The effect of proatherogenic microbes on macrophage cholesterol homeostasis in apoE-deficient mice.

BACKGROUND: Pathogens such as Aggregatibacter actinomycetemcomitans (Aa) and Chlamydia pneumoniae (Cpn) associate with an increased risk for cardiovascular diseases by inducing inflammation. We hypothesized that the pathogens affect the vascular wall by disturbing cholesterol homeostasis and endothelial function. METHODS: Aa- and Cpn-infections were induced in apoE-deficient mice by intravenous and intranasal applications, respectively. Cholesterol efflux from mouse peritoneal macrophages to apo(lipoprotein)A-I was assessed. The efflux capacity of mouse sera as acceptors of cholesterol from RAW264.7-macrophages was determined. Additionally, endothelial function was studied by following the relaxation capacity of rat mesenteric arteries after incubation in the conditioned culture media of the peritoneal macrophages isolated from the mice. RESULTS: Infection increased serum phospholipid transfer protein (PLTP) and lipopolysaccharide (LPS) activity, as well as serum amyloid A (SAA) and TNF-α concentrations. Peritoneal macrophages of mice with Aa-infection showed increased cholesterol uptake and reduced cholesterol efflux. Sera of Cpn and Cpn + Aa-infected mice had reduced cholesterol efflux capacity from RAW264.7-macrophages. Conditioned macrophage medium from mice with chronic C. pneumoniae infection induced endothelial dysfunction. Additionally, concentrations of serum adhesion molecules, intercellular adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM) in Cpn-groups and E-selectin in Cpn + Aa-group, were elevated. The serum markers of endothelial function correlated positively with SAA. CONCLUSIONS: Aa- and Cpn-infections may generate proatherogenic changes in the vascular wall by affecting the macrophage cholesterol homeostasis and endothelial function.

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.

Ile-Pro-Pro and Val-Pro-Pro tripeptide-containing milk product has acute blood pressure lowering effects in mildly hypertensive subjects.

Casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) lower blood pressure (BP) in long-term clinical studies. Their acute effects on BP and vascular function, important for daily dosing scheme, were studied in a placebo-controlled double-blind crossover study using a single oral dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro as well as plant sterols. Twenty-five subjects with untreated mild hypertension received in random order 250 g of study product (25 mg peptides and 2 g plant sterols) or placebo. Ambulatory BP was monitored for 8 h post-dose and arterial stiffness measured by pulse wave analysis at 2, 4, and 8 h. Blood and urine samples were analyzed for markers of the renin-angiotensin system (RAS) and endothelial function. Baseline adjusted treatment effect for systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial BP was -2.1 mmHg (95% CI: -4.1 to -0.1, p = 0.045), -1.6 mmHg (95% CI: -3.1 to -0.1, p = 0.03), and -1,9 mmHg (95% CI: -3-3 to -0.4, p = 0.0093), respectively, in favor of the active treatment for 8 h post- dose. No significant differences between the treatments were seen in brachial or aortic augmentation index, pulse wave velocity, or markers of RAS. Urinary excretion of cGMP, the second messenger of endothelial nitric oxide, was higher in the active group vs. placebo (p = 0.01). The results indicate that a single dose of a fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro and plant sterols acutely lowers brachial SBP and DBP in mildly hypertensive subjects.

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.

Casein-derived tripeptide Ile-Pro-Pro improves angiotensin-(1-7)- and bradykinin-induced rat mesenteric artery relaxation.

AIMS: Milk casein-derived bioactive tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro) lower blood pressure in animal models of hypertension and humans. In some studies, their angiotensin-converting enzyme (ACE)-inhibitory effect has been demonstrated. Besides classical ACE-angiotensin II-AT(1)-receptor pathway (ACE-Ang II- AT(1)), the significance of ACE2-angiotensin-(1-7)-Mas-receptor (ACE2-Ang-(1-7)-Mas) axis in the blood pressure regulation has now been acknowledged. The present study was aimed to further evaluate the renin-angiotensin system (RAS)-related vascular effects of Ile-Pro-Pro in vitro using rat mesenteric arteries. MAIN METHODS: Superior mesenteric arteries of spontaneously hypertensive rat (SHR) were isolated, cut into rings and mounted in standard organ bath chambers. Endothelium-intact arterial rings were incubated in Krebs solution either with Ile-Pro-Pro, proline-proline (Pro-Pro), isoleucine (Ile), proline (Pro) or captopril for 6h at +37°C and vascular reactivity was measured. KEY FINDINGS: In the presence of AT(1)-antagonist valsartan, Ang II induced vasodilatation, which was more pronounced in the arteries incubated with Ile-Pro-Pro (P<0.05) compared to the other compounds. Ang-(1-7)-induced vasodilatation was augmented by Ile-Pro-Pro or Pro (P<0.001 vs. control). Mas-receptor antagonist A-779 did not alter the responses. Ile-Pro-Pro and Pro augmented also bradykinin-induced relaxations (P<0.001 vs. control). Control arteries and arteries incubated with captopril showed only slight relaxations at higher bradykinin concentrations. SIGNIFICANCE: Casein-derived tripeptide Ile-Pro-Pro and amino acid Pro enhance the vasodilatory effect of Ang-(1-7) and bradykinin. The role of ACE2-Ang-(1-7)-Mas axis in the modulation of vascular tone by these compounds seems probable.