Chios mastic improves blood pressure haemodynamics in patients with arterial hypertension: Implications for regulation of proteostatic pathways

Chios mastic (CM) is the resin of Pistacia lentiscus var. chia, exclusively produced in the Greek Aegean island of Chios. Accumulating evidence suggests a cardioprotective role of this resin by way of its antioxidant and anti-inflammatory properties.1,2 These attributes may also convey favourable effects on prohypertensive mechanisms,3 but its impact on blood pressure (BP) haemodynamics has not been investigated in clinical or experimental settings. We therefore sought to assess the acute effects of CM on peripheral and aortic haemodynamics and associated changes in gene expression of molecules involved in pathways related to hypertension. The study population consisted of 27 consecutive eligible volunteers (13 hypertensive patients) who responded to a call for participation in the study. Exclusion criteria were history of cardiovascular disease, current treatment with statins, history of allergy to CM, current use of vitamins or antioxidant supplements, dieting at the time of the study, history of eating disorders, acute or chronic inflammatory disease and active cancer. All volunteers gave written informed consent. In a randomised double-blind case controlled crossover design, volunteers were assessed at two consecutive visits one week apart. Participants were randomly assigned to first visit oral administration of 2800 mg of CM (four tablets of 700 mg IASIS pharma Hellas S.A., Athens, Greece) or placebo. At each visit, haemodynamic parameters were assessed at three distinct time points (baseline, 2 and 3 hours after administration). Haemodynamic assessment included non-invasive measurement of aortic systolic blood pressure (SBP) and diastolic blood pressure and aortic augmentation index normalised for heart rate at 75 bpm by using radial artery tonometry (Sphygmocor System; Atcor Medical, Sydney, Australia) as previously described.4 We also studied expression levels of genes involved in proteome stability, proteasome and chaperones, cellular pro-oxidant responses, mitochondrial–metabolic pathways and inflammation (Figure 1) using isolated peripheral blood mononuclear cells (PBMCs). Gene expression analysis was performed in samples from four hypertensive patients with available samples of isolated PBMCs and the highest observed decrease in peripheral SBP 2 hours after CM administration, and was compared to three control normotensive subjects without decrease in peripheral SBP after CM administration. Univariable and multivariable analyses using linear mixed models with random effects were implemented to assess the effect of CM administration on longitudinal changes in parameters of interest. Statistical analysis was performed using the STATA package, version 11.1 (StataCorp, College Station, TX, USA). The level of statistical significance was set at P < 0.05 except for multiple comparisons (n = 2) for which it was set at P = 0.025 after Bonferroni adjustment. Heat map summarising changes in relative mRNA expression levels of the indicated genes in peripheral blood mononuclear cells of controls (normotensive subjects), hypertensive patients (Htn), and Htn versus control donors, treated with Chios mastic. *P<0.1, **P<0.05 (in other cases colours indicate a noted trend in gene expression pattern). Hypertensive patients presented acute decreases in peripheral and aortic SBP and in peripheral pulse pressure after CM administration (Table 1). After adjustment for age, gender and body mass index (BMI), these differences retained significance while as compared to placebo, a favourable effect of treatment with CM was observed (Table 1). In normotensive subjects, no significant changes in any haemodynamic parameter after CM administration were observed. A group effect between hypertension status and CM administration was observed in aortic SBP and pulse pressure (at 2 and 3 hours post-CM, Pfor interaction<0.05 for all) even after multivariable adjustment for age, gender and BMI. Finally, from gene expression analyses, CM administration in hypertensive patients tended to reduce the expression of proteasomal (PSMB6, PSMB7, RPN6), chaperone HSP27 and the pro-oxidant NOX2 genes (P < 0.1 for all, Figure 1); as compared to controls, in hypertensive patients PSMB6 and NOX2 expression was significantly reduced (P < 0.05, Figure 1) suggesting that CM exerts disease-specific regulatory effects on genes involved in proteostatic and pro-oxidant pathways. Longitudinal changes in haemodynamic and vascular parameters of interest in hypertensive patients and normotensive subjects across three distinct time points, before and after the administration of CM. T0: baseline; T1: 2 hours after the baseline; T2: 3 hours after the baseline. All variables are presented as mean ± standard deviation, except AI in which median (interquartile range) is provided. Observed statistical significance for the interaction term between treatment (CM vs. placebo) and time in corresponding linear mixed models (adjusted for age, gender and BMI) for longitudinal measurements of haemodynamic and vascular markers. Significant (P < 0.025) unadjusted difference from baseline (T0). Significant (P < 0.025) difference from baseline (T0) after adjustment for age, gender and BMI. The prespecified level of statistical significance for differences between T0 and T1, T2 was set at P = 0.025 after Bonferroni adjustment for multiple (n = 2) comparisons. CM: chios mastic; BMI: body mass index; pSBP: peripheral systolic blood pressure; pDBP: peripheral diastolic blood pressure; aSBP: aortic systolic blood pressure; aDBP: aortic diastolic blood pressure; pPP: peripheral pulse pressure; aPP: aortic pulse pressure; AI: augmentation index. Longitudinal changes in haemodynamic and vascular parameters of interest in hypertensive patients and normotensive subjects across three distinct time points, before and after the administration of CM. T0: baseline; T1: 2 hours after the baseline; T2: 3 hours after the baseline. All variables are presented as mean ± standard deviation, except AI in which median (interquartile range) is provided. Observed statistical significance for the interaction term between treatment (CM vs. placebo) and time in corresponding linear mixed models (adjusted for age, gender and BMI) for longitudinal measurements of haemodynamic and vascular markers. Significant (P < 0.025) unadjusted difference from baseline (T0). Significant (P < 0.025) difference from baseline (T0) after adjustment for age, gender and BMI. The prespecified level of statistical significance for differences between T0 and T1, T2 was set at P = 0.025 after Bonferroni adjustment for multiple (n = 2) comparisons. CM: chios mastic; BMI: body mass index; pSBP: peripheral systolic blood pressure; pDBP: peripheral diastolic blood pressure; aSBP: aortic systolic blood pressure; aDBP: aortic diastolic blood pressure; pPP: peripheral pulse pressure; aPP: aortic pulse pressure; AI: augmentation index. The novel finding of this study is that CM acutely improves both aortic and peripheral arterial BP in hypertensive patients in a placebo controlled, double-blind, randomised, crossover design. We did not identify any haemodynamic changes in normotensive subjects, which is suggestive of a disease-specific effect of CM in hypertension. This concept was further supported by our observation that gene expression pathways related to hypertension were affected differently in hypertensive patients as compared to normotensive subjects. These findings provide important clinical and therapeutic implications in hypertension, because CM is a natural resin free of serious side effects and without known interactions with major antihypertensive drug classes, which we found to be effective on BP lowering using an easy to prescribe stable tablet form with fixed concentration. Furthermore, considering that the magnitude of BP drop after CM administration matches effect sizes observed with first-line antihypertensive drugs,5 the observed improvement of BP haemodynamics by CM implies that this resin may be of clinical utility in hypertension management and the subsequent reduction of total cardiovascular risk,6 exerting cardiovascular protective effects. In support, we observed, similar to peripheral BP, changes in aortic BP after CM administration. As compared to peripheral, aortic pressures are more strongly associated with target organ damage in hypertension, are more sensitive to changes after drug or non-pharmaceutical interventions and confer superior prognostic value for adverse cardiovascular events in comparison to peripheral haemodynamics.7 Notably, baseline SBP in hypertensive patients was higher with CM than placebo. Although this difference may imply probable regression to the mean bias, implementation of a randomised crossover design, which is considered the main method to minimise this type of bias,8 suggests that the observed treatment effect is true. We further addressed this issue by using linear mixed models instead of simple linear regression analysis, which corrected and normalised for baseline differences between groups with appropriate interaction terms between treatment status and time. Although the mechanisms involved in BP lowering by CM have not been investigated, several of its beneficial attributes can be seen in our gene expression analyses. Interestingly, we observed that PBMC gene expression of NOX2, a potent superoxide-producing enzyme, and of several proteasome components decreased in hypertensive responders after CM administration. Given that accumulating evidence supports genetic control of BP early in life9 and overexpression of both NOX2 and proteasome components are interrelated and adversely affect processes in hypertension,10 our findings support the hypothesis of a hypotensive effect of CM by means of downregulation of NADPH oxidase and proteostatic mechanisms. In conclusion, this is the first study to demonstrate favourable effects of CM on peripheral and aortic BP haemodynamics in hypertensive patients. Our gene expression analysis pointed towards downregulation of the proteasome system and the NOX2 pro-oxidant pathway, which merits further investigation. Taken together, these findings should trigger further research of the role of CM in the management of hypertension. Chios mastic and placebo tablets were provided by IASIS pharma Hellas S.A., Athens, Greece. CK and GG drafted the manuscript. KL, EDP, VKP, IB, AM, AL and AK contributed to the acquisition of data for the work. KS, IB, IPT and KK contributed to the conception and/or design of the work. GG contributed to the analysis of data for the work. KS, CK, GG and IPT contributed to the interpretation of data for the work. KK, IPT and KS critically revised the manuscript. The first two authors contributed equally for 1st authorship and the last two authors contributed equally for senior authorship. All authors gave final approval and agree to be accountable for all aspects of the work ensuring integrity and accuracy. The author(s) declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article. The author(s) received no financial support for the research, authorship and/or publication of this article.