Small dense HDLs display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

Several studies aiming to elucidate the molecular deter minants of HDLmediated vasodilation reveal that S1P plays a pivotal role in endothelial cell integrity via activating NO production (23)(24)(25)(26)(27). Although HDL carries the majority of S1P in plasma (28), there is a considerable variation in S1P content among different HDL subclasses, which is elevated in small dense proteinrich HDL3 relative to large light lipidrich HDL2 (29)(30)(31). These data suggest that HDL par ticles may differ in their capacity to induce vasorelaxation, with small dense HDL3 displaying potent vasorelaxing prop erties as a result of elevated S1P content, enhanced activa tion of S1P receptors, and increased NO production.
In order to evaluate this hypothesis, we examined five HDL subpopulations isolated from healthy normolipidemic subjects for their ability to counteract the inhibitory effects of oxLDL on acetylcholineinduced vasodilation in isolated rab bit aortic rings. In a candidatebased approach to define spe cific constituents associated with the vasodilatory response, we assessed the impact of reconstituted HDLs (rHDLs) con taining apoAI and phosphatidylcholine (PC) in the absence or presence of other biologically active components known to be enriched or depleted in HDL3 (32,33). Our data reveal that small dense HDL3 particles displayed potent protective effects against oxidative stressinduced endothelium dys function, whereas large light HDL2 particles did not induce aorticring relaxation. Consistent with these data, HDL associated S1P was a potent vasorelaxant in aortic rings. These findings indicate that elevated content of S1P may account for the potent vasodilatory activity of HDL3.

Blood samples
Blood samples were obtained from four healthy blood donors. EDTA plasma was prepared by 15 min lowspeed centrifugation at 4°C. Sucrose, at a final concentration of 0.6%, was added to the plasma as a cryoprotectant for lipoproteins; samples were ali quoted and frozen at 80°C under nitrogen; each aliquot was thawed only once directly before use.

HDL subfractionation and characterization
First, total HDL was isolated from plasma by sequential flo tation ultracentrifugation, as described previously (34). Subse quently, plasma HDL subfractions, including HDL2b, 2a, 3a, 3b, and 3c, were isolated by a singlestep isopycnic density gradient ultracentrifugation of the total HDL fraction at 15°C at 274,000 g for 44 h, as described previously (35). All HDL subfractions were extensively dialyzed against PBS treated with Chelex 100 ion exchange resin (pH 7.4) at 4°C in the dark, stored at 4°C, and used within 10 days (35).
All HDL subfractions were analyzed for the content of major HDL lipid components, including total cholesterol (TC), free cholesterol (FC), phospholipid, and triglyceride (TG), using commercially available enzymatic colorimetric kits (CHOPPAP; Biomerieux, France). Cholesterol ester content was calculated by multiplying the difference between TC and FC by 1.67 (35). Total protein was measured in HDL using the BCA assay. apoAI was assessed by immunoturbidimetry using an automated Konelab 60i analyzer (Thermo Fisher Scientific, VillebonsurYvette, France). Paraoxonase1 (PON1) activity was determined photometrically in the presence of CaCl 2 using phenylacetate as a substrate (36). Albumin and apoM content were evaluated in HDL subpopula tions by LC/MS/MS, as described previously (32).

S1P quantification
An HPLCbased method was used to determine the S1P con tent of HDL, as previously described (30). Briefly, 100 l of iso lated HDL subfractions containing 0.5 to 2.0 mg of total HDL mass per milliliter of buffer were added to 1 ml of acidified meth anol and supplemented with 15 pmol of an internal standard [DerythroS1P (C17 base); Avanti, InstruChemie]. The S1Pcon taining phase was isolated with 1 ml chloroform, 200 l NaCl (4 M), and 100 l NaOH (3 M) in a twostep procedure. Subsequently, the organic phase was evaporated and dissolved in 50 l of metha nol and 0.07 M K 2 HPO 4 (9:1 v/v). Five microliters of the de rivatization mixture containing 10 mg ophthaldialdehyde, 200 l ethanol, 10 l 2mercaptoethanol, and 10 ml boric acid (3% v/w) were added to the lipid followed by a 15 min incubation at room temperature. One microliter of the derivatized sample was ana lyzed with a Hewlett Packard HPLC system using an RP 18 Kromasil column (2.1 mm inner diameter × 150 mm) and an isocratic elu ent containing methanol:K 2 HPO 4 (0.07 M) (78:22 v/v) at a flow rate of 0.25 ml/min maintained at 45°C. Derivatives were de tected using a Hewlett Packard spectrofluorometer at 340 nm and 456 nm as excitation and emission wavelengths, respectively. S1P quantification was done by comparison of its fluorescent signal with that of the derivative of the internal standard (coefficient of variation, <5%). The values of S1P were reported per mole and per gram of HDL protein, as previously described by us (29). To calculate HDL molarity, the following molecular masses of HDL subfractions were used: HDL2b, 404 kDa; HDL2a, 270 kDa; HDL3a, 215 kDa; HDL3b, 199 kDa; and HDL3c, 163 kDa (29).

rHDL preparation and loading with S1P
rHDL was prepared by cholate dialysis from a mixture of hu man apoAI, isolated from EDTA plasma, with egg yolk PC at the molar ratio of 1:80 (37). rHDLs were then incubated with or with out S1P (40 mol/l), at a concentration of 100 mg HDL protein per deciliter at 37°C for 2 h, under constant stirring. Subse quently, the rHDLs were extensively dialyzed against PBS (pH 7.4) to remove excessive S1P. S1P concentration was measured in the S1Penriched rHDL by HPLC with fluorometric detection, as described above. S1P-loading of albumin S1P, at a concentration of 2 or 40 M, was added to a 0.1% w/v solution of human serum albumin, a second major physiologic S1P carrier in human plasma (38), and incubated at 37°C for 2 h. According to previously reported data (15), a maximum of 0.5 mol of S1P can be loaded per gram of human serum albumin. Because, in our experiments, the S1P/albumin ratios were 0.02 and 0.4 mol/g, we assumed that all added S1P was bound to albumin at the end of the incubation. Nevertheless, both S1Pen riched and control albumin samples incubated in parallel in the absence of S1P were extensively dialyzed against PBS (pH 7.4) to remove unbound S1P if any.

TG enrichment of HDL
To prepare TGenriched HDLs, isolated HDL3a and 3b sub populations (650 l) were incubated for 3 h at 37°C in the pres ence of human VLDL (570 l) and lipoproteindeficient serum as a source of lipid transfer proteins (80 l), freshly isolated from three healthy normolipidemic donors by a singlestep isopycnic density gradient ultracentrifugation, as described above. The amount of VLDL included in the incubation was sufficient to reach a final TG concentration of 1 mM. TGenriched HDL was then reisolated by a singlestep isopycnic density gradient ultra centrifugation, as described.

Preparation of oxLDL
LDL (density 1.019-1.063 g/ml) was isolated from human EDTA plasma by sequential flotation ultracentrifugation, as previ ously described (39). LDL was oxidized by incubating freshly pre pared LDL, adjusted to a final concentration of 1.

Ex vivo vasoreactivity of rabbit aortic rings
Vasoreactivity experiments were performed on rabbit aortic rings in accordance with principles of laboratory animal care and the protocol was approved by the local Ethics Committee for ani mals at the University of Burgundy (Dijon, France). As previously described (7-9), the descending aorta was rapidly removed and transferred into Krebs solution bubbled with 95% O 2 plus 5% CO 2 . The aortas were cut into 3 mm rings and suspended horizontally between two wire hooks in 20 ml jacketed organ baths containing oxygenated Krebs solution (composition: NaCl, 119 mmol/l; KCl, 4.7 mmol/l; KH 2 PO 4 , 1.18 mmol/l; MgSO 4 , 1.17 mmol/l; CaCl 2 , 2.5 mmol/l; EDTA, 0.027 mmol/l; glucose, 11 mmol/l; and NaHCO 3 , 25 mmol/l) maintained at 37°C. In brief, aortic rings were initially contracted by 0.3 mol/l artere nol hydrochloride at a concentration resulting in 75% of the maximal contraction, and acetylcholine in the 1 nmol/l to 0.01 mmol/l concentration range was added cumulatively to relax the rings. After a washout step and a further 30 min recovery period, aortic rings were incubated for 2 h with oxLDL alone or with oxLDL in the presence of different HDL subfractions, control rHDL, S1Penriched rHDL, S1Penriched albumin, or rHDL supplemented with recombinant human apoJ (0.038 M) or apoCIII (3 M) (BioVision Inc.). We used a coincubation model that more adequately reflected physiological conditions where both HDL and oxLDL are simultaneously present in the same environment. All HDL preparations were compared at a constant concentration of 1 g protein per liter. Subsequently, aortic ring segments were recontracted with arterenol hydrochloride, and then were relaxed again progressively by adding sodium nitroprus side (NO) to verify the exclusive involvement of endothelium dependent relaxation response.
The maximal relaxation (E max ) evoked by acetylcholine and ex pressed as a percentage of the contraction to arterenol hydrochloride (0.3 mol/l) was calculated from experimental data. To reduce variation among experiments resulting from different intrinsic properties of aortic vessels, three different aortic rings were used and the average results were reported (40).

NO production in endothelial cells
Simian vacuolating virus 40 (SV40)transformed murine endo thelial cells (SVECs) (ATCC number: CRL2181) were cultured in DMEM supplemented with 10% FBS to subconfluency. After over night starvation, cells were incubated with 4,5diaminofluorescein diacetate (DAF2) (1 M; Cayman Chemical) for 30 min before adding HDL (50 g protein per milliliter) for 15 min. Triazolo fluorescein fluorescence was measured under conditions provided by the manufacturer using excitation/emission wavelengths of 485/520 nm as an indicator of NO production (12).

Antioxidative activity of HDL
Antioxidative activity of HDL3a and 3b subpopulations was evaluated toward oxidation of reference LDL, obtained from a healthy normolipidemic donor, by 2,2′azobis(2amidinopro pane) hydrochloride, an azo initiator of lipid peroxidation. Ac cumulation of conjugated dienes in the samples was measured at 234 nm and average oxidation rate in the propagation phase was calculated, as previously described (36,41).

Statistical analysis
Betweengroup differences were analyzed by the nonparametric Wilcoxon's matchedpair for continuous variables or Chisquare test for discontinuous variables using StatView statistical software (SAS Institute Inc., Berkeley, CA). Spearman's correlation coeffi cient was calculated to assess relationships between continuous variables. All results are expressed as mean ± SD except E max val ues, which are expressed as mean ± SEM; P < 0.05 was considered statistically significant.

Compositional characterization of HDL particle subpopulations
As expected, the content of major lipid classes showed a distinct trend to decrease in parallel with increment in HDL total protein and apoAI [percentage by weight (wt%)] from large light lipidrich HDL2b to small dense proteinrich HDL3c, with only a minor degree of variation for most lipid classes relative to total lipid ( Table 1). In marked contrast, S1P, a minor bioactive lipid, showed significant variation across HDL subpopulations with almost 4fold enrichment in small dense HDL3 (0.54-0.94 mol/g protein HDL), as compared with HDL2 particles (0.08-0.24 mol/g protein HDL), as reported earlier (30). The lowest S1P content was measured in the HDL2a subpopulation (Table 1). In a similar fashion, PON1 activity and apoM were predomi nantly associated with small dense HDL3 particles, as doc umented earlier by us and others (32, 36) (ratios of the levels measured in HDL3c relative to HDL2a of 66 and 4.5, respectively).

Protective effects of HDL particles toward oxLDL-enhanced vasoconstriction in arterenol hydrocholoride-contracted rings
Preincubation of isolated rabbit aortic rings with oxLDL markedly inhibited acetylcholineinduced vasorelaxation, as compared with rings preincubated with a control buffer (E max = 50%, P < 0.02). We compared the effects of differ ent HDL subpopulations that were matched for their pro tein content, but differed in S1P content. Large light HDL2b exhibited no effect in counteracting the oxLDL induced inhibitory effects on the endothelial vasorelax ation response and HDL2ainduced vasoconstriction (Fig. 1). In contrast, HDL3 particle subpopulations potently re stored endotheliumdependent vasorelaxation, with the greatest effects observed in the presence of the small dense HDL3c subfraction (E max = +28%, P < 0.05) followed by HDL3b (E max = +22%, P < 0.05) and HDL3a (E max = +20%, P < 0.05) (Fig. 1). As a result, the capacity of HDL particles to protect vessels was proportional to their density (Fig. 2).
In addition, strong positive correlations were found be tween the aortic ring vasorelaxation and both S1P (r 2 = 0.54, P = 0.0019) and apoAI (r 2 = 0.64, P = 0.0003) content of HDL (Fig. 3A, B), whereas TG content of HDL revealed a negative correlation (r 2 = 0.75, P < 0.0001; Fig. 3C). In parallel, S1P and TG contents in HDL subfractions were negatively corre lated (r = 0.36, P = 0.02). In order to adjust for the effects of TG and apoAI, the ratios of S1P/TG and S1P/(apoAI + TG) were calculated and plotted against the maximal endothe liumdependent relaxation induced by HDL subpopulations.

Influence of rHDL on the inhibitory effect of oxLDL on vasorelaxation of rabbit aortic rings
To identify the constituents of HDL particles associated with their vasoprotective effects, the potential role of sev eral candidates displaying vasoprotective properties, such as apoAI, S1P, and apoJ (clusterin), as well as those associ ated with HDL dysfunction, such as apoCIII, was assessed.
In the first set of experiments, rHDLs containing only apoAI and PC, and S1Penriched rHDL were used to in vestigate the influence of apoAI and S1P on aortic ring vasorelaxation.

Influence of albumin loaded with S1P on the inhibitory effect of oxLDL toward vasorelaxation of rabbit aortic rings
In order to further characterize the distinct role of S1P in the vasorelaxation response induced by HDL, albumin, as a second major plasma carrier of S1P (38), was loaded with S1P and incubated with aortic rings in the presence of oxLDL. As shown in Fig. 4C, such S1Penriched albumin was able to sig nificantly counteract the inhibitory effect of oxLDL on the vasorelaxation of rabbit aortic rings (up to 1.6fold, P < 0.01), Values are the means of two measurements performed in preparations of HDL particles obtained from three healthy donors. Lipid composition of HDL expressed as wt% relative to total lipid is shown in parentheses. consistent with the specific capacity of S1P to induce vasore laxation independent of other HDL components.

Increased S1P1-dependent NO generation in murine aortic endothelial cells (SVECs) treated with small dense HDL3
To characterize the potential role of NO in the vasodila tory effects of HDL subpopulations, HDLdependent NO generation in murine endothelial cells was evaluated by measuring the kinetics of the increase in the fluorescence of DAF2, a fluorescent probe for NO. Total HDL at a con centration of 50 g protein per milliliter showed the most reproducible NO increment under our experimental con ditions after 15 min incubation time (1.68fold vs. baseline at time 0; Fig. 5A); this condition was therefore chosen to analyze the effects of individual HDL subpopulations. Small dense HDL3c induced NO production levels in SVECs com parable to those induced by total HDL (1.50fold vs. 1.55 fold increases, respectively, relative to incubations in the absence of HDL after 15 min; Fig. 5B). The mechanism un derlying potent vasodilatory effects of HDL3 was further as sessed using the S1P receptor antagonist, VPC 23019 (42). In the presence of VPC 23019, both total HDL and the small, dense HDL3c subpopulation lost their stimulatory effects on endothelial NO production in murine aortic en dothelial cells, with a stronger effect on HDL3c (Fig. 5B), indicative of the key role of S1P1/S1P3 receptors in the HDL3cinduced NO production.

Influence of TG enrichment on antioxidative activity of HDL
To obtain insight into the negative relationship between TG content and vasodilatory activity of HDL, we examined antioxidative activity of TGenriched HDL particles toward LDL oxidation. Incubation of small dense HDL3a and HDL3b in the presence of isolated VLDL and lipopro teindeficient serum for 3 h at 37°C markedly (by 57 ± 9%) increased TG content of HDL in three independent experiments performed in three individual HDL samples. Such TG enrichment caused a significant reduction in an tioxidative activity of HDL3a and HDL3b particles, evaluated as inhibition of LDL oxidation by 2,2′azobis(2amidino propane) hydrochloride in the propagation phase (up to 36% and 22%, respectively; P < 0.05).

DISCUSSION
The present study, for the first time, demonstrates distinct effects of small dense proteinrich HDL3 in counteracting the inhibitory effects of oxLDL on endotheliumdependent vasorelaxation in rabbit aortic rings. Our data reveal that small dense HDL3 subpopulations enriched in S1P exert po tent vasorelaxing activity, thereby suggesting a role of this bioactive constituent for the enhanced vasodilatory activity. In a candidatebased approach, no consistent effect was ob served for several other components enriched or depleted in HDL3. Furthermore, small dense HDL3 particles potently induced NO generation in endothelial cells, an effect that was blocked by a S1P receptor antagonist, documenting the involvement of S1P1/S1P3 receptors in the HDL3mediated vasorelaxation.
Due to the high complexity of the lipid and protein com position of HDL, structurefunction relationships across HDL subpopulations are complex. Our studies have previ ously shown the superior atheroprotective functionality of HDL3 particles relative to HDL2 (53). Potential heteroge neity of the vasodilatory properties of HDL was, however, not evaluated in earlier studies. In particular, it remained unclear as to whether S1P enrichment renders superior va sodilatory activity to HDL3. Earlier, HDLinduced endo thelial vasorelaxation in aortic segments was shown to be directly related to HDL content of S1P (24). In the present study, small dense proteinrich HDL3 was more potent to prevent oxLDLinduced inhibitory effects on endothe liumdependent vasorelaxation relative to large light HDL2, suggesting beneficial functional consequences of S1P en richment in HDL3.
Although HDL3 particles are enriched in S1P relative to HDL2, demonstration of a direct link between S1P content and vasorelaxation activity can be complicated by the impact of other components enriched or depleted in HDL3. In a proofofconcept experiment, S1Penriched rHDL consistently counteracted oxLDLinduced inhibi tory effects toward endotheliumdependent vasorelaxation of rabbit aortic rings, supporting the key role of S1P in the potent vasodilatory properties of HDL3. By contrast, the vasodilatory effect of other HDL components, such as apoJ (10) and apoCIII (11), were either weak or inconsistent with their HDL content (33,54). Indeed, while apoJ did not significantly enhance vasodilation induced by HDL, the vasodilatory effect of apoCIII was observed, which was inconsistent with its enrichment in large versus small HDL (32,33).
Further along this line, although PON1 activity was en riched in HDL3 and correlated with the vasodilatory effect of HDL subpopulations, this correlation was weaker, as com pared with those calculated for S1P and apoAI. Importantly, in our study, HDLs were derived from EDTA plasma and as such are well known to display greatly diminished PON1 ac tivity as a result of PON1 inhibition by EDTA (55). We there fore believe that although PON1 activity might, in principle, contribute to the vasodilatory effects of HDL via reducing both oxidative stress and NO inactivation by reactive oxygen species, this pathway is of rather minor importance.
apoM, as the main carrier of S1P, is also enriched in small dense HDL (32,33). The significant correlation of apoM content with the vasodilatory effect of HDL sub populations provides an additional argument in support of the major role of S1P in the HDLinduced vasorelaxation. However, the association of HDL S1P and apoM remains incompletely clarified. Despite the fact that apoM was ini tially proposed as the only interaction partner for S1P in HDL (56), S1P has later been reported to be enriched in HDL independent of apoM (27,57,58). Our data obtained using rHDL and albuminassociated S1P are consistent with a vasodilatory activity of S1P, which can be indepen dent of apoM and HDL binding (59).
Indeed, the magnitude of the vasodilatory effect of S1P against vasoconstriction enhanced by oxLDL in arterenol hydrochloridecontracted rings was preserved following S1P conjugation to albumin instead of HDL, consistent with an S1Pspecific mechanism. It is of note that albumin associated S1P was unlikely to contribute to the vasodila tory properties of HDL in our study, as no albumin was detected by LC/MS/MS in HDL subpopulations isolated by the singlespin isopycnic density gradient ultracentrifu gation approach, as previously reported (32).These data imply that albuminbound S1P may act as a natural substi tute of HDLassociated S1P under conditions of HDL and/ or apoM deficiency (59).
In addition to S1P, HDL content of apoAI and TG showed significant positive and negative correlations with endotheliumdependent relaxation activity, respectively, suggesting potential roles of apoAI and TG in HDLmedi ated vasorelaxation. This conclusion is in accordance with our previous studies showing impaired vasodilatory effects of HDL in type 2 diabetes or abdominal obesity in parallel with decreased HDL content of apoAI and increased con tent of TG (7,8). In contrast to S1P, apoAI content does not considerably differ across HDL subpopulations. Indeed, Fig. 3. Correlations between S1P (A), apoAI (B), and TG (C), apoAI/TG ratio (D), S1P/TG ratio (E), and S1P/(apoAI + TG) ratio (F) in HDL subpopula tions and the maximal endotheliumdependent relax ation of rabbit aortic rings after incubation with HDL subpopulations and oxLDL. Values are from three HDL preparations isolated from the plasma of three healthy donors. the concentration of apoAI was similar across HDL sub populations when HDL concentration was fixed at 1 g total protein per liter in our aortic ring system. These data sug gest that it is conformational status rather than the abun dance of apoAI in small dense HDLs that can favor their enhanced biological functionality (60). Indeed, apoAI conformation is peculiar in small dense HDL, potentially enhancing its biological activity (22,60). It is reasonable to consider that such conformational modifications may en hance HDL interactions with receptors involved in vaso dilation, such as with scavenger receptor class B type I (SRBI), which favors NO synthesis (23), or with ABCG1, which promotes NO production by increasing cholesterol efflux (61).
It is important, in this regard, that the negative correla tion between the vasodilatory properties of HDL subpopu lations and their content of TG can be explained by the role of TG in both altering the conformation of apoAI (22) and decreasing the antioxidative activity of HDL. In deed, vascular oxidative stress is wellestablished to dimin ish endothelial NO bioavailability (3). HDL antioxidative activity can therefore counteract the inhibitory effect of oxLDL on vasorelaxation. In the present study, we found that TGenriched small dense HDL displayed significantly reduced antioxidative activity, consistent with the negative correlation between TG content and vasodilatory activity of HDL.
The underlying mechanism by which HDLassociated S1P mediates vasorelaxation has been attributed to the ac tivation of eNOS and subsequent NO generation in endo thelial cells (24,62). In our study, the HDL3c subpopulation strongly induced NO production, suggesting the potential role of HDL3associated S1P in the intracellular signaling pathway activation. Moreover, we showed that a S1P1/S1P3 receptor antagonist effectively blocked NO production in murine aortic endothelial cells incubated with HDL3c, Fig. 4. Influence of S1Penriched rHDL (A), S1Ploaded albumin (B), and apoCIII or apoJloaded rHDL (C) on the inhibitory effect of oxLDL toward endotheliumdependent vasorelaxation of rabbit aortic rings. The graph shows mean doseresponse curves for acetylcholine (Ach) in aortas contracted with 0.3 mol/l arterenol hydrochloride and preincubated for 2 h with Krebs buffer, oxLDL alone, oxLDL and S1Penriched rHDL, and oxLDL and rHDL containing only apoAI and PC (A); oxLDL and rHDL supplemented with apoJ or apoCIII, and oxLDL and rHDL containing only apoAI and PC (B); oxLDL and albumin (1g/l) preincubated with S1P (2 and 40 M) (C). All HDL and oxLDL preparations were used at a final concentration of 1 g protein per liter. Data are presented as the mean of four independent experiments.
implying that the potent vasodilatory effect of HDL3 is as sociated with enhanced NO production through the S1P1/ S1P3 receptor pathway. Together with the results of aortic ring studies, these findings highlight the importance of the HDL lipid moiety in endothelial vasorelaxation.
Reduced S1P content of HDL in coronary artery disease and myocardial infarction may further reflect the cardio protective impact of HDLassociated S1P (15,16,63,64). Such a reduction in S1P content of HDL can be linked to oxidative modification of HDL particles (15). Indeed, in vitro oxidation of HDL from healthy subjects resulted in the loss of HDLS1P with a concomitant reduction in HDL capacity to uptake S1P in vitro (15). Consistent with this observation, oxidation of HDL from healthy subjects re duced its ability to counteract the inhibitory effect of ox LDL on endotheliumdependent vasorelaxation (40).
The protective effect of HDL against cardiovascular dis ease is wellrecognized. However, circulating HDL choles terol concentration, which is commonly used in clinical practice, does not accurately estimate the cardioprotective functionality of HDL (65). Thus, identification of HDLre lated factors involved in, or serving as, biomarkers to cardiovascular disease remains in the focus of research. Available data identify HDLassociated S1P as a potential biomarker of cardiovascular risk. In addition, our findings may have important implications in developing HDLtar geted therapy as novel therapeutic strategies can be envis aged by loading HDL particles with S1P.
In summary, the present study reveals that marked S1P enrichment in small dense HDL3 particles is associated with their potent ability to counteract the inhibitory effects of oxLDL on endotheliumdependent vasorelaxation and to induce NO generation in endothelial cells, suggesting the pivotal role of S1P to the distinct vasodilatory and NO stimulatory effects of HDL3, which may contribute to HDL mediated vasoprotection.