The results of this study provide potentially important insights into plasma proteome changes accompanying outcome to chronic vasodilator therapy in pediatric IPAH. As there is no cure for IPAH, there is a clear and present need for increased understanding of the mechanisms leading to response to therapy and thus outcome. To that end, we embarked on the first study to directly interrogate differences in plasma proteomes from PAH patients responsive/nonresponsive to long-term treatment. We identified several plasma proteins associated with differential response, some of which have been previously identified as being distinctly expressed in PAH patients versus controls.
SAA is a family of conserved acute phase proteins expressed by macrophages, vascular smooth muscle cells, and endothelial cells in response to stimulation by TNF alpha and IL-6 [37
]. SAA-1 and SAA-2 expression can increase 10-to 1000-fold during acute phase periods [38
]. SAA-3 is a pseudogene not expressed in humans and SAA-4 is constituitively expressed [39
]. In the systemic vasculature, SAA stimulates vascular proteoglycan synthesis in a pro-atherogenic manner [40
]. Such a role constitutes an early event in the “response to retention” hypothesis of atherosclerosis development [41
]. Higher serum SAA levels decrease the activity of PON-1, an enzyme with well-established potent anti-oxidative and anti-inflammatory properties [42
]. Here, we too found that children with a good outcome had lower SAA-4 levels than those with a poor outcome and higher risk of death, both before and after therapy, and that higher SAA-4 in nonresponders with poor outcome was associated with decreased PON-1 (). Low PON-1 predicts arterial stiffness in renal transplant patients [43
]. Intriguingly, vascular stiffening is a major patho-biological component of IPAH [44
], and is a better predictor of outcome in pediatric IPAH compared to measurement of pulmonary vascular resistance alone [45
]. Although we did not directly test PON-1 activity, levels of PON-1 appear to correlate to its activity [46
]. Recently, SAA has been shown to stimulate vascular smooth muscle cell calcium entry and downstream signaling associated with coronary muscle dysfunction under inflammatory conditions such as atherosclerosis [47
]. Similarly, we recently reported that plasma endothelin-1 induces pulmonary artery smooth muscle cells to produce hyaluronic acid to which THP-1 monocytes readily adhere [48
]. These studies raise the possibility that blood borne factors in PAH patients modulate vascular remodeling in PAH through their effects on both resident vascular cells and peripheral blood mononuclear cells, though local production of SAA at sites of lung inflammation might also be critically important [49
We also demonstrated a small yet statistically significant increase in SAP in responders with good outcome after treatment with PAH specific therapy. This result (1.27-fold) was verified by ELISA, which showed a 2-fold difference. There was no significant difference in responders versus nonresponders with regard to SAP plasma levels. SAP was first identified as a plasma glycoprotein that is a component of systemic amyloid deposits [50
], and has recently been identified at high concentration in arterial atherosclerotic lesions [51
]. SAP binds Ca2+
-dependent ligands (danger- or damage-associated molecular patterns) at sites of injury such that Fc gamma receptor (FcγR) expressing cells can be activated to phagocytose [52
]. In this way, SAP likely facilitates the local restricted innate immune cell activation at sites of tissue damage. Therapeutic administration of SAP has been shown to reduce fibrocyte numbers in bleomycin lung fibrosis [53
], and inhibit kidney fibrosis associated with monocyte-macrophage regulatory mechanisms by binding primarily to FcγRIII and FcγRI [54
]. Fibrocytes are pro-inflammatory/pro-fibrotic cells that differentiate from peripheral blood mononuclear cells, traffic to pulmonary blood vessels, and participate in the ensuing remodeling [55
]. We recently found increased numbers of fibrocytes in pediatric IPAH patients compared to controls [57
]. Our present study is the first to demonstrate quantitative changes in SAP in IPAH. Although our finding that SAP is increased in responders after therapy may be associative, it is tempting to suggest that increased SAP might limit fibrocyte differentiation, which could, in turn, decrease the extent of inflammation and fibrosis.
The limitations of the study include the relatively small number of samples tested, sample bias, as well as the DIGE method chosen for the proteomic analysis. To the first point, pediatric IPAH is a rare disease, and the number of accessible patients available is limited. We had an obvious difference in mean age between those with a good versus poor outcome, as has been previously reported [23
]. Future studies will certainly include larger study samples to expand the list of differentially expressed proteins and to examine differences in age, gender, disease class, and even protein posttranslational modification. Furthermore, there was an inherent selection bias in choosing the samples for the study. All samples were chosen in retrospect based on known outcome. Some of the pretreatment samples were from patients that had recently begun therapy and therefore were not completely treatment naïve, and patients were determined to be responders in a “post hoc” fashion. Thus, our study should be considered as primarily hypothesis generating. In our small study, no differences in age or gender with regard to SAP or SAA-4 could be discerned, and there were no apparent correlations between age and plasma levels of either SAA-4 or SAP. However, since age appears to be a critical factor determining response to long-term therapy in PAH patients, this requires further study. The prospect of serial analysis of plasma protein profile changes over time within individuals is particularly exciting in this context. To the latter point, no single proteomic technique is presently capable of examining the entire plasma proteome, with its ~1012
dynamic range of protein concentration [58
]. We attempted to minimize this limitation by depleting samples of albumin and IgG prior to analysis. Since the fold changes we observed by DIGE using depleted samples were nearly identical to those we obtained by ELISA using raw plasma, it is unlikely that the depletion protocol introduced quantitative errors.
The choice of sample size in our study was based on previous investigations into the plasma proteome [5
], as well as other proteomes [7
]. The carefully controlled selection of subjects in this study, as well as standardized blood collection, processing and storage protocol, collectively minimized the variation of external factors. Some of the proteins contributing to this concept of a “responder” plasma proteome are known to be involved in inflammation and regulation of monocytes [48
], as well as plasma protein transport [59
] and even angiogenesis [60
]. This implies that the response to vasodilators, with regard to circulating proteins, is almost certainly a multifactorial, complex process that likely involves the contribution of many cell types. Indeed, our finding of reduced haptoglobin and hemopexin in responders versus nonresponders after therapy suggests that within nonresponders an ongoing intravascular hemolysis may be present [61
]. Such a phenomenon may or may not impact the biology of the peripheral blood mononuclear cells. Ergo, cautious optimism must be exercised whenever conclusions are made using results from one tissue compartment (blood) and applied to other tissues (lung). Furthermore, the plasma proteome changes we observed in response to therapy may be related to unaccounted-for variability in therapeutic regimen, age, gender, diet, etc.
Despite its limitations, our study suggests that differences in the plasma proteome can potentially differentiate outcome to PAH therapy. The ability to discriminate response to therapy has major implications for our understanding of the disease process and for our ability to durably treat it. In addition, this study provides the basis for identification of more potential biomarkers and therapeutic targets for IPAH from a serially accessible resource. By understanding the differences in the plasma proteome, future investigations can focus on disease related processes in this complex biological fluid.