Discussion
SU5416 was the first VEGF receptor 2 inhibitor to enter clinical development for cancer therapy.26 It has been shown that inhibiting the VEGF- and endothelial-dependent proliferation will result in structural changes known as plexiform lesions.10,26-29 Allowing for the selection of an apoptosis resistant, proliferating endothelial cell phenotype by the combination of blockade of VEGF receptor 2 and hypoxia, severe PAH will develop.26 In this study, we reasoned that a pulmonary insult such as pneumonectomy followed by injection with SU5416 will yield similar results. In fact, our SuPNx model was shown to cause severe angio-obliterative PAH associated with increased cell proliferation and proapoptotic signaling, resulting in neointimal and medial remodeling.11 In addition, unlike the hypoxia model where partial reversibility of pulmonary hypertension is seen after returning to normoxia, the SuPNx model is independent of hypoxic vasoconstriction and hemoconcentration.11
In our study, the RVSP in the SUPNx42 rats increased significantly, which may favor the assessment of drug effects in preclinical trials. The RVSP improved significantly in both the early and late DIZE treatment protocols, suggesting the effectiveness of hemodynamic changes by DIZE in PAH. However, although the Fulton index did increase in the SuPNx42 rats, there were only trends of improvement with DIZE in either the early or the late treatment protocols. It is possible that, due to the severe pathologic changes in this chronic PAH model, the observed hypertrophy in cardiomyocytes was irreversible, leading to heart failure.
In addition to endothelin, RAS has also been implicated as a causative factor in PAH.1 Ang II, a principal effector peptide of the RAS, can exert deleterious effects on the pulmonary vasculature resulting in vasoconstriction, proliferation, and inflammation, all of which are contributable to the development of PAH. However, it is difficult to measure the plasma and tissue levels of Ang II due to its very short half-life (16 ± 1 s in mice).30 In contrast, ACE, which catalyzes the conversion of Ang I to Ang II, is abundant in the small pulmonary arteries and is therefore more easily to be detected 31. Thus, measuring ACE, instead of Ang II levels in the lung, provides a more practical method for assessing the associated hemodynamic changes. In our study, there were no significant changes in pulmonary Ang II levels among the four animal groups. However, the expression of pulmonary ACE was increased in SuPNx42 rats and ameliorated by DIZE in both early and late treatment group, suggesting that ACE may be a representative marker in in this animal model of PAH.
In the RAS, ACE/Ang II/AT1 constitutes the vasopressor arm, which is counterbalanced by the ACE2/Ang-(1-7)/Mas receptor axis.32 By converting Ang II to the vasodilatory peptide Ang-(1–7), ACE2 provides a negative feedback on the RAS and protects the major organs such as heart and kidneys from being damaged by excessive Ang II generated during the development of PAH.33,34 Interestingly, in the present study, the RV and pulmonary levels of Ang-(1-7) were not significantly altered in the SuPNx42 group when compared with the sham-operated rats. However, the RV levels of Ang-(1-7) were significantly elevated in both the early and late DIZE treatment groups, and the pulmonary levels of this peptide were also significantly increased in the early DIZE treatment group.
It should be noted that, besides the action of ACE2, Ang-(1-7) can also be formed by other biochemical pathways. It can be generated from hydrolysis of angiotensin I by neprilysins (NEPs) or cleavage of the Ang-(1-9) by ACE.35-38  With respect to its metabolism, Ang-(1-7) can be subsequently degraded by ACE to form Ang-(1-5), by dipeptidyl peptidase 3 (DPP3) to produce Ang-(3-7) and Ang-(5-7), or by aminopeptidase A (APA) to generate Ang-(2-7).38 Affecting the activity of any of the aforementioned enzymes will undoubtedly result in a change of the levels of Ang-(1-7). To this end, it is speculated other alternative pathways may also influence the formation of Ang-(1-7) in our animal model. Further studies are needed to fully elucidate the involved biochemical pathways.
It has been reported that Ang-(1–7) can stimulate the releases of endothelial derived nitric oxide (eNOS) and vasodilator prostaglandins as well as potentiate the vasodilatory effect of bradykinin.39-41 Consistent with these results, eNOS was significantly elevated in our early DIZE treatment group when compared to SuPNx42 rats (Figure 5B), inferring that Ang-(1-7) may stimulate eNOS release in this animal model. The increased expression of eNOS could contribute to the lowering of pulmonary artery pressure observed herein (Figure 2A).
In summary, our model of hypertensive pulmonary vascular disease in pneumonectomized, SU5416-injected rats resemble the neointimal proliferation and vascular occlusion by smooth muscle cells that occurs in human PAH. The efficacy of DIZE in the early and late treatment group suggests its ability to rescue animals from established hypertensive pulmonary vascular disease. However, the exact mechanisms by which DIZE exerts its beneficial effects remain to be investigated. From our results, it is likely that attenuation of PAH by DIZE involves a combination of antiproliferative effects on pulmonary vascular smooth muscle cells through production of Ang-(1-7), suppression of the growth of vascular smooth muscle cells, and also induction of endothelial cell eNOS expression.41,42