further enhanced. In amlodipine group, the rat hemodynamics (LVSP, LVEDP and ±dp/dt_max, et al.) and vascular damage were significantly reduced, while in amlodipine-FA group, the heart function were further improved, and myocardial and vascular hypertrophy were significantly reduced.

Conclusions

As compared to amlodipine alone, amlodipine -FA can lower both blood pressure and plasma Hcy, significantly enhancing vascular endothelial function to protect the heart and blood vessel in renal hypertensive rats with HHcy.

KEYWORDS:

• Amlodipine
• folic acid
• hypertension
• homocysteine
• cardiovascular protection

Introduction

Recently, a screening involving more than 1.7 million of Chinese residents aged from 35 to 75 years old shows that the rate of hypertension is 37.2%, while that of hypertension’s awareness, treatment, and effective control are only 36.0%, 22.9% and 5.7% (1). Hypertension is a major risk for cardio-cerebrovascular diseases. Importantly, the risk of onset of cardiovascular diseases in hypertensive patients depends not only on blood pressure (BP) level, but also on the presence of various complications (2). For example, as a complication of hypertension, stroke has become a vital threat to public health worldwide, being the first cause of death in China (3). Therefore, effective treatment for hypertension is critical for the prevention of onset of related comorbidities such as stroke (4).

Homocysteine (Hcy) is an intermediate metabolite of methionine. Prospective studies suggest that high Hcy is an independent risk factor for cardio-cerebrovascular diseases, and has synergistic effects with hypertension in causing related complications (5–7). Moreover, both the guidelines for the primary prevention of stroke and secondary prevention of stroke from the American Heart Association/American Stroke Association pointed out that “elevated levels of plasma Hcy are associated with a 2 to 3-fold increased risk for atherosclerotic vascular disease, including stroke” (8,9).

Amlodipine is a dihydropyridine calcium channel blocker, which selectively acts on vascular smooth muscle and is used to treat hypertension and stable angina. Folic acid (FA), an essential nutrient for the growth and reproduction of cells, is an important coenzyme of methionine cycle. It can be converted into tetrahydrofolate in cells, and re-synthesized methionine to provide methyl donor during Hcy degradation, reducing Hcy production (10,11). A large number of studies have shown that FA is the most effective drug to reduce Hcy (12), which is used to treat hyperhomocysteinemia (HHcy). Therefore, FA may enhance the antihypertensive and cardiovascular protective effects of amlodipine. The effects of FA, amlodipine and amlodipine-FA on BP, Hcy, endothelial cell function, cardiac hemodynamics, and cardiac and vascular morphology were observed in renal hypertensive rats with elevated Hcy in our study, which provided an experimental basis for clinical application and further development of amlodipine-FA fixed dose combinations.

Materials and methods

Animals

Sprague Dawley rats (Experimental animal center of Nanjing medical university, SCXK（Su）2002–0031) were housed in a temperature-controlled room (temperature,18–25°C, relative humidity, 40–70%) with a 12-hour day/night cycle and were maintained with ad libitum access to tap water and normal rat chow during experiments. The procedures followed in this study were approved by the institutional animal care and use committee at the Anhui University of Chinese medicine.

Drugs and main reagents

Amlodipine (Zhejiang Xinsaike pharmaceutical Co., Ltd) was dissolved in 0.5% Sodium carboxymethyl cellulose (CMC-Na). FA (Jiangsu Linhai pharmaceutical Co., Ltd) was dissolved in 0.5% CMC-Na. Methionine (Tianjin Taihe pharmaceutical Co., Ltd) was dissolved in water. ET-1 radioimmunoassay diagnostic kit, Institute of radioimmunoassay, science and technology development center, PLA general hospital; Kit for radioimmunoassay of α₁-microglobulin, Beijing north institute of biotechnology; NO kit, Nanjing Jiancheng bioengineering research institute.

Experimental protocol

Male SD rats, weighing 120–160 g, were anesthetized with urethane and placed a 0.2 mm silver clip on the left renal artery to establish the two-kidneys one-clip (2K1C) Goldblatt model (13). After 8–10 weeks, rats with the systolic BP reached or exceeded 140 mmHg were considered as the model of hypertension. These rats were randomly divided into model group (0.5% CMC-Na), FA group (0.08 mg/kg), amlodipine group (0.5 mg/kg) and amlodipine-FA group at different doses (0.5 + 0.04 mg/kg, 0.5 + 0.08 mg/kg, 0.5 + 0.16 mg/kg), n = 14, respectively. The other normotensive rats were used as normal control group (n = 14). The normal control group drank tap water routinely, while the other groups drank 0.5% methionine solution to induce HHcy (14,15). Drugs were administered intragastrically once a day for 26 weeks.

BP measurement

Non-invasive BP measurement was achieved via using Mouse Rat Blood Pressure (MRBP) from IITC life science Inc., USA. The determination of BP was performed by tail-cuff method before and the 1^st, 5^th, 9^th, 13^th, 18^th, 22^nd, 26^th weeks after drug administration.

Determination of plasma Hcy

Plasma Hcy level was measured at the 13^th week and the 26^th week by HPLC respectively.

Measurement of endothelial cell function

At the end of the 26^th week, blood samples were taken to measure the levels of plasma nitric oxide (NO) and endothelin-1 (ET-1) according to the instructions of the assay kit.

Hemodynamic measurements

At the end of the 26^th week, the rats were anesthetized with 20% urethane and fixed in the supine position. The right common carotid artery was separated and a catheter filled with heparin was inserted into the left ventricle (LV). Hemodynamic parameters were examined by RM6240BD multi-channel physiological signal monitoring (Chengdu instrument factory). LV end-diastolic pressure (LVEDP), LV systolic pressure (LVSP) and the maximum rate of LV pressure rise (dp/dt_max) were measured to evaluate the effects on cardiac function. A micro-transducer catheter was inserted into the femoral artery to measure the systolic and diastolic pressure (16).

Measurement of cardiac weight index and ventricular weight index

After hemodynamic measurements, the rats were sacrificed by dislocation of cervical vertebra, their hearts were picked out, washed with precooled normal saline, separated the left ventricle (including ventricular septum), weighed the whole heart and the left heart, then the heart weight index (whole heart weight/body weight, Heart/BW) and the ventricular weight index (left ventricle/body weight, LV/BW) were calculated.

Quantitative morphological analysis of abdominal aorta

After euthanasia the abdominal aorta of rats were taken, fixed in 10% formalin, dehydrated and embedded, sectioned continuously, stained by HE method, observed by light microscope, analyzed by computer morphological image analysis system (Jiangsu Jieda Technology Development Co., Ltd.), measured the media thickness (MT) and lumen diameter (LD) of the vascular, and calculated the ratio of MT/LD × 100%.

Histological analysis

After weighing, the left hearts were fixed in 4% paraformaldehyde and embedded in paraffin immediately. Sections of 3 μm were stained with hematoxylin and eosin (H&E), and imaged with Nikon microscope.

Statistical analysis

Data are expressed as Mean±SEM. Statistical analyses were performed using the SAS 8.0 (SAS Institute, Cary, NC). One-way analysis of variance was used to compare groups (One-way ANOVA), Q test was used for pairwise comparison between groups (Students-NewmanKeuls，SNK). In all cases, P < .05 was considered significant.

Results

Effects of amlodipine-FA on BP in 2K1C rats with HHcy

In this study, the BP and plasma Hcy level in the model group were significantly higher compared to normal rats all through the entire experimental. Amlodipine successfully reduced the augmented BP in 2K1C rats with HHcy. However, BP levels in model rats treated with amlodipine were still significantly higher compared to that in normal rats. There was no significant difference in BP between FA group and the model group. Interestingly, supplementation of FA (0.16 mg/kg) further enhanced the anti-hypertension effect of amlodipine, starting from the 13^th week to the end (Figure 1). Moreover, at the 26^th week, BP levels in model rats administrated with amlodipine-FA (0.16 mg/kg) were even close to the normal rats (128 ± 3.49 mmHg vs 121.5 ± 2.22 mmHg, P = .125). Therefore, FA alone demonstrates certain potential in lowering BP, while its supplementation may significantly enhance BP hypotensive action of amlodipine in the context of hypertension and HHcy.

Figure 1. BP response to combination of amlodipine and FA in 2K1C rats with HHcy.

Note: Alterations in BP along the 26 weeks. FA group (0.08 mg/kg); Amlodipine group (0.5 mg/kg); Am-FA low group (0.5+0.04 mg/kg); Am-FA med group (0.5+0.08 mg/kg); Am-FA high group (0.5+0.16 mg/kg). Data were represented as Mean±SEM, N=10~14. ^#P<.05 compared with the amlodipine group.

Effects of amlodipine-FA on plasma Hcy and vascular endothelial function

Plasma Hcy was significantly elevated in 2K1C rats fed with methionine at the 13^th week till the end compared with control rats. FA alone greatly reduced plasma Hcy induced by methionine in 2K1C rats, whereas amlodipine insignificantly affected it. The amlodipine-FA therapy showed a comparable effect to FA alone in affecting HHcy (Figure 2a-2b). Therefore, FA but not amlodipine could be responsible for lowering plasma Hcy.

Figure 2. Changes in plasma Hcy, ETand NO 2K1C rats with HHcy treated with amlodipine and FA.

Note: Plasma levels of Hcy at the 13^th (a) and 26^th week (b). Plasma levels of ET-1 (c) and NO (d).FA group (0.08 mg/kg); Amlodipine group (0.5 mg/kg); Am-FA low group (0.5+0.04 mg/kg); Am-FA med group (0.5+0.08 mg/kg); Am-FA high group (0.5+0.16 mg/kg). Data were represented as Mean±SEM, N=10–14. *P<.05; **P<.01

Endothelial cell dysfunction is an early indicator of atherosclerosis and vascular diseases. To evaluate endothelial cell function, plasma ET-1 and NO were measured. Plasma ET-1 was upregulated in 2K1C rats with HHcy, indicating endothelial cell dysfunction. Amlodipine significantly decreased the level of plasma ET-1, although the level was still significantly higher than that in normal rats. Notably, supplementation of FA (0.08 to 0.16 mg/kg/day) to amlodipine successfully reversed the augmented level of plasma ET-1 to that in normal rats (Figure 2c), but there is no difference between the amlodipine group and amlodipine-FA groups in ET-1. Hypertension plus HHcy also significantly decreased the level of plasma NO. Interestingly, FA individually and partly reversed the level of plasma NO, while amlodipine only exhibited a trend to upregulate it. FA supplementation to amlodipine at medium dosage showed further improvement in plasma NO compared to amlodipine or FA alone (Figure 2d).

Hemodynamic effects of amlodipine-FA in 2K1C rats with HHcy

In order to evaluate cardiac function, LVSP (reflecting cardiac afterload), LVEDP (reflecting cardiac preload) and ±dp/dt_max were measured. LVSP and LVEDP were significantly higher in hypertensive 2K1C rats with HHcy compared to that in normal rats, amlodipine but not FA could be able to significantly reverse the LVSP and LVEDP in model rats. Meanwhile amlodipine-FA showed no further enhancement in affecting LVSP and LVEDP (Figure 3a-3b). It was a same case for the alterations in -dp/dt_max (Figure 3c). Interestingly, FA alone significantly improved the decreased +dp/dt_max 6 h post-last administration of drugs. Additionally, the combination of FA at medium dosage (0.08 mg/kg) to amlodipine further strengthened its action of affecting this parameter both 3 h and 6 h post-last drug administration (Figure 3d).

Figure 3. Hemodynamic alterations in 2K1C rats with HHcy treated with amlodipine and FA.

Note: (a) LVSP at 3h and 6h, (b) LVEDP at 3h and 6h, (c) +dp/dtmax at 3h and 6h, (d) –dp/dtmax at 3h and 6h, (e) SBP at 3h and 6h, (f) DBP at 3h and 6h. FA group (0.08 mg/kg); Amlodipine group (0.5 mg/kg); Am-FA low group (0.5+0.04 mg/kg); Am-FA med group (0.5+0.08 mg/kg); Am-FA high group (0.5+0.16 mg/kg). Data were represented as Mean±SEM, N=10–14. *P<.05; **P<.01.

Hypertension combined with HHcy dramatically increased systolic blood pressure (SBP) to around 170 mmHg in rats. Amlodipine alone can significantly reduce SBP to about 140 mmHg, but still higher than that in normal rats. Although FA alone showed no anti-hypertensive effect in model rats, supplementation of FA at both medium and high dosages to amlodipine significantly reversed the elevated SBP in model rats to the level of normal rats (Figure 3e). In addition, amlodipine – FA therapy strategy affected DBP in a similar way to SBP (Figure 3f).

Effects of amlodipine-FA on cardiac hypertrophy in 2K1C rats with HHcy

In hypertensive rats with HHcy, the ratio of LV/BW or heart/BW were significantly higher than that in normal rats, indicating cardiac hypertrophy. Amlodipine but not FA could significantly ameliorate the cardiac hypertrophy. However, supplementation of FA exhibited no further amelioration in the prevention of cardiac hypertrophy compared to amlodipine individually (Figure 4a-4b). Nevertheless, the results of H&E staining revealed myocyte hypertrophy, as well as large and abnormal nuclei in model rats with coronary atherosclerosis also detected. Amlodipine significantly attenuated these pathological changes, while the amlodipine-FA therapy (FA at the dosage of 0.08 to 0.16 mg/kg/day) further reversed these deteriorations (Figure 4c).

Figure 4. Histological changes in heart in response to the combination of amlodipine and FA in 2K1C rats with HHcy.

Note: (a) H&E staining of heart was observed with a light microscope (200 ×) and scale bar represented 50 μm. (b) The ratio of LV to BW. (c) The ratio of total heart to BW. FA group (0.08 mg/kg); Amlodipine group (0.5 mg/kg); Am-FA low group (0.5+0.04 mg/kg); Am-FA med group (0.5+0.08 mg/kg); Am-FA high group (0.5+0.16 mg/kg). Data were represented as Mean±SEM, N=10–14. *P<.05; **P<.01.

Effects of amlodipine-FA on morphology of abdominal aorta in 2K1C rats with HHcy

Compared with the normal group, the aortic wall of the model group was relatively thicker, the smooth muscle in the middle of the artery was hypertrophy, the elastic fiber cells were loose, the LD was decreased, and the ratio of MT/LD was increased. Amlodipine but not FA could reduce the MT and increase the MT/LD ratio. The combination of FA and amlodipine could enhance the effect of amlodipine (Figure 5).

Figure 5. Abdominal aorta alterations in response to the combination of amlodipine and FA in 2K1C rats with HHcy.

Note: (a)H&E staining of abdominal aorta was observed with a light microscope (200×) and scale bar represented 50 μm (b) media thickness(MT) of the vascular (c) lumen diameter(LD) of the vascular (d) The ratio of MT/LD×100%.FA (0.08 mg/kg); Amlodipine group (0.5 mg/kg); Am-FA low group (0.5+0.04 mg/kg); Am-FA med group (0.5+0.08 mg/kg); Am-FA high group (0.5+0.16 mg/kg) Data were represented as Mean±SEM, N=10–14. P<.05; *P<.05; **P<.01.

Discussion

Hypertension can result in large and medium arteries damaged and atherosclerosis. Hcy is an independent risk element for cardiovascular disease and induces endothelial dysfunction and arterial stiffening. High Hcy and hypertension have synergistic effect on the risk of atherosclerotic vascular disease, which may be the two most important risk factors leading to the high incidence of cardiac-cerebral vascular diseases (7). Reducing the level of hypertension and Hcy, simultaneously, may be an important measure to effectively control the high incidence of stroke in China. In the mouse abdominal aortic aneurysm model, the inflammatory cell infiltration, vascular injury and the occurrence of abdominal aortic aneurysm caused by angiotensin II can be significantly intensified by the increase of Hcy with a clear dose-response effect. However, FA can significantly alleviate this pathological effect of Hcy (17). In the stroke primary prevention trial in China, 20702 patients with essential hypertension were randomly treated with enalapril-FA tablets or enalapril tablets for 4.5 years (median), and the results showed that compared with enalapril based antihypertensive therapy, enalapril-FA based antihypertensive therapy could further significantly reduce the risk of first stroke by 21%, compound cardiovascular event by 20% and ischemic stroke by 24% (18).

It has been reported that the concentration of Hcy increased 5 μmol·L⁻¹, the risk of stroke increased by 59%, and the risk of ischemic heart disease increased by 32%. Consistently, the concentration of Hcy decreased 3 μmol·L⁻¹, the risks of stroke and ischemic heart disease by 24%（15%–33%）and 16% (11%–20%), respectively (19). HHcy greatly increased the incidence of angiotensin II-induced abdominal aortic aneurysm (AAA) and aortic dissection in apolipoprotein E-deficient mice. Histology indicated HHcy markedly exaggerated aortic adventitial inflammation. Increased levels of proinflammatory interleukin-6 and monocyte chemoattractant protein-1 were preferentially colocalized within adventitial fibroblasts in HHcy plus angiotensin II mice, which suggested the importance of adventitial fibroblasts activation in Hcy-aggravated AAA. Hcy sequentially stimulated adventitial fibroblasts transformation into myofibroblasts, secretion of interleukin-6 and monocyte chemoattractant protein-1, and consequent recruitment of monocytes/macrophages to adventitial fibroblasts, which was abolished by the NADPH oxidase inhibitor diphenyliodonium. FA supplement markedly reduced HHcy-aggravated angiotensin II-induced AAA formation in apolipoprotein E-deficient mice (17).

Folic acid and its active metabolite 5-methyltetrahydrofolate(5-MTHF) are essential nutrients in humans and play a significant role in nucleotide synthesis and methylation reactions. Folic acid can reverses endothelial dysfunction of endocardium in homocysteinemic hypertensive rats (20). Hcy-induced endothelial dysfunction in humans was improved with oral administration of FA. FA may also have beneficial effects on BP by increasing nitric oxide synthesis in endothelial cells, or by reducing plasma Hcy. Two prospective cohort studies showed that higher total folate intake was associated with a decreased risk of incident hypertension, particularly in younger women (21). On the contrary, folate deficiency can promote oxidative stress and multiple features of the metabolic syndrome that are associated with increased risk for diabetes and cardiovascular disease (22).

Clinical studies have shown that long-term oral use of FA can control the level of plasma Hcy and may reduce cardiovascular events (23), such like effectively reducing the risk of hypertension, mending arterial endothelial structure and avoiding the happening of atherosclerosis in hyperhomocysteine patients (24). In addition, FA’s beneficial effects on endothelial structure are not completely dependent on the Hcy-lowering effects. 5-MTHF, an active form of FA, reduced superoxide generation and provided antioxidant potential, thus restoring impaired endothelial structure. Another explanation for the beneficial effects of FA on endothelial structure refers to its effects on the enzyme endothelial nitric oxide synthase.

In our study, after 8 to 10 weeks of stenosis of the left renal artery in rats with 0.2 mm silver clip, stable hypertension can be formed. The level of plasma Hcy in rats with renal hypertension can be significantly increased by orally administrated with 0.5% methionine solution at the same time. Our study showed that the BP of rats in FA group decreased slightly, and there was no significant difference compared with the model group. The BP in amlodipine group decreased significantly, and further decreased in amlodipine-FA group, with significant differences at 9^th–26^th weeks. It showed that FA can further enhance the antihypertensive effect of amlodipine in 2K1C rats with HHcy. Compared with the normal group, the level of plasma Hcy in the model group increased significantly, while that in the FA group and amlodipine-FA group decreased greatly. There was no significant change in Hcy in the amlodipine group, indicating that FA had the effect of reducing Hcy, and amlodipine had no significant effect on the level of plasma Hcy. A recent clinical study in China further demonstrated that individualized FA administration combined with amlodipine can not only effectively reduce blood pressure and Hcy levels in patients with H-type hypertension, but also reduce clotting factor levels and significantly improve the pre-thrombotic state (25).

Endothelial dysfunction, act as an early marker of cardiovascular damage has been widely concerned. Endothelial cells can produce a wide variety of substrates in response to various physical and chemical stimuli, such as vasodilator substrate nitric oxide and vasoconstrictor substrate ET-1 (26), which have been acknowledged as markers of endothelial activity. High Hcy level associating with risks of cardio-cerebrovascular diseases might be partially accounted by inductions of endothelial dysfunctions (27,28).

In the current study, plasma NO level of the model group was decreased with ET-1 level was increased compared to control group, indicating that model rats developed dysfunctions in endothelium. Both of amlodipine and FA demonstrated endothelial protection caused by hypertension and HHcy. Furthermore, combination of the two medicines enhanced this action. The effect of FA is consistent with the literature reports (29).

Bloor et al. (2010) showed that folate reduced neointimal thickness and superoxide formation and increased microvessel density in diabetic animals. In addition to folate’s protective effects on vascular endothelial cells, this evidence suggests its beneficial effects on vascular smooth muscle cells and a possible role in the prevention of atherosclerosis. Further research shows that folate upregulated p21 and p27 expression and inactivated homocysteine-mediated AKT1 phosphorylation, resulting in antiproliferative effects in homocysteine-challenged rat aortic smooth muscle cells (30). In our study, abnormal alterations in parameters reflecting cardiac states (LVSP, LVEDP and ±dp/dtmax) and vascular morphology parameters (MT, LD and MT/LD), indicated cardiovascular damage in model rats. Despite that FA alone induced no significant amelioration on hemodynamic alterations in model rats, LVSP and LVEDP were further decreased, and ±dp/dtmax was further increased owing to amlodipine-FA combination in comparison to amlodipine. FA has the tendency to reduce the vascular remodeling. The combination of FA and amlodipine can enhance the effect of amlodipine on heart and vascular remodeling in hypertensive rats. No obvious changes were found in the heart and vascular in the normal rat. In the model rat, cardiac myocytes were hypertrophied, nuclei were large and deeply stained, coronary artery was sclerotic, aortic wall was thickened, smooth muscle in the middle of artery was hypertrophied, and elastic intercellular cells were loose. Amlodipine-FA can significantly reduce the pathological changes of the heart and blood vessels and improve the reconstruction of the heart and blood vessels and the thickness of arterial media as well as the improvement of vascular remodeling. Enalapril maleate and folic acid tablet can maintain the normal state of cells via the alleviation of ERS and vascular damages, reduction of Hcy (31). This is similar to the results of our study.

In summary, Amlodipine combined with folic acid can reduce both blood pressure and Hcy. Compared with amlodipine alone, Amlodipine-FA strengthen the protective effect on vascular endothelium and significantly improve cardiac and vascular lesions.

To date, our results provide experimental basis for the clinical application of amlodipine-FA tablets, which have been approved into market in China recently. This is the second drug approved for the treatment of hypertension and hyperhomocysteinemia (H-type hypertension). It is a better choice for patients with H-type hypertension.

Conflicts of interest

The authors declared no conflict of interest.

Author contributions

Conceived and designed the experiments: Pr. Guangliang Chen and Haipeng Liu; Performed the experiments: Li li, Xiaohui Tong and Zebing Ma; Performed the pathological examination: Lei Lv and Zebin Ma; Analysed the data: Tong Xiaohui; Wrote the manuscript: Tong Xiaohui Tong and Pr.Guangliang Chen.

Additional information

Funding

This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” [Grant Number: [2012] 52–401013], the National Major Scientific and Technological Special Project for “Safety evaluation of clinical study of amlodipine folic acid tablets” [Grant Number: 2018ZX09739010–006].