Treatment With Low-dose Atorvastatin,Losartan,and Their Combination Increases Expression of Vasoactive-Related Genes in Rat Aortas

Abstract

Recently it has been shown that statins and angiotensin receptor blockers (ARBs) at low doses express beneficial pleiotropic vascular effects. We aimed to explore whether these drugs at low doses induce the expression of vasoactive-related genes. Sixty adult Wistar rats were treated with low-dose atorvastatin (2 mg/kg), low-dose losartan (5 mg/kg), their combination or saline daily for 4, 6, or 8 weeks. Expression of the vasoactive-related genes endothelin receptor type A (EDNRA), endothelial nitric oxide synthase 3 (NOS3), inducible nitric oxide synthase 2 (NOS2), and angiotensin II receptor type 1 (AGTRL1a) was measured in isolated thoracic aortas. Expression of EDNRA gradually decreased, the lowest values being obtained after 8 weeks (low-dose atorvastatin, losartan [1.6- and 1-7-fold vs controls, respectively; both P < .05], and the combination [2.3-fold vs control, P < .001]). The highest values of NOS3 were obtained after 6 weeks (low-dose atorvastatin, losartan, and their combination, 3.1-fold, P < .01; 3.4-fold, P < .001; and 3.6-fold, P < .001 vs controls, respectively) and then declined after 8 weeks. The combination was more effective in inducing total NOS3 expression when compared to the separate drugs (1.4-fold; P < .05). Importantly, expression of NOS3 was associated with increased plasma NO levels and positively correlated with thoracic aorta relaxation. No changes in expression of NOS2 and AGTRL1a were observed. We showed that low-dose atorvastatin or losartan and especially their combination increases the expression of NOS3 and decreases the expression of EDNRA. These findings are valuable in explaining the effectiveness of the “low-dose pharmacological approach” for improvement in arterial function.

Keywords

vascular pharmacology vasoactive-related gene expression atorvastatin losartan chronic low-dose treatment

Introduction

Despite numerous preventive and progressively improving treatment strategies, cardiovascular diseases maintain their primacy in morbidity and mortality in developed countries.^1,2 Statins and renin–angiotensin system inhibitors have proven beneficial effects on the cardiovascular system. These effects, also known as pleiotropic effects, occur in addition to both drugs’ primary effects (cholesterol or blood pressure reduction, respectively).^3,4 Both drug groups show the ability to induce cardiovascular protection through improvement in endothelial function and consequent vasodilation, as well as through antioxidant, immunomodulatory, inhibition of cell migration, proliferation, and plaque stabilization mechanisms.⁵

The beneficial cardiovascular pleiotropic effects of statins and angiotensin receptor blockers (ARBs) were shown at therapeutic doses.^6
–8 Notably, only a few studies examined low doses of statins and ARBs, but these revealed the existence of pleiotropic effects in the absence of their primary effects.^9,10 We showed that subtherapeutic, low doses of a statin (fluvastatin), an ARB (valsartan), and particularly their combination, substantially improved both functional and structural properties of the arterial wall in healthy middle-aged males.^11
–13 Furthermore, in a previous animal study, we demonstrated that atorvastatin or losartan in low doses had important protective cardiovascular effects (increased thoracic artery vasodilation, coronary flow increase, and serum nitric oxide [NO] concentration increase) and that the combination of both drugs was even more efficient than the separate drugs.¹⁴

Endothelial cells maintain vascular tone mainly through the balance between NO and endothelin ([ET] in the cardiovascular system by endothelin 1 [ET-1]) production. Nitric oxide is a potent vasodilator causing relaxation of vascular smooth muscle cells primarily through the activation of soluble guanylate cyclase and elevation of intracellular cyclic guanosine monophosphate (cGMP). The production of NO is catalyzed by different nitric oxide synthases (NOS): endothelial NOS (eNOS), encoded by NOS3 gene and inducible NOS (iNOS) encoded by NOS2 gene.¹⁵ On the other hand, ET-1 is a potent vasoconstrictor, exerting its effects through the activation of specific ET receptors of type A (ET_A; encoded by the EDNRA gene) within the vascular smooth muscle cells, or type B (ET_B) located on both endothelium and vascular smooth muscle cells.¹⁶ It is well known that NO and ET-1 interact via various mechanisms. Endogenous NO suppresses ET-1 production via cGMP-dependent and -independent pathways.^16
–18

The aim of the present study was to explore some of the mechanisms underlying the protective cardiovascular effects of low-dose statins and ARBs, through analyzing the expression of selected vasoactive-related genes in the thoracic aortas of rats treated with low-dose atorvastatin, low-dose losartan, or their low-dose combination. The genes chosen were endothelin receptor type A gene (EDNRA), endothelial NOS3 gene, inducible NOS2 gene, and angiotensin II receptor type 1 gene (AGTRL1a).

Materials and Methods

Animals, Drugs, and Study Design

Sixty adult Wistar rats of both sexes weighing 230 to 280 g were obtained from the Faculty of Medicine, Ljubljana. They were bred under constant housing conditions and fed with standard rat chow in the form of pellets (Altromin No. 1320, Lage, Germany). Rats were randomly assigned to 1 of 4 experimental groups: (1) rats that received tap water (controls); (2) rats that received atorvastatin (2 mg/kg/d; orally po); (3) rats that received losartan (5 mg/kg/d; po), and (4) rats that received a combination of atorvastatin and losartan (2 mg/kg/d po and 5 mg/kg/d po, respectively). After 4, 6, or 8 weeks of treatment, the animals were sacrificed. Each group was composed of the same number of male and female rats. Atorvastatin (atorvastatin calcium) and losartan (losartan potassium) were generously provided by Krka Pharmaceuticals (Krka, d. d., Novo mesto, Slovenia). The drugs were diluted in distilled water to prepare solutions that were applied orally for 4 to 8 weeks prior to the organ isolation. The same animals were used also for another study¹⁴ in which different parameters were measured.

All experiments were conducted in accordance with the guidelines set down by the Veterinary Administration of the Republic of Slovenia (permit No. 34401-23/2009/3), which conforms to the Guide for the Care and Use of Laboratory Animals from the Institute for Laboratory Animal Research, National Research Council, Washington DC (National Academy Press, 1996) and to EU Directive 2010/63/EU.

The rats were anaesthetized with an intraperitoneal (ip) injection of urethane (130 mg urethane/100 mg body weight; Sigma-Aldrich, St Louis, Missouri). Heparin in a dosage of 1000 IU (Krka, Novo mesto, Slovenia) was also injected ip. The thoracic aortas were then isolated, snap frozen in liquid nitrogen and stored at −80°C until RNA isolation. The samples used for the analysis of gene expression came from animals used in a previous study in which other parameters were measured (thoracic aorta vasorelaxation, isolated heart coronary flow, laboratory analysis of blood serum).¹⁴

RNA Isolation

Approximately 200 mg of tissue was pulverized in liquid nitrogen and total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, California), according to the manufacturer’s recommendations. RNA was dissolved in RNase-free water. Total RNA quantity and purity was assessed by measuring A₂₆₀ and A₂₈₀. Total RNA integrity was determined by 2% agarose gel electrophoresis using an Agilent 2100 Bioanalyser (Agilent Technologies, Palo Alto, California).

Reverse Transcription and Quantitative Polymerase Chain Reaction

First-strand reverse transcription complementary DNA (cDNA) synthesis was performed on 10 μg of total RNA at a final volume of 100 μL using a High-Capacity cDNA Archive kit (Applied Biosystems, Foster City, California), according to the manufacturer’s instructions. The cDNA was stored at −80°C until the samples were processed. Quantitative polymerase chain reaction (PCR) was performed on 9 ng of cDNA template (referring to the amount of total RNA used in the reverse transcription reaction) from each sample for angiotensin II receptor type 1 (AGTR1a), EDNRA, inducible NOS2, endothelial NOS3, and hydroxymethylbilane synthase (HMBS) as a reference gene, using predesigned TaqMan Gene Expression Assays Rn00578456_m1, Rn00561137m_1, Rn00561646_m1, Rn01423590_m1, Rn02132634_s1, Rn00561129_m1, and Rn00565886_m1, respectively (Applied Biosystems). Reactions were performed with a LightCycler Probes Master (Roche Applied Science, Mannheim, Germany) on a LightCycler 480 thermal cycler (Roche Applied Science). Cycling conditions comprised preincubation at 95°C for 10 minutes, followed by 50 cycles of denaturation at 95°C for 10 seconds and primer annealing and elongation at 60°C for 30 seconds. The quantity of messenger RNA (mRNA) in the sample was estimated using a standard curve performed on each plate and obtained by stepwise dilution of the stock mRNA processed as described above. The reactions were performed in triplicate for samples and in quadruplicate for standards. The amounts of target genes were normalized to the amount of reference gene. The reference gene was selected from 9 candidates obtained by reviewing the literature. Twelve samples representing untreated animals and animals with all variant treatments were used to determine the gene expression of all candidate genes as described above, and the selection of the most appropriate reference gene (HMBS) was based on the smallest geometric average threshold cycle (Ct) of individual candidates and results from geNorm.¹⁹ In the case of NOS3, the contribution of DNA to the quantitation of mRNA was negligible, as assessed by running a PCR reaction with samples from participants but omitting the reverse transcription.

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 5.0 (GraphPad Software, San Diego, California). Values were expressed as means ± standard error of the mean (SEM). The differences in gene expression between groups were analyzed by 1-way analysis of variance (ANOVA) with Bonferroni posttest used to make intergroup comparisons. The Spearman rank order correlation test was used to study relations between variables. A value of P < .05 was considered significant.

Results

Time-Dependent Expression of Selected Genes

Week-by-week gene expression was examined in thoracic aortas of rats treated for 4, 6, or 8 weeks with low-dose atorvastatin, low-dose valsartan, or their low-dose combination. The genes analyzed were EDNRA, NOS3, NOS2, and AGTR1a. The values of the week-by-week gene expression are presented in Table 1.

Endothelin receptor type A gene

The highest expression of EDNRA gene was in the fourth week of treatment, progressively declining after that period until the eighth week of treatment. In the atorvastatin and losartan groups, EDNRA expression was significantly lower only after 8 weeks of treatment compared to the control group; low-dose atorvastatin diminished EDNRA concentration by 1.6-fold and low-dose losartan by 1.7-fold (both P < .05). Their combination was even more effective; in the latter group, EDNRA expression was diminished already after 4 and 6 weeks of treatment (1.6-fold and 1.8-fold, respectively; both P < .05) and also after 8 weeks of treatment (2.3-fold; P < .001) compared to the control group (Table 1).

Table 1.

Gene Expression of Selected Genes After 4, 6, or 8 Weeks of Treatment With Low-dose Atorvastatin, Low-Dose Losartan, or Their Combination and in the Control Group.^a

	Control	Atorvastatin	Losartan	Combination
EDNRA	4 w: 1.77 ± 0.23	4 w: 1.56 ± 0.23	4 w: 1.57 ± 0.17	4 w: 1.07 ± 0.04^b
	6 w: 1.76 ± 0.35	6 w: 1.43 ± 0.11	6 w: 1.17 ± 0.48	6 w: 0.96 ± 0.02^b
	8 w: 1.78 ± 0.15	8 w: 1.11 ± 0.06^b	8 w: 1.05 ± 0.01^b	8 w: 0.76 ± 0.10^c
NOS3	4 w: 0.75 ± 0.15	4 w: 1.26 ± 0.26	4 w: 1.41 ± 0.07	4 w: 1.19 ± 0.01
	6 w: 0.80 ± 0.28	6 w: 2.51 ± 0.43^d	6 w: 2.74 ± 0.89^c	6 w: 2.84 ± 0.26^c
	8 w: 0.82 ± 0.19	8 w: 1.33 ± 0.03	8 w: 1.58 ± 0.15	8 w: 1.44 ± 0.08
NOS2	4 w: 0.117 ± 0.097	4 w: 0.009 ± 0.002	4 w: 0.018 ± 0.006	4 w: 0.004 ± 0.001
	6 w: 0.125 ± 0.024	6 w: 0.012 ± 0.003	6 w: 0.015 ± 0.001	6 w: 0.008 ± 0.001
	8 w: 0.105 ± 0.037	8 w: 0.015 ± 0.003	8 w: 0.018 ± 0.005	8 w: 0.032 ± 0.007
AGTR1a	4 w: 1.13 ± 0.15	4 w: 1.19 ± 0.03	4 w: 1.25 ± 0.02	4 w: 0.97 ± 0.12
	6 w: 1.16 ± 0.21	6 w: 1.19 ± 0.09	6 w: 1.26 ± 0.31	6 w: 1.02 ± 0.09
	8 w: 1.17 ± 0.17	8 w: 1.03 ± 0.13	8 w: 1.00 ± 0.02	8 w: 0.97 ± 0.11

Abbreviations: EDNRA, endothelin receptor type A gene NOS3, endothelial nitric oxide synthase 3 gene NOS2, inducible nitric oxide synthase gene AGTR1a, angiotensin II receptor, type 1a gene 4 w, 4 weeks 6 w, 6 weeks 8 w, 8 weeks.

^aAll values are means ± SEM.

^b P < .05.

^c P < .001.

^d P < .01, all compared to the control group.

Nitric oxide synthase 3 gene

Expression of NOS3 was low after 4 weeks of treatment with low-dose atorvastatin, low-dose losartan, or their combination, reaching maximum values after 6 weeks of treatment and then declining again after 8 weeks of treatment. After 6 weeks of treatment, atorvastatin increased NOS3 expression up to 3.1-fold (P < .01), losartan up to 3.4-fold (P < .001), and their combination up to 3.6-fold (P < .001) compared to the control group (Table 1).

Nitric oxide synthase 2 gene

The expression of NOS2 gene was lower in all treatment groups compared to the control from the fourth to eighth week of treatment, but the differences did not reach the level of significance. The expression in separate groups remained constant throughout the treatment period (Table 1).

Angiotensin II receptor type 1a gene

The expression of AGTR1a gene remained constant in all separate groups throughout the treatment period. The lowest values were observed in the combination group, though not reaching significance compared to the control group (Table 1).

Overall Expression of Selected Genes

The expression of selected genes in the low-dose atorvastatin, low-dose losartan, or low-dose combination group was analyzed for the overall treatment period in separate treatment groups (from fourth until eighth week of treatment). The results are presented in Figure 1. The expression of EDNRA was decreased during the overall treatment period compared to the control group; in the low-dose atorvastatin and losartan groups by 1.3-fold (both P < .05) and in the combination group by 1.9-fold (P < .001) compared to controls. Expression of NOS3 gene was increased in the low-dose atorvastatin and losartan groups by 1.9-fold (both P < .01); while in the combination group, the expression was increased 2.6-fold (P < .001) compared to the control group. The low-dose atorvastatin and losartan combination was even more effective when compared to the separate drugs; NOS3 expression was increased 1.4-fold (both P < .05) compared to the atorvastatin or losartan group. No significant differences were observed between treatment groups and controls in NOS2 and AGTR1a expression.

Figure 1.

Overall gene expression of selected genes in low-dose atorvastatin, low-dose losartan, and their combination groups and in the control group of rats. N represents the number of animals in the experimental group. All values are means ± SEM. *P < .05, **P < .01, and ***P < .001 compared to the control group; ⁺ P < .05 compared to the combination group. EDNRA indicates endothelin receptor type A gene; NOS3, endothelial nitric oxide synthase 3 gene; NOS2, inducible nitric oxide synthase gene; AGTR1a, angiotensin II receptor, type 1a gene.

Relations of Gene Expression With NO Plasma Levels and Isolated Organ Variables

In a previous study, we investigated the effects of low-dose atorvastatin, losartan, or their combination on coronary flow of isolated rat heart, thoracic aorta relaxation, and serum levels of NO.¹⁴ In the present work, we explored the relations of these variables with vasoactive-related genes. This analysis showed that when NOS3 expression was increased, it was continuously accompanied with a serum NO level increase, though the correlation was not significant. Expression of NOS3 positively correlated with thoracic aorta relaxation (R = .82; P < .05) and isolated heart coronary flow (R = .26; P = .05). Expression of the EDNRA gene negatively correlated with serum NO concentration (R = −.58; P < .05). Expression of AGTR1a gene negatively correlated with isolated heart coronary flow (R = −.60; P < .05).

Discussion

In the present study, we demonstrated that the protective vascular effects of low-dose atorvastatin, losartan, and their low-dose combination are mediated through activation of the eNOS pathway and inhibition of the ET system pathway. This was demonstrated through the increase in NOS3 and reduction of EDNRA expression in aortas from rats treated with low-dose atorvastatin, losartan, or their low-dose combination up to 8 weeks. The combination of both drugs was significantly more effective than any of the drugs alone in upregulating NOS3 and nonsignificantly downregulating EDNRA.

In a previous related study, we investigated the effects of chronic (4 to 8 weeks) treatment with low-dose atorvastatin, losartan, or their combination on the coronary flow of isolated rat hearts exposed to ischemic–reperfusion injury and on thoracic aorta relaxation.¹⁴ While focusing on serum NO analysis previously, we further upgraded our research in the present study, exploring gene expression on stored thoracic aorta samples of the same rats. We sought to elucidate the mechanistic background of previously obtained protective cardiovascular effects in a thoracic aorta and isolated heart ischemic–reperfusion injury model. Since increased vasodilatation capacity was revealed in previous experiments, we analyzed genes that encode vasoactive molecules, comprising the endothelial and inducible NOS pathway, the ET receptor, and angiotensin receptor. Interestingly, we found that the maximal coronary flow increase and thoracic aorta relaxation were observed after 6 weeks of treatment, declining later on. A similar “up-and-down phenomenon” was observed in NOS3 expression, which was the highest after 6 weeks of treatment. In contrast, the expression of EDNRA gene decreased with therapy duration, the lowest values being achieved after 8 weeks of treatment.

The expression of numerous genes associated with cardiovascular protection in regard to treatment with statins^20

–23 and ARBs^24
–26 have been explored and proved in previous studies. There is some data on the influence of atorvastatin and losartan on the expression of cardiovascular system–associated genes. Atorvastatin was shown to attenuate the expression of matrix metalloproteinases^27,28 and several proangiogenic mediators, such as plasminogen activator inhibitor 1 and thrombospondin 1; on the other hand, it stimulated the expression of angiopoietin 2 and even that of eNOS.²⁹ Losartan was shown in a rat model to downregulate the receptor for advanced glycation end products and NF-kappaB, thereby improving the endothelial function.³⁰ Despite numerous publications, only a few studies exist which explored the effect of subtherapeutic, low doses of any statin or ARB on genes that are important in the cardiovascular system. Pitavastatin in low doses was shown to induce eNOS phosphorylation, activate Akt phosphorylation, and increase NO production in endothelial cells, but it failed to increase eNOS mRNA expression.³¹

The effect of statins and ARBs on genes similar to those investigated in our study was also reported but notably only for therapeutic dosages. Atorvastatin in high doses (10 mg/kg/d) was reported to induce eNOS mRNA expression and inhibit the expression of inducible NO synthase mRNA in the aorta of rats with sepsis.³² The expression of ET-1 was significantly lowered after 12 weeks losartan treatment.³⁰ Fluvastatin increased eNOS RNA expression, eNOS protein production, nitrite production, and reduced the production of ET-1 mRNA expression in the Human Umbilical Vein Endothelial Cells (HUVEC) cell line;³³ similar results were also obtained for simvastatin, rosuvastatin,³⁴ and atorvastatin.³⁵ Several statins were shown to inhibit iNOS expression in murine cells.³⁶ Cerivastatin and fluvastatin were shown to downregulate AT(1)-R expression and attenuate the biological function of angiotensin II in vascular smooth muscle cells.³⁷ A similar effect was also shown for simvastatin³⁸ and losartan in animal and human studies.^39
–41 The efficiency of pravastatin, irbesartan, and their combination in therapeutic doses was proven through an increase in lnQ quotient (calculated from expression of eNOS and C-type natriuretic peptide, divided by expression of oxidized low-density lipoprotein receptor and NAD(P)H oxidase subunit gp91phox) in the left internal mammary arteries of patients undergoing coronary artery bypass grafting surgery, where each drug increased lnQ, but their combination was proven to be more efficient, thus providing greatest endothelial protection.⁴²

As mentioned above, only scanty data exist in regard to the effect of low-dose statins and ARBs on the expression of genes associated with cardiovascular protection, particularly the vasoactive genes. Nevertheless, similar studies at therapeutic doses in humans seem to prove that both drugs have the ability to express a vasoactive reference gene. In this regard, we showed that the potential of the mentioned drugs, as far as their pleiotropic effects are concerned, is much greater than one would expect. Low, subtherapeutic doses as used in the present study induced the expression of NOS3 and reduced the expression of EDNRA. In contrast to our results, in the study of Wang et al, pitavastatin in low dose induced only eNOS phosphorylation but failed to increase eNOS mRNA expression.³¹

Despite our best efforts and a thorough literature review, we could not find any research article similar to the present one. Therefore, it could be claimed that, for the first time, we showed that subtherapeutic, low doses of statins and ARBs have the capacity to influence the expression of vasoactive genes. This is a novel finding in vascular pharmacology. The importance of these results lies in their proof that vascular gene expression can also be mediated through low doses of statins and ARBs that are below the range producing their primary action, namely, decreasing lipids and blood pressure (as shown in our previous study¹⁴). Of course, other genes that were not included in our study might also be affected by the treatment used and could contribute to obtaining improved vasorelaxation. In future studies, it would be of value to test the effects of low doses of statins and ARBs on the expression of genes involved in other cardioprotective functions, such as immunomodulatory, antioxidative, and antiproliferative effects.

Overall, based on the results presented in this article, one could speculate that low doses might be used for purposes of pure vascular protection, without influencing blood pressure and lipid levels. Putting together the results of our studies performed on healthy volunteers and our previous animal studies, we could confirm the possible potential of the new so-called low-dose vascular pharmacology approach for cardiovascular prevention. The results of the present study further strengthen this conclusion. In this regard, the low-dose combination of a statin and an ARB is of particular interest. Clearly, further studies are needed to clarify the full potential of this approach.

Conclusion

In conclusion, we showed for the first time that low-dose atorvastatin or losartan, and especially their combination, have the capacity to influence the expression of vasoactive genes. Low-dose drugs increased NOS3 expression, related to vasodilation, and decreased EDNRA expression, related to vasoconstriction, both considered as vasoprotective effects. These findings could (partially) explain the beneficial vascular effects of the “low-dose” approach establishing the use of low doses of statins, ARBs, and particularly their low-dose combination, for improvement in arterial function.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The present study was supported by the Slovenian Research Agency, Ljubljana Slovenia [research project L3-2293].

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