Adverse effects of an aldosterone synthase (CYP11B2) inhibitor, fadrozole (FAD286), on inflamed rat colon.

Recently, we described local aldosterone production in the murine large intestine. Upregulated local aldosterone synthesis in different tissues has been linked with inflammatory conditions, which have been attenuated by the aldosterone synthase (CYP11B2) inhibitor, fadrozole (FAD286). Therefore, we investigated the effect of inhibition of intestinal aldosterone synthesis on the development of intestinal inflammation. Sprague-Dawley rats were administered 5% (v/w) dextran sodium sulphate (DSS) for 7 days with or without daily FAD286 (30 mg/kg/d) subcutaneous injections on 3 days before, during and one day after DSS. Tissue aldosterone concentrations were evaluated by ELISA, CYP11B2 by Western blot and RT-qPCR. FAD286 halved adrenal aldosterone production but, intriguingly, increased the colonic aldosterone concentration. The lack of inhibitory effect of FAD286 in the colon might have been affected by the smaller size of colonic vs. adrenal CYP11B2, as seen in Western blot. When combined with DSS, FAD286 aggravated the macroscopic and histological signs of intestinal inflammation, lowered the animals' body weight gain and increased the incidence of gastrointestinal bleeding and the permeability to iohexol in comparison to DSS-animals. To conclude, FAD286 exerted harmful effects during intestinal inflammation. Local intestinal aldosterone did not seem to play any role in the inflammatory pathogenesis occurring in the intestine.

Ketogenic Diet High in Saturated Fat Promotes Colonic Claudin Expression without Changes in Intestinal Permeability to Iohexol in Healthy Mice.

Ketogenic diets (KDs) have been studied in preclinical models of intestinal diseases. However, little is known of how the fat source of these diets influences the intestinal barrier. Herein, we studied the impact of four-week feeding with KD high either in saturated fatty acids (SFA-KD) or polyunsaturated linoleic acid (LA-KD) on paracellular permeability of the intestine to iohexol in healthy male C57BL/6J mice. We investigated jejunal and colonic tight junction protein expression, histological changes, and inflammatory markers (Il1b, Il6, Tnf, and Lcn2), as well as the activity and expression of intestinal alkaline phosphatase (IAP) in feces and jejunal tissue, respectively, and plasma lipopolysaccharide. KDs did not change intestinal permeability to iohexol after two or twenty-six days of feeding regardless of fat quality. SFA-KD, but not LA-KD, upregulated the colonic expression of tight junction proteins claudin-1 and -4, as well as the activity of IAP. Both KDs resulted in increased epithelial vacuolation in jejunum, and this was pronounced in SFA-KD. Jejunal Il1ß expression was lower and colonic Il6 expression higher in LA-KD compared to SFA-KD. In colon, Tnf mRNA was increased in LA-KD when compared to controls. Overall, the results suggest that KDs do not influence intestinal permeability to iohexol but elicit changes in colonic tight junction proteins and inflammatory markers in both jejunum and colon. Future research will show whether these changes become of importance upon proinflammatory insults.

Local aldosterone synthesis in the large intestine of mouse: An ex vivo incubation study.

OBJECTIVE: To investigate the regulation of local aldosterone synthesis by physiological stimulants in the murine gut. METHODS: Male mice were fed for 14 days with normal, high (1.6%) or low (0.01%) sodium diets. Tissue liver receptor homolog-1 and aldosterone in the colon and caecum were detected using an enzyme-linked immunosorbent assay (ELISA). Released corticosterone and aldosterone in tissue incubation experiments after stimulation with angiotensin II (Ang II) and dibutyryl-cAMP (DBA; the second messenger of adrenocorticotropic hormone) were assayed using an ELISA. Tissue aldosterone synthase (CYP11B2) protein levels were measured using an ELISA and Western blots. RESULTS: In incubated colon tissues, aldosterone synthase levels were increased by a low-sodium diet; and by Ang II and DBA in the normal diet group. Release of aldosterone into the incubation buffer was increased from the colon by a low-sodium diet and decreased by a high-sodium diet in parallel with changes in aldosterone synthase levels. In mice fed a normal diet, colon incubation with both Ang II and DBA increased the release of aldosterone as well as its precursor corticosterone. CONCLUSION: Local aldosterone synthesis in the large intestine is stimulated by a low-sodium diet, dibutyryl-cAMP and Ang II similar to the adrenal glands.

Renin-angiotensin system in intestinal inflammation-Angiotensin inhibitors to treat inflammatory bowel diseases?

Inflammatory bowel diseases (IBDs) are chronic disorders of the gastrointestinal tract, which manifest in recurring gastrointestinal inflammation. The current treatment options of IBD are not curative and are lacking in aspects like prevention of fibrosis. New treatment options are needed to fulfil the unmet needs and provide alternatives to drugs with resistances and side effects. Drugs targeting the renin-angiotensin system (RAS), besides being antihypertensive, also possess anti-inflammatory and antifibrotic properties and could offer an inexpensive alternative to control inflammation and fibrosis in the gut. RAS inhibitors have been effective in preventing and alleviating colitis in preclinical studies, but available human data are still sparse. This review outlines the pathophysiological functions of RAS in the gut and summarizes preclinical studies utilizing pharmacological RAS inhibitors in the treatment of experimental colitis. We discuss the alterations in intestinal RAS and the available evidence of the benefits of RAS inhibitors for IBD patients. Retrospective studies comparing IBD patients using ACE inhibitors or angiotensin II receptor blockers have provided optimistic results regarding a milder disease course and fewer hospitalizations and corticosteroid use in patients using RAS inhibitors. Prospective studies are needed to evaluate the effectiveness of these promising medications in the treatment of IBD.

Randomised Controlled Trial: Partial Hydrolysation of Casein Protein in Milk Decreases Gastrointestinal Symptoms in Subjects with Functional Gastrointestinal Disorders.

Unspecific gastrointestinal symptoms associated with milk consumption are common. In addition to lactose, also other components of milk may be involved. We studied whether the partial hydrolysation of milk proteins would affect gastrointestinal symptoms in subjects with functional gastrointestinal disorders. In a randomised, placebo-controlled crossover intervention, subjects (n = 41) were given ordinary or hydrolysed high-protein, lactose-free milkshakes (500 mL, 50 g protein) to be consumed daily for ten days. After a washout period of ten days, the other product was consumed for another ten days. Gastrointestinal symptoms were recorded daily during the study periods, and a validated irritable bowel syndrome-symptom severity scale (IBS-SSS) questionnaire was completed at the beginning of the study and at the end of both study periods. Blood and urine samples were analysed for markers of inflammation, intestinal permeability and immune activation. Both the IBS-SSS score (p = 0.001) and total symptom score reported daily (p = 0.002) were significantly reduced when participants consumed the hydrolysed product. Less bloating was reported during both study periods when compared with the baseline (p < 0.01 for both groups). Flatulence (p = 0.01) and heartburn (p = 0.03) decreased when consuming the hydrolysed product but not when drinking the control product. No significant differences in the levels of inflammatory markers (tumor necrosis factor alpha, TNF-α and interleukin 6, IL-6), intestinal permeability (fatty acid binding protein 2, FABP2) or immune activation (1-methylhistamine) were detected between the treatment periods. The results suggest that the partial hydrolysation of milk proteins (mainly casein) reduces subjective symptoms to some extent in subjects with functional gastrointestinal disorders. The mechanism remains to be resolved.

Local ocular renin-angiotensin-aldosterone system: any connection with intraocular pressure? A comprehensive review.

The renin-angiotensin system (RAS) is one of the oldest and most extensively studied human peptide cascades, well-known for its role in regulating blood pressure. When aldosterone is included, RAAS is involved also in fluid and electrolyte homeostasis. There are two main axes of RAAS: (1) Angiotensin (1-7), angiotensin converting enzyme 2 and Mas receptor (ACE2-Ang(1-7)-MasR), (2) Angiotensin II, angiotensin converting enzyme 1 and angiotensin II type 1 receptor (ACE1-AngII-AT1R). In its entirety, RAAS comprises dozens of angiotensin peptides, peptidases and seven receptors. The first mentioned axis is known to counterbalance the deleterious effects of the latter axis. In addition to the systemic RAAS, tissue-specific regulatory systems have been described in various organs, evidence that RAAS is both an endocrine and an autocrine system. These local regulatory systems, such as the one present in the vascular endothelium, are responsible for long-term regional changes. A local RAAS and its components have been detected in many structures of the human eye. This review focuses on the local ocular RAAS in the anterior part of the eye, its possible role in aqueous humour dynamics and intraocular pressure as well as RAAS as a potential target for anti-glaucomatous drugs.KEY MESSAGESComponents of renin-angiotensin-aldosterone system have been detected in different structures of the human eye, introducing the concept of a local intraocular renin-angiotensin-aldosterone system (RAAS).Evidence is accumulating that the local ocular RAAS is involved in aqueous humour dynamics, regulation of intraocular pressure, neuroprotection and ocular pathology making components of RAAS attractive candidates when developing new effective ways to treat glaucoma.

Vascular Calcification, Vitamin K and Warfarin Therapy - Possible or Plausible Connection?

Atherosclerosis is a pathological process underpinning many cardiovascular diseases; it is the main cause of global mortality. Atherosclerosis is characterized by an invasion of inflammatory cells, accumulation of lipids and the formation of fatty streaks (plaques) which subsequently allow accumulation of calcium and other minerals leading to a disturbance in the vascular endothelium and its regulatory role in arterial function. Vascular calcification is a different process, stringently regulated mainly by local factors, in which osteoblast-like cells accumulate in the muscular layer of arteries ultimately taking on the physiological appearance of bone. The elevated stiffness of the arteries leads to severe vascular complications in brain, heart and kidneys. Recently, evidence from animal experiments as well as clinical and epidemiological results suggests that long-term treatment with warfarin, but not with the novel direct anticoagulants, can increase the risk or even induce vascular calcification in some individuals. Gamma-carboxylation is an enzymatic process not only needed for activation of vitamin K but also other proteins which participate in bone formation and vascular calcification. Thus, reduced expression of the vitamin K-dependent proteins which physiologically inhibit calcification of cellular matrix could be postulated to lead to vascular calcification. Published clinical data, describing at present a few thousand patients, need to be supplemented with controlled studies to confirm this interesting hypothesis.

Many Faces of Renin-angiotensin System - Focus on Eye.

The renin-angiotensin system (RAS), that is known for its role in the regulation of blood pressure as well as in fluid and electrolyte homeostasis, comprises dozens of angiotensin peptides and peptidases and at least six receptors. Six central components constitute the two main axes of the RAS cascade. Angiotensin (1-7), an angiotensin converting enzyme 2 and Mas receptor axis (ACE2-Ang(1-7)-MasR) counterbalances the harmful effects of the angiotensin II, angiotensin converting enzyme 1 and angiotensin II type 1 receptor axis (ACE1-AngII-AT1R) Whereas systemic RAS is an important factor in blood pressure regulation, tissue-specific regulatory system, responsible for long term regional changes, that has been found in various organs. In other words, RAS is not only endocrine but also complicated autocrine system. The human eye has its own intraocular RAS that is present e.g. in the structures involved in aqueous humor dynamics. Local RAS may thus be a target in the development of new anti-glaucomatous drugs. In this review, we first describe the systemic RAS cascade and then the local ocular RAS especially in the anterior part of the eye.

A Single Drop in the Eye - Effects on the Whole Body?

INTRODUCTION: Although the local adverse effects of ophthalmic drugs, including allergic reactions, are well recognized, less is known about the systemic side- effects of eye drops, especially during pregnancy, breast-feeding and early childhood. Ophthalmologists should also be aware of unusual, in some cases even life-threatening, effects of commonly used eye drops. CONCLUSION: This brief review outlines the routes of systemic absorption and the kinetics of active components present in eye drops, and identifies the clinically relevant systemic adverse effects.

Local corticosterone production and angiotensin-I converting enzyme shedding in a mouse model of intestinal inflammation.

AIM: To investigate local corticosterone production and angiotensin-I converting enzyme (ACE) protein expression and their interaction in healthy and inflamed intestine. METHODS: Acute intestinal inflammation was induced to six weeks old male Balb/c mice by administration of either 3% or 5% dextran sodium sulfate (DSS) in drinking water for 7 d (n = 12 in each group). Healthy controls (n = 12) were given tap water. Corticosterone production and ACE protein shedding were measured from ex vivo incubates of the small and large intestine using EIA and ELISA, respectively. Morphological changes of the intestinal wall were assessed in hematoxylin-eosin stained tissue preparations of jejunum and distal colon. Effects of angiotensin II, captopril and metyrapone on corticosterone production was assessed by incubating pieces of small intestine of healthy mice in the presence of 0.1, 1 or 10 µmol/L angiotensin II, 1, 10 or 100 µmol/L captopril or 1, 10 or 100 µmol/L metyrapone solutions and measuring corticosterone released to the incubation buffer after 90 min (n = 5 in each group). RESULTS: Both concentrations of DSS induced inflammation and morphological changes in large intestines but not in small intestines. Changes were observed as distortions of the crypt structure, mucosal erosion, immune cell infiltration to the mucosa and submucosal edema. Ex vivo corticosterone production (2.9 ± 1.0 ng/mL vs 2.0 ± 0.8 ng/mL, P = 0.034) and ACE shedding (269.2 ± 97.1 ng/mL vs 175.7 ± 52.2 ng/mL, P = 0.016) were increased in small intestines in 3% DSS group compared to the controls. In large intestine, corticosterone production was increased compared to the controls in both 3% DSS (229 ± 81 pg/mL vs 158 ± 30 pg/mL, P = 0.017) and 5% DSS groups (366 ± 163 pg/mL vs 158 ± 30 pg/mL, P = 0.002). Large intestine ACE shedding was increased in 5% DSS group (41.5 ± 9.0 ng/mL vs 20.9 ± 5.2 ng/mL, P = 0.034). Angiotensin II treatment augmented corticosterone production in small intestine at concentration of 10 µmol/L (0.97 ± 0.21 ng/mg protein vs 0.40 ± 0.09 ng/mg protein, P = 0.036). CONCLUSION: Intestinal ACE shedding is increased by DSS-induced intestinal inflammation and parallels local corticosterone production. ACE product angiotensin II stimulates corticosterone formation in healthy intestine.

Effect of fermented milk product containing lactotripeptides and plant sterol esters on haemodynamics in subjects with the metabolic syndrome--a randomised, double-blind, placebo-controlled study.

We investigated the effects of fermented milk product containing isoleucine-proline-proline, valine-proline-proline and plant sterol esters (Pse) on plasma lipids, blood pressure (BP) and its determinants systemic vascular resistance and cardiac output. In a randomised, double-blind, placebo-controlled study, 104 subjects with the metabolic syndrome (MetS) were allocated to three groups in order to receive fermented milk product containing (1) 5 mg/d lactotripeptides (LTP) and 2 g/d plant sterols; (2) 25 mg/d LTP and 2 g/d plant sterols; (3) placebo for 12 weeks. Plasma lipids and home BP were monitored. Haemodynamics were examined in a laboratory using radial pulse wave analysis and whole-body impedance cardiography in the supine position and during orthostatic challenge. There were no differences between the effects of the two treatments and placebo on the measurements of BP at home or on BP, systemic vascular resistance index and cardiac index in the laboratory, neither in the supine nor in the upright position. The changes in plasma LDL-cholesterol concentration were - 0.1 (95% CI - 0.3, 0.1 and - 0.3, 0.0) mmol/l in the 5 and 25 mg/d LTP groups, respectively, and +0.1 (95% CI - 0.1, 0.3) mmol/l during placebo (P= 0.024). Both at baseline and at week 12, the increase in systemic vascular resistance during head-up tilt was lower in the 25 mg/d LTP group than in the 5 mg/d LTP group (P< 0.01), showing persistent differences in cardiovascular regulation between these groups. In subjects with the MetS, intake of LTP and Pse in fermented milk product showed a lipid-lowering effect of borderline significance, while no antihypertensive effect was observed at home or in the laboratory.

Does the cis/trans configuration of peptide bonds in bioactive tripeptides play a role in ACE-1 enzyme inhibition?

BACKGROUND: The milk casein-derived bioactive tripeptides isoleucine-proline-proline (IPP) and valine-proline-proline (VPP) have been shown to prevent development of hypertension in animal models and to lower blood pressure in moderately hypertensive subjects in most but not all clinical trials. Inhibition of angiotensin-converting enzyme 1 (ACE-1) has been suggested as the explanation for these antihypertensive and beneficial vascular effects. Previously, human umbilical vein endothelial cells (HUVEC) have not been used to test ACE-1 inhibiting properties of casein derived tripeptides in vasculature. PURPOSE: We focused on the cis/trans configurations of the peptide bonds in proline-containing tripeptides in order to discover whether the different structural properties of these peptides influence their activity in ACE-1 inhibition. We hypothesized that the configuration of proline-containing peptides plays a significant role in enzyme inhibition. METHODS: AutoDock 4.2 docking software was used to predict suitable peptide bond configurations of the tripeptides. Besides modeling studies, we completed ACE-1 activity measurements in vitro using HUVEC cultures. RESULTS: In HUVEC cells, both IPP and VPP inhibited ACE-1. Based on molecular docking studies, we propose that in ACE-1 inhibition IPP and VPP share a similar cis configuration between the first aliphatic (isoleucine or valine) and the second (proline) amino acid residues and more different configurations between two proline residues. In vivo experiments are needed to validate the significance of the present findings.

Low serum enterolactone concentration is associated with low colonic Lactobacillus-Enterococcus counts in men but is not affected by a synbiotic mixture in a randomised, placebo-controlled, double-blind, cross-over intervention study.

The aims of the present study were to assess the possible differences in faecal microbiota between men with a low serum enterolactone concentration and those with a high concentration, and to investigate the impact of a synbiotic mixture on serum enterolactone concentration in men with a low concentration. We compared faecal microbiota between ten men with the lowest serum enterolactone concentration and ten men with the highest concentration at recruitment (n 84). Furthermore, we carried out a randomised, double-blind, placebo-controlled, cross-over intervention study (6-week intervention periods and 4-week washout period) to investigate the impact of a synbiotic mixture (two Lactobacillus strains, one Bifidobacterium strain, one Propionibacterium strain and galacto-oligosaccharides (32 g/l)) on serum enterolactone concentration in fifty-two men who had a concentration < 20 nmol/l. Serum sensitive C-reactive protein (CRP) concentration was measured at the end of the first intervention period. Men with a low serum enterolactone concentration when compared with those with a high concentration had less faecal bacteria, especially those belonging to the Lactobacillus-Enterococcus group (median 8·2 (interquartile range 7·8-8·4) log10 colony-forming units/g v. median 8·8 (interquartile range 8·5-8·9) log10 colony-forming units/g, P= 0·009). The synbiotic mixture that was used did not have a significant effect on serum enterolactone (synbiotic v. placebo ratio 0·96 (95 % CI 0·76, 1·22), P= 0·724) or serum sensitive CRP (synbiotic v. placebo ratio 0·99 (95 % CI 0·74, 1·33), P= 0·954) concentration. Men with a low serum enterolactone concentration harbour less colonic bacteria, especially those belonging to the Lactobacillus-Enterococcus group. A synbiotic mixture does not increase serum enterolactone concentration.

[Renin-angiotensin system (RAS) in the bowel]. / Reniini-angiotensiinijärjestelmä (RAS) suolessa - mitä se siellä?

The significance of the renin-angiotensin system (RAS) in blood pressure regulation is indisputable. In addition to the heart and circulatory system, its components have recently been shown to be expressed also in the digestive tract, pancreas, brain and eyes, raising the idea that the significance of RAS for the functions of the body may be much more complex than currently thought. Its effects in the digestive tract could involve regulation of smooth muscle of the blood vessels and bowel, changes in permeability of the gut mucosa, or functioning as a digestive enzyme.

Antihypertensive effects of bioactive tripeptides-a random effects meta-analysis.

A meta-analysis of possible antihypertensive effects of small doses of bioactive tripeptides isoleucine-proline-proline and valine-proline-proline in commercial milk products or tablets was carried out. A random effects model was used on 19 randomized, placebo-controlled clinical intervention trials (published 1996-October 2010) consisting of about 1500 prehypertensive or mildly hypertensive subjects.The overall blood pressure lowering for systolic blood pressure was -4.0 mmHg (95% CI -5.9 to -2.1 mmHg, P < 0.001) and for diastolic blood pressure -1.9 mmHg (95% CI -3.1 to -0.8 mmHg, P < 0.001). However, a positive effect was not reported in all the studies. The results suggest that rather small daily doses of the lactotripeptides in different functional food products may offer a valuable option as a non-pharmacological treatment of prehypertension or mild hypertension as part of life-style advice.

Plant sterols and casein-derived tripeptides attenuate blood pressure increase in spontaneously hypertensive rats.

In this study, we investigated the synergistic effects of plant sterols (PS) and casein-derived tripeptides on arterial tone and blood pressure in experimental hypertension. We hypothesized that PS and tripeptides could have positive, synergistic effects on the development of hypertension and endothelial dysfunction in young spontaneously hypertensive rats (SHR). Six-week-old male SHR were divided into 3 groups to receive milk products containing PS, or PS with tripeptides, or a control containing no active components for 8 weeks. Systolic blood pressure (SBP) was measured weekly, and vascular reactivity measurements with isolated mesenteric arteries were performed at the end of the study. Biochemical measurements for several parameters were performed by enzyme-linked immunosorbent assay using plasma samples. Levels of angiotensin-converting enzyme 1, cyclooxygenase-2, endothelial nitric oxide synthase, and P-selectin messenger RNA expressions were determined from aortic tissue by real-time polymerase chain reaction. The study showed that long-term treatment with PS + tripeptides attenuated the development of hypertension in SHR (SBP, 187 ± 5 mm Hg vs 169 ± 4 mm Hg in control group; P < .01). Plant sterols alone did not affect SBP significantly. Endothelial dysfunction was observed in all SHR; however, treatment with PS resulted in poorer endothelium-dependent and nitric oxide-mediated relaxation compared with other groups. Aortic cyclooxygenase-2 and P-selectin were significantly down-regulated in PS and PS + tripeptides groups when compared with the control group. The expression of endothelial nitric oxide synthase was significantly lower in PS than in PS + tripeptides group. In conclusion, long-term treatment with PS has a slight but not significant antihypertensive effect. Plant sterols do not provide any beneficial effects on endothelial function in hypertensive rats; however, treatment with both PS and tripeptides showed mild anti-inflammatory effects.

Flavonoids eupatorin and sinensetin present in Orthosiphon stamineus leaves inhibit inflammatory gene expression and STAT1 activation.

Cytokines and other inflammatory mediators, such as prostaglandin E2 (PGE2) and nitric oxide (NO) produced by cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), respectively, activate and drive inflammation and therefore serve as targets for anti-inflammatory drug development. Orthosiphon stamineus is an indigenous medicinal plant of Southeast Asia that has been traditionally used in the treatment of rheumatoid arthritis, gout, and other inflammatory disorders. The present study investigated the anti-inflammatory properties of Orthosiphon stamineus leaf chloroform extract (CE), its flavonoid-containing CE fraction 2 (CF2), and the flavonoids eupatorin, eupatorin-5-methyl ether (TMF), and sinensetin, identified from the CF2. It was found that CE (20 and 50 µg/mL) and CF2 (20 and 50 µg/mL) inhibited iNOS expression and NO production, as well as PGE2 production. Eupatorin and sinensetin inhibited iNOS and COX-2 expression and the production of NO (IC50 5.2 µM and 9.2 µM for eupatorin and sinensetin, respectively) and PGE2 (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively) in a dose-dependent manner. The extracts and the compounds also inhibited tumor necrosis factor α (TNF-α) production (IC50 5.0 µM and 2.7 µM for eupatorin and sinensetin, respectively). Eupatorin and sinensetin inhibited lipopolysaccharide (LPS)-induced activation of transcription factor signal transducers and activators of transcription 1α (STAT1α). Furthermore, eupatorin (50 mg/kg i. p.) and sinensetin (50 mg/kg i. p.) inhibited carrageenan-induced paw inflammation in mice. The results suggest that CE and CF2, as well as the known constituents of CF2, i.e., eupatorin and sinensetin, have meaningful anti-inflammatory properties which may be utilized in the development of novel anti-inflammatory treatments.

A spread containing bioactive milk peptides Ile-Pro-Pro and Val-Pro-Pro, and plant sterols has antihypertensive and cholesterol-lowering effects.

Lifestyle intervention is recommended as the primary treatment for mild hypertension and hypercholesterolemia. We studied the effects of a spread containing bioactive milk peptides IPP and VPP, as well as plant sterols, on cardiovascular risk factors in 104 hypertensive, hypercholesterolemic subjects in a randomised, placebo-controlled double-blind intervention. Middle-aged subjects consumed 20 g day⁻¹ of a spread containing 4.2 mg of IPP and VPP as well as 2 g of plant sterols for 10 weeks after a 2 week run-in period. Blood pressure was measured at home 3 times a week. Office blood pressure and 24 h ambulatory blood pressure measurements were performed at the end of the run-in and intervention periods. Blood samples were analysed for serum lipids, plasma glucose and inflammation markers. A significant decrease (-4.1 mmHg vs. -0.5 mmHg, p = 0.007) in systolic blood pressure was seen in the active group, compared to placebo at home measurements. Office blood pressure and 24 h nighttime or daytime ambulatory systolic or diastolic pressure did not differ between the groups. Total (-0.16 vs. 0.25 mmol l⁻¹, p = 0.005) and LDL cholesterol (-0.16 vs. 0.18 mmol l⁻¹, p = 0.006) decreased significantly in the active group compared to the placebo. No significant differences between groups were seen for plasma glucose or inflammation markers. The results thus suggest that milk peptides IPP and VPP and plant sterols, in a low-fat spread matrix, produce a clinically significant reduction in systolic blood pressure as well as serum total and LDL cholesterol without adverse effects. Functional foods that affect 2 major risk factors offer a safe and convenient way to reduce the risk of cardiovascular disease by supporting lifestyle intervention.

Downregulation of Bradykinin type 2 receptor expression in cardiac endothelial cells during senescence.

OBJECTIVES: Bradykinin type 2 receptor (BK-2R) knockout mice develop microvascular dysfunction and cardiac hypertrophy. In aged human cardiac microvascular endothelium, dysfunction develops before heart failure symptoms. Since endothelial aging is an independent risk factor for cardiovascular disease, we aimed to clarify the role of kinin receptors in age-related endothelial senescence. METHODS AND RESULTS: Using qRT-PCR, a downregulation of BK-2Rs during senescence of cultured human coronary artery endothelial cells (HCAECs) and rat cardiac microvascular endothelial cells (RCMECs) was observed. BK-2R downregulation was associated with a decreased cell proliferation rate, with a growth arrest phenotype and reduced angiogenic potential. By staining senescence-associated ß-galactosidase, RCMECs from old spontaneously hypertensive rats (SHRs) were found to be significantly more senescent than those derived from age-matched WKY rats, albeit their telomere lengths were similar. Despite downregulation of BK-2Rs and BK-1Rs, a novel family member GPR-100 was highly expressed in HCAECs throughout the culture period. CONCLUSIONS: Aging cardiac endothelial cells gradually lose their capacity to express BK-2Rs, and this loss appears to be parallel with a loss of the angiogenic potential of the aging cells. Since RCMECs from hypertensive rats showed premature senescence, hypertension may predispose to cardiac dysfunction by accelerating endothelial aging.

High blood pressure-lowering and vasoprotective effects of milk products in experimental hypertension.

Milk casein-derived angiotensin-converting enzyme (ACE)-inhibitory tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) have been shown to have antihypertensive effects in human subjects and to attenuate the development of hypertension in experimental models. The aim of the present study was to investigate the effect of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols on already established hypertension, endothelial dysfunction and aortic gene expression. Male spontaneously hypertensive rats (SHR) with baseline systolic blood pressure (SBP) of 195 mmHg were given either active milk (tripeptides and plant sterols), milk or water ad libitum for 6 weeks. SBP was measured weekly by the tail-cuff method. The endothelial function of mesenteric arteries was investigated at the end of the study. Aortas were collected for DNA microarray study (Affymetrix Rat Gene 1.0 ST Array). The main finding was that active milk decreased SBP by 16 mmHg compared with water (178 (SEM 3) v. 195 (SEM 3) mmHg; P < 0.001). Milk also had an antihypertensive effect. Active milk improved mesenteric artery endothelial dysfunction by NO-dependent and endothelium-derived hyperpolarising factor-dependent mechanisms. Treatment with active milk caused mild changes in aortic gene expression; twenty-seven genes were up-regulated and eighty-two down-regulated. Using the criteria for fold change (fc) < 0.833 or > 1.2 and P < 0.05, the most affected (down-regulated) signalling pathways were hedgehog, chemokine and leucocyte transendothelial migration pathways. ACE expression was also slightly decreased (fc 0.86; P = 0.047). In conclusion, long-term treatment with fermented milk enriched with tripeptides and plant sterols decreases SBP, improves endothelial dysfunction and affects signalling pathways related to inflammatory responses in SHR.