Constitutive expression of pregnancy-associated plasma protein-A in arterial smooth muscle reduces the vascular response to injury in vivo

Abstract

Pregnancy-associated plasma protein-A (PAPP-A) functions to increase local IGF-I bioactivity. In this study, we used transgenic mice that constitutively express human PAPP-A in arterial smooth muscle to test the hypothesis that overexpression of PAPP-A enhances vascular smooth muscle cell (SMC) response to IGF-I in vivo. PAPP-A transgenic (Tg) and wild-type (WT) mice underwent unilateral carotid ligation, a model of injury-induced SMC hyperplasia and neointimal formation. In both WT and PAPP-A Tg mice, endogenous PAPP-A mRNA expression showed peak elevation 5 days after carotid ligation. However, PAPP-A Tg mice had 70-75% less neointima than WT at 5 and 10 days postligation, with a significant reduction in occlusion of the ligated artery. WT and PAPP-A Tg mice had equivalent increases in medial area and vessel remodeling postligation. There was little change in medial area and no evidence of neointima in the contralateral carotid of WT or PAPP-A Tg mice. Both WT and PAPP-A Tg carotids exhibited signs of dedifferentiation of SMC, which precedes the increase in proliferation and migration that results in neointimal formation. However, the number of proliferating cells in the media and neointima of the ligated PAPP-A Tg artery was reduced by 90% on day 5 postsurgery compared with WT. This decrease was associated with a significant decrease in an in vivo marker of IGF-I bioactivity and reduced IGF-I-stimulated receptor phosphorylation ex vivo. These data suggest differential effects of chronic (transgenic) and transient (endogenous) PAPP-A expression on neointimal formation following vascular injury that may be due in part to the differential impact on IGF-I signaling.

Fig. 1.

Endogenous pregnancy-associated plasma protein-A (PAPP-A) gene expression in wild-type mice (dashed line) and PAPP-A transgenic (Tg) mice (solid line) following unilateral carotid ligation. Mouse PAPP-A mRNA levels were measured by real-time PCR, and ligated values are expressed relative to the unligated (sham) artery. Results are means ± SE of 4–6 mice. There were no significant differences between WT and PAPP-A Tg mice.

Fig. 2.

Medial (A) and neointimal (B) area in wild-type (WT; gray bars) and PAPP-A Tg mice (black bars) following unilateral carotid ligation. Results are means ± SE of 8–12 mice. Dashed line indicates sham values in medial area. *Significant difference between WT and PAPP-A Tg mice at P < 0.0001. C: sections of carotids 10 days following unilateral ligation stained with Verhoff von Giessen. Thick black arrows indicate the neointima, thick white arrows indicate the internal elastic lamina (IEL), and thin black arrows indicate the external elastic lamina (EEL). Neointimal area was defined by IEL minus luminal area. Medial area was defined by EEL minus IEL.

Fig. 3.

Vessel remodeling in WT (gray bars) and PAPP-A Tg mice (black bars) following unilateral carotid ligation. Vascular remodeling was calculated as EEL ligated divided by EEL unligated times 100. Results are means ± SE of 8–12 mice.

Fig. 4.

Expression of vascular differentiation markers in WT and PAPP-A Tg mice following carotid ligation. mRNA levels were measured by real-time PCR. Results are means (n = 5 or 6 mice) expressed relative to day 0. αSMA, α-smooth muscle actin; Myhll, smooth muscle myosin heavy chain; SM22α, smooth muscle 22α.

Fig. 5.

Ki-67 immunostaining of carotid arteries in WT (gray bars) and PAPP-A Tg mice (black bars) following ligation. Results are means ± SE (n = 4–6 mice). *Significant difference between WT and PAPP-A Tg mice at P < 0.001.

Fig. 6.

IGF-I-stimulated IGF-I receptor (IGF-IR) phosphorylation ex vivo. Two days postligation, carotids from WT and PAPP-A Tg mice were harvested and then treated without (gray bars) or with 50 nM IGF-I (black bars) for 10 min. Western immunoblotting for phosphorylated IGF-IR was performed and analyzed as described in materials and methods. Results are means ± SE (n = 5 mice). *Significant IGF-I effect at P < 0.05.