Association between Resistant Arterial Hypertension, Type 2 Diabetes, and Selected microRNAs

Introduction: In recent years, a very close relationship between miRNA and cardiovascular diseases has been found. RAH and T2DM are accompanied by a change in the microRNA expression spectrum. Objectives: This study aimed to evaluate the clinical characteristics and expression of selected microRNAs in patients with idiopathic RAH and T2DM. Patients and methods: A total of 115 patients with RAH were included in this study. Among them were 53 patients (46.09%) with T2DM. miRNA levels were determined using quantitative real-time polymerase chain reaction. The expression of the examined genes was calculated from the formula RQ = 2−ΔΔCT. Results: Analysis using the Mann–Whitney U test showed a statistically significant (p < 0.05) difference in the expression of MIR1-1 (p = 0.031) and MIR195 (p = 0.042) associated with the occurrence of T2DM in the subjects. The value of MIR1-1 gene expression was statistically significantly higher in patients with T2DM (median: 0.352; mean: 0.386; standard deviation: 0.923) compared to patients without T2DM (median: 0.147; mean: −0.02; standard deviation: 0.824). The value of MIR195 gene expression was statistically significantly higher in patients with T2DM (median: 0.389, mean: 0.442; standard deviation: 0.819) compared to patients without T2DM (median: −0.027; mean: 0.08; standard deviation: 0.942). Conclusions: The values of MIR1-1 and MIR195 gene expression were statistically significantly higher in patients with RAH and T2DM compared to patients with RAH and without T2DM. Further studies are necessary to precisely clarify the roles of miRNAs in patients with RAH and T2DM. They should demonstrate the utility of these genetic markers in clinical practice.


Introduction
Arterial hypertension (AH) is a disease that is caused by a combination of genetic susceptibility and environmental factors.It is a well-established fact that AH is a major contributor to cardiovascular and cerebrovascular diseases, with a global prevalence projected to increase from 26.4% in 2000 to 29.2% in 2025 [1].The pathogenesis of AH is caused by several mechanisms, which are of utmost importance.These mechanisms include increased activity of the sympathetic nervous system, overactivation of the renin-angiotensinaldosterone system (RAAS), dysfunction of the vascular endothelium, impaired platelet function, thrombogenesis, vascular smooth muscle, cardiac hypertrophy, and altered angiogenesis [2].According to the latest definition, resistant arterial hypertension (RAH) is diagnosed when systolic blood pressure (SBP) and diastolic blood pressure (DBP) values are ≥140 mm Hg and ≥90 mm Hg, respectively, despite the use of three antihypertensive drugs in the maximum recommended and tolerated doses: an inhibitor of the RAAS system (angiotensin-converting enzyme inhibitor-ACE inhibitor or receptor antagonist Isolation of PBMCs was conducted using Ficoll-Paque Plus™ via density gradient centrifugation from whole blood samples (Cytiva, Uppsala, Sweden).Whole-blood samples obtained from study individuals were collected into 2.7 mL S-Monovette vials (Sarstedt, Germany) containing K3 ethylenediaminetetraacetic acid (EDTA) as an anticoagulant factor.Samples were directly submitted to PBMC isolation via density gradient centrifugation using Ficoll-Paque Plus™ (Cytiva, Uppsala, Sweden).Whole-blood samples were diluted in equal volumes of PBS (phosphate-buffered saline, without calcium and magnesium ions, Biomed-Lublin, Lublin, Poland) in sterile 15 mL Eppendorf conical tubes (Eppendorf, Germany).Diluted blood was carefully layered on 3 mL Ficoll-Paque Plus™ in a new, sterile, 15 mL conical tube.Tubes were centrifuged at 400× g for 30 min at 20 • C (5810 R Centrifuge, Eppendorf, Germany), and the brake was turned off.The layer of mononuclear cells was transferred to a new, sterile, 15 mL Eppendorf conical tubes.Cells were gently resuspended in 3 volumes of PBS by pipetting.The tubes were centrifuged at 450× g for 10 min at 20 • C (5810 R Centrifuge, Eppendorf, Germany).The supernatant was removed.This step was repeated twice.The cell pellet was resuspended in 1 mL of PBS.The entire volume was distributed to several sterile 1.5 mL centrifuge tubes (DNA LoBind Tube, Eppendorf, Germany) and centrifuged at 400× g for 10 min at 4 • C (5415 R Centrifuge, Eppendorf, Germany).After the last centrifugation, the supernatant was removed and the tubes with PBMC pellets were stored at −80 • C till the isolation of RNA.

Total RNA Isolation
Total RNA was isolated from PBMCs using the mirVana™ miRNA Isolation Kit (Invitrogen by Thermo Fisher Scientific, Vilnius, Lithuania), according to the manufacturer's protocol.The quantity and quality assessment of isolated total RNA was performed using a NanoDrop 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).For all samples analyzed, the A260/A280 ratio was between 1.8 and 2.0.
The expression of the examined genes was calculated from the formula RQ = 2 −∆∆CT [13].RNU48 snRNA was used as a normalizer during the analysis.Expression Suite Software 1.0.3 was used to calculate the gene expression level (Life Technologies, Waltham, MA, USA).

Statistical Analysis of Data
Statistica v.13.3 was used in the statistical analysis and graphic design (p < 0.05 was assumed statistically significant).The histograms and Shapirro-Wilk test were used to check the normality of the data distributions.Because the distributions of the analyzed variables deviated from the normal distributions, non-parametric statistical methods were used.The U Mann-Whitney test was used to calculate the differences in the expression level and the r-Spearman coefficient was used for correlation analysis.

Results
Baseline characteristics regarding demographics, biochemical parameters, comorbidities, and echocardiography parameters were collected.The Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation was used to calculate the estimated glomerular filtration rate (eGFR) and assess patients' kidney function.Each participant underwent a routine echocardiographic examination according to the standard protocol.
The patients included in this study were treated with pharmacotherapy according to the recommendations of the European Society of Hypertension and the European Society of Cardiology [3,14].All groups of hypertensive drugs were administered.Table 2 shows the pharmacotherapy of RAH.This study included 28 women (52.83%) and 25 men (47.17%) with RAH and T2DM.Tables 3 and 4 show the characteristics of the study group divided into two subgroups: RAH without T2DM and RAH with T2DM.When comparing patients with RAH and T2DM and without T2DM, the more frequent occurrence of atrial fibrillation in the RAH group without T2DM is noteworthy.More than 90% of patients with RAH and T2DM had diagnosed chronic heart failure with preserved left ventricular ejection fraction (HfpEF), while in the RAH group without T2DM, this percentage was 74.19%.In the case of cerebrovascular disease, a higher percentage of patients with previous stroke history was noted in the RAH and T2DM group (16.98% vs. 12.90%).Also in the RAH and T2DM group, there was a higher percentage of patients with a history of myocardial infarction (22.64% vs. 8.06%), PCI (45.28% vs. 24.19%),and CABG (15.09% vs. 1.61%).Table 5 shows cardiovascular morbidity in the studied patient groups.The analysis showed that patients with RAH and T2DM had higher values of the parameters of left atrial enlargement (LA dimension in LAX: 40 mm vs. 39.42 mm; LA surface area in Ap4CH: 21.72 mm vs. 20.75mm; LA volume: 99.41 mL vs. 90.74mL, LAVI: 48.08 mL/m 2 vs. 44.74mL/m 2 ) and left ventricle hypertrophy (LVMI: 113 g/m 2 vs. 108.69g/m 2 , LVMM: 221.37 g vs. 211.81g, RWT: 0.4560 vs. 0.4565) compared to patients with RAH and without T2DM (Tables 6 and 7).In the group of patients with RAH and T2DM, a higher percentage of diastolic dysfunction was found (92.45%) compared to patients with RAH without T2DM (77.41%).

Statistical Analysis
Analysis using the Mann-Whitney U test showed a statistically significant (p < 0.05) difference in the expression of MIR1-1 (p = 0.031) and MIR195 (p = 0.042) associated with the occurrence of type 2 diabetes in the subjects.The value of MIR1-1 gene expression was statistically significantly higher in patients with type 2 diabetes (median: 0.352; mean: 0.386; standard deviation: 0.923) compared to patients without type 2 diabetes (median: 0.147; mean: −0.02; standard deviation: 0.824) (Figure 1).The value of MIR195 gene expression was statistically significantly higher in patients with type 2 diabetes (median: 0.389, mean: 0.442; standard deviation: 0.819) compared to patients without type 2 diabetes (median: −0.027; mean: 0.08; standard deviation: 0.942) (Figure 2).A possible trend toward significance was found in the expression of the MIR126 gene (p = 0.051), the MIR133A1 gene (p = 0.076), the MIR143 gene (p = 0.08), and the MIR320A gene (p = 0.095) in patients with type 2 diabetes.The value of MIR126 gene expression showed a statistical trend toward significance in patients with type 2 diabetes (median: 0.332; mean: 0.427; standard deviation: 0.753) compared to patients without type 2 diabetes (median: 0.112; mean: 0.116; standard deviation: 0.798).The value of MIR133A1 gene expression showed a statistical trend toward significance in patients with type 2 diabetes (median: 0.147; mean: 0.233; standard deviation: 1.056) in contrast to patients without type 2 diabetes (median: −0.168; mean: −0.106, standard deviation: 0.883).The value of MIR143 gene expression showed a statistical trend toward significance in patients with type 2 diabetes (median: 0.547; mean: 0.494; standard deviation: 1.047) compared to patients without type 2 diabetes (median: 0.205; mean: 0.071; standard deviation: 1.148).The value of MIR320A gene expression showed a trend close to significance in patients with type 2 diabetes (median: 0.045; mean: 0.144; standard deviation: 0.786) in contrast to patients with no diagnosis of type 2 diabetes (median: −0.210, mean: −0.139; standard deviation: 0.844).In the case of the remaining microRNAs (MIR145, MIR155, MIR21, MIR26b, and MIR-208A), the differences between the two study groups were statistically insignificant.Additionally, a Spearman test was performed.In patients with RAH who had not been diagnosed with type 2 diabetes, there was a negative correlation between the duration of RAH and the expression of miR-145 and miR-208 (r Spearman= −0.306, p < 0.05; r Spearman= −0.290, p < 0.05).

Discussion
miRNAs play a crucial role in many physiological and pathological processes and regulate various functions in the cardiovascular system, kidneys, and liver.However, the specific targets of most microRNAs are still not well understood.These microRNAs can repress and inhibit the expression of genes.Even subtle changes in the levels of miRNAs can disrupt biochemical and physiological pathways, leading to the onset of various disorders.
In the literature, data confirm the relationship between MIR1, MIR195, and the coexistence of AH and T2DM.MIR195 plays an important role in regulating cardiovascular diseases.In a recently published work, Hao et al. showed the role of MIR195a-5p in blood pressure regulation via electrolyte excretion involving Na + -K + -2Cl − cotransporter isoform A (NKCC2A).They suggested that MIR195a-5p is involved in the mechanism by which tumor necrosis factor-α (TNF-α) restricts increases in sodium retention and blood pressure that are dependent on NKCC2A after a rise in salt consumption.NKCC2A expression follows in the thick ascending limb of the Henle loop The statistical analysis carried out showed no difference in the value of miR (hsa-miR-1, hsa-miR-126*, hsa-miR-133a, hsa-miR-143, hsa-miR-145*, hsa-miR-155, hsa-miR-195, hsa-miR-208, hsa-miR-21, hsa-miR-26b, hsa-miR-320) expressions in women and men (Table 8).
Additionally, a Spearman test was performed.In patients with RAH who had not been diagnosed with type 2 diabetes, there was a negative correlation between the duration of RAH and the expression of miR-145 and miR-208 (r Spearman = −0.306,p < 0.05; r Spearman = −0.290,p < 0.05).

Discussion
miRNAs play a crucial role in many physiological and pathological processes and regulate various functions in the cardiovascular system, kidneys, and liver.However, the specific targets of most microRNAs are still not well understood.These microRNAs can repress and inhibit the expression of genes.Even subtle changes in the levels of miRNAs can disrupt biochemical and physiological pathways, leading to the onset of various disorders.
In the literature, data confirm the relationship between MIR1, MIR195, and the coexistence of AH and T2DM.MIR195 plays an important role in regulating cardiovascular diseases.In a recently published work, Hao et al. showed the role of MIR195a-5p in blood pressure regulation via electrolyte excretion involving Na + -K + -2Cl − cotransporter isoform A (NKCC2A).They suggested that MIR195a-5p is involved in the mechanism by which tumor necrosis factor-α (TNF-α) restricts increases in sodium retention and blood pressure that are dependent on NKCC2A after a rise in salt consumption.NKCC2A expression follows in the thick ascending limb of the Henle loop (TAL).TNF increases MIR195a-5p expression, which in turn interacts with the 3 ′ UTR of NKCCA2 to regulate NaCl excretion.This attenuates NaCl-induced increases in blood pressure [15].One of the most common complications of improperly controlled AH (RAH) is cardiac hypertrophy.MIR195 plays a significant role in this case.Zheng et al. showed that inhibited MIR195-5p expression attenuated hypertrophy in streptozocin-induced diabetic mice.In their study, overexpression of MIR195-5p significantly promoted AngII-induced cardiac hypertrophy.Therapeutic silencing of MIR195 improves myocardial function in diabetes by reducing oxidative damage, inhibiting apoptosis, and promoting endothelial cell angiogenesis.The authors concluded that MIR195 may represent an alternative therapeutic target for diabetic heart diseases [16].Previous publications have shown that MIR195 inhibits the gene expression of B cell leukemia/lymphoma 2 (BCL-2) and Sirtuin 1 (SIRT1) in cardiomyocytes, leading to decreased levels of these proteins in diabetic hearts.Decreased MIR195 expression increased levels of BCL-2 protein, which is a known anti-apoptotic protein.SIRT1, which is activated by resveratrol, causes improved cardiac function in animal models of diabetes.MIR195 can contribute to diabetic cardiomyopathy by affecting systemic inflammation, insulin sensitivity, and causing liver damage [17,18].BCL-2 also regulates another important anti-apoptotic protein-heat shock protein 60 (Hsp60).Increased BCL-2 expression levels lead to decreased Hsp60 levels in the myocardium of diabetic rats.MIR1 plays an important role in inducing high glucose-based apoptosis of cardiac cells [19].In an interesting study, it was found that MIR1 mediates insulin-like growth factor (IGF-1), which is a well-known anti-apoptotic factor.When H9C2 cells were administered, high glucose, mitochondrial dysfunction, and apoptosis were observed due to high expression of MIR1.However, IGF-1 blocked these effects by regulating MIR1 expression [20].In another publication, Liu et al. investigated the effect of MIR1 expression on vascular smooth muscle cells (VSMCs).Prolonged high blood pressure can cause mechanical stretch of the vascular wall, leading to changes in VSMCs.These changes may include increased proliferation, synthesis of collagen and extracellular matrix, migration of cells, and decreased expression of contractility markers, which are typically present in this type of muscle cell.MIR1 inhibits the proliferation of muscle cells such as cardiomyocytes.In the above-mentioned publication, the downregulation of miR1 was responsible for a higher rate of VSMC proliferation.The anti-proliferative effects of MIR1 are mediated through IGF-1 [21,22].
Hu Y. et al. studied the relationship between MIR195 expression and Dopamine receptor D1 function (DRD1) in a group of patients with AH and T2DM.This receptor belongs to the superfamily of G-protein-coupled receptors (GPCRs).The primary importance of this subtype receptor is sodium reuptake and renal sodium excretion.Dysfunction of DRD1 leads to essential hypertension.MIR195-5p was found to efficiently suppress DRD1 expression by binding to the two 3 ′ UTRs.The study also identified two single nucleotide polymorphisms (SNPs), namely 231T-A and 233C-G, in the MIR195-5p binding sites of the DRD1 3 ′ -UTR.The results suggest the potential clinical significance of DRD1 regulation by MIR195-5p in patients with AH and concurrent type 2 diabetes (T2DM) [23].
Data from the literature point to the fact that up-regulation of MIR195 contributes to cardiac hypertrophy-induced arrhythmia by targeting calcium and potassium channels.miR-195 can directly target the CACNB1 gene (calcium voltage-gated channel auxiliary subunit beta 1), KCNJ2 gene (potassium voltage-gated channel subfamily J member 2), and KCND3 gene (potassium voltage-gated channel subfamily D member 3) to regulate Cavβ1, Kir2.1, and Kv4.3 protein domain expression.There are elevated levels of a certain substance, denoted by MIR195, in a failing heart muscle.These elevated levels regulate the acetylation of two important enzymes, pyruvate dehydrogenase (PDH) and ATP synthase.This in turn suppresses the activity of another important enzyme called Sirtuin 3 (SIRT3), which inhibits enzymatic activity.This presents a new pathway for research on MIR195.Xuan et al. have demonstrated that overexpression of MIR195 inhibits the expression of three proteins, namely Cavβ1, Kir2.1, and Kv4.3.However, when a MIR195 inhibitor is added, this downregulated protein expression is reversed [24,25].
In the study of arterial hypertension, cardiomyocyte apoptosis is closely related to AH.The balance between cell death and cell survival is a precisely controlled process.Accumulated data support the role of cardiomyocyte apoptosis in the development of various heart diseases.This includes the transition from hypertensive compensatory hypertrophy to heart failure [26].Huang X et al. studied the relationship between MIRNA1 level and the expression of Bcl-2 and explored the role of MIRNA1 in cardiomyocytes in hypertensive rats.In their study, silencing MIR1 activated the mitogen-activated protein kinase (MAPK) pathway, promoted autophagy, and reduced the degree of myocardial fibrosis caused by hypertension [27].
In our study, in patients with RAH and without diagnosed T2DM, a negative correlation was found between the duration of RAH and the expression of miR-145 and miR-208.The role of MIR145 in the pathogenesis and regulation of AH (RAH) is extremely complex.Wang Y. et al. demonstrated the role of MIR145 in spontaneous hypertensive rats (SHRs) and rat vascular endothelial cells.Higher expression of MIR145 in hypertensive rats was associated with an important decrease in the production of nitric oxide.The direct target for MIR145 is solute carrier family 7 member 1 (SLC7A1).A decrease in the expression of MIR145 causes increased expression of SLC7A1 and phosphorylated endothelial nitric oxide synthase [28].Chen et al. showed the role of complement 3 (C3) in the development of AH in SHRs.Simultaneous suppression of MIR145 and an increased expression of C3 induce the expression of Kruppel-like factor 5 (KLF5), which changes the type of VSMCs from the contractile to the synthetic phenotype.Furthermore, activation of C3 leads to the stimulation of liver X receptor α (LXRα), which in turn activates the renin-angiotensin system and platelet-derived growth factor A (PDGF-A) as well as transforming growth factor β1 (TGF-β1) in vascular smooth muscle cells (VSMCs) [29].It also turns out that MIR145 may be responsible for VSMC remodeling through disintegrating and metalloprotease 17 (ADAM17), which is a protein involved in the shedding of several membrane proteins that are important for inflammation and immunity, namely TNF-α, intercellular adhesion molecule-1 (ICAM-1), and angiotensin convertase enzyme 2 (ACE2).A direct effect of ADAM17 on ACE2 has been demonstrated, leading to the shedding of ACE2 into the extracellular matrix.MIR145 attenuates the transformation of VSMCs from the contractile to synthetic type.Its molecular pathway includes the secretion of ACE2 in VSMCs mediated by ADAM17.Finally, the activation of the ACE2-Ang-(1-7)-Mas axis may yield vascular structural remodeling in metabolic hypertension [30].
There are also reports linking MIR208 with cardiac hypertrophy in patients with arterial hypertension.Huang X et al. examined the expression levels of MIR208 and sexdetermining region Y-box 6 (SOX6) in patients with cardiac hypertrophy.The expression of MIR208 was significantly higher in patients with left ventricular hypertrophy (LVH) than in those without LVH.In an in vitro model, SOX6 expression levels were statistically significantly decreased in phenylephrine-stimulated cardiomyocytes compared to normal cardiomyocytes.The authors concluded that the expression levels of MIR208 are associated with LVH via negative regulation of SOX6 [31].In our study, the parameters of left ventricle hypertrophy were more pronounced in the RAH and T2DM group.
Referring the study results to the studied population, it should be stated that patients with RAH and T2DM and increased expression of MIR1-1 and MIR-195 have increased cardiovascular morbidity, including the occurrence of heart failure and a history of stroke.An increased incidence of ischemic heart disease, including a number of myocardial infarctions and a history of PCI and CABG, was also observed in this group.The statistical analyses carried out so far showed no difference in the value of miR expressions in women and men.

Study Limitations
There are several limitations in our study.Firstly, in T2DM patients, muscle or adipose tissue samples would have been more relevant; however, we had no IRB approval for tissue sample collection.Secondly, it was a single-center study, performed on a relatively small number of patients; therefore, caution is needed when interpreting our findings with regard to the general RAH or T2DM populations.Thirdly, we used a statistical significance value of p < 0.05.Due to the possibility of false-positive results, a threshold of <0.0001 might have been preferable.

Conclusions
In conclusion, we found that the values of MIR1-1 and MIR195 gene expression were statistically significantly higher in patients with RAH and T2DM compared to patients with RAH and without T2DM.Further studies are necessary to precisely clarify the roles of miRNAs in patients with RAH and T2DM.They should also demonstrate the utility of these genetic markers in clinical practice.

Table 3 .
Baseline characteristics of patients with RAH without T2DM.

Table 4 .
Baseline characteristic patients with RAH and T2DM.

Table 5 .
Cardiovascular morbidity in the studied patient groups.

Table 6 .
Echocardiographic parameters in patients with RAH without T2DM.

Table 7 .
Echocardiographic parameters in patients with RAH and T2DM.