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2004 Genetic and Environmental Determinants of Pulmonary Endothelial Cell Function


Conference Organizer - Troy Stevens  

Program Committee: Troy Stevens, Jahar Bhattacharya, David Cornfield, Joe G N Garcia, Mark N Gillespie, Charles A Hales, Asrar B Malik, Sharon I Rounds, Mary I Townsley, Norbert F Voelkel, Wiltz W Wagner Jr, E Kenneth Weir

Executive Summary – Wiltz W Wagner Jr and  Belen Wagner  

American Heart Association and the conference organizing Committee gratefully acknowledge the educational Grants provided for the support of this Conference by:

Pulmonary Circulation Foundation, American Thoracic Society, National Institutes of Health

And the members of the Pharmaceutal Roundtable: Bristol-Meyers Squibb, AstraZeneca, GlaxoSmithKline, Merck, Novartis, Pfizer, Sanofi Synthelabo, Takeda  



Session I  Endothelium in Vascular Development

Chairs:  Sharon I Rounds and Ivan F McMurtry

8:15 AM         Molecular Anatomy of the Developing Circulations  

George D Yancopoulos  

The Eph receptor tyrosine kinases and their membrane-tethered ephrin ligands provide critical guidance cues at points of cell-to-cell contact. It has recently been reported that the ephrin-B2 ligand is a molecular marker for the arterial endothelium at the earliest stages of embryonic angiogenesis, while its receptor EphB4 reciprocally marks the venous endothelium. These findings suggested that ephrin-B2 and EphB4 are involved in establishing arterial versus venous identity and perhaps in anastamosing arterial and venous vessels at their junctions. By using a genetically engineered mouse in which the lacZ coding region substitutes and reports for the ephrin-B2 coding region, we demonstrate that ephrin-B2 expression continues to selectively mark arteries during later embryonic development as well as in the adult. However, as development proceeds, we find that ephrin-B2 expression progressively extends from the arterial endothelium to surrounding smooth muscle cells and to pericytes, suggesting that ephrin-B2 may play an important role during formation of the arterial muscle wall. Furthermore, although ephrin-B2 expression patterns vary in different vascular beds, it can extend into capillaries about midway between terminal arterioles and postcapillary venules, challenging the classical conception that capillaries have neither arterial nor venous identity. In adult settings of angiogenesis, as in tumors or in the female reproductive system, the endothelium of a subset of new vessels strongly expresses ephrin-B2, once again contrary to earlier views that such new vessels lack arterial/venous characteristics and derive from postcapillary venules. While earlier studies had focused on a role for ephrin-B2 during the earliest embryonic stages of arterial/venous determination, our current findings using ephrin-B2 as an arterial marker in the adult challenge prevailing views of the arterial/venous identity of quiescent as well as remodeling adult microvessels and also highlight a possible role for ephrin-B2 in the formation of the arterial muscle wall. Ephrin-B2 selectively marks arterial vessels and neovascularization sites in the adult, with expression in both endothelial and smooth-muscle cells. Gale, N. W., Baluk, P., Pan, L., Kwan, M., Holash, J., DeChiara, T. M., McDonald, D. M., Yancopoulos, G. D.  Dev Biol 230, 2, 2001, Pg 151-160 © 2001 Academic Press. 

9:00 AM         Discussion  

9:20 AM         Angiogenesis and Vasculogenesis in Lung Vascular Development  

                        Daphne deMello  

Despite its relevance to a variety of congenital anomalies, the earliest stages of lung vascular development are poorly understood. In other organs, two processes have been identified: vasculogenesis, the development of blood lakes in mesenchyme, and angiogenesis, the branching of new vessels from preexisting ones. In the present study we established the events in the development of the lung's vasculature in Swiss-Weber mouse fetuses between 9 and 20 days gestation, using light microscopy (LM), transmission electron microscopy (TEM), barium-gelatin angiograms, and scanning electron microscopy (SEM) of Mercox (methyl methacrylate) vascular casts. Three features were identified: (1) central sprouting or angiogenesis for up to approximately seven generations (counting the artery to each lung as first generation); (2) the formation of peripheral lakes by vasculogenesis; and (3) the development of communications between the central and peripheral systems. At 9 days gestation, intercellular spaces were apparent in the lung mesenchyme; these were formed by discharge of vesicles from mesenchymal cells, which then regrouped to provide "endothelial" cells lining the spaces. The isolated lakes coalesced to form sinusoidal spaces of irregular profile. At 12 days gestation, the earliest time at which were able to make a cast, sprouting of arteries and veins from the central pulmonary vascular trunks was apparent. Between 13 and 14 days gestation the earliest connection between the peripheral and central spaces was identified. Such connections became more numerous and dense by term. Similar images seen on examination of human fetal lung sections by LM indicated that similar processes occur in the vascular development of the human lung. deMello, D. E., Sawyer, D., Galvin, N., Reid, L. M. Am J Respir Cell Mol Biol, 16:  568-581, 1997  

9:40 AM         Discussion  

10:00 AM       Break  

10:30 AM       Human Lung Vascular Development  

                         Sheila G Haworth

Recent studies on the morphogenesis of the pulmonary arteries have focused on nonhuman species such as the chick and the mouse. Using immunohistochemical techniques, we have studied 16 lungs from human embryos and fetuses from 28 d of gestation to newborn, using serial sections stained with a panel of antibodies specific for endothelium, smooth muscle, and extracellular matrix proteins. Cell replication was also assessed. Serial reconstruction showed a continuity of circulation between the heart and the capillary plexus from at least 38 d of gestation. The intrapulmonary arteries appeared to be derived from a continuous expansion of the primary capillary plexus that is from within the mesenchyme, by vasculogenesis. The arteries formed by continuous coalescence of endothelial tubes alongside the newly formed airway. Findings were consistent with the pulmonary arterial smooth muscle cells being derived from three sites in a temporally distinct sequence: the earliest from the bronchial smooth muscle, later from the mesenchyme surrounding the arteries, and last from the endothelial cells. Despite their different origins, all smooth muscle cells followed the same sequence of expression of smooth muscle-specific cytoskeletal proteins with increasing age. The order of appearance of these maturing proteins was from the subendothelial cells outward across the vessel wall and from hilum to periphery. The airways would seem to act as a template for pulmonary artery development. This study provides a framework for studying the signaling mechanisms controlling the various aspects of lung development. Hall, S. M., Hislop, A. Aierce, C. M., Haworth, S. G. Am J Respir Cell Mol Biol, 23:  194-203, 2000  

10:50 AM     Discussion  

11:10 AM    Lung Vascular Development in eNOS Knockout Mice

                       Duncan J Stewart

Endothelium-derived NO plays a critical role in the regulation of cardiovascular function and structure, as well as acting as a downstream mediator of the angiogenic response to numerous vascular growth factors. Although endothelial NO synthase (eNOS)-deficient mice are viable, minor congenital cardiac abnormalities have been reported and homozygous offspring exhibit high neonatal mortality out of proportion to the severity of these defects. The aim of the present report was to determine whether abnormalities of the pulmonary vascular development could contribute to high neonatal loss in eNOS-deficient animals. We now report that eNOS-deficient mice display major defects in lung morphogenesis, resulting in respiratory distress and death within the first hours of life in the majority of animals. Histological and molecular examination of preterm and newborn mutant lungs demonstrated marked thickening of saccular septae, with evidence of reduced surfactant material. Lungs of eNOS-deficient mice also exhibited a striking paucity of distal arteriolar branches and extensive regions of capillary hypoperfusion, together with misalignment of pulmonary veins, which represent the characteristic features of alveolar capillary dysplasia. We conclude that eNOS plays a previously unrecognized role in lung development, which may have relevance for clinical syndromes of neonatal respiratory distress. Han, R. N., Babaei, S., Robb, M., Lee, T., Ridsdale, R., Ackerley, C., Post, M., Stewart, D. J., Defective lung vascular development and fatal respiratory distress in endothelial NO synthase-deficient mice: a model of alveolar capillary dysplasia?  Circulation Research. 2004;94:1115-1123 Published online before print March 11, 2004, doi: 10.1161/01.RES.0000125624.85852.1E
© 2004 American Heart Association, Inc.

11:30 AM     Discussion

 11:50 AM     Homeobox Genes and the Control of Pulmonary Endothelial Cell Behavior in and Disease  

                      Peter L Jones

Herein, we show that the paired-related homeobox gene, Prx1, is required for lung vascularization. Initial studies revealed that Prx1 localizes to differentiating endothelial cells (ECs) within the fetal lung mesenchyme, and later within ECs forming vascular networks. To begin to determine whether Prx1 promotes EC differentiation, fetal lung mesodermal cells were transfected with full-length Prx1 cDNA, resulting in their morphological transformation to an endothelial-like phenotype. In addition, Prx1-transformed cells acquired the ability to form vascular networks on Matrigel. Thus, Prx1 might function by promoting pulmonary EC differentiation within the fetal lung mesoderm, as well as their subsequent incorporation into vascular networks. To understand how Prx1 participates in network formation, we focused on tenascin-C (TN-C), an extracellular matrix (ECM) protein induced by Prx1. Immunocytochemistry-histochemistry showed that a TN-C-rich ECM surrounds Prx1-positive pulmonary vascular networks both in vivo and in tissue culture. Furthermore, antibody-blocking studies showed that TN-C is required for Prx1-dependent vascular network formation on Matrigel. Finally, to determine whether these results were relevant in vivo, we examined newborn Prx1-wild-type (+/+) and Prx1-null (-/-) mice and showed that Prx1 is critical for expression of TN-C and lung vascularization. These studies provide a framework to understand how Prx1 controls EC differentiation and their subsequent incorporation into functional pulmonary vascular networks. Ihida-Stansbury, K., McKean, D. M., Gebb, S. A., Martin, J. F.,Stevens, T., Nemenoff, R., Akeson, A., Vaughn, J., Jones, P. L. Circ Res, 94:  1507-1514, 2004  

12:10 PM     Discussion  

12:30 PM     Lunch  

Session II    Endothelium in Vascular Development

Chairs:  Stephen H Abman and David N Cornfield

2:00 PM       VEGF Control of Lymphatic and Pulmonary Vascular Development

                      Ann Akeson  

During development, the lung mesenchyme has a dynamic relationship with the branching airway. Embryonic lung mesenchyme is loosely packed and composed of indistinguishable cells, yet it is the source of vascular progenitors that will become endothelial cells, smooth muscle cells and fibroblasts. In the lung, vessel development in the periphery proceeds first through vasculogenesis, the migration and assembly of cells into a primitive network, and subsequently, through angiogenesis, the sprouting of vessels from this network. As a way to assess the cellular and molecular mechanisms of lung vascularization, we have isolated and cloned cell lines from mouse fetal lung mesenchyme (MFLM). Two of these MFLM cell lines, MFLM-4 and MFLM-91U, display characteristics of an endothelial lineage. RNA analysis demonstrates transcripts for the vascular endothelial growth factor receptors R1 and R2, the receptor tyrosine kinases, Tie-1 and Tie-2, as well as the Tie-2 ligands, Ang-1 and -2. The MFLM cell lines form extensive networks of capillary-like structures with lumens when cultured on a reconstituted basement membrane. In vivo, following blastocyst injection, the MFLM cells chimerize endothelium of the lung and areas of the heart vasculature. The results from these studies suggest that MFLM-4 and MFLM-91U, derived from embryonic lung mesenchyme, can function in vitro and in vivo as endothelial precursors and as models of cardiopulmonary vascularization. Akeson, A. L., Wetzel, B., Thompson, F. Y., Brooks, S. K., Paradis, H., Gendron, R. L., Greenberg, J. M. Dev Dyn, 217:  11-23, 2000  

2:20 PM         Discussion

2:40 PM         VEGF and Lung Vascular Patterning

Sarah A Gebb  

Extensive study has provided considerable insight into the mechanisms governing branching morphogenesis and developmental maturation of the pulmonary epithelium. The process by which the vascular tree arises in the mesodermal mesenchyme of the developing lung, however, is not known. Because normal epithelial branching and differentiation have been shown to be dependent on interactions with the lung mesenchyme, we hypothesized that the developing pulmonary vasculature is dependent on a reciprocal interaction with pulmonary epithelium. In this study we have defined the temporal and spatial expression of flk-1 mRNA, which encodes an endothelial cell-specific vascular endothelial growth factor (VEGF) receptor, in fetal and neonatal rat lung. Flk-1-positive cells were observed in the lung at every prenatal stage from fetal day 11 through birth, demonstrating that vascularization has been initiated as soon as the lung evaginates from the foregut epithelium. The spatial distribution of vascular precursors was distinct and consistent in early lung (fetal days 11-16): clusters of flk-1-positive cells were localized in the mesenchyme closely apposed to the developing epithelium. This spatial relationship between vascular precursors and the developing epithelium suggested that vascular development in the lung may be dependent on interactions between the two tissue types. To investigate this possibility, day-13 distal lung mesenchyme was cultured in the presence and absence of lung epithelium. Lung mesenchyme cultured in the absence of epithelium degenerated significantly, and few flk-1-positive cells were maintained. In contrast, lung mesenchyme recombined with lung epithelium contained abundant flk-1-positive cells, and their spatial distribution mimicked that observed in vivo. These studies provide the first detailed information regarding the temporal and spatial pattern of pulmonary vascularization in early development and suggest that tissue interactions play an important role in growth and maintenance of the developing lung vasculature. Gebb, S. A., Shannon, J. M. Dev Dyn, 217:159-160, © Wiley-Liss, Inc.

2:25    PM      Discussion

2:45   PM       New Insights in Vascular Formation Revealed by Lung Allograft Models  

Thiennu Vu  

The vasculature forms an intrinsic functional component of the lung and its development must be tightly regulated and coordinated with lung epithelial morphogenesis. Vascular endothelial growth factor (VEGF) and its receptors are highly expressed in a complementary pattern in the lungs during embryonic development. VEGF is expressed by epithelium and the receptors in the surrounding mesenchyme. To determine the function of VEGF in lung formation, we inhibited its activity using a soluble receptor in lung renal capsule grafts. Inhibition of VEGF results in inhibition of vascular development and significant alteration in epithelial development. Epithelial proliferation is inhibited, sacculation is impaired, and the epithelium undergoes apoptosis. Interestingly, when VEGF is attenuated, epithelial differentiation still proceeds, as shown by acquisition of both proximal and distal markers. These data show that VEGF co-ordinates epithelial and vascular development. It is required for the development of the lung vasculature and the vasculature is necessary for epithelial proliferation and morphogenesis, but not for cell differentiation. Zhao, L., Wang, K., Ferrara , N. Vu , T. H. Mech Dev, 122:  877-886, 2005  

4:40 PM       Discussion

5:00 PM       Summary 

Bruce R Pitt  

Session III   Signaling in Phenotypically Diverse Endothelia

Chairs:  David Rodman and James C Parker  

8:15 AM         The Endothelial Cell Response to Shear  

Peter F Davies  

The relationships between blood flow, mechanotransduction, and the localization of arterial lesions can now be advanced by the incorporation of new technologies and the refinement of existing methods in imaging modalities, computational modeling, fluid dynamics, and high throughput genomics and proteomics. When combined with traditional cell and molecular technologies, a powerful palette of investigative approaches is available to address shear stress biology of the endothelium at levels extending from nanoscale subcellular detailed mechanistic responses through to higher organizational levels of regional endothelial phenotypes and heterogeneous vascular beds. Davies, P. F., Spaan, J. A., Krams, R., Ann Biomed Eng, 33:  1714-1718, 2005  

9:00 AM         Discussion

9:20 AM         Flow-Adaptation in Lung Endothelia  

Shampa Chatterjee  

We have shown previously that acute ischemia leads to depolarization of pulmonary microvascular endothelial cells that is prevented with cromakalim, suggesting the presence of ATP-sensitive K+ (KATP) channels in these cells. Thus KATP channel expression and activity were evaluated in rat pulmonary microvascular endothelial cells (RPMVEC) by whole cell current measurements, dot blot (mRNA), and immunoblot (protein) for the inwardly rectifying K+ channel (KIR) 6.2 subunit and fluorescent ligand binding for the sulfonylurea receptor (SUR). Low-level expression of a KATP channel was detected in endothelial cells in routine (static) culture and led us to examine whether its expression is inducible when endothelial cells are adapted to flow. Channel expression (mRNA and both KIR6.2 and SUR proteins) and inwardly rectified membrane current by patch clamp increased significantly when RPMVEC were adapted to flow at 10 dyn/cm2 for 24 h in either a parallel plate flow chamber or an artificial capillary system. Induction of the KATP channel with flow adaptation was also observed in bovine pulmonary artery endothelial cells. Flow-adapted but not static RPMVEC showed cellular plasma membrane depolarization upon stop of flow that was inhibited by a KATP channel opener and prevented by addition of cycloheximide to the medium during the flow adaptation period. These studies indicate the induction of KATP channels by flow adaptation in pulmonary endothelium and that the expression and activity of this channel are essential for the endothelial cell membrane depolarization response with acute decrease in shear stress. Chatterjee, S., Al-Mehdi, A. B., Levitan, I. , Stevens, T., Fisher, A. B. Am J Physiol, 285:  C959-967, 2003

9:40 AM       Discussion

10:00 AM     Break

10:30 AM     Programming Endothelial Cell Gene Expression  

William C Aird  

An important limitation of standard transgenic assays is that multiple copies of the transgene are inserted randomly into the mouse genome, resulting in line-to-line variation in expression. One way to control for these variables is to target a single copy of the transgene to a defined locus of the mouse genome by homologous recombination. In the present study, we have used such an approach to target the promoters of 2 different genes, namely von Willebrand factor (VWF) and Flt-1, to the hypoxanthine phosphoribosyltransferase (Hprt) gene locus. Consistent with previous findings in standard transgenic animals, we report that the VWF promoter contains information for expression in a subset of endothelial cells in the heart, skeletal muscle, and brain. In contrast, the Flt-1 promoter directs expression in all vascular beds except for the liver. The Flt-1 transgene was active in the endothelium of tumor xenografts, whereas the VWF promoter was not. Under in vitro conditions, conditioned medium from tumor cells resulted in a significant up-regulation of Flt-1 mRNA and promoter activity, but no change in VWF levels. Taken together, these results suggest that (1) Hprt locus targeting is a valuable tool for studying vascular bed-specific gene regulation, (2) the VWF and Flt-1 promoters are regulated by distinct transcriptional mechanisms in the intact endothelium, and (3) tumor angiogenesis results in the differential activation of endothelial cell-specific promoters. Minami, T., Donovan, D. J., Tsai, J.C., Rosenberg , R. D., Aird, W. C., Blood, 100:  4019-4025, 2002  

10:50 AM     Discussion  

11:10 AM     Endothelial Heterogeneity:  The Systemic Circulation of the Lung  

                        Elizabeth Wagner  

In vivo models of airway inflammation suggest that most protein transudation occurs from bronchial microcirculation. However, due to technical limitations in the isolation and culture of bronchial endothelial cells, most studies of lung vascular permeability have focused on pulmonary endothelium. Thus conditions for culture of sheep bronchial artery endothelial cells (BAEC) and bronchial microvascular endothelial cells (BMVEC) were established. The bronchial artery and the mainstem bronchi, stripped of epithelium, were dissected, and endothelial cells were isolated by enzymatic treatment. BAEC and BMVEC demonstrated positive staining for factor VIII-related antigen, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-labeled low-density lipoprotein, and PECAM-1. Radioligand binding studies confirmed equivalent numbers of bradykinin B(2) receptors on BAEC and BMVEC. Permeability of BAEC and BMVEC was determined after treatment with bradykinin and thrombin by comparing the translocation of FITC-dextran (mol wt 9,500) across confluent monolayers (n = 10-12). Bradykinin caused a maximal increase in permeability in BAEC (165% increase) and BMVEC (144% increase) by 15 min compared with vehicle controls. Thrombin treatment altered BMVEC permeability only, reaching a maximal response at 60 min (109% increase). These results demonstrate bronchial endothelial cell heterogeneity and establish methods to determine intracellular mechanisms contributing to airway disease in relevant cell systems. Moldobaeva, A., Wagner, E. M., Am J Physiol, 283:  L520-527, 2002  

11:30 AM      Discussion  

11:50 AM      Transdifferentiation of Pulmonary Endothelium

Kurt Stenmark

Vascular endothelium, even at a specific site, appears to be a very heterogeneous tissue, with existence of specific subsets of EC capable of enhanced proliferative capabilities, enhanced secretion of growth-promoting factors, and/or even endothelial–mesenchymal transdifferentiation . These attributes of at least some EC, even if considered highly unusual or extreme, could contribute directly to the development of vascular lesions in various forms of pulmonary hypertension. Frid, M. G., Aldashev, A. A., Crossno, J. T., Jorgensen, J. M., Kale, V. A., Stenmark, K. R. Yin and Yang of an endothelial cell: from normal to the extreme in growth, secretion, and transdifferentiation capabilities. Paediatric Respiratory Vol 5, Sup1,2004, S253-S257

12:10 PM      Discussion  

12:30 PM      Lunch  

Session IV  Signaling in Phenotypically Distinct Endothelia

Chairs: Paul M Hassoun and Charles A Hales  

2:00 PM         Calcium Signaling in Capillaries

Jahar Bhattacharya  

Elevation of lung capillary pressure causes exocytosis of the leukocyte adhesion receptor P-selectin in endothelial cells (ECs), indicating that lung ECs generate a proinflammatory response to pressure-induced stress. To define underlying mechanisms, we followed the EC signaling sequence leading to P-selectin exocytosis through application of real-time, in situ fluorescence microscopy in lung capillaries. Pressure elevation increased the amplitude of cytosolic Ca(2+) oscillations that triggered increases in the amplitude of mitochondrial Ca(2+) oscillations and in reactive oxygen species (ROS) production. Responses to blockers of the Ca(2+) oscillations and of mitochondrial electron transport indicated that the ROS production was Ca(2+) dependent and of mitochondrial origin. A new proinflammatory mechanism was revealed in that pressure-induced exocytosis of P-selectin was inhibited by both antioxidants and mitochondrial inhibitors, indicating that the exocytosis was driven by mitochondrial ROS. In this signaling pathway mitochondria coupled pressure-induced Ca(2+) oscillations to the production of ROS that in turn acted as diffusible messengers to activate P-selectin exocytosis. These findings implicate mitochondrial mechanisms in the lung's proinflammatory response to pressure elevation and identify mitochondrial ROS as critical to P-selectin exocytosis in lung capillary ECs. Ichimura, H., Parthasarathi, K., Quadri, S., Issekutz, A. C., Bhattacharya, J., J Clin Invest, 111: 691-699, 2003  

2:20 PM         Discussion  

2:40 PM         Calcium Signals that Mediate Adhesion of Circulating Cells

                        Songwei Wu

In the present study, we demonstrate that lung microvascular endothelial cells express a Cav3.1 (alpha1G) T-type voltage-gated Ca2+ channel, whereas lung macrovascular endothelial cells do not express voltage-gated Ca2+ channels. Voltage-dependent activation indicates that the Cav3.1 T-type Ca2+ current is shifted to a positive potential, at which maximum current activation is -10 mV; voltage-dependent conductance and inactivation properties suggest a "window current" in the range of -60 to -30 mV. Thrombin-induced transitions in membrane potential activate the Cav3.1 channel, resulting in a physiologically relevant rise in cytosolic Ca2+. Furthermore, activation of the Cav3.1 channel induces a procoagulant endothelial phenotype; eg, channel inhibition attenuates increased retention of sickled erythrocytes in the inflamed pulmonary circulation. We conclude that activation of the Cav3.1 channels selectively induces phenotypic changes in microvascular endothelial cells that mediate vaso-occlusion by sickled erythrocytes in the inflamed lung microcirculation. Wu, S.,Haynes, J., Jr., Taylor , J. T., Obiako, B. O., Stubbs, J. R., Li, M., Stevens, T. Circ Res, 93:  346-353, 2003  

3:00 PM         Discussion  

3:20 PM         Terry Wagner Lecture Introduction  

3:25 PM         Oxygen and Oxidant Signaling in Lung Endothelia

Mark Gillespie  

In rat cultured pulmonary arterial (PA), microvascular, and venous endothelial cells (ECs), the rate of mitochondrial (mt) DNA repair is predictive of the severity of xanthine oxidase (XO)-induced mtDNA damage and the sensitivity to XO-mediated cell death. To examine the importance of mtDNA damage and repair more directly, we determined the impact of mitochondrial overexpression of the DNA repair enzyme, Ogg1, on XO-induced mtDNA damage and cell death in PAECs. PAECs were transiently transfected with an Ogg1-mitochondrial targeting sequence construct. Mitochondria-selective overexpression of the transgene product was confirmed microscopically by the observation that immunoreactive Ogg1 colocalized with a mitochondria-specific tracer and, with an oligonucleotide cleavage assay, by a selective enhancement of mitochondrial Ogg1 activity. Overexpression of Ogg1 protected against both XO-induced mtDNA damage, determined by quantitative Southern analysis, and cell death as assessed by trypan blue exclusion and MTS assays. These findings show that mtDNA damage is a direct cause of cell death in XO-treated PAECs. Dobson, A. W., Grishko, V., LeDoux, S. P., Kelley, M. R., Wilson, G. L., Gillespie, M. N.Am J Physiol, 283:  L205-L210, 2002  

3:45  PM    Discussion  

4:15 PM     Summary

                    John T Reeves

5:15 PM     Dinner

 Session IV   Signaling in Phenotypically Distinct Endothelia

 Chairs: Asrar B Malik and E Kenneth Weir  

8:15 AM         In Vivo Phage Display and Targeting:  Describing organ and Segment-Specific Endothelium 

                        Renata Pasqualini  

The vascular endothelium expresses differential receptors depending on the functional state and tissue localization of its cells. A method to characterize this receptor heterogeneity with phage display random peptide libraries has been developed. Using this technology, several peptide ligands have been isolated that home to tissue-specific endothelial cell receptors following intravenous administration. Such peptide ligands, or antibodies directed against specific vascular receptors, can be used to target therapeutic compounds or imaging agents to endothelial cells in vitro and in vivo. Recent advances in the field include identification of novel endothelial receptors expressed differentially in normal and pathological conditions and the isolation of peptides or antibody ligands to such receptors in in vitro assays, in animal models and in a human patient. These milestones, which extend the 'functional map' of the vasculature, should lead to clinical applications in diseases such as cancer and other conditions that exhibit distinct vascular characteristics. Trepel, M., Arap, W., Pasqualini, R. Curr Opin Chem Biol, 6:  399-404, 2002  

9:00 AM         Discussion  

9:20 AM         Intravascular Metastasis to Lung:  Role of Microvascular Endothelium

Abu-Bakr Al-Mehdi  

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium. Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., Muschel, R. J.Nat Med, 6:  100-102, 2000  

9:40 AM       Discussion  

10:00 AM     Break  

10:30  AM    Circulating Endothelial Cells In Vascular Repair  

                       Takayuki Asahara  

Postnatal neovascularization has previously been considered synonymous with angiogenesis, but the finding that circulating endothelial progenitor cells (EPCs) may home to sites of neovascularization and there differentiate into endothelial cells (ECs) is consistent with "vasculogenesis," through which the primordial vascular network is established in the embryo. Our findings suggest that growth and development of new blood vessels in the adult are not restricted to angiogenesis but encompass vasculogenesis as well, although the proportional contributions remain to be clarified. Likewise, augmented or retarded neovascularization probably involves enhancement or impariment of the vasculogenesis process. Isner, J. M., Kalka, C., Kawamoto, A., Asahara, T. Ann N Y Acad Sci, 953:  75-84, 2001  

10:50 AM      Discussion

11:10 AM      Vessel Differentiation and Angiogenesis  

                       James B Hoying  

We have previously demonstrated the ability to construct 3-dimensional microvascular beds in vitro via angiogenesis from isolated, intact, microvessel fragments that retain endothelial cells and perivascular cells. Our objective was to develop and characterize an experimental model of tissue vascularization, based on the implantation of this microvascular construct, which recapitulated angiogenesis, vessel differentiation, and network maturation. METHODS AND RESULTS: On implantation in a severe combined-immunodeficient mouse model, vessels in the microvascular constructs rapidly inosculated with the recipient host circulation. Ink perfusion of implants via the left ventricle of the host demonstrated that vessel inosculation begins within the first day after implantation. Evaluation of explanted constructs over the course of 28 days revealed the presence of a mature functional microvascular bed. Using a probe specific for the original microvessel source, 91.7%+/-11% and 88.6%+/-19% of the vessels by day 5 and day 28 after implantation, respectively, were derived from the original microvessel isolate. Similar results were obtained when human-derived microvessels were used to build the microvascular construct. CONCLUSIONS: With this model, we reproduce the important aspects of vascularization, angiogenesis, inosculation, and network remodeling. Furthermore, we demonstrate that the model accommodates human-derived vessel fragments, enabling the construction of human-mouse vascular chimeras. Shepherd, B. R.,  Chen, H. Y., Smith, C. M., Gruionu, G., Williams, S. K., Hoying, J. B. Arterioscler Thromb Vasc Biol, 24:  898-904, 2004  

11:30 AM       Discussion  

11:50 AM       Microarray Profiling Endothelial Cell Phenotypes  

Jen-Tsan Chi  

The vascular system is locally specialized to accommodate widely varying blood flow and pressure and the distinct needs of individual tissues. The endothelial cells (ECs) that line the lumens of blood and lymphatic vessels play an integral role in the regional specialization of vascular structure and physiology. However, our understanding of EC diversity is limited. To explore EC specialization on a global scale, we used DNA microarrays to determine the expression profile of 53 cultured ECs. We found that ECs from different blood vessels and microvascular ECs from different tissues have distinct and characteristic gene expression profiles. Pervasive differences in gene expression patterns distinguish the ECs of large vessels from microvascular ECs. We identified groups of genes characteristic of arterial and venous endothelium. Hey2, the human homologue of the zebrafish gene gridlock, was selectively expressed in arterial ECs and induced the expression of several arterial-specific genes. Several genes critical in the establishment of left/right asymmetry were expressed preferentially in venous ECs, suggesting coordination between vascular differentiation and body plan development. Tissue-specific expression patterns in different tissue microvascular ECs suggest they are distinct differentiated cell types that play roles in the local physiology of their respective organs and tissues. Chi, J. T., Chang, H. Y., Haraldsen, G., Jahnsen, F. L., Troyanskaya, O. G., Chang, D. S., Wang, Z., Rockson, S. G., van de Rijn, M., Botstein, D., Brown, P. O., Proc Natl Acad Sci, 100:  10623-10628, 2003  

12:10 PM      Discussion  

12:30 PM      Summary

                        Rubin M Tudor  

12:50  PM      Lunch  

Session V   Posters

Facilitators: Edward C Dempsey, Asar B Malik, and John T Reeves  

2:30 PM       Posters

5:30 PM       Summary

                      Mary I Townsley

6:00 PM       Dinner  

 Session VI   Endothelium in Medicine: Clinical Targets

Chair:  D  Michael Shasby

8:15 AM        Endothelium as a Clinical Target  

Joe G N Garcia  

Substances released by platelets during blood clotting are essential participants in events that link hemostasis and angiogenesis and ensure adequate wound healing and tissue injury repair. We assessed the participation of sphingosine 1-phosphate (Sph-1-P), a biologically active phosphorylated lipid growth factor released from activated platelets, in the regulation of endothelial monolayer barrier integrity, which is key to both angiogenesis and vascular homeostasis. Sph-1-P produced rapid, sustained, and dose-dependent increases in transmonolayer electrical resistance (TER) across both human and bovine pulmonary artery and lung microvascular endothelial cells. This substance also reversed barrier dysfunction elicited by the edemagenic agent thrombin. Sph-1-P-mediated barrier enhancement was dependent upon G(ialpha)-receptor coupling to specific members of the endothelial differentiation gene (Edg) family of receptors (Edg-1 and Edg-3), Rho kinase and tyrosine kinase-dependent activation, and actin filament rearrangement. Sph-1-P-enhanced TER occurred in conjunction with Rac GTPase- and p21-associated kinase-dependent endothelial cortical actin assembly with recruitment of the actin filament regulatory protein, cofilin. Platelet-released Sph-1-P, linked to Rac- and Rho-dependent cytoskeletal rearrangement, may act late in angiogenesis to stabilize newly formed vessels, which often display abnormally increased vascular permeability. Garcia, J. G., Liu, F., Verin, A. D., Birukova, A., Dechert, M. A., Gerthoffer, W. T., Bamberg , J. R., English, D., J. Clin. Invest., 108:  689-701, 2001

9:00 AM        Discussion  

9:20 AM        Genetic Determination of Endothelial Biology  

Robert P Hebbel  

Abnormal tissue factor (TF) expression has been demonstrated on blood monocytes and circulating endothelial cells in humans with sickle cell anemia. We have now studied sickle transgenic mice to help define the biology of endothelial TF expression in sickle disease. Using immunostaining of tissue sections, we find that this is confined almost exclusively to the pulmonary veins. About 15% and 13% of these exhibit TF-positive endothelium in the wild-type normal mouse and the normal human hemoglobin (HbA)-expressing control transgenic mouse, respectively. The mild sickle mouse is indistinguishable from normal (approximately 14% positive), but TF expression is significantly elevated in the moderate and severe mouse models of sickle disease (approximately 29% and approximately 41% positive, respectively). Exposure of the mild sickle mouse to hypoxia for 3 hours, followed by reoxygenation, converted its TF expression phenotype to that of the severe sickle mouse (approximately 36% positive). Pretreatment with lovastatin eliminated excessive expression of TF in the posthypoxic mild sickle mouse (approximately 16% positive) and in the more severe mouse at ambient air (approximately 21% positive). In addition to identifying tissue expression of endothelial TF in the sickle lung, these studies implicate reperfusion injury physiology in its expression and suggest a rationale for use of statins in sickle disease. Solovey, A. Kollander, R., Shet, A., Milbauer, L. C., Choong, S., Panoskaltsis-Mortari, A., Blazar, B. R., Kelm, R. J., Jr., Hebbel, R. P. Blood, 104:  840-846, 2004  

9:40 AM        Discussion

10:00 AM       Summary

                        John V Weil  

10:20 AM     Introduction: The Estelle B Grover Lecture

                       Wiltz W Wagner Jr  

10:25 AM     Severe Angioprolierative Pulmonary Hypertension: The Cancer Concept  

                      Norbert F Voelkel

We believe that the monoclonal cell expansion in primary pulmonary hypertension is the result of autonomous growth of stem cell-like endothelial cells, whereas the polyclonal proliferation in secondary pulmonary hypertension occurs as a response of endothelial cells to exogenous stimuli (like viral infection or high shear stress). In this context, we propose that different transcriptional and translational events govern the growth and expansion of monoclonal when compared with polyclonal pulmonary endothelial cells. The availability of antibodies directed against specific tyrosine kinase proteins involved in vasculogenesis/angiogenesis now permits the identification and localization of the components of such a misguided angiogenesis cell proliferation program in the pulmonary hypertensive vascular lesions. Voelkel, N. F., Cool, C., Lee, S. D., Wright, L., Geraci, M. W., Tuder, R. M. Chest, 114, 225S-230S, 1998  

10:45 AM       Discussion  

11:00 AM       Closing Summary  

                        Troy Stevens  

11:30 AM       Conference Adjourns