This study showed a significant difference in the reliance on glycolysis between CTEPH-EC and PAH-EC. PAH-EC presented higher expression levels of glycolytic and glutamine-related enzymes compared to CTEPH-EC and healthy ECs. The increase in glycolytic enzymes in PAH-EC compared to CTEPH-EC was further accompanied by an increase in PDHA1 mRNA levels. Furthermore, protein levels of oxidative phosphorylation complexes I, II and IV were higher in PAH-EC compared to CTEPH-EC. Accordingly, a substantially different metabolic profile at the level of glycolysis, oxidative phosphorylation and glutamine metabolism is present in PAH-EC compared to CTEPH-EC and suggests differences in molecular mechanisms and regulatory pathways that could be important in the disease pathology and treatment.
In this study, a similar HOXD gene expression pattern between CTEPH-EC and PAH-EC was shown. Toshner et al.  described that, considering the HOXD expression patterns, ECs could be clustered based on the type of blood vessel that they were derived from. Their study showed that high expression of HOXD3, HOXD8 and HOXD9 was associated with ECs that are microvascular in origin . In the present study, the expression profiles of all 3 HOXD genes, both in CTEPH-EC and PAH-EC, were similar to HPAEC. Based on this data it can be concluded that CTEPH-EC and PAH-EC used in the present study origin both from a macrovascular lineage.
It is known that, in PAH, vascular ECs adopt metabolic changes associated with vascular hyperproliferation and resistance to apoptosis. PAH-EC are found to increase glycolysis in order to assure proliferation [13, 24]. Although previous studies in CTEPH have shown an hyperproliferative dysfunctional phenotype in CTEPH endothelial cells [25, 26], not much is known about metabolic alterations in CTEPH-EC. Based on recent in vitro and in vivo studies from our group, CTEPH-EC seem to be associated with impairments in their metabolism that point towards lower glycolysis and glutaminolysis in CTEPH-EC compared to healthy ECs [18, 27]. In accordance with those observations, the present study showed the upregulation of glycolytic genes GLUT1, HK2 and LDHA in PAH-EC compared to CTEPH-EC indicating different metabolic profiles between both PH diseases. Glycolytic regulator PFKFB3 was not found differently expressed between all EC types studied. An explanation could be that PFKFB3 activity is dependent mainly on post-translational modifications [28, 29].
Another important key feature of metabolic adaptations in ECs from PAH patients found in this study, is the increased expression of PDHA1 compared to CTEPH-EC and healthy ECs. PDK1 phosphorylates PDHA1, which blocks mitochondrial oxidative phosphorylation and further promotes glycolysis [30, 31]. The present study showed increased gene and protein expression of PDHA1 in PAH-EC compared to CTEPH-EC and healthy ECs, but no difference in gene or protein expression of PDK1. Contrary to previous results16 and regardless an increased glycolysis, we could not find an upregulation of PDK1 in PAH-EC or a downregulation of mitochondrial activity. Protein levels of oxidative phosphorylation complexes I, II and IV were increased in PAH-EC compared to CTEPH-EC and healthy ECs indicative for an elevated functional mitochondrial respiration and could explain the increase in active PDHA1 which is in line with the increased use of glycolysis.
Besides glycolysis and oxidative phosphorylation, glutamine metabolism is thought to be involved in PAH pathology and has been shown to be altered in CTEPH-EC [18.32]. Glutamine metabolism is essential in EC proliferation and is driven by the expression of GLS1 and GLUD1 . The current study showed increased gene expression of GLUD1 but not GLS1 in PAH-EC compared to CTEPH-EC. This observation implies a role of glutamine metabolism in PAH-EC but, also, further confirms a difference in metabolism between PAH-EC and CTEPH-EC that needs deeper attention. At last, the oxidative arm of the PPP, important for maintaining cell viability under high rates of proliferation , was not found different between CTEPH-EC and PAH-EC, neither between PAH-EC and healthy ECs.
Based on the EC metabolic and viability profile between CTEPH and PAH, ECs were treated with metabolic inhibitors to understand whether differences in metabolism could be translated into differences in viability upon inhibition. All ECs studied showed a dose-dependent reduction in viability after incubation with metabolic inhibitors 3PO, DCA, BPTES and UK-5099. Particularly, PAH-EC were further affected when cultured with 3PO, DCA, BPTES compared to HPAEC and CTEPH-EC. These results are in line with the higher expression level of glycolytic and glutamine-related enzymes found in PAH-EC compared to CTEPH-EC and healthy ECs. Previous studies have shown beneficial effects of orphan small molecule DCA in both human PAH and experimental PAH [15, 16]. DCA is a pyruvate analogue which inhibits all four PDK isoforms and activates PDH activity. Although there was a dose response in CTEPH-EC when cultured with DCA, it did not reach statistically significance and suggest that DCA treatment may be better suited in PAH patients than CTEPH. Inhibition of UK-5099, is a potent inhibitor that blocks pyruvate transportation into mitochondria. This inhibitor affected all cell lines without a clear difference among groups. On the other hand, it has been recently demonstrated that 3PO does not directly inhibit the enzymatic activity of key glycolytic enzymes such as HK, LDHA or PFKFB3 . Further studies need to test other glycolytic inhibitors to confirm these results.
Based on the results of this study, showing an endogenous glycolic difference in CTEPH-EC compared to PAH-EC, blocking glycolysis may not be beneficial in CTEPH-EC and requires further investigation for the development of novel CTEPH treatments.
This study presents some limitations. PAH patients were significantly younger and mostly female compared to CPEPH patients which is in line with the observed female predominance of PAH  and the earlier onset of disease . The significant increase in hemodynamic parameters in PAH patients compared to CTEPH patients could be explained by the more severe disease state of PAH patients included in this study. Differences in disease severity could potentially contribute to the differences in metabolism and viability between endothelial cells isolated from PAH and CTEPH patients. Additionally, our sample size is limited, and these results should be verified in a bigger patient group.