Endosialin (TEM1, CD248) is a marker of stromal fibroblasts and is not selectively expressed on tumour endothelium

Fibroblasts are a diverse cell type and display clear topographic differentiation and positional memory. In a screen for fibroblast specific markers we have characterized four monoclonal antibodies to endosialin (TEM1/CD248). Previous studies have reported that endosialin is a tumour endothelium marker and is localized intracellularly. We demonstrate conclusively that endosialin is a cell surface glycoprotein and is predominantly expressed by fibroblasts and a subset of pericytes associated with tumour vessels but not by tumour endothelium. These novel antibodies will facilitate the isolation and classification of fibroblast and pericyte lineages as well as the further functional analysis of endosialin.     

Molecular characterization of the mouse Tem1/endosialin gene regulated by cell density in vitro and expressed in normal tissues in vivo

Human tumor endothelial marker 1/endosialin (TEM1/endosialin) was recently identified as a novel tumor endothelial cell surface marker potentially involved in angiogenesis, although no specific function for this novel gene has been assigned so far. It was reported to be expressed in tumor endothelium but not in normal endothelium with the exception of perhaps the corpus luteum. Here we describe the cDNA and genomic sequences for the mouse Tem1/endosialin homolog, the identification and characterization of its promoter region, and an extensive characterization of its expression pattern in murine and human tissues and murine cell lines in vitro. The single copy gene that was mapped to chromosome 19 is intronless and encodes a 92-kDa protein that has 77.5% overall homology to the human protein. The remarkable findings are 1) this gene is ubiquitously expressed in normal human and mouse somatic tissues and during development, and 2) its expression at the mRNA level is density-dependent and up-regulated in serum-starved cells. In vitro, its expression is limited to cells of embryonic, endothelial, and preadipocyte origin, suggesting that the wide distribution of its expression in vivo is due to the presence of vascular endothelial cells in all the tissues. The ubiquitous expression in vivo is in contrast to previously reported expression limited to corpus luteum and highly angiogenic tissues such as tumors and wound tissue.     

Molecular cloning and characterization of endosialin, a C-type lectin-like cell surface receptor of tumor endothelium

Endosialin, the antigen identified with monoclonal antibody FB5, is a highly restricted 165-kDa cell surface glycoprotein expressed by tumor blood vessel endothelium in a broad range of human cancers but not detected in blood vessels or other cell types in many normal tissues. Functional analysis of endosialin has been hampered by a lack of information about its molecular structure. In this study, we describe the purification and partial amino acid sequencing of endosialin, leading to the cloning of a full-length cDNA with an open reading frame of 2274 base pairs. The endosialin cDNA encodes a type I membrane protein of 757 amino acids with a predicted molecular mass of 80.9 kDa. The sequence matches with an expressed sequence tag of unknown function in public data bases, named TEM1, which was independently linked to tumor endothelium by serial analysis of gene expression profiling. Bioinformatic evaluation classifies endosialin as a C-type lectin-like protein, composed of a signal leader peptide, five globular extracellular domains (including a C-type lectin domain, one domain with similarity to the Sushi/ccp/scr pattern, and three EGF repeats), followed by a mucin-like region, a transmembrane segment, and a short cytoplasmic tail. Carbohydrate analysis shows that the endosialin core protein carries abundantly sialylated, O-linked oligosaccharides and is sensitive to O-sialoglycoprotein endopeptidase, placing it in the group of sialomucin-like molecules. The N-terminal 360 amino acids of endosialin show homology to thrombomodulin, a receptor involved in regulating blood coagulation, and to complement receptor C1qRp. This structural kinship may indicate a function for endosialin as a tumor endothelial receptor for as yet unknown ligands, a notion now amenable to molecular investigation.     

Cardiovascular ephrinB2 function is essential for embryonic angiogenesis

EphrinB2, a transmembrane ligand of EphB receptor tyrosine kinases, is specifically expressed in arteries. In ephrinB2 mutant embryos, there is a complete arrest of angiogenesis. However, ephrinB2 expression is not restricted to vascular endothelial cells, and it has been proposed that its essential function may be exerted in adjacent mesenchymal cells. We have generated mice in which ephrinB2 is specifically deleted in the endothelium and endocardium of the developing vasculature and heart. We find that such a vascular-specific deletion of ephrinB2 results in angiogenic remodeling defects identical to those seen in the conventional ephrinB2 mutants. These data indicate that ephrinB2 is required specifically in endothelial and endocardial cells for angiogenesis, and that ephrinB2 expression in perivascular mesenchyme is not sufficient to compensate for the loss of ephrinB2 in these vascular cells.     

Rudhira is a cytoplasmic WD40 protein expressed in mouse embryonic stem cells and during embryonic erythropoiesis

We describe a novel murine gene rudhira that is expressed at high levels in embryonic stem cells and is restricted to blood islands and the erythroid lineage during embryonic development. Rudhira is expressed in angiogenic precursors but is excluded from the differentiated endothelium. Rudhira-expressing cells are seen in close proximity to endothelial cells in angiogenic blood vessels. Rudhira encodes a predicted cytoplasmic WD40 protein that is 98% identical to human BCAS3. The gene encoding BCAS3 maps to a breakpoint of hematological neoplasms on human chromosome 17q23, but its expression and function remain to be determined. We demonstrate that mouse Rudhira is a novel marker for analysis of the erythroid lineage.     

Protocadherin 12 (VE-cadherin 2) is expressed in endothelial, trophoblast, and mesangial cells

Protocadherin 12 protein (PCDH12, VE-cadherin 2) is a cell adhesion molecule that has been isolated from endothelial cells. Here, we have used Northern and Western blots, immunohistology, and flow cytometry to examine the distribution of PCDH12 in mouse tissues. It is an N-glycosylated protein of 150-kDa mass. In the endothelium, PCDH12 immunoreactivity was variable and dependent upon the vascular bed. In both the embryo and embryonic stem cell differentiation system, signals were localized in vasculogenic rather than angiogenic endothelium. In addition, the protein was strongly expressed in a subset of invasive cells of the placenta, which were identified as glycogen-rich trophoblasts. In adult mice, strong PCDH12 signals were observed in mesangial cells of kidney glomeruli whereas expression was not detected in other types of perivascular cells. As opposed to most protocadherins, PCDH12 is not expressed in early embryonic (day 12.5) and adult brains. As a first approach to obtain insight into PCDH12 function, we produced transgenic mice deficient in PCDH12, which were viable and fertile. They did not display any obvious histomorphological defects. We conclude that PCDH12 has a unique expression pattern and that its deficiency does not lead to conspicuous abnormalities. Moreover, PCDH12 is the first specific marker for both glycogen-rich trophoblasts and mesangial cells.     

Mesenchymal stem cell marker Stro-1 is a 75 kd endothelial antigen

Stro-1 is the best-known mesenchymal stem cell (MSC) marker. However, previous studies have observed its expression in the endothelium. In the present study we performed immunofluorescence (IF) staining for Stro-1, using endothelial marker vWF as reference. In the liver, both proteins were expressed in the endothelium of the central veins and hepatic sinusoids. In the lung, both were expressed in the endothelium of pulmonary blood vessels, but while vWF was absent in the alveolar capillaries, Stro-1 was present. In the kidney, both were expressed in the endothelium of renal arterial branches, but while vWF was strongly expressed in the glomeruli, Stro-1 only scantly. IF staining in cultured endothelial cells also showed extensive overlaps between Stro-1 and vWF. Western blot analysis with Stro-1 antibody detected a single protein band of 75kd in endothelial cells but not smooth muscle cells, fibroblasts, or B cells. Cancer cell lines PC3, DU145, MCF7, and K562 were also positive. Adipose-derived stem cells (ADSCs) expressed higher levels of Stro-1 when cultured beyond the first passage or when induced to differentiate into endothelial cells. These data, together with previous studies, indicate that Stro-1 is intrinsically an endothelial antigen, and its expression in MSC is probably an induced event.     

Pericytes and vascular stability

Newly formed endothelial tubes are initially unstable and subsequently become stabilized through the formation of a perivascular matrix and the association with pericytes. The presence of pericyte per se is not sufficient for vascular stability. Instead, specific qualities of the cells are required that seem to correlate with marker expression and the nature of the endothelial-pericyte contacts. Most likely, specific intercellular signals are required as mediators of endothelial and pericyte cell function and vascular stability. Several ligand-receptor systems have been implicated in endothelial-pericyte interactions. Here, we discuss the role of some of these signaling systems in the regulation of vascular stability.     

Transient Expression of Iron Transport Proteins in the Capillary of the Developing Rat Brain

Iron is essential for normal brain function and its uptake in the developing rat brain peaks during the first two weeks after birth, prior to the formation of the blood–brain barrier (BBB). The first step of iron transport from the blood to the brain is transferrin receptor (TfR)-mediated endocytosis in the capillary endothelial cells. However, the subsequent step from the endothelium into interstitium has not been fully described. The goal of this study was to examine the expression of iron transport proteins by immunodetection and RT–PCR in the developing rat brain. Tf and TfR are transiently expressed in perivascular NG2+ cells of the capillary wall during the early postnatal weeks in the rat brain. However, MTP-1 and hephaestin were expressed in endothelial cells, but not in the NG2+ perivascular cells. Immunoblot analysis for these iron transfer proteins in the developing brain generally confirmed the immunochemical findings. Furthermore, the expression of Tf and TfR in the blood vessels precedes its expression in oligodendrocytes, the main iron-storing cells in the vertebrate brain. RT–PCR analysis for the primary culture of endothelial cells and pericytes revealed that Tf and TfR were highly expressed in the pericytes while MTP-1 and hephaestin were expressed in the endothelial cells. The specific expression of Tf and TfR in brain perivascular cells and MTP-1 and hephaestin in endothelial cells suggest the possibility that trafficking of elemental iron through perivascular cells may be instrumental in the distribution of iron in the developing central nervous system.     

Stem cell antigen-1 expression in the pulmonary vascular endothelium

Although the function of the cell surface protein stem cell antigen-1 (Sca-1) has not been identified, expression of this molecule is a characteristic of bone marrow-derived hematopoietic stem cell populations. Expression of Sca-1, however, is not restricted to hematopoietic tissue. By RT-PCR and Western analysis, we found that Sca-1 is expressed in the adult mouse lung. Sca-1 immunohistochemistry revealed a linear staining pattern on the endothelial surface of large and small pulmonary arteries and veins and alveolar capillaries. Expression of Sca-1 in the pulmonary endothelium was confirmed by dual fluorescent microscopy on lung sections and by fluorescence-activated cell sorting analysis of digested lung tissue; each of these methods showed colocalization with the endothelial marker platelet/endothelial cell adhesion molecule-1. In the kidney, Sca-1 expression was also noted in large vessels, but, in contrast to the lung, was not observed in capillaries. Overall, our data indicate that Sca-1 expression helps define the surface phenotype of endothelial cells throughout the pulmonary vasculature.     

Generation and characterization of rgs5 mutant mice

Regulators of G-protein signaling (RGS) are involved in a wide variety of functions, including olfaction, vision, and cell migration. RGS5 has a perivascular expression pattern and was recently identified as a marker for brain pericytes. This suggests a role for RGS5 in vascular development and pericyte biology. We have created a mouse line which lacks the rgs5 gene and replaced it with a green fluorescent protein (GFP) reporter (rgs5(GFP/GFP)). The mice are viable and fertile and display no obvious developmental defects, and the vasculature appears to develop normally with proper pericyte coverage. Also, no differences were observed in the vasculature under pathological conditions, such as tumor growth and oxygen-induced retinopathy. The GFP expression in pericytes of rgs5(GFP) mice allows detection and sorting of these cells, thereby providing a valuable novel tool for pericyte research.     

Differential distribution and modulation of expression of alpha 1/beta 1 integrin on human endothelial cells

In this paper we report that the integrin complex alpha 1/beta 1, a laminin/collagen receptor, is expressed on cultured foreskin microvascular endothelium, but is absent on endothelial cells from large vessels such as the aorta and umbilical and femoral veins. The restricted expression of integrin alpha 1/beta 1 to microvascular endothelium was also demonstrated in vivo, by immunohistochemical staining of human tissue sections. Alpha 1 specific antibodies reacted strongly with endothelial cells of small blood vessels and capillaries in several tissues, but not with endothelium of vein and arteries of umbilical cord. Expression of integrin alpha 1 can be induced in cultured umbilical vein endothelial cells by treatment with 5 ng/ml tumor necrosis factor alpha (TNF alpha). Induction of alpha 1 subunit expression also occurred after treatment of umbilical vein endothelium with 10(-5) M retinoic acid or with 10 nM PMA; Maximal induction of alpha 1 integrin was reached after 48 h of treatment and costimulation with TNF alpha and PMA resulted in a synergistic effect. The induction of alpha 1 integrin changed the adhesive properties of umbilical vein endothelial cells, by increasing the adhesiveness to collagen, laminin, and laminin fragment P1, while adhesion to fibronectin and laminin fragment E8 remained constant. The alpha 1 integrin is thus a marker of a specific population of endothelial cells and its expression confers distinctive properties of interaction with the underlying basal membrane.     

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells,but Not in Mature Vasculature

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Transgenic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization. (J Histochem Cytochem 58:721–730, 2010)     

Epidermal growth factor‐like domain 7 is a marker of the endothelial lineage and active angiogenesis

Epidermal growth factor‐like domain 7 (Egfl7) expression in the developing embryo is largely restricted to sites of mesodermal progenitors of angioblasts/hemangioblasts and the vascular endothelium. We hypothesize that Egfl7 marks the endothelial lineage during embryonic development, and can be used to define the emergence of endothelial progenitor cells, as well as to visualize newly‐forming vasculature in the embryo and during the processes of physiologic and pathologic angiogenesis in the adult. We have generated a transgenic mouse strain that expresses enhanced green fluorescent protein (eGFP) under the control of a minimal Egfl7 regulatory sequence (Egfl7:eGFP). Expression of the transgene recapitulated that of endogenous Egfl7 at sites of vasculogenesis and angiogenesis in the allantois, yolk sac, and in the embryo proper. The transgene was not expressed in the quiescent endothelium of most adult organs. However, the uterus and ovary, which undergo vascular growth and remodeling throughout the estrus cycle, expressed high levels of Egfl7:eGFP. Importantly, expression of the Egfl7:eGFP transgene was induced in adult neovasculature. We also found that increased Egfl7 expression contributed to pathologic revascularization in the mouse retina. To our knowledge, this is the first mouse model that enables monitoring of endothelial cells at sites of active vasculogenesis and angiogenesis. This model also facilitated the isolation and characterization of EGFL7⁺ endothelial cell populations by fluorescence activated cell sorting (FACS). Together, our results demonstrate that the Egfl7:eGFP reporter mouse is a valuable tool that can be used to elucidate the mechanisms by which blood vessels form during development and under pathologic circumstances. genesis 52:657–670, 2014. © 2014 Wiley Periodicals, Inc.     

The alpha-isoform of caveolin-1 is a marker of vasculogenesis in early lung development.

Caveolin-1 is a scaffolding protein component of caveolae, membrane invaginations involved in endocytosis, signal transduction, trans- and intracellular trafficking, and protein sorting. In adult lung, caveolae and caveolin-1 are present in alveolar endothelium and Type I epithelial cells but rarely in Type II cells. We have analyzed patterns of caveolin-1 expression during mouse lung development. Two caveolin-1 mRNAs, full-length and a 5' variant that will translate mainly into caveolin-1alpha and -beta isoforms, are detected by RT-PCR at embryonic day 12 (E12) and afterwards in the developing and adult lung. Immunostaining analysis, starting at E10, shows caveolin-1alpha localized in primitive blood vessels of the forming lung, in an overlapping pattern to the endothelial marker PECAM-1, and later in all blood vessels. Caveolin-1alpha is not detected in fetal or neonatal lung epithelium but is detected in adult epithelial Type I cells. Caveolin-1 was previously shown to be expressed in alveolar Type I cells. These data suggest that expression of caveolin-1 isoforms is differentially regulated in endothelial and epithelial cells during lung development. Caveolin-1alpha is an early marker for lung vasculogenesis, primarily expressed in developing blood vessels. When the lung is fully differentiated postnatally, caveolin-1alpha is also expressed in alveolar Type I cells.     

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

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, alphaSMA, PDGFR-beta). 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.     

Synoviocyte-derived CXCL12 is displayed on endothelium and induces angiogenesis in rheumatoid arthritis

CXCL12 (stromal cell-derived factor-1) is a potent CXC chemokine that is constitutively expressed by stromal resident cells. Although it is considered a homeostatic rather than an inflammatory chemokine, CXCL12 has been immunodetected in different inflammatory diseases, but also in normal tissues, ant its potential functions and regulation in inflammation are not well known. In this study, we examined the cellular sources of CXCL12 gene expression and the mechanism and effects of its interactions with endothelial cells in rheumatoid arthritis synovium. We show that CXCL12 mRNA was not overexpressed nor induced in cultured rheumatoid synoviocytes, but it specifically accumulated in the rheumatoid hyperplastic lining layer and endothelium. CXCL12 gene expression was restricted to fibroblast-like synoviocytes, whereas endothelial cells did not express CXCL12 mRNA, but displayed the protein on heparitinase-sensitive factors. CXCL12 colocalized with the angiogenesis marker alpha(v)beta(3) integrin in rheumatoid endothelium and induced angiogenesis in s.c. Matrigel plugs in mice. The angiogenic activity of rheumatoid synovial fluid in vivo was abrogated by specific immunodepletion of CXCL12. Our results indicate that synoviocyte-derived CXCL12 accumulates and it is immobilized on heparan sulfate molecules of endothelial cells, where it can promote angiogenesis and inflammatory cell infiltration, supporting a multifaceted function for this chemokine in the pathogenesis of rheumatoid arthritis.     

Pericyte deficiencies lead to aberrant tumor vascularizaton in the brain of the NG2 null mouse

Tightly regulated crosstalk between endothelial cells and pericytes is required for formation and maintenance of functional blood vessels. When the NG2 proteoglycan is absent from pericyte surfaces, vascularization of syngeneic tumors growing in the C57Bl/6 mouse brain is aberrant in several respects, resulting in retardation of tumor progression. In the NG2 null mouse brain, pericyte investment of the tumor vascular endothelium is reduced, causing deficiencies in both pericyte and endothelial cell maturation, as well as reduced basal lamina assembly. While part of this deficit may be due to the previously-identified role of NG2 in β1 integrin-dependent periyte/endothelial cell crosstalk, the ablation of NG2 also appears responsible for loss of collagen VI anchorage, in turn leading to reduced collagen IV deposition. Poor functionality of tumor vessels in NG2 null brain is reflected by reduced vessel patency and increased vessel leakiness, resulting in large increases in tumor hypoxia. These findings demonstrate the importance of NG2-dependent pericyte/endothelial cell interaction in the development and maturation of tumor blood vessels, identifying NG2 as a potential target for anti-angiogenic cancer therapy.     

Expression of alphaVbeta3 integrin in the chick embryo aortic endothelium

The integrin chain alphaV, expressed in association with beta3, by cells of the megakaryocytic/thrombocytic and endothelial lineages is thought to play an important role in angiogenesis. alphaVbeta3 expression by endothelial cells is not constitutive but induced by various stimuli in avian and human models. Here the developmental pattern of alphaVbeta3 expression was analysed in the chick embryo by immunocytochemistry, using a specific monoclonal antibody. On day 2 of development alphaVbeta3 expression was restricted to rare cells in the blood stream, in the embryo proper and in the yolk sac blood islands. AlphaVbeta3 expression by endothelial cells became detectable on day 3 and was restricted to the dorsal aorta. Interestingly it was absent from the intra-aortic hemopoietic clusters (E3.5) which, as we have showed previously, express the alphaIIbbeta3 integrin and display progenitor potentialities. However the endothelium underlying intra-embryonic hemopoietic clusters expressed this integrin. In contrast E6-7 para-aortic hemopoietic foci contained numerous alphaVbeta3 positive cells. Both alphaVbeta3 and alphaIIbbeta3 were expressed in these latter hemopoietic sites, while alphaVbeta3 was still selectively expressed by the aortic endothelium until E6. Thereafter, at E7 the pulmonary artery also expressed it. Since alphaIIbbeta3 is expressed by avian and murine multilineage hemopoietic progenitors, we then studied the hemopoietic potentialities of alphaVbeta3/alphaIIbeta3 double positive cells from embryonic bone marrow differentiating in vitro in erythro-myeloid conditions. Thrombocytic, erythroid and myeloid progenitor potentialities were found within the cell population expressing both beta3 integrins.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.