endothelin-1 mechanotransduction pathway

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Matrix stiffening induces endothelial dysfunction via the TRPV4/microRNA-6740/ET-1 mechanotransduction pathway Xiang Song , Zhenwei Sun , Gan Chen , Pan Shang , Guoxing You , Jingxiang Zhao , Sisi Liu , Dong Han , Hong Zhou PII: DOI: Reference:

S1742-7061(19)30689-0 https://doi.org/10.1016/j.actbio.2019.10.013 ACTBIO 6398

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Acta Biomaterialia

Received date: Revised date: Accepted date:

1 April 2019 16 September 2019 4 October 2019

Please cite this article as: Xiang Song , Zhenwei Sun , Gan Chen , Pan Shang , Guoxing You , Jingxiang Zhao , Sisi Liu , Dong Han , Hong Zhou , Matrix stiffening induces endothelial dysfunction via the TRPV4/microRNA-6740/ET-1 mechanotransduction pathway, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.10.013

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Matrix

stiffening

induces

endothelial

dysfunction

via

the

TRPV4/microRNA-6740/ET-1 mechanotransduction pathway

Xiang Song 1‡, Zhenwei Sun2, Gan Chen1*, Pan Shang1, Guoxing You1, Jingxiang Zhao1, Sisi Liu3, Dong Han4*, Hong Zhou1*

1

Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences,

Beijing 100039, China 2

Department of Blood Transfusion, The 988 hospital of PLA, Zhengzhou 450042, China

3

Institute of Biomechanics and Medical Engineering, Department of Engineering Mechanics,

Tsinghua University, Beijing 100084 4

‡ *

National Centre for Nanoscience and Technology, Beijing, China 100190

These authors contributed equally to this work Corresponding authors;

Gan Chen, [email protected]; Dong Han, [email protected]; Hong Zhou, [email protected].

Abstract 1

Vascular stiffening is associated with the prognosis of cardiovascular disease (CVD). Endothelial dysfunction, as shown by reduced vasodilation and increased vasoconstriction, not only affects vascular tone, but also accelerates the progression of CVD. However, the precise effect of vascular stiffening on endothelial function and its mechanism is unclear and a possible underlying has not been determined. In this study, we found that increasing substrate stiffness promoted endothelin-1 (ET-1) expression and inhibited endothelial nitric oxide synthase expression in human umbilical vein endothelial cells. Additionally, miR-6740-5p was identified as a stiffness-sensitive microRNA, which was downregulated by a stiff substrate, resulting in increased ET-1 expression. Furthermore, we found that substrate stiffening reduced the expression and activity of the calcium channel TRPV4, which subsequently enhanced ET-1 expression by inhibiting miR-6740-5p. Finally, analysis of clinical plasma samples showed that plasma miR-6740-5p levels in patients with carotid atherosclerosis were significantly lower than those in healthy people. Taken together, our findings show a novel mechanically regulated TRPV4/miR-6740/ET-1 signaling axis by which substrate stiffness affects endothelial function. Our findings indicate that vascular stiffening induces endothelial dysfunction, thereby accelerating progression of CVD. Furthermore, this study indicates that endothelial dysfunction induced by improper biophysical cues from cardiovascular implants may be an important reason for complications arising from the use of cardiovascular implants.

Statement of significance Cardiovascular disease is the leading cause of morbidity and mortality worldwide. The incidence of cardiovascular disease is accompanied by increased vascular stiffness. Our work indicated that increased vascular stiffness accelerates the development of cardiovascular disease by inducing endothelial dysfunction, which is a key contributor to the pathogenesis of cardiovascular disease. In addition, we identified a novel underlying molecular pathophysiological mechanism by which increased stiffness induce endothelial dysfunction. Our work could help determine the pathogenesis of cardiovascular disease induced by 2

biomechanical factors.

Key words Stiffness, cardiovascular disease, endothelin-1, miR-6740-5p, TRPV4

1. Introduction Cardiovascular disease (CVD), which includes atherosclerosis, coronary heart disease, and hypertension, is the leading causes of death worldwide [1, 2]. The incidence of CVD is strongly associated with increased vascular stiffness [3-6], which is a maker of early vascular disease [7] and an independent predictor of all-cause mortality in CVD [8]. Endothelial cells (ECs) cover the inner wall of blood vessels and form a barrier separating the circulating blood from the vessel wall. Endothelial dysfunction is not only a clinical marker for predicting cardiovascular events [9, 10], but also an important contributor to the pathogenesis of CVD [11, 12]. Clinical investigations have indicated that increased vascular stiffness is closely related to endothelial dysfunction in CVD [13, 14]. Extracellular matrix stiffness affects endothelial proliferation, adhesion, and expression of regular reference genes [15-18]. However, direct experimental evidence that shows exactly how vascular stiffening affects endothelial dysfunction is still limited. Additionally, the molecular mechanisms by which substrate stiffness affects endothelial function are still unclear. Endothelial dysfunction is characterized by an imbalance between vasoconstrictive and vasodilatory molecules. These molecules are synthesized and released by ECs and include nitric oxide (NO), prostacyclin and endothelin-1 (ET-1). NO and prostacyclin are effective vasodilators and are produced by endothelial nitric oxide synthase (eNOS) and prostacyclin synthase (PGIS), respectively, while ET-1 is a potent vasoconstrictor [19]. Increased tissue 3

levels of ET-1 contribute to hypertension and formation of active coronary atherosclerotic plaques [20, 21]. Additionally, endothelium-restricted overexpression of ET-1 causes endothelial dysfunction and a decrease in NO bioavailability [22, 23]. MicroRNAs (miRNAs) are a class of non-coding RNA that can regulate cellular behavior in response to mechanical cues, including shear stress [24, 25], cyclic stretch [26], and stiffness [27]. This indicates that miRNAs are sensitive to mechanical cues, which indicates a potential association between miRNAs and ECM stiffness in regulating endothelial function. The mechanism by which substrate stiffness is transduced into mechanosensitive endothelial function remains largely unknown. Transient receptor potential cation channel subfamily V member 4 (TRPV4) is a member of the TRP superfamily of cation channels. TRPV4 responds to not only exogenous and endogenous chemical stimuli, but also to physical stimuli, including moderate heat and shear stress [28-30]. However, whether TRPV4 acts as a mechano-sensor to sense ECM stiffness and modulate endothelial function still needs to be investigated. In this study, the effect of substrate stiffening on endothelial function and its regulatory mechanism were investigated in human umbilical vein endothelial cell (HUVECs). Polyacrylamide hydrogels with three different stiffnesses (4, 25, and 50kPa) were prepared to mimic pathophysiological states on the basis of the published range of a healthy vascular basement membrane compliance of 2.5-8 kPa [31, 32].

2. Materials and Methods 2.1 Cell culture HUVECs were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in Endothelial Cell Growth Medium (EGM-2 Bulletkit; Lonza, Switzerland). HEK 293T cells were cultured in Dulbecco’s modified Eagle’s medium (Thermo Scientific, USA) containing 10% fetal bovine serum (ExCell Bio, China). Cells were cultured at 37°C in an atmosphere with 5% CO2 and 95% humidity. Media were changed daily. For experiments, 4

cells were plated on type I collagen (Sigma-Aldrich, USA)-coated (100 µg/mL) hydrogels (4, 25, and 50 kPa; Matrigen Life Technologies, USA) and cultured for up to 3 days. 2.2 miRNA microarray Total RNA extracted from HUVECs cultured on soft (4 kPa) and stiff (50 kPa) hydrogel substrates for 72 h was used for Agilent SurePrint gene-chip analysis (Agilent Technologies, USA). Compliant data were submitted to Gene Expression Omnibus for miRNA array analysis. Data collection and array normalization were performed using Agilent Feature Extraction Software (Agilent Technologies). Percentile normalization and principal component analysis were conducted by Gene spring 12.0 (Agilent Technologies). 2.3 Quantitative real-time polymerase chain reaction analyses of miRNA and mRNA Total RNA was extracted from HUVECs using the Ultrapure RNA Kit (CWbio, China) and then reverse transcribed using the Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s instructions. ET-1, eNOS, and PGIS mRNA levels were determined using SYBR® Green Realtime PCR Master Mix (Toyobo, Japan) and the Real-Time PCR Detection System (Bio-Rad, USA). Beta-2-microglobulin was used for normalization of the results. Primer sequences are listed in Table S1. HUVECs and plasma miRNA extraction were performed using the miRNA Purification Kit (CWbio). The MiRcute Plus miRNA First-Strand cDNA Synthesis Kit (Tiangen Biotech, China) was used to obtain cDNA. Quantitative real-time polymerase chain reaction (qPCR) was conducted using the miRcute Plus miRNA qPCR Detection Kit (Tiangen Biotech) to analyze the expression of hsa-miR-6740-5p and hsa-miR-214-3p. U6 was used as an internal control for gene expression. 2.4 Cell transfection HUVECs were seeded in 24-well plates at 6×104 cells per well. Mimics and inhibitors of hsa-miR-6740-5p were purchased from GenePharma (Shanghai, China). According to the manufacturer’s instructions, transfection was conducted with synthetic RNA at a final concentration of 300 nM using Lipofectamine RNAiMAX (Thermo Scientific). Twenty-four 5

hours after transfection, total RNA and culture medium were collected for quantitative analysis. 2.5 ET-1 measurement Cell culture medium was collected to measure secreted levels of ET-1 using an ET-1 ELISA kit (Abcam, UK) following the manufacturer’s protocol. 2.6 Immunofluorescence For immunofluorescence staining, HUVECs were fixed and then blocked before being incubated with primary anti-vinculin and anti-eNOS primary antibodies (Cell Signaling Technology, Inc., USA). This was followed by secondary antibody (Cell Signaling Technology, Inc.) incubation and DAPI (Invitrogen, USA) staining. Actin filaments were stained with phalloidin (AAT Bioquest, USA). Images were obtained using a Nikon A1 laser scanning confocal microscope (Nikon, Japan). 2.7 Electrophysiology Whole-cell currents of HUVECs were recorded using a Multiclamp 700B amplifier and digitized by an Axon DigiData 1550 (Molecular Devices, USA). Data were collected at 10 kHz and filtered at 5 kHz. Current was elicited by −80 mV to 80 mV ramps from the holding potential of −70 mV. The standard extracellular recording solution contained the following: 3 mM KCl, 1.8 mM CaCl2, 150 mM NaCl, 1 mM MgCl2, 5.5 mM D-glucose, and 10 mM HEPES. The pipette solution contained the following: 10 mM KCl, 1 mM MgCl2, 135 mM K-gluconate, 4 mM Mg-ATP, 0.6 mM Na-GTP, 10 mM HEPES, and 2 mM BAPTA. The pH was adjusted to 7.3 with potassium hydroxide and osmolarity was adjusted to 295-300 mOsm with sucrose. Cumulative currents and density were recorded in HUVECs treated with an agonist, 40 nM GSK101790A (GSK101), or an antagonist, 40 nM GSK2193874, at ±80 mV. All experiments were performed at room temperature. 2.8 Calcium imaging Cytosolic [Ca2+]i concentrations in HUVECs were measured using the Fluo-4 Calcium Imaging Kit (Life Technologies). In brief, HUVECs were incubated with 2 mM Fluo-4/AM (Life 6

Technologies) in EGM-2 at 37°C for 10 min, followed by washing four times with Live Cell Imaging Solution (LCIS; Life Technologies). Cells were then bathed in opti-MEM with 1% fetal bovine serum and observed using an Olympus IX71 inverted microscope (Olympus, Japan). Fluorescence was excited at 494 nm and emitted at 506 nm, and then recorded, stored digitally, and analyzed using the TILLvisION 4.0 program (TILL Photonics, Germany). 2.9 Luciferase reporter assay EDN1 (ET-1 mRNA) was predicted to be directly regulated by miR-6740-5p because it contains one miR-6740-5p binding site according to TargetScan. Fragments containing the 3′-UTR of EDN1 with the miR-6740-5p binding site (GAAGGAA; wild-type) or a mutated site (GTTGGAA; mutated type) were inserted into the pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega Corporation, USA). For reporter assays, HEK293 cells were co-transfected with 50 nM miR mimics and 100 ng luciferase reporter vectors using Lipofectamine 2000 Reagent (Thermo Scientific). Twenty-four hours after transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega Corporation) according to the manufacturer’s instructions. 2.10 Plasma collection All blood samples were collected from patients and healthy volunteers from The 988 Hospital of the Chinese People's Liberation Army after obtaining informed consent and approval by the ethics committee. Plasma was extracted by centrifuging whole blood at 966 × g for 2 min at room temperature. Plasma samples were collected and divided into two aliquots, which were then stored at −80°C for analysis. 2.11 Statistical analysis GraphPad Prism 6.0 (GraphPad Software, USA) and Origin Pro 8.5 (OriginLab Corporation, USA) were used for statistical analysis. Data are expressed as the mean ± SD from at least three independent experiments. The unpaired t-test was used to compare two groups and one-way ANOVA was used to test multiple groups. P values of less than 0.05 were considered to be significant. 7

3. Results 3.1 Increasing substrate stiffness promotes endothelial adhesion

Figure 1. Cytoskeleton formation and gene expression in HUVECs cultured on substrates with varying stiffness. (A) Fluorescence images of vinculin (red), F-actin (green), and the nucleus (blue) in HUVECs cultured on soft (4 kPa), medium (25 kPa), and stiff (50 kPa) substrates for 48 h. Scale bar: 50 μm. (B-D) mRNA levels of eNOS, PGIS and ET-1 in HUVECs cultured on soft, medium, and stiff substrates for 48 h were determined by qPCR (n=3). (E-G) Levels of NO, PGI2, and ET-1 in the culture medium of HUVECs cultured on soft (4 kPa), medium (25 kPa), and stiff (50 kPa) substrates for 48 h (n=3). Data represent the mean ± SD and asterisks indicate a significant difference compared with the soft group (*P<0.05; **P<0.01; 8

***P<0.001). (H) Immunofluorescence images of eNOS (red) and the nucleus (blue) in HUVECs cultured on soft, medium, and stiff substrates for 48 h. Scale bar: 50 μm.

Dynamic regulation of the filamentous actin (F-actin) cytoskeleton is crucial for cell migration. Formation of F-actin in HUVECs that were cultured on hydrogel substrates with of three different substrate stiffnesses were was compared after staining with fluorescein isothiocyanate-conjugated phalloidin. We found that substrate stiffening increased formation of F-actin in HUVECs (Figure 1A), which is in line with previous studies [33, 34]. Vinculin, as a prominent member of the focal adhesions (FAs), is involved in linking the actin cytoskeleton to the plasma membrane. Immunofluorescent staining of vinculin in HUVECs showed that rigid substrates increase vinculin expression in HUVECs (Figure 1A). These results indicate that increased substrate stiffness strengthens the cytoskeleton in HUVECs.

3.2 Increasing substrate stiffness induces endothelial dysfunction ET-1 mRNA levels were significantly higher in HUVECs grown on a stiff substrate than on a soft substrate (Figure 1D). Similarly, substrate stiffening was accompanied by increased ET-1 levels in the culture medium (Figure 1G). In contrast, substrate stiffening reduced eNOS and PGIS mRNA levels (Figure 1B, C) and downstream NO and PGI 2 production (Figure 1E, F). Immunofluorescent staining showed reduced eNOS expression in HUVECs grown on the stiff substrate (Figure 1H). These results indicate that a stiff substrate facilitates induced endothelial dysfunction by upregulating ET-1 expression and simultaneously downregulating eNOS and PGIS expression.

3.3 miR-6740 is a stiffness-sensitive miRNA that is downregulated by a stiff substrate To identify mechanosensitive miRNAs in HUVECs in vitro, miRNA array analysis was conducted using endothelial-enriched miRNAs from HUVECs cultured on soft and stiff substrates (4 and 50 kPa). We integrated these two genome-wide datasets using a systems biology 9

approach to help us identify relevant miRNA target gene associations. The microarray data showed that 30 endothelial miRNAs had at least a 1.5-fold expression change between the two groups (22 up- and 8 down-regulated). These miRNAs were considered differentially expressed (Figure 2A and Table S2). Kyoto encyclopedia of genes and genomes pathway analysis showed that these stiffness-responsive miRNAs were involved in focal adhesion, and the PI3K-Akt and Hippo signaling pathways (Figure 2B). Additional independent RNA samples were used to validate miR-6740-5p expression using qPCR. We found that a stiff substrate induced significantly lower miR-6740-5p levels compared with a soft substrate, which was consistent with the microarray analysis (Figure 2C). Additionally, miR-6740-5p expression was detected in exosomes extracted from the culture medium. A stiff substrate also induced significantly lower miR-6740-5p levels compared with a soft substrate (Figure S1A) indicating that a soft substrate promoted miR-6740-5p secretion in vesicles. The exosomes were identified according to size and positive rates of surface markers. These findings indicate high reliability of the exosomal miRNA detection results (Figure S1C).

10

Figure 2. miR-6740-5p is a mechanosensitive miRNA that is downregulated by a stiff substrate. (A) Heat map of representative stiffness-induced miRNAs in HUVECs. Red represents high expression, while blue represents low expression. Thirty miRNAs (22 up-, 8 down-regulated after culture for 48 h) were selected on the basis of a 1.5-fold change. (B) Kyoto encyclopedia of genes and genomes analysis shows the biological processes that selected microRNAs are involved in. (C) Expression changes of miR-6740-5p and miR-214-3p were validated by qPCR after culture for 48 h (n=3). Data represent the mean ± SD and asterisks indicate a significant difference (***P<0.001).

3.4 EDN1 is targeted and directly repressed by miR-6740 A TargetScan search showed EDN1 to be a potential novel target of miR-6740-5p. The 11

EDN1 binding site for miR-6740-5p is shown in Figure 3A. To validate EDN1 as a miR-6740-5p target, the 3′-UTR of wild-type and mutant genes was cloned in the pmirGLO vector (Figure 3B). HEK293T cells were co-transfected with wild-type or mutant pmirGLO plasmids and mimics or inhibitors of miR-6740-5p. The presence of miR-6740-5p mimics and wild-type vectors inhibited luciferase expression, which indicated that EDN1 was a target of miR-6740-5p (Figure 3C). Furthermore, an effect of miR-6740-5p inhibition was an increase in EDN1 mRNA and protein levels (Figure 3D, E). These data provide compelling evidence of a novel target for miR-6740-5p.

Figure 3. ET-1 is a novel direct target of miR-6740-5p in HUVECs. (A) Potential miR-6740-5p binding sites in the 3′-UTR of human EDN1. (B) The design for wild-type and mutant fragments of the EDN1 3′-UTR. (C) Luciferase activity of wild-type or mutated EDN1 upon addition of 1 µg of either negative control miRNA or miR-6740 mimics (n=3). (D, E) ET-1 mRNA and protein production in HUVECs cultured on soft or stiff substrates with negative control miRNA, miR-6740 mimics, or inhibitors (n=3). Data represent the mean ± SD and asterisks indicate a significant difference (*P<0.05; **P<0.01; ***P<0.001).

3.5 Substrate stiffness regulates the miR-6740/ET-1 pathway via TRPV4 TRPV4 plays a vital role in sensing shear stress, osmotic pressure, and hypotonic cell swelling. However, whether TRPV4 has the same role in response to stiffness in ECs is unknown. 12

We found that a soft substrate enhanced TRPV4 mRNA and protein levels (Figure 4A, B). To assess the interplay between TRPV4 and the miR-6740/ET-1 pathway in stiffness-dependent gene regulation, HUVECs were treated with 40 nM GSK1016790A (GSK101), a TRPV4-specific agonist, and GSK2193874 (GSK219), a TRPV4-specific antagonist. GSK101 increased miR-6740-5p expression and suppressed ET-1 in HUVECs cultured on both soft and stiff substrates (Figure 4C, E). In contrast, GSK219 inhibition of the TRPV4 channel reduced miR-6740 expression and induced ET-1 production (Figure 4D, F). These results indicate that TRPV4 may be a novel mechanosensor that responds to substrate stiffness and subsequently regulates the downstream miR-6740/ET-1 pathway. In addition, treatment with agonist GSK101 (2 nM) induces PGI2 production in the HUVEC culture medium (Fig S2). This indicates that PGI2 synthesis can be reduced by TRPV4 channel activity. The mechanism of this regulation warrants future study.

Figure 4. Effects of substrate stiffness on TRPV4 expression and TRPV4-induced changes in miR-6740 and ET-1 expression in HUVECs. (A) TRPV4 mRNA levels in HUVECs cultured on 13

soft, medium, and stiff substrates for 48 h were determined by qPCR (n=3). Data are shown as the mean ± SD and asterisks indicate a significant difference compared with the soft group (***P<0.001). (B) Immunofluorescence images of TRPV4 (purple) and the nucleus (blue) in HUVECs cultured on soft, medium, and stiff substrates for 48 h. Scale bar: 50 μm. (C, D) miR-6740-5p levels in HUVECs cultured on soft and stiff substrates in the presence of GSK101 or GSK219 (2 nM) were measured by qPCR. (E, F) ET-1 in the culture medium of HUVECs cultured on soft and stiff substrates in the presence of GSK101 or GSK219 (2 nM) for 24 h. Data represent the mean ± SD and asterisks indicate a significant difference (*P<0.05; **P<0.01; ***P<0.001).

To assess

the effects

of substrate stiffness

on TRPV4 channel activity,

an

electrophysiological patch clamp test was conducted to assess TRPV4 activity in HUVECs cultured on stiff and soft substrates. During voltage ramps (±80mV > 1s), GSK101 evoked inward and outward transmembrane currents, which were abolished by GSK219. A soft substrate triggered a GSK101-evoked current response that was characterized by elevated cumulative currents (Figure 5A, B), a quick increase toward baseline (Figure 5C), and abrupt peak currents (Figure 5D) compared with a stiff substrate. Compared with HUVECs cultured on stiff substrates, calcium oscillations in HUVECs cultured on soft substrates were significantly enhanced both in frequency and amplitude under the same GSK101 dose, which was abrogated by GSK219 (Figure 6A, B). These results indicate that the soft substrate was capable of enhancing TRPV4 expression and activity, indicating that substrate stiffness regulates the miR-6740/ET-1 pathway via the TRPV4 channel.

14

Figure 5. TRPV4 activity in response to substrate stiffness. (A, B) Cumulative currents (left) and density (right) for 40 nM GSK101 and GSK101+blocker (GSK219) at ±80 mV. (C) Representative traces of GSK101-evoked currents, which were blocked by the antagonist GSK219 (40 nM), on soft (left) and stiff (right) substrates. (D) Peak current (left) and its density (right) of TRPV4 in the presence of GSK101 on soft (n=6) and stiff (n=5) substrates. Data represent the mean ± SD and asterisks indicate significant differences (*P<0.05; **P<0.01; ***P<0.001).

15

Figure 6. Soft substrate increases cytosolic Ca2+ oscillations which could be abrogated by TRPV4 antagonist GSK219 in cultured HUVECs (A) Representative images showing cytosolic Ca2+ fluorescence in HUVECs treated with GSK101 (40 nM) or GSK219 (40 nM) on soft and stiff substrates. Scale bar: 50 μm. (B) Summary of the average cytosolic Ca 2+ maximal Fluo-4/AM intensity changes in HUVECs on soft and stiff substrates (cell quantity: 50-60). Data are shown as the mean ± SD and asterisks indicate significant differences (*P<0.05; **P<0.01; ***P<0.001).

3.6 Low plasma miR-6740 levels in patients with carotid atherosclerosis Finally, based on the in vitro findings that miR-6740 is a stiffness-sensitive miRNA, we aimed to evaluate whether humans suffering from carotid atherosclerosis (i.e., displaying vascular stiffening [35, 36]) have lower miR-6740-5p expression in plasma. Carotid atherosclerosis was defined as carotid intima-media thickness (IMT) >1.2 mm and the presence of plaques according to previous studies [37, 38]. Plaque was defined as the presence of focal lesion resulting in a thickness greater than 1.5 mm [39]. The control group included 16

volunteers with a carotid intima-media thickness (IMT) <1.2 mm and no plaques. Clinical characteristics for all participants are shown in Table 1. Age, sex, body mass index, and plasma levels of triglyceride, total cholesterol, high-density, and low-density lipoprotein cholesterol were not significantly different between the case and control groups. The case group, however, showed significantly lower levels of plasma miR-6740-5p compared with the control group (Figure 7). This finding indicates that miR-6740-5p is a novel, promising, prognostic biomarker for carotid atherosclerosis. Table1. Baseline characteristics of carotid atherosclerosis (AS) cases and controls. Case (n=21)

Control (n=15)

P value 0.115

64  4

62  6

Gender male, n (%)

9 (42.86)

5 (33.33)

Body mass index, kg/m2

24.1  2.8

25.2  2.9

Triglyceride (mmol/L)

1.4  0.8

1.5  0.6

0.699

Total cholesterol (mmol/L)

5.1  1.2

4.9  1.3

0.612

HDL-C (mmol/L)

1.3  0.3

1.2 0.3

0.483

LDL-C (mmol/L)

2.8  0.9

2.7 0.8

0.837

Smokers, n (%)

3 (14.29)

2 (13.33)

Alcohol drinkers, n (%)

1 (4.76)

1 (6.67)

CVD family history, n (%)

3 (14.29)

1 (6.67)

Statin history, n (%)

6 (28.57)

3 (20.00)

Age, years

a b

a,b

Data are shown as the mean  SD or number (%). Smoking was defined as having one or more cigarettes in one month. Alcohol drinker refers to drinking more than

twice in a month. CVD family history was defined as at least one family member suffering cardiovascular disease. Statin history was defined as receiving statin-like drug treatment.

17

Figure 7. Plasma miR-6740 levels in carotid atherosclerosis. Plasma miR-6740 levels in the control group and in patients with carotid atherosclerosis (case group) are shown. Data represent the mean ± SD and asterisks indicate a significant difference (***P<0.001).

Figure 8. Representative diagram of how matrix stiffening induces endothelial dysfunction via the TRPV4/miRNA-6740/ET-1 signaling pathway.

4. Discussion 18

Clinical evidence shows that vascular stiffening is associated with the prognosis of CVD. However, the precise mechanism of this association has not been investigated. In this study, we found

that

substrate

stiffening

induced

endothelial

dysfunction

through

a

novel

TRPV4/miR-6740/ET-1 mechanotransduction pathway. This molecular evidence advances understanding of the molecular pathogenesis of CVD. Our findings also provide a basis for developing novel preventive and therapeutic strategies for CVD via targeting the TRPV4/miR-6740/ET-1 axis. Vascular stiffening, which is a consequence of structural alterations of the vessel wall, is a marker of aging or early vascular disease [7], and is an independent predictor of all-cause mortality in CVD [8]. Clinical investigations have indicated that vascular stiffening is closely related to endothelial dysfunction in hypertension, diabetes, and coronary artery disease [13, 14]. However, direct experimental evidence that shows precisely how vascular stiffening affects endothelial dysfunction is still limited. Peng et al. found that increased wall stiffness suppresses Akt and eNOS phosphorylation in pulse-perfused endothelium [40]. Chang et al. further showed that NO production in endothelial cells decreases with increased stiffness of culture substrates [41]. Our results indicated that the increasing substrate stiffness reduced the mRNA and protein expression of eNOS. Simultaneously, we found for the first time, that increasing substrate stiffness promoted ET-1 expression, which plays a major role in endothelial function, via a novel mechanotransduction pathway. These results provide direct experimental evidence for the concept that vascular stiffening induces endothelial dysfunction, and suggest that vascular stiffness is a potential therapeutic target in CVD treatment. TRPV4, a Ca2+-permeant cation channel, is expressed in endothelial cells and is involved in the regulation of vascular tone [42]. Previous research demonstrated that TRPV4 channel plays a pivotal role in the maintenance of cardiovascular homeostasis and promotes vascular smooth muscle cell proliferation and migration [43-45]. TRPV4 is activated by both chemical and physical stimuli, including pressure [46], moderate warmth [47], membrane stretch [48], hypotonic cell swelling [49], and shear stress [50]. TRPV4 expression is significantly decreased 19

in the arteries of stroke-prone spontaneously hypertensive rats [51]. In addition, the hypertension in these rats is associated with arterial stiffening [5, 52]. These studies indicate that increased vascular stiffness may inhibit TRPV4 expression in vivo, although this requires verification. This study improves our understanding of the interactions between endothelial function and the extracellular microenvironment and also provides insight for the design of cardiovascular implants. Promoting formation of an endothelial cell monolayer and endothelial function on implant materials is an effective approach for preventing complications of post-implantation [53, 54]. Not enough attention has been paid to providing a physiologically compatible mechanical environment that facilitates endothelial function and prevents complications. We suggest that more research is needed to improve the mechanical properties of implant materials to promote endothelial cell function and thereby reduce implantation complications. In summary, our study shows that the TRPV4/miR-6740/ET-1 mechano-transduction pathway is an important mechanism by which substrate stiffness affects endothelial function (Figure 8). Additionally, plasma miR-6740-5p levels have the potential to be used as a biomarker in CVD. These findings add to our understanding of the interaction between vascular stiffening and endothelial dysfunction in the pathogenesis of CVD and complications of cardiovascular implants. Moreover, this study may also provide a basis for developing novel preventive and therapeutic strategies for CVD treatment.

Acknowledgments This work has been supported by grants from the National Natural Science Foundation of China (Grant Nos. 81600148 and 81700181).

Disclosure The authors declared no potential conflicts of interest with respect to the research, 20

authorship, and publication of this article.

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Graphical abstract

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