PAH is a multifactorial disease involving abnormal vascular tone, endothelial dysfunction, inflammation, dysregulated angiogenesis, and enhanced thrombosis.[9
] In this study, we postulated that circulating markers of angiogenic activity or inflammation might serve as sensitive markers of PAH disease severity and prognosis, and due to their ability to reflect vascular remodeling activity, perform better than conventional biomarkers. Widely used markers such as BNP and NT-proBNP correlate closely with functional status, therapeutic responses, and outcomes in PAH,[22
] but are limited by the fact that right ventricular distention reflected by these markers occurs in relatively advanced disease. In fact, the potent anti-angiogenic molecule sEng had the best overall performance based upon ROC analysis among mildly symptomatic (NYHA Class I-II) patients, suggesting sensitivity even with modest disease burden. sEng, sVEGFR1, and NT-proBNP predicted functional class and disease severity and, along with CRP, predicted transplant-free survival in PAH by univariate analysis. After adjusting for NT-proBNP levels, sEng, sVEGFR1, and CRP each still predicted survival in PAH, indicating that these biomarkers predict prognosis in PAH above and beyond right ventricular compromise. Only sEng and CRP independently predicted survival based on multivariate analysis, however. Importantly, endoglin expression was enhanced in the microvascular endothelium of IPAH and HPAH lung tissues as compared to non-PAH control lungs, and was intensely expressed in plexiform lesions. Endothelial-derived anti-angiogenic proteins such as sEng and sVEGFR1, known to be released in the contexts of endothelial activation and vascular remodeling, appear to be more sensitive and specific in Group I PAH than previously described markers, and may represent surrogates of angiogenic remodeling activity in PAH. The elevation of anti-angiogenic markers in Group I PAH of diverse etiologies suggests dysregulated angiogenesis may be a common feature of these conditions.
Endoglin and VEGFR1 are expressed in the vascular endothelium where they transduce prosurvival and proproliferative signals required for vascular homeostasis and angiogenesis.[30
] In contrast, the soluble extracellular domains of endoglin and VEGFR1 appear to exert potent antiangiogenic effects. Endoglin, induced with endothelial activation, serves as a coreceptor for bone morphogenetic protein (BMP) ligands such as BMP9 and BMP10 which promote endothelial survival and quiescence.[33
] sEng, which blocks signaling of these ligands, potently inhibits tumor xenograft vascularization in vivo
] Similarly, sVEGFR1 is a truncated splice variant of VEGFR1 that inhibits angiogenesis and induces endothelial dysfunction by sequestering VEGF and placental growth factor.[32
] Both sEng and sVEGFR1 are markedly elevated in patients with pre-ecclampsia, and in addition to being sensitive biomarkers of this disease, appear to contribute to the underlying pathophysiology.[25
] The elevation of sEng and sVEGFR1 seen in PAH may also reflect endothelial activation and proliferation common to these two conditions. While sVEGFR1 has been found to be elevated in patients with PH associated with sickle cell disease,[38
] and sEng was found to be elevated among individuals with scleroderma and PAH, we found increased levels of sEng and sVEGFR1 in diverse Group I PAH patients, including those with minimal symptoms. The observation of markedly enhanced endoglin expression in plexiform lesions, thought to be sites of dysregulated angiogenesis in PAH,[9
] would support the interpretation that circulating sEng reflects endothelial activation, proliferation, and active pulmonary vascular remodeling. sEng may be released from sites of active remodeling by the activity of matrix metalloproteinase-14 (MMP-14), reported to regulate endoglin cleavage in carcinoma tissues.[29
] We did not find elevated vascular expression of MMP-14 in normal or PAH lung tissues (data not shown), suggesting that other mechanisms might contribute. Endoglin is expressed in the failing left ventricle, and its soluble isoform is detected in the circulation after subjecting the left ventricle to pressure overload.[39
] Circulating endoglin can also be detected in the context of endothelial microparticles, as others have observed in PAH.[40
] Our current approach, however, did not distinguish between the expression of sEng as a secreted isoform versus proteolytic cleavage product, or as a ventricular- or endothelium-derived fragment.
In our study, levels of circulating sEng and sVEGFR1 in PAH patients were 2- to 3-fold increased as compared to controls, lower than levels reported in pre-ecclampsia.[25
] If these antiangiogenic proteins derive from a pulmonary vascular bed that is obliterated due to extensive remodeling in PAH, the observed increases in these proteins may reflect proportionally greater expression by a diminished arteriolar tree. Since hypoxia itself can induce endoglin cleavage, elevated levels of endoglin associated with PAH could simply reflect hypoxemia. We did not observe increased expression of endoglin in the vessels of lungs obtained from individuals with known hypoxemic airway disease without PAH, as compared to lung tissues without airway disease or PAH, decreasing the chance of such a nonspecific finding ().
The performance of sEng as a biomarker might suggest a pathophysiologic role in the progression of PAH, but further work in appropriate models will be needed to ascertain whether antiangiogenic factors such as sEng are causal, protective, or secondary to pulmonary vascular remodeling. Future studies are also needed to determine whether risk stratification based on the novel antiangiogenic biomarkers should impact the therapeutic approach taken for individual patients.
Elevated OPG levels have previously been reported among individuals with IPAH,[41
] whereas OPG was only elevated among APAH-CTD patients in our cohort. OPG levels predict mortality associated with systemic cardiovascular disease, atherosclerosis, or diabetes mellitus.[26
] OPG, being a key modulator of tumor necrosis factor-α signaling and osteoclast function, may reflect states of vascular inflammation and bone remodeling, as well as vascular calcification, itself a marker of cardiovascular risk.[47
] IL-15, an inflammatory cytokine observed to be elevated in systemic sclerosis with thoracic involvement,[27
] is also thought to contribute to the pathophysiology of atherosclerosis and systemic vascular disease.[49
] Since the levels of these markers were not measured in CTD patients lacking PAH, it is possible that the elevation of OPG and IL-15 among APAH-CTD patients reflects systemic inflammation associated with autoimmunity.
In contrast, CRP, an acute phase reactant and established biomarker of cardiovascular risk used in stratification of patients for coronary disease primary prevention,[52
] was a sensitive marker of Group I PAH disease, consistent with previous reports.[53
] While some groups have not found CRP to predict survival in PAH,[54
] our study validates CRP as an important predictor of long-term prognosis in PAH. CRP and sEng were independent predictors of transplant-free survival, suggesting that these markers may reflect distinct aspects of PAH progression or severity, a notion supported by the lack of correlation between these two markers ().
None of the biomarkers assayed were significantly elevated in the relatively small cohort of asymptomatic first-degree relatives of IPAH and HPAH patients, suggesting these biomarkers may be surrogates of disease activity rather than disease potential. However, several of the biomarkers exhibited variability among controls and first-degree relatives, with CRP trending toward being elevated in first-degree relatives. Monitoring long-term outcomes in a larger number of first-degree relatives, combined with screening for genetic risk-factors such as bone morphogenetic protein type II receptor (BMPR2) mutations, could determine if these markers correlate with subsequent disease in susceptible populations.
Several factors limit the interpretation of these results. While our study contained a relatively large cohort of Group I PAH patients, conclusions about the relevance of these markers to specific etiologies of PAH or to first-degree relatives of IPAH patients are limited by the relatively small sample size for each category. Our study exhibited a higher female predominance than other PAH cohorts such as the REVEAL registry (90% vs. 79%). This relative enrichment of female patients may have been a function of the self-referral mechanism for enrollment, or due to chance. It should be noted that our PAH cohort is similar to other published populations of Group I PAH with regard to age, etiology of disease, functional status, and hemodynamic variables. Moreover, the control cohort was chosen to match the female predominance of the disease cohort (). In a small number of patients, we were able to measure most, but not all, of the biomarkers (NT-proBNP, sVEGFR1, and IL-15) due to insufficient quantities of blood. Therefore, correlations were obtained by comparing only patients for which both values were available (). Since the majority of patients in this study were enrolled in a nonclinical setting (Pulmonary Hypertension Association Scientific Sessions), clinical imaging, functional class, and hemodynamic assessments were obtained contemporaneously (±3 months by our definition) rather than simultaneously with blood sampling, and carried a significant missing data rate (). Given that CRP and OPG are elevated in other forms of vascular disease, further studies would be needed to control for the presence or absence of diabetes, cerebrovascular disease, and coronary artery disease as confounding variables. Our finding that sEng or sVEGFR1 are useful diagnostic and prognostic tools for PAH will need to be confirmed in validation cohorts.
In summary, this study has identified the potential role of two angiogenic modulatory proteins, sEng and sVEGFR1, as biomarkers of Group I PAH, and validated the role of CRP in the assessment and prognosis of PAH. Our findings on sEng and sVEGFR1 provide further evidence of an important role for dysregulated angiogenesis in the pathophysiology of PAH. The ability of these novel biomarkers to detect PAH even in minimally symptomatic patients and to predict long-term survival suggests that they could reflect pulmonary vascular remodeling activity at earlier stages, and perhaps be used to expedite treatment during critical periods when the outcome of the disease may be most amenable to modification.