Egfl7, a novel epidermal growth factor-domain gene expressed in endothelial cells.

We report the cloning and characterization of a novel epidermal growth factor (EGF) domain gene that was identified in a retroviral gene entrapment screen and is expressed in endothelial cells. This gene encodes a protein of 278 amino acids with an amino-terminal signal peptide and two centrally located EGF-like domains. We have named this novel gene in accordance with the guidelines of the Mouse Genome Informatics group Egfl7, for EGF-like domain 7. Egfl7 mRNA is expressed in highly vascularized adult tissues such as the lung, heart, uterus, and ovary. In addition, Egfl7 is expressed early during mouse embryogenesis and in undifferentiated murine embryonic stem cells. The analysis of Egfl7 expression in embryonic day 9.5 embryos by in situ hybridization indicates that Egfl7 is expressed in vascular structures in both the embryo proper and the yolk sac and at sites of mesodermal precursors of angioblasts. Within the cell, EGFL7 protein is localized to the endoplasmic reticulum and Golgi apparatus, suggesting that the protein is targeted for secretion. Indeed, recombinant EGFL7 is readily detectable in the supernatant media of transiently transfected HEK293 cells. We also report the identification of an Egfl7 paralog, Egfl8, and show that EGFL8 protein shares similar domains and molecular weight with EGFL7.     

Control of apoptosis during angiogenesis by survivin expression in endothelial cells

Mechanisms controlling endothelial cell survival during angiogenesis were investigated. Stimulation of quiescent endothelial cells with mitogens, including vascular endothelial growth factor and basic fibroblast growth factor, induced up to approximately 16-fold up-regulation of the cell cycle-regulated apoptosis inhibitor survivin. Mitogen stimulation rapidly increased survivin RNA expression in endothelial cells, which peaked after 6 to 10 hours in culture and decreased by 24 hours. Inflammatory cytokines, tumor necrosis factor alpha, and interleukin-1 did not induce survivin expression in endothelial cells. Formation of three-dimensional vascular tubes in vitro was associated with strong induction of survivin in endothelial cells, as compared with two-dimensional cultures. By immunohistochemistry, survivin was minimally expressed in endothelium of nonproliferating capillaries of normal skin, whereas it became massively up-regulated in newly formed blood vessels of granulation tissue in vivo. Recombinant expression of green fluorescent protein survivin in endothelial cells reduced caspase-3 activity and counteracted apoptosis induced by tumor necrosis factor alpha/cycloheximide. These findings identify survivin as a novel growth factor-inducible protective gene expressed by endothelial cells during angiogenesis. Therapeutic manipulation of survivin expression and function in endothelium may influence compensatory or pathological (tumor) angiogenesis.     

VE-Cadherin-Cre-recombinase transgenic mouse: a tool for lineage analysis and gene deletion in endothelial cells.

The ability to target gene deletion to a specific cellular compartment via the Cre/loxP system has been a powerful tool in the analysis of broadly expressed genes. Here, we report the generation of a transgenic mouse line in which expression of Cre-recombinase is under the regulatory control of the VE-Cadherin promoter. Temporal distribution and activity of the enzyme was evaluated with two independent Cre reporter lines. Histological analysis was performed throughout development and in the adult. Recombination of lox P sites with subsequent expression of beta-galactosidase or GFP was detected as early as E7.5 in endothelial cells of the yolk sac. Progressive staining of the embryonic vasculature was noted from E8.5-13.5; however, more contiguous reporter expression was only seen by E14.5 onward in all endothelial compartments including arteries, veins, and capillaries. In addition, we found Cre activity in lymphatic endothelial cells. Unlike other endothelial-specific Cre mice, this model showed expression in the adult quiescent vasculature. Furthermore, the constitutive nature of the VE-Cadherin promoter in the adult can be advantageous for analysis of gene deletion in pathological settings.     

Expression of the hyaluronan receptor LYVE-1 is not restricted to the lymphatic vasculature; LYVE-1 is also expressed on embryonic blood vessels

Expression of the hyaluronan receptor LYVE-1 is one of few available criteria used to discriminate lymphatic vessels from blood vessels. Until now, endothelial LYVE-1 expression was reported to be restricted to lymphatic vessels and to lymph node, liver, and spleen sinuses. Here, we provide the first evidence that LYVE-1 is expressed on blood vessels of the yolk sac during mouse embryogenesis. LYVE-1 is ubiquitously expressed in the yolk sac capillary plexus at E9.5, then becomes progressively down-regulated on arterial endothelium during vascular remodelling. LYVE-1 is also expressed on intra-embryonic arterial and venous endothelium at early embryonic stages and on endothelial cells of the lung and endocardium throughout embryogenesis. These findings have important implications for the use of LYVE-1 as a specific marker of the lymphatic vasculature during embryogenesis and neo-lymphangiogenesis. Our data are also the first demonstration, to our knowledge, that the mouse yolk sac is devoid of lymphatic vessels.     

Paladin (X99384) is expressed in the vasculature and shifts from endothelial to vascular smooth muscle cells during mouse development

Background: Angiogenesis is implicated in many pathological conditions. The role of the proteins involved remains largely unknown, and few vascular‐specific drug targets have been discovered. Previously, in a screen for angiogenesis regulators, we identified Paladin (mouse: X99384, human: KIAA1274), a protein containing predicted S/T/Y phosphatase domains. Results: We present a mouse knockout allele for Paladin with a β‐galactosidase reporter, which in combination with Paladin antibodies demonstrate that Paladin is expressed in the vasculature. During mouse embryogenesis, Paladin is primarily expressed in capillary and venous endothelial cells. In adult mice Paladin is predominantly expressed in arterial pericytes and vascular smooth muscle cells. Paladin also displays vascular‐restricted expression in human brain, astrocytomas, and glioblastomas. Conclusions: Paladin, a novel putative phosphatase, displays a dynamic expression pattern in the vasculature. During embryonic stages it is broadly expressed in endothelial cells, while in the adult it is selectively expressed in arterial smooth muscle cells. Developmental Dynamics 241:770–786, 2012. © 2012 Wiley Periodicals, Inc.     

VE-statin/Egfl7 expression in malignant glioma and its relevant molecular network

This study investigated VE-statin/Egfl7 expression and its role and regulatory mechanism in malignant glioma progression. Forty-five paraffin-embedded glioma (grade I-II: n=24; grade III-IV: n=21) were examined. VE-statin/Egfl7 protein expression was detected via immunohistochemistry, and its correlation with pathological grade was evaluated. Three-dimensional cell culture was then performed to investigate the influence of VE-statin/Egfl7 on the angiogenesis of umbilical vein endothelial cells. Microarray detection was used to molecularly profile VE-statin/Egfl7 and relevant signaling pathways in malignant glioma (U251 cells). Data showed that VE-statin/Egfl7 protein was mainly expressed in the cytoplasm of cancer and vascular endothelial cells and was significantly related to the degree of malignancy (t=4.399, P<0.01). Additionally, VE-statin/Egfl7 expression was low in certain gray-matter neurons but undetectable in glial cells. VE-statin/Egfl7 gene silencing significantly inhibited angiogenesis in umbilical vein endothelial cells. The following microarray results were observed in VE-statin/Egfl7-silenced U251 cells: 1) EGFR family members showed the highest differential expression, accounting for 5.54% of differentially expressed genes; 2) cell survival-related signaling pathways changed significantly; and 3) the integrin ανβ3 signaling pathway was markedly altered. Thus, malignant glioma cells and glioma vascular endothelial cells highly express VE-statin/Egfl7, which is significantly correlated with the degree of malignancy. Moreover, VE-statin/Egfl7 plays an important role in glioma angiogenesis. Microarray results indicate that VE-statin/Egfl7 may regulate EGFR and integrins to influence the FAK activity of downstream factors, triggering the PI3K/Akt and Ras/MAPK cascades and subsequent malignant glioma development.     

Avian embryonic coronary arterio‐venous patterning involves the contribution of different endothelial and endocardial cell populations

Background: Coronary vasculature irrigates the myocardium and is crucial to late embryonic and adult heart function. Despite the developmental significance and clinical relevance of these blood vessels, the embryonic origin and the cellular and molecular mechanisms that regulate coronary arterio‐venous patterning are not known in detail. In this study, we have used the avian embryo to dissect the ontogenetic origin and morphogenesis of coronary vasculature. Results: We show that sinus venosus endocardial sprouts and proepicardial angioblasts pioneer coronary vascular formation, invading the developing heart simultaneously. We also report that avian ventricular endocardium has the potential to contribute to coronary vessels, and describe the incorporation of cardiac distal outflow tract endothelial cells to the peritruncal endothelial plexus to participate in coronary vascular formation. Finally, our findings indicate that large sinus venosus‐independent sections of the forming coronary vasculature develop without connection to the systemic circulation and that coronary arterio‐venous shunts form a few hours before peritruncal arterial endothelium connects to the aortic root. Conclusions: Embryonic coronary vasculature is a developmental mosaic, formed by the integration of vascular cells from, at least, four different embryological origins, which assemble in a coordinated manner to complete coronary vascular development. Developmental Dynamics 247:686–698, 2018. © 2017 Wiley Periodicals, Inc.     

Nestin expression by newly formed human blood vessels

Nestin is a type VI intermediate filament protein originally described in neural stem cells. Here we report that immature endothelial cells generated in the course of angiogenesis express nestin. Endothelial cells of embryonic capillaries destined to vascularize growing organs also express this intermediate filament protein. Whereas nestin was sporadically expressed in mature adult human endothelial cells sporadically express nestin, this protein was consistently expressed in adult angiogenic vasculature. Nestin expression was also detected in capillaries of the corpus luteum, which replenishes itself by angiogenesis. Nestin-immunoreactive vessels were also observed in the infarcted hearts where transient ischemia triggered regeneration accompanied with neovascularization of the myocardium. Nestinpositive endothelial cells lined vessels nourishing solid growing tumors, including melanoblastomas and glioblastomas. Our data provide definitive evidence that endothelial precursors express the neural stem cell marker nestin and that this protein participates in formation of the cytoskeleton of newly formed endothelial cells. Because nestin expression was recognized under all conditions of vascular development, nestin represents a novel and reliable marker of neovascularization.     

Thrombospondin-1-deficient mice exhibit increased vascular density during retinal vascular development and are less sensitive to hyperoxia-mediated vessel obliteration.

Thrombospondin-1 (TSP1) is a natural inhibitor of angiogenesis. Its expression is most prominent during the late stages of vascular development and in the adult vasculature. Our previous studies have shown that TSP1 expression promotes a quiescent, differentiated phenotype of vascular endothelial cells. However, the physiological role TSP1 plays during vascular development and neovascularization requires further delineation. Here, we investigated the role of TSP1 during development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy. The retinal vascular density was increased in TSP1-deficient (TSP1-/-) mice compared with wild-type mice. This finding was mainly attributed to increased number of retinal endothelial cells in TSP1-/- mice. During oxygen-induced ischemic retinopathy, the developing retinal vasculature of TSP1-/- mice was less sensitive to vessel obliteration induced by hyperoxia but exhibited a similar level of neovascularization induced by normoxia compared with wild-type mice. This finding is consistent with the similar pattern of VEGF expression detected in wild-type and TSP1-/- mice. Furthermore, the increased expression of TSP1 during development of retinal vasculature was not affected by oxygen-induced ischemic retinopathy. In addition, the regression of ocular embryonic (hyaloid) vessels, as well as the newly formed retinal vessels during oxygen-induced ischemic retinopathy, was delayed in TSP1-/- mice. Therefore, TSP1 is a modulator of vascular homeostasis and its expression is essential for appropriate remodeling and maturation of retinal vasculature.     

Egfl7 Represses the Vasculogenic Potential of Human Endothelial Progenitor Cells

Egfl7 (VE-statin) is a secreted protein mostly specific to the endothelial lineage during development and in the adult and which expression is enhanced during angiogenesis. Egfl7 involvement in human postnatal vasculogenesis remains unresolved yet. Our aim was to assess Egfl7 expression in several angiogenic cell types originating from human bone marrow, peripheral blood, or cord blood. We found that only endothelial colony forming cells (ECFC), which are currently considered as the genuine endothelial precursor cells, expressed large amounts of Egfl7. In order to assess its potential roles in ECFC, Egfl7 was repressed in ECFC by RNA interference and ECFC angiogenic capacities were tested in vitro and in vivo. Cell proliferation, differentiation, and migration were significantly improved when Egfl7 was repressed in ECFC in vitro, whereas miR-126-3p levels remained unchanged. In vivo, repression of Egfl7 in ECFC significantly improved post-ischemic revascularization in a model of mouse hind-limb ischemia. In conclusion, ECFC are the sole postnatal angiogenic cells which express large amounts of Egfl7 and whose angiogenic properties are repressed by this factor. Thus, Egfl7 inhibition may be considered as a therapeutic option to improve ECFC-mediated postnatal vasculogenesis and to optimize in vitro ECFC expansion in order to develop an optimized cell therapy approach.     

Activin receptor-like kinase 1 is essential for placental vascular development in mice

Activin receptor-like kinase 1 (ALK1) is involved in the pathogenesis of hereditary hemorrhagic telangiectasia type II (HHT2) and pulmonary arterial hypertension. We have previously shown that Alk1 is predominantly expressed in the arterial endothelium and plays a pivotal role in the formation of embryonic blood vessels. At present, however, little is known about the precise expression pattern and function of ALK1 during extra-embryonic vascular development. Using previously generated lacZ reporter lines, we sought to examine the expression pattern and role of Alk1 during placental development in mice. Alk1 expression was restricted to endothelial cells of fetal vessels from the emergence of chorioallantoic fusion to the late gestational period, and no detectable Alk1 expression was observed in syncytiotrophoblasts or spongiotrophoblasts. Predominant arterial expression was observed in the umbilical and fetal placental vessels as well as in embryonic vessels. Morphological analysis of Alk1-null embryos indicates that Alk1 is essential for the development of distinct umbilical arteries and veins. The invasion of chorioallantoic mesoderm into the forming labyrinth layer was largely unaffected in the Alk1-null placenta, but chorioallantoic vessels appeared to be severely dilated and fused. Results from this study provide valuable information regarding the role of ALK1 in the development of placental vasculature as well as insights into the pathogenesis of HHT.     

Tissue‐specific venous expression of the EPH family receptor EphB1 in the skin vasculature

Background : The major arteries and veins are formed early during development. The molecular tools to identify arterial and venous endothelial cells improve our understanding of arterial–venous differentiation and branching morphogenesis. Compared with arterial differentiation, relatively little is known about what controls venous development, due to lack of definitive molecular markers for venous endothelial cells. Results: Here we report that the antibody against EphB1, an EphB class receptor, makes it possible to establish a reliable whole‐mount immunohistochemical analysis of venous identity with greater resolution than previously possible in embryonic and adult skin vasculature models. EphB1 expression is restricted to the entire venous vasculature throughout embryonic development to adulthood, whereas the previously established venous marker EphB4 is also detectable in lymphatic vasculature. This venous‐restricted expression of EphB1 is established after the vascular remodeling of the primary capillary plexus has occurred. Compared with its venous‐specific expression in the skin, however, EphB1 is not restricted to the venous vasculature in yolk sac, trunk and lung. Conclusions: These studies introduce EphB1 as a new venous‐restricted marker in a tissue‐specific and time‐dependent manner. Developmental Dynamics 242:976–988, 2013. © 2013 Wiley Periodicals, Inc.     

Angiopoietins: a link between angiogenesis and inflammation

The angiopoietin (Ang)-Tie ligand-receptor system has a key regulatory role in regulating vascular integrity and quiescence. Besides its role in angiogenesis, it is an important regulator in numerous diseases including inflammation. Ang-1-mediated Tie2 activation is required to maintain the quiescent resting state of the endothelium. Agonistic Ang-1 functions are antagonized by Ang-2, which is believed to inhibit Ang-1-Tie2 signaling. Ang-2 destabilizes the quiescent endothelium and primes it to respond to exogenous stimuli, thereby facilitating the activities of inflammatory (tumor necrosis factor and interleukin-1) and angiogenic (vascular endothelial growth factor) cytokines. Intriguingly, Ang-2 is expressed weakly by the resting endothelium but becomes strongly upregulated following endothelial activation. Moreover, endothelial cells store Ang-2 in Weibel-Palade bodies from where it can be made available quickly following stimulation, suggesting a role of Ang-2 in controlling rapid vascular adaptive processes. This suggests that Ang-2 is the dynamic regulator of the Ang-Tie2 axis, thereby functioning as a built-in switch controlling the transition of the resting quiescent endothelium towards the activated responsive endothelium.     

Transgenic induction of vascular endothelial growth factor-C is strongly angiogenic in mouse embryos but leads to persistent lymphatic hyperplasia in adult tissues

Vascular endothelial growth factor-C (VEGF-C) is the quintessential lymphangiogenic growth factor that is required for the development of the lymphatic system and is capable of stimulating lymphangiogenesis in adults by activating its receptor, VEGFR-3. Although VEGF-C is a major candidate molecule for the development of prolymphangiogenic therapy for defective lymphatic vessels in lymphedema, the stability of lymph vessels generated by exogenous VEGF-C administration is not currently known. We studied VEGF-C-stimulated lymphangiogenesis in inducible transgenic mouse models in which growth factor expression can be spatially and temporally controlled without side effects, such as inflammation. VEGF-C induction in adult mouse skin for 1 to 2 weeks caused robust lymphatic hyperplasia that persisted for at least 6 months. VEGF-C induced lymphangiogenesis in numerous tissues and organs when expressed in the vascular endothelium in either neonates or adult mice. Very few or no effects were observed in either blood vessels or collecting lymph vessels. Additionally, VEGF-C stimulated lymphangiogenesis in embryos after the onset of lymphatic vessel development. Strikingly, a strong angiogenic effect was observed after VEGF-C induction in vascular endothelium at any point before embryonic day 16.5. Our results indicate that blood vessels can undergo VEGF-C-induced angiogenesis even after down-regulation of VEGFR-3 in embryos; however, transient VEGF-C expression in adults can induce long-lasting lymphatic hyperplasia with no obvious side effects on the blood vasculature.     

Arterial macrophages and regenerating endothelial cells express P-selectin in atherosclerosis-prone apolipoprotein E-deficient mice

P-selectin expression has been reported in platelets, endothelial cells, and vascular smooth muscle cells in response to vascular injury. Here, we report P-selectin expression on macrophages in the arterial wall after carotid denudation injury and spontaneous atherosclerosis in atherosclerosis-prone apoE-deficient (apoE(-/-)) mice. Double-immunofluorescence staining revealed robust P-selectin expression in macrophage-rich regions of both denudation-induced carotid neointimal lesions and innominate atherosclerotic plaques. Co-localization of P-selectin with macrophages was verified at the single cell level using double immunostaining plus 4,6-diamidino-2-phenylindole (for nuclei) counterstaining. No platelet staining was seen in association with the macrophage staining, excluding platelet contamination. Furthermore, P-selectin mRNA expression was readily detectable in macrophage-rich plaques of atherosclerotic innominate arteries and blood monocyte-derived macrophages from apoE(-/-) mice. Strong P-selectin expression was also seen in the areas of regenerated endothelium after arterial injury. In addition, co-localization of P-selectin with vascular smooth muscle cells was readily observed in denudation-injured carotid arteries at 7 and 14 days. We conclude that macrophages in carotid injury-induced neointimal lesions and spontaneous atherosclerotic plaques of the innominate artery acquire the ability to express P-selectin, as does regenerating endothelium. These findings provide a potential new paradigm in macrophage-mediated vascular inflammation, atherosclerosis, and neointimal hyperplasia after arterial injury.     

Regulation of embryonic lung vascular development by vascular endothelial growth factor receptors,Flk‐1 and Flt‐1

The biological effects of vascular endothelial growth factor A (VEGF‐A) are mediated by fetal liver kinase‐1 (Flk‐1) and fms‐like tyrosine kinase‐1 (Flt‐1). In lung tissue, VEGF‐A is diffusely expressed throughout the embryonic stages, whereas the development of vascular endothelial cells is not uniform. Noting the signaling properties of the two receptors, we hypothesized that Flk‐1 and Flt‐1 regulate the embryonic development of lung vasculature. We herein show the spatiotemporal expression and experimental inhibition of Flk‐1 and Flt‐1 of embryonic mouse lung tissue. When Flk‐1 was predominantly expressed (embryonic day [E] 9.5–E13.5), then vascular endothelial cells actively proliferated. When Flt‐1 was enhanced (E14.5–E16.5), these cells less actively proliferated, thereby constituting organized networks. The treatment of cultured lung buds (E11.5) with antisense oligonucleotides complementary to Flk‐1 inhibited branching of capillaries and proliferation of endothelial cells. In contrast, the inhibition of Flt‐1 promoted the branching of capillaries and enhanced proliferation of endothelial cells. Of interest, inhibition of Flt‐1 promoted Flk‐1 expression. These results suggest that the two VEGF‐A receptors regulate pulmonary vascular development by modulating the VEGF‐A signaling. Anat Rec, 290:958–973, 2007. © 2007 Wiley‐Liss, Inc.     

Inducible expression of human endoglin during inflammation and wound healing in vivo

OBJECTIVE: Because angiogenesis and inflammation are intimately associated and endoglin is required for angiogenesis, we wished to determine whether it also plays a role in inflammation. METHODS: Using an immunohistochemical approach, we examined spatial and temporal changes in endoglin expression during inflammation and angiogenesis. RESULTS: We found low levels of endoglin expression in quiescent endothelium in a range of normal adult human tissues. However, constitutive levels of expression are higher in dermal capillaries surrounding hair follicles and in alveolar capillaries as well as in the high endothelial cells of lymph tissue. During inflammatory disease, endoglin expression is strongly upregulated and is consistently associated with an infiltrate of inflammatory cells. For the first time, we have determined the relative changes in endoglin expression from the normal quiescent state through the inflammatory changes and angiogenesis that occur during dermal wound healing in vivo using a timed human wound healing model. Endoglin expression increases rapidly, reaching a peak of expression co-incident with maximal T cell infiltrate and persisting at an elevated level for at least 28 days in both activated and proliferating endothelial cells. CONCLUSION: Enhanced endoglin expression is not limited to angiogenesis, it is also associated with inflammation.     

Cell Type-Specific Expression of Angiopoietin-1 and Angiopoietin-2 Suggests a Role in Glioblastoma Angiogenesis

Glioblastomas are highly vascular tumors which overexpress the angiogenesis factor vascular endothelial growth factor (VEGF). VEGF and its receptors, VEGF-R1 and VEGF-R2, have been shown to be necessary for embryonic angiogenesis as well as for tumor angiogenesis. Recently, the angiopoietin/Tie2 receptor system has been shown to exert functions in the cardiovascular system that are distinct from VEGF but are also critical for normal vascular development. To assess the potential role of Tie2 and its ligands angiopoietin-1 and angiopoietin-2 in tumor vascularization, we analyzed their expression pattern in human gliomas. Tie-2 was up-regulated in tumor endothelium compared to normal human brain tissue. We further observed cell type-specific up-regulation of the message for both angiopoietin-1 and angiopoietin-2 in gliomas. Whereas Ang-1 mRNA was expressed in tumor cells, Ang-2 mRNA was detected in endothelial cells of a subset of glioblastoma blood vessels. Small capillaries with few periendothelial support cells showed strong expression of Angiopoietin-2, whereas larger glioblastoma vessels with many periendothelial support cells showed little or no expression. Although the function of Tie2 and its ligands in tumor angiogenesis remains a subject of speculation, our findings are in agreement with a recently proposed hypothesis that in the presence of VEGF, local production of Ang-2 might promote angiogenesis.     

Maintenance and repair of the lung endothelium does not involve contributions from marrow-derived endothelial precursor cells

Lung endothelium is believed to be a quiescent tissue with the potential to exhibit rapid and effective repair after injury. Endothelial progenitor cells derived from the bone marrow have been proposed as one source of new endothelial cells that may directly contribute to pulmonary endothelial cell homeostasis and repair. Here we use bone marrow transplantation models, using purified hematopoietic stem cells (HSCs) or unfractionated whole marrow, to assess engraftment of cells in the endothelium of a variety of tissues. We find scant evidence for any contribution of bone marrow-derived cells to the pulmonary endothelium in the steady state or after recovery from hyperoxia-induced endothelial injury. Although a rare population of CD45-/CD31+/VECadherin+ bone marrow-derived cells, originating from HSCs, can be found in lung tissue after transplantation, these cells are not readily found in anatomic locations that define the pulmonary endothelium. Moreover, by tracking transplanted bone marrow cells obtained from donor transgenic mice containing endothelial lineage-selective reporters (Tie2-GFP), no contribution of bone marrow-derived cells to the adult lung, liver, pancreas, heart, and kidney endothelium can be detected, even after prolonged follow-up periods of 11 months or after recovery from hyperoxic pulmonary endothelial injury. Our findings argue against any significant engraftment of bone marrow-derived cells in the pulmonary vascular endothelium.