Vascular endothelial growth factor receptor 3 signaling contributes to angioobliterative pulmonary hypertension

Abstract

The mechanisms involved in the development of severe angioobliterative pulmonary arterial hypertension (PAH) are multicellular and complex. Many of the features of human severe PAH, including angioobliteration, lung perivascular inflammation, and right heart failure, are reproduced in the Sugen 5416/chronic hypoxia (SuHx) rat model. Here we address, at first glance, the confusing and paradoxical aspect of the model, namely, that treatment of rats with the antiangiogenic vascular endothelial growth factor (VEGF) receptor 1 and 2 kinase inhibitor, Sugen 5416, when combined with chronic hypoxia, causes angioproliferative pulmonary vascular disease. We postulated that signaling through the unblocked VEGF receptor VEGFR3 (or flt4) could account for some of the pulmonary arteriolar lumen-occluding cell growth. We also considered that Sugen 5416-induced VEGFR1 and VEGFR2 blockade could alter the expression pattern of VEGF isoform proteins. Indeed, in the lungs of SuHx rats we found increased expression of the ligand proteins VEGF-C and VEGF-D as well as enhanced expression of the VEGFR3 protein. In contrast, in the failing right ventricle of SuHx rats there was a profound decrease in the expression of VEGF-B and VEGF-D in addition to the previously described reduction in VEGF-A expression. MAZ51, an inhibitor of VEGFR3 phosphorylation and VEGFR3 signaling, largely prevented the development of angioobliteration in the SuHx model; however, obliterated vessels did not reopen when animals with established PAH were treated with the VEGFR3 inhibitor. Part of the mechanism of vasoobliteration in the SuHx model occurs via VEGFR3. VEGFR1/VEGFR2 inhibition can be initially antiangiogenic by inducing lung vessel endothelial cell apoptosis; however, it can be subsequently angiogenic via VEGF-C and VEGF-D signaling through VEGFR3.

Keywords: MAZ51; Sugen 5416; VEGF isoforms; VEGF receptor 3; capillary rarefaction; chronic hypoxia; right heart failure; sFlt-1.

Figure 1

A, Lung tissue expression of the vascular endothelial growth factor (VEGF) isoform and of VEGF receptor proteins by Western blot analysis. There is no change in the expression of VEGF-A and VEGF-B proteins, while VEGF-C and VEGF-D are increased in expression in Sugen 5416/chronic hypoxia (SuHx) animals (B–E). VEGFR3 protein expression is increased when referenced to β-actin (G). The concentration of soluble VEGFR1 (sFlt-1) in the lung tissue is reduced (H), while the sFlt-1 levels trended toward an increase in the serum of SuHx animals (I). SPARC protein expression is reduced in whole-lung tissue protein extracts from SuHx rats (J). Asterisks indicate P < 0.05. n = 4. Hx: hypoxia-only rats.

Figure 2

Proteins expressed in right ventricle (RV) tissues. All vascular endothelial growth factor (VEGF) isoform proteins are reduced in expression in the RV of Sugen 5416/chronic hypoxia (SuHx) rats (A–E). Immunohistochemistry (magnification, ×10; scale = 50 μm) shows loss of staining in the SuHx RV tissues when applying an antibody specifically directed against VEGF-C (F). There is only a trend toward an increased concentration of the sFlt-1 protein in the SuHx RV. Asterisks indicate P < 0.05. n = 4. Hx: hypoxia-only rats.

Figure 3

Immunohistochemistry of lung tissue sections (magnification, ×40; scale = 20 μm). Vascular endothelial growth factor (VEGF)–C protein is expressed in bronchial epithelium in normal control lungs; in lungs of chronically hypoxic rats there is faint staining of some cells of the adventitia, while arteriolar endothelial cells express VEGF-C in the lungs of Sugen 5416/chronic hypoxia (SuHx) rats (A). VEGFR3 protein is expressed in endothelial cells in normal, chronically hypoxic, and SuHx animals and in macrophages. VEGFR3 expression is found in bronchus-associated lymphoid tissue in large and small pulmonary artery endothelial cells (B). Luminal endothelial cells in SuHx animals express VEGFR3 at 3 weeks of the study protocol but not any longer in the lungs of animals that had received Sugen 5416 and been exposed to chronic hypoxia and killed 6 weeks after the Sugen 5416 implantation (C). Asterisks indicate P < 0.05. n = 4. Hx: hypoxia-only rats.

Figure 4

Double immunofluorescence staining of vascular endothelial growth factor receptor 3 (VEGFR3) and von Willebrand factor (vWF) for lung tissue sections. Control and hypoxia sections shows some of the endothelial cells of a medium vessel >50 μm expressing VEGFR3. The section from the Sugen 5416/chronic hypoxia (SuHx) rat lung shows abundant VEGFR3 expression in and around the lesion. 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain. Magnification, ×40.

Figure 5

Concomitant treatment of Sugen 5416/chronic hypoxia (SuHx) rats with the vascular endothelial growth factor receptor 3 (VEGFR3) blocker MAZ51 reduced right ventricular systolic pressure (RVSP; A), the number of obliterated lung arterioles (C–D), the degree of perivascular cell accumulation (E), and the size of the bronchoalveolar lymph cell aggregate (F). Asterisks indicate P < 0.05. n = (4–7). Magnification, ×10. Scale = 50 μm. RV/LV+S: ratio of right ventricular weight to left ventricular plus septal weight; BALT: bronchus-associated lymphoid tissue.

Figure 6

Treatment of animals with established Sugen 5416/chronic hypoxia (SuHx)–triggered pulmonary hypertension with the vascular endothelial growth factor receptor 3 (VEGFR3) inhibitor MAZ51 for 2 weeks worsened the degree of pulmonary hypertension when assessed by measurement of right ventricular systolic pressure (RVSP; A). In addition to the persistence of small arteriole occlusion, the vessels appear highly muscularized (C). Asterisks indicate P < 0.05. n = 3. Magnification, ×2.5. Scale = 100 μm.