The effect of lipoprotein apheresis (Direct Adsorption of Lipids, DALI) (LA) on plasma levels of pentraxin 3 (PTX3), an inflammatory marker that reflects coronary plaque vulnerability, and expression of PTX3 mRNA was evaluated in patients with hyperLp(a)lipoproteinemia and angiographically defined atherosclerosis/coronary artery disease. Eleven patients, aged
The development and progression of atherosclerosis involves the immune response and inflammation. Among the major markers of inflammation are two proteins of the pentraxin superfamily: the acute phase proteins C-reactive protein (CRP) and pentraxin 3 (PTX3). The pentraxins are acute phase proteins comprising five units in a cyclic pentameric structure held together by noncovalent bonds. All pentraxins share a characteristic C-terminal domain (pentraxin domain) of 200 amino acids that has within it 8 amino acids that are highly conserved (pentraxin signature) and are structurally different according to their short and long chain.
Short pentraxins found in humans include the serum amyloid P component (SAP) and CRP [
Eleven patients (10 males and 1 female), aged
Patient demographics and diagnosis and details of lipoprotein apheresis treatment.
Patient |
Age, years | Male ( |
Treatment frequency | Processed volume, mL | DALI configuration | Diagnosis | CAD |
---|---|---|---|---|---|---|---|
1 | 47 | Male | Weekly | 10000 | 1250 | HyperLp(a) | AWA |
2 | 62 | Male | Weekly | 10000 | 1250 | HyperLp(a) | Yes |
3 | 52 | Male | Biweekly | 10000 | 1250 | HyperLp(a) | Yes |
4 | 43 | Male | Biweekly | 10000 | 1250 | HyperLp(a) | Yes |
5 | 49 | Male | Biweekly | 10000 | 1000 | HyperLp(a) | Yes |
6 | 52 | Female | Biweekly | 10000 | 1250 | HyperLp(a) | Yes |
7 | 50 | Male | Weekly | 10000 | 1250 | HyperLp(a) | Yes |
8 | 73 | Male | Biweekly | 10000 | 1000 | HyperLp(a) | Yes |
9 | 51 | Male | Weekly | 10000 | 1250 | HyperLp(a) | Yes |
10 | 63 | Male | Weekly | 10000 | 1250 | HyperLp(a) | Yes |
11 | 66 | Male | Weekly | 10000 | 1250 | HyperLp(a) | Yes |
DALI, Direct Adsorption of Lipids; HyperLp(a), hyperLp(a)lipoproteinemia; CAD, coronary artery disease; AWA, arterial wall atherosclerosis assessed by angiography but no CAD.
Written informed consent was obtained from all patients, according to the recommendations of the Declaration of Helsinki, guiding physicians in biomedical research involving human subjects (adopted by the 18th World Medical Association General Assembly, Helsinki, Finland, June 1964, and amended by the 29th World Medical Assembly, Tokyo, Japan, October 1975, the 35th World Medical Assembly, Venice, Italy, October 1983, and the 41st World Medical Assembly, Hong Kong, September 1989).
The DALI system is based on the binding of positively charged apolipoprotein B100 to negatively charged polyacrylic acid covalently bound to polymethacrylamide. In the present study, DALI apheresis procedures were performed weekly, with all patients subjected to the same standardized protocol (adsorber volume 500 mL and/or 750 mL gel; flow rate Qb 60 mL/min). Anticoagulation was achieved using an initial bolus dose of heparin 20 IU/kg, followed by infused citric acid 1 mL/20–40 mL blood. Vascular access was via the antecubital veins, and the mean volume processed per session was ~10000 mL.
Four samples from each of the 11 patients were collected and stored. The 4 samples for each patient correspond to the following: blood collected before first apheresis treatment (considered as baseline), PRE1; blood collected after first apheresis treatment, POST1; blood collected before second apheresis treatment, PRE2; blood collected after second apheresis treatment, POST2.
Two naïve samples (one from a nonsick patient and one from a sick patient) were collected and processed under the same procedure, for downstream treatment and analysis procedures adjustments and validation. After collection was completed, the samples were shipped under dry ice to LabOmics S.A. (Nivelles, Belgium) for proteomics and transcriptomics analysis.
The SYBR Green assay was capable of detecting ≥0.01 pg of PTX3 mRNA ≥0.1 ng of total RNA with high specificity and reproducibility (coefficient of variation for delta Ct <0.9%).
The ELISA was performed using a commercial ELISA kit purchased from Cusabio (
CRP levels in the study samples were analyzed at the Altamedica SpA Laboratory, Rome, Italy, by nephelometry using the Siemens N Cardio Phase hsCRP, 150 tests (Siemens Healthcare, Milan, Italy) and according to the manufacturer’s instructions.
All values were expressed as mean and standard deviation. The comparison of averages was performed using the Pearson test for correlation between paired values. Missing data and values out of range were excluded from the analysis. The values were significant for
A statistically significant downregulation of PTX3 mRNA expression was observed in all 11 patients (
Effect of LA treatment on PTX3 mRNA expression (a), change in expression (b), and change in expression by LA schedule (c). LA, lipoprotein apheresis; PTX3, pentraxin 3; QW, once weekly; Q2W, biweekly; PRE1, blood sample collected before first LA session; POST1, blood sample collected after first LA session; PRE2, blood sample collected before second LA session; POST2, blood sample collected after second LA session.
Plasma levels of soluble PTX3 peptide oscillated over the course of the two LA sessions. After a slight increase after the first LA session, from 0.08 ng/mL PRE1 to 0.1 ng/mL POST1, plasma soluble PTX3 levels (PTX peptide expression, assessed by ELISA) fell to 0.008 ng/mL PRE2 but climbed again after the second LA session (to 0.112 ng/mL POST2), as shown in Figure
Effect of LA treatment on PTX3 peptide expression (ELISA) (a) and change in expression in all patients (b) and by LA schedule (c) in patients with HyperLp(a) (
C-reactive protein (CRP) levels were also progressively reduced, starting from the first LA session (from
Effect of LA treatment on CRP expression (a), change in expression (b).
We studied the effects of a sustained reduction of apoB100-containing lipoproteins, achieved via LA with the DALI system, on the expression of mRNA which codifies PTX3 synthesis and on plasma levels of soluble PTX3 protein and CRP, in patients diagnosed with HyperLp(a) and AWA/CAD. The increase in plasma levels of soluble PTX3 observed at the first treatment, present in all patients in this study, including those treated every 7 days or every 15 days, might be due to an initial proinflammatory effect of the treatment. It has previously been noted that hemodialysis increases plasma levels of PTX3. A potential relationship between PTX3 and various cytokines removed during extracorporeal treatment might exist [
In our study, PTX3 mRNA expression was elevated but soluble plasma protein levels were reduced by the apheresis sessions but rebounded after session. This suggests a different impact of DALI-LA on mRNA encoding PTX3 and the product of gene signaling action at cellular level. Other authors obtained a reduction of PTX3 with the HELP-LA system [
The apparent clearance of CRP by DALI in the absence of PTX3 clearance is worth noting. The two proteins have different molecular weight despite similar structural conformation. hsCRP is a member of the small pentraxins family with a molecular weight of 25,106 Da versus 40,165 Da for PTX3. The MW of LDL is much smaller at 514 kD. The pore size of the polyacrylate gel used for DALI might play a role in this effect, as could inflammatory effects caused by leachates from filters, tubing, and so forth. Certainly the picture at present is confusing, but the potential impacts on inflammatory process in the atherosclerotic plaque are intriguing.
C-reactive protein is an acute phase protein that appears in circulation in response to inflammatory cytokines, such as interleukin-6, and serves as a biomarker for systemic inflammation [
In this study, LA (DALI system) showed essentially no effect on soluble PTX3 levels in plasma. However, importantly, it was associated with an intense downregulation of expression of PTX3 mRNA, which codifies PTX3 synthesis, suggesting a significant change/modulation at a posttranscriptional rather than posttranslational level that has not been previously reported in patients with severe genetically defined dyslipidemia and associated CAD. This is particularly evident because downregulation of PTX3 mRNA expression was not only linearly correlated with treatment (LA procedure), but also intensified if treatment was weekly, rather than biweekly. This may suggest that the observed downregulation is more intense when LA is more frequently applied. Thus, the conclusion of this study is that LA directly affects expression of mRNA codifying for PTX3, probably (but not definitely) by reducing apoB100-containing lipoproteins. In the light of the role of PTX3 as a proinflammatory and proatherogenic marker, it is possible that the application of sustained lipid-lowering treatment may induce antiatherogenic and (probably) anti-inflammatory effects that are linked not only to a reduction of apoB100-containing lipoproteins, but also to the direct removal of soluble PTX3 protein in plasma exerting a protective endothelial effect. In clinical practice, this might be achieved by LA treatment and also possibly by the most effective available cholesterol-lowering drugs (HMGCoA-reductase inhibitors/statins). The effects of the latest generation of lipid-lowering drugs, including proprotein convertase subtilisin/kexin type 9 inhibitors, cholesteryl ester transfer protein inhibitors, microsomal triglyceride transfer protein inhibitors, and apolipoprotein B synthesis inhibitors, should also be investigated. One last issue for consideration is that in the light of these findings, and from the evidence available from both short- and long-term studies, whether the inhibition of PTX3 achieved by sustained therapeutic lipid-lowering is transitory or stable should be investigated and confirmed. Thus, additional follow-up of patients with genetically determined dyslipidemia and CAD is required, with well-planned, continuous, long-term treatment to further investigate the therapeutic potential of lowering PTX3 directly or through gene-silencing treatments. In particular, a longitudinal study could offer further insights into cardiovascular hard endpoints, including acute myocardial infarction, stroke, and need for revascularization interventions.
In conclusion, LA via the DALI method reduced the expression of PTX3 at the level of mRNA, and this may contribute to the apparent anti-inflammatory effects of the procedure.
Claudia Stefanutti had consulting agreements with Kaneka NV Europe and Fresenius Medical Care Germany, as speaker. She has received research grants from the same companies. Fabio Mazza has no conflict of interests. Michael Steiner has no conflict of interests. Gerald F. Watts has no conflict of interests. Joel De Nève is an employee of LabOmics. Daniela Pasqualetti has no conflict of interests. Juergen Paal was employee of Fresenius Medical Care GmBH at the time of writing.
The authors would like to thank both Fresenius Medical Care Deutschland GmbH, Else-Kröner-Straße 1, Bad Homburg, Germany, that supported the study and Eastmond Medicomm Ltd. that provided editorial support funded by Fresenius Medical Care Deutschland GmbH.