Immunoelectron microscopic study of PECAM-1 expression on lymphatic endothelium of the human tongue

The expression of platelet-endothelial cell adhesion molecule-1 (PECAM-1) on lymphatic and blood vessels of the human tongue was examined with fluorescence and transmission electron microscopy (TEM). The study used anti-desmoplakins antiserum for light microscopic identification of the lymphatic vessels, plus a pre-embedding immunogold electron microscopic technique for TEM observations. Before making TEM observations, cryostat serial sections were immunostained with anti-desmoplakins or anti-PECAM-1 and then embedded. Semithin sections from each cryostat section were photographed under a light microscope and compared in order to identify the lymphatic vessels expressing PECAM-1. In fluorescence microscopy, PECAM-1 expression on lymphatic vessels was weaker than that on blood vessels. TEM observations showed that PECAM-1 expression on the blood vessels was observed only on the luminal surface of the endothelium. In lymphatic vessels, PECAM-1 expression was found both on the luminal and abluminal surfaces of the endothelium. The density of the PECAM-1 reaction products was lower in lymphatic vessels than in blood vessels. The density of PECAM-1 reaction products on the luminal surface of lymphatic vessels was higher than on the abluminal surfaces. The results suggest that blood vessels are more active than lymphatic vessels in leukocyte migration. The expression of PECAM-1 on the abluminal surface of lymphatic endothelium may allow leukocytes to adhere to the endothelium and interact in their migration from tissue into lymphatic vessels.     

Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.     

Ultrastructural localization of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium.

The distribution of platelet endothelial cell adhesion molecule (PECAM-1, CD31) in vascular endothelium has been disputed. Originally reported to be highly concentrated at interendothelial cell contacts, recent studies have claimed that CD31 is distributed evenly over the entire endothelial cell surface. We re-investigated this question with two different murine anti-CD31 antibodies (MEC 13.3 and M-20), using a pre-embedding immunonanogold electron microscopic procedure that allowed precise label quantitation. MEC 13.3 reacted strongly with the luminal and abluminal plasma membranes of the endothelial cells lining microvessels in normal tissues and in angiogenic vessels induced by a tumor and vascular endothelial growth factor (VEGF-A164). Lateral plasma membranes were significantly less labeled. Conversely, M-20 strongly labeled the cytoplasmic face of the lateral plasma membranes of endothelial cells, although sparing specialized junctions, and only weakly labeled the luminal and abluminal plasma membranes. Both antibodies stained a significant minority of vesicles and vacuoles comprising the vesiculovacuolar organelle (VVO). Neither antibody was reactive in CD31-null mice. We conclude that CD31 is distributed over the entire endothelial cell surface, exclusive of specialized junctions, and in VVOs, but is not equally accessible to different antibodies in all locations.     

Enhancement of angiogenic effectors through hypoxia-inducible factor in preterm primate lung in vivo

Development of lung microvasculature is critical for distal airway formation. Both processes are arrested in the lungs of preterm newborns with bronchopulmonary dysplasia (BPD), a chronic form of lung disease. We hypothesized that activation of hypoxia-inducible factors (HIFs) augments lung vascular development. Pulmonary angiogenic factors were assessed by quantitative real-time PCR, Western blot, and immunohistochemistry in preterm baboons (125 days+14 days pro re nata O2 model) treated for 14 days with intravenous FG-4095, an inhibitor of prolyl hydroxylase domain-containing proteins (PHDs) that initiates HIF degradation. HIF-1alpha, but not HIF-2alpha, mRNA and protein were increased (8- and 3-fold, respectively) in FG-4095-treated baboons relative to untreated controls. Expression of PHD-1, -2, and -3 was unchanged. Of note, mRNA and/or protein for platelet-endothelial cell adhesion molecule 1 (PECAM-1) and vascular endothelial growth factor (VEGF) were increased by FG-4095. Moreover, PECAM-1-expressing capillary endothelial cells detected by immunohistochemistry were augmented in FG-4095-treated baboons to levels comparable to those in fetal age-matched controls. Alveolar septal cell expression of Ki67, a proliferative marker, and VEGF were similar in untreated controls and FG-4095-treated neonates. These results indicate that HIF stimulation by PHD inhibition enhances lung angiogenesis in the primate model of BPD.     

Distal angiogenesis: a new concept for lung vascular morphogenesis

Although several molecular players have been described that play a role during the early phases of lung development, it is still unknown how the vasculature develops in relation to the airways. Two opposing models describe development of lung vasculature: one suggests that both vasculogenesis and angiogenesis are involved, whereas the second describes vasculogenesis as the primary mechanism. Therefore, we examined the development of the murine pulmonary vasculature through a morphological analysis from the onset of lung development [9.5 days postcoital (dpc)] until the pseudoglandular stage (13.5 dpc). We analyzed fetal lungs of Tie2-LacZ transgenic mice as well as serial sections of wild-type lungs stained with endothelial-specific antibodies (Flk-1, Fli-1, and PECAM-1). Embryos were processed with intact blood circulation to maintain the integrity of the vasculature; hence individual vessels could be identified with accuracy through serial section analysis. Furthermore, circulating primitive erythrocytes, formed exclusively by the blood islands in the yolk sac, are trapped in vessels during fixation, which proves the connection with the embryonic circulation. We report that from the first morphological sign of lung development, a clear vascular network exists that is in contact with the embryonic circulation. We propose distal angiogenesis as a new concept for early pulmonary vascular morphogenesis. In this model, capillary networks surround the terminal buds and expand by formation of new capillaries from preexisting vessels as the lung bud grows. The fact that at an early embryonic stage a complete vascular network exists may be important for the general understanding of embryonic development.     

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.     

Attenuation of retinal vascular development and neovascularization in PECAM-1-deficient mice

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1−/−) mice. A significant decrease in retinal vascular density was observed in PECAM-1−/− mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1−/− mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1−/− mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1−/− mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1−/− mice. In addition, retinal neovascularization was attenuated in PECAM-1−/− mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1−/− mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.     

Kinetic expression of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) during embryonic stem cell differentiation

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is widely used as a marker during vasculogenesis and angiogenesis from embryonic stem (ES) cells. However, the expression of PECAM-1 isoforms in ES cells has not been determined. The present study was designed to determine the role of PECAM-1 isoforms during in vitro endothelial differentiation of ES cells. It was found that undifferentiated ES cells expressed high level of PECAM-1, which primarily located at cell-cell junction, but the expression of PECAM-1 was sharply down-regulated during early ES cell differentiation. In addition, undifferentiated ES cells were found the expressed all eight known alternatively spliced PECAM-1 isoforms, among them the expression of PECAM-1 isoforms lacking exon 15 or 14&15 was predominant. Quantitative analysis revealed a significant increase in the expression of PECAM-1 isoform lacking exon 12&14&15 as vascular development of ES cells. These results indicate a constitutive expression of PECAM-1 in undifferentiated murine ES cells and suggest a developmental role of PECAM-1 isoform changes during vasculogenesis and angiogenesis.     

Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung

Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. Expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes.