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Influence of dietary phytosterols and phytostanols on diastolic blood pressure and the expression of blood pressure regulatory genes in SHRSP and WKY inbred rats

Published online by Cambridge University Press:  24 November 2008

Qixuan Chen
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9
Heidi Gruber
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9
Eleonora Swist
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9
Catherine Pakenham
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9
Walisundera M. N. Ratnayake
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9
Kylie A. Scoggan*
Affiliation:
Nutrition Research Division, Food Directorate, Health Products and Food Branch, Health Canada, Banting Research Centre, 251 Sir Frederick Banting Driveway, Ottawa, Ontario, CanadaK1A 0K9 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, CanadaK1H 8M5
*
*Corresponding author: Kylie A. Scoggan, fax: +1 613 941 6182, email kylie_scoggan@hc-sc.gc.ca
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Abstract

The aim of the present study was to determine the impact of increased consumption of phytosterols or phytostanols on blood pressure and renal blood pressure regulatory gene expression in stroke-prone spontaneously hypertensive (SHRSP) and normotensive Wistar–Kyoto (WKY) inbred rats. SHRSP and WKY inbred rats (10/group) were fed a control diet or a diet supplemented with phytosterols or phytostanols (2·0 g/kg diet). After 5 weeks, SHRSP rats demonstrated higher systolic and diastolic blood pressures than WKY inbred rats. SHRSP rats that consumed the phytosterol or phytostanol supplemental diets displayed a 2- or 3-fold respective increase in the diastolic blood pressure than those that consumed the control diet. Angiotensinogen (Agt), angiotensin I-converting enzyme 1 (Ace1), nitric oxide synthase (Nos) 1, Nos3, cyclooxygenase 2 (Cox2) and THUMP domain containing 1 were expressed at higher levels in SHRSP compared with WKY inbred rats. Renin and angiotensin II receptor type 1a were expressed at lower levels in SHRSP than WKY inbred rats. Phytostanol supplementation up-regulated the expression of Ace1 and Nos3 in SHRSP rats. Phytosterol supplementation increased the mRNA levels of Nos1 and spondin 1 (Spon1) in SHRSP and WKY inbred rats. Cox2 mRNA levels were elevated in both phytosterol- and phytostanol-supplemented SHRSP and WKY inbred rats. Therefore, the increased blood pressure in SHRSP rats may be partly due to altered renal expression of blood pressure regulatory genes. Specifically, up-regulation of Ace1, Nos1, Nos3, Cox2 and Spon1 were associated with the increased diastolic blood pressure observed in phytosterol- or phytostanol-supplemented SHRSP rats.

Type
Full Papers
Copyright
Copyright © The Authors 2008

Consumption of phytosterols and phytostanols at the level of 1·5–3·0 g/d has been documented to reduce blood LDL cholesterol between 8 and 15 %(Reference Katan, Grundy and Jones1, Reference Law2). The average western diet provides 150–400 mg phytosterols and 20–50 mg phytostanols daily(Reference Jones, MacDougall and Ntanios3). Therefore, a variety of foods have been supplemented with phytosterols and/or phytostanols to provide high enough intakes of phytosterols and phytostanols in order to market these novel foods as an aid to lower blood cholesterol levels. Phytostanol ester-supplemented margarine was first launched in Finland in 1995. Now several commercially available phytosterol/phytostanol-enriched products are available in the European Union member states(4) and in the USA.

However, whether or not phytosterols or phytostanols elicit a net beneficial effect on vascular disease remains controversial. Various national and international bodies continue to struggle with the safety issues of phytosterols and phytostanols in foods, because there are growing concerns for their potential excessive daily intake and hazards, as outlined below. First, rare individuals with phytosterolaemia, caused by mutations in either the ATP-binding cassette G5 or the ATP-binding cassette G8 half-transporter genes(Reference Lee, Lu and Hazard5), over-absorb phytosterols and phytostanols and develop accelerated atherosclerosis and premature coronary artery disease(Reference Salen, Horak and Rothkopf6). Second, epidemiological studies have demonstrated that even a slight elevation in the serum levels of phytosterols or phytostanols is associated with an increased risk of CHD, independent of the serum cholesterol levels(Reference Assmann, Cullen and Erbey7Reference Glueck, Speirs and Tracy9). Third, the use of phytosterol- or phytostanol-enriched food products was found to elevate phytosterol and phytostanol levels in the serum of a free-living population(Reference Fransen, de Jong and Wolfs10) and in healthy subjects(Reference Clifton, Mano and Duchateau11), as well as in the serum and aorta of patients with aortic stenosis(Reference Weingärtner, Lütjohann and Ji12). Fourth, phytosterol ester supplementation to wild-type mice increased their phytosterol, but not cholesterol, levels in plasma; endothelial function was also impaired and experimental cerebral lesion size was increased(Reference Weingärtner, Lütjohann and Ji12). Fifth, in stroke-prone spontaneously hypertensive rats (SHRSP) that have a mutation in the Abcg5 gene(Reference Scoggan, Gruber and Lariviere13), dietary phytosterols or phytostanols induced the accumulation of phytosterols or phytostanols in the serum and tissues, decreased the deformability of red blood cells, accelerated the onset of stroke and reduced the lifespan(Reference Ratnayake, Plouffe and L'Abbé14Reference Ratnayake, Plouffe and Hollywood16).

The hypertensive SHRSP strain, a model for essential hypertension and haemorrhagic stroke(Reference Carswell, McBride and Graham17), was derived from the spontaneously hypertensive rat (SHR) strain that was previously developed from the normotensive Wistar–Kyoto (WKY) inbred strain(Reference Okamoto, Yamori and Nagaoka18). We have determined that both SHRSP and its ‘parental’ normotensive WKY inbred rats have a mutation in the Abcg5 gene(Reference Scoggan, Gruber and Lariviere13). SHRSP rats are hypertensive, whereas WKY inbred rats are normotensive. The precise cause of hypertension is unclear, but is likely to be multifactorial and to involve genetic predisposition, related primarily to the kidney(Reference Satoh, Owada and Ishida19), and responses to environmental factors, such as high salt intake and obesity. In the present study, we further investigated the molecular mechanism(s) by which phytosterols and phytostanols accelerated the onset of stroke and reduced the lifespan via measuring blood pressure, and the expression of several renal genes known to be involved in blood pressure regulation in SHRSP and WKY inbred rats fed diets containing high levels of phytosterols or phytostanols.

Materials and methods

Preparation of phytosterols and phytostanols

A mixture of phytosterols was extracted from rapeseed oil deodoriser distillate (CanAmera Foods, Altona, MB, Canada), as described previously(Reference Ratnayake, L'Abbé and Mueller15). The final phytosterol product contained 22·0 % brassicasterol, 31·9 % campesterol and 43·2 % β-sitosterol as determined by GC (6890 Network GC system; Agilent Technologies, Santa Clara, CA, USA). A portion of the phytosterol product was converted to phytostanols via catalytic hydrogenation at the pilot plant facility of the POS Pilot Plant Corporation (Saskatoon, Sask., Canada). The final phytostanol product contained 54·7 % campestanol and 44·8 % sitostanol.

Experimental diets

Three diets were tested (Table 1). Each diet contained 10 % fat and was prepared according to the AIN-93G recommendations for rodent diets(20). The diets were made in pellet form and stored at 4°C. The final total phytosterol and phytostanol content of the diets was 0·25, 2·13 and 1·96 g/kg in the control, phytosterol and phytostanol diets, respectively (Table 1). These dietary levels of phytosterols and phytostanols were used because our previous studies demonstrated that 2·0 g/kg diet of phytosterols and phytostanols significantly accelerated the onset of stroke and reduced the lifespan of SHRSP rats(Reference Ratnayake, Plouffe and L'Abbé14Reference Ratnayake, Plouffe and Hollywood16). These dietary levels of phytosterols and phytostanols are similar to the amounts suggested for cholesterol lowering in humans on a dietary fat basis(Reference Law2, Reference Jones, MacDougall and Ntanios3). For example, consumption of 2·0 g per day of dietary phytosterols or phytostanols in an individual who consumes 100 g fat per day (average fat intake) represents 2·0 % of that person's fat intake. Our rats were also fed 2·0 g phytosterols or phytostanols in a diet that contained 100 g fat (soyabean oil) per kg of diet, which represents 2·0 % of the rat's dietary fat intake.

Table 1 Composition of test diets (g/kg)*

* Based on the AIN-93G diet(20).

Vitamin-free test casein (Harlan Teklad, Madison, WI, USA).

Phytosterols isolated from rapeseed oil deodoriser distillate.

§ Phytosterols isolated from rapeseed oil deodoriser distillate were hydrogenated to produce phytostanols.

93G-MX Mineral mix (35·0 g/kg), 93-VG Vitamin mix (10·0 g/kg), l-cystine (3·0 g/kg), choline bitartrate (2·5 g/kg) and tert-butylhydroquinone (0·014 g/kg).

Includes phytosterols, phytostanols and cholesterol.

Animals

SHRSP and WKY inbred rats were obtained from Charles River Laboratories (Kingston, NY, USA). Thirty-five-day-old male SHRSP and WKY inbred rats were each randomly assigned to one of the three diet groups (n = 10/group). They had free access to drinking-water containing NaCl (10 g/l) and one of the three diets (Table 1) for 5 weeks. NaCl loading was applied to accelerate the progress of hypertension(Reference Ratnayake, Plouffe and Hollywood16) for pronouncing the difference in the effects of the treatment.

Rats were housed individually in wire-bottom cages in an environmentally controlled room maintained at 22°C and 60 % relative humidity with a 12 h day/12 h night cycle. Body weight, food intake and water consumption of the rats were recorded twice weekly. After 5 weeks on the test diets, 12-h food-deprived rats were killed via exsanguination from the abdominal aorta while under 3 % isoflurane anaesthesia. Kidneys were removed and frozen immediately in liquid nitrogen. One kidney from each rat was ground under liquid nitrogen for RNA extraction. The animal protocol was approved by the Institutional Animal Care Committee at Health Canada.

Blood pressure measurements

Blood pressure was measured at the beginning, middle and end (weeks 1, 3 and 5) of the feeding phase of the study by the tail-cuff method using a II TC Model 31 Blood Pressure Apparatus (IITC Inc./Life Science Instruments, Woodland Hills, CA, USA) as per the manufacturer's instructions. Only week 1 and 5 data are presented. Briefly, the blood pressure apparatus was stored in a separate, larger, warmer room (26–27°C). The pressure channels were calibrated every day and the chamber was kept at 29–30°C. Rats were trained on the apparatus every day for 1 week before the experiment. The rats were transferred to the blood pressure room 30 min before handling to allow them to gradually get used to the heat. Each rat was placed in the chamber for 5 min before starting the measurements, allowing the rats to acclimatise. The same investigator monitored the computer screen and the animal in order to detect artefacts such as animal distress or movement. The systolic and mean blood pressures were measured by the same investigator every time. The diastolic blood pressure was calculated by the equation, diastolic = (3 × mean − systolic)/2, as per the manufacturer's instructions. The blood pressure of each rat at each measuring point was obtained as an average of three readings.

Analyses of sterols

The sterol composition of the diets was analysed by GC, as described previously(Reference Ratnayake, L'Abbé and Mueller15).

RNA isolation and real-time quantitative RT-PCR

Procedures described previously were followed(Reference Scoggan, Gruber and Chen21). Total RNA was extracted from each kidney sample using two passes of TRIzol reagent (Invitrogen Life Technologies, Burlington, ON, Canada). The isolated RNA was then purified and DNase I treated on RNeasy mini columns (Qiagen, Mississauga, ON, Canada). The quality of the purified RNA was assessed by a Bioanalyzer (Agilent 2100 Bioanalyzer; Agilent Technology, Waldbronn, Germany) and by a conventional agarose gel.

The purified RNA was quantified using RiboGreen RNA Quantitation Reagent and Kit (Molecular Probes, Eugene, OR, USA) and subsequent cDNA synthesis was performed with Retroscript Kit (Ambion, Austin, TX, USA). Real-time quantitative PCR was performed on an M × 4000 Multiplex Quantitative PCR System using the Brilliant SYBR Green QPCR Core Reagent Kit (Stratagene, La Jolla, CA, USA) or TaqMan Gene Expression Assay (Applied Biosystems, Foster city, CA, USA). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was selected to normalise the expression of the target genes. The primers were synthesised by Integrated DNA Technologies (Coralville, IA, USA) and described in Table 2(Reference Yu, Pandit and Klett22Reference Nakazawa, Tanaka and Takahashi24). For each experiment, a non-template reaction and a no-RT reaction were included as negative controls. The specificity of the PCR was confirmed by dissociation curve analysis of the products as well as by size verification of the amplicons in a conventional agarose gel.

Table 2 Primer sequences for real-time quantitative PCR (rat)

The relative amounts of each gene-of-interest were normalised to Gapdh expression levels in the kidney as an endogenous internal standard. A serial dilution of a standard was run on each plate for each gene-of-interest and Gapdh. The relative levels of mRNA (gene-of-interest/Gapdh) were then calibrated to the control group, namely control rats (WKY inbred) fed the control diet (set as 1·0).

Statistical analysis

Data are presented as the means with their standard errors. All data were evaluated for equality of variance before statistical analysis. Variables with skewed distribution were log transformed. Statistical analyses were performed using Statistica 8.0 software (StatSoft, Tulsa, OK, USA). Two-way ANOVA was used to determine the effects of strain (two levels: SHRSP and WKY inbred), diet (three levels: control, high phytosterols and high phytostanols) and their interactions. Comparisons between groups were made by Tukey's honestly significant differences (HSD) test. Differences were considered significant when P < 0·05.

Results

Body weight, food intake and water consumption

Body weight of the animals increased steadily during the 5-week feeding phase of the study. There were no effects of the diet on body weight gain, food efficiency and total water consumption (Table 3). However, SHRSP rats had less initial body weight (P = 0·0069), more weight gain (P = 0·0002), more food intake (P < 0·0001) and less food efficiency (P < 0·0001) compared with WKY inbred rats. SHRSP rats also consumed less, 1 % salt water, than WKY inbred rats (P < 0·0001).

Table 3 Growth, food and water intake of the rats

(Mean values with their standard errors)

a,b,c Mean values within a row with unlike superscript letters are significantly different (P < 0·05, Tukey's HSD test).

* No diet treatment at the beginning of the experiment.

Blood pressure

There were no differences in the initial systolic and diastolic blood pressures among groups (Table 4). After consuming the diets for 5 weeks, SHRSP rats demonstrated higher mean systolic (P < 0·0001) and diastolic (P < 0·0001) blood pressures as well as a greater increase in the systolic (P < 0·0001) and diastolic (P < 0·0001) blood pressures when compared with WKY inbred rats. SHRSP rats that consumed the phytosterol or phytostanol supplemental diets displayed a 2- or 3-fold respective increase in the diastolic blood pressure than those that consumed the control diet (overall diet effect P = 0·0016).

Table 4 Systolic and diastolic blood pressure of the rats

(Mean values with their standard errors)

BP, blood pressure. Acontrol v. sterols; Bcontrol v. stanols; Csterols v. stanols (P < 0·05, Tukey's HSD test).

a,b,c Mean values within a row with unlike superscript letters are significantly different (P < 0·05, Tukey's HSD test).

* No diet treatment at the beginning of the experiment.

Renal mRNA expression of genes involved in blood pressure regulation

SHRSP rats had significantly higher mRNA expression of angiotensinogen (Agt; P < 0·0001), angiotensin I-converting enzyme 1 (Ace1; P < 0·0001), nitric oxide synthase 1 (Nos1; P < 0·0001), Nos3 (P < 0·0001), cyclooxygenase 2 (Cox2; P < 0·0001) and THUMP domain containing 1 (Thumpd1; P < 0·0001) than WKY inbred rats (Table 5). Whereas WKY inbred rats had slightly higher mRNA expression of renin (P = 0·0003) and angiotensin II receptor type 1a (Agtr1a; P = 0·007) than SHRSP rats.

Table 5 Renal gene expression of the rats

(Mean values with their standard errors)

A Control v. sterols; Bcontrol v. stanols; Csterols v. stanols (P < 0·05, Tukey's HSD test).

a,b,c,d Mean values within a row with unlike superscript letters are significantly different (P < 0·05, Tukey's HSD test).

Phytostanol supplementation up-regulated the expression of Ace1 and Nos3 in SHRSP rats (overall diet effect: P = 0·029 and 0·005, respectively). Phytosterol supplementation significantly increased the mRNA levels of Nos1 and spondin 1 (Spon1) in SHRSP and WKY inbred rats (overall diet effect: P = 0·017 and 0·0005, respectively). Cox2 mRNA levels were significantly elevated in both phytosterol- and phytostanol-supplemented SHRSP and WKY inbred rats (overall diet effect: P = 0·001).

There were no strain and diet effects on the mRNA expression of Ace2. There was a strain–diet interaction on the mRNA expression of Ace1 (P = 0·006) and Agtr1a (P = 0·016). These interactions indicated that the high phytostanol diet up-regulated the Ace1 and Agtr1a expression in SHRSP rats, but not in WKY inbred rats.

Discussion

In the present study, we demonstrated that SHRSP rats have both higher systolic and diastolic blood pressures compared with WKY inbred rats. We also demonstrated, for the first time, that a high phytosterol or phytostanol diet (2 g/kg diet) induces a greater increase in the diastolic blood pressure in SHRSP rats (Table 4). In our previous studies, diets containing vegetable oils fortified with phytosterols (2 g/kg diet)(Reference Ratnayake, L'Abbé and Mueller15)and diets containing commercial margarines fortified with either phytosterols (14 g/kg diet) or phytostanols (11 g/kg diet)(Reference Ratnayake, Plouffe and L'Abbé14)induced the accumulation of phytosterols or phytostanols in the serum and tissues, decreased the deformability of red blood cells, accelerated the onset of stroke and reduced the lifespan of SHRSP rats. Taken together, these data suggest that increased consumption and subsequent tissue retention of either phytosterols or phytostanols exacerbates hypertension, which may contribute to the early onset of stroke and reduced lifespan of salt-loaded SHRSP rats. Our findings are supported by Ogawa et al. (Reference Ogawa, Yamamoto and Kamisako25), who reported that a diet containing soyabean oil fortified with phytosterols (4·5 g/kg diet) elevated systolic blood pressure and promoted the onset of stroke in salt-loaded SHRSP rats. Similarly, studies from Naito et al. (Reference Naito, Nagata and Takano26, Reference Naito, Kasama and Yoshida27) indicated that feeding rapeseed oil, but not soyabean oil, elevated systolic blood pressure in SHRSP, SHR and WKY rats. The higher levels of phytosterols present in rapeseed oil compared with soyabean oil(Reference Ratnayake, L'Abbé and Mueller15, Reference Naito, Nagata and Takano26) were speculated to contribute to this increased blood pressure.

In order to elucidate the molecular mechanism(s) by which phytosterols and phytostanols resulted in an increase in the blood pressure in SHRSP rats, the expression of several renal genes known to be involved in blood pressure regulation were investigated (Table 5). We report for the first time that phytosterols and/or phytostanols up-regulated the mRNA expression of Ace1, Nos1, Nos3, Cox2 and Spon1 in SHRSP rats. We also found that SHRSP rats have higher mRNA levels of Agt, Ace1, Nos1, Nos3, Cox2 and Thumpd1 and lower mRNA levels of renin and Agtr1a compared with WKY inbred rats.

The renin–angiotensin–aldosterone system (RAAS) is a hormone system that helps regulate long-term blood pressure and extracellular volume in the body(Reference Hammoud, Vaccari and Nagamia28). In response to stimuli, the kidneys release the enzyme renin, which converts Agt into angiotensin I, and the latter is converted to angiotensin II by angiotensin I-converting enzyme 1 (Ace1). Angiotensin II, the major bioactive product of the RAAS, reacts mainly with angiotensin II receptor (Agtr) and results in increased blood pressure by mediating a powerful vasoconstrictor effect, leading to aldosterone secretion and sodium reabsorption in the kidney, and stimulating sympathetic activity(Reference Hammoud, Vaccari and Nagamia28). In the present study, SHRSP rats had significantly higher mRNA expression of Agt (P < 0·0001) and Ace1 (P < 0·0001) than WKY inbred rats. In addition, phytostanol supplementation further up-regulated the Ace1 mRNA expression in SHRSP but not in WKY inbred rats. Elevation of the Agt and Ace1 mRNA levels of SHRSP rats in the present study was consistent with the study by Obata et al. (Reference Obata, Nakamura and Takano29), who reported that SHRSP rats had significantly higher mRNA expression of Agt and Ace than WKY rats. These results are also supported by Takemori(Reference Takemori, Ishida and Ito30), who demonstrated that SHRSP rats had higher plasma angiotensin II levels than WKY rats. Therefore, the increased expression of these genes may contribute, at least in part, to the pathogenesis of hypertension in SHRSP rats (Fig. 1).

Fig. 1 Summary of the proposed mechanisms by which phytosterols and phytostanols exacerbate the development of hypertension in SHRSP rats. Overall, ingestion of high levels of phytosterols and phytostanols may exacerbate the development of hypertension in SHRSP rats by directly or indirectly activating RAAS, stimulating excessive peroxynitrite formation, increasing PGH2/TXA2 activity and decreasing the availability of NO and PGI2. +/ − , activated/inhibited by ONOO− ; *, elevated in SHRSP rats in the present study; †, up-regulated by phytosterols or phytostanols in the present study.

The present study also found that SHRSP rats had slightly lower mRNA levels of renin (1·16-fold, P = 0·0003) and Agtr1a (1·10-fold, P = 0·007) than WKY inbred rats. These results are not consistent with those of Obata et al. (Reference Obata, Nakamura and Takano29), who demonstrated a higher mRNA expression of Agtr1a in SHRSP than WKY rats. While the reason for this difference is unclear, it may be related to the different tissue samples used to isolate total RNA in these studies (whole kidney in the present study v. >95 % glomeruli in the study of Obata et al. (Reference Obata, Nakamura and Takano29) or the different methods used for measuring mRNA expression (real-time QPCR in the present study v. RNase protection assay in the study of Obata et al. (Reference Obata, Nakamura and Takano29)). Based on the present results, it is possible that SHRSP rats down-regulate the renin and Agtr1a expression in order to compensate for the up-regulation of Agt and Ace1. However, this compensatory mechanism is not sufficient to prevent the increase in blood pressure when compared with WKY inbred rats.

A reduced bioavailability of nitric oxide (NO) has also been postulated to contribute to the development of hypertension(Reference Schulz, Jansen and Wenzel31). NO is biosynthesised from arginine and oxygen by a family of Nos, including neuronal Nos (Nos1 or nNos), inducible Nos (Nos2 or iNos) and endothelial Nos (Nos3 or eNos). In the present study, SHRSP rats had higher mRNA expression of Nos1 (P < 0·0001) and Nos3 (P < 0·0001) than WKY inbred rats. Also, phytosterol and phytostanol supplementation up-regulated the mRNA expression of Nos1 and Nos3, respectively. However, in addition to synthesising NO, Nos can also generate superoxide(Reference Arehart, Gleim and Kasza32), which has been found to react with NO to produce peroxynitrite (ONOO− )(Reference Xia, Dawson and Dawson33). Thus, increased scavenging of NO by superoxide could lead to a decrease in NO availability despite increased synthesis (Fig. 1). The present results and the proposed explanation are supported by the studies from Kerr et al. (Reference Pryor and Squadrito34) and others(Reference Zou35, Reference Bachschmid, Thurau and Zou36), who reported that Nos3 mRNA expression and superoxide generation were significantly higher in SHRSP than in WKY counterparts.

In the present study, SHRSP rats had significantly higher Cox2 mRNA expression than WKY inbred rats and both phytosterol and phytostanol supplementation increased Cox2 mRNA levels. Prostanoids (including PG, prostacyclin and thromboxane (TXA2)) have been demonstrated to play an important role in the regulation of blood pressure(Reference Upmacis, Deeb and Hajjar37). Cox (also known as PGH2 synthase) converts arachidonic acid to PGH2, the precursor of the series-2 prostanoids that cause dilation (such as PGI2) or constriction (such as TXA2) in vascular smooth muscle cells. Peroxynitrite, produced from a reaction of NO with superoxide (as mentioned earlier), has been shown to activate Cox(Reference Harris, Zhang and Cheng38), causing the accumulation of PGH2, which was able to activate the TXA2/PGH2 receptor and mediate smooth muscle contraction(Reference Cosentino, Sill and Katusić39). Peroxynitrite has also been shown to be a potent oxidant(Reference McBride, Brosnan and Mathers40), which inhibits PGI2 synthase(Reference Cosentino, Sill and Katusić39, Reference McBride, Brosnan and Mathers40) leading to a decrease in PGI2 and subsequently reducing muscle relaxation. Cox enzyme itself can also generate superoxide(Reference Kukreja, Kontos and Hess41, Reference Kerr, Brosnan and McIntyre42), which may further promote peroxynitrite formation. Excessive expression of Cox2 may also stimulate RAAS, leading to increased arterial pressure and vascular lesions(Reference Hamilton, Miller and Al-Benna43). Therefore, excessive peroxynitrite formation from the reaction of NO with superoxide may contribute to hypertension in SHRSP rats by decreasing the availability of NO and PGI2, and increasing vasoconstrictors, PGH2/TXA2 and RAAS (Fig. 1).

The observed phytosterol and phytostanol up-regulation of Cox2 mRNA expression in the present study was supported by the results from Ohara et al. (Reference Ohara, Kasama and Naito44), who reported that rapeseed oil increased Cox2 protein expression in SHR rats, when compared with soyabean oil. Recent studies have also shown that ingestion of rapeseed oil significantly increased glucose-6-phosphate dehydrogenase activity in SHR(Reference Ohara, Kasama and Naito44) and WKY(Reference Naito, Kasama and Yoshida27) rats, compared with soyabean oil. Glucose-6-phosphate dehydrogenase, the first enzyme in the pentose phosphate pathway, provides NADPH for the generation of NO by Nos(Reference Bermúdez, Bermúdez and Acosta45, Reference Matsui, Xu and Maitland46) and for the generation of NADPH oxidase-derived superoxide(Reference Lassègue and Clempus47, Reference Van Heerebeek, Meischl and Stooker48). As mentioned earlier, superoxide reacts with NO leading to a decrease in NO availability and the generation of peroxynitrite. These data suggest that NADPH production by glucose-6-phosphate dehydrogenase in response to phytosterols and phytostanols could contribute to the mechanisms by which NO availability is diminished and excessive peroxynitrite is produced, leading to an increase in blood pressure (Fig. 1). This proposed mechanism is also supported by the studies which showed that rapeseed oil lowered the activities of anti-oxidative enzymes, superoxide dismutase and catalase in SHR(Reference Ohara, Kasama and Naito44) and WKY rats(Reference Naito, Kasama and Yoshida27).

Recently, Clemitson et al. (Reference Clemitson, Dixon and Haines49) identified Spon1 as a candidate hypertension gene. They reported that the mRNA levels of Spon1 in the kidney, heart and aorta were significantly higher in SHR than in WKY rats. In the present study, strain effects on Spon1 expression in the kidney were not observed between SHRSP and WKY inbred rats (P = 0·117). However, phytosterol supplementation up-regulated renal Spon1 mRNA levels in both SHRSP and WKY inbred rats. Therefore, the role of Spon1 in hypertension is unclear and its significance in phytosterol/phytostanol-induced blood pressure elevation warrants further study.

Thumpd1 was also recently identified as a candidate hypertension gene(Reference Clemitson, Dixon and Haines49, Reference Hubner, Wallace and Zimdahl50) and was shown to have higher mRNA expression in hypertensive SHR rats compared with normotensive Brown Norway rats. Similarly, we found that SHRSP rats have a significantly higher mRNA expression of Thumpd1 (1·99-fold, P < 0·0001) than WKY inbred rats; however, diet effects were not demonstrated. Our data suggest that although Thumpd1 may contribute to hypertension in SHRSP rats, it does not appear to be involved in the phytosterol/phytostanol-induced blood pressure increase in SHRSP rats.

In conclusion, we determined that SHRSP rats have higher systolic and diastolic blood pressures compared with WKY inbred rats. SHRSP rats fed phytosterol- or phytostanol-supplemented diets demonstrated a significant increase in the diastolic blood pressure compared with rats fed the control diet. Increased blood pressure in SHRSP rats may be due, in part, to altered renal expression of blood pressure regulatory genes. Specifically, the up-regulation of Ace1, Nos1, Nos3, Cox2 and Spon1 is associated with the increased diastolic blood pressure observed in the phytosterol- or phytostanol-supplemented SHRSP rats. Overall, we speculate that ingestion of high levels of phytosterols and phytostanols may exacerbate the development of hypertension and related conditions in SHRSP rats by directly or indirectly activating RAAS, stimulating excessive peroxynitrite formation, increasing PGH2/TXA2 activity and decreasing the availability of NO and PGI2. Therefore, it would be prudent to determine whether foods fortified with high levels of phytosterols or phytostanols may have a potential health risk to individuals predisposed to hypertension.

Acknowledgements

The authors thank Dr Don Caldwell and the members of the Animal Resources Division of Health Canada for their technical assistance with the animal phase of the present study. Q. C. performed data analysis and interpretation and contributed to the drafting of the paper; H. G., E. S. and C. P. performed data analysis; W. M. N. R. and K. A. S. designed the study and contributed to the drafting of the paper. Q. C. is a recipient of a Health Canada Office of the Chief Scientist Postdoctoral Fellowship. This research was funded by Health Canada. The authors declare no conflict of interest.

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Figure 0

Table 1 Composition of test diets (g/kg)*

Figure 1

Table 2 Primer sequences for real-time quantitative PCR (rat)

Figure 2

Table 3 Growth, food and water intake of the rats(Mean values with their standard errors)

Figure 3

Table 4 Systolic and diastolic blood pressure of the rats(Mean values with their standard errors)

Figure 4

Table 5 Renal gene expression of the rats(Mean values with their standard errors)

Figure 5

Fig. 1 Summary of the proposed mechanisms by which phytosterols and phytostanols exacerbate the development of hypertension in SHRSP rats. Overall, ingestion of high levels of phytosterols and phytostanols may exacerbate the development of hypertension in SHRSP rats by directly or indirectly activating RAAS, stimulating excessive peroxynitrite formation, increasing PGH2/TXA2 activity and decreasing the availability of NO and PGI2. +/ − , activated/inhibited by ONOO− ; *, elevated in SHRSP rats in the present study; †, up-regulated by phytosterols or phytostanols in the present study.