Original Article Monocyte Low-Density Lipoprotein Receptor-Related Protein 1 (LRP1) Expression Correlates with cIMT in Mexican Hypertensive Patients Ricardo Gamboa,1 María José Jaramillo-Estrella,1 María del Rocio Martínez-Alvarado,1 Maria Elena Soto,1 Yazmin Estela Torres-Paz,1 David de Gonzalo-Calvo,2 Leonardo Del Valle-Mondragón,1 Rebeca López-Marure,1 Vicenta C. Llorente-Cortés,2 Claudia Huesca-Gómez1 Instituto Nacional de Cardiologia Ignacio Chavez,1 Ciudad de México - Mexico Hospital de Sant Pau - Lipids and Cardiovascular Pathology Group,2 Barcelona, Catalunya – Spain Abstract Background: Arterial hypertension (HTA) represents a major risk factor for cardiovascular morbidity and mortality. It is not yet known which specific molecular mechanisms are associated with the development of essential hypertension. Objective: In this study, we analyzed the association between LRP1 monocyte mRNA expression, LRP1 protein expression, and carotid intima media thickness (cIMT) of patients with essential hypertension. Methods: The LRP1 monocyte mRNA expression and protein levels and cIMT were quantified in 200 Mexican subjects, 91 normotensive (NT) and 109 hypertensive (HT). Statistical significance was defined as p < 0.05. Results: HT patients group had highly significant greater cIMT as compared to NT patients (p=0.002) and this correlated with an increase in the expression of LRP1 mRNA expression (6.54 vs. 2.87) (p = 0.002) and LRP1 protein expression (17.83 vs. 6.25), respectively (p = 0.001). These differences were maintained even when we divided our study groups, taking into account only those who presented dyslipidemia in both, mRNA (p = 0.041) and proteins expression (p < 0.001). It was also found that Ang II mediated LRP1 induction on monocytes in a dose and time dependent manner with significant difference in NT vs. HT (0.195 ± 0.09 vs. 0.226 ± 0.12, p = 0.046). Conclusion: An increase in cIMT was found in subjects with hypertension, associated with higher mRNA and LRP1 protein expressions in monocytes, irrespective of the presence of dyslipidemias in HT patients. These results suggest that LRP1 upregulation in monocytes from Mexican hypertensive patients could be involved in the increased cIMT. (Arq Bras Cardiol. 2021; 116(1):56-65) Keywords: Monocytes; LRP1; mRNA; Hypertension/epidemiology; Mexico; Carotid Intima Media Thickness. Introduction classic LDL receptors.5 These receptors are not regulated by cholesterol and allow a massive uptake of modified LDL, Arterial hypertension (HTA) is a chronic and multifactorial causing intracellular lipid accumulation. disease that constitutes a serious public health problem.1 Hypertension rarely causes symptoms in the early stages; it is The low-density lipoprotein receptor–related protein a silent killer, causing accelerated atherosclerosis, damage to 1 (LRP1), which is a transmembrane multiligand receptor6 belonging to the LDLR family, is expressed in different cells such major organs, disability, and death from cardiovascular diseases.2 as neurons, fibroblasts, tumoral cells, hepatocytes, vascular Atherosclerotic lesions include altered endothelial smooth muscle cells, and monocytes and macrophages.7, 8 It cells, circulating monocytes, vascular smooth muscle cells is known to participate in the uptake of modified LDL9 and is (VSMC) migration, and foam cell development.3 The altered over expressed in atherosclerotic plaques in both animal and endothelium allows the entrance and retention of low density human models.10, 11 lipoprotein (LDL) into the intima layer.4 Once LDL is trapped Furthermore, LRP1 gene expression is increased in in the arterial intima, it undergoes changes, such as oxidation mononuclear cells from patients with coronary occlusion.12,13 and aggregation, that facilitate its uptake by intimal monocytes- In monocytes and macrophages, LRP1 contributes to the macrophages and VSMC through their recognition by non- uptake of modified aggregated LDL.14,15 Nevertheless, the effects of hypertension on LRP1 expression in humans are not exactly known. Therefore, obtaining circulating monocytes Mailing Address: Claudia Huesca-Gómez • made it possible to study the mechanisms of their participation Instituto Nacional de Cardiologia Ignacio Chavez – Physiology - “. Juan in the formation of atherosclerotic plaque.16 In another way, Badiano No 1, Col. Sección XVI Mexico 14080 – Mexico the cIMT is considered an excellent non-invasive marker E-mail: claudia.huesca@cardiologia.org.mx Manuscript received August 09, 2019, revised manuscript February 07, for cardiovascular disease; it has been associated with 2020, accepted March 16, 2020 atherosclerosis and cardiovascular risk factors17,18 and the prevalence of cardiovascular disease, proving it is useful in the DOI: https://doi.org/10.36660/abc.20190535 diagnosis of atherosclerosis.19-21 Accordingly, the purpose of this 56 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article paper was to study the LRP1 mRNA levels and protein expression Biochemical Determinations in monocytes from patients with essential arterial hypertension Blood samples were collected after a 12-hour fasting and their correlation with carotid intima media thickness. period; glucose, total cholesterol (TC), triglycerides (TG), and high density lipoprotein cholesterol (HDL-C) were measured in fresh samples (fasting plasma) using standardized Methods enzymatic procedures in a Hitachi 902 analyzer (Hitachi Ltd,Tokio, Japan); low density lipoprotein cholesterol Study Population and Design (LDL-C) was estimated using the DeLong et al. formula.23 A total of 200 unrelated Mexican subjects (109 patients All assays were under an external quality control scheme diagnosed with essential hypertension and 91 normotensive (Lipid Standardization Program, Center for Disease Control subjects) were recruited at the Instituto Nacional de in Atlanta, GA, USA). Cardiología “Ignacio Chávez”. The inclusion criteria for both Ang II serum concentrations were evaluated by capillary groups were: to be Mexican by birth with at least 3 previous zone electrophoresis as previously described.24 Total high- generations, be older than 40 years, and to agree to participate sensitivity C-reactive protein (hs-CRP) levels were determined in the study by signing an informed consent. Controls were by immunonephelometry on a BN Pro Spec nephelometer apparently healthy, asymptomatic individuals, without a family (Dade Behring Marburg GmbH, Germany). Inter-assay history of hypertension or premature cardiovascular disease, coefficient of variation (CV) values were <6 % for all of these with blood pressure ≤120/80 mmHg. For the hypertensive assays. Non-HDL-cholesterol (non HDL-C) was calculated by group, subjects had blood pressure ≥140/90 mmHg or had subtracting HDL-C from total cholesterol. The dyslipidemia been previously diagnosed with essential hypertension. The value was defined according to conventional cardiovascular exclusion criterion was suffering from a chronic degenerative risk factors: (TC) ≥200 mg/dL and/or HDL-C ≤ 40mg/dL and/ disease. All participants answered standardized and validated or LDL-C ≥130 mg/dL and/or TG ≥150mg/dL. questionnaires to obtain information on their family and medical history, alcohol and tobacco consumption, eating Separation of Peripheral Blood Monocyte habits, and physical activity. Collected whole-blood in tubes with EDTA was diluted The ethics committee of the Instituto Nacional de 1:1 with PBS 1×–1% heparin; Histopaque 1077 (10771, Cardiología “Ignacio Chavez” approved the project; the Sigma-Aldrich) was subsequently added. Peripheral blood patients gave written informed consent prior to the study. All mononuclear cells (PBMCs) were obtained from the central procedures were in agreement with the Helsinki Declaration white band of the gradient after centrifugation. Next, of 1975, as revised in 2013. monocytes were obtained by directly enriching for CD14+ cells by the magnetic sorting system (MACS; Miltenyi Biotec, Anthropometric Measurement Bergisch-Gladbach, Germany). 1 mL aliquot of TripureTM The selected subjects underwent anthropometric reagent (Roche Molecular Biochemicals) was then added for measurements to determine their height in meters (m) and collecting the monocytes. Cells were stored at −80 °C. weight in kilograms (kg). Blood pressure was measured using a mercury sphygmomanometer, following the Cell Line THP-l Culture recommendations of the VII Joint National Committee on Human monocytic leukemia cells were maintained in Prevention, Detection, Evaluation, and Treatment of High a suspension culture of RPMI-1640 medium (Gibco-BRL) Blood Pressure (JNC VII). containing 2 mM glutamine, 25 mM HEPES, 1.5 g/L sodium bicarbonate, 50 U/mL penicillin, and 50 μg/mL streptomycin Carotid Intima Media Thickness (Sigma), supplemented with 10% fetal bovine serum (FBS), at A specialist in sonography resolution assessed the carotid 37°C, in 5% CO2. Arrested THP-1 cells were pre-incubated with intima media thickness (cIMT); all measurements were Ang II (1 µmol/L) for increasing periods of time to analyze the performed with A Sonosite Micromax ultrasound coupled to effect of Ang II on LRP1 expression in the monocytes. The dose a 13 MHz multifrequency high-resolution linear transducer. of angiotensin II was selected on the basis of previous studies in Measurements were made on the common carotid after our group and provides a plasma concentration of angiotensin II similar to that reported in patients with hypertension.25 the examination of a 10-mm longitudinal section at a 2-cm distance from the bifurcation, the anterior or proximal wall, and the posterior or distal wall were measured on the lateral, RNA Extraction and cDNA Synthesis anterior, and posterior projections, followed by an axis Total RNA was extracted using monocyte TripureTM Isolation perpendicular to the artery to discriminate two lines: one Reagent (Roche Molecular Diagnostics, Indianapolis, USA), for the intima-blood interface and the other to the media- according to the manufacturer’s instructions. RNA yield and adventitious interface. Five measurements were obtained quality were assessed by 1% agarose gel electrophoresis; of the right carotid and five of the left carotid, using average RNA was stored at -80°C until analysis. Reverse transcription (average cIMT) and maximum values (maximum cIMT), reaction was performed using 1 µg of total RNA for cDNA automatically calculated by the software. cIMT was considered synthesis according to High Capacity cDNA Reverse abnormal with values greater than or equal to 75 percentile Transcription kit (Applied Biosystems Foster City, CA, USA). by age and sex.22 The cDNA was stored at -80°C. Arq Bras Cardiol. 2021; 116(1):56-65 57 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article Gene Expression Assays of approximately 0.08 in the cases and 0.02 in the controls LRP1 gene expression (Hs00233899_m1) and HPRT with a ∆ = 0.06, with a statistical power of 95%, p <0.05. (Hs99999909_m1) (endogenous gene) were performed According to the following formula our value of n was =79 via semi-quantitative real-time reverse-transcriptase polymerase chain reaction (RT-PCR), using a commercial kit. The “TaqMan Gene Expression” was performed using 1 µl reverse transcription products mixed with 10 µl of TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 1 µl 20x assays and 8 µl nuclease-free water. After gentle mixing, the mixture was transferred to a real-time po= Probability that LRP1 expression occurs in cases PCR microplate, using 7300 Real Time PCR System (Applied q0= Probability that LRP1 expression doesn’t occur in cases Biosystems) equipment. pi= Probability that LRP1 expression occurs in controls The used conditions were: 50°C for 2 min and 10 min at qi= Probability that LRP1 expression doesn’t occur in 95°C, followed by 40 cycles at 95°C for 15s, and 60°C for controls 1min. Expression levels were measured in duplicate and the threshold cycle [Ct] values were determined and normalized using the endogenous gene expression (HPRT). 1.96= value <0.05 1.28= power (0.84) Western Blot analysis Total protein was isolated from monocytes using TriPureTM Isolation Reagent (Roche Molecular Diagnostics), according to the manufacturer’s instructions. The protein was quantified using Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA, USA). Equivalent amounts of total protein (25 µg) were loaded onto 10% (v/v) SDS-polyacrylamide gels under reducing conditions. The samples were electrotransferred Results to nitrocellulose membranes, which were saturated at room temperature for 1 h in TTBS (20 mM Tris–HCl, pH 7.5, 500 Characteristics of the Study Population mM NaCl, 0.01% Tween 20 and 5% non-fat milk). Western A population of 200 Mexican subjects was studied, of which blot analyses were performed using specific monoclonal 91 were normotensive (NT) and 109 were hypertensive (HT) antibodies against human LRP1 (85kDa -chain, clone 8B8 subjects. The biochemical and anthropometric characteristics RDI 61067, dilution 1:40) and the corresponding secondary of the studied population are shown in Table 1. Out of the antibodies (1:10,000 dilution; Dako; Glostrup, Denmark). total population, 62.5% was female and 37.5% was male. The QuantityOne software (Bio-Rad, Hercules, CA, USA) Age, body mass index (BMI), cIMT, HDL-C, C-reactive protein, was used to quantify the bands present in the membranes Ang II, and LDL-C/HDL-C, TC/HDL-C, TG/HDL-C indexes via densitometry, and they were detected using ECL Prime were statistically different between groups. These parameters Western Blotting Detection Reagent (Amersham). The were higher in the hypertensive group as compared to the expression levels were measured in duplicate and normalized normotensive group, except for HDL-C levels, which were by comparing them with the concentration of a loading lower in the hypertensive group. Obesity prevalence was protein control. The results were expressed as arbitrary units 19.8% in normotensive and 44.1% in hypertensive subjects. of intensity. No significant differences were found when the comparison was made between both genders of the same parameters. Statistical Analysis Also, we compared our groups according to dislipidemia levels according to ATP III; however, significant differences were only Data were analyzed using the SPSS v19 software (SPSS found in HDL-C (≤40 mg/dL), (NT= 16.5% vs HT= 32.7%, Inc. Chicago USA). The results were expressed as the mean p=0.001) and triglycerides (≥150mg/dL) (NT= 42.7% vs HT= ± standard deviation (SD) in the continuous variables and 57.3%, p=0.001) (data no shown). percentages for categorical variables. The Shapiro-Wilk test used to assess normality. The comparison between groups was performed using the unpaired Student’s t-test for continuous Correlation Between Hypertension and Expression of LRP1 variables and chi square test for categorical variables. The in Monocytes correlation analysis was done according to the Pearson With the purpose of ascertaining the levels of mRNA and method. Multiple logistic regressions were used to explore protein expression, an LRP1 analysis was performed for both the associations between cIMT and LRP1 expression. Data groups (Figure 1). Significant differences were found between is presented as odds ratios (OR) with a confidence interval NT versus HT groups in mRNA expression (P=0.002) and of 95%. A p <0.05 value was considered as statistically for protein expression (p=0.001). When men and women significant. The sample size was calculated taking the reference subjects were compared, the only signficiant difference of Schulz 2002,13 according to proportions of independent found was in LRP1 mRNA in hypertensive subjects and samples, taking into account an incidence of the LRP1 gene there was an overexpression in women as compared to men 58 Arq Bras Cardiol. 2021; 116(1):56-65 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article Table 1 – Anthropometric, clinical, and biochemical characteristics of study patients Normotensive Hypertensive Parameters p (n=91) (n=109) Age (years) 46.0±11.35 50.36±11.57 0.007 Gender (W/M) (%) 61.5/37.5 64/36 0.313 Weight (kg) 71.44±14.30 75.21±12.71 0.056 Height (cm) 161.99±9.81 159.39±9.06 0.057 BMI (Kg/m2) 26.92±4.06 29.36±3.77 <0.001 SBP (mmHg) 110.23±9.07 142.78±10.82 <0.001 DBP (mmHg) 69.90±75.85 91.94±7.72 <0.001 cIMT mean (mm) 0.587±0.16 0.729±0.16 0.002 cIMT max (mm) 0.606±0.18 0.787±0.16 0.008 Total cholesterol (mg/dL) 197.32±40.41 198.91±37.42 0.772 Triglycerides (mg/dL) 166.56±94.45 192.95±98.43 0.001 Log TG 2.16±0.22 2.23±0.19 0.010 HDL-C (mg/dlL) 52.51±13.25 46.66±13.59 0.002 LDL-C (mg/dL) 117.02±33.20 122.23±31.74 0.258 LDL/HDL 2.36±0.84 2.76±0.91 0.001 Non HDL-C 144.80±41.28 152.67±36.54 0.154 CT/HDL 3.96±1.20 4.50±1.26 0.003 TG/HDL 3.62±2.73 4.65±3.21 0.017 Glucose (mg/dL) 89.36±7.78 89.18±8.91 0.877 C Reactive Protein (mg/dL) 2.37±2.06 3.87±2.85 0.011 Smoking 1.83±0.38 1.67±0.51 0.491 The values are expressed as mean ± SD or percentages for categorical values. Unpaired Student T Test and chi-square test for categorical values were used. BMI: body mass index; SBP: Systolic blood pressure; DBP: Diastolic blood pressure; cIMT mean: carotid intima media thickness mean; IMT max: Intima-media thickness maximal; HDL-C: High density lipoprotein cholesterol; LDL-C: Low density lipoprotein cholesterol. (p=0.044). Moreover, an increase in the LRP1 mRNA and Association between monocytes LRP1 expression and protein expression was found in hypertensive dyslipidemic carotid intima/media thickness from patients with subjects as compared to normotensive dyslipidemic subjects hypertension. (data no shown). To know if there was a relationship between the thickness Conversely, to examine if others factors like cIMT and of the cIMT and LRP1 mRNA expression and/or LRP1 protein Ang II variable were analyzed to determine whether they expression, multiple logistic regressions adjusted by lipid could participate in the blood pressure values (Table 2). A profile, age, and gender were conducted (Table 3). A significant significant difference was found between NT versus HT for difference was found between cIMT and the LRP1 mRNA cIMT (p=0.002) and Ang II (p=0.046), respectively. However, expression levels (p=0.047) and LRP1 protein levels (p=0.039) when subjects were broken down by gender, no differences in hypertensive patients. were found in either of the two parameters. Therefore, an adjusted logistic regression for lipids was performed to analyze whether dyslipidemia could influence the association between LRP1 and cIMT in hypertensive Angiotensin II Effect on Monocyte LRP1 Expression Levels patients (Table 3, Models 1-4). An association between To study the effect of Ang II mediated LRP1 induction on cIMT and LRP1 mRNA expression with the entire set of lipid monocytes, the THP1 monocyte cell line was incubated with parameters was found: Model 1 (p=0.046), the association Ang II for 4h and 8h, with concentrations of 1 and 10 µM. In was maintained after adjusting each of the lipid parameters, the THP1 monocyte cell line, Ang II increased LPR1 mRNA Model 2 adjusted by total cholesterol (p= 0.053), Model 3 expression in a dose and time dependent manner, being more adjusted by triglycerides (p=0.049), Model 4 adjusted by evident at 8 hours of incubation (Figure 2). HDL-C (p=0.038), and Model 5 adjusted by LDL-C (p=0.052). Arq Bras Cardiol. 2021; 116(1):56-65 59 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article NT HT NT Women NT Men HTA Women HTA Men Normalized Arbitrary LRPI Protein expression to HPRT LRP1 mRNA expression units 8.0 25.0 6.0 20.0 4.0 15.0 2.0 5.0 0.0 0.0 NT HT NT Women NT Men HT Women HTA Men NT HT NT Women NT Men HT Women HTA Men * P= 0.002 ** P= 0.044 * P= 0.001 Figure 1 – Quantification of LRP1 expression in total subjects and broken down by genders. (A) Comparison of LRP1 expression in monocytes from normotensive and hypertensive subjects. Real-time PCR analysis of LRP1 mRNA expression. Data were processed with a specially designed software, based on the Ct value of each sample, and normalized to HPRT1 (B) Western blot analysis showing LRP1 protein expression in monocytes. Table 2 – Values of cIMT and Ang II broken down by gender NT NT HTA HTA NT HTA p p p Women Men Women Men IMT (mm) 0.568 ± 0.16 0.715 ± 0.16 0.002 0.553 ± 0.149 0.583 ± 0.178 0.303 0.692 ± 0.14 0.719 ± 0,19 0.643 Ang II (pmol/ml) 0.195± 0.09 0.226 ± 0.12 0.046 0.200 ±0.090 0.186 ± 0.090 0.468 0.220 ± 0.11 0.238 ± 0,14 0.482 NT: Normotensive; HTA: Hypertensive; IMT: mean intima-media thickness, Ang II: Angiotensin II. Unpaired Student T Test. Figure 2 – Effect of angiotensin II on the LRP1 expression in THP1 cells. 60 Arq Bras Cardiol. 2021; 116(1):56-65 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article Table 3 – Association between the expression of LRP1 and cIMT However, we did not observe an association between cIMT adjusted for lipid parameters in patients with hypertension and LRP1 protein expression when adjusting the complete mRNA OR [CI 95%] p set of lipid parameters, Model 1 (p=0.066). Nevertheless, when we adjusted with each lipid parameter, an association Adjustment [-] 0.308 [0.230 – 38.650] 0.047 was found: Model 2 adjusted by total cholesterol (p=0.040), Model 1 0.310 [0.340 - 38.887] 0.046 Model 3 adjusted by Triglycerides (p=0.041), Model 4 adjusted by HDL-C (p=0.038), and Model 5 adjusted by Model 2 0.303 [-0.280 - 38.511] 0.053 LDL-C (p=0.040). Model 3 0.308 [0.131 – 38.832] 0.049 Afterwards, a linear regression was made between cIMT and expression levels of both LRP1 mRNA and protein Model 4 0.312 [0.150 – 38.33] 0.038 expression adjusted by lipid profile; a positive correlation Model 5 0.301 [-0.181 - 38.19] 0.052 between these variables was maintained (Figure 3). Protein OR [CI 95%] p Discussion adjustment [-] 0.312 [1.771 - 65.319] 0.039 As expected, our results showed that the average cIMT was Model 1 0.294 [-2.150 - 65.208] 0.066 higher in hypertensive subjects than in normotensive subjects. However, this value was associated in an important way with Model 2 0.211 [1.544 - 65.637] 0.040 the LRP1 overexpression in circulating monocytes. Model 3 0.313 [1.445 - 65.77] 0.041 cIMT is considered an atherosclerosis marker and an Model 4 0.317 [2.020 - 66.015] 0.038 excellent predictor of death and cardiovascular events.26 In hypertensive patients with coronary artery disease, increased Model 5 0.313 [1.528 - 65.6689] 0.040 cIMT is closely associated with atherosclerosis.27 Our data Model 1: adjusted by all lipid parameters. Model 2: adjusted by total Cholesterol. showed a strong association between hypertension and Model 3: adjusted by Triglycerides. Model 4: adjusted by HDL-C. Model 5: cIMT. These results agree with previously published data in adjusted by LDL-C. Multiple logistic regressions analysis. studies made in patients and animal models. In a study of cIMT (mm) cIMT (mm) P=0.047 P=0.039 r=0.308 r=0.312 LRP1 mRNA expression LRP1 Protein Expressão expression de proteína LRP1 (normalized to HPRT) (arbitrary (unidades units) arbitrárias) Figure 3 – Correlation between cIMT and the expression levels of mRNA and LRP1 protein adjusted for CT, TG, HDL-C, and LDL-C. P<0.005 is considered as statistically significant. Arq Bras Cardiol. 2021; 116(1):56-65 61 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article young people with borderline hypertension (130-140/85-89 could be: a) the association between sLPR1 and cholesterol mmHg), an increase in the cIMT in the brachial arteries was was performed in hypercholesterolemic populations (severe observed when patients were compared to normotensive hypercholesterolemia); b) the LRP1 could be expressed in a subjects; an association between cIMT and ambulatory SBP wide range of tissues and the specificity could be different; in of 24 hours was found.27 In addition, hypertension, diabetes, our case, the LRP1 expression in monocytes was measured; and age are considered independent prognostic factors for c) the populations are very different; whereas our study was intima hyperplasia in the radial artery.28-30 In a hypertension done using a mixture of indigenous American [65%], European animal model, a significant thickening of the intima-media [31%], and African [3%] subjects, the other study consisted was reported as the direct cause of the illness.31,32 solely of Caucasians.40 Hypertension is among the main risk factors in the Our data indicate that the expression of LRP1 in monocytes etiology of atherosclerotic vascular disease.33,34 However, from hypertensive patients correlates with increased cIMT. the mechanisms by which arterial pressure increases the Adjusted logistic regression shows that the correlation between incidence of atherosclerosis are not completely clear. Studies cIMT and LRP1 mRNA expression is maintained even after that focus on elucidating these mechanisms are critically adjusting lipid parameters. However, this association was lost important. There is a strong association between hypertension when the adjustment was done with LRP1 protein. These and the LRP1 expression in the vascular wall of a rat model.35 results can be explained by the strong positive effect of The upregulation of LRP1 by hypertension has functional modified LDL on the stability of LRP1 protein.41,42 Therefore, consequences as it promotes intracellular lipid accumulation dyslipidemia probably contributes to maintaining a high LRP1 and, thus, the formation of foam cells. Hypertension also protein expression in monocytes from hypertensive patients. has a high impact on vascular remodeling, chronic changes This could justify why the association between cIMT and LRP1 in hemodynamic forces, and structural alterations in the protein expression after adjustment for lipid profile is lost. vascular wall.36 Our results show overexpression on both mRNA and Conclusions protein expression of the LRP1 receptor in monocytes from Our findings suggest that the effect of hypertension on hypertensive patients. They also show that Ang II increased atherosclerosis might occur through the overexpression of the expression of LRP1 in cultures of THP-1 in a time and dose LRP1 in circulating monocytes. Ang II induced monocyte LRP1 dependent manner. Therefore, the mechanism through which upregulation, and it may play an important role in the increased high blood pressure regulates the expression of LRP1 could cIMT associated with cardiovascular risk factor induction of be mediated by the angiotensin II effect, which is considered atherosclerotic lesion progression. These results reinforce the one of the main hypertension mediators. It has also been high relevance of LRP1 overexpression in the formation and reported that angiotensin induces the activity of Sp1/Sp3 progression of atherosclerotic plaques in humans. transcription factors, which are involved in the recognition of LRP1 promoter,13 causing LRP1 overexpression at a vascular level and favoring the formation of foam cell in human vascular Author Contributions smooth muscle cells.33 Conception and design of the research: Llorente-Cortés VC, Huesca-Gómez C; Acquisition of data: Gamboa R, In addition to angiotensin II, blood flow acts on the function Jaramillo-Estrella MJ, Martínez-Alvarado M del R, Torres-Paz and structure of the endothelium through the modulation YE, Gonzalo-Calvo D, Del Valle-Mondragón L, López-Marure of the gene expression.37 The functional changes that are R; Analysis and interpretation of the data: Gamboa R, experienced by monocytes due to continuous changes in Jaramillo-Estrella MJ, Soto ME, Huesca-Gómez C; Statistical blood flow might have a positive influence on LRP1 expression, analysis: Soto ME, Huesca-Gómez C; Obtaining financing: thus stimulating LDL uptake and causing an increase in cIMT. Huesca-Gómez C; Writing of the manuscript: Gamboa R, In addition to a high prevalence of obesity, the Mexican Jaramillo-Estrella MJ, Llorente-Cortés VC, Huesca-Gómez C; population is facing a serious problem of dyslipidemia, which is Critical revision of the manuscript for intellectual content: explained by an interaction of genetic and environmental factors.38 Gamboa R, Llorente-Cortés VC. In the analysis of dyslipidemia subjects according to conventional cardiovascular risk factors, we found an increase Potential Conflict of Interest in the LRP1 mRNA and protein expression in hypertensive dyslipidemic subjects as compared to normotensive No potential conflict of interest relevant to this article was dyslipidemic subjects, which could mean that LRP1 is reported. overexpressed by hypertension regardless of dyslipidemia. Previous studies have shown that circulating soluble Sources of Funding low-density lipoprotein receptor-related protein 1 This study was funded by Complementary Support for (sLRP1) concentration may be intimately associated Researchers in the Consolidation Process SNI-1-2009 de with hypercholesterolemia (LDL-C>200 mg/dL) and CONACyT number 119410. an upregulating effect of hypercholesterolemia on the expression of LRP1 in cells of the vascular wall in in vitro and in vivo models.39 Despite observing a high percentage Study Association of hypercholesterolemia in normotensive and hypertensive This article is part of the thesis of master submitted by María subjects, our results found no significant differences between José Jaramillo-Estrella, from Universidad Nacional Autonoma both groups. A possible explanation for these differences de México. 62 Arq Bras Cardiol. 2021; 116(1):56-65 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article References 1. Bhadoria AS, Kasar PK, Toppo NA, Bhadoria P, Pradhan S, Kabirpanthi V. 18. Lind L, Andersson J, Rönn M, Gustavsson T, Holdfelt P, Hulthe J, et al. Brachial Prevalence of hypertension and associated cardiovascular risk factors in artery intima-media thickness and echogenicity in relation to lipids and Central India. J Family Community Med. 2014;21(1):29-38. markers of oxidative stress in elderly subjects:-the prospective investigation of the vasculature in Uppsala Seniors (PIVUS) Study. Lipids. 2008;43(2):133-41. 2. Solberg LA, Strong JP. Risk factors and atherosclerotic lesions. A review of autopsy studies. Arteriosclerosis, 1983;3(3):187-98. 19. Koyoshi R, Miura S, Kumagai N, Shiga Y, Mitsutake R, Saku K. Clinical significance of flow-mediated dilation, brachial intima-media thickness and 3. Hoffman RP. Vascular endothelial dysfunction and nutritional compounds pulse wave velocity in patients with and without coronary artery disease. in early type 1 diabetes. Curr Diabetes Rev. 2014;10(3):201-7. Circ J. 2012;76(6):1469-75. 4. Meljarejo E. Memorias: el papel del óxido nítrico en la enfermedad 20. Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, Guize L, et al. Aortic aterosclerótica. Act Med Colomb. 2001;26(4):200-1. stiffness is an independent predictor of all-cause and cardiovascular mortality in hypertensive patients. Hypertension. 2001;37(5):1236-41. 5. Badimon L, Vilahur G, Padró T. Lipoproteins, platelets and atherothrombosis. Rev Esp Cardiol. 2009;62(10):1161-78. 21. Hafner F, Kieninger A, Meinitzer A, Gary T, Froehlich H, Haas E, et al. Endothelial dysfunction and brachial intima-media thickness: long term 6. Luoma J, Hiltunen T, Särkioja T, Moestrup SK, Gliemann J, Kodama T, et cardiovascular risk with claudication related to peripheral arterial disease: al. Expression of alpha 2-macroglobulin receptor/low density lipoprotein a prospective analysis. PLoS One. 2014;9(4):e93357. receptor-related protein and scavenger receptor in human atherosclerotic lesions. J Clin Invest. 1994;93(5):2014-21. 22. Stein JH, Korcarz CE, Hurst RT, Lonn E, Kendall CB, Mohler ER, et al. Use of carotid ultrasound to identify subclinical vascular disease and evaluate 7. Gonias SL, Campana WM. LDL receptor-related protein-1: a regulator of cardiovascular disease risk: a consensus statement from the American inflammation in atherosclerosis, cancer, and injury to the nervous system. Society of Echocardiography Carotid Intima-Media Thickness Task Force Am J Pathol. 2014;184(1):18-27. Endorsed by the Society for Vascular Medicine. J Am Soc Echocardiography. 2008;21(2):93-111. 8. Ferrer DG, Jaldin-Fincati JR, Amigone JL, Capra RH, Collino, CJ, Albertini RA, et al. Standardized flow cytometry assay for identification of human 23. DeLong DM, DeLong ER, Wood PD, Lippel K, Rifkind BM. A comparison monocytic heterogeneity and LRP1 expression in monocyte subpopulations: of methods for the estimation of plasma low- and very low-density decreased expression of this receptor in non classical monocytes. Cytom. lipoprotein cholesterol. The Lipid Research Clinics Prevalence Study. JAMA. Part A. 2014;85(7): 601-10. 1986;256(17):2372-7. 9. Handschug K, Schulz S, Schnürer C, Köhler S, Wenzel K, Teichmann W, 24. Tenorio-López FA, Zarco-Olvera G, Sánchez-Mendoza A, Rosas-Peralta M, et al. Low-density lipoprotein receptor-related protein in atherosclerosis Pastelín-Hernández G, Valle-Mondragón L. Simultaneous determination of development: up-regulation of gene expression in patients with coronary angiotensins II and 1-7 by capillary zone electrophoresis in plasma and urine obstruction. J Mol Med (Berl). 1998;76(8):596-600. from hypertensive rats. Talanta. 2010;80(5):1702-12. 10. Hiltunen TP, Luoma JS, Nikkari T, Ylä-Herttuala S: Expression of LDL receptor, 25. Sendra J, Llorente-Cortés V, Costales P, Huesca-Gómez C, Badimon L. Angiotensin II upregulates LDL receptor-related protein (LRP1) expression VLDL receptor, LDL receptor-related protein, and scavenger receptor in in the vascular wall: a new pro-atherogenic mechanism of hypertension. rabbit atherosclerotic lesions: marked induction of scavenger receptor Cardiovasc Res. 2008;78(3):581-9. and VLDL receptor expression during lesion development. Circulation. 1998;97(11):1079-86. 26. Zielinski T, Dzielinska Z, Januszewicz A, Rynkun D, Makowiecka Ciesla M, Tyczynski P, et al. Carotid intima-media thickness as a marker of 11. Llorente-Cortes V, Otero-Viñas M, Berrozpe M, Badimon L. Intracellular lipid cardiovascular risk in hypertensive patients with coronary artery disease. accumulation, low-density lipoprotein receptor-related protein expression, Am J Hypertens. 2007;20(10):1058-64. and cell survival in vascular smooth muscle cells derived from normal and atherosclerotic human coronaries. Eur J Clin Invest. 2004;34(3):182-90. 27. Amato M, Montorsi P, Ravani A, Oldani E, Galli S, Ravagnani PM, et al. Carotid intima-media thickness by B-mode ultrasound as surrogate 12. Schulz S, Birkenmeier G, Schagdarsurengin U, Wenzel K, Müller-Werdan of coronary atherosclerosis: correlation with quantitative coronary U, Rehfeld D, et al. Role of LDL receptor-related protein (LRP) in coronary angiography and coronary intravascular ultrasound findings. Eur Heart J. atherosclerosis. Int J Cardiol. 2003;92(2-3):137-44. 2007;28(17):2094-101. 13. Schulz S, Schagdarsurengin U, Greiser P, Birkenmeier G, Müller-Werdan 28. Toikka JO, Laine H, Ahotupa M, Haapanen A, Viikari JS, Hartiala JJ, et al. U, Hagemann M, et al. The LDL receptor-related protein (LRP1/A2MR) Increased arterial intima-media thickness and in vivo LDL oxidation in young and coronary atherosclerosis--novel genomic variants and functional men with borderline hypertension. Hypertension. 2000;36(6):929-33. consequences. Hum Mutat. 2002;20(5):404. 29. Chowdhury UK, Airan B, Mishra PK, Kothari SS, Subramaniam GK, Ray R, et 14. Llorente-Cortés V, Royo T, Otero-Viñas M, Berrozpe M, Badimon L. Sterol al. Histopathology and morphometry of radial artery conduits: basic study regulatory element binding proteins downregulate LDL receptor-related and clinical application. Ann Thorac Surg. 2004;78(5):1614-21. protein (LRP1) expression and LRP1-mediated aggregated LDL uptake by 30. Ruengsakulrach P, Sinclair R, Komeda M, Raman J, Gordon I, Buxton B. human macrophages. Cardiovasc Res. 2007;74(3):526-36. Comparative histopathology of radial artery versus internal thoracic artery 15. Sakr SW, Eddy RJ, Barth H, Wang F, Greenberg S, Maxfield FR, et al. The and risk factors for development of intimal hyperplasia and atherosclerosis. uptake and degradation of matrix-bound lipoproteins by macrophages Circulation. 1999;100(19 Suppl):II139-44. require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase 31. Kowala MC, Cuénoud HF, Joris I, Majno G. Cellular changes during activity. J Biol Chem. 2001;276(40):37649-58. hypertension: a quantitative study of the rat aorta. Exp Mol Pathol. 1986;45(3):323-35. 16. Llorente-Cortés V, Gonzalo-Calvo D, Orbe J, Páramo JA, Badimon L. Signature of subclinical femoral artery atherosclerosis in peripheral blood 32. Rossi MA, Colombini-Netto M. Chronic inhibition of NO synthesis per mononuclear cells. Eur J Clin Invest. 2014;44(6):539-48. se promotes structural intimal remodeling of the rat aorta. J Hypertens. 2001;19(9):1567-79. 17. Halcox JP, Donald AE, Ellins E, Witte DR, Shipley MJ, Brunner EJ, et al. Endothelial function predicts progression of carotid intima-media thickness. 33. Simon A, Levenson J. Stratification of vascular risk in hypertension and Circulation. 2009;119(7):1005-12. therapeutic perspective. Am J Hypertens. 1995;8(10 Pt 2):45S-48. Arq Bras Cardiol. 2021; 116(1):56-65 63 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article 34. Violi F, Criqui M, Longoni A, Castiglioni C. Relation between risk factors and 39. Gonzalo-Calvo D, Cenarro A, Martinez-Bujidos M, Badimon L, Bayes-Genis cardiovascular complications in patients with peripheral vascular disease. A, Ordonez-Llanos J, et al. Circulating soluble low-density lipoprotein Results from the A.D.E.P. study. Atherosclerosis. 1996;120(1):25-35. receptor- related protein 1 (sLRP1) concentration is associated with hipercolesterolemia: a new potential biomarker for atherosclerosis. Int J 35. Aledo R, Costales P, Ciudad C, Noé V, Llorente-Cortes V, Badimon L. Cardiol. 2015 Dec 15;201:20-9. Molecular and functional characterization of LRP1 promoter polymorphism c.1-25 C>G (rs138854007). Atherosclerosis. 2014;233(1):178-85. 40. Johnson NA, Coram NA, Shriver MD, Romieu I, Barsh GS, London SJ, et al. Ancestral components of admixed genomes in a Mexican cohort. PloS 36. Baumbach GL, Heistad DD. Remodeling of cerebral arterioles in chronic Genet. 2011;7(12):e1002410. hypertension. Hypertension. 1989;13(6 Pt 2):968-72. 41. Llorente-Cortes V, Casani L, Cal R, Llenas A, Juan-Babot O, Camino-López 37. Davies PF, Tripathi SC. Mechanical stress mechanisms and the cell. An S, et al. Cholesterol-lowering strategies reduce vascular LRP1 overexpression endothelial paradigm. Circ Res. 1993;72(2):239-45. induced by hypercholesterolaemia. Eur J Clin Invest. 2011;41(10):1087-97. 38. Aguilar-Salinas CA, Gómez-Pérez FJ, Rull J, Villalpando S, Barquera S, Rojas 42. Cal R, García-Arguinzonis M, Revuelta-López E, Castellano J, Padró T, R. Prevalence of dyslipidemias in the Mexican National Health and Nutrition Badimon L, et al. Aggregated low-density lipoprotein induces LRP1 Survey 2006. Salud Pública Mex. 2010;52(Suppl 1):S44-53. stabilization through E3 ubiquitin ligase CHFR downregulation in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2013;33(2):369-77. 64 Arq Bras Cardiol. 2021; 116(1):56-65 Gamboa et al. LRP1 correlates with cIMT in Hypertention Original Article This is an open-access article distributed under the terms of the Creative Commons Attribution License Arq Bras Cardiol. 2021; 116(1):56-65 65