Antihypertensive treatment differentially affects vascular sphingolipid biology in spontaneously hypertensive rats.
ABSTRACT We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A(2), cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A(2). This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR.
For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20-25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A(2). The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction.
In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.
- SourceAvailable from: atvb.ahajournals.org[show abstract] [hide abstract]
ABSTRACT: Sphingolipids and their metabolic products are now known to have second-messenger functions in a variety of cellular signaling pathways. Lactosylceramide (LacCer), a glycosphingolipid (GSL) present in vascular cells such as endothelial cells, smooth muscle cells, macrophages, neutrophils, platelets, and monocytes, contributes to atherosclerosis. Large amounts of LacCer accumulate in fatty streaks, intimal plaque, and calcified intimal plaque, along with oxidized low density lipoproteins (Ox-LDLs), growth factors, and proinflammatory cytokines. A possible role for LacCer in vascular cell biology was suggested when this GSL was found to stimulate the proliferation in vitro of aortic smooth muscle cells (ASMCs). A further link of LacCer in atherosclerosis was uncovered by the finding that Ox-LDLs stimulated specifically the biosynthesis of LacCer. Ox-LDL-stimulated endogenous synthesis of LacCer by activation of UDP-Gal:GlcCer,beta1-4galtransferase (GalT-2) is an early step in this signaling pathway. In turn, LacCer serves as a lipid second messenger that orchestrates a signal transduction pathway, ultimately leading to cell proliferation. This signaling pathway includes LacCer-mediated activation of NADPH oxidase that produces superoxide. Such superoxide molecules stimulate the GTP loading of p21(ras). Subsequently, the kinase cascade (Raf-1, Mek2, and p44MAPK [mitogen-activated protein kinase]) is activated. The phosphorylated form of p44MAPK translocates from the cytoplasm to the nucleus and engages in c-fos expression, proliferating cell nuclear antigen (PCNA) such as cyclin activation, and cell proliferation takes place. Interestingly, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of GalT-2, can abrogate the Ox-LDL-mediated activation of GalT-2, the signal kinase cascade noted above, as well as cell proliferation. Additional studies have revealed that LacCer mediates the tumor necrosis factor-alpha (TNF-alpha)-induced nuclear factor-kappaB expression and intercellular adhesion molecule (ICAM-1) expression in vascular endothelial cells via the redox-dependent transcriptional pathway. LacCer also stimulates the expression of CD11/CD8, or Mac-1, on the surface of human neutrophils. Collectively, this phenomenon may contribute to the adhesion of neutrophils or monocytes to the endothelial cell surface and thus initiate the process of atherosclerosis. In addition, the LacCer-mediated proliferation of ASMCs may contribute to the progression of atherosclerosis. On the other hand, programmed cell death (apoptosis) by proinflammatory cytokines such as TNF-alpha, interleukin-1, and high concentrations of Ox-LDL occur via activation of a cell membrane-associated neutral sphingomyelinase (N-SMase). N-SMase hydrolyzes sphingomyelin into ceramide and phosphocholine. In turn, ceramide or a homologue serves as an important stress-signaling molecule. Interestingly, an antibody against N-SMase can abrogate Ox-LDL- and TNF-alpha-induced apoptosis and therefore may be useful for in vivo studies of apoptosis in experimental animals. Because plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke, or heart failure. Interestingly, in human liver cells, N-SMase action mediates the TNF-alpha-induced maturation of the sterol regulatory-element binding protein. Moreover, a cell-permeable ceramide can reconstitute the phenomenon above in a sterol-independent fashion. Such findings may provide new avenues for therapy for patients with atherosclerosis. The findings described here indicate an important role for sphingolipids in vascular biology and provide an exciting opportunity for further research in vascular disease and atherosclerosis.Arteriosclerosis Thrombosis and Vascular Biology 11/1998; 18(10):1523-33. · 6.34 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Ceramide has been shown to be critically involved in many forms of apoptosis including death triggered by receptors, e.g. CD95 or the tumor necrosis factor receptor, during development, or stress. Stress stimuli that employ ceramide to induce apoptosis include irradiation, heat shock or UV-light. The release of ceramide by these stimuli is mediated by the acid sphingomyelinase (ASM), an enzyme that is rapidly activated by many apoptotic stimuli. Studies on CD95 may serve as paradigm for the regulation of the ASM and showed that stimulation via this receptor triggers a rapid translocation of the ASM from intracellular stores onto the extracellular leaflet of the cell membrane. This seems to be mediated by a fusion of ASM containing vesicles with the cell membrane resulting in cell surface exposure of the ASM. Surface ASM releases ceramide from sphingomyelin that rapidly forms ceramide-enriched platforms in the cell membrane. These ceramide-enriched membrane domains serve as signaling platforms that cluster receptor molecules and transmit the apoptotic stimuli into the cell. Identical mechanisms seem to be operative in the signaling of apoptosis by other death receptors or stress suggesting a general role of ceramide-enriched platforms in apoptosis.Pharmacological Research 06/2003; 47(5):393-9. · 4.35 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: Sphingosine 1-phosphate is formed in cells in response to diverse stimuli, including growth factors, cytokines, G-protein-coupled receptor agonists, antigen, etc. Its production is catalysed by sphingosine kinase, while degradation is either via cleavage to produce palmitaldehyde and phosphoethanolamine or by dephosphorylation. In this review we discuss the most recent advances in our understanding of the role of the enzymes involved in metabolism of this lysolipid. Sphingosine 1-phosphate can also bind to members of the endothelial differentiation gene (EDG) G-protein-coupled receptor family [namely EDG1, EDG3, EDG5 (also known as H218 or AGR16), EDG6 and EDG8] to elicit biological responses. These receptors are coupled differentially via G(i), G(q), G(12/13) and Rho to multiple effector systems, including adenylate cyclase, phospholipases C and D, extracellular-signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase and non-receptor tyrosine kinases. These signalling pathways are linked to transcription factor activation, cytoskeletal proteins, adhesion molecule expression, caspase activities, etc. Therefore sphingosine 1-phosphate can affect diverse biological responses, including mitogenesis, differentiation, migration and apoptosis, via receptor-dependent mechanisms. Additionally, sphingosine 1-phosphate has been proposed to play an intracellular role, for example in Ca(2+) mobilization, activation of non-receptor tyrosine kinases, inhibition of caspases, etc. We review the evidence for both intracellular and extracellular actions, and extensively discuss future approaches that will ultimately resolve the question of dual action. Certainly, sphingosine 1-phosphate will prove to be unique if it elicits both extra- and intra-cellular actions. Finally, we review the evidence that implicates sphingosine 1-phosphate in pathophysiological disease states, such as cancer, angiogenesis and inflammation. Thus there is a need for the development of new therapeutic compounds, such as receptor antagonists. However, identification of the most suitable targets for drug intervention requires a full understanding of the signalling and action profile of this lysosphingolipid. This article describes where the research field is in relation to achieving this aim.Biochemical Journal 08/2000; 349(Pt 2):385-402. · 4.65 Impact Factor
Antihypertensive Treatment Differentially Affects
Vascular Sphingolipid Biology in Spontaneously
Le ´on J. A. Spijkers1, Ben J. A. Janssen2, Jelly Nelissen2, Merlijn J. P. M. T. Meens2, Dayanjan Wijesinghe3,
Charles E. Chalfant3, Jo G. R. De Mey2, Astrid E. Alewijnse1, Stephan L. M. Peters1*
1Department of Pharmacology & Pharmacotherapy, Academic Medical Center, Amsterdam, The Netherlands, 2Department of Pharmacology, Maastricht University,
Maastricht, The Netherlands, 3Department of Biochemistry, Virginia Commonwealth University, Richmond, Virginia, United States of America
Background: We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR),
is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of
ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A2,
cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A2. This phenomenon is almost absent in
vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse
elevated ceramide levels and reduce ceramide-mediated contractions in SHR.
Methods and Findings: For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist
losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 19167 mmHg,
losartan: 12565 mmHg and hydralazine: 113614 mmHg). The blood pressure lowering was associated with a 20–25%
reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups.
Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While
both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of
calcium-independent phospholipase A2. The latter finding may explain the effect of losartan treatment on
sphingomyelinase-induced vascular contraction.
Conclusion: In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and
specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not
blood pressure lowering per se, reduces ceramide-mediated arterial contractions.
Citation: Spijkers LJA, Janssen BJA, Nelissen J, Meens MJPMT, Wijesinghe D, et al. (2011) Antihypertensive Treatment Differentially Affects Vascular Sphingolipid
Biology in Spontaneously Hypertensive Rats. PLoS ONE 6(12): e29222. doi:10.1371/journal.pone.0029222
Editor: Joseph Najbauer, City of Hope National Medical Center and Beckman Research Institute, United States of America
Received August 5, 2011; Accepted November 22, 2011; Published December 15, 2011
This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for
any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.
Funding: This study was performed within the framework of Top Institute Pharma project T2-108. CEC and DSW acknowledge support by grants from the
Veteran’s Administration (VA Merit Review I to CEC, a Research Career Scientist Award to CEC and a CDA1 to DSW), from the National Institutes of Health
(HL072925) (CEC), CA117950 (CEC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Sphingolipids are a class of bioactive lipids with important roles
in cell signaling as they control cell growth, migration and
differentiation [1–4]. It is becoming increasingly evident that these
lipids play an important physiological role in the cardiovascular
system. In the vasculature for instance, the sphingolipid sphingo-
sine-1-phosphate (S1P) is known to regulate endothelial function
via activation of nitric oxide synthase [5,6] or inhibition of
endothelium-derived hyperpolarizing factors .
More recently it was shown that sphingolipids also play a
pathological role in hypertension. For instance, a recent genetic
analysis by Fenger et al. suggested the involvement of the
sphingolipid system in the regulation of blood pressure and
hypertension on a genetic basis . Moreover, Yogi et al. have
recently shown in vitro that S1P is a potent inducer of pro-
inflammatory signaling pathways through epidermal growth factor
receptor and platelet-derived growth factor trans-activation, a
pathway that is up-regulated in spontaneously hypertensive stroke-
prone rats . We have previously shown that sphingolipids are
involved in the pathophysiology of hypertension in vivo . This
latter study showed that vascular and plasma levels of the bioactive
sphingolipid ceramide were significantly higher in spontaneously
hypertensive rats (SHR) than in normotensive Wistar Kyoto
(WKY) rats. In addition, we demonstrated that also in humans
with essential hypertension, plasma ceramide levels correlated
positively to the level of blood pressure. The pathophysiological
relevance of this finding was demonstrated by the observation that
pharmacological elevation of ceramide in isolated carotid arteries
of SHR but not WKY, leads to endothelium-dependent arterial
contractions by thromboxane A2 (TXA2) and increases blood
pressure in SHR in vivo . Thus sphingolipids are not only
involved in the regulation endothelium-derived relaxing factors
but also the production of endothelium-derived contracting
PLoS ONE | www.plosone.org1December 2011 | Volume 6 | Issue 12 | e29222
factors, especially in the setting of hypertension, and may thus
contribute to endothelial dysfunction.
Aforementioned data clearly indicate that hypertension is
associated with marked alterations in vascular sphingolipid
biology. In the present study we investigated whether these
alterations in sphingolipid biology could be reversed by antihy-
pertensive treatment. We investigated the effects of chronic (4-
week) treatment with the angiotensin II type 1 antagonist losartan
or the non-selective vasodilator hydralazine in SHR on ceramide
levels in vascular tissue and blood plasma. Furthermore, we
investigated the effects of both antihypertensive treatment
regimens on ceramide-mediated, endothelium-dependent arterial
contractions induced by exogenously applied sphingomyelinase
(SMase). In this study, we observed that both antihypertensive
drugs significantly lowered arterial ceramide levels in concurrence
with the reduced blood pressure, and that losartan, but not
hydralazine, inhibited SMase-induced contractions of isolated
carotid arteries, most likely by decreasing the expression of
endothelial calcium-independent PLA2(iPLA2).
The animal experiments performed in this study followed a
protocol that was specifically approved by the Animal Ethics
Committee of Maastricht University, Maastricht, The Netherlands
(approval number: 2010-050), and was in accordance with EU
guidelines (2010/63/EU) on the care and use of laboratory animals.
Compounds and antibodies
Acetyl-b-methylcholine (methacholine; MCh) and phenylephrine
(Phe) were purchased from Sigma-Aldrich Chemical Co. (St. Louis,
MO, USA) and neutral sphingomyelinase C (SMase; from
Staphylococcus aureus) from Biomol International L.P. (Plymouth, PA,
USA). Antibodies against cyclooxygenase-1 (order#160109; 1/400
dilution used) and thromboxane synthase (#160715; 1/200) were
purchased from Cayman Chemical; calcium-independent phospho-
lipase A2antibody (#ab23706; 1/400) from Abcam (Cambridge,
UK) and Von Willebrand factor antibody (GTX74830; 1/200) from
GeneTex (Irvine, CA, USA). Alexa Fluor 488-labeled (#A-11029:
1/400) and Alexa Fluor 546-labeled secondary antibodies (#A-
11010: 1/400) were from Invitrogen (Carlsbad, CA, USA).
Animals and treatment
Adult six month old male spontaneously hypertensive rats
(SHR) were purchased from Charles River (L’Arbresle, France).
Rats were anesthetized with isoflurane and osmotic minipumps
(2ML4; Alzet, California, USA) were subcutaneously implanted.
The minipumps were filled with losartan (dissolved in saline), or
hydralazine (dissolved in saline). The concentrations were chosen
to obtain the continuous 4 weeks release of losartan at 20 mg/
kg.day and hydralazine at 9 mg/kg.day. Because the maximal
solubility of hydralazine was reached in saline, additional
hydralazine was added to the drinking water to increase the dose
to 20 mg/kg.day. In untreated SHR a dummy device (PE tube of
the same size as the 2ML4 pumps) was implanted subcutaneously.
Blood pressure measurements
Conscious tail cuff blood pressure measurements were per-
formed 28 days after the initiation of the drug treatments using the
system (Kent Scientific Corporation, CT, USA).
Differences in tail cuff systolic blood pressures (SBP) were verified
by intra-arterial measurements when rats were anesthetized with
2.5% isoflurane. For this purpose a PE-10 canula was inserted into
the abdominal aorta via the femoral artery. The arterial pressure
was recorded at 2.5 kHz using IDEEQ data acquisition software
(Maastricht, The Netherlands). When blood pressure was
stabilized, baseline values of blood pressure were recorded and
averaged over 10–15 minutes. Hereafter, blood plasma, organs
and blood vessels were collected and processed.
Liquid chromatography - mass spectrometry on blood
Post-anesthesia, the thoracic region was opened and 5 mL of
blood was collected by abdominal aorta puncture using a 21G
needle (BD Microlance 3) and collected in a pre-chilled (0uC)
polypropylene blood collection tube containing PECT solution as
described in Spijkers et al. . Blood plasma was prepared by
centrifugation for 20 min at 16006 g, 4uC within 10 min after
collection and stored at 280uC. Furthermore, the thoracic aorta
was isolated and snap-frozen in liquid nitrogen. For blood plasma
samples, lipids were extracted from 33 mL blood plasma as
described by and Merrill et al.  Wijesinghe et al.  with slight
modifications. Briefly; to 33 mL of plasma 167 mL water, 1 mL
methanol and 0.5 mL chloroform were added together with an
internal standard containing 500 pmol of the following; d17:1
sphingosine, sphinganine, sphingosine-1-phosphate and sphinga-
nine-1-phosphate, and d18:1/12:0 ceramide, ceramide-1-phos-
phate, sphingomyelin and glucosylceramide. The mixture was
sonicated and incubated at 48uC overnight. The following day,
extracts were subjected to base hydrolysis for 2 h at 37uC using
150 mL of 1 M methanolic potassium hydroxyde. Following base
hydrolysis the extract was completely neutralized by the addition of
6 mL glacial acetic acid. The neutralization was confirmed by pH
measurement. Half of the extract was dried down and resuspended
in reversed phase sample buffer (60%A:40%B) (A=methanol:water
60:40 with 5 mM ammonium formate and 1% formic acid,
B=methanol with 5 mM ammonium formate and 1% formic
acid). To the remainder of the extract 1 mL chloroform and 2 mL
water were added, and the lower phase was transferred to another
tube, dried down and brought up in normal phase sample buffer
(98%A:2%B). Sphingosine, sphinganine, sphingosine-1-phosphate
sphinganine-1-phosphate and ceramide-1-phosphate were quanti-
fied via reversed phase HPLC ESI-MS/MS using a Discovery C18
column attached to a Shimadzu HPLC (20AD series) and subjected
to mass spectrometric analysis using a 4000 Q-Trap (Applied
Biosystems) as described by Wijesinghe et al. . Ceramides,
sphingomyelins and monohexosyl ceramides were quantified via
normal phase HPLC ESI-MS/MS using an amino column (Sigma)
as described by Merrill et al. . For aorta samples, lipids were
extracted from 500 mL of a 10% homogenate of the tissue in PBS
according to Merrill et al.  and Wijesinghe et al.  with slight
modifications. Briefly to 500 mL of the 10% tissue homogenate
2 mLofmethanoland 1 mL ofchloroformwas added togetherwith
an internal standard and processed as described above. The inter-
day variability is less than 5% while the intraday variability is less
than 7%. The accuracy has been previously verified .
Arterial preparation and isometric force recording
Carotid artery segments were isolated from the rats and mounted
into a wire myograph for isometric tension measurements as
described by Mulders et al. . In brief, vessels were allowed to
equilibrate and organ bath buffers were replaced every 15 min with
carbogen aerated (95% O2, 5% CO2) Krebs-Henseleit buffer
(pH 7.4; in mM: 118.5 NaCl, 4.7 KCl, 25.0 NaHCO3, 1.2 MgSO4,
1.8 CaCl2, 1.1 KH2PO4and 5.6 glucose). Two high K+-containing
Krebs buffer contractions were performed (pH 7.4; in mM: 23.2
NaCl, 100 KCl, 25 NaHCO3, 1.2 MgSO4, 1.8 CaCl2, 1.1KH2PO4
Blood Pressure Reduction and Sphingolipid Biology
PLoS ONE | www.plosone.org2December 2011 | Volume 6 | Issue 12 | e29222
and 5.6 glucose) with 30 min washout in between. Then, 0.3 mM
phenylephrine was applied to gain a stabile contraction of .60% of
theK+-induced contraction,and 10 mMofmethacholinewas added
to assess endothelial integrity. After 30 min another high K+-Krebs
buffer contraction was performed. After 30 min wash-out, the
enzyme sphingomyelinase (SMase; 0.1 U/mL) was applied to the
organ baths to measure alterations in vasomotor tone during
1 hour. In other arteries, concentration-response curves for
methacholine were generated in half-log concentration increments
during phenylephrine-induced contractions.
Immunohistochemical protein staining and subsequent fluores-
cence intensity quantification in carotid artery segments of SHR
were performed as described previously . In brief, carotid artery
segments were collected in ice-cold Krebs buffer directly after
dissection and cleaning, rapidly submerged in OCT compound
(Sakura, TissueTek) and frozen in liquid nitrogen with subsequent
storage at 280uC. Frozen sections (5 mm thick) were cut on a Leica
CM3050S cryostat and cold-air dried. Slides were fixed in 100%
acetone for 1 min, washed shortly in PBS and incubated with
blocking buffer (2% BSA/PBS) for 30 min at room temperature.
After a short wash (in0.1%BSA/PBS),slideswereincubated at 4uC
overnight with the primary antibody (dissolved in 0.1% BSA/PBS).
Following a triple wash in 0.1% BSA/PBS for 15 min, the
appropriate Alexa Fluor 546-labeled secondary antibody was
applied for 1 hour at room temperature. The antibody against
von Willebrand Factor (vWF) was applied for 1 hour at room
temperature as endothelium marker and finally Alexa Fluor 488-
labeled secondary antibody was applied. Vessel slides were
embedded in DAPI-containing mounting medium and imaged
using a Nikon Eclipse TE2000-U fluorescence microscope (Plan
Fluor ELWD 206 objective, Nikon DXM1200F digital camera)
with NIS Elements AR 2.30 software. Quantification of fluores-
cence (fluorescent light units; FLU) was performed using NIS
Elements. Using the vWF endothelial marker region, mean
fluorescence intensity of the protein of interest was quantified for
EC area. Then, the tunica media was selected and mean
fluorescence intensity was determined for smooth muscle cells.
For both determinations, an intensity threshold as low as possible
was selected to exclude background fluorescence and restricting the
area of interest to mere tissue. All settings and exposure times were
equally applied to all tissue slides.
Statistical data analysis
SBP, heart rate, organ weights, protein quantification, aortic and
blood plasma sphingolipid content, and isometric tension measure-
ments in carotid artery segments are presented as means6SEM
performed by one-way ANOVA followed by Bonferroni multiple
comparisons test (95% confidence interval). Full concentration
response curves were analyzed by one-way repeated measures
ANOVA. All statistical analyses were performed using Prism
(GraphPad Prism Software, San Diego, CA, USA). Values of
p,0.05 were considered to be statistically significant.
Antihypertensive effects of losartan and hydralazine in
The 4-week treatment with either losartan or hydralazine had
no significant effect on body weight (Table 1). In untreated
conscious SHR, SBP was 19167 mmHg. The 4 weeks treatment
with losartan and hydralazine (at 20 mg/kg.day) reduced tail
cuff SBP towards normotensive levels (12565 mmHg and
113614 mmHg, respectively) (n=4–6, P,0.05). Qualitatively
similar results were obtained when blood pressures were verified
by intra-arterial measurements under isoflurane-anesthetized
conditions (12866, 9765 and 10666 mmHg for untreated,
losartan and hydralazine treated groups, respectively) (n=6–8,
P,0.05). No significant differences in blood pressure were found
between the two treatment groups. Treatment did not significantly
change heart rate, although a trend existed for an increased heart
rate in the hydralazine group as reported before  (Fig. 1B).
Table 1. General characteristics of treated and untreated
spontaneously hypertensive rats.
Body weight before treatment (g)33561033467 33567
Body weight after treatment (g)3566935367 36268
Normalized heart weight (mg/g) 4.660.2 3.760.2* 4.260.1
Normalized kidney weight (mg/g)3.360.1 3.260.13.360.1
Carotid artery media/lumen ratio0.4660.010.3960.01*0.3860.01*
Heart and kidney weight normalized to rat body weight. n=6–8,
*p,0.05 compared to control.
Figure 1. Losartan and hydralazine lower blood pressure in
SHR. Hemodynamic parameters of SHR after four weeks untreated or
treated with losartan or hydralazine. (A) systolic blood pressure; (B)
corresponding heart rate. Data are expressed as mean6SEM, n=4–6,
* p,0.05 compared to untreated.
Blood Pressure Reduction and Sphingolipid Biology
PLoS ONE | www.plosone.org3 December 2011 | Volume 6 | Issue 12 | e29222
Treatment with losartan was associated with a significantly greater
reduction in normalized heart weight than observed in hydral-
azine-treated or untreated SHR. No differences in kidney weights
were found between the three groups.
Losartan and hydralazine reduce vascular ceramide levels
Lipidomic analysis on isolated aortic tissue of SHR indicated
that the levels of ceramide were reduced by approximately 20–
25%, 4-weeks after treatment with either losartan or hydralazine
(Fig. 2A upper panel; n=8, p,0.05). Most, but not all ceramide
subspecies were decreased in vascular tissue after both treatments
(Fig. 2A lower panel). This reduction in vascular ceramide levels
was not associated with a concomitant reduction of plasma
ceramide levels. Only hydralazine reduced (mostly long chain)
ceramide levels rather modestly, while losartan had no effect
Effects of losartan and hydralazine on endothelial
function in isolated carotid arteries
Myograph studies on isolated carotid arteries indicated a
significantly improved relaxation in response to methacholine in
arteries from animals treated with losartan and hydralazine
compared to untreated SHR (Fig. 3A). In phenylephrine pre-
contracted carotid arteries of untreated SHR, methacholine
(10 mM) induced a maximal relaxation of 5562% (Fig. 3B). In
contrast, in arteries isolated from both losartan-treated and
hydralazine-treated SHR, endothelium-dependent relaxations to
10 mM methacholine were significantly enhanced (7562% and
6862% respectively, n=7–8, p,0.05). Although losartan seemed
to be somewhat more effective in restoring endothelial function
than hydralazine, the difference between the two treatments did
not reach statistical significance.
When methacholine is added to the organ bath as a single
concentration of 10 mM, in isolated vessels from SHR the
endothelium-dependent relaxation is followed by an endotheli-
um-dependent contractile response due to the release of an
endothelium-derived contracting factor (EDCF) as depicted
schematically in Figure 3C. The ratio between the endothelium-
dependent contraction and relaxation provides a more accurate
representation of endothelial function. We observed that this ratio
was significantly lower in arteries of losartan-treated rats (1.260.1)
than in arteries of hydralazine-treated (1.860.3) or untreated rats
(2.060.2, n=7–8, p,0.05) (Fig. 3D).
Losartan, but not hydralazine treatment, prevents SMase-
induced contraction in isolated carotid arteries of SHR
Pharmacological elevation of ceramide by exogenous addition
of SMase induced a strong contraction of isolated carotid arteries
in untreated SHR (1.960.3 mN/mm) which was similar to the
contractions described previously  (Fig. 4). Losartan treatment
largely preventedthese contractile
(0.460.1 mN/mm, n=5, p,0.05). The contractile response to
SMase was not altered by hydralazine treatment (1.360.3 mN/
mm, n=6, p.0.05) (Fig. 4).
Losartan, but not hydralazine treatment, reduces
We previously described the involvement of calcium-indepen-
dent phospholipase A2(iPLA2), cyclooxygenase-1 (COX-1) and
thromboxane synthase (TXAS) in the SMase-induced arterial
contractions. These enzymes are up-regulated in the endothelium
of carotid arteries from SHR compared to those from normoten-
sive WKY rats . Whereas both losartan as well as hydralazine
treatment substantially reduced COX-1 expression in endothelium
and smooth muscle cells (Fig. 5B; EC: 4768 and 4565 vs 79611
relative fluorescence (FLU), respectively; VSMC: 8668 and 8965
vs 12067 FLU, respectively; n=7–8, p,0.05), only losartan
reduced endothelial iPLA2expression (Fig. 5A; 4565 vs 7568 and
77611 FLU, respectively; n=6–7, p,0.05).
Figure 2. Losartan and hydralazine lower vascular but not plasma ceramide levels in SHR. Ceramide levels (top: total ceramide, bottom:
ceramide subspecies) were measured in (A) aorta and (B) blood plasma of untreated, losartan-treated or hydralazine-treated SHR. Data are presented
as mean6SEM, n=8, * p,0.05. Ceramide subspecies depicted as separated by different tail length. DH: dihydroceramide.
Blood Pressure Reduction and Sphingolipid Biology
PLoS ONE | www.plosone.org4December 2011 | Volume 6 | Issue 12 | e29222
The thromboxane A2 synthase (TXAS) expression was
unaltered after losartan or hydralazine treatment compared to
the untreated group in both endothelium (Fig. 5C; 41610, 46610
and 45610 FLU, respectively; n=7–8, p.0.05) and smooth
muscle cell layer (6965, 8169 and 7568 FLU, respectively;
We have previously shown that hypertension is associated with
marked alterations in sphingolipid biology; increased ceramide
levels and a predisposition to ceramide-induced endothelium-
dependent contractile responses . Here we show that blood
pressure lowering by either losartan or hydralazine lowers vascular
ceramide levels, and that losartan, but not hydralazine treatment
reverses the predisposition to ceramide-induced contractile
The 4-week treatment of SHR with losartan as well as
hydralazine, significantly decreased blood pressure as expected,
and this was associated with a concomitant lowering of vascular
ceramide levels. Since both drugs decreased blood pressure to the
same extent but likely via unrelated mechanisms, it can be
assumed that the vascular ceramide levels are subject to, and a
reflection of, the prevailing blood pressure. The reduction in
vascular ceramide levels was not, or only marginally, reflected in
plasma ceramide levels. This marginal effect could be the result of
detection at an early phase, and a stronger effect may be elicited
after prolonged antihypertensive treatment. In addition, it could
be that ceramide is released from endothelial cells abluminally,
resulting in an increase in basolateral ceramide levels and less
systemic spillover towards blood plasma. The exact mechanism of
the decreased vascular ceramide levels by blood pressure lowering
is currently unknown. Angiotensin signaling has been linked to
increases in cellular ceramide levels (for review see Berry et al.
). However, since these ceramide increasing effects were
attributed to AT2receptor stimulation, this does not explain the
effects of losartan in the present study. Furthermore, since in
addition to losartan also hydralazine was effective in reducing
vascular ceramide levels, the reduction in ceramide was, as
mentioned before, most likely a consequence of the decreased
blood pressure itself. In this regard it is noteworthy that
endogenous SMase activity may be potentiated by high shear
stress to initiate ceramide production . This may elevate
ceramide in hypertension (which on itself may further increase
vascular tone in hypertensive subjects) and therefore lower
ceramide levels are detected after blood pressure lowering.
Although we have measured ceramide levels and investigated
sphingolipid biology in larger vessels (i.e., aorta and carotid
arteries), we know from our previous in vivo experiments  that
these changes also affect other vascular (resistance) beds. The
beneficial effect of losartan and hydralazine on endothelial
function of SHR isolated carotid arteries is reflected by the
improved potency and efficacy of methacholine-induced relaxa-
tion. This finding is in accordance with previous studies showing
improvements of endothelial function by treatment with either
ACE inhibitors or AT1blockers in several vascular beds [16,17].
Interestingly, losartan markedly decreased SMase-induced
vascular contractions, from which we know that these are
endothelium-dependent and most likely mediated by TXA2since
these can be inhibited by thromboxane synthase inhibition and
TP-receptor antagonism . Hydralazine was ineffective in this
respect, suggesting that not blood pressure lowering per se is
responsible for this effect. We have previously established that
SMase-induced ceramide elevation in the carotid artery of SHR
Figure 3. Losartan and hydralazine improve endothelial function in isolated carotid arteries of SHR. (A) Concentration-response-curve
of methacholine-induced relaxation on phenylephrine pre-constriction. (B) Maximal relaxation potential after the addition of a single concentration
(10 mM) of methacholine on phenylephrine-induced pre-constriction. (C) Schematic representation of a SHR carotid artery response towards a single
10 mM methacholine (MCh) addition on phenylephrine (Phe) precontraction resulting in endothelium-derived relaxation (EDR) and subsequent an
additional endothelium-derived contraction (EDC). (D) Quantification of the normalized EDC/EDR ratio after 10 mM methacholine addition on
phenylephrine pre-contraction as depicted in C. Data are expressed as mean6SEM, n=7–8, * p,0.05 compared to untreated SHR.
Blood Pressure Reduction and Sphingolipid Biology
PLoS ONE | www.plosone.org5 December 2011 | Volume 6 | Issue 12 | e29222
leads to vasoconstriction via an iPLA2, cyclooxygenase-1 and
thromboxane synthase-dependent mechanism. Thus, the present
observation that losartan, but not hydralazine, decreased SMase-
mediated endothelium-dependent vascular contractions may be
explained by differential effects of these drugs on the expression of
the aforementioned proteins in the thromboxane synthesis
pathway. Immunohistochemical analysis indicated that losartan
as well as hydralazine treatment decreases cyclooxygenase-1
expression in the carotid artery. iPLA2expression, however, was
only lowered by losartan treatment. The latter finding may explain
the reduced SMase-induced vascular contraction after losartan
One possible explanation why losartan selectively reduces
vascular iPLA2expression could be that protein kinase C (PKC)
increases both the activity  and the expression  of iPLA2.
Since the angiotensin II type 1 receptor is a potent activator of
PKC, and angiotensin II can activate iPLA2in a variety of cell
types , it is conceivable that angiotensin II increases iPLA2
expression in the vasculature. For that reason one would expect
that at least angiotensin II receptor antagonists and angiotensin
converting enzyme inhibitors would reduce iPLA2 expression.
Considering the clinical importance of AT1receptor blockade, this
new aspect associated with losartan treatment warrants further
Interestingly, the AT1 receptor antagonists losartan and
irbesartan, have been shown to possess TP-receptor antagonistic
properties [21,22]. Although this phenomenon may partly explain
the beneficial effects of losartan described in the in vivo part of this
study, it is very unlikely that this interferes with SMase-induced
contractions in the isolated carotid arteries. This is because the
residual losartan concentrations will be very low once the artery
segments are mounted into the organ baths because the drug easily
diffuses out of the isolated arteries during myography experiments
that last several hours. This has been proven experimentally in a
study of Matsumoto et al. , where losartan-treated rats showed
no residual antagonistic effect in isolated arteries on the TP
receptor, as indicated by unaffected U46619 concentration-
response curves compared to controls in comparable myography
This study demonstrates a clear link between hypertension and
sphingolipid biology and as such it represents a new pathophys-
iological mechanism in endothelial dysfunction and blood pressure
regulation. This pathophysiological mechanism might also be of
relevance for new drugs entering the market that target the
sphingolipid system (such as the recently approved immunosup-
pressant FTY720 ) or drugs that modulate sphingolipid
metabolism and increase ceramide levels as a side effect (such as
VEGF antagonists ). Indeed, these drugs are known to
increase blood pressure in both experimental and clinical settings.
This study also demonstrates that losartan has some unique
properties that prevent ceramide-mediated endothelium-depen-
dent vasoconstriction in arteries from SHR and thus may improve
endothelial function, which receives a growing interest in
hypertension treatment [26–29], via this alternative pathway.
This phenomenon gives rise to some interesting new questions
such as whether this is a unique property of losartan or that other
AT1 blockers, RAAS inhibitors or unrelated antihypertensive
drugs share the same properties. Future studies are warranted to
answer these questions and whether the effect of losartan, and
possibly other drugs, on reducing iPLA2expression are a beneficial
contribution to antihypertensive treatment options, especially in
conjunction with disease states that have been reported to be
associated with increased ceramide levels and signaling such as
diabetes and obesity [30,31].
In conclusion, this study corroborates the association between
blood pressure and alterations in sphingolipid biology by showing
that vascular ceramide levels are sensitive to antihypertensive
therapy. In addition, it demonstrates that losartan can improve
endothelial function via inhibition of ceramide-mediated endo-
Figure 4. Losartan, but not hydralazine, reduces ceramide-
mediated endothelium-dependent contractions in isolated
carotid arteries of SHR. (A) Typical tracing of 0.1 U/mL sphingomy-
elinase (SMase)-induced endothelium-dependent contraction in carotid
arteries of untreated, losartan-treated and hydralazine-treated SHR.
(B) Quantification of peak ‘‘EDCF-mediated contractile responses’’ to
SMase. Data are expressed as mean6SEM, n=7–8, * p,0.05 compared
to untreated SHR.
Blood Pressure Reduction and Sphingolipid Biology
PLoS ONE | www.plosone.org6December 2011 | Volume 6 | Issue 12 | e29222
The authors would like to thank Jacques Debets, Peter Leenders and
Agnieszka Brouns for their technical assistance and Bart Heijnen for tissue
Conceived and designed the experiments: LJAS BJAJ JGRDM AEA
SLMP. Performed the experiments: LJAS BJAJ JN MJPMTM DW.
Analyzed the data: LJAS BJAJ DW CEC JGRDM AEA SLMP. Wrote the
paper: LJAS AEA SLMP.
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