In this study, we isolated endothelial cell lines from material collected at PEA, and generated an in vitro model of endothelial pathology in CTEPH. CTEPH-EC presented an abnormal hyperproliferative phenotype, a reduced angiogenic capacity and eNOS expression, a dysregulated mitochondrial function, and an overexpression of key adhesion molecules.
Isolation and characterization of CTEPH-EC. CTEPH-EC showed typical cobblestone morphology and stained positive for a wide range of endothelial markers and negative for muscular or hematopoietic markers. Our results indicate that isolated CTEPH-EC are positive for the macrovascular homeobox-containing (HOX) genic pattern excluding the possibility of a peripheral microcirculation origin. HOX genes are well conserved among mammalian species and good predictors of EC identity[15]. Toshner et al., identified a differential expression of certain HOX genes between lung microvascular ECs and pulmonary artery macrovascular ECs[15].
It has been recently shown, that ECs derived from patients with PAH (PAH-EC) have an hyperproliferative and an apoptosis-resistant phenotype contributing to the progression of the disease[16]. In this study, we showed that CTEPH-EC also grew at a consistently faster rate and presented higher number of Ki-67+ cells when compared to healthy endothelial cells. This result is in line with a recent publication by Naito et al in which they showed greater proliferative potential of CTEPH-EC[17]. In this study, we additionally demonstrated that CTEPH-EC had a significantly higher clonogenic potential and could survive for many more cell culture passages without losing their proliferative potential and phenotype. As reported for PAH-EC, CTEPH-EC also presented a significant decrease in apoptotic caspase related genes compared to controls. However, the expression of survival factors such as Bcl2 or tumorigenic-associated genes p53 and p21 did not differ between patient and control cells. Additionally, CTEPH-EC maintained a smaller cell size consistent with a less differentiated phenotype. Altogether, these results strongly support that CTEPH-EC present a more viable, proliferative phenotype compared to pulmonary ECs derived from healthy subjects.
CTEPH-EC overexpression of adhesion molecules. CTEPH-EC showed a significant increase in expression of adhesion molecules (CD31, VCAM-1, ICAM.1, CD44) compared to healthy HPAE cells. Several studies have shown the role of CD31 in modulating apoptosis and cell growth under stress conditions[18]. Cheung et al., showed that in response to apoptotic stimuli, CD31 engages a pro-survival pathway that in some cancers influences tumor immuno-resistance[19]. Tsuneki et al., showed that CD44 could regulate endothelial proliferation and apoptosis by modulating the expression of CD31 and VE-cadherin[20]. The increased expression of adhesion molecules in CTEPH-EC suggests a possible correlation with CTEPH-EC pro-survival characteristics. Indeed, Ataam et al., has recently suggested the important role of ICAM-1 overexpression in regulating abnormal EC growth via phosphorylation of downstream effectors such as SRC, p38, ERK[21]. Targeting adhesion molecules as early indicators of endothelial dysfunction deserves further study to restore endothelial function in CTEPH.
CTEPH-EC function. Functionally, CTEPH-EC had reduced capacity to form tube-like structures and decreased migration capacity in response to wounding. In agreement with this, studies reported an impairment of angiogenesis in CTEPH patients. Alias et al., showed a downregulation of mRNA levels of VEGFR2 in PEA specimens[22]. Also, Zabini et al., reported that homogenized PEA specimens contained several cytokines that inhibit angiogenesis[23]. We comprehensively assessed angiogenesis, demonstrating that the formation of tubular structures by CTEPH-EC is functionally impaired both in vitro and in vivo. Additionally, our results also demonstrate reduced cellular migration in CTEPH-EC compared to control cells. CD31 overexpression has been shown to inhibit migration of ECs through a PECAM-1/γ-catenin/desmoplakin/vimentin mechanism[18]. Overexpression of adhesion molecules in CTEPH-EC could significantly affect their angiogenic potential, migration and contribute to the thrombogenic response and vessel wall remodeling.
Mitochondrial dysfunction in CTEPH-EC. Endothelial metabolism is closely linked to EC function[24]. Accordingly, a better understanding of the metabolic changes in CTEPH-EC is a crucial step in resolving CTEPH pathogenesis. It has been shown that PAH-EC have an altered mitochondrial-metabolic phenotype with a metabolic shift towards glycolysis and an increased mitochondrial fusion/fission imbalance[25].Compounds stimulating TCA cycle are under investigation for PAH treatment [26, 27]. The question is whether such molecules could also be applied to CTEPH patients. Our results show that fusion regulatory GTPases MFN1, MFN2 and OPA1 were downregulated in CTEPH-EC compared to control cells. However, unlike in PAH-EC, fission regulatory protein DRP1 was not changed. Downregulation of MFN2 in CTEPH-EC indicates mitochondrial unbalance and damage.
On the other hand, our electron microscopy results showed abnormal mitochondria with irregular inner membrane and cristae in CTEPH-EC. Cristae shape determines the assembly and stability of respiratory chain super-complexes, impacting mitochondrial respiratory efficiency[28]. In our study, CTEPH-EC displayed increased mitochondrial respiration activity in all the respiratory chain complexes. Increased oxygen consumption could be due to a rise of mitochondrial mass or due to proton leakage. As we did not find an increase in number of mitochondria, we imply the observed uncoupling leakage as main causative factor for oxidative alterations in CTEPH-EC. In agreement with these results we confirmed the presence of higher levels of mitochondrial reactive oxygen species (mROS) production in CTEPH-EC. Taken together, our results show that CTEPH-EC mitochondria are dysfunctional with a fusion/fission imbalance, an increased mitochondrial respiration and uncoupling leakage. Such mitochondrial dysfunction is significantly different from the imbalance previously reported in PAH-EC. This acknowledges different mitochondrial dynamics between PAH and CTEPH needed to be taken into account for future therapeutic strategies[25].
Oxidative stress. Higher levels of protein carbonyl groups and 8-OHdG, indicators for the oxidative status of proteins and DNA damage, were observed in CTEPH-EC in comparison to healthy ECs. Oxidative stress is caused by elevated ROS production or reduced cellular detoxification[29]. Antioxidant enzyme SOD2 is first line of defense against superoxide accumulation and cell death[29, 30]. In PAH levels of SOD2 are downregulated causing ionic dysregulation and downstream pulmonary vasoconstriction[31]. Our results show that SOD2 mRNA and protein levels are also significantly downregulated in CTEPH-EC. Epigenetic attenuation of SOD2 could be one of the keys behind CTEPH-EC dysfunctionalities.
Decreased eNOS activity has also been associated with mitochondrial impairment and dysregulated angiogenesis in PH[32]. In agreement with these findings, our study showed that increased levels of mROS in CTEPH-EC were accompanied by a significant reduction in eNOS and caveolin-1 expression. Understanding the underlying signaling in oxidative/nitrative stress-induced pathways in CTEPH, merits further attention [33].
Correlation with clinical features. Significantly, eNOS and VCAM-1 mRNA levels were related with clinical risk parameters. Higher expression levels of the adhesion molecule VCAM-1 were observed in patients with higher levels of pulmonary vascular resistance and lower CTEPH-EC eNOS mRNA levels were associated with patients classified in worst WHO-functional class. eNOS and VCAM-1 patient levels merits further investigation as potential predictive and diagnostic tools in CTEPH.
Limitations. The use of primary cell cultures involves involuntary selection of cells with the highest growth potential. To minimize this limitation, all CTEPH-EC were isolated strictly following the same protocol and all experiments were carried out in early passage cells (unless otherwise stated). We confirmed that CTEPH-EC derived from material collected at PEA are dysfunctional. However, it is not possible to conclude that functional differences found in CTEPH-EC are a primary cause or a secondary consequence to the haemodynamic perturbations caused by the pulmonary vascular obstruction. Further studies would need to be designed to answer this specific subject.