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Cystathionine γ-Lyase Modulates Flow-Dependent Vascular Remodeling

Originally publishedhttps://doi.org/10.1161/ATVBAHA.118.311402Arteriosclerosis, Thrombosis, and Vascular Biology. 2018;38:2126–2136

Abstract

Objective—

Flow patterns differentially regulate endothelial cell phenotype, with laminar flow promoting vasodilation and disturbed flow promoting endothelial proinflammatory activation. CSE (cystathionine γ-lyase), a major source of hydrogen sulfide (H2S) in endothelial cells, critically regulates cardiovascular function, by both promoting vasodilation and reducing endothelial activation. Therefore, we sought to investigate the role of CSE in the endothelial response to flow.

Approach and Results—

Wild-type C57Bl/6J and CSE knockout (CSE−/−) mice underwent partial carotid ligation to induce disturbed flow in the left carotid. In addition, endothelial cells isolated from wild-type and CSE−/− mice were exposed to either laminar or oscillatory flow, an in vitro model of disturbed flow. Interestingly, laminar flow significantly reduced CSE expression in vitro, and only disturbed flow regions show discernable CSE protein expression in vivo, correlating with enhanced H2S production in wild-type C57BL/6J but not CSE−/− mice. Lack of CSE limited disturbed flow-induced proinflammatory gene expression (ICAM-1[intercellular adhesion molecule 1], VCAM-1 [vascular cell adhesion molecular 1]) and monocyte infiltration and CSE−/− endothelial cells showed reduced NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and proinflammatory gene expression in response to oscillatory flow in vitro. In addition, CSE−/− mice showed reduced inward remodeling after partial carotid ligation. CSE−/− mice showed elevated vascular nitrite levels (measure of nitric oxide [NO]) in the unligated carotids, suggesting an elevation in baseline NO production, and the NO scavenger 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide normalized the reduced inward remodeling, but not inflammation, of ligated carotids in CSE−/− mice.

Conclusions—

CSE expression in disturbed flow regions critically regulates both endothelial activation and flow-dependent vascular remodeling, in part through altered NO availability.

Introduction

Blood vessels are constantly exposed to mechanical stimuli from circulating blood. The movement of blood exerts a frictional force, namely fluid shear stress, parallel to the vascular wall. Patterns of blood flow in circulation vary along the hierarchical tree of blood vessels. In most arterial regions, blood flow is unidirectional, with high-velocity, and laminar. However, bifurcations, branch points, and curvatures in vessels cause disturbed flow profiles characterized by low-velocity shear stress and changes in flow direction. These distinct patterns of shear stress differentially regulate vascular heterogeneity and homeostasis. Although high-velocity, laminar shear maintains endothelial cells quiescence, low-velocity, oscillatory shear activates endothelial cells resulting in enhanced monocyte recruitment.1 Laminar flow and disturbed flow are sensed by the endothelial cell layer resulting in disparate cellular responses. Exposing endothelial cells to oscillatory flow in vitro activates NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells), enhances expression of adhesion molecules, and induces cell proliferation and apoptosis, whereas laminar flow inhibits inflammatory signaling and reduces endothelial cell turnover.24 In addition to these flow patterns, vessels also respond to changes in blood flow. Elevated flow induces transient vasodilation and outward remodeling if the changes become chronic, whereas reductions in flow result in transient vasoconstriction and inward remodeling, should flow reductions be sustained.

Hydrogen sulfide (H2S) is an important signaling molecule in the cardiovascular system where CSE (cystathionine γ-lyase) is a major source of it.5 H2S and CSE play critical roles in endothelial function and vascular health. Our previous studies have shown that CSE-derived polysulfides, oxidative products of H2S, promotes angiogenesis in ischemic limbs and increases endothelial permeability.6,7CSE knockout mice were also shown to have increased atherosclerotic plaque formation after high-fat diet, whereas CSE transgenic mice on the apolipoprotein E knockout background are protected against plaque formation.8,9 Although, vascular remodeling is the basis of neointimal formation and stenosis, it can be largely affected by dyslipidemia in models of atherosclerosis. It is not known whether CSE is involved in the physiological vascular adaptive response to shear stress in the absence of lipid metabolism disorders. In this study, we used CSE knockout mice on the C57BL/6J background to investigate the role of CSE in flow-mediated vascular remodeling.

Materials and Methods

The authors declare that all supporting data are available within the article and in the online-only Data Supplement.

Animals

All mice used in this study were housed in accordance with the National Research Council’s Guide for the Care and Use of Laboratory Animals. Studies were approved by Louisiana State University Health Science Center-Shreveport institutional animal care and use committee. Partial carotid ligations were performed on 12- to 18-week-old male C57BL/6J wild-type and CSE knockout mice. As we described in our previous study, the left external carotid, internal carotid, and occipital arteries were ligated with suture, sparing the superior thyroid artery, whereas the right side was left intact as the nonligated control.3 Ultrasound measurements were taken under anesthesia with a VisualSonics VEVO 3100 system before and 1 or 6 day(s) after ligation to confirm the reduction in blood flow. Mice were euthanized 2 days and 7 days after ligations to collect their carotid arteries. Endothelial mRNA samples were obtained 2 days postligation by flushing TRIzol through the artery. The 7-day samples were used for immunohistochemistry and the biochemical measurements.

Cell Culture and Shear Stress

Human aortic endothelial cells were purchased from Lonza and maintained at 37°C in MCDB 131 media (Sigma, M8537) with 10% fetal bovine serum, 2 mmol/L glutamine, 10 U/mL penicillin, 10 μg/mL streptomycin, 60 μg/mL heparin, and 170 μg/mL bovine brain extract. Mouse aortic endothelial cells were isolated from 3 wild-type or CSE knockout mice as previously described.6 In brief, aortic endothelial cells were collected by Matrigel invasion (Corning, 354234) and enriched with Dynabeads (Invitrogen, 11205D) coupled with a CD105 antibody (eBioscience, 13-1051-85). The resultant endothelial cells were immortalized with a temperature sensitive large T-antigen. Mouse aortic endothelial cells were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 10 U/mL penicillin, and 10 μg/mL streptomycin. The shear stress experiments were performed using parallel plate flow chambers and peristaltic pumps as previously reported.10,11 Cells were exposed to either laminar flow (12 dynes/cm2) or oscillatory flow (±5 dynes/cm2 with 1 dynes/cm2 superimposed for nutrient/waste exchange). After shear treatments, cells were lysed directly in 2× laemmli buffer for Western blotting or fixed in 4% formaldehyde for immunocytochemistry.

Immunoblotting

Proteins in the cell lysate were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% dry milk in tris-buffered saline with 0.1% Tween-20 and blotted using antibodies listed in the Major Resource Table in the online-only Data Supplement.

Immunocytochemistry

Fixed endothelial cells were permeabilized with 0.1% Triton-X in PBS and stained for the NF-κB p65 subunit (1 μg/mL, Santa Cruz, Sc-109). The primary antibody was probed with Alexa Fluor 488 conjugated secondary antibody. Nuclei were visualized using 4’,6-diamidino-2-phenylindole staining. Fluorescent images were taken from at least 5 random fields on each slide.

Tissue Staining

Carotid tissue was fixed with 4% formaldehyde and embedded in paraffin. 5-μm sections, 150 to 450 μm proximal from the ligation site, were used for immunohistochemistry and Russell-Movat Pentachrome staining as previously described.12 Carotid medial thickness and circumference with Nikon Element software. Lumen size is calculated according to circumference. Primary antibodies used for immunostaining are listed in the Major Resource Table in the online-only Data Supplement.

Quantitative Real-Time Polymerase Chain Reaction

mRNA was isolated from TRIzol samples according to the manufacturer's instruction. The iScript cDNA synthesis kit (Bio-rad, 1708890) was used to transcribe mRNA. Quantitative real-time polymerase chain reaction was performed using SYBR Green Master Mix (Bio-rad, 1708882), and gene expression was quantified using the 2−ΔΔCT method. All genes of interest were normalized to the housekeeping genes B2M and RPL13A. The primers used in polymerase chain reaction reactions are listed in the Major Resource Table in the online-only Data Supplement.

Sulfide Measurement

H2S and its metabolites were measured using a monobromobimane-based method as we previously reported.13 Carotid arteries were homogenized in 250 μL reaction buffer (0.1 mmol/L diethylenetriaminepentaacetic acid in 100 mmol/L Tris buffer, pH 9.5) with 0.1% Triton-X. H2S was derivatized with monobromobimane. The pools of sulfane sulfurs and acid-labile sulfurs were released from the sample in reducing and acidic environments respectively, followed by monobromobimane derivatization. The resultant product sulfide dibimane was quantified by reverse phase high-performance liquid chromatography.

Nitric Oxide Bioavailability

Nitric oxide (NO) metabolites (NOx) were measured as we previously reported.14 Carotid arteries were homogenized in 250 μL NO preservation buffer (1.25 mol/L potassium ferricyanide, 56.9 mmol/L N-ethylmaleimide, 6% Nonidet™ P 40 substitute in PBS). Nitrite is reduced with the triiodide method. NO is measured by the ozone-based chemiluminescent assay (Sievers Nitric Oxide Analyzer 280i).

Statistical Analysis

Data were shown as mean±SE of the mean. Data were tested for normality using GraphPad Prism software and analyzed for statistical significance using either parametric (Student t test, 2-way ANOVA with Tukey post hoc test) or nonparametric tests (Mann-Whitney U test) as indicated in the figure legends. P values <0.05 were considered as statistical significance.

Results

Flow Patterns Regulate Endothelial CSE Expression

Flow patterns critically regulate endothelial activation and endothelial-dependent vasodilation,1517 where H2S signaling has been shown to be important.7,18,19 Because CSE is the predominant source of sulfide in endothelial cells, we asked whether its expression is regulated by flow. Human aortic endothelial cells were exposed to long-term (18 hours) laminar or oscillatory flow, and CSE expression was assessed by Western blotting. Surprisingly, laminar flow significantly reduced CSE expression compared with oscillatory flow (Figure 1A). In comparison, the expression of CBS (cystathionine β-synthase), another sulfide producing enzyme reported to regulate endothelial function,20 was not altered by flow patterns. To study the effects of shear stress on CSE expression in vivo, we analyzed CSE staining the aortic arch, which experiences high-velocity laminar flow on the greater curvature and disturbed flow on the lesser curvature. Consistent with our cell culture models, the aortic lesser curvature showed higher CSE expression compared with the greater curvature (Figure 1B), suggesting that endogenous sites of laminar and disturbed flow show a similar flow pattern-specific CSE expression. To validate this in an acute model of flow disturbances, we performed partial carotid ligation to induce low, oscillatory flow in the left carotid arteries, with the right carotid serving as an internal control for high laminar flow.3 Alterations in flow dynamics were verified by ultrasound after the surgery, showing the flow rate was reduced 80% in the left carotid artery (Figure I in the online-only Data Supplement). As shown in Figure 1C, CSE expression was increased in the ligated carotid artery compared to the contralateral side. Specificity of this staining pattern was verified using IgG controls, secondary antibody-only controls, and CSE knockout tissue (Figure II in the online-only Data Supplement). Importantly, the increased CSE expression largely colocalized with endothelial cells (von Willebrand factor–positive) in the vessel intima and adventitia. The microvessels in adventitia, known as vasa vasorum, appeared to undergo expansion in the ligated vessels. Although CSE is known to regulate angiogenesis in a variety of models,21 we did not observe any effect of CSE deletion on vasa vasorum expansion in this model (Figure III in the online-only Data Supplement).

Figure 1.

Figure 1. Shear stress regulates CSE (cystathionine γ-lyase) expression. Human aortic endothelial cells were exposed to laminar shear stress (LSS) or oscillatory shear stress (OSS) for 16 hours. A, CBS (cystathionine β-synthase) and CSE expression were assessed by Western blotting. Representative images are shown. (n=5; ***P<0.001 by 1-way ANOVA). CSE protein expression was assessed by immunostaining at sites of laminar (greater curvature) or disturbed flow (lesser curvature). Scale bar=200 μm; n=5; **P<0.01 by Mann-Whitney U test. C, Partial carotid ligation was performed in wild-type mice to induce disturbed flow in the ligated left carotid compared to the unligated right carotid. Scale bar=100 μm, n=7. vWF indicates von Willebrand factor. DAPI indicates 4’,6-diamidino-2-phenylindole; and Exp., expression.

Flow Patterns and Sulfide Metabolism

Free H2S can be metabolized to sulfane sulfurs and acid-labile sulfurs, which serve as biological reservoirs of H2S. Because laminar flow-induced CSE suppression is lost in areas of disturbed flow, we wanted to know whether this led to the elevation of H2S and its metabolites. Sulfide pools from the carotid arteries were measured 7 days after ligation (Figure 2). In wild-type mice, both free H2S and sulfane sulfurs were increased by the ligation, whereas the acid-labile pool was not affected. CSE knockout mice showed a complex change in sulfur species. In nonligated carotid arteries, CSE knockout led to a trend toward basal reduction of sulfane sulfur content but an unexpected increase of free sulfide, although neither reached statistical significance. Acid-labile sulfur was unchanged. Of particular importance, the augment in sulfane sulfur after ligation was blunted in CSE knockout mice, whereas the change of free sulfide was not affected. Total sulfide levels showed a negative trend in the CSE knockout mice but did not reach statistical significance (P=0.08). Together, these data suggest disturbed flow increases CSE-derived sulfane sulfur, which may be involved in flow-mediated vascular responses.

Figure 2.

Figure 2. Partial ligation enhances free and sulfane sulfur in wild-type (WT) but not CSE−/−(cystathionine γ-lyase knockout) carotid arteries. Distinct sulfide pools, including free H2S (A), acid-labile sulfur (B), sulfane sulfur (C), and total sulfide (D) were measured by monobromobimane 7 d after partial carotid ligation in WT and CSE−/− mice. (n=6–8; *P<0.05 comparing right carotid (RC) to left carotid (LC); ##P<0.01 comparing WT to CSE−/− by 2-Way ANOVA).

CSE and Flow-Mediated Vascular Inflammation

Disturbed flow patterns promote endothelial proinflammatory activation through enhanced NF-κB signaling, whereas laminar flow limits endothelial activation.22 In wild-type mice, the partial carotid ligation increased Mac-2 positive macrophages 7 days after the surgery (Figure 3A–3C), whereas the contralateral right carotids were devoid of macrophage staining (Figure IV in the online-only Data Supplement). Surprisingly, this macrophage recruitment was completely blunted in CSE knockout mice (Figure 3A–3C). To study endothelial proinflammatory gene expression, we isolated intimal mRNA 2 days after ligation as previously described.3,23 VCAM-1 (vascular endothelial adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) are normally upregulated on the surface of endothelial cells by disturbed flow to recruit leukocytes from the circulation. Upregulation of VCAM-1 and ICAM-1 mRNA is blunted in CSE knockout intima (Figure 4A). To assess the proinflammatory response to flow in vitro, we analyzed NF-κB activation and proinflammatory gene expression in response to acute (<2 hours) or chronic (18 hours) oscillatory flow. CSE knockout endothelial cells showed reduced NF-κB activation, as assessed by phosphorylation of the p65 subunit (hereafter NF-κB), in response to acute oscillatory flow (Figure 4B). However, extracellular signal-regulated kinase 1/2 (ERK1/2) activation by oscillatory flow was unaffected (Figure V in the online-only Data Supplement), suggesting that CSE is not required for all flow-induced responses. Consistent with this pattern, chronic oscillatory flow–induced NF-κB activation, assessed by NF-κB phosphorylation (Figure 4C) and nuclear translocation (Figure 4D), was similarly blunted in CSE knockout endothelial cells. In addition, CSE knockout endothelial cells show diminished oscillatory flow–induced ICAM-1 expression (Figure 4E) and VCAM-1 expression (Figure 4F). Thus, CSE critically regulates flow-induced NF-κB signaling to drive ICAM-1/VCAM-1 expression and macrophage recruitment.

Figure 3.

Figure 3. Absence of macrophages in ligated CSE−/−(cystathionine γ-lyase knockout) carotids. A, 7 d after partial carotid ligation in wild-type (WT) and CSE−/− mice, mouse carotid arteries were immunostained for macrophages (Mac-2) and smooth muscle actin (SMA). B/C, Macrophages in (B) tunica intima, media, and (C) adventitia were quantified by Mac-2 positive area (scale bar=100 μm; n=7–8; P<0.05 by (B) Mann-Whitney U Test and (C) Student t test).

Figure 4.

Figure 4. Shear-induced NF-κB (nuclear factor–κB) activation and adhesion protein expression are reduced in CSE−/− (cystathionine γ-lyase knockout) endothelium. A, Intimal mRNA was collected 48 h after partial ligation and assessed for VCAM-1 (vascular cell adhesion molecular 1) and ICAM-1 (intercellular adhesion molecule 1) expression. n=4 to 8. B, NF-κB phosphorylation after short-term oscillatory shear stress (OSS; 0–120 min) was prevented in CSE−/− mouse aortic endothelial cells (MAECs). n=4. C/D, NF-κB activation after chronic OSS (16 h) was assessed by (C) phosphorylation and (D) nuclear translocation in wild-type (WT) and CSE−/− MAECs. For nuclear translocation, at least 80 cells were scored for nuclear NF-κB staining per condition within each experiment. n=4. E/F, Chronic OSS-induced (E) ICAM-1 and (F) VCAM-1 expression was assessed by Western blotting. Representative images are shown. n=4; * P<0.05, ** P<0.01, *** P<0.001 by Student t test (A) or 2-way ANOVA (B–F). ERK indicates extracellular signal-regulated kinase 1/2; Exp., expression; LC, left carotid; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; p-ERK, phosphorylated ERK; p-NF-κB, phosphorylated NF-κB; and RC, right carotid.

CSE and Flow-Dependent Vascular Remodeling

Reductions in blood flow cause inward vascular remodeling to reduce lumen diameter and maintain shear forces within a physiological range. In the absence of hypercholesterolemia, the reductions in blood flow observed in the partial carotid ligation model results in inward remodeling with reduced lumen volume, medial thickening, and adventitial expansion.24,25 Because endothelial CSE was upregulated in vessels with low/disturbed flow, we examined whether CSE expression affects the remodeling of these vessels in response to reduced flow. In wild-type mice, thickness of the tunica media was increased 7 days after partial carotid ligation (Figure 5A and 5B). In comparison, CSE knockout mice exhibited reduced medial thickness compared to wild-type mice in both ligated and nonligated carotid arteries (Figure 5A and 5B). Meanwhile, partial carotid ligation resulted in a reduced overall vessel area and smaller lumen size in the wild-type mice. However, CSE knockout mice did not show a significant change in vessel or lumen area following partial ligation (Figure 5A and 5C), and lumen area was significantly larger in CSE knockout mice. Although these data suggest that CSE deficient mice show defective inward remodeling, CSE deficiency did not fully prevent the ligation induced medial thickening (Figure 5B). Therefore, CSE contributes to flow-mediated vascular remodeling, whereas the lack of CSE significantly blunts this process.

Figure 5.

Figure 5. Impaired vascular remodeling in CSE−/− (cystathionine γ-lyase knockout) carotids. A, Movat’s stain was performed 7 d after carotid partial ligation. B and C, Medial thickness, vessel and lumen areas were quantified (scale bar=100 μm; n=7–8; P<0.05 by 2-way ANOVA). LC indicates left carotid; RC, right carotid; and WT, wild-type C57BL/6J.

Flow Patterns and NO Metabolism

H2S has been shown to interact with NO signaling on multiple levels.21 Here, we examined NO availability in the carotid artery samples after partial carotid ligation (Figure 6A). Ligation increased Nox concentration in wild-type mice but not CSE knockout mice. Meanwhile, CSE knockout mice showed an elevated basal level of NOx in carotid artery tissue. These patterns correlate with the change of free H2S and sulfane sulfurs. H2S can increase NO synthesis by activating eNOS (endothelial nitric oxide synthase).26,27 In cell culture, short-term laminar shear activates eNOS activity by the phosphorylation of S1176. However, the eNOS phosphorylation by shear was not different between wild-type and CSE knockout mouse aortic endothelial cells (Figure 6B).

Figure 6.

Figure 6. Increased NO availability in CSE−/− (cystathionine γ-lyase knockout) carotid arteries. A, Nitrite levels in carotid arteries were measured by triiodide chemiluminescence 7 d after partial ligation (n=5–7; P<0.05 by 2-way ANOVA). B, Laminar shear stress (LSS) induced eNOS (endothelial nitric oxide synthase) phosphorylation was not affected in CSE−/− mouse aortic endothelial cells (n=4; *indicated time points vs static condition, P<0.05 by 2-way ANOVA). WT indicates wild-type C57BL/6J. LC indicates left carotid. RC indicates right carotid. p-eNOS indicates phosphorylated eNOS.

The Role of NO in the Remodeling of CSE Deficient Carotid Artery to Disturbed Flow

CSE knockout carotid arteries exhibited a higher level of NOx and an inability to reduce lumen size under reduced flow. To investigate whether this dilated phenotype was because of the excessive NO in the vasculature, we treated CSE knockout mice with 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (c-PTIO), a NO scavenger, before and after the ligation. To verify NO scavenging, we measured plasma nitrite levels and observed a significant reduction after c-PTIO treatment (Figure VI in the online-only Data Supplement). The ligated carotid artery in CSE knockout mice regained the ability to reduce lumen diameter with the c-PTIO treatment (Figure 7C). However, NO scavenging did not alter the medial thickening induced by ligation. Furthermore, c-PTIO treatment did not significantly enhance macrophage recruitment after partial carotid ligation in CSE knockout mice (Figure VIIA–VIIC in the online-only Data Supplement) and did not restore oscillatory flow-induced NF-κB activation or ICAM-1 expression in CSE knockout endothelial cells (Figure VIID–VIIE in the online-only Data Supplement). These data suggest that the elevated NO bioavailability in CSE knockout carotid arteries contributes to the dilated phenotype but not the increased medial thickness or the reduced inflammation.

Figure 7.

Figure 7. 2-(4-carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (c-PTIO) prevented carotid artery dilation in CSE−/−(cystathionine γ-lyase knockout) mice. CSE−/− mice were treated with c-PTIO (1 mg/kg) for 10 consecutive d. Partial carotid ligation was performed on day 4. Carotids were collected after another 7 d. A, Movat’s stain was performed 7 d after partial carotid ligation in CSE−/− mice. B and C, Medial thickness, vessel, and lumen areas were quantified (scale bar=100 μm; n=6–8; P<0.05; ***P<0.001 using 2-way ANOVA. LC indicates left carotid; PBS, phosphate buffered saline; and RC, right carotid.

Discussion

Blood flow critically regulates vascular homeostasis and function. In embryonic development, maturation of normal blood vessels requires hemodynamic force.28 In adults, shear stress regulates vasodilation, vascular remodeling, and susceptibility to atherosclerotic plaque formation. In large arteries, such as the aorta and carotids, the vascular wall is mostly exposed to a high-velocity laminar flow. This laminar flow is beneficial for vascular health by suppressing inflammatory genes, enhancing eNOS activity and nitric oxide availability, and reducing reactive oxidative species.29 In comparison, disturbed flow in vessel curvatures, branch points, and bifurcations stimulates an atherosusceptible endothelial phenotype, with reduced NO production, enhanced oxidant stress, and increased proinflammatory gene expression.

As the predominant source of H2S in the cardiovascular system, CSE critically regulates multiple aspects of vascular function, including endothelial permeability and ischemic vascular remodeling.6,7 In this study, we found CSE expression is regulated by shear stress. In the lesser aortic curvature and the ligated carotid artery where blood flow is disturbed, endothelial CSE is highly expressed (Figure 1). However, in vitro flow models suggest that laminar flow–induced CSE downregulation plays a dominant role in this differential expression pattern. We also noticed that microvasculature in the vasa vasorum shows strong CSE expression in both ligated and nonligated carotid arteries and this staining increased upon vasa vasorum expansion after vessel ligation. These data suggest that CSE may play a larger role in regulating microvascular function under physiological conditions, as it is only expressed in large vessel endothelial cells at atherosclerosis-prone sites. Interestingly, NF-κB critically regulates CSE expression,30 and laminar flow suppresses NF-κB- dependent proinflammatory gene expression,31 suggesting a potential NF-κB-dependent regulatory mechanism.

Increased CSE expression in the ligated carotid arteries is associated with elevated sulfane sulfur content, which is blunted by genetic CSE deficiency (Figure 2). Sulfane sulfur species include oxidative products of H2S, such as persulfides and polysulfides, which can not only serve as a biological reservoir for H2S but are highly reactive themselves. These reactive sulfur species, like reactive oxygen species, can modify protein cysteines and subsequent protein functions.32 Mounting evidence suggests it is sulfane sulfurs that mediate many biological effects originally attributed to free H2S.21 It has also been shown that, in addition to H2S oxidation, H2S producing enzymes, such as CSE can be a direct source of sulfane sulfur.33 In the current study, we showed that CSE induced by disturbed flow favored the production of sulfane sulfur rather than free H2S because the latter was not affected by the absence of CSE. However, it is not clear whether this is because of increased oxidative stress under disturbed flow or preferentially regulated by CSE activity toward sulfane sulfur generation.

H2S has been described as having both anti-inflammatory and proinflammatory functions, potentially because of the dose or pool of sulfur species involved.34CSE deletion prevented the recruitment of macrophages in the ligated carotid artery possibly by suppressing the activation of NF-κB pathway and the expression of adhesion molecules in endothelium (Figures 3 and 4). NF-κB plays a critical role in disturbed flow-associated inflammation and is critical for macrophage recruitment at atheroprone regions.29,35 However, the role of CSE and H2S in NF-κB regulation may depend on the cell type, spatial signaling, and dose. In endothelial cells, addition of exogenous H2S prevented TNF-α (tumor necrosis factor α)–induced NF-κB activation and expression of ICAM-1 and VCAM-1.18 Although the inhibitory effect of exogenous H2S on NF-κB phosphorylation, nuclear translation, and DNA binding was shown to depend on p65 sulfhydration at Cys38, enhanced H2S production by endogenous CSE promotes NF-κB activation, DNA binding, and antiapoptotic gene expression through a similar Cys38 sulfhydration-dependent mechanism.36,37 Consistent with a role for endogenous CSE-derived H2S in proinflammatory responses, CSE knockdown significantly diminished lipopolysaccharide-induced NF-κB activation in macrophages,38 and CSE knockout mice show reduced NF-κB activity and inflammation in response to sepsis.39 However, CSE knockout mice also show enhanced inflammation in atherosclerosis models,9 suggesting that this anti-inflammatory effect may be context-dependent.

Previously, CSE has been shown to be protective against atherosclerosis.8,9,40 After 12 weeks of high fat diet feeding, CSE knockout mice had increased atherosclerotic lesion area in both wild-type (C57BL/6J/129SvEv) and apolipoprotein E knockout background.9 The accelerated atherosclerosis was inhibited by either exogenous H2S or global CSE overexpression on CSE knockout mice.8,9 In addition, exogenous H2S reduced ICAM-1 expression in TNF-α treated endothelial cells in culture.41 Collectively, these results demonstrate anti-inflammatory roles for CSE and H2S. However, the development of atherosclerosis involves multiple factors, such as hyperglycemia, hyperlipidemia, and leukocyte recruitment, where the role of CSE and H2S can be complicated. In our model, we focused on the physiological roles of CSE on vascular remodeling in adaption to different flow patterns.

With regards to this matter, Yang et al40 showed increased neointima formation in CSE deficient mice after 4-week carotid artery ligation. They also showed that smooth muscle cells isolated from these mice exhibited increased oxidative stress and ICAM-1 expression. However, it is well known that distinct mouse strains have different vascular remodeling phenotypes.42 The CSE deficient mice used in early studies were on the C57BL/6J/129SvEv mixed background. The strain of 129/SvEv has 2 copies of renin genes in the same chromosome. which may contribute to their age-dependent hypertension.9 In addition, hypertension predisposes the mice to atherosclerosis, making it difficult to study the role of disturbed flow in vascular remodeling. In our current study, we established fully congenic CSE genetic mutation on the C57BL/6J background, which does not develop spontaneous hypertension.43 Moreover, our partial carotid ligation model lasted for only one week, because we focused on early vascular response to disturbed flow. In contrast to CSE knockout mice on a mixed background, we saw CSE deficiency partially prevented ligation induced medial thickening, indicating CSE contributes to, rather than protects against, the inward vascular remodeling induced by disturbed flow (Figure 5).

It is thought that blood vessels have a shear stress set-point. The alteration of shear stress results in the change of vessel diameter to restore the original magnitude of shear stress.44 However, this regulatory mechanism is compromised in the CSE knockout carotid artery, which remained dilated under reduced shear after ligation (Figure 2). Endothelium-derived NO and eNOS activity is essential in the regulation of vascular tone. It is also well known that eNOS is phosphorylated at S1177 (the human equivalent to mouse S1176) and activated by shear stress.45 Previous studies have shown that H2S and NO work in a synergistic fashion.26,27 One study showed that silencing CSE in human umbilical vascular endothelial cells attenuated eNOS phosphorylation at S1177 under shear stress.46 However, we show that endothelial cells isolated from CSE knockout mice did not show compromised eNOS activation by shear stress (Figure 6B). In vivo, carotid ligation increased the NOx level in the wild-type mice, which is concurrent with monocyte infiltration in partially ligated carotid arteries, suggesting the involvement of iNOS (inducible NO synthase; Figure 6A). In comparison, the NOx level was not altered by ligation in the CSE knockout mice. Instead, CSE knockout mice showed basally elevated NOx in carotid arteries. This upregulation of basal NO availability is not likely a contribution of iNOS, because monocyte infiltrates were absent in the CSE knockout carotid artery. On the contrary, nNOS may play a compensatory role in CSE knockout vessels. As a nitric oxide synthase mainly found in the nervous system, nNOS (neuronal NO synthase) in innervating nerve fibers can be important for normal vascular function.47,48 Although it is undetectable in normal vessels, nNOS is upregulated in vascular smooth muscle cells and endothelial cells and plays a protective role during atherosclerosis.4951

Aside from a possible role of nNOS, it has been reported that sulfide and its metabolites (eg, hydropersulfides) can react with NO to form a range of chemical products, including unique S-N hybrid species, such as nitroso-persulfide (SSNO) with concomitant abilities to regulate vascular reactivity.52,53 Given that CSE deficient vessels had significantly blunted sulfane sulfur, it is possible that NOx species accumulate because of the absence of a substantial chemical reaction pathway as hydropersulfide or polysulfide levels are known to exist in micromolar quantities.54 Future studies are required to confirm the source of NO in CSE knockout vessels and to determine how CSE is regulating it. Regardless of its source, the increase in NO availability prevented the carotid artery from constriction after ligation as the NO scavenger c-PTIO was able to rescue the dilated phenotype (Figure 7). However, NO scavenging did not change the thickness of the tunica media, suggesting medial thickening and lumen narrowing are differentially regulated in CSE knockout mice.

In conclusion, CSE plays an important role in flow-induced vascular remodeling. CSE expression and sulfane sulfur production are enhanced by disturbed flow in conduit vessels. The enhanced CSE expression correlates with macrophage recruitment to these areas, which is possibly through an NF-κB dependent pathway. CSE knockout mice exhibit a complex change of vascular remodeling under disturbed flow, including reduced medial thickening and inability to narrow lumen size. The dilated phenotype in CSE knockout mice was NO-dependent but not because of eNOS dysfunction.

Nonstandard Abbreviations and Acronyms

CBS

cystathionine β-synthase

CSE

cystathionine γ-lyase

eNOS

endothelial nitric oxide synthase

H2S

hydrogen sulfide

ICAM-1

intercellular adhesion molecule 1

NF-κB

nuclear factor κB

NO

nitric oxide

NOx

nitric oxide metabolites

VCAM-1

vascular cell adhesion molecule 1

Footnotes

*These authors contributed equally to this article.

The online-only Data Supplement is available with this article at https://www.ahajournals.org/doi/suppl/10.1161/ATVBAHA.118.311402.

Correspondence to A. Wayne Orr, PhD, Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Sciences Center, Shreveport, LA, Email or Christopher G. Kevil, PhD, Center for Cardiovascular Diseases and Sciences, Louisiana State University Health Sciences Center, Shreveport, LA, Email

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Highlights

  • Vascular CSE (cystathionine γ-lyase) expression and sulfane sulfur are enhanced by disturbed flow.

  • Enhanced CSE derived sulfane sulfur under disturbed flow promotes endothelial cell activation, macrophage recruitment, and vascular inward remodeling.

  • CSE deficiency does not affect flow-induced eNOS (endothelial nitric oxide synthase) phosphorylation but enhances nitrite levels in the carotid artery (both ligated and nonligated) resulting in a dilated vascular phenotype in a NO-dependent manner.