Pneumonectomy combined with SU5416 or monocrotaline pyrrole does not cause severe pulmonary hypertension in mice

Abstract

In the field of pulmonary hypertension (PH), a well-established protocol to induce severe angioproliferation in rats (SuHx) involves combining the VEGF-R inhibitor Sugen 5416 (SU5416) with 3 wk of hypoxia (Hx). In addition, injecting monocrotaline (MCT) into rats can induce inflammation and shear stress in the pulmonary vasculature, leading to neointima-like remodeling. However, the SuHx protocol in mice is still controversial, with some studies suggesting it yields higher and reversible PH than Hx alone, possibly due to species-dependent hypoxic responses. To establish an alternative rodent model of PH, we hypothesized mice would be more sensitive to hemodynamic changes secondary to shear stress compared with Hx. We attempted to induce severe and irreversible PH in mice by combining SU5416 or monocrotaline pyrrole (MCTP) injection with pneumonectomy (PNx). However, our experiments showed SU5416 administered to mice at various time points after PNx did not result in severe PH. Similarly, mice injected with MCTP after PNx (MPNx) showed no difference in right ventricular systolic pressure or exacerbated pulmonary vascular remodeling compared with PNx alone. These findings collectively demonstrate that C57/B6 mice do not develop severe and persistent PH when PNx is combined with either SU5416 or MCTP.NEW & NOTEWORTHY We attempted to establish a mouse model of severe and irreversible pulmonary hypertension by substituting hypoxia with pulmonary overcirculation. To do so, we treated mice with either SU5416 or monocrotaline pyrrole after pneumonectomy and performed hemodynamic evaluations for PH. Despite this "two-hit" protocol, mice did not exhibit signs of severe pulmonary hypertension or exacerbated pulmonary vascular remodeling compared with PNx alone.

Keywords: Sugen 5416; mice model; monocrotaline pyrrole; pneumonectomy; pulmonary hypertension.

Graphical abstract

Figure 1.

Pneumonectomy combined with SU5416 does not exacerbate pulmonary hypertension or pulmonary vascular remodeling in mice. A: SuPNx protocol of experiment 1. B: right heart catheterization revealed a mild but significantly elevated right ventricle systolic pressure (RVSP) in PNx and SuPNx mice compared with control (Con), but no difference between PNx and SuPNx (P = 0.7503). C: Fulton index was increased in PNx and SuPNx mice compared with Con, but no difference was seen between the two PNx groups (P = 0.8563). D: representative images of precision-cut lung slices after immunofluorescence staining focusing on distal arterioles (CD31: green, SMA: white, DAPI: blue). Scale bar: 100 μm. All data are presented as means ± SD; one-way ANOVA followed by Tukey’s multiple comparison was applied. d42, postoperative day 42; PNx, left pneumonectomy; SuPNx, SU5416 administration + left pneumonectomy.

Figure 2.

Pneumonectomy combined with SU5416 does not induce pulmonary hypertension or pulmonary vascular remodeling at postoperative day 70. A: SuPNx protocol of experiment 2. B: right ventricle systolic pressure (RVSP) measured by right heart catheterization reveals no difference between control (Con) and SuPNx mice. C and D: echocardiogram reveals no difference in cardiac function or right ventricle remodeling between the two groups. E: representative images of Elastica van Gieson (EvG) staining in lung tissues from Con and SuPNx mice. Scale bar: 50 μm. F: quantification of EvG staining revealing unchanged intima and media layer thickness between Con and SuPNx mice. G: progression of pulmonary artery acceleration time/cardiac length (PAAT/CL), cardiac index (CI), and right ventricle dilation diameter (RVDd) from week 2 to week 10 measured by echocardiogram. Data are presented as means ± SD; unpaired t test was applied. Closed triangle: male mice; open triangle: female mice. CI, cardiac index; PNx, left pneumonectomy; SuPNx, SU5416 administration + left pneumonectomy; TAPSE, tricuspid annular plane systolic excursion.

Figure 3.

Pneumonectomy combined with monocrotaline pyrrole does not exacerbate pulmonary hypertension or right ventricular dysfunction. A: MPNx protocol for experiment 3. B: right ventricle systolic pressure (RVSP) was mildly elevated 14 days after PNx and in the 21-day MPNx group, while it remained unaltered in the MPNx group on day 14 (P = 0.1377). No difference was found between the PNx and MPNx groups (P = 0.8678 or 0.5086, respectively). C: echocardiogram revealed a reduced right ventricle fractional area change (RVFAC) in the 14-day PNx group, 14-day and 21-day MPNx groups. No difference was found between the PNx and MPNx groups (P = 0.5563 or 0.9876, respectively). D: MPNx protocol of experiment 4. E: RVSP was elevated in PNx and MPNx groups on day 42, while no difference was found between the two PNx groups (P = 0.8714). F: echocardiogram revealed a reduced RVFAC in the 42-day PNx and MPNx groups, but no significant difference in RVFAC between PNx mice and MPNx mice (P = 0.9379). All data are presented as means ± SD; one-way ANOVA followed by Tukey’s multiple comparison. Con, control; d7, 14, 21, 42: postoperative day 7, 14, 21, 42; MPNx, MCTP + left pneumonectomy; PNx, left pneumonectomy.