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