Leaf extracts of Campomanesia xanthocarpa positively regulates atherosclerotic-related protein expression

EDUARDO B.B. CUNHA NATÁLIA F. DA SILVA JEAN DE LIMA JULIA A. SERRATO CARLOS A.M. AITA ROBERTO H. HERAI About the authors

Abstract

Atherosclerosis is caused by a monocyte-mediated inflammatory process that, in turn, is stimulated by cytokines and adhesion molecules. Monocytes are then differentiated into macrophages, leading to the formation of arterial atherosclerotic plaques. Recently, guavirova leaf extracts from Campomanesia xanthocarpa (EG) have shown potential effects on the treatment of plaque formation by reducing cholesterol, LDL levels and serum oxidative stress. We evaluated the effect of EG on the viability of human monocytic and endothelial cell lines at three time points (24, 48 and 72 hours) and whether it can modulate the migration and in vitro expression of CD14, PECAM-1, ICAM-1, HLA-DR and CD105. Cell viability was affected only at higher concentrations and times. We observed decreased ICAM-1 expression in cells treated with 50 μg/ml EG and CD14 expression with IFN-γ and without IFN-γ. CD14 also decreased endothelial cell expression in the presence of IFN-γ and GE. We also found decreased expression of PECAM-1 when treated with EG and IFN-γ. In addition, EG-treated endothelial cells showed higher migration than the control group. Reduced expression of these markers and increased migration may lead to decreased cytokines, which may be contributing to decreased chronic inflammatory response during atherosclerosis and protecting endothelial integrity.

Key words
Campomanesia xanthocarpa; plant extract; atherosclerosis; in vitro cell treatment; protein expression

Introduction

Atherosclerosis is a chronic inflammatory disease, and it is characterized by the formation of atherosclerotic plaques. When ruptured and depending on where it is lodged, plaques can cause acute myocardial infarction (Zhu et al. 2017Zhu YN, Fan WJ, Zhang C, Guo F, Li W, Wang YF, Jiang ZS & Qu SL. 2017. “Role of Autophagy in Advanced Atherosclerosis (Review).” Mol Med Rep 15(5): 2903-2908.).

At cellular level, the atherogenesis is caused by the oxidized low-density lipoprotein (ox-LDL), and along with minimally modified low-density lipoprotein (mmLDL), they stimulate the appearance of leukocyte adhesion molecules, such as the vascular cell adhesion protein 1 (VCAM-1), the intercellular adhesion molecule 1 (ICAM-1) and the platelet endothelial cell adhesion molecule (PECAM-1), and together are responsible for attracting monocytes and lymphocytes within the intima (Eisenhardt et al. 2012Eisenhardt SU, Starke J, Thiele JR, Murphy A, Björn Stark G, Bassler N, Sviridov D, Winkler K & Peter K. 2012. “Pentameric CRP Attenuates Inflammatory Effects of MmLDL by Inhibiting MmLDL-Monocyte Interactions.” Atherosclerosis 224(2): 384-393.). In this process, when a monocyte penetrates the endothelium, there is an increase in expression of PECAM-1 and a decrease of the LV-cadherin expression, thus causing a weakening of the endothelial integrity (Hashimoto et al. 2011Hashimoto K ET AL. 2011. “Monocyte Trans-Endothelial Migration Augments Subsequent Transmigratory Activity with Increased PECAM-1 and Decreased VE-Cadherin at Endothelial Junctions.” Int J Cardiol 149(2): 232-239.). In addition, overexpression of PECAM-1 also directly induces ICAM-1 overexpression. This protein, through the leukocyte trans-endothelial migration pathway, plays an essential role in the process of adhesion and transmigration of monocytes into the intima (Privratsky & Newman 2014Privratsky JR & Newman pj. 2014. “PECAM-1: Regulator of Endothelial Junctional Integrity.” Cell Tissue Res 355(3): 607-619.), and thus causing the formation of atherosclerotic plaques inside blood vessels. During the cell migration and plaque formation process, the mmLDL lipoprotein stimulates the onset of leukocyte adhesion molecules, and also stimulates the expression of tool-like receptor 4 (TLR4) (Estruch et al. 2013Estruch m, Bancells c, Beloki l, Sanchez-Quesada JL, Ordóñez-Llanos J & Benitez S. 2013. “CD14 and TLR4 Mediate Cytokine Release Promoted by Electronegative LDL in Monocytes.” Atherosclerosis 229(2): 356-362.). This molecule precisely with CD14 (co-receptor required for the activation of TLR4) (Estruch et al. 2013Estruch m, Bancells c, Beloki l, Sanchez-Quesada JL, Ordóñez-Llanos J & Benitez S. 2013. “CD14 and TLR4 Mediate Cytokine Release Promoted by Electronegative LDL in Monocytes.” Atherosclerosis 229(2): 356-362.) is responsible for the production of pro-inflammatory cytokines (interleukin 1 beta (IL-1β), interleukin 6 (IL6), tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ)) and VCAM-1. The production of IFN-γ also modulates an increase in the HLA-DR expression by the cells (Hansson & Jonasson 2009Hansson GK & JONASSON L. 2009. “The Discovery of Cellular Immunity in the Atherosclerotic Plaque.” Arterioscler Thromb Vasc Biol 29: 1714-1717.). Another important molecule in the context of the atherosclerotic process is the CD105. It is a transmembrane protein, being one of the main glycoproteins expressed on the surface of the endothelial cells, playing a fundamental role in the cardiovascular system (López-Novoa & Bernabeu 2010López-Novoa JM & Bernabeu C. 2010. “The Physiological Role of Endoglin in the Cardiovascular System.” Am J Physiol Heart Circ Physiol 299(4): 959-974.). Genetic alterations in the gene that codes for this protein can cause hereditary hemorrhagic (telangiectasia), besides being involved in pre-eclampsia and in several types of cancer (Di Paolo et al. 2018DI Paolo V et al. 2018. “Evaluation of Endoglin (CD105) Expression in Pediatric Rhabdomyosarcoma.” BMC Cancer 18(1): 31., Metcalfe et al. 2018Metcalfe E, Arik D, Oge T, Etiz D, Yalcin OT, Kabukcuoglu S, Pasaoglu O & Ozalp SS. 2018. “CD105 (Endoglin) Expression as a Prognostic Marker of Angiogenesis in Squamous Cell Cervical Cancer Treated with Radical Radiotherapy.” J Cancer Res Ther 14(6): 1373-1378.). Recent evidence shows that CD105 also plays an important role in platelet adhesion within the endothelium by interacting with integrins (Rossi et al. 2018Rossi E et al. 2018. “Human Endoglin as a Potential New Partner Involved in Platelet–Endothelium Interactions.” Cell Mol Life Sci 75(7): 1269-1284.). Together, these molecules directly contribute to the increase of the inflammatory process that culminates in atherosclerosis (de Vries et al. 2014de Vries MA, Klop B, Eskes SA, van der Loos TL, Klessens-Godfroy FJ, Wiebolt J, Janssen HW, Westerman EM & Castro Cabezas M. 2014. “The Postprandial Situation as a Pro-Inflammatory Condition.” Clinica e Investigacion En Arteriosclerosis 26(4): 184-192., Rocha et al. 2016Rocha DM, Caldas AP, Oliveira LL, Bressan J & Hermsdorff HH. 2016. “Saturated Fatty Acids Trigger TLR4-Mediated Inflammatory Response.” Atherosclerosis 244: 211-215.).

It was previously demonstrated that PECAM-1 is directly involved in the development of atherosclerotic lesions (Hashimoto et al. 2007Hashimoto K, Kataoka N, Nakamura e, Tsujioka k & Kajiya f. 2007. “Oxidized LDL Specifically Promotes the Initiation of Monocyte Invasion during Transendothelial Migration with Upregulated PECAM-1 and Downregulated VE-Cadherin on Endothelial Junctions.” Atherosclerosis 194(2): e9-17.). In another work in the offspring of PECAM-1 deficient mice, it was also observed a decrease in atherosclerotic lesion and leukocyte migration and reduced expression of the proteins VCAM-1, ICAM-1, P-selectin, and decreased activation of NF-kB (Tzima et al. 2005Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H & Schwartz MA. 2005. “A Mechanosensory Complex That Mediates the Endothelial Cell Response to Fluid Shear Stress.” Nature 437(7057): 426-431.).

Recently, it was demonstrated that the use of plant leaves of the Myrtaceae group, such as guavirova (Campomanesia xanthocarpa Berg), have the potential to be used as a natural treatment for atherosclerosis (Dickel et al. 2007Dickel ML, Stela MR & Mara RR. 2007. “Plants Popularly Used for Loosing Weight Purposes in Porto Alegre, South Brazil.” J Ethnopharmacol 109(1): 60-71., Klafke et al. 2016aKlafke JZ, Porto FG, de Almeida AS, Parisi mm, Hirsch GE, Trevisan g & Viecili PRN. 2016a. “Biomarkers of Subclinical Atherosclerosis and Natural Products as Complementary Alternative Medicine.” Current Pharmaceutical Design 22(3): 372-382.). In hypercholesterolemic patients, the administration of guavirova leaves have been shown to reduce plasma oxidative stress (Viecili et al. 2014Viecili PRN et al. 2014. “Effects of Campomanesia Xanthocarpa on Inflammatory Processes, Oxidative Stress, Endothelial Dysfunction and Lipid Biomarkers in Hypercholesterolemic Individuals.” Atherosclerosis 234(1): 85-92., Klafke et al. 2010Klafke JZ et al. 2010. “Effects of Campomanesia Xanthocarpa on Biochemical, Hematological and Oxidative Stress Parameters in Hypercholesterolemic Patients.” J Ethnopharmacol 127(2): 299-305.) and decreased total cholesterol and LDL levels by inhibiting the concentration-dependent activity of the precursor cholesterol synthesis enzyme HMGR (Klafke et al. 2010Klafke JZ et al. 2010. “Effects of Campomanesia Xanthocarpa on Biochemical, Hematological and Oxidative Stress Parameters in Hypercholesterolemic Patients.” J Ethnopharmacol 127(2): 299-305., Islam et al. 2015Islam b, Sharma c, Adem a, Aburawi e & Ojha s. 2015. “Insight into the Mechanism of Polyphenols on the Activity of HMGR by Molecular Docking.” Drug Des Devel Ther 9: 4943-4951.). In animal models, guavirova leaves showed antithrombotic and fibrinolytic effects (Klafke et al. 2012Klafke JZ et al. 2012. “Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia Xanthocarpa.” Evidence-Based Complementary and Alternative Medicine 2012: 954748.), and also decreased levels of nitric oxide (Viecili et al. 2014Viecili PRN et al. 2014. “Effects of Campomanesia Xanthocarpa on Inflammatory Processes, Oxidative Stress, Endothelial Dysfunction and Lipid Biomarkers in Hypercholesterolemic Individuals.” Atherosclerosis 234(1): 85-92.) and proinflammatory cytokines such as IL-1, IL6, TNF-α and IFN-γ (Klafke et al. 2016bKlafke JZ et al. 2016b. “Study of Oxidative and Inflammatory Parameters in LDLr-KO Mice Treated with a Hypercholesterolemic Diet: Comparison between the Use of Campomanesia Xanthocarpa and Acetylsalicylic Acid.” Phytomedicine 23(11): 1227-1234.). In an in vitro study, it was also demonstrated that the guavirova leaves can act as an antiplatelet formation (Klafke et al. 2012Klafke JZ et al. 2012. “Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia Xanthocarpa.” Evidence-Based Complementary and Alternative Medicine 2012: 954748.).

Although the previous studies have correlated positive effects of C. xanthocarpa in patients with hypercholesterolemia, it is still missing studies describing, at cellular and molecular levels, the role of EG in regulating the molecules involved in hypercholesterolemia and plaque formation.

In this work, we investigated whether EG is able to modulate in vitro atherosclerotic-related cellular phenotypes. We show that leaf extracts of C. xanthocarpa significantly affect cellular viability under different concentrations and at different time intervals. We also show that EG significantly induces endothelial cell migration. At molecular level, we demonstrate that EG also modulates atherosclerotic-related proteins (such as CD14, ICAM-1, VCAM-1 and CD105, HLA-DR) that are involved in the inflammatory process correlated with the plaque formation in atherogenesis. Together, our findings suggest that EG can modulate distinct cellular phenotypes that protects cells against atherosclerosis (Figure 1).

Figure 1
Guavirova plant extract associated with the regulation of molecules involved in atherosclerosis. Schematic summary of the main mechanisms of atherosclerosis and molecules modulated by aqueous extract of C. xanthocarpa (EG) leaves. Continuous exposure to risk factors leads to endothelial dysfunction, making the endothelium more permeable for the entrance of LDL within intimal region, also inducing increased expression of selectin on the surface of monocytes and endothelial cells. This process culminates with increased monocyte migration (dependent on binding with PECAM-1, which is EG-modulated) to the intimal region, that differentiates into dendritic (DC) cells (highly specialized in the activation of TH1-secreting TH lymphocytes). IFN-γ: EG decreases the amount of IFN-γ) and / or macrophages that begin to phagocyte LDL. The interactions between dendritic cells, macrophages and smooth muscle cells (SMC) with LDL produce reactive oxygen species (ROS), which is decreased by EG. LDL molecules can be transformed into oxLDL (EG-decreased molecules) and mmLDL. These mmLDL are capable of binding to the CD14-TLR4 complex (EG decreases CD14), increasing the production of IL-6, IL-1β, IFN-γ and ICAM-1 (both negatively modulated molecules by EG), that together potentiates the inflammatory process. This process favors the transformation of macrophages into foam cells and leads to the proliferation of MCS from the middle layer to the intima of the vessel, which secretes extracellular matrix, forming the fibrous capsule. This imbalance within the intima leads to apoptosis of these cells, leading to the formation of the necrotic nucleus plaque, where DNA / RNA fragments are present and stimulates the activation of TH1 lymphocytes by dendritic cells, leading to increased IFN-γ production. Exposure of this plaque content triggers platelet activation and aggregation, leading to thrombus formation, with the participation of PECAM-1, by binding to platelets / endothelium and platelets / platelets.

MATERIALS AND METHODS

Extract preparation

The leaves of Campomanesia xanthocarpa were collected during the year 2010 in the city of Cruz Alta (RS, Brazil) and an exsicata number 1088 was deposited in the Herbarium of the University of Cruz Alta (RS, Brazil). The collected material was subjected to a cleaning process using sodium hypochlorite at 0.4%, immediately followed by washing in running potable water for 15 min. Then, the material was dried at 40-45 °C and ground to a fine powder (Klafke et al. 2010Klafke JZ et al. 2010. “Effects of Campomanesia Xanthocarpa on Biochemical, Hematological and Oxidative Stress Parameters in Hypercholesterolemic Patients.” J Ethnopharmacol 127(2): 299-305.).

For the tests, an aqueous extract of leaves of Campomanesia xanthocarpa (EG) was prepared. Initially, 50 g of dried leaves were added to 500 ml of distilled water, 37 °C under constant stirring for 30 min. Then the solution was filtered, frozen and lyophilized to determine the total dry matter content. The final powder was diluted in Milli-Q water, filtered on a 20 μm filter and adjusted to the desired concentration (25, 50, 100 and 500 μg/ml) to perform the tests (Klafke et al. 2012Klafke JZ et al. 2012. “Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia Xanthocarpa.” Evidence-Based Complementary and Alternative Medicine 2012: 954748.).

Cellular lines

Cells from the THP-1 human monocytic strain were cultured and expanded in RPMI medium containing 10% fetal bovine serum (FBS), 1% antibiotic (penicillin and streptomycin) at 37o C, in 5% CO2 atmosphere until used in experiments.

EA.hy926 is a permanent cell line derived by fusing human umbilical vein endothelial cells (HUVECs) with cell line A549 (Edgell et al. 1983Edgell CJ, McDonald & Graham JB. 1983. “Permanent Cell Line Expressing Human Factor VIII-Related Antigen Established by Hybridization.” Proc Natl Acad Sci USA 80(12): 3734-3737.) lineage were obtained from the Rio de Janeiro Cell Bank (BCRJ, RJ, Brazil) and ATCC (American Type Culture Collection) CRL 2922. Cells were cultured and expanded in Dulbecco’s modified eagle medium (DMEM) supplemented with 4000 mg/L glucose, 4 mM glutamine, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate and 10% FBS, 1% antibiotic at 37 °C, in 5% atmosphere of CO2 until used in the experiments.

Cellular viability

Viability measured using MTT (3-4,5-dimetiltiazol-2yl-2,5-difenil brometo de tetrazolina) assay (1x104 cells/well) in 96-well plates, incubated for 24, 48 and 72 hours at 37 °C with different concentrations of EG (25, 50, 100 and 500 μg/ml in 2% RPMI). Then 10 μl of the MTT solution (5 mg / ml) was added for a further 3 h of incubation and the reaction was stopped by the addition of dimethylsulfoxide (DMSO). The optical density (OD) reading will be performed by spectrophotometry at 570 nm.

Analysis of the expression of cell markers

For flow cytometry experiments, cells were initially maintained for at least 24 hours in culture medium containing 2% FBS. Next, the cells were distributed in 24-well plates at a concentration of approximately 1x106 cells / well. To induce activation of the cells incubated with 1000 U/ml IFN-γ for 30 minutes. The cells were then incubated with the EG at 50 μg/ml for 24 hours. At the end of the incubation the cells were washed 2x with phosphate-buffered saline (PBS) and transferred to citometry tubes.

Immunophenotyping of THP-1 cells was performed with the use of fluorochrome-conjugated monoclonal antibodies (FITC, PE or APC) directed against human CD14 (cluster of differentiation 14), ICAM-1, human HLA-DR molecules. Immunophenotyping of EA-hy926 cells was performed using fluorochromes-conjugated monoclonal antibodies (FITC, PE or APC) directed against human PECAM-1, CD105, ICAM-1 and HLA-DR molecules. As controls was used isotypic monoclonal antibodies conjugated to FITC, PE and APC markers. Cells will be incubated with the specific antibodies for 30 min at 40°C and then washed with PBS, centrifuged for 5 min at 400xg, resuspended and fixed in 500 μl PBS with 2% formaldehyde (Merck) for flow cytometry.

Cell analysis was performed on FACSCalibur flow cytometer (BD Biosciences, USA). The equipment was adjusted to the conditions of analysis of cell size and complexity, and the fluorescence adjustment was performed using the FITC, PE and APC isotype controls. Data acquisition and analysis will be performed using the software CellQuest Pro (BD Biosciences, USA).

Cellular migration

For cellular migration the scratch healing-wound method was used, which is based on the creation of a wound in the cellular monolayer. In this work, the cells were analyzed by observation in an inverted phase microscope (Liang et al. 2007Liang CC, Park AY & Guan JL. 2007. “In Vitro Scratch Assay: A Convenient and Inexpensive Method for Analysis of Cell Migration in Vitro.” Nat Protoc 2 (2): 329-333.).

For the migration analysis, EA-hy926 cells were cultured and expanded in DMEM supplemented with 4,000 mg/L glucose, 4 mM glutamine, 1 mM sodium pyruvate, 1500 mg/L sodium bicarbonate and 2% FBS at 37 °C, in an atmosphere of 5% CO2. These cells were plated at 5x105 cells, in triplicates for each experimental group. After checking the confluence of the monolayer, the wound was performed with the aid of a p200-type pipette tip, after which a complete medium wash (2% FBS, 1% antibiotic) was done to remove the debris. Then the plates received 2 ml of complete medium (control) and EG at the concentrations of 50 μg/ml and 100 μg/ml of the extract. In all, 8 trials were performed for each condition evaluated. Scratch wound area was measured using TScratch software version 1.0 (CSE Lab, Switzerland).

Statistical analysis

For the normality analysis the Shapiro-Wilk test was used, for the parametric data the student T test and for the nonparametric data of Wilcoxon. All tests were two-tailed and the p value was considered significant when less than 0.05. For simplicity of presentation, outputs the results on average of percentages. To analyze the data, the IBM SPSS 20 Statistics for Windows software was used.

Results

C. xanthocarpa alters cellular viability under different extract concentrations and time intervals

To assess whether EG interferes with cell viability, the MTT method was applied to two cell lines: monocytic THP-1 (Figure 2) and endothelial EA-hy926 (Figure 3).

Figure 2
Cell viability analysis of GE-treated THP-1 lineage cells. Cells were treated with different concentrations of C. xanthocarpa (25, 50, 100 and 500 μg/ml) and subsequently submitted to MTT cell viability analysis at 24, 48 and 72 hours. The treated groups were compared to the control group C (without extract), and the variations were expressed as percentage and mean (µ) and standard deviation (σ). **p-value = 0.012 compared group C48 (48 hours) with group T500 (µ = 81, σ = 0.02) (500 μg/ml), ***p-value < 0.000 compared group C72 (72 hours) with group T100 (µ = 72, σ = 0.09) (100 μg/ml) and C T500 (µ = 59, σ = 0.08) (500 μg/ml). Six triplicates were performed.
Figure 3
Cell viability analysis of EA-HY926 line cells treated with GE. Cells were treated with different concentrations of C. xanthocarpa (25, 50, 100 and 500 μg/ml) and subsequently submitted to MTT cell viability analysis at 24, 48 and 72 hours. The treated groups were compared to the control group C (without extract), and the variations were expressed as percentage and mean (µ) and standard deviation (σ). **p-value = 0.019 compared group C48 (48 hours) with group T500 ( µ = 77, σ = 0.04) (500 μg/ml), ***p-value < 0.000 compared group C72 (72 hours) with group T100 (µ = 72, σ = 0.09) (100 μg/ml) and C T500 (µ = 58, σ = 0.10) (500 μg/ml). Six triplicates were performed.
Figure 4
Analysis of cell migration of EA-HY926 strain treated with GE. Cells were treated with different concentrations of C. xanthocarpa (50 and 100 μg/ml), and then quantified the number of cells after 24 hours. The treated groups were compared to the control group c (without extract), and the variations were represented as mean of cells (µ) and standard deviation (σ). a: represents the moment of injury; b: 24 hours after injury in the control group (µ = 31.55, σ = 3.71); c: 24 hours after injury in the group treated with 50 μg/ml (µ = 43.3, σ = 3.58) of C. xanthocarpa. *p-value = 0.042 compared to group c. Eight assays were performed.

In monocytes, we did not detect significant alterations in cell viability in 24 hours of incubation with EG, at concentrations of 25, 50, 100 and 500 μg/ml of EG (Figure 2). Only after 48 hours of incubation of the cells with 500 μg/ml of EG, a significant decrease (p-value = 0.012) of 19% of cell viability was observed for monocytic cells (Figure 2). This same observation was made after 72 hours of cell incubation at concentrations of 100 and 500 μg/ml, in which a significant decrease of 28% (p-value = 0.000) and 41% (p-value = 0.000) were observed, respectively (Figure 2).

Additionally, we also found that at 48 hours of incubation, there was a significant decrease (p-value = 0.019) in cell viability at the concentration of 500 μg/ml for both investigated cell lines (Figures 2, 3). After 72 hours of incubation, it was also observed a significant decrease (p-value = 0.000) in cell viability at 100 and 500 μg/ml, the highest used concentrations of EG (Figures 2, 3).

C. xanthocarpa reduces expression of proteins involved with inflammatory process of atherosclerosis

To assess whether EG alters the expression of proteins involved with inflammatory process of atherosclerosis, we performed flow cytometry experiments over the same cells lines we used in the previous experiments: monocytic THP-1 and endothelial EA-hy926 (Figure 5).

Figure 5
Immunophenotypic analysis of CD14 expression in cell lines (THP-1) treated with EG. Results are expressed as mean fluorescence (µ) and standard error (). a: Isotype control; b: Expression of CD14 by THP-1 cells (µ = 56.51, = 6.73); c: Expression of CD14 by THP-1 cells in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 46.38, = 7.64), *p-value = 0.018 compared to group b; d: Isotypic control stimulated with IFNγ; e: Expression of CD14 by IFNγ-stimulated THP-1 cells (µ = 65.15, = 8.03); f: CD14 expression by IFNγ-stimulated THP-1 cells and in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 54.98, = 9.04 ), *p-value = 0.045 compared to group e. Six assays were performed, and 10,000 events were collected in each trial.

In monocytic cells, EG at the concentration of 50 μg/ml significantly decreased (p-value = 0.02771) the percentage expression of CD14 protein (mean = 46.38%) (Figure 5c) when compared to the control group (mean = 56.51%) (Figure 5b). In these same cells, the percentage of ICAM-1 expression did not show significant alteration (p-value > 0.05) in the treated cells (Figure 6c) when compared to control cells (Figure 6b).

Figure 6
Immunophenotypic analysis of ICAM-1 expression in cell lines treated with EG. Results are expressed as mean fluorescence (µ) and standard error (). a: Isotype control; b: Expression of ICAM-1 by THP-1 cells (µ = 30.26, = 12.95); c: Expression of ICAM-1 by THP-1 cells in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 18.95, = 12.82); d: Isotypic control stimulated with IFNγ; e: Expression of CD54 by IFNγ-stimulated THP-1 cells (µ = 66.90, = 8.01); f: Expression of ICAM-1 by IFNγ-stimulated THP-1 cells and in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 58.57, = 7.08), *p-value = 0.005 compared to group e. Six assays were performed, and 10,000 events were collected in each trial.

We then stimulated the monocytic cells by IFN-γ using the same concentration of 50 μg/ml of EG. We also observed significant decrease (p-value = 0.04484) in the percentage expression of CD14 protein (mean = 54.98%) (Figure 5e) when compared to the control group (mean = 65.13%) (Figure 5f). We also observed a significant decrease (p-value < 0.05) in the percentage expression of ICAM-1 protein (mean = 58.57%) (Figure 6f) when compared to relative controls (Figure 6e) (mean = 66.90%).

Next, we investigated similar experiments in endothelial cells incubated by EG at 50 μg/ml. The expression of the protein PECAM-1 did not show significant change (p-value > 0.05) in the treated cells (Figure 7c) when compared to control cells (Figure 7b). The percentage of the proteins CD14 (Figures 8b, 8c), HLA-DR and CD105 also did not show significant change in treated cells when compared to relative controls.

Figure 7
Immunophenotypic analysis of PECAM-1 expression in EG cell lines EA.HY926 treated with EG. Results are expressed as mean fluorescence (µ) and standard error (). a: Isotypic control; b: Expression of PECAM-1 by EA.HY926 cells (µ = 46.12, = 9.79); c: Expression of CD31 by EA.HY926 cells in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 40.32, = 8.34); d: Isotypic control stimulated with IFNγ; e: Expression of PECAM-1 by IFNγ-stimulated EA.HY926 cells (µ = 57.45, = 7.91); f: Expression of PECAM-1 by IFNγ stimulated cells EA.HY926 and in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 49.93, = 6.10), *p-value = 0.032 compared to group e. Six assays were performed, and 10,000 events were collected in each trial.

We then stimulated the endothelial cells by IFN-γ using the same concentration of 50 μg/ml of EG. We observed a significant decrease (p-value = 0.032) in the percentage of the protein PECAM-1 expression in treated cells (mean = 49.93%) (Figures 7e, 7f) when compared to relative controls (mean = 57.45%). Similarly, we also found a significant decrease (p-value = 0.036) in the percentage of expression of the protein CD14 in treated cells (mean = 11.92%) (Figure 8f) when compared to relative controls (Figure 8e) (mean = 20.84%). However, the percentage of the CD105 HLA-DR protein expression did not show significant change (p-value > 0.05).

Figure 8
Immunophenotypic analysis of CD14 expression in EG-treated cell lines EA.HY926. Results are expressed as mean fluorescence (µ) and standard error (). a: Isotype control; b: CD14 expression by EA.HY926 cells (µ = 19.63, = 7.97); c: Expression of CD14 by EA.HY926 cells in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 17.62, = 6.69); d: Isotypic control stimulated with IFNγ; e: CD14 expression by IFN-γ stimulated cells EA.HY926 (µ = 20.84, = 6.49); f: CD14 expression by the IFNγ stimulated cells EA.HY926 and in the presence of the extract of C. xanthocarpa at 50 μg/ml (µ = 11.92, = 3.58), *p-value = 0.036 compared to group e. Six assays were performed, and 10,000 events were collect

Migration of endothelial cells are induced by C. xanthocarpa

The use of EG at the concentration of 50 μg/ml with cultured endothelial cells induced a significant increase (p-value = 0.042) in the cell migration (mean = 43.30%) when compared to the control experiment without use of the extract (mean = 31.55%), corresponding to an average of 1.5 fold increase. However, when EG concentration was increased to 100 μg/ml, no change in endothelial cell migration was observed compared to controls (Figure 4), 7 independent trials were performed.

Discussion

In this work (Figure 1), we investigated how extracts of guavirova leaves (EG) can modulate cellular phenotypes that are correlated with the onset of the atherosclerotic process. We have demonstrated that EG significantly reduced ICAM-1 protein expression in monocytic cell lineages, and the reduction of PECAM-1 protein in endothelial cells when stimulated with IFN-γ. It has been previously reported that the decreased expression of ICAM-1 (Liu et al. 2017Liu CW et al. 2017. “Resveratrol Attenuates ICAM-1 Expression and Monocyte Adhesiveness to TNF-α-Treated Endothelial Cells: Evidence for an Anti-Inflammatory Cascade Mediated by the MIR-221/222/AMPK/P38/NF-7kappa;B Pathway.” Sci Rep 7: 44689.) and the unstable expression of PECAM-1 can increase the permeability of the endothelium as they act as key molecules for this phenotype (Liu et al. 2011Liu G, Vogel SM, Gao X, Javaid K, Hu G, Danilov SM, Malik AB & Minshall RD. 2011. “Src Phosphorylation of Endothelial Cell Surface Intercellular Adhesion Molecule-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation.” Arterioscler Thromb Vasc Biol (6): 1342-1350., Woodfin et al. 2007Woodfin A, Voisin MB & Nourshargh S. 2007. “PECAM-1: A Multi-Functional Molecule in Inflammation and Vascular Biology.” Arterioscler Thromb Vasc Biol 27(12): 2514-2523.). This observation was corroborated by a recent study that has revealed that PECAM-1 may have anti-inflammatory and proinflammatory roles in the cells (Malik et al. 2019Malik G, Wilting J, Hess CF, Ramadori G & Malik IA. 2019. “PECAM-1 Modulates Liver Damage Induced by Synergistic Effects of TNF-α and Irradiation.” J Cell Mol Med 23(5): 3336-3344.). In addition to this process, the increase in vascular permeability is dependent on the binding of monocytes to endothelial cells, causing activation of the ICAM-1-mediated Src Kinase signaling protein (Liu et al. 2012Liu G, Place AT, Chen Z, Brovkovych VM, Vogel SM, Muller WA, Skidgel RA, Malik AB & Minshall RD. 2012. “ICAM-1-Activated Src and ENOS Signaling Increase Endothelial Cell Surface PECAM-1 Adhesivity and Neutrophil Transmigration.” Blood 120(9): 1942-1952.).

The expression modulation of the PECAM-1 molecule may be one of the ways in which EG acts to promote the antithrombotic and fibrinolytic capacity that has previously been observed by in vivo experiments with mice (Klafke et al. 2012Klafke JZ et al. 2012. “Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia Xanthocarpa.” Evidence-Based Complementary and Alternative Medicine 2012: 954748.), and its antiplatelet property supported by in vitro experiments (Klafke et al. 2012Klafke JZ et al. 2012. “Antiplatelet, Antithrombotic, and Fibrinolytic Activities of Campomanesia Xanthocarpa.” Evidence-Based Complementary and Alternative Medicine 2012: 954748., Otero et al. 2017Otero JS et al. 2017. “Inhibitory Effect of Campomanesia Xanthocarpa in Platelet Aggregation: Comparison and Synergism with Acetylsalicylic Acid.” Thromb Res 154: 42-49.). By modulating these molecules, we hypothesize that EG can act reducing leukocyte transmigration to the intima layer of a blood vessel.

In addition, we also detected that EG significantly reduced CD14 expression in the two cell lines tested (endothelial and monocytic), especially after stimulation with IFN-γ. Chavez-Sanchez and colleagues demonstrated that increases the activation of CD14 by mmLDL increases the secretion of IL-1 and IL-6, and after blocking CD14 these same molecules reduced their secretion (Chavez-Sanchez et al. 2010Chavez-Sanchez L, Chavez-Rueda K, Legorreta-Haquet MV, Zenteno E, Ledesma-Soto Y, Montoya-Diaz E, Tesoro-Cruz E, Madrid-Miller A & Blanco-Favela F. 2010. “The Activation of CD14, TLR4, and TLR2 by MmLDL Induces IL-1beta, IL-6, and IL-10 Secretion in Human Monocytes and Macrophages.” Lipids Health Dis 9: 117.). Corroborated with these findings, Klafke et al. (2016b)Klafke JZ et al. 2016b. “Study of Oxidative and Inflammatory Parameters in LDLr-KO Mice Treated with a Hypercholesterolemic Diet: Comparison between the Use of Campomanesia Xanthocarpa and Acetylsalicylic Acid.” Phytomedicine 23(11): 1227-1234. observed that guavirova decreases serum levels of IL-1, IL-6 (Klafke et al. 2016bKlafke JZ et al. 2016b. “Study of Oxidative and Inflammatory Parameters in LDLr-KO Mice Treated with a Hypercholesterolemic Diet: Comparison between the Use of Campomanesia Xanthocarpa and Acetylsalicylic Acid.” Phytomedicine 23(11): 1227-1234.). Together, our data suggest that EG can also control the inflammatory process that is associated with the development of atherosclerosis and plaque formation.

Another important molecule in the context of the atherosclerotic process is CD105. In our experiments with endothelial cell lines, EG did not significantly interfere in the expression of CD105. Such transmembrane protein is one of the major glycoproteins expressed on the surface of endothelial cells and plays a key role by ensuring homeostasis of the cardiovascular system (López-Novoa & Bernabeu 2010López-Novoa JM & Bernabeu C. 2010. “The Physiological Role of Endoglin in the Cardiovascular System.” Am J Physiol Heart Circ Physiol 299(4): 959-974.). Recent evidences also show that CD105 plays an important role in platelet adhesion to the endothelium by interacting with integrins (Rossi et al. 2018Rossi E et al. 2018. “Human Endoglin as a Potential New Partner Involved in Platelet–Endothelium Interactions.” Cell Mol Life Sci 75(7): 1269-1284.). When this function is impaired, it may be related to abnormal bleeding and thrombosis (Rossi et al. 2018Rossi E et al. 2018. “Human Endoglin as a Potential New Partner Involved in Platelet–Endothelium Interactions.” Cell Mol Life Sci 75(7): 1269-1284.), and also could affect the vascular integrity, which is crucial to avoid the progress of atherosclerosis (Torres et al. 2015Torres N, Guevara-Cruz M, Velázquez-Villegas LA & Tovar AR. 2015. “Nutrition and Atherosclerosis.” Arch Med Res 46(5): 408-426.). In our data, the fact that EG did not significantly affects the expression of CD105 (which is required by the molecules of the atherosclerotic-related inflammation process (de Vries et al. 2014de Vries MA, Klop B, Eskes SA, van der Loos TL, Klessens-Godfroy FJ, Wiebolt J, Janssen HW, Westerman EM & Castro Cabezas M. 2014. “The Postprandial Situation as a Pro-Inflammatory Condition.” Clinica e Investigacion En Arteriosclerosis 26(4): 184-192., Rocha et al. 2016Rocha DM, Caldas AP, Oliveira LL, Bressan J & Hermsdorff HH. 2016. “Saturated Fatty Acids Trigger TLR4-Mediated Inflammatory Response.” Atherosclerosis 244: 211-215.) in EG treated cells versus controls, might suggest that the EG is acting as a protective treatment to atherosclerosis.

Due to the importance of vascular endothelial integrity in atherosclerosis, we also investigated whether EG altered cellular migration. We demonstrated that EG promoted increased endothelial cell migration, and at a higher concentration did not cause significant changes. As previously described, endothelium integrity is extremely important to avoid atherosclerosis (Torres et al. 2015Torres N, Guevara-Cruz M, Velázquez-Villegas LA & Tovar AR. 2015. “Nutrition and Atherosclerosis.” Arch Med Res 46(5): 408-426.), and migration of endothelial cells is one of the key processes for maintaining vascular integrity (Vitorino et al. 2011Vitorino P, Hammer M, Kim J & Meyer T. 2011. “A Steering Model of Endothelial Sheet Migration Recapitulates Monolayer Integrity and Directed Collective Migration.” Mol Cell Biol 31(2): 342-350.). As previously demonstrated, in the same cell line used by this study (EA.hy926), atorvastatin promoted increased endothelial migration at concentrations up to 0.1 μM, but inhibited cell migration at higher doses (1 μM) (Korybalska et al. 2012Korybalska k, Kawka E, Breborowicz A & Witowski J. 2012. “Atorvastatin Does Not Impair Endothelial Cell Wound Healing in an in Vitro Model of Vascular Injury.” J Physiol Pharmacol 18: 389-395.). At low doses, statin-derived drugs, such as atorvastatin, can inhibit cholesterol synthesis but do not impair the synthesis of farnesyl and geranylgeranyl pyrophosphates, key intermediates in cell growth pathways. However, at high doses they inhibit synthesis of prenyl radicals, in addition to cholesterol synthesis, resulting in inhibition of cell migration and proliferation (Korybalska et al. 2012Korybalska k, Kawka E, Breborowicz A & Witowski J. 2012. “Atorvastatin Does Not Impair Endothelial Cell Wound Healing in an in Vitro Model of Vascular Injury.” J Physiol Pharmacol 18: 389-395.).

CONCLUSION

Together, our findings suggest that EG can directly modulate atherosclerotic-related molecules, protecting the cells against markers that are altered during the early onset of the disease. The presented results are also supported by the literature, and although additional experiments are required, they suggest that EG could be used as an alternative for hypercholesterolemia treatment (Figure 1).

ACKNOWLEGMENTS

We thank Dr. Andrea Stinghen from Universidade Federal do Paraná (UFPR, PR, Brasil) by providing the EA.hy926 endothelial cell lines. The present work was carried out with the support of the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brazil) - Financing Code 001. Eduardo del Bosco Brunetti Cunha is recipient of a PhD fellowship from CAPES. Roberto H. Herai is supported by Fundação Araucária (grant #CP09/2016).

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Publication Dates

  • Publication in this collection
    20 Nov 2020
  • Date of issue
    2020

History

  • Received
    5 Dec 2019
  • Accepted
    13 Apr 2020
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