CVD is the leading cause of death worldwide, and its major risk factor is hypertension( Reference Lopez, Mathers and Ezzati 1 , Reference Gu, Reynolds and Wu 2 ). Hypertension affects approximately 25 % of the adult population worldwide, and its prevalence is predicted to increase by 60 % by 2025( Reference Kearney, Whelton and Reynolds 3 ). The important role in the pathophysiology of hypertensive disease was played by myocardial remodelling( Reference Zhang, Shao and Ma 4 ). In addition, cardiac hypertrophy and fibrosis that develop as an adaptive response to pressure overload ends with progressive heart disease( Reference Devereux and Roman 5 , Reference Katz 6 ).
Oxidative stress had an important role in the pathogenesis and development of CVD( Reference Higashi, Noma and Yoshizumi 7 ). Recent studies have shown that systemic arterial hypertension was induced by chronic administration of l-arginine analogue such as N ω -nitro-l-arginine methyl ester hydrochloride (l-NAME) to rats by blocking the endothelial nitric oxide synthase (NOS) isoform and in turn reducing the NO biosynthesis. This leads to endothelial dysfunction followed by impaired vasodilation and inflammatory phenotypic changes in the coronary vascular wall( Reference Sanada, Node and Minamino 8 , Reference Rossi and Colombini-Netto 9 ). Earlier reports highlight the crucial role of vascular endothelial cells in the pathogenesis of the arterial wall alterations and hypertension( Reference Ferreira, Rocha and da Silva Torres 10 ). Inhibition of oxidative stress maintains bioavailability of NO, and therefore the agents with antioxidant properties can improve regulation of vascular tone by inactivating free radicals and increasing NO bioavailability( Reference Kumar, Saravanakumar and Raja 11 ).
In recent years, increased consumption of dietary agents such as fruits, vegetables, whole grains and fish has a crucial role in the control of CVD including hypertension( Reference Retelny, Neuendorf and Roth 12 ). The protective effects of plant intake can be because of the presence of phenolic compounds and flavonoids. Phenolic acids are hydroxylated derivatives of benzoic and cinnamic acids( Reference Kumar, Prahalathan and Raja 13 ), and veratric acid (VA) (Fig. 1), one of the major benzoic acid derivatives from vegetables and fruits that occurs naturally in Tabebuia impetiginosa, is reported to have antibacterial, antifungal, anti-inflammatory, antispasmodic( Reference Narasimhan, Ohlan and Ohlan 14 ) and antioxidant activities( Reference Szwajgier, Pielecki and Targonski 15 ). Phenolic acids have a beneficial role in the prevention of many human diseases, particularly atherosclerosis and cancer, because of their antioxidant properties( Reference Mattila and Kumpulainen 16 ). In our previous phase of experiments, we reported the antihypertensive, free-radical-scavenging and anti-hyperlipidaemic effects of VA in l-NAME-induced hypertensive rats( Reference Saravanakumar and Raja 17 , Reference Saravanakumar and Raja 18 ). In this study, we evaluated the effects of VA on ventricular function, endothelium-dependent acetylcholine (ACh)-induced relaxation in aorta, oxidative stress, heart and aortic structural remodelling in a rat model of arterial hypertension induced by l-NAME.
Fig. 1 Structure of veratric acid; the chemical structure was obtained from the PubChem database (pubchem.ncbi.nlm.nih.gov).
Methods
Animals and chemicals
Healthy male albino Wistar rats (180–220 g) were obtained from the Central Animal House, Department of Experimental Medicine, Rajah Muthiah Medical College and Hospital, Annamalai University, India. They were housed (three rats per cage) in polypropylene cages (47×34×20 cm) lined with husk, renewed every 24 h and maintained in an air-conditioned room (25 (sd 3)°C) with a 12 h light–12 h dark cycle. Animals received the standard pellet diet (Kamadhenu Agencies) and water ad libitum. The whole experiment was carried out according to the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, New Delhi, India and approved by the Institutional Animal Ethics Committee of Rajah Muthiah Medical College and Hospital (Reg No. 160/1999/CPCSEA, approval number: 747), Annamalai University, Annamalainagar.
l-NAME and VA were purchased from Sigma-Aldrich. All other chemicals used in this study were of analytical grade obtained from Merck and HiMedia.
Animal model of N ω -nitro-l-arginine methyl ester hydrochloride-induced hypertension and veratric acid treatment
Animals were given l-NAME in drinking water at a dosage of 40 mg/kg body weight (b.w.) for 4 weeks. VA was dissolved in 5 % dimethyl sulfoxide (vehicle) and administered to rats orally everyday using an intragastric tube for a period of 4 weeks.
Experimental protocol
On the basis of our previous study, the dosage of VA was fixed as 40 mg/kg for this study( Reference Saravanakumar and Raja 17 ). Each of the following groups consisted of six animals – Group I: control; Group II: rats were treated with VA (40 mg/kg b.w.); Group III: rats were given l-NAME (40 mg/kg b.w.); Group IV: rats were co-administered with l-NAME (40 mg/kg b.w.) and VA (40 mg/kg b.w.). Vehicle alone was administered to control (Group I) and l-NAME control rats (Group III) orally using an intragastric tube daily for 4 weeks.
Preparation of tissue homogenates
After the completion of the experimental period, the rats were anaesthetised and killed by cervical dislocation. Aortic tissues were sliced into pieces and homogenised in 0·1 m-Tris-HCl buffer in cold condition (pH 7·4) to give 20 % homogenate (w/v). The homogenate was centrifuged at 560 g for 10 min at 4°C. The supernatant was separated and used for various biochemical estimations.
Lipid peroxidation products and antioxidants
The levels of thiobarbituric acid-reactive substances (TBARS) and lipid hydroperoxides (LOOH) in aorta were estimated by the methods of Niehaus & Samuelsson( Reference Niehaus and Samuelsson 19 ) and Jiang et al.( Reference Jiang, Hunt and Wolff 20 ), respectively. The activities of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in aorta were estimated by the methods of Kakkar et al.( Reference Kakkar, Das and Viswanathan 21 ), Sinha( Reference Sinha 22 ) and Rotruck et al.( Reference Rotruck, Pope and Ganther 23 ), respectively. The levels of non-enzymatic antioxidants such as GSH, vitamin C and vitamin E in aorta were estimated by the methods of Ellman( Reference Ellman 24 ), Roe & Kuether( Reference Roe and Kuether 25 ) and Baker et al.( Reference Baker, Frank and DeAngelis 26 ), respectively.
Langendorff isolated heart study
The left ventricular function of the rat heart was assessed using the Langendorff isolated heart preparation. Briefly, after anaesthesia, the heart was excised and placed in cooled (4°C) Krebs–Henseleit bicarbonate solution (composition (mm): 118 NaCl, 4·7 KCl, 1·2 MgSO4, 1·2 KH2PO4, 2·3 CaCl2, 25·0 NaHCO3 and 11·0 glucose). The heart was then attached to the cannula through the aorta and retrogradely perfused with the Krebs solution maintained at 37°C and continuously gassed with a mixture of 95 % O2–5 % CO2. Perfusion pressure was kept constant at 80 mmHg. An elastic water-filled balloon was introduced into the left ventricle through a left atrial incision. Isovolumetric recordings of the rate of pressure development (+dp/dt) and rate of pressure decline (−dp/dt) were obtained from the ventricular balloon connected via fluid-filled tubing to a Pressure Transducer (ADInstruments) linked with a PowerLab data acquisition unit (ADInstruments)( Reference Fenning, Harrison and Rose'meyer 27 ).
Tension recording: organ bath study
After anaesthesia, the thoracic aortas were carefully removed and transferred to cold modified Krebs–Henseleit solution (MKHS) of the following composition (mm): 118 NaCl, 4·7 KCl, 1·2 MgSO4, 1·2 KH2PO4, 11·1 d-glucose, 11·9 NaHCO3 and 2·5 CaCl2, pH 7·4. Aortas were cleared of connective tissue and cut into rings of about 2–3 mm length. The aortic ring segments were mounted on two stainless steel hooks and suspended in 10 ml organ baths containing MKHS, maintained at 37°C and aerated continuously with 95 % O2–5 % CO2 mixture. A passive tension of 1·5 g was applied during the equilibration period of 90 min, during which the bath solution was changed every 15 min. Tension was continuously recorded using a high-sensitivity force transducer and recorded in a computer using a Chart version 5.4.1 software programme (PowerLab).
After the equilibration period, tissue viability was examined by recording the contraction to high K+ (80 mm)-depolarising solution, in which part of the NaCl in the MKHS was replaced with an equimolar concentration of KCl. Cumulative concentration–response curves to relaxants were elicited in arterial segments pre-contracted with 1–3 μm-phenylephrine. Endothelial integrity was examined by adding 3 μm-ACh at the plateau phase of phenylephrine contraction. In endothelium-intact rings, ACh (3 μm) caused 80 % relaxation. To examine the influence of membrane depolarisation on the vasodilator responses to ACh, arterial rings were pre-contracted with K+ (40 mm)-depolarising solution. Separate arterial rings were used for control and treatment groups for the assessment of vasodilator responses to ACh.
Histopathology of heart and aorta
The heart and aorta samples obtained from all experimental groups were cleared of blood and immediately fixed in 10 % buffered neutral formalin. Heart and aorta sections (5 μm) were prepared from processed paraffin-embedded samples. Aortic sections were stained with Masson’s Trichrome. Heart sections were stained with Van Gieson’s staining. Tissue sections were examined under a light microscope and photomicrographs were taken.
Immunohistochemistry
Paraffin-embedded tissue sections were dewaxed and rehydrated through grade ethanol to distilled water. Endogenous peroxidase was blocked by incubation with 3 % H2O2 in methanol for 10 min. The antigen retrieval was achieved by microwave in citrate buffer solution (2·1 g citric acid/l D.H2O; 0·37 g EDTA/l D.H2O; 0·2 g trypsin) (pH 6·0) for 10 min, followed by washing step with Tris-buffered saline (8 g NaCl; 0·605 g Tris) (pH 7·6). The tissue sections were then incubated with blocking reagent for 15 min at room temperature to block non-specific binding sites. The tissue sections were then incubated with the polyclonal anti-fibronectin antibody with 1:500 dilution overnight at 4°C. The bound primary antibody was detected by incubation with the secondary antibody conjugated with horseradish peroxidase for 30 min at room temperature. After rinsing with Tris-buffered saline, the antigen–antibody complex was detected using 3,3′-diaminobenzidine, the substrate of horseradish peroxidase. Each slide was microscopically analysed, and the percentage of the positively stained area was enumerated semi-quantitatively. The percentage of positive area was scored as follows: 3+=strong staining, >50 % of area was stained; 2+=moderate staining, between 20 and 50 % of area was stained; 1+=weak staining, between 1 and 20 % of area was stained; 0=negative, <1 % of area was stained( Reference Rajasekaran, Manoharan and Silvan 28 ).
Statistical analysis
Data were analysed by one-way ANOVA followed by Duncan’s multiple-range test using SPSS version 20.0. For aortic relaxation, data were analysed by two-way ANOVA followed by Bonferroni’s post hoc test. Values are represented as mean values and standard deviations of six animals in each group. Values were considered significant when P<0·05.
Results
Effect of veratric acid on lipid peroxidation and antioxidants
Table 1 shows the effect of VA on the levels of TBARS and LOOH in the aortic tissues of l-NAME-induced hypertensive rats. The l-NAME rats exhibited a significant increase in the levels of TBARS and LOOH. The administration of VA reduced the levels of TBARS and LOOH significantly (P<0·05).
Table 1 Effect of veratric acid (VA) on lipid peroxidation and antioxidants in the aorta of experimental hypertensive rats (Mean values and standard deviations; six animals)
l-NAME, N ω -nitro-l-arginine methyl ester hydrochloride; TBARS, thiobarbituric acid-reactive substances; LOOH, lipid hydroperoxides; Ua, enzyme concentration required to inhibit the chromogen produced by 50 % in 1 min under standard condition; Ub, micromoles of H2O2 consumed per minute; Uc, micrograms of GSH utilised per minute.
* Mean values were significantly different compared with control (P<0·05; Duncan’s multiple-range test).
† Mean values were significantly different compared with l-NAME (P<0·05; Duncan’s multiple-range test).
Table 1 illustrates the effect of VA on the activities of SOD, CAT and GPx in the aortic tissues of l-NAME hypertensive rats. The activities of SOD, CAT and GPx decreased significantly (P<0·05) in l-NAME rats, and the administration of VA significantly increased these enzymatic antioxidants.
Table 1 portrays the effect of VA on the levels of vitamin C, vitamin E and GSH in the aortic tissues of control and l-NAME-induced hypertensive rats. The levels of GSH, vitamin C and vitamin E decreased significantly in l-NAME rats, and administration of VA significantly (P<0·05) increased the levels of these non-enzymatic antioxidants.
Effect of veratric acid on cardiac function: Langendorff study
The systolic contractility of the isolated heart was measured by the first temporal derivative of the left ventricular pressure (LVP) positive development (+dp/dt, mmHg/s), and the isovolumetric relaxation was measured by the first temporal derivative of the LVP negative development (−dp/dt, mmHg/s). In the heart of l-NAME rats, the rate of LVP rise (+dp/dt, mmHg/s) and the rate of LVP decline (−dp/dt, mmHg/s) were significantly (P<0·05) reduced. VA treatment significantly (P<0·05) promoted ventricular function in l-NAME rats (Fig. 2).
Fig. 2 Veratric acid improved cardiac ventricular function in experimental hypertensive rats. Values are means (six animals), and standard deviations represented by vertical bars. * Mean values were significantly different compared with control (P<0·05; Duncan’s multiple-range test). † Mean values were significantly different compared with N
ω
-nitro-l-arginine methyl ester hydrochloride (l-NAME) (P<0·05; Duncan’s multiple-range test). 
, Control; 
, control+veratric acid; 
, l-NAME; 
, l-NAME+veratric acid.
Effect of veratric acid on vascular function: organ bath study
Fig. 3 and Table 2 show concentration-dependent relaxation to ACh in phenylephrine pre-contracted endothelium-intact rings from aorta of experimental rats. Sensitivity of the aortic rings to ACh from rats given l-NAME was significantly (P<0·05) reduced. When aortic tissues from l-NAME rats given VA were challenged with ACh, the vasodilation was almost restored.
Fig. 3 Veratric acid recovered vascular function in experimental hypertensive rats. Values are means (six animals), and standard deviations. * Mean values were significantly different compared with control (P<0·05; Bonferroni’s post hoc test). † Mean values were significantly different compared with N
ω
-nitro-l-arginine methyl ester hydrochloride (l-NAME) (P<0·05; Bonferroni’s post hoc test). ACh, acetylcholine; ■, control; 
, control+veratric acid; 
, l-NAME; 
, l-NAME+veratric acid.
Table 2 Effect of veratric acid (VA) on acetylcholine-induced relaxations in endothelium-intact aorta of experimental hypertensive rats (Mean values and standard deviations; six animals)
l-NAME, N ω -nitro-l-arginine methyl ester hydrochloride.
* Mean values were significantly different compared with control (P<0·05; Bonferroni’s post hoc test).
† Mean values were significantly different compared with l-NAME (P<0·05; Bonferroni’s post hoc test).
Masson’s Trichrome-stained histopathology of aorta
Fig. 4(a)–(d) illustrates photomicrographs of Masson’s Trichrome staining of the thoracic aorta of control and l-NAME-administered rats. Aorta from control rats showed normal architecture (Fig. 4(a)). Control group treated with VA also showed normal thoracic aorta (Fig. 4(b)). Fig. 4(c) showed that l-NAME-treated rats exhibited increased endothelial cells and interstitial collagen accumulation (stained blue). VA administered to l-NAME rats revealed reduced collagen accumulation (stained blue) as compared with the l-NAME rats (Fig. 4(d)).
Fig. 4 Photomicrographs of Masson’s Trichrome-stained sections of thoracic aorta from control and N ω -nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats (40×). (a) Control rats showing normal pathological staining. (b) Control+veratric acid (VA) (40 mg/kg)-treated rats showing negative staining without any pathological alterations. (c) l-NAME-induced hypertensive rats showing increased endothelial cells and interstitial collagen accumulation (stained blue). (d) l-NAME+VA (40 mg/kg) revealed reduced collagen accumulation (stained blue) as compared with the hypertensive rats.
Van Gieson’s stained histopathology of the heart
Fig. 5(a)–(d) demonstrates the Van Gieson’s staining of the heart. l-NAME hypertensive rats exhibited increase in the interstitial collagen accumulation and fibrosis stained red (Fig. 5(c)). VA supplementation (40 mg/kg) in l-NAME rats showed reduction in the collagen accumulation (Fig. 5(d)). Control and control treated with VA showed normal cardiac architecture without any pathological changes (Fig. 5(a) and (b)).
Fig. 5 Photomicrographs of Van Gieson’s stained sections of heart from control and N ω -nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats (40×). (a) Control rats showing normal pathological staining. (b) Control+veratric acid (VA) (40 mg/kg)-treated rat showing negative staining without any pathological alterations. (c) l-NAME-induced hypertensive rats showing pathological alteration with positive staining (fibrosis and collagen deposition). (d) l-NAME+VA (40 mg/kg) rats showing reduced positive staining.
Effect of veratric acid on fibronectin expression: immunohistochemical analysis
In l-NAME-induced hypertensive rats, the expression of heart fibronectin (FN) was higher when compared with control rats (Fig. 6). Treatment with VA (40 mg/kg) decreased the expression of heart FN in l-NAME-induced hypertensive rats.
Fig. 6 Representative photomicrographs of immunohistochemistry for fibronectin in heart of control and N ω -nitro-l-arginine methyl ester hydrochloride (l-NAME)-induced hypertensive rats (40×). (a) Control rats showing normal architecture without any pathological alterations (0=negative, <1 % of area staining). (b) Control+veratric acid (VA) (40 mg/kg)-treated rats showing near normal architecture (0=negative, <1 % of area staining). (c) l-NAME hypertensive rats showing increased fibronectin-positive staining (2+=moderate staining, between 20 and 50 % of area was stained). (d) l-NAME+VA (40 mg/kg)-treated rat showing diminished fibronectin expression (1+=weak staining, between 1 and 20 % of area was stained).
Discussion
NO participates in the modulation of vascular tone, and it is synthesised and released by endothelial cells( Reference Katsumi, Nishikawa and Hashida 29 ). In addition, NO is important in cellular events such as vascular smooth muscle cell proliferation( Reference Sanada, Node and Minamino 8 ). It is well established that arterial hypertension was caused by the chronic inhibition of NO biosynthesis by administration of l-NAME, an l-arginine analogue( Reference Boe, Eren and Murphy 30 ), characterised by cardiac remodelling( Reference Yang, Yang and Chen 31 ), dysfunction of endothelial-dependent relaxation( Reference Ulker, McKeown and Bayraktutan 32 ) and renal function changes( Reference Ndisang and Chibbar 33 ). l-NAME-induced hypertension is a well-established model of experimental hypertension( Reference Paulis, Zicha and Kunes 34 ).
Oxidative stress has an important role in the development of hypertension, originated through excessive production of reactive oxygen species (ROS)( Reference Dikalov and Ungvari 35 ). Hypertension results in the production of large amounts of ROS such as superoxide, hydrogen peroxide and hydroxyl radicals( Reference Kumar, Prahalathan and Raja 13 ). Our results showed that the lipid peroxidation products, measured as TBARS and LOOH, were increased in the aortic tissues of l-NAME-induced hypertensive rats. Lipid peroxidation is an important pathogenic event in hypertension, and its complications were associated with the accumulation of LOOH( Reference Kumar, Prahalathan and Raja 13 ). l-NAME rats show increased levels of lipid peroxides, which might be because of free-radical-mediated membrane damage. Oral administration with VA (40 mg/kg) significantly decreased the levels of TBARS and LOOH in l-NAME-treated rats. This reveals the anti-lipidperoxidative role of VA that is probably mediated by its ability to scavenge free radicals( Reference Saravanakumar and Raja 17 ).
The first line of cellular defence against oxidative injury was offered by free-radical-scavenging enzymes such as SOD, CAT and GPx, decomposing superoxide and hydrogen peroxide, otherwise interacting to form the more reactive hydroxyl radical( Reference Rodrigo, Libuy and Feliú 36 ). The important process responsible for the effective removal of oxidative stress in intracellular organelles is the equilibrium between the enzymatic antioxidants and free radicals( Reference Senthil, Veerappan and Ramakrishna Rao 37 ). Our previous study suggested that the activities of antioxidant enzymes SOD, CAT and GPx were significantly decreased in the erythrocytes and tissues of l-NAME-induced hypertensive rats and increased after treatment with VA( Reference Saravanakumar and Raja 17 ). The result of the present study is parallel with our previous report showing that VA administration significantly improved the activities of SOD, CAT and GPx in the aortic tissue. The increased activities of these enzymes in VA-treated rats might be because of its free-radical-scavenging ability( Reference Saravanakumar and Raja 17 ).
The residual free radicals escaping from decomposition enzymes were scavenged by the non-enzymatic antioxidants, namely vitamin C, vitamin E and GSH( Reference Seifi, Kadkhodaee and Karimian 38 ). Vitamin C present in the aqueous environment reduces free radicals present on the surface of the membrane owing to its multiple antioxidant properties( Reference Karthikeyan and Rani 39 ). The most effective lipid-soluble antioxidant in the biological system is vitamin E. GSH is directly involved in the removal of superoxide radicals, peroxyl radicals and singlet oxygen( Reference Abidi, Afaq and Arif 40 ).
In the current study, the levels of non-enzymatic antioxidants were declined in l-NAME rats. Increased use of these non-enzymatic antioxidants for the neutralisation of free radicals might be the reason for its decreased levels( Reference Newaz and Nawal 41 ). Our previous report explored the protective effect of VA on non-enzymatic antioxidant system( Reference Saravanakumar and Raja 17 ). Consistent with the previous report, in this study VA treatment enhanced the levels of non-enzymatic antioxidants, which suggests that this compound might be potentially useful in counteracting free-radical-mediated oxidative stress caused by lipid peroxidation( Reference Saravanakumar and Raja 17 ).
Hiremath et al.( Reference Hiremath, Bauer and Aguirre 42 ) indicated that fibrosis was induced by oxidative stress generated during hypertensive condition. The exaggeration in the rate of synthesis and stabilisation of matrix and/or decrease in the capacity of degradation lead to the development of fibrosis( Reference Chatziantoniou and Dussaule 43 ). The accumulation of collagen in the heart wall, an adaptive-compensatory mechanism to increased haemodynamic load, leads to a loss of wall compliance, and eventually to heart failure( Reference Olzinski, McCafferty and Zhao 44 ). There are early reports of increased collagen synthesis in the arterial wall in hypertension that occurred globally in l-NAME hypertensive rats( Reference de Gasparo, Hess and Nuesslein-Hildesheim 45 , Reference Mattson, Kunert and Roman 46 ). In this study, Masson’s Trichrome staining of the thoracic aorta and Van Gieson’s staining of the heart of l-NAME hypertensive rats showed collagen accumulation. VA supplementation throughout the experimental period considerably reduced the collagen deposition in l-NAME rats. These histological observations exhibited the protective role of VA in tissues of l-NAME hypertensive rats with its antioxidant property( Reference Saravanakumar and Raja 17 ).
Mulvany et al.( Reference Mulvany, Baumbach and Aalkjaer 47 ) reported that arterial hypertension can reduce vessel and cardiac lumen size through hypertrophy, with wall thickening, and remodelling, with an increase in the thickness of the arterioles. Left ventricular hypertrophy and remodelling are frequently seen in hypertensive subjects( Reference Nadruz 48 ). Cardiac contractile function impairment is one of the major pathogenic features of cardiac remodelling. Hypertension-induced ventricular dysfunction was assessed by the Langendorff isolated heart study. It was previously known that enhanced stiffness of the myocardium impedes ventricular contraction and relaxation caused by excess production and accumulation of extracellular matrix (ECM) structural proteins, or fibrosis, leading to distorted architecture and diastolic and systolic dysfunction of the heart( Reference Díez, Querejeta and López 49 , Reference Manabe, Shindo and Nagai 50 ). From the antioxidant point of view, Mapanga et al.( Reference Mapanga, Rajamani and Dlamini 51 ) explored that oleanolic acid, an antioxidant agent, blunts hyperglycaemia-induced contractile dysfunction. Comparable with the previous report, in this study, l-NAME induction impairs the contractile function of the heart and VA treatment restores the ventricular function with its antioxidant property( Reference Saravanakumar and Raja 17 ).
Reduced production or activity of NO contributes to several human diseases, especially hypertension( Reference Versari, Daghini and Virdis 52 , Reference Török 53 ). A previous report showed that l-NAME treatment causes deficiency in NO production, which leads to the development of hypertension( Reference Sanada, Node and Minamino 8 ). In the aorta of l-NAME-induced hypertensive rats, the endothelium-dependent relaxant response to ACh was markedly blunted( Reference Chen, Shi and Yu 54 ). Long-term inhibition of NOS results in decreased production of NO, and an increased release of vasoconstrictors in endothelial cells eventually leads to endothelial dysfunction( Reference Kung, Moreau and Takase 55 ). In the present work, the ACh-induced relaxation in endothelium-intact aorta was significantly augmented in the rats treated with VA when compared with that of the l-NAME-treated rats. It shows that VA, to an extent, preserves the functional capacity of the endothelium owing to its antioxidant capacity( Reference Saravanakumar and Raja 17 ).
In hypertensive heart disease, increased FN expression could contribute to ECM cell attachment and promote collagen deposition, as well as elevated gelatinase levels, leading to reactive myocardial fibrosis, which increases myocardial stiffness and reduces compliance( Reference Fan, Takawale and Lee 56 ). It was already explored that, in deoxycorticosterone acetate plus salt rats with endothelin-dependent hypertension, the cardiac extracellular remodelling was associated with increased FN, matrix metalloproteinase activity and up-regulation of inflammatory mediators( Reference Ammarguellat, Gannon and Amiri 57 ). Moreover, in the models of pressure overload, the cardiac FN expression is elevated, and hypertrophy is caused by FN expression in the cultured cardiac myocytes( Reference Chen, Huang and Stewart 58 ). In this study, immunohistochemical analysis explored that VA administration decreased the expression of FN in the heart of hypertensive rats, which might be because of the free-radical-scavenging ability of VA( Reference Saravanakumar and Raja 17 ), which could exert a beneficial action against pathophysiological alterations caused by free radicals.
Oxidative stress has been identified as one unifying mechanism in the pathogenesis of CVD. Antioxidants may need to be given as a pharmacotherapy, with the aim of reducing the burden of CVD. In conclusion, major findings from the present study demonstrated that VA supplementation could effectively prevent the cardiovascular dysfunction, lipid peroxidation, cardiac fibrosis, aortic fibrosis and FN expression. Further, VA has the potential to modulate alteration at the antioxidant level in l-NAME rats (Fig. 7). Our findings illustrate that in future VA may be used as a beneficial molecule in the treatment of hypertension-associated cardiovascular remodelling.
Fig. 7 Preventive role of veratric acid on cardiovascular remodelling. iNOS, inducible nitric oxide synthase.
Acknowledgements
The authors thank Dr Madhavan R. Nirmal, Department of Oral Pathology, Rajah Muthiah Dental College and Hospital, Annamalai University, Tamil Nadu, India for carrying out the immunohistochemistry study.
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
The authors’ contributions are as follows: B. R. and M. S. designed the research; M. S., J. M. and T. S. performed the experiments; P. P., S. K. and S. K. M. analysed the data; M. S., J. M. and T. S. wrote the paper; B. R. and S. K. M. revised the paper. All authors read and approved the final manuscript.
None of the authors has any conflicts of interest to declare.
