Endothelial cell (EC) apoptosis is a critical trigger for pulmonary arterial hypertension (PAH), resulting the loss of fragile distal lung arterioles either directly, by a degenerative mechanism, and/or indirectly, by promoting the emergence of growth dysregulated vascular cells.
We developed a murine model for EC apoptosis via diphtheria toxin (DT) exposure in order to study its downstream effects.
48-72 hours after the intra-tracheal dministration of 10ng DT, binary transgenic mice (CRE+DTR+) harbouring the human DT receptor (hDTR) targeted to ECs showed an increase in lung endothelial TUNEL and activated caspase-3 staining associated with an incrrase lung vascular permeability (Evans-Blue assay). Flow-cytometric analysis showed a >50% loss of hDTR+, CD144+, CD31+ and CD34+ ECs at 72 hrs, with an 80% decrease in NG5+pericytes, leading to 35% decrease in total lung microvascular volume by Micro-CT, mainly in vessels <150um in diameter. Right ventricular systolic pressure (RVSP) (n=19) was elevated at 72 hours after a single dose of DT compared with saline (33.7±1 vs. 23.9±0.35 mmHg, respectively; p<0.0001), consistent with pulmonary hypertension (PH), returning to control levels by 1 week (n=20).
In contrast, right ventricular hypertrophy (RVH) was only seen at 1 week post DT (n=15) (0.27±0.006) vs. control (0.24±0.004; p=0.001). Resolution of PH was accompanied by recovery in lung vascular permeability and volume, as well lung EC and pericyte populations. Interestingly, a marked increase in CD11b+Ly6G+ neutrophils was seen at 72 hours post DT (1414±293.5) vs. saline (109±29; p=0.004), with no change in M1 or M2 macrophage numbers, returning to control levels at 1 week, suggesting a potential role of inflammatory cells in the regenerative/recovery process in this model.
These data suggest that sustained loss of ECs is needed to maintain a PAH-phenotype and that the balance between injury and repair may be an important determinant in the pathogenesis of PAH.