Vasoinhibin, an N-terminal Prolactin Fragment, Directly Inhibits Cardiac Angiogenesis in Three-dimensional Heart Culture

Abstract

Vasoinhibins (Vi) are fragments of the growth hormone/prolactin (PRL) family and have antiangiogenic functions in many species. It is considered that Vi derived from PRL are involved in the pathogenesis of peripartum cardiomyopathy (PPCM). However, the pathogenic mechanism of PPCM, as well as heart angiogenesis, is not yet clear. Therefore, the aim of the present study is to clarify whether Vi act directly on angiogenesis inhibition in heart blood vessels. Endothelial cell viability was decreased by Vi treatment in a culture experiment. Furthermore, expression of proangiogenic genes, such as vascular endothelial growth factor, endothelial nitric oxide synthase, and VE-cadherin, were decreased. On the other hand, apoptotic factor gene, caspase 3, and inflammatory factor genes, tumor necrosis factor α and interleukin 6, were increased by Vi treatment. In three-dimensional left ventricular wall angiogenesis assay in mice, Vi treatment also inhibited cell migration, neovessel sprouting, and growth toward collagen gel. These data demonstrate that Vi treatment directly suppresses angiogenesis of the heart and support the hypothesis that Vi induce PPCM.

Keywords: angiogenesis; heart; peripartum cardiomyopathy; prolactin; vasoinhibin.

Figure 1

Effects of vasoinhibins (Vi) on cell viability. Effects of Vi on endothelial cell (A) and cardiomyocyte (B) viablity in 10- and 26-h culture. The white bar shows absorbance in the control. The light gray bar shows it in the 0.5 nM-Vi-treated group, the dark gray bar shows it in the 5 nM-Vi-treated group, and the black bar shows it in the 50 nM-Vi-treated group. Vi: vasoinhibin (n = 3, Mean ± SEM, *P < 0.05, **P < 0.01 vs. control).

Figure 2

Effects of vasoinhibins (Vi) on gene expression in endothelial cells by real-time PCR. The white bar shows relative expression levels in the control and the black bar shows those in the Vi-treated group. eNOS, endothelial nitric oxide synthase; VEGFA, vascular endothelial growth factor A; TNF, tumor necrosis factor α; IL6, interleukin 6; CASP3, caspase 3; Vi, vasoinhibin 50 nM, N.D., not detectable (n = 3, Mean ± SEM, *P < 0.05, **P < 0.01, ***P < 0.001 vs. control).

Figure 3

Lectin staining of the left ventricular wall in three-dimensional culture. Lectin staining of the three-dimensional cultured left ventricular wall using endothelial cell-specific lectin (green) and DAPI (blue). The heart segments were treated with VEGF under hypoxia in the absence or presence of Vi. Arrows indicate neovessel and arrow heads indicate migrated lectin-positive cell. (A,B) White bar = 200 μm. (C,D) White bar = 100 μm. Vi, vasoinhibin 100 nM.

Figure 4

Angiogenesis analysis of lectin-stained left ventricular wall in three-dimensional culture. The white bar shows the numerical values in the control and the black bar shows those in the vasoinhibins (Vi)-treated group. The heart segments were treated with VEGF under hypoxia in the absence or presence of Vi. The procedures of measuring values are described in Section “Materials and Methods.” Vi, vasoinhibin 100 nM (n = 3, mean ± SEM, *P < 0.05 vs. control). (A) Number of migrated cells, (B) number of neovessels, (C) Length of neovessels, and (D) proportion of lectin-positive cell.