The role of circulating thrombospondin-1 in patients with precapillary pulmonary hypertension
The presented study showed, that levels of circulating TSP-1 differ significantly between patients with PH compared to subjects without PH. The subgroup analysis demonstrated the importance of the specific subtype of PH for the interpretation of this biomarker. Furthermore, associations of circulating TSP-1-levels and pulmonary hemodynamics were elaborated. And finally, the association of elevated TSP-1 with increased mortality became evident.
As Bauer and Isenberg showed, TSP-1 is abundantly expressed in lung tissue of patients with PH. In pulmonary endothelium, CD47 controls endothelial NO-synthase. Binding of TSP-1 uncouples eNOS via CD47 and promotes progress of hypoxic PH in animal models [15]. Furthermore, TSP-1 has been shown to deteriorate shear stress dependent vasodilation under hypoxic conditions and is induced by hypoxia in pulmonary vascular endothelium [16].
In this study we demonstrated a significant increase in plasma levels of TSP-1 in patients with various types of PH. While levels of TSP-1 varied in different subtypes of PH, the common denominator was their hemodynamic state. The univariate non-linear regression analysis showed a significant association of circulating TSP-1 with PVR and CO. This supports the initial hypothesis, that the increase of PVR increases shear stress resulting in a release of TSP-1 from pulmonary endothelial cells. However, hemodynamic parameters to determine shear stress in humans remain scarce. The extent of shear stress in a specific pulmonary vessel is influenced by both right ventricular function and properties of the vascular bed of the lung. PVR and cardiac output remain the best surrogate variables of standard right heart catheterization for estimation of shear stress.
On the other hand it has been shown, that TSP-1 production is minimal at optimal shear rates [17], explaining nonlinear U-shaped relation of shear stress and TSP-1 release. In this study we showed first clinical evidence for this complex relationship (Fig. 3), but had to omit possibly confounding factors (i.e. age, medication) due to limited data points.
Induction of TSP-1 in endothelial cells has been shown previously [18] and was confirmed for PAH [16]. Our data contrasts these findings, as an association of TSP-1 with mixed central venous oxygen saturation, which reflects peripheral oxygen depletion, could not be confirmed. Furthermore, the vascular endothelium might not be the only source of TSP-1 in PH, as increasing right ventricular load might release TSP-1 from cardiomyocytes as hemodynamics deteriorate. The release of TSP-1 from the myocardium especially due to ischemia-reperfusion injury has been shown previously [19] and might limit specificity of TSP-1 as a marker for diagnosis of PH.
Experimental data supported the hypothesis of TSP-1 interfering with the NO-pathway [20]. This abrogates soluble guanylate cyclase and protein kinase G as well as adenylate cyclase [21–23], resulting in direct pulmonary arterial vasoconstriction, disease progression [16] and disruption of key pharmacological targets of specific therapy.
Examination of drug regimes suggested lower TSP-1 levels on treatment with PDEIs, higher levels on PC-treatment and intermediate levels under ERA treatment. This observation might lead to the assumption, that treatment regime was influential on circulating TSP-1. On the other hand, PC therapy is an invasive therapeutic concept and often withheld until treatment goals are not sufficiently achieved by oral medications. Therefore, these observations might reflect the finding, that TSP-1 was elevated with deteriorating hemodynamics and increased functional class. The influence of medical therapy on TSP-1 levels could not be determined in this study and warrants longitudinal trials observing TSP-1 levels before and after initiation of new drug regimes.
As this study was designed as a cross-sectional observation, we included patients in different stages of disease. While some had newly diagnosed PH, others were in the final stage of their illness. Thus a non-matched comparison of TSP-1 levels between groups seems a serious limitation for this study. The comparison of incident and prevalent PH demonstrated slightly higher levels of TSP-1 in incident PH, statistical significance was not reached. While the groups had similar hemodynamics, the influence of medication, the course of disease and adaptive processes might lead to this finding. The reasons and possibly influence on the prognostic value of TSP-1 was beyond the scope of this cross-sectional pilot study.
Based on the data linking TSP-1 to hemodynamics and the possibility of its negative impact on pathophysiological mechanisms of PH, we sought to gain insight of the prognosis attached to TSP-1 as a biomarker. At the end of the study, survivor and non-survivors were compared and in all groups non-survivors had significantly higher levels of TSP-1 when entering the study (Fig. 4). CTEPH patients demonstrated relatively higher levels in both survivors and non-survivors compared to the other subgroups. It is therefore mandatory to exactly classify the type of PH before using TSP-1 as a potential biomarker.
Survival analysis in this study was based on follow-up in regular intervals up to five years. The patients had received standard therapy according to current guidelines. Cardiovascular mortality was chosen as the primary endpoint, but in this dataset it was identical with all cause mortality. We were able to show a significantly increased mortality in patients with high TSP-1 levels. This data is limited by the sample size of 93 patients and inclusion of further confounding factors or multivariate analysis with established predictors of mortality is obsolete. Nevertheless, the optimized cut-off at 2051 ng/mL yielded a highly significant difference between groups. Though, as discussed above, the circulating levels of TSP-1 might vary between subgroups and therefore a different cut-off can be expected especially in patients with CTEPH. Furthermore, serial measurements are needed to evaluate the true impact on survival and various clinical events in more complex frailty models.
Nevertheless, this data provides valuable information on the relation of TSP-1 with pulmonary hemodynamics, a clinical application of in vitro observations considering crucial pathways in PH and indicates an impact on the prognosis of a serious disease. These observations are the clinical implication of previous studies on the role of TSP-1 in experimental PH [15, 24].
The presented data might form the basis for evaluation of TSP-1 as an indicator for the choice of medication and might facilitate therapeutic decisions. TSP-1 interacts significantly with the NO pathway, thereby leading to uncoupling of specific therapeutic drugs [22]. Observational studies should evaluate TSP-1 as a marker for possible non-responders before initiation of therapy.