Pers reverse angiotensin II -induced vascular smooth muscle cell proliferation by targeting cyclin E expression via inhibition of the MAPK signaling pathway

ABSTRACT The circadian rhythm of blood pressure (BP) is believed to be regulated by the clock system, which is closely linked to levels of angiotensin II (Ang II). This study aimed to investigate whether Ang II mediates the proliferation of vascular smooth muscle cells (VSMCs) through the interaction between the clock system and the mitogen-activated protein kinase (MAPK) signaling pathway. Primary rat aortic VSMCs were treated with Ang II, with or without MAPK inhibitors. VSMC proliferation, expression of clock genes, CYCLIN E, and MAPK pathways were assessed. Ang II treatment resulted in increased VSMC proliferation and rapid upregulation of clock gene Periods (Pers) expression. Compared to the non-diseased control (NC) group, VSMCs incubated with Ang II displayed a noticeable delay in the G1/S phase transition and downregulation of CYCLIN E upon silencing of Per1 and Per2 genes. Importantly, silencing Per1 or Per2 in VSMCs led to decreased expression of key MAPK pathway proteins, including RAS, phosphorylated mitogen-activated protein kinase (P-MEK), and phosphorylated extracellular signal-regulated protein kinase (P-ERK). Moreover, the MEK and ERK inhibitors, U0126 and SCH772986, significantly attenuated the Ang II-induced proliferation of VSMCs, as evidenced by an increased G1/S phase transition and decreased CYCLIN E expression. The MAPK pathway plays a critical role in regulating VSMC proliferation in response to Ang II stimulation. This regulation is controlled by the expression of circadian clock genes involved in the cell cycle. These findings provide novel insights for further research on diseases associated with abnormal VSMC proliferation.


Introduction
Hypertension, the leading contributing factor to allcause global mortality (Mills et al. 2016), has witnessed a steady increase in the number of patients worldwide over the past 40 years (Stafoggia et al. 2014). It is projected that by 2025, the global number of hypertensive patients will reach 1.56 billion (Forouzanfar et al. 2017). Although the circadian rhythm of blood pressure (BP) in humans has been known for several decades, the underlying mechanisms behind the disruption of this rhythm and its association with adverse cardiovascular outcomes remain unclear (Douma et al. 2018). The objective of this study is to explore the pathological mechanisms that drive abnormal blood pressure rhythms, with the aim of facilitating the development of improved anti-hypertensive drugs or treatments.
In mammals, various behavioral and physiological processes, including metabolism, energy homeostasis, heart rate, BP, and cytokine secretion, exhibit circadian rhythms that are governed by a clock system (Bollinger and Schibler 2014). This system comprises the central circadian clock located in the hypothalamic suprachiasmatic nucleus (SCN) and peripheral clocks present in multiple peripheral tissues (Lin et al. 2014). The central clock receives photic information from the retina and synchronizes peripheral clocks with the external light/ dark cycles through neural and humoral pathways (Shostak 2017). At the molecular level, four families of core clock genes (Clock, Bmal1, Per, and Cry) form a transcription-translation feedback loop that operates on a nearly 24-hour cycle in all nucleated cells (Gerber et al. 2013;Roenneberg and Merrow 2016). This feedback loop involves a positive limb (Bmal1 and Clock) and a negative limb (Per and Cry). The Bmal1/Clock heterodimer binds to the E-boxes within the promoters of Cry and Per, initiating their transcription. Subsequently, the PER and CRY proteins form a complex that inhibits the positive limb, leading to rhythmic oscillations (Panda et al. 2002;Takahashi 2017).
Vascular smooth muscle cells (VSMCs) play a crucial role in maintaining the structural and functional integrity of blood vessel walls, including processes such as contraction, relaxation, growth, development, remodeling, and repair (Nonaka et al. 2001). Abnormal proliferation of VSMCs is closely associated with vascular remodeling and stiffening, contributing to the initiation and progression of various vascular diseases, including hypertension, atherosclerosis, myocardial hypertrophy, and renal failure (Kirschenlohr et al. 1996;Sun et al. 2015). Furthermore, VSMCs exhibit a circadian oscillation system, and the activity of circadian clock genes contributes to the daily regulation of VSMC proliferation and vasoconstriction Nonaka et al. 2001;Su et al. 2012;Xie et al. 2015). Angiotensin II (Ang II), a peptide hormone that binds to the corresponding type 1 (AT1) receptor, induces phenotypic changes and abnormal proliferation of VSMCs, leading to impaired vasoconstriction and increased blood pressure (Qu et al. 2016). Interestingly, Ang II is also involved in the regulation of circadian rhythm. Nonaka et al. demonstrated that Ang II can induce oscillations of clock genes in VSMCs (Nonaka et al. 2001). Furthermore, our research indicated that the inhibition of AT1 receptors can modulate the circadian rhythm of Ang II and restore the expression of circadian clock genes in the abdominal aorta, thereby reversing the non-dipper blood pressure pattern . Notably, administering valsartan (VAL) during sleep time, as opposed to awake time, was found to be more effective in blocking Ang II, leading to distinct circadian rhythms of Per1 and Per2 in VSMCs and ultimately correcting MLC20 phosphorylation, which may be involved in regulating circadian blood pressure patterns . However, the precise mechanism underlying the regulation of clock genes induced by Ang II remains incompletely understood.
The MAPK signaling cascade serves as a crucial mechanism for inter-and intra-cellular communication, facilitating various fundamental cell functions such as growth, survival, and differentiation (Ambrosio et al. 1989;Degirmenci et al. 2020;Mulcahy et al. 1985). Research has demonstrated that the core protein mitogen-activated protein kinase (MEK)/RAS/extracellular signal-regulated protein kinase (ERK) orchestrates these cellular processes (Ambrosio et al. 1989;Degirmenci et al. 2020;Mulcahy et al. 1985). Moreover, studies have revealed the activation of the MAPK signaling pathway upon the combination of Ang II and AT1 receptor, resulting in VSMC proliferation (Eguchi et al. 1996;Liao et al. 2012). Furthermore, evidence suggests that the MAPK signaling pathway also contributes to the regulation of circadian rhythms (Coogan and Piggins 2004). Circadian variations in the phosphorylation levels of ERK have been observed in various tissues, such as the mouse SCN, the rat pineal gland, the mouse liver, and the bullfrog retina. The circadian oscillation of ERK protein phosphorylation is under the control of circadian core clock components (Akashi and Nishida 2000;Ho et al. 2003;Obrietan et al. 1998;Sanada et al. 2000;Tsuchiya et al. 2013). In mammalian circadian rhythms, the MAPK pathway plays a significant role in clock resetting (Akashi and Nishida 2000). However, the precise involvement of the MAPK pathway in the circadian regulation of VSMCs remains poorly understood to date.
In this study, our hypothesis centered on the notion that Ang II-induced clock gene expression plays a pivotal role in the phenotypic changes and proliferation of VSMCs, primarily mediated through the MAPK pathway. To investigate this, we assessed the circadian expression patterns of clock genes, RAS/ERK proteins, and CYCLIN E in VSMCs. Simultaneously, we analyzed the cell cycle distribution and cell viability of VSMCs. Our findings revealed that Ang II regulates the timedependent expression of Per1 and Per2 in VSMCs, while also activating the MAPK pathway, which subsequently leads to abnormal proliferation of VSMCs. This work not only contributes to the understanding of the pathological mechanisms underlying abnormal circadian rhythms in hypertension but also identifies potential therapeutic targets for the clinical treatment of hypertension.

Cell culture
The substrate-attached explant methods were applied to culture primary rat VSMCs obtained from the abdominal aorta of eight-week-old Sprague-Dawley (SD) rats. This animal experiment was permitted by Ethics Committee of Wannan Medical College (NO: LLSC−2022-078). All operations comply with regulations and regulations. The cells were cultured at 37°C in 5% CO 2 /95% air. After about seven days, the VSMCs began to migrate from the explants. At 80% −90% confluence, the cells were passaged by 0.25% trypsin-Ethylene Diamine Tetraacetic Acid (EDTA) solution. VSMCs were isolated from the tissue block after 10 days of culture ( Figure S1A,B). The identity of the cells was verified by the highly expressed VSMC α-actin (α-SMA) (Abcam, Massachusetts, USA) via immunohistochemistry ( Figure S1C,D). Six to eight passages of cells were used for the following study.

Serum shocking
The cell culture media was substituted with Dulbecco's modified eagle medium (DMEM) plus 50% fetal bovine serum (FBS) to induce circadian-clock-gene oscillations. When the VSMCs were began to incubated with 50% serum shocking, this time point was recorded as Zeitgeber time [ZT] 0. After 2 hours of cultivation with 50% serum shocking, it is recorded as ZT2. This procedure induces alterations in circadian rhythms of affected cells Lin et al. 2014;Nonaka et al. 2001). Serum-free DMEM containing gradient concentration Ang II (Santa Cruz, CA, USA) or other inhibitors were added at ZT2 and cell were first collected after 1 h at ZT3 after washed twice with phosphate buffered saline (PBS). Then the cells were collected at an interval of every 4 h. 1 μM valsartan (Santa Cruz, CA, USA), MEK\ERK protein inhibitors 10 μM U0126 (APExBIO, Houston, USA), and 500 nM SCH772984 (APExBIO, Houston, USA) were used as inhibitors in this experiment.

RNA extraction and real-time polymerase chain reaction (RT-PCR)
RNA was extracted from isolated VSMCs using TRIzol (Beyotime, Shanghai, China) according to the manufacturer's instructions. The RNA concentration was determined through photometric measurement on the Nanodrop 2000 (BioTeke Corp, Beijing, China). Quantitative RT-PCR was performed on the Bio-Rad CFX96 system (Bio-Rad, Hercules, CA, USA) according to the manufacturer's manual. The relative quantification of gene expression was analyzed from the measured threshold cycles (CT) using the 2 −ΔΔCt method (Lin et al. 2014). All primers were purchased from Generary Biotech Co., Ltd. (Shanghai, China). The target gene names and their primer sequences are shown in Table S1. RT-PCR was performed as previously described ).

Western blotting
Samples were homogenized in lysis buffer. A protein assay kit bicinchoninic acid (BCA; Beyotime, Shanghai, China) was used to measure the total protein in the supernatant. Total protein was separated in SDS-PAGE (Beyotime, Shanghai, China) and transferred onto 0.2-μm polyvinylidene fluoride (PVDF) membranes (Beyotime, Shanghai, China). Information about the antibodies used in this study is shown in Table S2. Western blotting was performed as previously described ) the information about antibodies used in this study were shown in Table S2.

Cell transfection
Rat Per1 and Per2 cDNA was cloned and inserted into p3×FLAG-CMV−10 plasmid (GENERAL BIOL CO., LTD, Chuzhou, China). VSMCs were cultured until 90% confluence in six-well plates. According to the manufacturer's protocol, the VSMCs were transfected with sequence-specific SiRNA (RIBOBIO CO., LTD, Guangzhou, China) using Lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China). The working concentration of SiRNA and plasmid in the cell experiments were 50 nmol/L. Non-Targeting SiRNA was used as negative control (Shen et al. 2022). After 48 h of incubation, the transfected VSMCs were collected. Total RNA was extracted and the transfection efficiency was verified by RT-PCR. The target sequences of Per1 and Per2 SiRNA were used in this study is shown in Table S3. For Per1 gene, SiRNA−1 show the best interference efficiency among three SiRNA sequences and for Per2 gene, SiRNA−2 show the best interference efficiency. But the efficiency of co-knockdown/cooverexpression is decreased compare to single Per1 or Per2 knockdown/overexpression, respectively.

Cell vitality assay
We used 3-(4,5-dimethylthiazol−2-yl)−2,5-diphenyltetrazolium bromide (MTT) to analyze the proliferation activity of VSMCs. Twenty-four hours after cell transfection, the VSMCs were inoculated into the 96-well plates at 1000 cells/well. Then, we fostered them for 24 h and determined the cell viability. We added 20-μl MTT (5 mg/ml) to each well and discarded the culture after normal fostering for 4 h. We next added 100-μl dimethyl sulfoxide (DMSO) to each well and incubated in the dark for 10 min. We tested the absorbance at 490 nm using a microplate reader. At least three separate repeated experiments were performed.

Cell cycle analysis
For cell cycle analysis, the cells were harvested with trypsin, the cell concentration was adjusted to 1 million/ml, and 1 ml was collected for standby. The staining solution was prepared according to the instructions of the cell cycle detection kit, and 500 μL of working solution was required for each sample. After centrifugation, the supernatant was carefully removed. Next, we added 1 ml of precooled 75% ethanol to resuspend and fix overnight at − 20°C. We washed the fixative with PBS before staining, and centrifuged at 1500 rpm for 3 min. Next, we added 500 μL of dyeing solution, prepared in advance, and incubated in the dark for 1 h at room temperature. Following incubation, the staining solution was removed with PBS. The fractions of cells present in each phase of the cell cycle (G 0 /G 1 , S, and G 2 /M) were determined by flow cytometry using the Attune NxT flow cytometer (Thermo Fisher Scientific, Shanghai, China) and FlowJo software.

Statistical method
All experiments were repeated at least three times and data from the results were expressed as mean ± standard deviation (SD), and SPSS 26.0 and GraphPad Prism 8.0 were utilized for the data analysis. The comparison between the two groups was measured by the one-way analysis of variances (ANOVA), followed by a leastsignificant difference (LSD)-test or Dunnett's T3, and values of P < 0.05 suggested the differences were statistically significant. The presence of circadian rhythms is detected by JTK_CYCLE, an algorithm that operates in the statistical language R.

Measurement of Per1 and Per2 expression rhythm in VSMCs
To confirm the rhythmic expression of clock genes following a 50% serum shock, we conducted an analysis of Per1 and Per2 expression at 4-hour intervals over a duration of 35 hours. The results indicated that VSMCs displayed cyclic oscillations, with peak expression occurring at approximately 24 hours and exhibiting a period of approximately 24 hours ( Figure S2). Furthermore, the oscillations exhibited a progressively increasing trend. These findings provide compelling evidence that VSMCs possess a circadian rhythm following the 50% serum shock.

Ang II increases the proliferation of VSMCs might related with Pers gene
To assess the impact of Ang II on VSMCs, cells were subjected to various concentrations of Ang II for a 24hour period. The results demonstrated a dosedependent increase in VSMC proliferation upon Ang II treatment ( Figure 1A). Additionally, there was a notable elevation in the percentage of VSMCs in the S-phase of the cell cycle ( Figure 1B,C). Notably, the administration of VAL effectively reversed the effects induced by Ang II on VSMCs.
Subsequently, SiRNA was utilized to individually or concurrently knockdown the Per1 and Per2 genes. The knockdown efficacy for Per1 and Per2 was approximately 50% and 82%, respectively. However, the co-knockdown efficacy of Per1 and Per2 (Per1/2) was slightly lower compared to the efficacy observed with single knockdown ( Figure 1D,E). Following the knockdown procedure, cell viability was significantly reduced in both the Per1 and Per2 single knockdown groups, as well as the Per1/2 coknockdown group, compared to the negative control (NC) group ( Figure 1F). Moreover, a notable increase in the percentage of VSMCs in the G1-phase was observed following Per1 and Per2 knockdown ( Figure 1G,H).

Ang II mediates Pers genes and proteins time-dependent expression in VSMCs
Following serum shocking for a duration of 23 hours, the administration of Ang II (100 nM) resulted in significant alterations in the mRNA expression of Per1 in VSMCs. Specifically, at ZT3 and ZT7, there was a notable increase, while at ZT19 and ZT23, a decrease was observed compared to the control group cells (Figure 2A). The expression of the Per2 gene exhibited a significant increase at ZT3 but decreased at ZT19 and ZT23 ( Figure 2A). These findings indicate that shortterm stimulation with Ang II leads to an increase in Per1 and Per2 mRNA expression, while prolonged stimulation results in a decrease. Notably, the effects of Ang II on Per1 and Per2 mRNA expression were effectively reversed by the administration of VAL.
Moreover, the relative expression of PER1 protein in VSMCs displayed significant down-regulation at ZT7 and ZT11, and up-regulation at ZT3, ZT19, and ZT23 in the Ang II group compared to the control group ( Figure 4B). Similar to PER1, the expression of PER2 protein in VSMCs exhibited significant down-regulation at ZT3 and ZT7, and up-regulation at ZT19 and ZT23 following Ang II incubation ( Figure 4C). These results demonstrate the timedependent regulation of clock gene expression by Ang II, as evidenced by changes in Per2 mRNA and PER2 protein levels. Furthermore, these findings suggest that Ang II can negatively feedback regulate the expression of Pers genes and the biogenesis of PERs proteins in VSMCs, and this effect can be reversed by the administration of VAL.

Pers might regulate Cyclin E expression in VSMCs
The 24-hour expression rhythm of Cyclin E mRNA in VSMCs is depicted in Figure 3(A). Comparing to the control group, the expression of Cyclin E in the Ang II group exhibited significant increases at ZT3 and ZT7. This observation suggests a potential relationship between Cyclin E and the Pers genes, as the Pers genes also showed increased expression shortly after Ang II stimulation. To investigate this further, we conducted overexpression experiments of the Per1 or Per2 gene, as well as co-overexpression experiments of the Per1/2 genes, in VSMCs. The overexpression of Per1 and Per2 genes resulted in approximately 9-fold and 17-fold increases in expression, respectively, compared to the negative control (NC) group cells after transfection. However, the transfection efficacy was relatively lower in the cooverexpression group cells ( Figure 3B,C).
Significant decreases in the relative expression of Cyclin E mRNA were observed following single or coknockdown of the Pers genes in VSMCs. Conversely, Figure 1. The phenotypic transformation of vascular smooth muscle cells (VSMCs) induced by Ang II might in relation to Pers. n = 3. Note. The VSMCs were initially treated with 50% FBS at ZT 0 (zeitgeber time 0). After a two-hour incubation period (ZT 2), the 50% FBS was removed, and the cells were exposed to serum-free DMEM containing varying concentrations of Ang II. Subsequently, the cells were divided into five groups: control group, Ang II gradient group, and Ang II gradient group + VAL (1 μM) group. Following a 24hour culture period, the cells were collected for analysis. (A) Cell proliferation rate was assessed for the control group, Ang II gradient group, and Ang II + VAL (1 μM) group. (B) The periodic distribution of the control group, Ang II gradient group, and Ang II + VAL (1 μM) group was examined. (C) Quantitative analysis was performed to evaluate the periodic distribution. (D) Relative expression of Per1 mRNA after SiRNA of Per1 and Per1/2; (E) Relative expression of Per2 mRNA after SiRNA of Per1 and Per1/2; (F) Inhibitor concentrations after silencing of Per1, Per2 and Per1/2 genes within 24 or 48 hours; (G) Periodic distribution of VSMCs after SiRNA of Per1, Per2 and Per1/2; (H) Quantitative analysis of periodic distribution. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 vs the untreated control. # P < 0.05 vs the Ang II (100 nM)-treated cells.
significant increases in the relative expression of Cyclin E mRNA were observed following single or cooverexpression of the Pers genes in VSMCs ( Figure 3D). Furthermore, the relative expression of CYCLIN E protein exhibited significant decreases following single or co-knockdown of the Pers genes in VSMCs. However, co-knockdown of Pers genes did not have an additive effect on CYCLIN E protein expression compared to the single Pers knockdown group cells ( Figure 3E). Instead, only cooverexpression of Pers genes led to an increase in CYCLIN E protein expression ( Figure 3F). These results indicate that the Pers genes may regulate Cyclin E expression.

Clock gene expression rhythm coordinate with that of RAS, P-MEK, and P-ERK
We investigated the expression rhythm of MAPK pathway proteins in VSMCs over a 24-hour period. In comparison to the control group, the Ang II group exhibited significant downregulation of RAS protein at ZT7 and ZT11, while it showed significant upregulation at ZT15 and ZT23 ( Figure 4A). Moreover, the Ang II group Note. When the cells reached approximately 80% confluence, they were treated with medium containing 50% fetal bovine serum (FBS) at ZT 0. After a two-hour incubation period (ZT 2), the medium containing 50% FBS was replaced with serum-free DMEM supplemented with Ang II (100 nmol). The samples were collected at specific time points: ZT 3, ZT 7, ZT 11, ZT 15, ZT 19, and ZT 23. (A) Relative expression levels of Per1 and Per2 mRNA were determined. (B) The relative expression of PER1 protein was assessed. (C) Relative expression levels of PER2 protein were evaluated. The data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs the untreated control; # P < 0.05, ## P < 0.01 vs the Ang II-treated cells. demonstrated significant downregulation of P-MEK protein at ZT3, ZT7, and ZT11, and upregulation at ZT15 and ZT19 ( Figure 4B). Additionally, the expression of P-ERK protein was significantly downregulated at ZT7 and ZT11, but upregulated at ZT19 and ZT23 ( Figure 4C). VAL partially reversed the effects of Ang II on the MAPK pathway. These findings indicate that Ang II decreases the expression of core MAPK proteins in the short term, but increases it over a longer duration, which is consistent with the expression patterns of PER1 and PER2 proteins in VSMCs.

Pers might regulate RAS, P-MEK, and P-ERK protein expression in VSMCs
The relative expression of RAS, P-MEK, and P-ERK proteins was significantly inhibited in cells from the Per1 SiRNA, Per2 SiRNA, and Per1/2 SiRNA groups compared to the NC group ( Figure 5A-C). Moreover, no additional inhibitory effect was observed in the Per1/ 2 SiRNA group cells compared to the single knockdown groups of Per1 or Per2 ( Figure 5A-C). Conversely, there were no significant differences in the relative expression of RAS, P-MEK, and P-ERK proteins between the single knockdown groups (Per1 or Per2) and the NC group ( Figure 5A-C). However, the relative expression of RAS, P-MEK, and P-ERK proteins was significantly increased in the Per1/2 group cells compared to the NC group cells ( Figure 5D-F). These findings suggest that Pers may play a regulatory role in the MAPK pathway in VSMCs.

MAPK pathway mediates VSMC proliferation through regulating Cyclin E expression
The MEK and ERK inhibitors, U0126 and SCH772986, significantly reduced the proliferation-promoting effect of Ang II ( Figure 6A). The distribution of the cell cycle revealed a significant increase in the percentage of S phase cells in the Ang II group compared to the control group cells. However, in the inhibitor group, the G1 phase was significantly increased, while the G2 phase was significantly decreased compared to the Ang II group (Figure 6B,C). The VSMCs exhibited G1 delay after being incubated with MEK and ERK inhibitors. Additionally, the MEK and ERK inhibitors significantly reversed the increased expression of Cyclin E gene and protein induced by Ang II (Figure 6D-F). These results suggest that the MAPK pathway might be involved in

Discussion
VSMC proliferation is closely associated with vascular remodeling and stiffening (Kirschenlohr et al. 1996;Sun et al. 2015). Ang II, the primary hormone influencing VSMCs, not only mediates immediate physiological effects such as vasoconstriction and BP regulation but also regulates the circadian clock gene rhythm in VSMCs through the AT1 receptor (Liao et al. 2012). In a study by Takaguri et al. (2020), it was discovered that the clock gene Bmal1 is involved in PDGF-BB-induced cell proliferation, partly through the ERK pathway in VSMCs. We speculated whether a similar mechanism occurs in VSMC proliferation mediated by Ang II. In our previous research, we demonstrated that Ang II Figure 4. The expression rhythm of RAS, P-MEK, and P-ERK proteins in VSMCs following incubation with angiotensin II (Ang II) with and without valsartan (VAL). n = 3. Note. The VSMCs were initially treated with medium containing 50% fetal bovine serum (FBS) at ZT 0. After a two-hour period (ZT 2), the medium containing 50% FBS was removed, and the cells were subsequently incubated with serum-free DMEM containing Ang II (100 nM), with or without VAL (1 μM) treatment. The samples were collected at six different time points: ZT 3, ZT 7, ZT 11, ZT 15, ZT 19, and ZT 23. (A) Relative expression of RAS protein was assessed after incubation with Ang II, with or without VAL treatment. (B) Relative expression of MEK phosphorylation was evaluated after incubation with Ang II, with or without VAL treatment. (C) Relative expression of ERK phosphorylation was analyzed after incubation with Ang II, with or without VAL treatment. The data is presented as mean ± standard deviation (SD). Statistical significance is denoted as follows: * P < 0.05, ** P < 0.01 vs Control group; # P < 0.05, ## P < 0.01 vs Ang II group.
significantly enhances VSMC proliferation, while VAL reverses its effects. The key findings of our present study are as follows: (i) Ang II regulates the rhythmic expression of clock genes Per1 and Per2, with negative feedback regulation between Pers mRNA and PERs protein; (ii) Per1 and Per2 might regulate the MAPK pathway; and (iii) the MAPK pathway is involved in the expression and regulation of CYCLIN E, thus influencing VSMC proliferation.
Ang II can regulate the rhythm of clock gene expression either directly or indirectly, influencing the 24hour rhythmic expression of clock genes in the SCN and VSMCs (Campos et al. 2013;Nonaka et al. 2001). Studies on Ang II-treated rats have shown a significant phase advance in the expression of Per2 and significant modifications in the expression of clock-controlled genes such as rev-erb alpha and dbp (Herichova et al. 2013). Acute changes in the renin-angiotensin system can also alter clock gene expression (Naito et al. 2003). Furthermore, Ang II-treated cells derived from circadian clock reporter mice demonstrated decreased Per2 promoter activity (Pati et al. 2016), highlighting the reciprocal relationship between angiotensin signaling and the circadian clock. It has also been reported that Ang II initially increases the activation of AT1 receptors, but chronic exposure leads to downregulation of these receptors, resulting in tissue desensitization to further Ang II stimulation (Nonaka et al. 2001). In our study, we observed that Ang II had a similar effect on regulating the circadian clock through the AT1 receptor. The expression of Per1 and Per2 mRNA rapidly increased after Ang II stimulation but decreased after long-term stimulation. We speculate that circadian rhythms may regulate the desensitization effect of Ang II on VSMCs; however, further studies are needed to better understand the complexities underlying desensitization and its impact on VSMC proliferation. Additionally, several studies have reported phase delays between clock gene mRNA and protein expression. For instance, in the SCN of SD rats under light-dark conditions, the clock protein cycles were delayed by 4-6 hours compared to the clock gene mRNA cycles (Li et al. 2007). Consistent with this, we observed a phase shift between the expression of mRNA and protein in our study.
Although we observed similar regulatory trends of Per1 and Per2 after Ang II stimulation, as well as a phase shift between mRNA and protein expression, there were slight differences in the time-dependent regulation of Per1 and Per2 at specific time points. We propose two possible reasons for these results. Firstly, existing literature has demonstrated that the timing of peak and trough in the periodic oscillations of Pers gene expression in in vitro experiments is time-dependent, with a constant anti-phase difference between limbs. The exact timing of phase and oscillations of the core clock, and their correlation with functional output oscillations, may vary from cell to cell, tissue to tissue, and from in vitro to in vivo. Secondly, Per1 and Per2 may play distinct roles in circadian oscillation. Ogawa et al. suggested that Per1 is part of a morning oscillator tracking dawn, while Per2 is part of an evening oscillator tracking dusk (Steinlechner et al. 2002). Furthermore, Riddle et al. demonstrated differential localization between Per1 and Per2 in the brain's master circadian clock. The central SCN exhibited the highest abundance of PER1 protein, while higher expression of PER2 protein was observed in the rostral, dorsal, and caudal aspects compared to PER1 (Riddle et al. 2017). Therefore, the differences in PER1 and PER2 expression at certain time points may arise from variations in their functions and structures. Figure 6. The expression of Cyclin E after incubation with Ang II with or without MAPK pathway inhibitor in VSMCs. n = 3. Note. The VSMCs were treated with MEK/ERK protein inhibitors, U0126 (10 μM) and SCH772984 (500 nM), which were added to the Ang II (100 nM) group at ZT 2. The cells were then divided into the following four groups: Control group, Ang II (100 nM) group, Ang II (100 nM) + U0126 (10 μM) group, and Ang II (100 nM) + SCH772984 (500 nM) group. (A) Proliferation rate of VSMCs after incubation with Ang II with or without the MAPK pathway inhibitor; (B) Periodic distribution of VSMCs after incubation with Ang II with or without the MAPK pathway inhibitor; (C) Quantitative analysis of periodic distribution; (D) Relative expression of CYCLIN E after incubation with Ang II with or without the MAPK pathway inhibitor. Data are presented as mean ± SD. *P < 0.05, **P < 0.01 vs Control group; # P < 0.05, ## P < 0.01 vs Ang II group.
MAPKs have been shown to influence the cycling of the circadian clock. Exposure to high doses of damaging UV light can activate conserved MAPK pathways through signal transduction mechanisms at any time of day (Allada and Meissner 2005). Supporting the role of ERK in light signaling to the clock, infusion of an ERK inhibitor into the mouse SCN prevented phase shifts in activity rhythms when administered during the subjective night (Butcher et al. 2002). Furthermore, MAPKs can physically and/or genetically interact with components of the molecular circadian oscillator (Goldsmith and Bell-Pedersen 2013). In mammals, the p38 MAPK pathway is believed to impact the circadian oscillator after activation by the inflammatory cytokine TNF-α (Petrzilka et al. 2009;Zarubin and Han 2005). Studies have also demonstrated that JNK kinase modulates the properties of the circadian oscillator by phosphorylating clock proteins in both the positive and negative branches of the oscillator (Uchida et al. 2012;Yoshitane et al. 2012). While most studies have focused on the input signal pathways of how exogenous signals regulate the clock system, few have examined the output pathway. In our study, we found evidence that the clock genes can regulate core proteins of the MAPK pathway, such as RAS, ERK, and MEK. These findings suggest a mechanism involving bidirectional regulation between the clock system and the MAPK pathway.
The MAPK pathway plays a significant role in VSMC proliferation. Multiple studies have reported the inhibitory effects of rubiarbonone C on VSMC proliferation and migration through the inhibition of the MAPK signaling pathway (Wan et al. 2018). Specifically, the suppression of p38 MAPK signaling pathway has been shown to inhibit VSMC proliferation (Wan et al. 2018). Furthermore, cyclins are known to be involved in VSMC proliferation, and the inhibition of the MAPK pathway has been demonstrated to suppress VSMC proliferation by regulating cell cycle proteins (Begum et al. 2011;Tao et al. 2011). Additionally, the MAPK signaling pathway regulates the expression of Cyclin D1, which leads to changes in the cell cycle (Qin et al. 2014). Our findings indicate that the MAPK signaling pathway can directly or indirectly modulate Ang II-induced VSMC proliferation through altered expression of circadian clock genes involved in the cell cycle.
Disruption of the circadian clock has been implicated in the pathogenesis of cardiovascular disorders . Notably, routine ingestion of antihypertensive drugs at bedtime in hypertensive patients has been shown to result in blood pressure decline and reduced occurrence of major cerebrovascular diseases (Hermida et al. 2020) However, the underlying mechanism of how the clock system and vessel function are integrated remains unclear. In this regard, our study suggests that Per1 and Per2 may reverse Ang II-induced VSMC proliferation by inhibiting the MAPK signaling pathway. These results contribute to a better understanding of the relationship between circadian clocks and the pathogenesis of cardiovascular disorders associated with VSMC proliferation. Nevertheless, research on the effects of clock genes on VSMC proliferation through the MAPK pathway has been limited. Takaguri et al. (Takaguri et al. 2020) reported the involvement of Bmal1 in PDGF-BB-induced cell proliferation, partially through ERK in VSMCs. However, their study did not investigate Ang II-induced VSMC proliferation and only focused on one MAPK pathway protein, ERK protein. In contrast, our present study provides a more comprehensive analysis of the mechanisms underlying clock gene regulation of Ang II-induced VSMC proliferation.
Our study has several limitations that should be acknowledged. Firstly, we focused on investigating the role of a single cyclin gene, CYCLIN E, in VSMC proliferation regulated by the circadian system. It is worth noting that other studies have demonstrated that drugs can inhibit VSMC proliferation by modulating the cell cycle through the detection of a different periodic gene, such as Cyclin D (Lee et al. 2013;Sun et al. 2019). Wang et al. have specifically identified the involvement of the CYCLIN E-CDK2 pathway in the regulation of VSMC proliferation and morphology . While our results regarding CYCLIN E are convincing, further investigations incorporating other cyclin genes would provide a more comprehensive understanding of the overall role of cyclins in VSMC proliferation. Another limitation of our study is that we solely conducted in vitro experiments, focusing on the detection of mRNA and protein expression. However, previous studies have explored the relationship between clock genes and VSMC function in vivo. For instance, Durgan et al. have demonstrated time-of-day-dependent oscillations in circadian clock genes and vascular function in the rat cerebral vasculature (Durgan et al. 2017). Sun et al. have also shown altered expression of circadian clock genes in the abdominal aorta of type 2 diabetic mice, which correlated with diurnal VSMC contractility (Sun et al. 2019). Therefore, future research should include animal experiments to better understand the complexities underlying the relationship between the clock system and VSMC proliferation. In summary, while our study provides valuable insights, it is important to address the limitations mentioned above and conduct further research to investigate the involvement of other cyclin genes and explore the in vivo relationship between clock genes and VSMC function. This will enhance our understanding of the intricate interplay between the clock system and VSMC proliferation.

Conclusions
Our findings provide valuable insights into the regulatory relationship between Ang II, the circadian clock, and VSMC proliferation. Specifically, we observed that Ang II can modulate the expression rhythm of clock genes in a time-dependent manner, suggesting a dynamic interaction between angiotensin signaling and the circadian clock. Moreover, we found evidence of desensitization between these two systems. Notably, we observed a delayed peak expression of clock proteins relative to clock mRNA in VSMCs following Ang II treatment, indicating a temporal discrepancy in the clock gene expression and protein synthesis process. Furthermore, our study revealed that the clock system exerts regulatory control over key proteins in the MAPK pathway, including RAS, ERK, and MEK phosphorylation. This highlights the involvement of the MAPK pathway in the regulation of VSMC proliferation under the influence of Ang II, which is mediated by the expression of circadian clock genes related to the cell cycle ( Figure 7). Overall, our findings contribute to a better understanding of the mechanisms underlying hypertension and cardiovascular diseases associated with abnormal VSMC proliferation. By elucidating the intricate interplay between Ang II, the circadian clock, and the MAPK pathway, our study opens up new avenues for future research and potential therapeutic interventions in these conditions.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The data that support the findings of this study are openly available in https://doi.org/10.6084/m9.figshare.14988309.v1 Figure 7. The mechanism of clock genes regulates the proliferation of vascular smooth muscle cells (VSMCs). Note. Angiotensin II (Ang II) induces circadian clock genes expression changes, while valsartan (VAL) can revert these changes, and then clock genes control VSMCs proliferation through regulating cell cycle via the mitogen-activated protein kinase (MAPK) signaling pathway. Ang II, angiotensin II; VAL, Valsartan; PER1, clock gene period1; PER2, clock gene period2; MEKs, ERKs, RAF, RAS, core protein of MAPK signaling pathway; GDP, Guanosine diphosphate; GTP, guanosine triphosphate; P, phosphorylation; CYCLIN E, E-type cyclins.