Influence of the Lactotripeptides Isoleucine–Proline–Proline and Valine–Proline–Proline on Systolic Blood Pressure in Japanese Subjects: A Systematic Review and Meta-Analysis of Randomized Controlled Trials

<div><p>Background</p><p>The lactotripeptides isoleucine–proline–proline (IPP) and valine–proline–proline (VPP) have been shown to decrease systolic blood pressure (SBP) in several populations, but the size of the effect varies among studies. We performed a meta-analysis including all published studies to evaluate the SBP-lowering effect of IPP/VPP in Japanese subjects more comprehensively.</p><p>Methods and Findings</p><p>Eligible randomized controlled trials were searched for within four bibliographic databases, including two Japanese ones. Eighteen studies (including a total of 1194 subjects) were included in the meta-analysis. A random effect model using the restricted maximum likelihood (REML) estimator was used for the analysis. The analysis showed that consumption of IPP/VPP induced a significant reduction in SBP as compared with placebo in Japanese subjects, with an estimated effect of -5.63 mm Hg (95% CI, -6.87 to -4.39, P<0.0001) and no evidence of publication bias. A significant heterogeneity between series was evident, which could be explained by a significant influence of the baseline blood pressure status of the subjects, the effect of IPP/VPP on SBP being stronger in hypertensive subjects (-8.35 mm Hg, P<0.0001) than in non-hypertensive subjects (-3.42mm Hg, P<0.0001). Furthermore, the effect of IPP/VPP on SBP remained significant when limiting the analysis to series that tested the usual doses of IPP/VPP consumed daily (below 5 mg/d), with estimated effects of -6.01 mm Hg in the overall population and -3.32 mm Hg in non-hypertensive subjects.</p><p>Conclusions</p><p>Results from this meta-analysis show that IPP/VPP lactotripeptides can significantly reduce office SBP in Japanese subjects with or without overt hypertension, and for doses that can potentially be consumed as an everyday supplement. This suggests that these peptides could play a role in controlling blood pressure in Japanese subjects. The systematic review protocol was published on the PROSPERO register (CRD42014014322).</p></div

Forest plot of treatment effects of isoleucine–proline–proline/valine–proline–proline in the meta-analysis of 33 series of findings of its effect on systolic blood pressure in Japanese subjects.

<p>FE: fixed effect. HT: hypertensive. NT: normotensive. PHT: pre-hypertensive. RE: random effect. SBP: systolic blood pressure.</p

Flow diagram of study selection.

<p>The list of the 27 articles selected for full text evaluation is available in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142235#pone.0142235.s006" target="_blank">S1 Table</a>, which also describes the outcome of the selection process for each article (including justification for exclusion). BP: blood pressure. IPP: isoleucine–proline–proline. SBP: systolic blood pressure. VPP: valine–proline–proline.</p

Effect of isoleucine–proline–proline and valine–proline–proline on systolic blood pressure at final endpoint.

<p>BP: blood pressure. IPP: isoleucine–proline–proline. HT: hypertensive. n: number of subjects. NA: not available. NT: normotensive. PHT: pre-hypertensive. SBP: systolic blood pressure. VPP: valine–proline–proline.</p><p>Effect of isoleucine–proline–proline and valine–proline–proline on systolic blood pressure at final endpoint.</p

Forest plot of treatment effects of isoleucine–proline–proline/valine–proline–proline on systolic blood pressure in the subgroup analysis according to the baseline blood pressure status of the subject.

<p>Data on SBP changes were available separately for NT, PHT and/or HT subjects for all studies except two, which were therefore excluded from this subgroup analysis (Mizushima et al 2004 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142235#pone.0142235.ref033" target="_blank">33</a>] and Yoshizawa et al 2010 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142235#pone.0142235.ref038" target="_blank">38</a>]). HT: hypertensive. NT: normotensive. PHT: pre-hypertensive. SBP: systolic blood pressure.</p

Funnel plot used to explore the potential for publication bias in the meta-analysis of 33 series for the effect of isoleucine–proline–proline/valine–proline–proline on systolic blood pressure in Japanese subjects.

<p>Series numbers are those indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142235#pone.0142235.t002" target="_blank">Table 2</a>. IPP: isoleucine–proline–proline. SBP: systolic blood pressure. VPP: valine–proline–proline.</p

Characteristics of the 18 studies included in the meta-analysis of randomized controlled trials of the effect of isoleucine–proline–proline and valine–proline–proline on systolic blood pressure in Japanese subjects.

<p>BP: blood pressure. D: double-blinded. IPP: isoleucine–proline–proline. HT: hypertensive. NT: normotensive. n: number of subjects. PHT: pre-hypertensive. S: single-blinded. VPP: valine–proline–proline. y: years.</p><p>Characteristics of the 18 studies included in the meta-analysis of randomized controlled trials of the effect of isoleucine–proline–proline and valine–proline–proline on systolic blood pressure in Japanese subjects.</p

Ankrd1/CARP upregulation in response to Angiotensin II stimulation.

<p><b><i>(A)</i></b> Representative images of cultured neonatal rat ventricular cardiomyocytes (NRVCs) stained with rhodamine phalloidin and 4′, 6-diamidino-2-phenylindole dihydrochloride (DAPI), and the cell size increased when treated with Angiotensin II (Ang II 1 µM) for 24h. <b><i>(B)</i></b> Hypertrophic marker atrial natriuretic peptide (ANP) detected by Real-time PCR using GAPDH as an internal control (*<i>P</i><0.05 vs. the control group). Expression changes of Ankrd1 in response to Ang II in cultured NRVCs were detected by PCR <b><i>(C)</i></b> or real-time PCR <b><i>(D)</i></b>. <b><i>(E)</i></b> Western blot analysis of time-dependent changes of CARP expression in NRVCs exposed to Ang II. *<i>P</i><0.05 vs. Ang II-untreated group, #<i>P</i><0.01 vs. Ang II 24 h group. Repeat times n = 3. Data are mean ± SEM.</p

Subcellular translocation of CARP in response to treatment with Ang II or Ad-Ankrd1.

<p><b><i>(A)</i></b> Expression changes of calpain 1 in response to Ang II stimulation in cultured neonatal rat ventricular cardiomyocytes (NRVCs) detected by real-time PCR, the insert represents amplification curve of calpain 1 and β-actin, *<i>P</i><0.05. <b><i>(B)</i></b> Confocal microscopic subcellular distribution of CARP (green) in response to Ang II stimulation in NRVCs. Immunostaining with F-actin (red) was used to confirm the cardiomyocytes, and DAPI (blue) was used to stain the nuclei. Bar = 10 µm. <b><i>(C)</i></b> Dose-dependent infective efficiency of Ad-Ankrd1 in cultured NRVCs detected by the green fluorescence of co-expressed EGFP. MOI: multiplicity of infection. <b><i>(D)</i></b> Western blot analysis of CARP protein levels in response to different dose of Ad-Ankrd1 infection (*<i>P</i><0.01 vs. Ad-EGFP group, n = 3). <b><i>(E)</i></b> Representative EGFP fluorescence microscopic photos of LV myocardium at 1 week after intramyocardial injection of vehicle (non-transfection), Ad-EGFP or Ad-Ankrd1 delivery. <b><i>(F)</i></b> Western blot analysis of CARP protein levels in response to intramyocardial injection of Ad-EGFP or Ad-Ankrd1 at 1 week after sham operation. <b><i>(G)</i></b> Subcellular location of forced Ankrd1 expression in Ad-Ankrd1 transfected cardiomyocytes. The green fluorescence was emitted from the report gene EGFP which was constructed in adenovirus carrying Ankrd1. Bar = 10 µm.</p

Olmesartan downregulates CARP and ameliorates cardiomyocyte hypertrophy.

<p><b><i>(A)</i></b> Treatment with RNH6270 (RNH 1 µM, active form of olmesartan) reduced Ang II (1 µM) -induced increased of CARP protein in NRVCs (*<i>P</i><0.05 vs. control, #<i>P</i><0.05 vs. Ang II. n = 3). <b><i>(B)</i></b> ANP mRNA expression in NRVCs exposed to Ang II (1 µM) stimulation in the presence/absence of RNH (1 µM). <b><i>(C)</i></b> Representative picture of whole heart from different group (scale bar = 2 mm). <b><i>(D)</i></b> HW/BW ratio was significantly lower in TAC mice treated with olmesartan medoxomil (OM) in comparison with untreated TAC mice, while addition of Ad-Ankrd1 partially blocked the antihypertrophic effect of OM. (*<i>P</i><0.01 vs. Sham, #<i>P</i><0.01 vs. TAC, &<i>P</i><0.05 vs. TAC + OM, n = 4–6 in each group). <b><i>(E)</i></b> Olmesartan treatment for 4 weeks reduced the myocardial expression of CARP in TAC mice (*<i>P</i><0.05 vs. Sham, #<i>P</i><0.05 vs. TAC). Data are mean ± SEM.</p