High blood pressure is a serious condition that can lead to coronary heart disease, stroke, kidney failure, and other health problems . Effects of phytosterols and phytostanols on blood pressure remain controversial. In human studies, consumption of a dietary portfolio (plant sterols, soybean protein, viscous fibres and almonds) for 1 year significantly reduced blood pressure in 66 hyperlipidemic subjects . Intake of a product containing sitostanol and campestanol for 4 weeks slightly decreased systolic blood pressure in 100 healthy men . However, elevated systolic blood pressure was observed in a phytosterolemic patient . In animal studies, in the presence of 1% salt-loading, our previous study demonstrated that, phytosterol or phytostanol supplemented diets (2.0 g/kg diet) induced a significant increase in diastolic blood pressure in SHRSP rats . Similarly, Naito et al. [22–24] showed that a diet containing 10% rapeseed oil (0.71 g total phytosterols/kg diet) induced higher systolic blood pressure than a diet containing soybean oil (0.29 g total phytosterols/kg diet) in SHRSP, SHR and WKY rats and thus thought that the higher content of phytosterols in rapeseed oil was related to the increased blood pressure. A diet containing soybean oil fortified with phytosterols (4.5 g/kg diet) for 24 days was also reported to elevate systolic blood pressure in SHRSP rats, but there were no significant differences on systolic blood pressure among diets containing 10% rapeseed oil, 10% soybean oil, and 10% soybean oil supplemented with 0.6 g total phytosterols/kg diet . A study by Chen et al.  demonstrated that consumption of a sitosterol diet (0.3 g/kg diet) for 6 weeks did not increase blood pressure in wild-type, heterozygous and homozygous (a mutation in Abcg5) female SHR inbred rats. In the absence of salt-loading, our present study found that dietary phytosterols or phytostanols (2.0 g/kg diet) significantly increased systolic and diastolic blood pressure in WKY inbred rats (Table 4). To our knowledge, this was the first study to investigate, in the absence of salt-loading, the effects of dietary phytosterols and phytostanols on blood pressure in rodent models. Our results were consistent with those reported by Naito et al.  who demonstrated that, in the absence of salt-loading, a diet containing 10% rapeseed oil for 4 weeks induced a higher systolic blood pressure in SHRSP rats compared to a diet containing 10% soybean oil. Taken together, our previous and present results suggest that increased intake of phytosterols or phytostanols may exacerbate hypertension in both hypertensive and normotensive rat models that over absorb phytosterols and phytostanols in the presence or absence of salt.
In order to elucidate the molecular mechanism(s) by which intake of phytosterols and phytostanols increased blood pressure in this rat model, the expression of several renal genes known to be involved in the renin-angiotensin-aldosterone system, nitric oxide, and cyclooxygenase-derived prostanoid pathways were investigated (Table 5). Spondin 1 and THUMP domain containing 1 were also measured because they were recently identified as candidate hypertension genes [40, 41]. Our present study demonstrated that, in the absence of salt-loading, the phytosterol diet slightly increased the mRNA expression of angiotensinogen (1.2-fold, P < 0.05, Table 5). There were no effects of phytosterols and phytostanols on the mRNA expression of renin, angiotensin I converting enzyme (Ace1) 1, Ace2, angiotensin II receptor 1a (Agtr1a), nitric oxide synthases (Nos) 1, Nos3, Cyclooxygenase 2, Spondin 1 and Thump domain 1 (P > 0.05). These results suggest that Nos, Cyclooxygenase 2, Spondin 1 and Thump domain 1 pathways do not play a critical role in the blood pressure regulation caused by the intake of phytosterols and phytostanols in WKY inbred rats. Also, the phytostanol diet increased blood pressure but did not change the mRNA levels of renal angiotensinogen. Therefore, the renin-angiotensin-aldosterone system may not play a leading role in the hypertensive responses caused by phytosterol and phytostanol diets in the absence of salt-loading in WKY inbred rats.
Currently there are no data indicating that consumption of phytosterol and phytostanol-enriched foods alters cardiovascular events, but epidemiological studies indicated a controversial association of phytosterols and phytostanols with cardiovascular risk. For example, the results from a cohort study in 1242 subjects older than 65 years in the Netherlands showed that plasma levels of phytosterols were significantly lower in patients with coronary heart disease . A prospective nested case-control study consisting of 373 cases and 758 controls in the United Kingdom indicated that there was no adverse relationship between plasma physiological levels of phytosterols and coronary heart disease risk in apparently healthy individuals . The study from 2542 subjects aged 30 to 67 years in the United States showed no significant difference in plasma sitosterol and campesterol levels between the individuals having a positive and a negative family history of CHD . However, studies in 595 subjects with hypercholesterolemia , in 48 postmenopausal women , in 53 patients undergoing coronary artery bypass graft surgery  and in 159 men that suffered a myocardial infarction in the Prospective Cardiovascular Munster study  suggest an association of elevated levels of phytosterols and phytostanols with an increased cardiovascular disease risk. In the present study dietary phytosterols and phytostanols (2.0 g/kg diet) were found, in the absence of salt-loading, to significantly increase systolic and diastolic blood pressure (P < 0.05, Table 4), to increase phytosterol and phytostanol accumulation in plasma, red blood cells, liver, aorta and kidney (P < 0.05), and to decrease cholesterol levels in liver, aorta, and kidney as well as plasma (only by phytostanols) (P < 0.05) (Table 3). These results suggest that, even though phytosterols and phytostanols lower cholesterol levels, their excessive accumulation in plasma and tissues may increase blood pressure thereby causing their net beneficial effects on cardiovascular events to remain questionable.
Our previous study indicated that a deficiency of membrane cholesterol due to phytosterol intake increases cell membrane rigidity, which might be a factor contributing to the shortened life span of SHRSP rats . Hamano et al.  also reported that a diet containing cholesterol (10 g/kg diet) delayed the onset of stroke and prolonged the life span of SHRSP rats, whereas diets with no added cholesterol greatly shortened post-stroke survival. In the present study supplementation of phytosterols and phytostanols produced significant decreases in the ratios of cholesterol to phytosterols and phytostanols in plasma, red blood cells, liver, aorta and kidney (Table 3) and increased blood pressure (Table 4) in WKY inbred rats. Therefore, the increased blood pressure caused by phytosterols and phytostanols in WKY inbred rats may be due to increased cell membrane rigidity. Other studies, in WKY rats in the presence of salt and in SHRSP rats in the absence of salt, have proposed that enhanced membrane activity of Na+, K+-ATPase by phytosterols in rapeseed oil may play a role in elevated blood pressure [21, 22]. Other mechanisms, such as endothelin and natriuretic peptides , have not been excluded from playing a role in hypertension caused by phytosterols and phytostanols in WKY inbred rats in this study and could be investigated in future studies. The molecular mechanisms of action of dietary phytosterols and phytostanols responsible for the absorption and accumulation of cholesterol, phytosterols and phytostanols have been previously discussed in our recently published paper .
Interestingly, it is generally speculated that phytostanol supplementation may be safer than phytosterol supplementation because the absorption of phytostanols (1-2%) is less than that of phytosterols (4-13%)  and the plasma levels of phytostanols are much less than that of phytosterols in humans after consumption of similar levels of either substance . However, in our present study, the phytostanol diet resulted in a significant increase in diastolic blood pressure compared to the phytosterol diet (P < 0.05, Table 4). This result is consistent with our previous study in which SHRSP rats fed phytostanol supplemented margarine had a slightly lower survival rate than rats fed phytosterol supplemented margarine .