Inhibition of Sphingomyelinase Attenuates Diet - Induced Increases in Aortic Stiffness

Abstract

Sphingomyelinases ensure ceramide production and play an integral role in cell turnover, inward budding of vesicles and outward release of exosomes. Recent data indicate a unique role for neutral sphingomyelinase (nSMase) in the control of ceramide-dependent exosome release and inflammatory pathways. Further, while inhibition of nSMase in vascular tissue attenuates the progression of atherosclerosis, little is known regarding its role on metabolic signaling and arterial vasomotor function. Accordingly, we hypothesized that nSMase inhibition with GW4869, would attenuate Western diet (WD) - induced increases in aortic stiffness through alterations in pathways which lead to oxidative stress, inflammation and vascular remodeling. Six week-old female C57BL/6L mice were fed either a WD containing excess fat (46%) and fructose (17.5%) for 16 weeks or a standard chow diet (CD). Mice were variably treated with GW4869 (2.0 μg/g body weight, intraperitoneal injection every 48 hours for 12 weeks). WD feeding increased nSMase2 expression and activation while causing aortic stiffening and impaired vasorelaxation as determined by pulse wave velocity (PWV) and wire myography, respectively. Moreover, these functional abnormalities were associated with aortic remodeling and attenuated AMP-activated protein kinase, Sirtuin 1, and endothelial nitric oxide synthase activation. GW4869 treatment prevented the WD-induced increases in nSMase activation, PWV, and impaired endothelium dependent/independent vascular relaxation. GW4869 also inhibited WD–induced aortic CD36 expression, lipid accumulation, oxidative stress, inflammatory responses, as well as aortic remodeling. These findings indicate that targeting nSMase prevents diet – induced aortic stiffening and impaired vascular relaxation by attenuating oxidative stress, inflammation and adverse vascular remodeling.

Graphical Abstract

1. Introduction

Excessive arterial stiffness, demonstrated clinically by an increase in pulse wave velocity (PWV), is a causal factor and independent prognostic indicator of cardiovascular disease (CVD)-related morbidity and mortality [1]. A Western diet (WD), characterized by excess intake of high fat, high fructose and refined carbohydrates, increases the prevalence of obesity and insulin resistance, which have emerged as important risk factors for arterial stiffening and related CVD [2, 3]. Clinical and animal data have shown that over-nutrition and/or obesity promotes accumulation of perivascular ectopic lipid, adverse vascular remodeling and stiffening and predicts CVD risk and mortality [2–5]. Indeed, our recent data showed that WD induced stiffening of the cortical actin cytoskeleton in endothelial cells, impaired endothelial nitric oxide production, oxidative stress, and inflammation, as well as adverse arterial remodeling, fibrosis and vascular stiffening [2, 3, 6].

Sphingomyelinases are a family of key enzymes in sphingomyelin metabolism that generate ceramide, which regulates different pathophysiologic processes, including cell growth, autophagy, oxidative stress, and inflammatory responses [7]. While the acid sphingomyelinase isoform is located in lysosomes involved in apoptosis signaling, the neutral sphingomyelinase (nSMase) isoform is located in the plasma membrane, endoplasmic reticulum, mitochondria, and nucleus to regulate oxidative stress, and inflammatory responses [7]. For instance, nSMase mediates angiotensin II – induced ceramide formation and related impaired arterial relaxation [8]. nSMase also mediates saturated fatty acid – induced oxidative stress, inflammation and endothelial dysfunction [9]. Conversely, in vivo inhibition of nSMase with GW4869 decreased development of atherosclerosis [10]. However, the impact of increased sphingomyelinases on arterial stiffening and underlying molecular mechanisms remains largely unexplored in diet – induced obesity. Because we have shown that 16 weeks of WD feeding induces an increase of pulse wave velocity (PWV) and aortic stiffening in female C57BL/6J mice [11], we used this model to further investigate whether inhibition of nSMase with GW4869 would prevent WD - induced aortic stiffness and impaired aortic relaxation by attenuating increased aortic lipid accumulation, oxidative and inflammatory responses, and associated aortic fibrosis and remodeling.

2. Material and methods

2.1. Animals and treatments

Five week old C57BL/6J female mice were purchased from Jackson Laboratories (Bar Harbor, ME) and cared for in accordance with National Institutes of Health guidelines. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri. At 6 weeks of age, mice were randomly distributed into four groups. Mice (2 groups) were fed a WD containing excess fat (46%) and fructose (17.5%) for 16 weeks. Control mice (2 groups) were fed a standard chow diet (CD). One group of WD and CD mice were treated with GW4869 (2.0 μg/g body weight, intraperitoneal injection every 48 hours for 12 weeks) to inhibit nSMase [12, 13]. Mice not receiving the antagonist received 200 μl of 3.75% DMSO saline (intraperitoneal injection at 48 hour intervals). The four experimental groups are designated as WD; WDGW; CD; and CDGW.

2.2. Structural, biochemical Parameters, tail cuff blood pressure measurements

Following the 16 week feeding/GW4869 protocol all mice underwent body composition analysis for whole body fat mass, lean mass and total body water by quantitative magnetic resonance analysis (EchoMRI-500; Echo Medical Systems, Houston, TX, USA) as previously described [3]. Plasma was collected at euthanasia, and samples were sent to University of Missouri Small Animal Veterinary Clinic for clinical chemistry analyses. Plasma nSMase activity was determined using a commercially available kit (#MAK152, Sigma-Aldrich, St. Louis, MO) as previously described [14]. Ceramide levels were determined by direct-binding Enzyme-Linked-Immunosorbent-Assay using 96-well polysorp black plates as previous described [15, 16]. Blood pressure was measured by tail cuff plesmythography (Koda 8; Kent Scientific Corporation, Torrington, Conn., USA) as previously described [3]. Detail methods are provided in the Online Supplement.

2.3. Aortic stiffness by PWV

Ultrasound PWV procedures were performed on isoflurane-anesthetized mice (1.75% in 100% oxygen). Quantification of PWV was based on the transit time method utilized to determine the difference in arrival times of a Doppler pulse wave at two locations along the aorta a known distance apart as previously described [2, 6].

2.4. Ex vivo aortic activity

For ex vivo aortic reactivity, a 2 mm segment of thoracic aorta was placed in ice-cold physiological salt solution (PSS) containing (in mM): 119 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.18 KH₂PO₄, 1.17 MgSO₄, 0.027 EDTA, 5.5 glucose, and 25 NaHCO₃, pH 7.4. Aortic segments were then mounted on 200 μm pins in a wire myograph (DMT, Ann Arbor, MI) for measurements of isometric tension. Viability of the aortic rings was tested by addition of KCl (80 mM•L⁻¹). Aortas were subsequently preconstricted with the thromboxane mimetic U46619 (20 nM). Relaxation of arterial rings to acetylcholine (1 nm to 10 mM) and sodium nitroprusside (1 nM to 10 mM) was assessed by cumulative addition of agonist to the vessel bath as previously described [2, 6].

2.5. Western-blot and quantitative PCR

Proteins were first separated by SDS-PAGE and then transferred to nitrocellulose membranes. Membranes were then incubated overnight at 4°C with primary antibodies against CD36, Sirtuin 1 (Sirt1), p-AMP-activated protein kinase (AMPK)α/AMPKα (1:1000 dilution, Catalog numbers: 14347, 3931S, 2535, and 2793, Cell Signaling Technology, Danvers, MA), pSer-endothelial nitric oxide synthase (eNOS)/eNOS (1:1000 dilution, #BDB610297, BDB612393, BD Biosciences, San Jose, CA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:2000 dilution, #5174s, Cell Signaling Technology, Danvers, MA). After rinsing, blots were incubated with secondary antibodies (1:5000 dilution of each antibody) for 1 hour at room temperature. Bands were visualized by chemiluminescence, and images were recorded using a Bio-Rad ChemiDoc XRS image-analysis system as previously described [2, 3, 6]. For quantitative PCR, total RNA was isolated using a Qiagen microRNeasy kit (Qiagen MD, USA). First-strand cDNA was synthesized from total RNA using the Improm-II reverse transcription kit (Promega, Madison, WI, USA) and quantitative real-time PCR was performed using a quantitative PCR Detection System (Biorad, Hercules, CA, USA). Primer sequences are listed in Supplemental Table S1. Results were normalized against the housekeeping gene GAPDH [2, 3, 6].

2.6. Immunohistochemistry and transmission electron microscopy (TEM)

A 2 mm segment of thoracic aorta was fixed in 3% paraformaldehyde, dehydrated in ethanol, paraffin embedded, and transversely sectioned in 5μm slices. Sections were then incubated with antibodies to nSMase2 (1:100 dilution, #MBC1558, Sigma, St Louis, MO), 3-nitrotyrosine (3-NT, 1:200 dilution, Millipore, Billerica, MA) and collagen I (1:100 dilution, Millipore, Billerica, MA) overnight at room temperature. After several washes the sections were incubated with appropriate secondary antibodies and observed using a confocal microscope (Leica, Buffalo Grove, IL). Verhoeff Van Gieson staining was used determine aortic medial thickness as previously described [2, 3, 6]. Oil Red O staining was performed on frozen aortic sections to detect the presence of lipid droplets as previously described [17]. For TEM, aortic samples were prepared, sectioned and stained as previously described. A JOEL 1400-EX transmission electron microscope (Joel Ltd. Tokyo, Japan) was utilized to capture and analyze three fields randomly chosen per mouse/aortic section [2]. Detailed methods for statistical analysis are further described in the Online Supplement.

2.7. Statistical Analysis

Data are reported as means ± SEM. Statistical differences in results were determined using two-way analysis of variance and Gabriel Students-Newman-Keuls post-test or paired t tests. Values were considered statistically significant when p<0.05. All analyses were performed using Sigma Plot (version 12) software (Systat Software).

3. Results

3.1. Characteristics of mice fed a WD with or without GW4869

Groups of mice receiving the CD exhibited similar body weights, whole body fat mass, cholesterol and triglyceride levels (Table 1). As expected, consumption of the WD for 16 weeks induced a significant increase in body weight (25% increase), whole body fat mass (177% increase), as well as in plasma cholesterol (88% increase) and ceramide (47% increase) levels. There were no significant differences in body weight, body composition and cholesterol levels between the two WD-fed groups. Compared to the WD alone group, GW4869 treatment reduced plasma triglyceride (32% decrease) and ceramide (25% decrease) levels. Of note, no significant differences in lean body weight, systolic and diastolic blood pressure, mean arterial pressures and pulse pressure were detected between any of the groups (Table 1).

Table 1.

Effect of GW4869 in mice fed a WD

Measures	CD	CDGW	WD	WDGW
Body weight (g)	23.05±0.28	22.21±0.16	28.87±0.64†	27.33±0.80†
Fat mass (g)	3.50±0.18	3.27±0.14	9.68±0.52†	8.76±0.31†
Lean mass (g)	16.53±0.25	16.11±0.11	16.33±0.22	15.77±0.27
Cholesterol (mg/dL)	69.38±6.81	55.38±4.23	130.25±5.40†	119.50±3.58†
Triglyceride (mg/dL)	75.63±9.24	61.38±6.00	69.75±9.29	47.38±2.40†‡
Plasma ceramide (FLU)	3.10±0.37	2.87±0.32	4.55±0.17†	3.40±0.19‡
Systolic	115.63±2.51	113.16±2.29	115.63±2.77	108.00±3.90
Diastolic	63.25±4.46	68.13±2.22	62.88±2.44	67.88±1.82
Mean arterial pressure	80.71±3.29	83.13±1.85	80.46±1.67	81.25±1.37
Pulse pressure	52.38±4.52	45.00±2.70	52.75±4.10	40.13±4.92

Values are mean ± SEM. Control Diet (CD), CD with GW4869 (CDGW), Western Diet (WD), and WD with GW4869 (WDGW). n=6–8.

^†p<0.05 compared with CD.

^‡p<0.05 compared with WD. FLU=Fluorescence light units.

3.2. Inhibition of nSMase prevents WD – induced excessive aortic stiffness

Our previous data demonstrated that 16 weeks of WD was associated with abnormal aortic stiffening [11]. The current study further found that 16 weeks of WD induced nSMase expression and activation in plasma and aortic tissue (Fig 1A–C) and an 21% increase of PWV compared to control mice (Fig 1D). However, GW4869 inhibited the WD – induced increases in both plasma nSMase activity and aortic nSMase2 protein expression, as well as reducing PWV (Fig 1A–D). To determine whether endothelial cell and vascular smooth muscle cell dysfunction occurred in response to WD, we measured isometric changes in aortic tension following exposure to acetylcholine and sodium nitroprusside. WD impaired both endothelium-dependent and -independent relaxation responses to both acetylcholine and sodium nitroprusside, suggesting diet-induced changes in endothelial and smooth muscle cell dysfunction. These defects were inhibited by treatment with GW4869 (Fig 1).

Fig 1. GW4869 prevents WD - induced increases in nSMase activity and aortic stiffening.

(A) WD increased plasma nSMase activity that was inhibited by GW4869. (B) GW4869 treatment inhibited WD - induced increases in aortic nSMase2 protein (C). Scales bars=50 μm. (D) In vivo aortic pulse wave velocity (PWV) as measured following 16 week feeding of a CD or WD. Relaxation responses of isolated aortic rings to the endothelium-dependent dilator, acetylcholine (E) and to the endothelium-independent vasodilator, sodium nitroprusside (F). n=6–8. †p<0.05 vs CD. ‡ p<0.05 vs WD groups.

3.3. Inhibition of nSMase decreases WD – induced ectopic lipid accumulation and CD36 expression in aorta

Overnutrition and obesity are related to aortic ectopic lipid accumulation that inhibits both aortic vasoreactivity and diastolic function [18]. As shown in Fig 2 and Supplemental Fig. S1, 16 weeks of WD increased aortic lipid accumulation (predominantly located in perivascular aortic tissues) that was correlated to increased CD36 expression. Of note, these abnormalities were blunted by GW4869 treatment, consistent with diet – induced increases in sphingomyelinase activity being associated with aortic lipid ectopic deposition and related increase in PWV and aortic stiffening.

Fig. 2. GW4869 treatment inhibits WD (16 wks) - induced increases in aortic ectopic lipid accumulation and CD36 expression.

(A) Oil Red O staining for aortic lipid droplets and corresponding quantitative analysis (B). Scales bars=50 μm. (C-D) Quantitative analysis showing GW4869 inhibition of WD – induced increases in CD36. n=5 – 6. †p<0.05 vs CD. ‡ p<0.05 vs WD groups.

3.4. Inhibition of nSMase prevents WD – induced aortic Sirt1, AMPKα, and eNOS inactivation.

In response to overnutrition and ectopic lipid accumulation stimulation, Sirt1 and AMPKα, the major nutrient sensing and metabolic signaling molecules, were inhibited in mice fed a WD. This change was associated with reduced eNOS activity (Fig 3). Treatment with GW4869 prevented both WD induced inhibition of Sirt1 and AMPKα activity. Further, GW4869 treatment prevented the WD – induced impairment in eNOS activity (Fig 3).

Fig 3. GW4869 prevents WD induced reductions in Sirt1, AMPKα and eNOS activity.

(A) Protein expression of Sirt1, eNOS, and AMPKα 1 in aortic tissues were measured with immunoblotting. (B) Quantitative analysis of protein expression in Sirt1, pS1177-eNOS, and pThr172 - AMPKα. n=6. †p<0.05 vs CD. ‡ p<0.05 vs WD groups.

3.5. Inhibition of nSMase decreases WD – induced aortic oxidative stress and inflammation responses

To delineate mechanisms by which GW4869 impacts WD-induced aortic oxidative stress and inflammatory responses, we evaluated the production of 3-NT, mRNA levels for NADPH oxidase 2 (NOX2) and NOX4, as well as expression of inflammatory cytokines. WD feeding led to significant increases of 3-NT production, a marker of oxidant stress from accumulation of the oxidant peroxynitrite (ONOO⁻). Similarly, WD feeding was associated with increased mRNA expression for NOX2 and NOX4 along with increased expression for the cytokines, intercellular adhesion molecular 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) (Fig 4). Importantly, GW4869 treatment attenuated the WD-induced increases in these measures of oxidative stress and inflammation (Fig 4).

Fig. 4. GW4869 inhibits WD-induced aortic oxidative stress and inflammatory response.

(A) Representative images of immunostaining for aortic 3 – nitrotyrosine with corresponding quantitative analysis in aortic tunica intima and adventitia (B). (C) mRNA expression of NOX2 and NOX4 in aortic tissues as measured by real-time PCR. (D) mRNA expression of inflammatory cytokines in ICAM-1, VCAM-1 and MCP-1. n=4 – 6. †p<0.05 vs CD. ‡ p<0.05 vs WD groups.

3.6. Inhibition of nSMase prevents WD – induced aortic remodeling

In the current study, we observed that 16 weeks of WD feeding promoted aortic ectopic lipid accumulation, excessive oxidative stress and inflammation, which were associated with maladaptive aortic issue remodeling as indicated by thickening of the aorta wall and fibrosis (Fig 5A–B). These findings were attenuated in GW4869 treated mice (Fig 5A–B). At the ultrastructural level, TEM further revealed that GW4869 inhibited WD - induced endothelial cell separating and thinning, as well as disorganization of fibrillary collagen (Fig 5C).

Fig. 5. GW4869 inhibits WD-induced aortic remodeling and ultrastructural abnormalities.

(A) Representative images showing medial wall thickening by VerhoeffVan Gieson staining (A) and periaortic fibrosis as indicated by immunohistochemical staining for collagen I (B) with corresponding quantitative analysis. Scales bars=50 μm. (D) TEM micrographs depicting aortic ultrastructure remodeling. Arrows indicate the endothelial surface. Scales bars=1 μm. n=4 – 8. †p<0.05 vs CD. ‡ p<0.05 vs WD groups

4. Discussion

The current results provide several novel findings that advance our understanding of the role played by sphingomyelinases in the development of excessive aortic stiffness in diet induced obesity. First, GW4869 prevented the increases in nSMase expression and activation, aortic stiffening and impaired relaxation which occurred in response to 16 weeks of WD feeding. The reduced PWV and improved aortic relaxation by GW4869 were associated with inhibition of ectopic lipid deposition, reduced CD36 expression and enhanced Sirt1, AMPKα, and eNOS activity. Meanwhile, GW4869 prevented WD - induced increases in aortic 3-NT production, NOX2, ICAM-1, VCAM-1, MCP-1, and associated aortic fibrosis and remodeling.

Accumulating evidence reveals that ceramide, a prototypical sphingolipid product of sphingomyelinase, is produced in vascular cells, found in human plasma, and is a risk factor for excessive oxidative stress, inflammation and atherosclerosis [19]. Related to this, the predominant mechanism for generation of ceramide occurs through sphingomyelin hydrolysis by nSMase [19, 20]. Increased ceramide is known to stimulate mitochondrial oxidative stress and related inflammation [19]. As a result, therapeutic strategies to target nSMase in prevention of CVD have been investigated recently. For instance, GW4869, a nSMase inhibitor, reversed excessive ceramide and oxidative stress – induced impaired flow mediated dilation in arterioles from patients with coronary heart disease [19]. In vivo GW4869 also decreased development of atherosclerosis [10]. Consistent with these data, our study showed in vivo GW4869 inhibited diet – induced increases in nSMase expression and activation, aortic stiffening, and improved aortic endothelium-dependent and -independent relaxation.

High fat diet feeding in rats has been shown to induce pronounced ceramide and sphingomyelin accumulation through increased activity of nSMase but not acid sphingomyelinase [21]. One study further found that nSMase mediated hyperosmolarity – induced ectopic lipid droplet formation [22]. Our data further showed that nSMase mediated 16 weeks of WD -induced increased CD36 expression and ectopic lipid accumulation in the aorta. Related to this, CD36 is a fatty acid translocase which promotes free fatty acid transfer and ectopic lipid accumulation. Increased CD36 has been found in obese rodents and patients [23–25]. Moreover, nSMase also mediated diet – induced reduced activity of AMPKα, Sirt1 and eNOS in the current study. AMPKα is a crucial regulator in maintaining metabolic homeostasis. Activated AMPKα increases the activity of Sirt1 to control the function of metabolic regulators by de-acetylation, leading to increased adiponectin and reduced inflammation responses [26]. Further, increases in AMPKα and Sirt 1 activation are necessary for phosphorylation and optimal activation of eNOS [27, 28]. As a consequence, repressed AMPKα and Sirt1 activity reduce eNOS activity and nitric oxide production that contribute to excessive aortic stiffness and impaired aortic relaxation in association with consumption of a WD. To this point, nSMase hydrolyzes sphingomyelin to produce ceramide that accumulates in tissues in response to obesity and high fat diet consumption [29]. Excessive accumulation of ceramides is thought to induce foam cell formation and promote toxicity in multiple types of cells, including endothelial cells and cardiomyocytes. As a consequence, ceramides are suggested to play roles in the pathogenesis of a number of disorders, such as diabetes, hypertension, heart failure, and atherosclerosis [10]. Indeed, recent clinical trials have shown that elevated circulating ceramide levels correlate strongly with cardiovascular events, including myocardial infarction and stroke [30]. Several studies have further reported that ceramide represses activation of eNOS [31], AMPK [32], and Sirt1 [33, 34]. Therefore, increased nSMase activation and ceramide levels, through inhibition of eNOS, AMPK, and Sirt1 activity, would be expected to lead to arterial stiffening and vascular dysfunction. Inhibiting sphingomyelinases with GW4869 may represent a potential therapeutic strategy in prevention of excessive arterial stiffness and associated CVD.

Increased nSMase activity promotes tissue oxidative stress and inflammatory responses [7] consistent with our data of increased 3-NT production, NOX2 and NOX 4 expression, as well as inflammatory cytokines, including ICAM-1, VCAM-1 and MCP-1. As we expected, GW4869 inhibited the aortic oxidative stress, inflammatory responses and vascular remodeling. In the present study, the mechanism in diet – induced nSMase activity and aortic stiffening is potentially related to altered exosome formation and release. To this point, recent studies indicated that ceramide is generated by nSMase and is involved in the inward budding of endosomes to form multivesicular bodies containing exosomes [35–37]. Emerging evidence strongly suggests that exosomes play an important role in mediating intercellular communication and inhibition of nSMase with GW4869 reduced release of exosomes and related CVD [10, 18, 38–40]. For instance, GW4869 prevented the effects of vascular adventitial fibroblasts-derived exosomes on vascular smooth muscle cell migration and related arterial remodeling [38]. GW4869 also inhibited the role macrophage – derived exosomes on triggering matrix metalloproteinase-2 expression and aortic aneurysm development.[39] Therefore, GW4869 may improve aortic stiffness through GW4869’s role in inhibition of exosome formation and release.

There are some limitations to this investigation. For example, we only evaluated one dose of GW4869. The dose chosen was based on data reported in mouse studies [12, 13], and future studies need to establish dose-response relationships for GW4869 with respect to arterial stiffening in diet induced obesity. In addition, the present study was only conducted in female mice. The rationale for having chosen female mice in this study is that obese and diabetic females lose CVD protection that is typically afforded by female sex and this may relate to an increased propensity to develop cardiovascular stiffness in obese female [41]. This is further supported by our previous work showing that female mice fed a WD display a more rapid onset of insulin resistance as well as aortic stiffness compared to males [6, 11, 41]. Future work, however, should include evaluation of GW4869 on diet induced arterial stiffening in males. Meanwhile, we did not investigate the role of nSMase in exosome formation and release that are potentially related to diet – induced arterial stiffening. Further, our study lacks direct information of changes in plasma high-density lipoproteins, low-density lipoproteins, free fatty acids, aortic matrix proteins and remodeling, tissue Sirt1 activity, as well as the oxidative stress production changes. Further targeted studies are required to directly verify these mechanisms.

Collectively, data presented here indicate a pivotal role of sphingomyelinases in development of excessive stiffness in diet – induced obesity. These vascular changes are associated with increases in aortic CD36 expression, ectopic lipid accumulation, oxidative stress, and inflammation, as well as reduced AMPKα, Sirt 1, and eNOS activation in the aorta of female mice fed a WD. These preclinical highly translational observations fill a gap in our knowledge of the role of nSMase in promotion of vascular stiffening.

Highlights.

• Sphingomyelinases promote excessive aortic stiffness and impaired vascular relaxation in association with consumption of a Western diet.

• Reduced AMP-activated protein kinase, Sirtuin 1, and endothelial nitric oxide synthase activation are related to elevated sphingomyelinase activation.

• Lipid metabolic disorders, oxidative stress, as well as inflammatory response are associated with arterial stiffening and cardiovascular disease.

• Targeting sphingomyelinases represents a potential therapeutic strategy in prevention of excessive arterial stiffness and associated cardiovascular disease.