Here, we report the existence of endothelial precursor (EPC) and stem cells in a distinct zone of the vascular wall that are capable to differentiate into mature endothelial cells, hematopoietic and local immune cells, such as macrophages. This zone has been identified to be localized between smooth muscle and adventitial layer of human adult vascular wall. It predominantly contains CD34-positive (+) but CD31-negative (-) cells, which also express VEGFR2 and TIE2. Only few cells in this zone of the vascular wall are positive for CD45. In a ring assay using the fragments of human internal thoracic artery (HITA), we show here that the CD34 + cells of the HITA-wall form capillary sprouts ex vivo and are apparently recruited for capillary formation by tumor cells. New vessels formed by these vascular wall resident EPCs express markers for angiogenically activated endothelial cells, such as CEACAM1, and also for mature endothelial cells, such as VE-cadherin or occludin. Vascular wall areas containing EPCs are found in large and middle sized arteries and veins of all organs studied here. These data suggest the existence of a 'vasculogenic zone' in the wall of adult human blood vessels, which may serve as a source for progenitor cells for postnatal vasculogenesis, contributing to tumor vascularization and local immune response.

The LINE-1 (L1) family of non-long terminal repeat retrotransposons is a major force shaping mammalian genomes, and its members can alter the genome in many ways. Mutational analyses have shown that coexpression of functional proteins encoded by the two L1-specific open reading frames, ORF1 and ORF2, is an essential prerequisite for the propagation of L1 elements in the genome. However, all efforts to identify ORF2-encoded proteins have failed so far. Here, applying a novel antibody we report the presence of proteins encoded by ORF2 in a subset of cellular components of human male gonads. Immunohistochemical analyses revealed coexpression of ORF1 and ORF2 in prespermatogonia of fetal testis, in germ cells of adult testis, and in distinct somatic cell types, such as Leydig, Sertoli, and vascular endothelial cells. Coexpression of both proteins in male germ cells is necessary for the observed genomic expansion of the number of L1 elements. Peptide mass fingerprinting analysis of a ϳ130-kDa polypeptide isolated from cultured human dermal microvascular endothelial cells led to the identification of ORF2-encoded peptides. An isolated ϳ45-kDa polypeptide was shown to derive from nonfunctional copies of ORF2 coding regions. The presence of both ORF1-and ORF2-encoded proteins in vascular endothelial cells and its apparent association with certain stages of differentiation and maturation of blood vessels may have functional relevance for vasculogenesis and/or angiogenesis. LINE-1 (long interspersed nuclear element) retrotransposons (L1)1 cover about 17% of the human genome (1) and play a significant role in shaping the mammalian genome, not only through their own expansion but also through the mobilization of non-L1 sequences. Although the average haploid human genome harbors ϳ516,000 L1 copies, the subgroup of active L1s is fairly small, encompassing 80 -100 elements (2, 3). So far, 82 retrotransposition-competent, full-length L1 elements were isolated and characterized (2-6). Remarkably, 84% of the retrotransposition capability of those elements was shown to be present in six highly active L1s (3). L1s affected the genome by (i) insertion of truncated L1s into new sites, (ii) intrachromosomal homologous recombination between L1s, (iii) transduction of 3Ј-flanking sequences during retrotransposition, (iv) aiding trans generation of processed pseudogenes and retrotransposition of Alu elements (7), and (v) by causing genome instability through substantial deletions (8, 9). A retrotransposition-competent, functional L1 element (RC-L1) covers ϳ6.1 kb and contains a 5Ј-untranslated region with an internal, CpG-rich promoter, a 1-kb ORF1 encoding a protein (p40) of approximately 40 kDa with RNA binding capability, followed by a 3.8-kb ORF2 coding for a protein (p150) with a predicted molecular mass of about 150 kDa with endonuclease (EN) and reverse transcription (RT) activities and a cysteine-histidine-rich domain. The 3Ј-end of L1 is terminated by a short 3Ј-untranslated region and a poly(A) tail (10) (Fig. 1A). L1 mRNAs ar...

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Here, we demonstrate the expression of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in angiogenic sprouts but not in large mother blood vessels within tumor tissue. Correspondingly, only human microvascular endothelial cells involved in in vitro tube formation exhibit CEACAM1. CEACAM1-overexpressing versus CEACAM1-silenced human microvascular endothelial cells were used in migration and tube formation assays. CEACAM1-overexpressing microvascular endothelial cells showed prolonged survival and increased tube formation when they were stimulated with vascular endothelial growth factor (VEGF), whereas CEACAM1 silencing via small interfering RNA blocks these effects. Gene array and LightCycler analyses show an up-regulation of angiogenic factors such as VEGF, VEGF receptor 2, angiopoietin-1, angiopoietin-2, tie-2, angiogenin, and interleukin-8 but a down-regulation of collagen XVIII/endostatin and Tie-1 in CEACAM1-overexpressing microvascular endothelial cells. Western blot analyses confirm these results for VEGF and endostatin at the protein level. These results suggest that constitutive expression of CEACAM1 in microvascular endothelial cells switches them to an angiogenic phenotype, whereas CEACAM1 silencing apparently abrogates the VEGF-induced morphogenetic effects during capillary formation. Thus, strategies targeting the endothelial up-regulation of CEACAM1 might be promising for antiangiogenic tumor therapy.Angiogenesis is defined as sprouting of new blood vessels from preexisting blood vessels and is a prerequisite for tumor growth and metastasis. It is regulated by angiogenic activators and inhibitors (1, 2). The structural formation and maturation of blood vessels during vasculogenesis and angiogenesis is a very complex process that runs in successive steps including proliferation and tube formation of endothelial cells, construction of the basement membrane, integration of peri-endothelial cells into the vascular wall, and embedding of blood vessels into the peri-vascular tissue (3-6). Numerous angiogenic factors including vascular endothelial growth factor (VEGF), 1 fibroblast growth factor-2, angiopoietin (Ang)-1, Ang2 (3, 7-9), and their receptors, which belong to the receptor tyrosine kinase family, are involved in several steps of this process (8). Several other factors such as angiogenin (12) and interleukin-8 (13-16) promote angiogenesis.Also, cell adhesion molecules such as integrins (17-19), VEcadherin (20, 21), I-CAM (22-24), and V-CAM (25, 26) play a crucial role for capillary morphogenesis and functional modulation of blood vessels, such as the regulation of vascular permeability (27). The human carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1), formerly known as biliary glycoprotein or CD66a in humans and C-CAM in rats, is a member of the carcinoembryonic antigen family, belongs to the immunoglobulin superfamily (28), and is a major carrier of SiLe X residues (29). It mediates cell adhesion via homophilic as well as heterophilic binding to other member...

ABSTRACT:Infants with glutaric aciduria type 1 (GA1) are subject to intracranial vascular dysfunction. Here, we demonstrate that the disease-specific metabolite 3-hydroxyglutaric acid (3-OH-GA) inhibits basal and vascular endothelial growth factor (VEGF)-induced endothelial cell migration. 3-OH-GA affects the morphology of VEGF-induced endothelial tubes in vitro because of partial disintegration of endothelial cells. These effects correlate with VE-cadherin loss. Remarkably, 3-OH-GA treatment of human dermal microvascular endothelial cells leads to disruption of actin cytoskeleton. Local application of 3-OH-GA alone or in combination with VEGF in chick chorioallantoic membrane induces abnormal vascular dilatation and hemorrhage in vivo. The study demonstrates that 3-OH-GA reduces endothelial chemotaxis and disturbs structural vascular integrity in vitro and in vivo. These data may provide insight in the mechanisms of 3-OH-GA-induced vasculopathic processes and suggest Nmethyl-D-aspartate receptor-dependent and -independent pathways in the pathogenesis of GA1.

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Our data confirmed that remodeling and growth potential of the small intestine submucosal biomaterial are time dependent. Additional experiments are required to investigate the stability of the patch material over a longer period.

Endostatin decreased vascular endothelial growth factor (VEGF)-induced formation of endothelial tubes and microvessels sprouting from aortic rings and blocked their network. After cessation of treatment, the survival time of endostatin plus VEGF-treated tubes was approximately doubled in comparison to VEGF alone. Endostatin antibody blocked VEGF-induced endothelial tube formation and disrupted existing tubes. Endostatin immunostaining was localized between endothelium and basement membrane and in inter-endothelial junctions of new, but not of quiescent, blood vessels. In tumors grown in SCID mice, endostatin immunostaining was stronger accompanying blood vessel maturation and was significantly prominent in vessels of tumor marginal zone where angiogenesis is highly active. These data indicate a new antiangiogenic action of endostatin stabilizing and maturating endothelial tubes of newly formed blood vessels. Thus, strategies accelerating vascular stabilization and maturation could be promising in tumor therapy.