Abstract
Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2–promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5–14.5) and later (E15.5–17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in the formation of the mural cell layer and weakening of vessel integrity. These results demonstrate that overexpression of progranulin in endothelial cells influences normal angiogenesis in vivo.
Highlights
Angiogenesis, the formation of new blood vessels from preexisting vessels, is critical whenever tissues undergo extensive remodeling processes, including during normal growth and development, wound healing [1] [2], in the female reproductive cycle [3], mammary gland development during pregnancy and lactation [4] and the expansion of fat mass during obesity [5] [6]
Serial sections stained for progranulin and the endothelial marker protein PECAM (CD31) show overlay of progranulin with endothelial cells in Tie2-gene for progranulin (Grn) positive mice (Fig. 1F)
Overproduction of progranulin by endothelial cells in the Tie2-Grn positive mice was demonstrated by immunohistological staining (Fig. 1G and H)
Summary
Angiogenesis, the formation of new blood vessels from preexisting vessels, is critical whenever tissues undergo extensive remodeling processes, including during normal growth and development, wound healing [1] [2], in the female reproductive cycle [3], mammary gland development during pregnancy and lactation [4] and the expansion of fat mass during obesity [5] [6]. Progranulin (which is called granulin-epithelin precursor, PC cell derived growth factor and acrogranin) is a pleiotropic regulatory protein that promotes cellular proliferation, cell migration and survival (reviewed in [12]) It is often overexpressed in cancers and contributes to tumor progression (reviewed in [13,14]) and stroma formation [15]. Progranulin stimulated proliferation and migration of endothelial cells in a mitogenactivated protein kinase (MAPK) and phosphatidyl-inositol-3kinase (PI3K)-dependent fashion [11]. It accelerated tubule formation of primary microvascular endothelial cells growing on Matrigel [11]. Tube-formation is an assay of organized selfassociation in vitro that recapitulates the formation of luminal endothelial structures during angiogenesis in vivo [32]
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