Isolated Anxa5⁺/Sca-1⁺ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo

Abstract

Pericytes are closely associated with endothelial cells, contribute to vascular stability and represent a potential source of mesenchymal progenitor cells. Using the specifically expressed annexin A5-LacZ fusion gene (Anxa5-LacZ), it became possible to isolate perivascular cells (PVC) from mouse tissues. These cells proliferate and can be cultured without undergoing senescence for multiple passages. PVC display phenotypic characteristics of pericytes, as they express pericyte-specific markers (NG2-proteoglycan, desmin, αSMA, PDGFR-β). They also express stem cell marker Sca-1, whereas endothelial (PECAM), hematopoietic (CD45) or myeloid (F4/80, CD11b) lineage markers are not detectable. These characteristics are in common with the pericyte-like cell line 10T1/2. PVC also display a phagocytoic activity higher than 10T1/2 cells. During coculture with endothelial cells both cell types stimulate angiogenic processes indicated by an increased expression of PECAM in endothelial cells and specific deposition of basement membrane proteins. PVC show a significantly increased induction of endothelial specific PECAM expression compared to 10T1/2 cells. Accordingly, in vivo grafts of PVC aggregates onto chorioallantoic membranes of quail embryos recruit endothelial cells, get highly vascularized and deposit basement membrane components. These data demonstrate that isolated Anxa5-LacZ⁺ PVC from mouse meninges retain their capacity for differentiation to pericyte-like cells and contribute to angiogenic processes.

Introduction

Blood vessels mature by the mutual interactions of endothelial cells and mural cells defining two distinct compartments, the lining endothelium and perivascular cells. As research in angiogenesis was mainly focused on the function of endothelial cells, it became obvious that perivascular cells may play a crucial role in normal and pathological angiogenesis [1]. Pericytes are recognized by their close association and the formation of a shared basement membrane with endothelial cells. This cell type regulates hemodynamic processes within the blood vessels and may also promote the survival of endothelial cells by producing angiogenic stimuli or serve as a pool of macrophage-like cells within the brain vasculature [2], [3]. Reduction of pericyte coverage is induced by the deficiencies of either the growth factor PDGF or its receptor PDGFR-β in mutant mouse models and results in cardiovascular dysfunction due to blood vessel dilatation, microvascular leakage, edemas and embryonic lethality [4]. In humans, the loss of pericytes is associated with diabetic retinopathy and their contribution to tumor-angiogenesis is discussed [5].

Perivascular cells (PVC), like pericytes and subpopulations of vascular smooth muscle cells, are assumed to be of mesenchymal origin derived from the neural crest although transdifferentiation from endothelial cells as well as derivation from hematopoietic lineages has also been proposed [6], [7]. Other data suggest that both endothelial cells and pericytes are derived from a common Flk-1⁺ progenitor cell [8], [9]. Pericytes reveal some features of mesenchymal stem cells as they retain the capacity for differentiation into distinct mesenchymal lineages. Previous studies showed that pericytes are able to differentiate into adipocytes, chondrocytes, fibroblasts as well as macrophages [10], [11].

The lack of highly specific markers for pericytes as well as technical difficulties in the isolation of these cells from mouse tissues hampered the use of pericytes. Previous methods for isolation and cultivation of pericytes were mostly based on the selective adherence of cell populations derived from different tissues of rat or bovine origin followed by a series of phenotypic characterizations [12], [13], [14], [15]. Therefore, the in vivo origin and identity of these cells remained uncertain [1]. Recently, we developed a method to purify a population of perivascular cells (PVC) from adult mouse meninges based on the cell specific expression of a targeted LacZ reporter in the mouse annexin A5 gene (Anxa5-LacZ) in adult PVC to detect and sort for viable Anxa5-LacZ⁺ perivascular cells [16]. We showed that Anxa5-LacZ⁺ PVC can be induced to form calcified matrices, undergo early steps of chondrogenesis or differentiate into adipogenic cells. Although these data demonstrate the stem cell-like capacity of the purified populations the characterization of their capacity for vascular differentiation remained unclear.

Here we analyze the capacity of Anxa5-LacZ⁺ PVC to contribute to vascular differentiation. The effects on vascular maturation was compared to the pericyte-like/vascular smooth muscle cell line 10T1/2 [3], [17], [18]. We could show that PVC lines and 10T1/2 express pericyte as well as stem cell markers on their cell surface. Both cell types have the potential for phagocytosis. PVC and 10T1/2 cells induce angiogenic effects after contact with endothelial cells in vitro. Newly formed vascular elements express increased amounts of typical markers of the vasculature as well as basement membrane proteins at the endothelial/pericyte interface. Taking into account their histological localization, plasticity of differentiation and their pericyte-like similarities to 10T1/2 cells, Anxa5-LacZ⁺ PVC define a novel source of vascular associated pericyte-like cells. Our results will allow the further characterization of the developmental origin as well as the functional role of poorly understood interactions between endothelial cells and pericytes in the mouse system, in particular related to diseases associated with pathological changes of vascular basement membranes.