INTRODUCTION

Hypertension, which is a serious chronic disease, is a key risk factor for cardiovascular adverse events. Vascular remodeling is the pathological basis of hypertension and includes histological or functional changes, such as thickening of the vessel wall, an increase in the vessel wall thickness/lumen ratio, an increase in the extracellular matrix and the phenotypic transformation of vascular smooth muscle cells (VSMCs) [1]. Many studies have shown that improving vascular remodeling can reduce blood pressure (BP) in animal models of hypertension.

Specialized pro-resolving lipid mediators (SPMs) are a kind of endogenous small molecular substances with anti-inflammatory and pro-inflammatory resolution effects that are produced by the metabolism of Ω-3 and Ω-6 polyunsaturated fatty acids (PUFAs) [2,3]. Some clinical studies have shown that supplementation with PUFAs can reduce BP in hypertensive patients and reduce the risk of hypertension in normal individuals [4–6]. As metabolites of PUFAs, SPMs may have stronger biological effects and antihypertensive effects. Resolvin D1 (RvD1) is an SPM derived from docosahexaenoic acids that has significant anti-inflammatory and pro-inflammatory resolution effects [2,3]. RvD1 can inhibit inflammation, reduce cardiomyocyte apoptosis and improve myocardial injury [7]. It can also resolve inflammation in the spleen and ventricle after myocardial infarction and improve cardiac function [8]. A recent study showed that RvD1 could prevent Ang II-induced hypertension [9]. However, the mechanism by which RvD1 improves hypertension is unclear.

Ras homolog gene family member A (RhoA) is a GTP-binding protein, and its downstream signaling pathways, including the mitogen-activated protein kinase (MAPK)-signaling pathway, can regulate cell proliferation, migration, differentiation and apoptosis [10,11]. During hypertension, the RhoA-related signaling pathway mainly affects the proliferation, migration and differentiation of VSMCs, thus affecting hypertension [12–14]. Interestingly, RvD1 has been shown to inhibit the activation of RhoA [15].

Therefore, we hypothesize that RvD1 plays an important role in vascular remodeling in hypertension by regulating the proliferation, migration and phenotypic transformation of VSMCs by inhibiting the RhoA-related signaling pathway.

MATERIALS AND METHODS Reagents

Ang II was purchased from Enzo Life Sciences (ALX-151–039-M025, Farmingdale, New York, USA). RvD1 was purchased from Cayman Chemical (10012554, Ann Arbor, Michigan, USA). The RhoA agonist U46619 [16] was purchased from ApexBio Technology (B6890, Houston, Texas, USA).

Animals

All animal care and experimental procedures were in accordance with the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 2011) and were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Wuhan, China).

Male C57BL/6J mice between 8 and 9 weeks old and weighing 23–25 g were obtained from GemPharmatech (Nanjing, China). The mice were maintained in a standard laboratory at the Cardiovascular Research Institute of Wuhan University. The animals were kept in cages with light/dark cycles of 12 h and had free access to food and water. After acclimating to the environment for 2 weeks, the animals were used for the experiments.

Ang II-induced hypertension model and treatments

The mice were anesthetized by an intraperitoneal injection (i.p.) of 3% amobarbital at a dose of 70 mg/kg. The hair on the left shoulder blade was shaved, a small incision was made in the middle of the shoulder blade, and the skin and fascia were separated by blunt dissection with hemostatic forceps. Then, an osmotic pump (Alzet, model 2004) was placed, and the wound was sutured and disinfected. The mice were allowed to wake up in the incubator before going back to their cages. For control mice, the wound was closed without implanting the pump. The mice were infused with Ang II (750 ng/min/kg) for 28 days by pumps, and saline or RvD1 (2 μg/kg/day, i.p.) was administered daily starting on the day of the surgery and lasting until the animals were euthanized. After 28 days, the mice were euthanized, and aortic tissue was removed for follow-up biological analysis.

Blood pressure measurement

A carotid catheter-calibrated tail-cuff system (CODA, Kent Scientific, Torrington, Connecticut, USA) was used. Every other day for 1 week prior to pump implantation, the mice were placed on the scaffold, the BP meter was activated for 10 min to acclimate, and the animals were then allowed to return to the cage. The day before surgery, the mice were tethered to the scaffold, and their tails were heated on a heating pad for 20 min before 15 cycles basal SBP, DBP, mean BP (MBP) and heart rate (HR) measurements was performed. This procedure was repeated at 7, 14 and 28 days after Ang II infusion.

Histological analysis

Aortic tissue was separated. After being fixed with 4% paraformaldehyde for 3 days, the tissue was embedded in paraffin and sliced into approximately 5-μm-thick sections. Aortic media thickness was analyzed by hematoxylin–eosin staining, and Masson trichrome staining was used to examine collagen deposition. The sections were then visualized, and images were captured by light microscopy (Olympus, Japan). Media thickness and the area of collagen deposition were measured using a quantitative digital image analysis system (ImagePro Plus, version 6.0).

Immunofluorescence analysis

The aortic tissue was fixed with 4% paraformaldehyde for 3 days and embedded in paraffin wax. The samples were sliced into approximately 5-μm-thick sections. After 5 min of high-pressure antigen repair (sodium citrate buffer, 100×, pH 6.0), the sections were incubated in PBS containing 10% fetal bovine serum (FBS) for 60 min. Then, the aortic sections were incubated overnight at 4 °C with the following primary antibodies: antialpha smooth muscle actin (α-SMA) (ab5694, Abcam, Cambridge, UK), antismooth muscle protein 22-α (SM22α) (GB11366, Servicebio, Wuhan, China), and antiosteopontin (OPN) (22952-1-AP, Proteintech, Wuhan, China). The sections were washed with PBS and incubated with appropriate secondary antibodies at 37 °C for 1 h. The nuclei were stained with 4,6 diamino-2-phenylindole (DAPI). Images were collected by a fluorescence microscope (Olympus, Japan) and DP2-BSW software (Version 2.2) and analyzed in a blinded manner by Image-Pro Plus (Version 6.0) software.

Cell culture and treatments

VSMCs were extracted from the aortic tissue of 4-week-old rats. The cells were cultured in Dulbecco's modified eagle medium/nutrient mixture F-12 (DMEM/F12) culture medium (SH30023.01, Cytiva) supplemented with 10% FBS (10099-141, Gibco, Grand Island, New York, USA) at 37 °C in a humidified 5% CO2 incubator. The culture medium was refreshed every 3 days. VSMCs were used at passages 3–7. The cells were exposed to Ang II (1000 nmol/l) or U46619 (100 nmol/l) for 24 h. Prior to Ang II treatment, VSMCs were pretreated with RvD1 (1, 10, and 100 nmol/l) for 30 min.

Cell proliferation assessment

Cell proliferation was measured using Cell Counting Kit-8 (ANT181, AntGene, Wuhan, China) and EdU imaging kits (K1075, ApexBio Technology) according to the manufacturer's instructions.

Wound healing assay

VSMCs were seeded into 6-well plates, grown to fill with wells, and incubated in FBS-free medium for 24 h before a sterile pipette tip was used to wound the cells. After being rinsed with PBS, the cells were treated with Ang II, U46619 and RvD1. The cells were observed under a light microscope (Olympus, Japan) at 0 and 24 h. Different images were randomly collected, the area of cell migration was determined, and the rate of cell migration was calculated by Image-Pro Plus (Version 6.0) software.

Western blotting

Aortic tissue and VSMCs were lysed in RIPA lysis buffer containing protease and phosphatase inhibitors. The samples were further homogenized by ultrasound and then centrifuged at 12 000g for 30 min. Total protein was collected, and the concentration was determined with a BCA protein assay kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA). The protein concentration was analyzed after normalization. The proteins (50 μg) were separated by 10% SDS–PAGE and transferred to Immobilon-FL polyvinylidene fluoride (PVDF) membranes (IPFL00010, Merck Millipore Ltd, Darmstadt, Germany). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated overnight at 4 °C on a shaker with the following primary antibodies: antiα-SMA (ab5694, Abcam), anti-SM22α (GB11366, Servicebio), anti-OPN (22952-1-AP, Proteintech), antiproliferating cell nuclear antigen (PCNA) (GTX100539, GeneTex, Irvine, California, USA), anti-RhoA (10749-1-AP, Proteintech), antiphospho-p44/42 MAPK (P-ERK1/2) (4370, Cell Signaling Technology, Danvers, Massachusetts, USA), antip44/42 MAPK (T-ERK1/2) (4695, Cell Signaling Technology), antiphospho-SAPK/JNK (P-JNK1/2) (4668, Cell Signaling Technology), anti-SAPK/JNK (T-JNK1/2) (9258, Cell Signaling Technology), antiphospho-p38 MAPK (P-P38) (4511, Cell Signaling Technology), antip38 MAPK (T-P38) (8690, Cell Signaling Technology), and antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) (AC027, Abclonal, Wuhan, China). In next day, the membranes were treated with goat antirabbit IgG (#7074, Cell Signaling Technology) or goat antimouse IgG (#7076, Cell Signaling Technology) secondary antibodies at room temperature for 1 h in the dark. After being washed three times in the dark, the membranes were scanned to measure the band intensities by an Odyssey Imaging System (LI-COR Biosciences, Lincoln, USA). The expression levels of the target proteins were normalized to GAPDH.

Statistical analysis

All data are expressed as the mean ± standard deviation (SD) and were analyzed by GraphPad Prism 7 software. Differences among groups were determined by one-way ANOVA or two-way ANOVA followed by Tukey's post hoc tests. P less than 0.05 was considered statistically significant.

RESULTS Resolvin D1 reduced Ang II-induced hypertension in mice

To clarify the role of RvD1 in hypertension, we established an Ang II-induced hypertension mouse model. The experimental protocol is shown in Fig. 1a. We measured BP once per week for 4 weeks after the mice were implanted with an osmotic pump filled with Ang II. We analyzed BP data measured at the same heart rate and found that the group treated with only Ang II had significantly higher SBP and DBP than the control group, and this increase in BP was suppressed by treatment with RvD1 (Fig. 1b–d). Apparently, RvD1 could reduce Ang II-induced hypertension in mice.

FIGURE 1:

The effects of resolvin D1 in Ang II-induced hypertension model in mice. (a) The experimental protocol. (b–d) SBP and DBP of the mice measured at similar HR once a week for 4 weeks after the mice were placed an osmotic pump prefilled with Ang II (n = 10–12) (SBP, DBP and HR was analyzed by two-way ANOVA followed by Tukey's post hoc tests). Hematoxylin–eosin (e and f) and Masson (g and h) staining were used for displaying the aortic medium thickness and vascular fibrosis, respectively (n = 5) (scale bar, 50 μm). Data are presented as the mean ± SD. ∗P < 0.05 compared with the Vehicle group, #P < 0.05 compared with the Ang II group. HR, heart rate.

Resolvin D1 inhibited Ang II-induced vascular remodeling in mice

We further examined vascular remodeling, which is the pathological basis of hypertension. Aortic tissues were sectioned and stained with hematoxylin–eosin. The results revealed that RvD1 treatment reduced Ang II-induced thickening of the aortic media (Fig. 1e–f). Masson trichrome staining was used to examine collagen deposition. Similarly, after Ang II treatment, fibrosis in the aorta was significantly aggravated, and RvD1 effectively reduced collagen deposition and improved vascular fibrosis (Fig. 1g–h).

In addition to morphological changes, phenotypic transformation of VSMCs was examined. Immunofluorescence was used to visualize changes in aortic tissues. We found that α-SMA and SM22α, which are markers of contractile VSMCs, were significantly downregulated, whereas OPN, which is a marker of synthetic VSMCs, was significantly upregulated, and these changes were reversed by RvD1 (Fig. 2a–d). To further verify this result, we examined the expression of related proteins in aortic tissue by Western blotting. As expected, the results were consistent with the immunofluorescence analysis (Fig. 2e–h).

FIGURE 2:

Resolvin D1 inhibited Ang II-induced phenotypic transformation of vascular smooth muscle cells in aortic. Immunofluorescence showed the expression of α-SMA, SM22α and OPN (a) in the heart and quantitative comparison among different groups (b–d) (n = 5) (scale bar, 50 μm). Protein expression of α-SMA, SM22α and OPN was tested by Western blotting (e). GAPDH was used as internal control and quantification of the α-SMA (f), SM22α (g) and OPN (h) level in the indicated groups (n = 4). Data are presented as the mean ± SD. ∗P < 0.05 compared with the Vehicle group, #P < 0.05 compared with the Ang II group.

Resolvin D1 inhibited the proliferation and migration of vascular smooth muscle cells after Ang II stimulation in vitro

To clarify the mechanism by which RvD1 improves Ang II-induced vascular remodeling, we examined the cellular function of VSMCs. We treated VSMCs with different concentrations of RvD1, and cell proliferation was determined by the Cell Counting Kit-8 assay. The results showed that 100 nmol/l RvD1 inhibited the proliferation of VSMCs after Ang II stimulation (Fig. 3a). Therefore, we used this concentration to stimulate the cells in subsequent experiments. EdU imaging kits showed that more proliferating cells were labeled after Ang II stimulation, and this effect was inhibited by treatment with RvD1 (Fig. 3b and c). In addition to cell proliferation, migration was also evaluated by using a wound healing assay. The ability of cells to migrate was significantly enhanced after stimulation with Ang II, and this effect was decreased by RvD1 treatment (Fig. 3d and e).

FIGURE 3:

Resolvin D1 inhibited the proliferation and migration of VSMCs after Ang II stimulation. (a) Different doses of RvD1 (1 nmol/l, 10 nmol/l, 100 nmol/l) were used to treat VSMCs and determined cell proliferation using the Cell Counting Kit-8 (n = 10). (b and c) EdU Imaging Kits (Cy3) were used to display and quantify proliferating cells for comparison between groups (n = 7–8) (scale bar, 50 μm). (d) Wound healing assay recorded the migration of VSMCs after 24 h of different stimulation (scale bar, 500 μm). (e) The rate of cell migration was calculated and compared (n = 4). Data are presented as the mean ± SD. ∗P < 0.05 compared with the PBS group, #P < 0.05 compared with the Ang II group.

Resolvin D1 inhibited the phenotypic transformation of vascular smooth muscle cells after Ang II stimulation in vitro

We confirmed that RvD1 attenuated Ang II-induced phenotypic transformation in VSMCs in vivo. Relevant parameters were also examined in vitro. After Ang II stimulation, the expression of α-SMA and SM22α was decreased, whereas that of OPN and PCNA was increased, and these effects were reversed by RvD1 (Fig. 4a–e).

FIGURE 4:

Resolvin D1 inhibited the phenotypic transformation of VSMCs after Ang II stimulation. Protein expression of α-SMA, SM22α, OPN and PCNA was tested by Western blotting (a). GAPDH was used as internal control and quantification of the α-SMA (b), SM22α (c), OPN (d) and PCNA (e) level in the indicated groups (n = 4). Data are presented as the mean ± SD. ∗P < 0.05 compared with the PBS group, #P < 0.05 compared with the Ang II group.

Resolvin D1 attenuated the proliferation, migration and phenotypic transformation of VSMCs after Ang II stimulation by inhibiting the RhoA/MAPK pathway

Studies have shown that RhoA/MAPK can promote the proliferation, migration and differentiation of VSMCs during hypertension [11,17,18]. Therefore, we examined the changes in the expression of this pathway in vitro and in vivo. The in-vivo results showed that the expression of RhoA was significantly increased in the Ang II group, and this effect was inhibited by treatment with RvD1 (Fig. 5a–b). The downstream MAPK signaling pathway also showed the same trend (Fig. 5a,c–e). In vitro, we obtained similar results (Fig. 5f–j).

FIGURE 5:

Resolvin D1 inhibited the expression of RhoA/MAPK pathway after Ang II treatment in vivo/in vitro. (a and f) Protein expression of RhoA/MAPK signaling pathway in vivo/in vitro was tested by Western blotting. GAPDH was used as internal control and quantification of RhoA (b and g). T-ERK, T-JNK and T-P38 were used as internal control and quantification of the P-ERK (c and h), P-JNK (d and i) and P-P38 (e and j) level in the indicated groups, respectively (n = 4). Data are presented as the mean ± SD. ∗P < 0.05 compared with the Vehicle/PBS group, #P < 0.05 compared with the Ang II group.

To further verify whether RvD1 improves vascular remodeling by inhibiting the RhoA/MAPK pathway, we used the RhoA agonist U46619 in vitro. RvD1 reduced Ang II-induced proliferation of VSMCs, the ameliorative effect was abrogated by U46619 (Fig. 6a–c). U46619 also counteracted RvD1-mediated inhibition of the migration of VSMCs (Fig. 6d–e). Similarly, RvD1 upregulated the markers of contractile VSMCs (α-SMA and SM22α) and downregulated the markers of synthetic VSMCs (OPN and PCNA), and these effects could be reversed by U46619 (Fig. 7a–e). Then, we examined the activation of the RhoA/MAPK pathway, and U46619 abolished the inhibitory effect of RvD1 on the activation of the RhoA/MAPK pathway (Fig. 7a,f–i). These results indicated that the RhoA/MAPK pathway could mediate the protective effects of RvD1 against the proliferation, migration and phenotypic transformation of VSMCs.

FIGURE 6:

U46619 reversed the inhibitory effect of resolvin D1 on the proliferation and migration of VSMCs after Ang II stimulation. (a) Cell Counting Kit-8 was used to determine cell proliferation (n = 15). (b and c) EdU Imaging Kits (Cy3) was used to examine the proportion of proliferating cells (n = 6) (scale bar, 50 μm). (d and e) Wound healing assay recorded the migration of VSMCs after 24 h of different stimulation (scale bar, 500 μm), the rate of cell migration was calculated and compared (n = 6). Data are presented as the mean ± SD. ∗P < 0.05 compared with the Ang II group, #P < 0.05 compared with the Ang II+RvD1 group.

FIGURE 7:

U46619 reversed the inhibitory effect of resolvin D1 on the phenotypic transformation and the expression of RhoA/MAPK pathway in VSMCs after Ang II stimulation. (a) Protein expression of α-SMA, SM22α, OPN, PCNA and RhoA/MAPK signaling pathway in vitro was tested by Western blotting. GAPDH was used as internal control and quantification of the α-SMA (b), SM22α (c), OPN (d), PCNA (e), RhoA (f). T-ERK, T-JNK and T-P38 were used as internal control and quantification of the P-ERK (g), P-JNK (h) and P-P38 (i) level in the indicated groups respectively (n = 4). Data are presented as the mean ± SD. ∗P < 0.05 compared with the Ang II group, #P < 0.05 compared with the Ang II+RvD1 group.

DISCUSSION

In this study, we explored the role of RvD1, an internal metabolite of PUFAs, in hypertension. This study showed that RvD1 treatment could inhibit Ang II-induced hypertension and vascular remodeling. Mechanistically, RvD1 was considered a novel inhibitor of RhoA, and it could inhibit RhoA/MAPK pathway activation to inhibit Ang II-induced proliferation, migration and phenotypic transformation of VSMCs, thereby reducing BP, thickening of the aortic media and vascular fibrosis (Fig. 8). Thus, this study provides compelling evidence supporting a protective effect of RvD1 against hypertension and vascular remodeling. RvD1 may be a new drug for treating hypertension or other vascular remodeling-related diseases.

FIGURE 8:

Resolvin D1 attenuates Ang II-induced hypertension through inhibiting the proliferation, migration and phenotypic transformation of vascular smooth muscle cells via blocking the RhoA/MAPK pathway.

Vascular remodeling is a typical pathological feature of many cardiovascular diseases, such as atherosclerosis, aortic dissection, arterial aneurysm and hypertension. SPMs are endogenous small molecules with anti-inflammatory and pro-inflammatory resolution effects, and an increasing number of SPM-related genetic models and pharmacological experiments have shown that SPMs protect against the occurrence and development of vascular remodeling [19]. The levels of SPMs, including RvD1, resolvin D2, and resolvin E1, are significantly reduced in atherosclerosis [20,21]. Numerous studies have shown that different SPMs, when combined with corresponding receptors, could improve vascular remodeling in atherosclerosis by inhibiting inflammation and oxidative stress, thereby reducing the activation of VSMCs or promoting the transformation of macrophages to repair phenotypes [21–25]. Moreover, SPMs can modulate the phenotype of VSMCs and are correlated with peripheral atherosclerosis [26]. Similarly, in aortic dissection and arterial aneurysm, SPMs alleviated vascular remodeling mainly through anti-inflammatory and pro-inflammatory resolution effects [27–29]. In addition, SPMs could inhibit the proliferation, migration and phenotypic transformation of VSMCs, thus reducing vascular remodeling after arterial injury [30,31].

Previous studies have shown that there is an imbalance between proinflammatory and resolution mediators in hypertension, and low SPM levels may be an indicator of the development of hypertension [32,33]. Resolvin D2 has been proven to improve Ang II-induced hypertension by modulating vascular factors, fibrosis and inflammation to protect cardiovascular structure and function [33]. RvD1 also plays a cardioprotective role in angiotensin II-induced hypertension and cardiac remodeling, but the mechanism by which RvD1 improves hypertension is unclear [9]. In this study, we used RvD1 prophylactically to examine its role in hypertension. This study further confirmed that RvD1 could attenuate Ang II-induced hypertension by inhibiting the proliferation, migration and phenotypic transformation of VSMCs.

The biological activities of SPMs are mediated by their cognate G protein-coupled transmembrane superfamily receptors (GPCRs) [34]. Two specific GPCRs for RvD1 have been identified: formyl peptide receptor 2 (FPR2) and G protein-coupled receptor 32 (GPR32) [34,35]. FPR2 and GPR32 are mainly expressed in immune cells, including polymorphonuclear cells, macrophages and T lymphocytes, and they are also expressed in endothelial cells and VSMCs [35]. As GPR32 has only been shown to be expressed in human cells and its homology has not been determined in mice, the effect of RvD1 on a mouse model could be mediated by FPR2 [35,36]. Although the role of RvD1 has been studied more extensively in immune cells than other cell types, recent evidence suggests that VSMCs express RvD1 receptors, and many direct effects of RvD1 have been observed in the vasculature. Multiple in-vivo and in-vitro models have demonstrated that RvD1 treatment can inhibit the proliferation, migration and phenotypic transformation of VSMCs [30,37–39]. These effects of RvD1 could affect the structure and function of vessels. Vascular remodeling is an important pathophysiological basis of hypertension, and VSMCs play an important role. Therefore, we focused on the effect of RvD1 on the cellular function of VSMCs. Notably, RvD1 may affect hypertension through other factors, such as various immune cells or endothelial cells, which needs to be further explored.

RhoA has been widely studied in hypertension. The role of RhoA-related signaling pathways in regulating cell proliferation, migration, differentiation, and apoptosis in hypertension has been examined by many studies [40,41]. As a downstream target of RhoA, the MAPK signaling pathway plays an important role in this process [18]. RvD1 promotes the targeting and clearance of necroptotic cells by inhibiting RhoA activation [15]. The MAPK signaling pathway can also be blocked by treatment with SPMs, including RvD1, in many diseases [7,42,43]. Similarly, we found RhoA/MAPK pathway was inhibited after the treatment with RvD1 in Ang II-induced hypertensive mice and Ang II-stimulated VSMCs. In addition, the contractility of VSMCs is also a key factor in hypertension. Previous studies have shown that SPMs, including RvD1, can inhibit the constriction of the rat thoracic aorta and human pulmonary artery induced by the thromboxane mimetic U46619 [44]. The contractility of VSMCs is related to cytosolic Ca2+ concentrations and the Ca2+ sensitivity of myofilaments, and it has been well documented that the RhoA-related signaling pathway is involved in this process [17,45]. Therefore, RvD1 improves hypertension not only by improving the cellular function of VSMCs but also by correcting the abnormal contractility of VSMCs.

This study used an Ang II-induced hypertension mouse model and Ang II-stimulated rat VSMCs to examine the role of RvD1 in vascular remodeling, which has led to limitations regarding species. Although the species used in vivo and in vitro are different, similar studies have been recognized and widely used in hypertension-related studies [46,47]. Therefore, our study is reliable. In addition, previous studies have shown that the expression levels of SPMs or their precursors are decreased in hypertensive patients or animal models of hypertension [32,48]. This indicates that the results of the present study are credible and provide a new target and important theoretical basis for the treatment of human hypertension. Of course, we will design further studies to verify the effect of RvD1 on humans.

Our study still has other limitations. First, we selected the dose of RvD1 based on previous studies. The dose-dependent effect of RvD1 on improving hypertension needs to be further examined. In this study, we used RvD1 prophylactically and did not examine its therapeutic effects, which may require further experiments. In addition, although we have established that RvD1 can improve hypertension by regulating the cellular function of VSMCs, it may also act on other cells, such as immune cells and endothelial cells, which is worth exploring.

CONCLUSION

This study provides direct evidence that RvD1 attenuates Ang II-induced hypertension by inhibiting the proliferation, migration and phenotypic transformation of VSMCs by blocking the RhoA/MAPK pathway. This provides new ideas for the diagnosis and treatment of hypertension.

ACKNOWLEDGEMENTS

Funding: this work was supported by grants from the National Natural Science Foundation of China (No. 82270454, 82100292 and 82070436) and Excellent Doctoral Program of Zhongnan Hospital of Wuhan University (ZNYB2022001).

Conflicts of interest

There are no conflicts of interest.

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