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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell Dev. Biol.</journal-id>
<journal-title>Frontiers in Cell and Developmental Biology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell Dev. Biol.</abbrev-journal-title>
<issn pub-type="epub">2296-634X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="publisher-id">829316</article-id>
<article-id pub-id-type="doi">10.3389/fcell.2022.829316</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cell and Developmental Biology</subject>
<subj-group>
<subject>Original Research</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Ferroptosis due to Cystathionine &#x3b3; Lyase/Hydrogen Sulfide Downregulation Under High Hydrostatic Pressure Exacerbates VSMC Dysfunction</article-title>
<alt-title alt-title-type="left-running-head">Jin et&#x20;al.</alt-title>
<alt-title alt-title-type="right-running-head">Ferroptosis Facilitates VSMC Dysfunction</alt-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Jin</surname>
<given-names>Ruxi</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1586175/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Yang</surname>
<given-names>Ruixue</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1631715/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cui</surname>
<given-names>Changting</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1524338/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Zhang</surname>
<given-names>Haizeng</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1643767/overview"/>
</contrib>
<contrib contrib-type="author">
<name>
<surname>Cai</surname>
<given-names>Jun</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1394613/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Geng</surname>
<given-names>Bin</given-names>
</name>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/19135/overview"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Chen</surname>
<given-names>Zhenzhen</given-names>
</name>
<xref ref-type="corresp" rid="c001">&#x2a;</xref>
<uri xlink:href="https://loop.frontiersin.org/people/1258435/overview"/>
</contrib>
</contrib-group>
<aff>
<institution>State Key Laboratory of Cardiovascular Disease</institution>, <institution>National Center for Cardiovascular Diseases</institution>, <institution>Hypertension Center</institution>, <institution>Fuwai Hospital</institution>, <institution>Chinese Academy of Medical Sciences and Peking Union Medical College</institution>, <addr-line>Beijing</addr-line>, <country>China</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>
<bold>Edited by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/946394/overview">Zhi Qi</ext-link>, Nankai University, China</p>
</fn>
<fn fn-type="edited-by">
<p>
<bold>Reviewed by:</bold> <ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/1587013/overview">Yongsheng Chang</ext-link>, Tianjin Medical University, China</p>
<p>
<ext-link ext-link-type="uri" xlink:href="https://loop.frontiersin.org/people/910602/overview">Jinwei Tian</ext-link>, The Second Affiliated Hospital of Harbin Medical University, China</p>
</fn>
<corresp id="c001">&#x2a;Correspondence: Bin Geng, <email>bingeng@hsc.pku.edu.cn</email>; Zhenzhen Chen, <email>chenzhenzhen@bjmu.edu.cn</email>
</corresp>
<fn fn-type="other">
<p>This article was submitted to Cell Death and Survival, a section of the journal Frontiers in Cell and Developmental Biology</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>03</day>
<month>02</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2022</year>
</pub-date>
<volume>10</volume>
<elocation-id>829316</elocation-id>
<history>
<date date-type="received">
<day>05</day>
<month>12</month>
<year>2021</year>
</date>
<date date-type="accepted">
<day>10</day>
<month>01</month>
<year>2022</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2022 Jin, Yang, Cui, Zhang, Cai, Geng and Chen.</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Jin, Yang, Cui, Zhang, Cai, Geng and Chen</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these&#x20;terms.</p>
</license>
</permissions>
<abstract>
<p>Hydrostatic pressure, stretch, and shear are major biomechanical forces of vessels and play critical roles in genesis and development of hypertension. Our previous work demonstrated that high hydrostatic pressure (HHP) promoted vascular smooth muscle cells (VSMCs) two novel subsets: inflammatory and endothelial function inhibitory VSMCs and then exacerbated VSMC dysfunction. However, the underlying mechanism remains unknown. Here, we first identified that aortic GPX4 (a core regulator of ferroptosis) significantly downregulated association with VSMC novel phenotype elevation in SHR rats and hypertension patients. In primary VSMCs, HHP (200&#xa0;mmHg) increased iron accumulation, ROS production, and lipid peroxidation compared with normal pressure (100&#xa0;mmHg). Consistently, the ferroptosis-related gene (<italic>COX-2</italic>, <italic>TFRC</italic>, <italic>ACSL4</italic>, and <italic>NOX-1</italic>) expression was also upregulated. The ferroptosis inhibitor ferrostatin-1 (Fer-1) administration blocked HHP-induced VSMC inflammatory (CXCL2 expression) and endothelial function inhibitory (AKR1C2 expression) phenotyping switch association with elevation in the GPX4 expression, reduction in the reactive oxygen species (ROS), and lipid peroxidation production. In contrast, the ferroptosis inducer RLS3 increased HHP-induced CXCL2 and AKR1C2 expressions. These data indicate HHP-triggering ferroptosis contributes to VSMC inflammatory and endothelial function inhibitory phenotyping switch. In mechanism, HHP reduced the VSMC GSH content and cystathionine gamma-lyase (CSE)/hydrogen sulfide (H<sub>2</sub>S)&#x2014;an essential system for GSH generation. Supplementation of the H<sub>2</sub>S donor-NaHS increased the VSMC GSH level, alleviated iron deposit, ROS and lipid peroxidation production. NaHS administration rescues both HHP- and RLS3-induced ferroptosis. Collectively, HHP downregulated VSMC CSE/H<sub>2</sub>S triggering GSH level reduction, resulting in ferroptosis, which contributed to the genesis of VSMC inflammation and endothelial function inhibitory phenotypes.</p>
</abstract>
<kwd-group>
<kwd>hydrostatic pressure</kwd>
<kwd>ferroptosis</kwd>
<kwd>hydrogen sulfide</kwd>
<kwd>VSMCs</kwd>
<kwd>hypertension</kwd>
</kwd-group>
</article-meta>
</front>
<body>
<sec id="s1">
<title>Introduction</title>
<p>Biomechanical forces within the vasculature contain wall shear stress, circumferential wall tensile stress, and hydrostatic pressure (<xref ref-type="bibr" rid="B29">Takayama et&#x20;al., 2013</xref>), and these biomechanics contribute to pathogenesis and development of hypertension and its complications (<xref ref-type="bibr" rid="B10">Hayashi and Naiki, 2009</xref>). Shear stress disorder impaired endothelial function and involved in vascular remodeling, atherosclerosis, plaque progression, and vascular malformations (<xref ref-type="bibr" rid="B43">Zhou et&#x20;al., 2014</xref>). The mechanical cyclic stretch force also contributes to vascular smooth muscle cell functions (e.g., apoptosis, proliferation, and migration), which is crucial in vascular remodeling during hypertension (<xref ref-type="bibr" rid="B7">Cheng et&#x20;al., 2012</xref>; <xref ref-type="bibr" rid="B21">Qi et&#x20;al., 2018</xref>), whereas little attention is paid to hydrostatic pressure regulation. A recent study has shown that high hydrostatic pressure (HHP)&#x2013;driving VSMCs differentiated into inflammatory subset (elevating CXCL2, CXCL3, and CCL2) and endothelial function inhibitory subset (upregulating AKR1C2, AKR1C3, and PEDF) by single-cell sequencing and exacerbated the VSMC dysfunction (<xref ref-type="bibr" rid="B6">Chen Z. et&#x20;al., 2021</xref>). However, the underlying mechanism remains unknown.</p>
<p>Ferroptosis is a novel type of programmed cell death, characteristic as iron overload, reactive oxygen species (ROS) accumulation, iron-dependent lipid peroxidation, and mitochondrial shrinkage (<xref ref-type="bibr" rid="B39">Wu et&#x20;al., 2021</xref>). Ferroptosis is triggered by the cystine/glutamate antiporter system (Xc<sup>&#x2212;</sup> system) dysfunction, glutathione (GSH) depletion, and glutathione-dependent antioxidant enzyme glutathione peroxidase 4 (GPX4) inactivation (<xref ref-type="bibr" rid="B42">Yang and Stockwell, 2016</xref>). GSH is an important antioxidant, synthesized from cysteine and oxidizes to oxidized glutathione (GSSG) dependent on GPX4 (<xref ref-type="bibr" rid="B1">Badgley et&#x20;al., 2020</xref>). Meanwhile, GPX4 also reduced cytotoxic lipid peroxide (L-OOH) into nontoxic alcohol L-OH in the presence of the cofactor GSH (<xref ref-type="bibr" rid="B26">Stockwell et&#x20;al., 2017</xref>). Thus, GPX4/GSH is an essential regulatory system in ferroptosis. Recently, many studies highlighted that ferroptosis is a crucial pathophysiological process in chronic diseases such as cancer, diabetes, and nervous system disease (<xref ref-type="bibr" rid="B23">Qiu et&#x20;al., 2020</xref>). Over iron deposits induced ferroptosis causing cardiomyopathy (<xref ref-type="bibr" rid="B9">Gujja et&#x20;al., 2010</xref>) or development of vulnerable plaques (<xref ref-type="bibr" rid="B34">Vinchi et&#x20;al., 2020</xref>). Recombinant human GPX4 (<xref ref-type="bibr" rid="B15">Liu et&#x20;al., 2021</xref>) and ferroptosis inhibitor Fer-1 (<xref ref-type="bibr" rid="B14">Li et&#x20;al., 2020</xref>) treatment attenuated myocardial injuries. These works indicate that ferroptosis contributes to the pathogenesis of cardiovascular diseases.</p>
<p>Cystathionine-&#x3b3;-lyase (CSE) endogenously produces hydrogen sulfide (H<sub>2</sub>S) exerting a cardiovascular protective role and as the key enzyme for <sc>l</sc>-cysteine (precursor of GSH) (<xref ref-type="bibr" rid="B11">Kimura, 2021</xref>). H<sub>2</sub>S plays an antioxidative role by facilitating the GSH content and removing ROS (<xref ref-type="bibr" rid="B27">Tabassum and Jeong, 2019</xref>), also inhibiting GPX4 activity and maintaining the Xc<sup>&#x2212;</sup> system stability to mitigate ferroptosis (<xref ref-type="bibr" rid="B5">Chen S. et&#x20;al., 2021</xref>; <xref ref-type="bibr" rid="B37">Wang et&#x20;al., 2021b</xref>). In the present study, we seek to clarify whether VSMC ferroptosis involve into the HHP-induced VSMC phenotyping switch. In mechanism, we investigate the CSE/H<sub>2</sub>S-dependent GSH level in ferroptosis in the HHP condition.</p>
</sec>
<sec sec-type="materials|methods" id="s2">
<title>Materials and Methods</title>
<sec id="s2-1">
<title>Human Samples</title>
<p>Human internal mammary arteries were obtained from patients undergoing off-pump coronary artery bypass graft. The internal mammary artery (about 5&#xa0;mm) was acquired from the patient&#x2019;s surgical donor artery; then, the paraffin slices were prepared for immunofluorescent staining. This study was approved by Fuwai Hospital Ethics Committee and performed in accordance with ethical standards.</p>
</sec>
<sec id="s2-2">
<title>Animal and Materials</title>
<p>Adult male Wistar&#x2013;Kyoto (WKY) and spontaneously hypertensive rats (SHR) aged 12&#x2013;16&#x20;weeks were housed in controlled temperature at a 12:12-h light&#x2013;dark cycle with free access to water and standard diet. All animal protocols complied with all relevant ethical regulations and were approved by the Institutional Animal Care and Use Committee, the Experimental Animal Center, Fuwai Hospital, and the National Center for Cardiovascular Diseases, China. The primary antibodies used in this study are as follows: anti-GPX4 (ab125066, Abcam); anti-CXCL2 (PA5-47015, Invitrogen); anti-AKR1C2 (ab166900, Abcam); anti-CSE (ab151769); ferrostatin-1 (S7243, Selleck); RLS3 (S8155, Selleck); and NaHS (161527, Sigma).</p>
</sec>
<sec id="s2-3">
<title>H<sub>2</sub>S Production Measurement</title>
<p>H<sub>2</sub>S production was measured by the modified methylene blue method, as mentioned previously (<xref ref-type="bibr" rid="B19">Niu et&#x20;al., 2021</xref>). In detail, the inner ring of the Erlenmeyer flask was added with zinc acetate and a filter paper to absorb H<sub>2</sub>S. The outer ring was added with an incubation buffer (potassium phosphate buffer, <sc>l</sc>-cysteine and pyridoxal 5&#x2032;-phosphate). The cell lysate in the potassium phosphate buffer was added into the outer ring of the conical flasks. The reaction was performed in 37&#xb0;C shaking water bath by incubation. After sufficient reaction, trichloroacetic acid was added into the outer ring and incubated for another 1&#xa0;h to terminate the reaction. Followed by adding N, N-dimethyl-<italic>p</italic>-phenylenediamine sulfate and 10% ammonium ferric sulfate, absorbance at 670&#xa0;nm was measured by spectrophotometry.</p>
</sec>
<sec id="s2-4">
<title>Cell Culture and Treatment</title>
<p>Primary human aorta vascular smooth muscle cells (HASMCs) were purchased from ScienCell Research Laboratories, Inc. HAMSCs were cultured in a smooth muscle cell medium (1,101, ScienCell Research Laboratories.) containing 2% FBS, 100 U/mL penicillin&#x2013;streptomycin, and 1% smooth muscle cell growth factor at 37&#xb0;C in a 5% CO<sub>2</sub> atmosphere. HASMCs in passages three to six were used in this study. We used a specific hydrostatic pressure device for hydrostatic pressure treatment, as described previously (<xref ref-type="bibr" rid="B6">Chen Z. et&#x20;al., 2021</xref>). HASMCs (&#x223c;70% confluence) were set in a hydrostatic pressure device at 100&#xa0;mmHg or 200&#xa0;mmHg for culturing for 24&#xa0;h. For inhibition or activation of ferroptosis, HASMCs were treated with Fer-1 (10&#xa0;&#x3bc;M, 24&#xa0;h) or RLS3 (0.1&#x20;&#x3bc;M, 6&#xa0;h) under hydrostatic pressure. For increasing exogenous H<sub>2</sub>S, HASMCs were treated with NaHS (100&#xa0;&#x3bc;M, 24&#xa0;h) under hydrostatic pressure.</p>
</sec>
<sec id="s2-5">
<title>Primary Mouse Vascular Smooth Muscle Cell Isolation</title>
<p>Aortic smooth muscle cells were isolated from wild-type male 6&#x2013;8-week and CSE-knockout mice. Mice were sacrificed by decapitation, followed by separating the aorta <italic>via</italic> removing the fatty tissue and vascular adventitia. Then, the mouse aorta was cut into pieces and allowed for digestion for 8&#xa0;h in the DMEM containing collagenase type 1. The obtained VSMCs were centrifuged at 300 xg for 5&#xa0;min, and the pellet was resuspended in the DMEM supplemented with 10% FBS, 2&#xa0;mM&#xa0;<sc>l</sc>-glutamine, and 100&#xa0;U/mL penicillin/streptomycin. The medium was replaced every 2&#xa0;days. All experimental procedures were conducted in an incubator at a temperature of 37&#xb0;C, in an atmosphere of 95% air and 5% CO<sub>2</sub>. Primary SMCs with the passage number three to eight were used in the&#x20;study.</p>
</sec>
<sec id="s2-6">
<title>Iron and GSH Detection</title>
<p>We used two different methods to detect the iron content. After treatment, the intracellular ferrous iron level was assessed with an iron colorimetric assay kit (ab83366, Abcam) according to the manufacturer&#x2019;s instruction protocol. For FeRhoNox-1 staining, 5&#xa0;&#x3bc;M of the FeRhoNox-1 staining working solution (MX4558 and MKBIO) was added and incubated in a 37&#xb0;C, 5% CO<sub>2</sub> incubator for 60&#xa0;min after treatment for 24&#xa0;h in a hydrostatic pressure chamber. After washing three times with PBS, cells were imaged with a confocal microscope. The cellular level of glutathione was determined using the Glutathione Assay Kit (S0035, Beyotime) according to the manufacturer&#x2019;s instruction protocol.</p>
</sec>
<sec id="s2-7">
<title>Reactive Oxygen Species (ROS) Measurement</title>
<p>ROS was measured by using a fluorometric intracellular ROS kit (MAK143, sigma). HASMCs were incubated with 10&#xa0;&#xb5;M DCFH-DA for 1&#xa0;h. After washing with PBS three times, fluorescence signals were observed by using a fluorescence microscope.</p>
</sec>
<sec id="s2-8">
<title>Mitochondrial Superoxide Measurement</title>
<p>We use the MitoSOX&#x2122; Red mitochondrial superoxide indicator (M36008, Invitrogen) to detect ROS in the mitochondria. After treatment, HASMCs were stained with 5&#xa0;&#xb5;M MitoSOX Red for 10&#x20;min, followed by washing with PBS. The images were observed by using a confocal microscope.</p>
</sec>
<sec id="s2-9">
<title>Lipid Peroxidation Detection</title>
<p>HASMCs were labeled with a lipid peroxidation sensor BODIPY&#x2122; 581/591 C11 (D3861, Invitrogen) at 2&#xa0;&#xb5;M in a living cell imaging solution for 1&#xa0;h at 37&#xb0;C. The images were observed by using a confocal microscope. The fluorescence signals correspond to the oxidized (orange) form of BODIPY 581/591 C11. Oxidation of BODIPY-C11 was calculated by the orange fluorescence intensity.</p>
</sec>
<sec id="s2-10">
<title>Immunofluorescence Staining</title>
<p>For aorta immunofluorescent staining, paraffin sections were incubated with 3% hydrogen peroxide to block the endogenous peroxidase activity. Antigen retrieval was performed for 20&#xa0;min at 97&#xb0;C in a citrate buffer, and sections were cooled to room temperature. For HAMSC immunofluorescent staining, cells were fixed with 4% paraformaldehyde for 15&#xa0;min at room temperature. After washed with PBS three times, cells were permeabilized with 0.5% Triton X-100 for 15&#xa0;min at room temperature. Subsequently, above tissue sections or cells were blocked with 1% BSA for 1&#xa0;h and incubated with the primary antibody at 4&#xb0;C overnight, followed by incubating with a fluorescent-labeled secondary antibody for 1&#xa0;h at room temperature according to the manufacturer&#x2019;s instructions. After washing three times with PBS, the nuclei were stained with Hoechst for 8&#xa0;min. The images of tissues or cells were collected using a fluorescence microscope or by confocal microscopy.</p>
</sec>
<sec id="s2-11">
<title>Western Blot</title>
<p>Cells were lysed in RIPA buffer containing protease- and phosphatase-inhibitor cocktails, followed by sonication and centrifugation. Total protein of cells was quantified by BCA assay. For the assay, 40&#xa0;&#x3bc;g protein samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Then, the PVDF membranes were blocked by 5% defatted milk for 1&#xa0;h and probed with the primary antibody at 4&#xb0;C overnight. After washing and incubating with the horseradish peroxidase&#x2013;conjugated secondary antibody for 1&#xa0;h at room temperature, the membranes were visualized using the chemiluminescence&#x20;kit.</p>
</sec>
<sec id="s2-12">
<title>RNA Extraction and qRT-PCR</title>
<p>Total RNA from HASMCs was extracted using TRIzol reagent (Invitrogen) according to the manufacturer&#x2019;s instructions. cDNA was reverse transcribed from 1&#xa0;&#xb5;g total RNA by using the cDNA synthesis kit (K1622, Thermo scientific). The real-time PCR was performed in a final volume of 20&#xa0;&#x3bc;l, which contained 10&#xa0;&#x3bc;l of the 2&#xd7;SYBR Mix (Yeasen, China), 1&#xa0;&#x3bc;l of forward and reverse primers, respectively, 4&#xa0;&#x3bc;l of template cDNA, and 5&#xa0;&#x3bc;l of RNase-free H<sub>2</sub>O. A sample without cDNA was subjected to an identical protocol as a negative control. The PCR amplification was accomplished with initial denaturation at 95&#xb0;C for 10&#xa0;min, followed by 40 cycles at 95&#xb0;C for 15&#xa0;s and 1&#xa0;min at 60&#xb0;C for primer annealing and extension. The relative expression of target genes was normalized to that of GAPDH and analyzed by the 2<sup>&#x2212;&#x394;&#x394;CT</sup> method. The primer sequences used for qRT-PCR are provided in <xref ref-type="sec" rid="s11">Supplementary Table&#x20;S1</xref>.</p>
</sec>
<sec id="s2-13">
<title>Statistical Analysis</title>
<p>All data are presented as the mean&#x20;&#xb1; SD. The statistical significance of differences between groups was analyzed by the <italic>t</italic>-test or by one-way analysis of variance (ANOVA) when more than two groups were compared. <italic>p</italic> values &#x3c;0.05 were considered as statistically significant.</p>
</sec>
</sec>
<sec sec-type="results" id="s3">
<title>Results</title>
<sec id="s3-1">
<title>Elevation of Ferroptosis in Aortic Media of SHR and Hypertensive Patients Associated With VSMC Novel Phenotypes</title>
<p>High hydrostatic pressure drives VSMC differentiation into two novel phenotypes: the inflammatory phenotype (marker genes are CXCL2/CXCL3/CCL2) and endothelial function inhibitory phenotype (marker genes are AKR1C2/AKR1C3/PEDF), and promote VSMC dysfunction (<xref ref-type="bibr" rid="B6">Chen Z. et&#x20;al., 2021</xref>). Compared with WKY rats, SHR exhibits CXCL2 and AKR1C2 increase in the media of aorta (<xref ref-type="fig" rid="F1">Figures 1A,B</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S1A</xref>). Similarly, CXCL2 and AKR1C2 expressions were significantly enhanced in the human internal mammary artery of hypertensive patients in comparison with normotensive patients (<xref ref-type="fig" rid="F1">Figures 1C,D</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S1B</xref>). To investigate ferroptosis involved in HHP-induced VSMC novel phenotypes, we measured the GPX4 (a key enzyme of ferroptosis) protein level. Here, we showed that aortic media GPX4 dramatically downregulated in the SHR and hypertensive patients compared with the related control (<xref ref-type="fig" rid="F1">Figures 1E,F</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S1A&#x2013;B</xref>). These results indicated that ferroptosis associated with the VSMC phenotype switch in the hypertensive animal model and hypertensive patients.</p>
<fig id="F1" position="float">
<label>FIGURE 1</label>
<caption>
<p>GPX4 expression positively correlated with the VSMC-specific phenotype in SHR and hypertensive patients. Immunofluorescent staining of CXCL2 <bold>(A)</bold> and <italic>AKR1C2</italic> <bold>(B)</bold> in the arterial media of SHR. Immunofluorescence of CXCL2 <bold>(C)</bold> and <italic>AKR1C2</italic> <bold>(D)</bold> on paraffin-embedded human normotension (NTN) and hypertension (HTN) internal mammary arteries. The GPX4 expression was detected by immunofluorescent staining in SHR <bold>(E)</bold> and human hypertensive patients <bold>(F)</bold>. Nuclei were counterstained with Hoechst. Scale bar &#x3d; 25&#xa0;&#xb5;M.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g001.tif"/>
</fig>
</sec>
<sec id="s3-2">
<title>High Hydrostatic Pressure Induces Ferroptosis</title>
<p>To seek whether HHP induces VSMC ferroptosis, we constructed a specific cell culture chamber and set 200&#xa0;mmHg as HHP and 100&#xa0;mmHg as normal (simulates hydrostatic pressure of the aortic wall in normotensive and hypertensive individuals). For evaluation ferroptosis, HHP (200&#xa0;mmHg)-treatment increased the VSMC total ROS production (<xref ref-type="fig" rid="F2">Figure&#x20;2A</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S2A</xref>), mitochondria-derived ROS releasing by MitoSOX Red staining (<xref ref-type="fig" rid="F2">Figure&#x20;2B</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S2B</xref>), lipid peroxidation production by BODIPY 588/591 C11 (<xref ref-type="fig" rid="F2">Figure&#x20;2C</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S2C</xref>), iron accumulation by FeRhoNox-1 staining (<xref ref-type="fig" rid="F2">Figure&#x20;2D</xref>), and iron content by the iron assay kit (<xref ref-type="fig" rid="F2">Figure&#x20;2E</xref>) in comparison to normal pressure. In line with the ROS enhancing and lipid peroxidation, the GPX4 protein (<xref ref-type="fig" rid="F2">Figure&#x20;2F</xref>) and mRNA level (<xref ref-type="fig" rid="F2">Figure&#x20;2G</xref>) also lowered under the HHP condition. Indeed, the ferroptosis-related gene (<italic>COX-2</italic>, <italic>TFRC</italic>, <italic>ACSL4</italic>, and <italic>NOX-1</italic>) mRNA expression was upregulated, and SLC7A11 mRNA was downregulated while exposed to HHP (<xref ref-type="fig" rid="F2">Figure&#x20;2H</xref>). Overall, these results indicate high hydrostatic pressure promotes VSMC ferroptosis.</p>
<fig id="F2" position="float">
<label>FIGURE 2</label>
<caption>
<p>High hydrostatic pressure promoted ferroptosis. HASMCs were treated with hydrostatic pressure for 24&#xa0;h. Immunofluorescent staining of total ROS, scale bar &#x3d; 50&#xa0;&#xb5;M <bold>(A)</bold>. MitoSOX Red staining of HASMCs for mitochondrial ROS, scale bar &#x3d; 25&#xa0;&#xb5;M <bold>(B)</bold>. BODIPY-C11 staining measured the lipid peroxidation; orange indicated oxidized C11, scale bar &#x3d; 25&#xa0;&#xb5;M <bold>(C)</bold>. Iron level was detected by FeRhoNox-1 staining <bold>(D)</bold> and iron assay kit <bold>(E)</bold>, scale bar &#x3d; 100&#xa0;&#xb5;M. Western blot assay measured the GPX4 protein level; the left panel is the representative image, and the right panel is the statistical graph <bold>(F)</bold>. The mRNA levels of GPX4 <bold>(G)</bold>, COX-2, TFRC, ACSL4, NOX-1, and SLC7A11 were measured by real-time PCR <bold>(H)</bold> under HHP conditions. Nuclei were counterstained with Hoechst. &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g002.tif"/>
</fig>
</sec>
<sec id="s3-3">
<title>Ferroptosis Contributes to the HHP-Induced Specific VSMC Phenotype Switch</title>
<p>To investigate the effect of ferroptosis on HHP-induced specific VSMC subsets, we treated HASMCs with the ferroptosis suppressor Fer-1. Fer-1 supplementation upregulated the VSMC GPX4 expression under normal or high hydrostatic pressure conditions (<xref ref-type="fig" rid="F3">Figure&#x20;3A</xref>). In line with GPX4 upregulation, Fer-1 administration also blocked HHP-stimulated ROS production (<xref ref-type="fig" rid="F3">Figure&#x20;3B</xref>), mitochondrial ROS production (<xref ref-type="fig" rid="F3">Figure&#x20;3C</xref>), and lipid oxidation (<xref ref-type="fig" rid="F3">Figure&#x20;3D</xref>), similar to other ferroptosis inducers (<xref ref-type="bibr" rid="B8">Dixon et&#x20;al., 2012</xref>). These results confirmed Fer-1 blocked HHP-associated ferroptosis. In association with ferroptosis inhibition, Fer-1 also blocked HHP-induced VSMC CXCL2 (<xref ref-type="fig" rid="F3">Figure&#x20;3E</xref>) and AKR1C2 (<xref ref-type="fig" rid="F3">Figure&#x20;3F</xref>) elevation.</p>
<fig id="F3" position="float">
<label>FIGURE 3</label>
<caption>
<p>Ferroptosis inhibitor Fer-1 mitigates the HHP-induced VSMC phenotype switch. HASMCs in a 100&#xa0;mmHg or 200&#xa0;mmHg incubator were treated with or without 10&#xa0;&#x3bc;M Fer-1 for 24&#xa0;h. The GPX4 protein level was evaluated by immunofluorescent staining; the upper panel is the representative image, and the lower panel is the statistical graph <bold>(A)</bold>. ROS production was determined by the ROS staining kit; the upper panel is the representative image, and the lower panel is the statistical graph <bold>(B)</bold>. Mitochondrial ROS was visualized by use of the fluorescent probe MitoSOX Red <bold>(C)</bold>. BODIPY 581/591 C11 lipid oxidation in HASMCs was measured by immunofluorescent staining; the statistical graph is the oxidized (orange signal) BODIPY 581/591 C11 fluorescence intensity <bold>(D)</bold>. Confocal images of the CXCL2 expression <bold>(E)</bold> and AKR1C2 expression <bold>(F)</bold> in HASMCs. Nuclei were counterstained with Hoechst. All scale bar &#x3d; 50&#xa0;&#x3bc;M &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g003.tif"/>
</fig>
<p>Since HHP-induced ferroptosis is in part of the GPX4/GSH pathway, we used a ferroptosis inducer RLS3 (GPX4 inactivation) to confirm its role in the VSMC novel phenotype switch. Under 200&#xa0;mmHg hydrostatic pressure condition, RLS3 ulteriorly reduced the GPX4 expression (<xref ref-type="fig" rid="F4">Figure&#x20;4A</xref>). Consistently, RLS3 exacerbated ferroptosis by increasing ROS production (<xref ref-type="fig" rid="F4">Figure&#x20;4B</xref>), mitochondrial ROS production(<xref ref-type="fig" rid="F4">Figure&#x20;4C</xref>), and lipid oxidation (<xref ref-type="fig" rid="F4">Figure&#x20;4D</xref>). Correspondingly, RLS3 further increases the HHP-induced VSMC CXCL2 (<xref ref-type="fig" rid="F4">Figure&#x20;4E</xref>) and AKR1C2 expression (<xref ref-type="fig" rid="F4">Figure&#x20;4F</xref>). Taken together, these data indicated that ferroptosis contributed to the pathophysiological process of HHP-induced VSMC-specific phenotypes.</p>
<fig id="F4" position="float">
<label>FIGURE 4</label>
<caption>
<p>Ferroptosis inducer RLS3 aggravates VSMC dysfunction in response to HHP. HASMCs were treated with RLS3 0.1&#xa0;&#x3bc;M for 6&#xa0;h in the presence of 200&#xa0;mmHg. Immunofluorescent staining of GPX4 (red) in HASMCs <bold>(A)</bold>. Intracellular ROS were measured by ROS staining; the upper panel is the representative image, and the lower panel is the statistical graph <bold>(B)</bold>. HASMCs were labeled with the MitoSOX Red probe to detect mitochondrial ROS <bold>(C)</bold>. Confocal images of the BODIPY 588/591 C11 staining; lipid oxidation was measured by the relative intensity of oxidized (orange fluorescence signal) BODIPY 581/591 C11 <bold>(D)</bold>. CXCL2 <bold>(E)</bold>, and AKR1C2&#x20;<bold>(F)</bold> protein levels were detected by immunofluorescent staining. All scale bar &#x3d; 50&#xa0;&#x3bc;M &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g004.tif"/>
</fig>
</sec>
<sec id="s3-4">
<title>CSE/H<sub>2</sub>S Alleviate HHP-Induced VSMC Ferroptosis</title>
<p>CSE/H<sub>2</sub>S plays a crucial role in GSH synthesis due to controlling the GSH precursor-<sc>l</sc>-cysteine generation, and CSE/H<sub>2</sub>S plays an essential protection in VSMC function (<xref ref-type="bibr" rid="B11">Kimura, 2021</xref>). To investigate whether CSE/H<sub>2</sub>S involved in the modulation of HHP-induced ferroptosis, we first measured the CSE/H<sub>2</sub>S changes exposed to HHP (200&#xa0;mmHg). As <xref ref-type="fig" rid="F5">Figure&#x20;5A</xref> showed, HHP treatment for 48&#xa0;h dramatically reduced the HASMC H<sub>2</sub>S production (about 27%). The CSE protein (<xref ref-type="fig" rid="F5">Figure&#x20;5B</xref>) and mRNA expression (<xref ref-type="fig" rid="F5">Figure&#x20;5C</xref>) were also downregulated under HHP. The association with CSE/H<sub>2</sub>S downregulation and the intracellular GSH level reduced about 28% (<xref ref-type="fig" rid="F5">Figure&#x20;5D</xref>).</p>
<fig id="F5" position="float">
<label>FIGURE 5</label>
<caption>
<p>CSE/H<sub>2</sub>S attenuates HHP-induced ferroptosis in VSMCs. H<sub>2</sub>S production was measured by the modified methylene blue method under 100 or 200&#xa0;mmHg <bold>(A)</bold>. Western blot assay analysis of the CSE expression under hydrostatic pressure; the left panel is the representative image, and the right panel is the statistical graph <bold>(B)</bold>. CSE mRNA level under hydrostatic pressure <bold>(C)</bold>. GSH assay kit measured the GSH level in response to hydrostatic pressure <bold>(D)</bold>. After NaHS administration (0.1mM, 24&#xa0;h) in the presence of 200&#xa0;mmHg, the GSH content was measured <bold>(E)</bold>. Representative image of GPX4 staining after NaHS treatment under HHP conditions <bold>(F)</bold>. Effect of NaHS on Fe<sup>2&#x2b;</sup> <bold>(G)</bold>, ROS <bold>(H)</bold>, mitochondrial ROS <bold>(I)</bold>, and lipid oxidation <bold>(J)</bold> under 200&#xa0;mmHg by immunofluorescent staining. In isolated primary mouse VSMCs, the effect of deletion of CSE on the GPX4 expression <bold>(K)</bold>, iron level <bold>(L)</bold>, ROS production <bold>(M)</bold>, mitochondrial ROS production <bold>(N)</bold>, and lipid peroxidation <bold>(O)</bold> under HHP conditions by immunofluorescent staining. All scale bar &#x3d; 50&#xa0;&#x3bc;M &#x2a;<italic>p</italic>&#x20;&#x3c; 0.05, &#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.01, &#x2a;&#x2a;&#x2a;<italic>p</italic>&#x20;&#x3c; 0.001.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g005.tif"/>
</fig>
<p>In contrast, supplementation H<sub>2</sub>S donor-NaHS under HHP culture, upregulated the GSH level and GPX4 expression (<xref ref-type="fig" rid="F5">Figures 5E,F</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3A</xref>) but lowered the HASMC iron concentration (<xref ref-type="fig" rid="F5">Figure&#x20;5G</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3B</xref>), ROS production (<xref ref-type="fig" rid="F5">Figure&#x20;5H</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3C</xref>), mitochondrial ROS production (<xref ref-type="fig" rid="F5">Figure&#x20;5I</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3D</xref>), and lipid peroxidative production (<xref ref-type="fig" rid="F5">Figure&#x20;5J</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3E</xref>). Next, we isolated primary CSE-knockout mouse VSMCs to confirm its role in HHP-induced ferroptosis. Under HHP conditions, CSE deficiency further decreased the GPX4 expression (<xref ref-type="fig" rid="F5">Figure&#x20;5K</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3F</xref>), enhanced iron accumulation (<xref ref-type="fig" rid="F5">Figure&#x20;5L</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3G</xref>), cellular and mitochondrial ROS (<xref ref-type="fig" rid="F5">Figure&#x20;5M&#x2013;N</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3H&#x2013;I</xref>), and lipid peroxidative production (<xref ref-type="fig" rid="F5">Figure&#x20;5O</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S3J</xref>). Our present data highlight HHP decreases CSE/H<sub>2</sub>S causing GSH level reduction attribution to pathogenesis of VSMC ferroptosis.</p>
<p>To confirm the effect of CSE/H<sub>2</sub>S on ferroptosis, we first used RLS3 to induce ferroptosis. In line with the above findings, NaHS administration also rescued the RLS3-induced GPX4 expression (<xref ref-type="fig" rid="F6">Figure&#x20;6A</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S4A</xref>), iron accumulation (<xref ref-type="fig" rid="F6">Figure&#x20;6B</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S4B</xref>), ROS production (<xref ref-type="fig" rid="F6">Figure&#x20;6C</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S4C</xref>), mitochondrial ROS production (<xref ref-type="fig" rid="F6">Figure&#x20;6D</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S4D</xref>), and lipid oxidation (<xref ref-type="fig" rid="F6">Figure&#x20;6E</xref> and <xref ref-type="sec" rid="s11">Supplementary Figure S4E</xref>). In summary, our result indicated that CSE/H<sub>2</sub>S contributes to the regulation of ferroptosis under HHP conditions.</p>
<fig id="F6" position="float">
<label>FIGURE 6</label>
<caption>
<p>CSE/H<sub>2</sub>S rescue RLS3-induced ferroptosis under HHP conditions. Under 200&#xa0;mmHg conditions, HASMCs were pretreated with NaHS 24&#xa0;h and followed by RLS3 treatment for another 6&#xa0;h. Immunofluorescent staining of GPX4&#x20;<bold>(A)</bold>. HASMCs were incubated with the FeRhNOX-1 probe for measuring iron <bold>(B)</bold>. HASMCs were labeled with the ROS probe to detect cellular ROS <bold>(C)</bold>, MitoSOX Red to detect mitochondrial ROS <bold>(D)</bold>, and BODIPY-C11 to detect lipid peroxidation <bold>(E)</bold> by immunofluorescent staining. All scale bar &#x3d; 50&#xa0;&#x3bc;M.</p>
</caption>
<graphic xlink:href="fcell-10-829316-g006.tif"/>
</fig>
</sec>
</sec>
<sec sec-type="discussion" id="s4">
<title>Discussion</title>
<p>Mechanical forces play an important role in vasculature and circulation, such as rapid regulation of vascular wall elasticity, administration of vascular remodeling, and the modulation of VSMC and endothelial function (<xref ref-type="bibr" rid="B22">Qiu et&#x20;al., 2014</xref>). Our previous study identifies a novel cellular taxonomy of VSMCs under hydrostatic pressure by single-cell RNA sequencing. HHP derived VSMCs into inflammatory and endothelial-function inhibitory VSMCs, resulting in VSMC dysfunction (cytokine secretion and angiogenesis inhibition) (<xref ref-type="bibr" rid="B6">Chen Z. et&#x20;al., 2021</xref>). However, the underlying mechanism is unclear. In the current study, we first demonstrated that HHP induced VSMC ferroptosis evidence as iron accumulation, ROS generation, and lipid peroxidation. Furthermore, inhibiting ferroptosis by Fer-1 alleviated, promoting ferroptosis by RLS3 increased inflammatory and endothelial function inhibitory VSMC phenotypes. For the mechanism, we demonstrated that HHP downregulated CSE/H<sub>2</sub>S and then lowered GSH generation, resulting in ferroptosis. Our findings highlight that ferroptosis is a novel pathophysiological regulatory mechanism for VSMC function and as a new druggable target for therapeutic hypertension concomitant vascular diseases.</p>
<p>Hypertension is tightly associated with oxidation stress (<xref ref-type="bibr" rid="B17">McMaster et&#x20;al., 2015</xref>; <xref ref-type="bibr" rid="B31">Touyz et&#x20;al., 2018</xref>). An imbalance between the oxidant and antioxidant system leads to ROS production exaggeration in the arterial media from hypertension patients with hypertension and experimental animal models of hypertension (<xref ref-type="bibr" rid="B32">Touyz and Schiffrin, 2001</xref>). Overproduced ROS promoted VSMC proliferation, induced VSMC migration, and switched to the differentiated phenotype (<xref ref-type="bibr" rid="B2">Badran et&#x20;al., 2020</xref>). Dedifferentiated VSMCs synthesize and secrete a repertoire of growth factors and inflammatory cytokines which is a key initiating factor for vascular remodeling. ROS promotes MMP2, MMP9, and collagen expressions and disturbs the extracellular matrix (<xref ref-type="bibr" rid="B24">Rajagopalan et&#x20;al., 1996</xref>; <xref ref-type="bibr" rid="B4">Branchetti et&#x20;al., 2013</xref>). Additionally, oxidation stress induces calcification, stiffness, and aging leading to VSMC dysfunction (<xref ref-type="bibr" rid="B28">Takaishi et&#x20;al., 2021</xref>). ROS overproduction, a center of oxidation stress, is a main characteristic of ferroptosis. Our study first identified GPX4, a key enzyme of ferroptosis, which was dramatically downregulated in arterial media of SHR. <italic>In vitro,</italic> VSMCs exhibited intracellular and mitochondrial ROS overproduction, lipid peroxidation, and iron accumulation under high hydrostatic stress. Ferroptosis upregulation under high hydrostatic stress is a novel pathophysiological process in VSMC dysfunction.</p>
<p>Compelling evidence has indicated that ferroptosis is an essential regulator of inflammation. GPX4 deficiency blocked T-cell survival and B-cell development (<xref ref-type="bibr" rid="B16">Matsushita et&#x20;al., 2015</xref>; <xref ref-type="bibr" rid="B18">Muri et&#x20;al., 2019</xref>). The ferroptosis inducer erastin promoted human peripheral blood mononuclear cells differentiating into B&#x20;cells and natural killer cells (<xref ref-type="bibr" rid="B30">Tang et&#x20;al., 2021</xref>). Ferroptotic death cells recruited macrophages by secreting CCL2 and CCL7 (<xref ref-type="bibr" rid="B36">Wang et&#x20;al., 2021a</xref>). Ferroptosis inhibitors exhibited the anti-inflammation effect by suppressing TNF-&#x3b1;, IL-6, and IL-1&#x3b2; releasing (<xref ref-type="bibr" rid="B33">Tsurusaki et&#x20;al., 2019</xref>). Consistent with the previous study, Fer-1 treatment attenuated, while RLS3 treatment augmented the HHP-stimulated CXCL2 (one marker gene of inflammatory VSMCs) protein level. In addition, high hydrostatic pressure also induced <italic>AKR1C2</italic> (one marker gene of endothelial function inhibitory VSMCs) which was lowered by Fer-1 but enhanced by RLS3. Studies indicated ferroptosis led to endothelial dysfunction which is a critical pathogenetic factor of hypertension. Iron accumulation resulted in endothelial damage (<xref ref-type="bibr" rid="B34">Vinchi et&#x20;al., 2020</xref>). Fer-1 administration attenuated ox-LDL-induced inflammation and endotheliocyte death, while erastin induced ROS production and ferroptotic death of HUVECs (<xref ref-type="bibr" rid="B40">Xiao et&#x20;al., 2019</xref>; <xref ref-type="bibr" rid="B3">Bai et&#x20;al., 2020</xref>). Combined with our results, ferroptosis may be a potential therapeutic target to hypertension <italic>via</italic> modulating endothelial function.</p>
<p>One important characteristic of ferroptosis is GSH depletion. GSH, an intracellular antioxidant, is the synthesis from the homocysteine/methionine cycle (<xref ref-type="bibr" rid="B25">Rodrigues and Percival, 2019</xref>). GSH precursor <sc>l</sc>-cysteine is also a main source of hydrogen sulfide. Growing evidence demonstrated that H<sub>2</sub>S enhances GSH production to attenuate oxidative stress. In a neurocyte, mitochondrial H<sub>2</sub>S production increases the GSH level and promotes its redistribution to the mitochondria to protect the neurocyte from oxidative stress (<xref ref-type="bibr" rid="B12">Kimura et&#x20;al., 2010</xref>). In a myotube, H<sub>2</sub>S promotes GSH synthesis to improve impaired glucose homeostasis (<xref ref-type="bibr" rid="B20">Parsanathan and Jain, 2018</xref>). H<sub>2</sub>S donor NaHS administration enhances GSH production to decrease oxidative stress and delay cell senescence (<xref ref-type="bibr" rid="B41">Yang et&#x20;al., 2013</xref>). Consistently, our study showed that exogenous (NaHS administration) H<sub>2</sub>S production significantly rescued GSH reduction in response to HHP. Thus, H<sub>2</sub>S downregulation&#x2013;mediated GSH reduction may be a novel mechanism of ferroptosis under&#x20;HHP.</p>
<p>Recently, studies showed the protective role of H<sub>2</sub>S is associated with ferroptosis inhibition. Wang and Chen et&#x20;al. identified H<sub>2</sub>S restrains ferroptosis <italic>via</italic> inhibiting ALOX12 acetylation and regulating the xCT (the functional submit of the Xc<sup>&#x2212;</sup> system) stability (<xref ref-type="bibr" rid="B5">Chen S. et&#x20;al., 2021</xref>; <xref ref-type="bibr" rid="B38">Wang et&#x20;al., 2021c</xref>). H<sub>2</sub>S donor GYY4137 treatment alleviates ferroptosis to attenuate acute lung injury (<xref ref-type="bibr" rid="B13">Li et&#x20;al., 2021</xref>). Inhibition of H<sub>2</sub>S production <italic>via</italic> the CBS inhibitor CH004 supplement aggravates ferroptosis in hepatocellular carcinoma (<xref ref-type="bibr" rid="B35">Wang et&#x20;al., 2018</xref>). These studies suggested H<sub>2</sub>S could restrain ferroptosis to exert a protective effect. However, experimental evidence in the cardiovascular system is still missing. Our present study illustrated that H<sub>2</sub>S donor NaHS administration significantly increased the GPX4 expression, downregulated ROS production, and lipid peroxidation, thus reversing high hydrostatic-induced ferroptosis. In addition, NaHS rescued RLS3-induced ferroptosis. Overall, H<sub>2</sub>S inhibits ferroptosis to attenuate HHP-induced VSMC dysfunction.</p>
<p>Taken together, the present study highlights the essential regulator role of ferroptosis in the HHP-triggered VSMC novel phenotype switch. The novel pathophysiological process also accounts for HHP, a direct biomechanical force, triggers VSMC ferroptosis, then drives vascular inflammation and limitation vascular relaxation, and contributes to vascular remodeling and aging even if calcified, thus exacerbating concomitant vascular damages and diseases (such as atherosclerosis). More interestingly, our study also indicates a new pathway about the ROS-GSH-iron-ferroptosis signal cascade in CSE/H<sub>2</sub>S cardiovascular protection.</p>
</sec>
</body>
<back>
<sec id="s5">
<title>Data Availability Statement</title>
<p>The original contributions presented in the study are included in the article/<xref ref-type="sec" rid="s11">Supplementary Material</xref>; further inquiries can be directed to the corresponding authors.</p>
</sec>
<sec id="s6">
<title>Ethics Statement</title>
<p>The studies involving human participants were reviewed and approved by the Institutional Animal Care and Use Committee, the Experimental Animal Center, Fuwai Hospital, National Center for Cardiovascular Diseases, China. The ethics committee waived the requirement of written informed consent for participation. The animal study was reviewed and approved by the Institutional Animal Care and Use Committee, the Experimental Animal Center, Fuwai Hospital, National Center for Cardiovascular Diseases, China.</p>
</sec>
<sec id="s7">
<title>Author Contributions</title>
<p>ZC and BG conceived the project and designed the experiments. RJ conducted most of the experiments and data analyses. RY conducted a part of experiments. CC and HZ provided the technical assistance. The manuscript was written by ZC and revised and edited by BG and&#x20;JC.</p>
</sec>
<sec id="s8">
<title>Funding</title>
<p>The study was supported by the CAMS Innovation Fund for Medical Sciences (CIFMS, 2021-I2M-1-007), the National Natural Science Foundation of China (81800367, 81870318, 81825002), the National Key R&#x26;D Program of China (2018YFC1312703), and the Beijing Outstanding Young Scientist Program (BJJWZYJH01201910023029).</p>
</sec>
<sec sec-type="COI-statement" id="s9">
<title>Conflict of Interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec sec-type="disclaimer" id="s10">
<title>Publisher&#x2019;s Note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors, and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
<sec id="s11">
<title>Supplementary Material</title>
<p>The Supplementary Material for this article can be found online at: <ext-link ext-link-type="uri" xlink:href="https://www.frontiersin.org/articles/10.3389/fcell.2022.829316/full#supplementary-material">https://www.frontiersin.org/articles/10.3389/fcell.2022.829316/full&#x23;supplementary-material</ext-link>
</p>
<supplementary-material xlink:href="DataSheet1.docx" id="SM1" mimetype="application/docx" xmlns:xlink="http://www.w3.org/1999/xlink"/>
</sec>
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