Knowledge of the effects of HDL on cultured vascular cells provides insight into how HDL functions in circulation. Previous studies on cultured endothelial11–14
and smooth muscle cells15–17
have shown that HDL has a number of anti-atherogenic properties. In contrast, we have discovered a HDL subclass in adults which is associated with a profound apoptotic effect on human ASMCs in vitro
, a property which could promote atherosclerotic plaque rupture, leading to thrombosis and myocardial infarction.21,23
The apoptotic effect was strongly associated with the presence of a higher molecular weight apoC-I isoform in the HDL particle, as well as generally higher apoC-I and HDL-C levels and a predominance of more buoyant HDL2 particles. To our knowledge, the promotion of ASMC apoptosis is one of the most potentially proatherogenic effects of HDL reported in the adult population to date.
This study confirms and extends the previous observations by Kolmakova et al
who first demonstrated that HDL isolated from LBW infants with an increased level of large HDL particles enriched in apoC-I could markedly induce ASMC apoptosis compared with apoC-I -poor HDL or pure apoC-I. Kwiterovich et al.21
hypothesized that if this HDL phenotype were to persist into adulthood, it could be a risk factor for CVD and explain the association between LBW and adult CVD originally described by Barker et al
Our data show that a nearly identical HDL phenotype is present in adults except it is associated with a higher molecular weight apoC-I mutation. This isoform of apoC-I was not observed in the matrix-assisted laser desorption ionization mass spectra of apoptotic apoC-I-enriched HDL from the LBW infants; but in that case, apoptosis was evaluated using pooled cord blood.21
Therefore, a direct correspondence with the apoC-I mass and apoptosis was not established as we have done in the experiments reported herein. Kwiterovich et al.21
reported the mass spectrum for only 1 of the 30 infants with the apoC-I-enriched HDL phenotype thus, the higher molecular weight apoC-I isoform may have been present in cord blood, but not detected. Although one participant with apoptotic HDL was aware they were ‘small and premature’, none of the other subjects knew any details of their birth history or their birth weight.
We did not find a statistically significant correlation with the total apoC-I level (or the apoA-I/apoC-I ratio) and the extent of ASMC apoptosis; this experimental aspect could not be addressed in the prior experiments by Kolmakova et al.20
because a pooled cord blood specimen was used. We believe that the absence of a correlation between the apoC-I concentration and apoptosis could be due to several factors. One is that the concentration in serum may not reflect the apoC-I enrichment in the HDL2 particle and/or levels of the higher molecular weight isoform which is closely associated with the apoptotic property of HDL2. Another explanation is the possibility that the apoptotic effect may not be due to the apoC-I isoform but rather a component closely correlated with the presence of the apoC-I isoform. The extent of apoptosis by ASMC morphology was confirmed by observing robust caspase-3 expression. This method correlates well with the TUNEL assay and has been shown to be a reliable indicator of apoptosis.40
We observed a ~3.5-fold enhanced caspase-3 expression for HDL2 fractions that stimulated apoptosis by DAPI staining compared with HDL2 from subjects with non-apoptotic HDL2, whereas Kolmakova et al.20
reported a 1.7-fold enhancement by apoC-I-enriched HDL compared with the cell medium, and no enhancement for apoC-I-poor HDL.
Multiple apoC-I polymorphisms have been described in the literature, but only one other structural isoform of apoC-I has been reported in humans. The T45S variant, which affects adipocyte regulation in aboriginal Canadians36
and in persons of American Indian or Mexican ancestry35
results in an additional protein product 14 Da lower than the normal C-I variant. To our knowledge, higher molecular weight variants of apoC-I have not been reported in humans. Although apoC-I is synthesized containing an additional 26 unit peptide, it is removed prior to secretion and no evidence of a precursor propeptide or a propeptide fragment has ever been detected in circulation. The complete absence of the normal apoC-I variant suggests this isoform is not a post-translational modification given such modifications result in a mixture of products (normal and modified). An alternate explanation we considered is that the higher molecular weight isoform is the result of a genetic mutation. Puppione et al
have discovered a pseudogene product corresponding to a higher molecular weight form of apoC-I product shifted by +58 Da in great apes. Although there is significant homology between human and great ape genes, the human pseudogene, located downstream from the apoC-I gene, contains an intervening stop codon in the protein coding sequence and no mRNA product has ever been detected.41,42
Thus, it is unlikely the higher molecular weight apoC-I isoform we observed is due to the human pseudogene product. Further experiments are in progress to identify the origin of the higher molecular weight isoform.
The physiological role of apoC-I is quite wide and varied. Most pertinent to our observations is that apoC-I accounts for most of the CETP-inhibitory action associated with HDL, but the mechanism of action is quite different from the CETP blockade by pharmacological inhibitors such as torcetrapib. Gautier et al.25
have shown that purified human apoC-I at concentrations above 2 µg/mL nearly abolishes CETP activity, and secondly that the inhibitory action of HDL on CETP is proportional to the C-I content. We observed that the CETP activity, measured in a subset of 11 subjects in this study with apoptotic HDL, was a factor of approximately four times lower compared with values reported from other studies utilizing the same commercial assay. For example, in the Multi-Ethnic Study of Atherosclerosis, CETP activity ranged from 35.1 to 55.9 pmol/µL/h43
and in the Framingham Heart Study the mean CETP activity was 149 ± 6.8 85 pmol/µL/h.44
We did not find a significant correlation between the apoC-I concentration and CETP activity in either the cohort with apoptotic HDL or the controls with normal HDL, a finding also reported by Kwiterovich et al
Using a different CETP activity assay, Pillois et al.45
recently reported a weak, but statistically significant negative correlation between the apoC-I level and CETP activity in a cohort of 240 patients with CHD (r
= 0.133, P
< 0.001); the correlation improved to 0.330 (P
< 0.001) in 101 normolipemic subjects within this cohort. Although they also observed a positive correlation with the apoC-I concentration and the HDL-C level, it was fairly weak (r
= 0.165, P
< 0.001) in the overall cohort and only slightly stronger in the normolipemic cohort (r
= 0.330, P
< 0.001). In contrast, the mean apoC-I concentration in the subjects reported herein was almost a factor of 2 higher than that of the cohort Pillois et al.
studied, and we observed a much stronger, statistically significant positive correlation between the apoC-I concentration and the HDL-C level (r
Finally, another intriguing role for apoC-I which merits note is that higher levels also appear to confer a survival benefit in patients with endotoxemia by stimulating TNF-α production.46
Plomgaard and Nielsen47
have suggested that this proinflammatory effect of apoC-I, while beneficial in the setting of sepsis, may ultimately be detrimental because it could promote the future development of atherosclerosis through enhanced inflammation.
There are several aspects of our experimental approach that differ from other investigations of how HDL affects vascular cells. One is that we used a relatively new ultracentrifugation method to isolate the HDL subclasses. When lipoproteins are isolated by ultracentrifugation, there is some concern that the HDL particle properties may be influenced by protein shedding during centrifugation. The literature suggests that the in vitro
functional properties of HDL subclasses isolated using conventional NaBr or KBr DGU12–14
are preserved. Compared with these methods, the DGU method utilizes a lower ionic strength gradient (~0.6 M for 0.3 M Cs2Cd (EDTA), vs. ~3 M for NaBr or KBr) and a reduced spin time (6 vs. 20–30 h) so the DGU method is less likely to perturb the HDL particle integrity. Reassuring evidence that the isolation method did not affect the in vitro
properties of HDL is provided by noting that the extent of ASMC apoptosis produced by HDL2 and HDL3 isolated by DGU is nearly identical to that observed for apoC-I-enriched HDL and apoC-I-depleted HDL, respectively, isolated by sequential immunoprecipitation in the original description of this HDL phenotype.20
Another difference with our approach is that most in vitro
studies have examined the effects of HDL on vascular cells using plasma. This includes Kolmakova's initial investigations of apoC-I-enriched HDL on ASMC.20
We have historically used serum since our initial experiments were performed using leftover serum from a standard lipid profile. Collins and Olivier48
determined that the only significant differences between the lipoprotein composition in plasma and in serum are primarily attributable to the absence of fibrinogen proteins as well as a slightly higher level of apoB-100 in serum compared with plasma. Thus, we believe the extent of ASMC apoptosis would be the same for HDL isolated from plasma.
We also acknowledge that a weakness of this study is the limited population diversity with respect to gender, ethnicity, and the cross-sectional design. The preponderance of women in this study was explained in part by our screening criteria which focused on subjects with a prominent HDL2 subclass distribution given this was the phenotypic signature of infants with apoC-I-enriched HDL independent of their gender.21
The circulating level of the HDL2 subclass is typically higher in adult women than men.49
The predominately female gender in our sample may have therefore biased the apparent association of apoC-I with higher HDL-C levels. Approximately 80% of subjects enrolled in the clinical studies supporting the serum library for this investigation were Caucasian. This in part reflects the demographics of the population but also the smaller number of minority populations agreeing to participate in studies.
In summary, our results demonstrate that some HDL2 subclasses containing a higher molecular weight isoform of apoC-I markedly enhance smooth muscle cell apoptosis in vitro
. Our findings corroborate the observations of Kwiterovich et al
and Kolmakova et al
pointing to the existence of an HDL subclass which may find its origins in an unfavourable intrauterine environment. HDL containing this apoC-I isoform may be expected to unfavourably impact CHD outcomes based upon additional supporting in vivo
evidence from Steen et al.22
demonstrating a compelling association of apoC-I with capsase-3 and ceramide at the site of atherosclerotic plaque rupture in a rabbit model and the proinflammatory effect of apoC-I reported by Plomgaard and Nielsen.47
Because individuals with this HDL subclass generally have higher HDL-C levels, likely resulting from the inhibitory effects of apoC-I on CETP, they may paradoxically be perceived to have a lower risk of CHD. This study therefore also offers an example of a mechanism associated with a potentially adverse effect of CETP inhibition on CHD risk.
We hypothesize that at the vascular level, this HDL subclass, with access to the subintimal space, may exert toxic effects on vascular cells which promote atherogenesis and contribute to plaque rupture based on our knowledge of the underpinning molecular mechanisms linking smooth muscle cell apoptosis and inflammation with enhanced plaque instability.23
Our findings further support a growing body of evidence demonstrating that measures of HDL function may be more informative with respect to the CHD risk than the HDL-C level. A failure to recognize this HDL subclass as a putative risk factor may result in an underestimation of an individual's risk for CHD, especially in those individuals having high HDL-C levels.