Pulmonary hypertension (PH) due to left heart disease (group 2 PH) is the most common type of PH (60-80% of patients). The exact underlying molecular mechanisms are still not completely understood. For disease manifestation the need of a second hit such as hypoxia has been proposed. To explore the role of a second hit, we combined group 2 PH induced by transverse aortic constriction (TAC) with hypoxia (HX). All experiments were approved by the local authorities (LANUV NRW).

Initially 10 week-old C57BL/6 mice received TAC (TAC group). By use of a 26G needle a standardized constriction of the aorta was generated and quantified intraoperatively by doppler measurements. To test the additional effect of HX on the onset of PH a second group of mice (TAC+HX) was exposed to hypoxic conditions (10% O2, System Oxycyler, Fa. BioSpherix) for 11 days after TAC. Sham operated mice, with or without HX, served as control groups. Functional analysis was performed 3 and 8 weeks after TAC and 3 weeks after TAC+HX, including echocardiography as well as right and left heart catheterization. Subsequently mice were sacrificed and hearts and lungs were harvested for histological analysis.

In the TAC only group pressure measurements in the right ventricle (RV) confirmed development of PH after 8 weeks in TAC mice (22.2±2.1mmHg, n=8 (sham) vs 26.3±1.1mmHg, n=7 (TAC), p<0.05) but not yet after 3 weeks. Left ventricular (LV) pressures were elevated already 3 weeks postoperatively and further increased until 8 weeks (89.9±6.5mmHg, n=8 (sham) vs 117±13.5mmHg, n=7 (TAC 8 wks), p<0.001). Echocardiography revealed an impairment of contractile function in the LV. The ratio of heart weight/tibia length was increased after 3 and 8 weeks, which was corroborated by the measurement of cross sectional areas of single cardiomyocytes (CSA) in the LV after 3 and 8 wks and in the RV after 8 wks. After 8 weeks, LV+RV showed increased myocardial fibrosis measured by collagen fraction (CF, 4.6±1.6%, n=7 (sham) vs 12.5±3%, n=7 (TAC), p<0.0001). Lung tissue also exhibited an increased interstitial CF, as well as elevated wall thickness of pulmonary arteries (25±2.9%, n=7 (sham) vs 32±3.4%, n=7 (TAC), p<0.001) after 8 weeks. If TAC was combined with HX, right heart catheter measurements revealed the development of PH already 3 weeks postoperatively (22.6±1mmHg, n=7 (sham) vs 28.6±1.8mmHg, n=8 (TAC+HX), p<0.001) while 11 days of HX without TAC had no impact on RV pressures. Consistent with altered hemodynamics already after 3 weeks TAC+HX mice showed increased CF in hearts (2.9±1.4%, n=7 (sham) vs 6.2±2.9%, n=11 (TAC+HX), p<0.05) and lungs, as well as an increased wall thickening of pulmonary arteries (25±3.2%, n=7 (sham) vs 30.2±3.1%, n=13 (TAC+HX), p<0.01).

In summary WT mice following TAC procedure develop type 2 PH over time, identified by increased RV pressures and cardiac as well as pulmonary fibrosis. In line with the postulated multifactorial genesis of type 2 PH the combination of TAC and HX could aggravate the onset and development of PH. Further experiments will include biochemical analysis and gene arrays of harvested tissue to elucidate underlying pathophysiological mechanisms and possible therapeutic targets.