INVITED REVIEW
NHLBI workshop report: endothelial cell phenotypes in heart, lung, and blood diseases

Troy Stevens1, Robert Rosenberg2, William Aird3, Thomas Quertermous4, Frances L. Johnson5, Joe G. N. Garcia6, Robert P. Hebbel7, Rubin M. Tuder8, and Susan Garfinkel9

1 Department of Pharmacology, University of South Alabama College of Medicine, Mobile, Alabama 36688; 2 Department of Cell Biology, Massachusetts Institute of Technology, Cambridge 02139; 3 Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; 4 Division of Cardiovascular Medicine, Stanford University School of Medicine, Stanford 94305; 5 Palo Alto Veterans Affairs Hospital, Palo Alto, California 94304; 6 Division of Pulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center, Baltimore, Maryland 21224; 7 Department of Medicine, University of Minnesota, Minneapolis, Minnesota 55455; 8 Department of Pathology, University of Colorado Health Sciences Center, Denver, Colorada 80262; and 9 National Heart, Lung, and Blood Institutes, Division of Lung Diseases, Bethesda, Maryland 20892


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Endothelium critically regulates systemic and pulmonary vascular function, playing a central role in hemostasis, inflammation, vasoregulation, angiogenesis, and vascular growth. Indeed, the endothelium integrates signals originating in the circulation with those in the vessel wall to coordinate vascular function. This highly metabolic role differs significantly from the historic view of endothelium, in which it was considered to be merely an inert barrier. New lines of evidence may further change our understanding of endothelium, in regard to both its origin and function. Embryological studies suggest that the endothelium arises from different sites, including angiogenesis of endothelium from macrovascular segments and vasculogenesis of endothelium from microcirculatory segments. These findings suggest an inherent phenotypic distinction between endothelial populations based on their developmental origin. Similarly, diverse environmental cues influence endothelial cell phenotype, critical to not only normal function but also the function of a diseased vessel. Consequently, an improved understanding of site-specific endothelial cell function is essential, particularly with consideration to environmental stimuli present both in the healthy vessel and in development of vasculopathic disease states. The need to examine endothelial cell phenotypes in the context of vascular function served as the basis for a recent workshop sponsored by the National Heart, Lung, and Blood Institute (NHLBI). This report is a synopsis of pertinent topics that were discussed, and future goals and research opportunities identified by the participants of the workshop are presented.


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ENDOTHELIAL CELLS were historically considered to be merely an inert barrier separating blood from interstitium. Recent developments, however, clearly indicate that endothelial cells play highly metabolic roles in the regulation of hemostasis, inflammation, vasoregulation, angiogenesis, and vascular growth. Moreover, we now recognize that these metabolic functions of endothelium differ between organs and even within the vascular tree of a single organ. Two causes of such heterogeneity have been considered. First, the environment in which a cell resides plays an important role in determining its function, or, stated another way, endothelium adapts to environmental signals. Second, data now indicate that the embryological origin of macro- and microvascular endothelium within certain organs is distinct, where macrovascular segments may arise from angiogenesis and microvascular segments may arise from vasculogenesis. Together, the environment in which an endothelial cell resides and its genetically "programmed" function establish cellular responses. Up until this time, limited consideration has been given to the collective nature of these influences on site-specific endothelial cell function, relevant to both the normal and injured vessel. A recent workshop sponsored by the National Heart, Lung, and Blood Institute addressed the interplay between environmental and genetic cues in regulation of endothelial cell function and dysfunction. The present report summarizes relevant findings presented at this workshop and is set in the context with available literature, but it is not written as a comprehensive review.

The workshop was organized to address new findings in endothelial biology that relate to heart, lung, and blood disorders. First, the embryological origin of endothelium in a developing organ was considered, in concert with environmental signals that differentially regulate endothelial function. Along these lines, specific consideration was given to how endothelium communicates with surrounding cells and how biochemical and mechanical stimuli determine endothelial cell heterogeneity. Second, discussion focused on putative circulating endothelial cells and the evidence that these blood-borne cells home to sites of vascular injury. Lung endothelium was considered separately, with emphasis given to segment-specific origin of endothelium and its implications to site-specific function, including control of the endothelial barrier, transcellular transport, and drug delivery. The drug delivery paradigm relates to the role of endothelium in disease states, including sickle cell disease, hemostasis and inflammation, and pulmonary hypertension with plexigenic arteriopathy. Finally, participants in the workshop provided specific recommendations for important areas of future investigation in vascular endothelial cell biology.


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Endothelial cell heterogeneity has been described at the level of morphology, function, antigen composition, and signaling networks. Far from being a giant monopoly of homogeneous cells, the endothelium represents a consortium of smaller enterprises of cells located within blood vessels of different tissues. While united in certain common features, each enterprise is uniquely adapted to meet the demands of its local environment. Evidence that endothelium in macro- and microvascular compartments arise from distinct embryological origins suggests that a developmentally determined phenotype may contribute to the cell's site-specific function. As an important corollary, the embryological origin and local environment of the endothelial cell may importantly contribute to both its normal function and its response to pathophysiological stimuli. Developmental origin and environmental cues likely represent interrelated contributions to the focal nature of vasculopathic disease states.

It is now well accepted that endothelial cells from different sites of the vascular tree differ in structure and function (13, 23, 44). Indeed, the expression of multiple endothelial cell markers, including thrombomodulin, P-glycoprotein, von Willebrand factor (vWf), and the family of selectin molecules, is quite diverse. Although investigations have established the existence of endothelial cell heterogeneity in vivo, they are limited by the relatively small number of endothelial cell-specific genes that have been cloned and characterized to date. One novel approach to identify vascular bed-specific phenotypes without a priori knowledge of the relevant genes uses an in vivo system in which phage home to different tissues. With the use of this screening approach, peptide sequences that confer selective phage homing to the vasculature have been recovered from 16 different organs and tumors, and endothelial cell specificity has been confirmed by immunohistochemistry (46, 61). These studies provide support for the existence of significant organ- and tissue-specific molecular heterogeneity of the endothelium, though the functional significance of such heterogeneity is still poorly understood.

The embryological origin of organ- and tissue-specific endothelium is still controversial. There are two possible mechanisms for the development of organ-specific endothelial cell phenotypes. In one case, endothelial cells would arise from the mesoderm with a predetermined phenotype and migrate to specific vascular beds in an organ, where they subsume site-specific function. In the second possibility, all endothelial cells would arise from the mesoderm and have a similar generic endothelial cell phenotype that would include universal markers such as CD31, but such cells would be devoid of vascular bed-specific endothelial cell markers. For this latter hypothesis to be correct, multipotent endothelial cells would undergo a terminal differentiation, under the influence of inductive signals from the involved organ.

The cardiac endothelial cell has been used as a model to explore its embryological beginning. Retroviral cell lineage studies of the chick embryo have demonstrated that coronary vessel and endocardial progenitors have distinct embryonic origins (38). Cells that generate coronary endothelium, vascular smooth muscle, and cardiac fibroblasts arise from the proepicardial organ located in the dorsal mesocardium. The proepicardial organ is induced to move to the heart, where it forms the epicardial mantle, by mechanisms that are poorly understood. Coronary progenitors then undergo an epithelial-mesenchymal transformation, delaminate from the growing epicardium, and migrate into the myocardium, steps necessary for the formation of coronary vessels. These migrating cells have varied potentials, giving rise to endothelium, smooth muscle, or fibroblasts of the coronary vessels. Coronary endothelial progenitors never intermingle with the endocardial endothelia, which go on to generate a daughter mesenchyme and eventually differentiate into cardiac cushion cells. An understanding of the molecules and cellular processes that generate and guide progenitors of the coronary vasculature is essential. Results from cell lineage studies may provide clues for identifying a reasonable source of coronary stem cells.

Many critical events must be completed to form a functioning coronary vasculature. While all coronary vessel progenitors comigrate and invade the subepicardial and myocardial matrix, only endothelial progenitors initiate coronary vasculogenesis. This occurs through the formation and fusion of channels to establish the capillary plexi. These capillary plexi then remodel to form distinct coronary vessels that insert into the forming aorta to complete the functional coronary vascular network. Each event in the overall process of coronary vasculogenesis is likely to be under complex genetic and cellular control. For example, vasculogenic processes can be altered by the local expression of myocyte-derived paracrine factors. However, little is known about how a specific group of capillary plexi differentiate into arteries, whereas others remain as venous or capillary endothelia. Finally, a subpopulation of endocardial endothelial cells and arterial endothelial cells becomes distinguishable from venous and capillary endothelia by their expression of unique sets of genes and secretion of unique paracrine factors. This functional heterogeneity within cardiac endothelial cells plays a critical role in inducing conduction cell differentiation within the embryonic myocardium. Therefore, clonal and functional heterogeneity of cardiac endothelial cells is vital not only for establishing the functional coronary blood circulation but also for regulating the diversification process of the cardiomyocyte lineage.

In addition to genetic determinants of endothelial cell function, environmental cues critically regulate endothelial cell heterogeneity. Such environmental cues can include the mechanical or biophysical parameters inherent to specific vascular loci, the presence or absence of inflammatory stimuli, and the unique microenvironment within different organ beds. Indeed, environmental cues are varied and can affect differential gene expression in endothelial cells. Aird and colleagues (1, 2, 28) have shown that different lengths of the human vWF and endothelial nitric oxide synthase (eNOS) promoters direct protein expression to distinct subsets of endothelial cells. Moreover, the expression of biologically active genes in the endothelium may be regulated by transcriptional networks, which themselves are controlled by vascular bed-specific mechanisms. One transcriptional network that has been studied involves the Egr-1 transcription factor, a member of the immediate-early gene family that couples short-term changes in the extracellular environment to long-term changes in gene expression. Egr-1 is regulated by distinct mechanisms in different subsets of endothelial cells, suggesting that it may mediate the differential expression of target genes within the endothelium and contribute to the establishment of phenotypic heterogeneity (37). Thus differential expression of genes within the endothelium is controlled, at least in part, by vascular bed-specific signaling pathways that begin in the local tissue environment and end at the level of the promoter. It remains important to identify the various components of these signaling pathways.


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Significant progress has been made in identifying 1) how endothelium communicates with surrounding cells and 2) how biochemical and mechanical stimuli determine endothelial cell phenotypes. These issues, though independent regulators of endothelial cell function, are interrelated environmental determinants of the blood vessel's site-specific milieu.

Cell-Cell Communication

Endothelial cells intimately contact other cells within the vessel wall; in particular, smooth muscle cells and pericytes wrap around endothelial cells and play a pivotal role in regulating angiogenesis (15). The role of cell-cell contact in establishing phenotype has been evaluated by using the 10t1/2 cell line, which has the capacity to develop into a number of different cell types, including vascular smooth muscle cells (29, 30). Experimental evidence from the use of 10t1/2 cells grown on Matrigel in the presence or absence of endothelial cells showed that only when 10t1/2 cells were cocultured with endothelial cells were they induced to form cords, and their expression of smooth muscle cell markers was dramatically upregulated. These data indicated that endothelial cells communicated with 10t1/2 cells and induced them to adopt a smooth muscle cell phenotype. This model system lends itself to characterization of endothelial cell-derived molecules that mediate the phenotypic modulation of the 10t1/2 cells. On the basis of previous work by D'Amore and colleagues (15, 30), transforming growth factor-beta (TGF-beta ) is a likely candidate. Indeed, recombinant soluble TGF-beta receptor introduced into the coculture system neutralized the contribution by 10t1/2 cells to cord formation. Thus the model suggested that endothelial cell production of TGF-beta induced the expression of vascular smooth muscle cell markers in a primitive mesodermal cell type when the cells were grown in apposition.

Brain endothelium has distinct characteristics important for its function. These cells express a group of organ-specific markers and are responsible for forming a uniquely impermeable blood-brain barrier. Like smooth muscle cells and pericytes, astrocytes wrap processes around endothelium in the blood-brain barrier, prompting questions regarding the role of this association on endothelial cell phenotype. Models of endothelial cell permeability have demonstrated that astrocytes increased endothelial cell adhesion, resulting in improved barrier function. Coculture of astrocytes with brain endothelial cells increased monolayer electrical resistance and upregulated transporter proteins such as gamma -glutamyl transferase (GTT). TGF-beta was found to be partially responsible for increased GTT expression. The important message from these studies is that differentiated organ-specific cells can modify the phenotype of endothelial cells in an ongoing fashion. Such data support the overall hypothesis that endothelial cell phenotype is under tonic modulation through secreted soluble factors, such as TGF-beta .

Indeed, in most organs, the formation of a functional vessel requires that endothelial cells coordinate their function with detection of stimuli from multiple cell types. This is especially true in the lung, where the microcirculation and organ parenchyma develop together in an intricate fashion, allowing for the potential of bidirectional communication between the developing parenchyma and the vasculature. The vascular endothelial cell growth factor (VEGF) 188 isoform was found to be specifically upregulated in mice at embryological days 17-18 in conjunction with alveolar formation and was shown to be expressed by type II alveolar epithelial cells at this stage. However, in mice engineered to express only the VEGF 120 isoform, alveolar vascularization was inhibited, the number of alveoli was greatly reduced, and the alveoli were not associated with vessels. Thus specific VEGF isoforms produced by alveolar epithelial cells are critical for correct pulmonary vascular development. These studies in the developing lung provide an extreme example of the importance of signaling between organ parenchymal cells and the endothelium.

Emerging data indicate that VEGF and the angiopoietins (Ang) work in complementary and coordinated fashion during normal vascular development and remodeling as well as in pathological angiogenesis. Both VEGF and Ang are important in early vascular development (21). In the presence of VEGF, the primary plexus forms, although additional factors are needed. Experiments with Ang-1 knockout or transgenic mice show that Ang-1 makes the plexus more stable and/or more mature, demonstrating that in embryonic development, Ang-1 critically remodels the primitive vascular plexus. Furthermore, experiments in transgenic animals with VEGF and/or Ang-1 show that the hypervascularity formed in the presence of excess VEGF is leaky and fragile, whereas the vessels made in the presence of excess Ang-1 are resistant to vascular leak induced by VEGF. Thus, in later life, Ang-1 is important for mediating and regulating the effects of VEGF (57). Interestingly, in both cases the common denominator may be the regulation of endothelial cell-smooth muscle cell communication.

The significance of cell-cell communication in vascular function is further illustrated by the ephrin B2 signaling network. The membrane-bound ligand ephrin B2 is specifically expressed in arteries and interacts with its membrane receptor ephrin B4, which is expressed by venous endothelial cells (21, 63). The current hypothesis regarding this vascular bed-specific expression is that ephrin signaling provides the map for how endothelial cells "know" to which type of vascular bed they belong. For example, direct attachment of an arterial endothelial angiogenic sprout to another arterial vessel or to a venous vessel without an intervening capillary must obviously be forbidden and requires a universal identification system. Ephrin B2 is first found on vascular smooth muscle cells at midgestation, when vascular continuity is being established. In preliminary tumor studies, ephrin B2 is induced in endothelial cells of tumor vessels. Such induction is specific for angiogenic sprouts in arterial vessels associated with the experimental tumor. Collectively, interaction of an endothelial cell with surrounding cells is critical for its differentiated vascular function, ranging from control of barrier properties to identifying appropriate connections to organize patent vessel formation.

Biochemical and Mechanical Stimuli

The role of biochemical signals in endothelial cell differentiation can be illustrated by using cells isolated from cardiac valves. Valve leaflets are coated with endothelial cells and contain poorly characterized interstitial cells. The valves arise in cardiac development from specialized areas of cardiac tissue termed endocardial cushions. A signal that is poorly understood, emanating from specific regions of the myocardial layer, induces adjacent endothelial cells to alter their gene expression, delaminate, and migrate into the cardiac jelly. This process is termed epithelial mesenchymal transformation to indicate the return to a less differentiated mesodermal phenotype. Although the molecular basis of this complex process is not well understood, a number of genes with a role in valve development have been identified through gene targeting in mice. This list includes several members of the TGF-beta signaling family.

Endothelial cells isolated from aortic and pulmonary valve leaflets give rise to cultured cells with significant heterogeneity that is reflected in their morphology. Early clonal populations have unique phenotypes with the expression of CD31 and/or smooth muscle cell alpha -actin (SMA), markers for endothelial cells or vascular smooth muscle cells, respectively. In later populations, smooth muscle myosin heavy chain was absent from the cloned cells, suggesting that the SMA-positive cells were not vascular smooth muscle cells but, rather, mesenchymal cells. To investigate whether expression of cell-specific markers could be regulated by soluble factors, multiple clones were evaluated in the presence of serum and TGF-beta . For one clone, SMA was upregulated by TGF-beta in the presence of serum. In a second clone, SMA was upregulated in CD31+ cells in response to reduced serum and removal of basic fibroblast growth factor (bFGF). A third clone coexpressed CD31 and SMA, and no change was evident with manipulation of culture conditions. These experiments collectively suggest that there are multiple phenotypes of clonal cell populations and that TGF-beta may be involved in cell transdifferentiation. However, such transdifferentiation was shown to be specific to subsets of valvular endothelium and not an inherent endothelial cell characteristic. Thus these findings support the idea that valve leaflets possess a specialized endothelial cell phenotype that can respond to external signals.

Blood flow across cardiac valves, through large arteries, and through the microcirculation differs substantially and has become a widely recognized determinant of endothelial cell phenotype. Under physiological conditions blood flow is involved in the regulation of vascular tone, vascular remodeling, and focal development of atherosclerotic lesions (27). Experimental models have linked blood flow with different modes of endothelial cell signaling, from control of signal transduction to the regulation of expression of many different genes. To begin to understand how hemodynamic forces alter vessel structure and function, a variety of gene discovery approaches have been utilized to investigate the patterns and repertoire of genes regulated by laminar vs. turbulent forces. Simple gene expression techniques, initially differential display, have been used to evaluate genes responsive to laminar flow. Two very interesting genes cloned through these studies were SMAD 6 and SMAD 7, which were predicted to encode factors capable of modulating TGF-beta signaling (59).

A hypothesis that has evolved from these studies is that physiological fluid mechanical forces may act as tonic "differentiative stimuli" partly responsible for maintenance of the endothelial phenotype in vivo (60). If true, then flow-sensitive genes specific for arterial endothelium could be identified. Recent experiments examined 50 clones from endothelial cell libraries that were generated after exposure of cells to laminar shear stress. The clones were randomly evaluated by hybridization with endothelial cell and non-endothelial cell RNAs and then probed with RNA from normal endothelial cells and endothelial cells exposed to shear stress. This process resulted in the identification of three novel clones that appeared to be endothelial cell specific, a finding confirmed by parallel in vivo studies.

The functional relevance of flow-sensitive, endothelial cell-specific genetic programs is still under evaluation. It is essential to determine the function of encoded proteins in the adult vascular wall and to avoid embryonic loss for factors that have a deleterious effect on embryonic development. To address these issues, recent experiments have been aimed at developing a system for regulated expression in the vascular wall and have employed the tetracycline-on system. Transgenic mice have been generated to selectively express the beta -galactosidase (lacZ) reporter gene, and data suggest that these transgenic lines express the reporter gene in an endothelial cell-restricted fashion. Virtually all of the endothelial cells stain positive for the reporter, especially in the murine aorta and the coronary arteries. Additional studies are now underway to express SMAD molecules in this system to investigate their role in the adult animal.


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Interestingly, not all endothelial cells are restricted to the tunica intima. Indeed, reendothelialization after balloon injury occurs rapidly, suggesting that the response to injury is more complex than simply proliferation and migration of endothelial cells adjacent to the lesion. The presence of cell islands in damaged vessels raises the question of whether there might be circulating cells that can contribute to the endothelial lineage, perhaps endothelial cell progenitors emanating from the bone marrow. In initial studies, endothelial cells were isolated from human blood using cell surface markers that distinguish a subset of circulating cells with an endothelial cell phenotype (6). When these cells were applied to a rabbit model of vascular disease, the cells were directly incorporated into vessels of ischemic limbs.

Thus it appeared that these cells were able to home to regions of tissue ischemia through a form of postnatal vasculogenesis, suggesting that circulating endothelial cells contribute to neovascularization. Indeed, there are two possible sources of endothelialization (e.g., of a graft), either the migration and co-option of existing vascular wall endothelial cells or the recruitment of circulating endothelial cells or their precursor cells (50). The putative circulating endothelial precursor (CEP) cells may reflect a phenotype of embryonic angioblasts, which are migratory endothelial cells with the capacity to circulate, proliferate, and differentiate into mature endothelial cells but have neither acquired characteristic markers of mature endothelium nor formed lumina. What remains to be determined is the proportion of new vessel growth that results from circulating stem cells vs. parent vessel endothelial cells. Moreover, the respective role of progenitor cells vs. differentiated endothelial cells in vascular repair remains to be completely determined.

It has become apparent that bone marrow-derived CEPs are likely involved in promoting postnatal physiological and pathophysiological processes, such as wound healing and tumor growth (6, 32). The use of a bone marrow transplantation model and the Tie2-b-lacZ transgenic mouse has supported this view. Experiments in which marrow was harvested from these transgenic mice and then transplanted into wild-type mice showed that the expression of the lacZ reporter gene was associated with developing vessels, and the cells were deduced to arise from the transplanted marrow (5). Such experiments were also conducted with the use of other injury models, including eye corneal injury, myocardial infarction, hindlimb ischemia, and the physiological angiogenesis associated with ovarian cycling. In the cornea model, staining for the reporter gene was seen in endothelial cells in vessels, and it was observed that VEGF accelerated the process of vascular repair. However, because VEGF is a poor mitogen in vitro, it was not clear how it could accelerate this process. Additional studies in mice demonstrated that administration of VEGF appeared to increase the number of endothelial cell progenitors (7). Additional studies in humans also provided support for the role of endothelial cell progenitors in angiogenesis. An increase in both circulating VEGF and endothelial cell progenitors was observed in humans undergoing VEGF gene therapy for peripheral vascular disease (31). Furthermore, the number of endothelial cell progenitors was increased in young vs. old patients undergoing VEGF gene therapy, where the increase in endothelial cell progenitors correlated with a better response in the younger patients. Younger patients appeared to have more endothelial cell progenitors at baseline and also appeared to show greater mobilization from the marrow. Experiments designed to evaluate the ability of endothelial cell progenitors to mediate or accelerate therapeutic angiogenesis have also been performed. CD34+ cells were harvested from human volunteers and administered to nude or athymic mice with ischemic hindlimbs. Laser-Doppler imaging revealed accelerated vascularization of ischemic limbs when human endothelial cell progenitors were delivered; indeed, by day 28, ischemic limbs looked normal. Subsequent studies suggested that these findings were also applicable to the myocardium. Ligation of a coronary artery in nude rats and subsequent injection with human endothelial cell progenitors all showed improved myocardial function. These data collectively support the contention that endothelial cell progenitors exist within the circulation and can target various vascular sites, thereby contributing to vascular repair. However, the physiological significance or contribution of CEPs in regulation of postnatal angiogenesis is not yet fully understood.

Although the presence of endothelial cells within the circulation is unequivocal, questions still remain regarding the ability to distinguish CEPs and circulating differentiated endothelial cells. Two major obstacles in isolation and characterization of CEPs are 1) the lack of specific markers to differentiate between mature vessel wall endothelial cells and circulating endothelial precursor cells and 2) the lack of specific functional markers to discriminate between hematopoietic progenitors and circulating endothelial precursor cells. Peichev and coworkers (45) have found that AC133, a novel hematopoietic stem cell marker, is expressed on subsets of CD34+ cells derived from various hematopoietic sources, but not on mature endothelial cells. Some CD34+ cells are AC133+, express high levels of VEGF2 and Tie-2, and are dependent on VEGF. Such cells, isolated from various sources, proliferate in an anchorage-independent manner and can be induced to differentiate into mature adherent, AC133-, endothelial monolayers. Injection of AC133+/VEGFR2+ cells into sublethally irradiated immunocompromised mice results in engraftment of human-derived CEPs onto the bone marrow and spleen. Thus these data support the concept that bone marrow-derived endothelial cells with the capacity to circulate and with vascular grafting potential may contribute to postnatal angiogenesis.

To assess the origins of circulating endothelial cells and of the endothelial outgrowth obtained from cultures of blood, Lin et al. (36) examined patients who had previously undergone sex-mismatched marrow transplantation. Using a fluorescence in situ hybridization assay to determine whether cells had XY or XX genotype, they found that almost all circulating endothelial cells were of recipient genotype, had limited growth capability, and, therefore, were presumably of vessel wall origin. In contrast, most of the late endothelial outgrowth from blood was of donor genotype, had a greater proliferative rate, and, therefore, was derived from a transplantable, marrow-derived cell.

Isolation and culture of circulating endothelial cells produces an enormous (10- to 18-fold) expansion of blood-derived cells, and therefore, it is likely that these cells could be useful for various biomedical/engineering purposes. The usefulness of blood-derived endothelial cells for gene therapy is being tested by using hemophilia A as a model. Lin et al. (36) have recently demonstrated that blood-derived endothelial cells can be stably transfected to express human FVIII, chemically selected, cryopreserved, and subsequently reexpanded to produce detectable and therapeutic FVIII levels for 97 days after injection into immunodeficient mice. It therefore appears that blood-derived endothelial cells may have a number of uses in diagnostics and in therapeutics.

It has been hypothesized that hematopoietic and endothelial cells have a common precursor, the hemangioblast. However, studies aimed at identification and characterization of the hemangioblast with the use of the normal mouse embryo are technically challenging. An alternate approach to studying early embryonic commitment is to utilize an in vitro differentiation model of embryonic stem (ES) cells. With the use of murine ES cells, it has been demonstrated that blast colony-forming cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast (10). These cells express the receptor tyrosine kinase flk-1 and form blast colonies in the presence of VEGF, an flk-1 ligand. Mice deficient in flk-1 are reported to be embryonic lethal, due to defects in blood island formation. However, early embryos do contain hematopoietic progenitors. The in vitro flk-1-/- ES cells are capable of generating both hematopoietic and endothelial cells. These data suggest that flk-1-expressing hemangioblasts are involved in hemangioblast migration to form blood islands in the yolk sac in embryonic development. Studies also demonstrate that flk-1-expressing cells are heterogeneous and could underlie the heterogeneity of endothelial cells. Monoclonal antibodies that uniquely recognize flk-1+ cells are being used to evaluate the expression pattern in embryonic and adult tissues. Moreover, in vitro generation of flk-1+ cells for blastocyst injection indicates these cells contribute to the hematopoietic compartment of the developing embryo; their contribution to circulating endothelial cells is currently being evaluated.


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The large lung microcirculation is required to accommodate 100% of the cardiac output and still maintain the low pulmonary vascular pressure and resistance necessary to optimize gas exchange. Historically, little consideration had been given to the unique functions of endothelium along the pulmonary vascular tree, and, indeed, pulmonary artery endothelial cells were generally utilized to establish in vitro models germane to the microcirculation. The general precept was that biochemical, mechanical, and anatomic organization of the macro- and microcirculations played dominant roles in establishing any known phenotype heterogeneity. Although not fully resolved (48), it has become apparent that endothelium of macro- and microvascular lung segments may arise from embryologically distinct origins, where macrovascular endothelium is derived from the pulmonary truncus through angiogenesis and the microvascular endothelium is derived from blood islands through vasculogenesis (16, 17). At midgestation, these distinct origins of vessels grow together to form a congruent circuit. Cohesion of angiogenic and vasculogenic sprouts occurs at vessels that are very roughly 100-200 µm in diameter in the fully differentiated organ (estimated from the adult rat). Interestingly, this size of vessel is near the terminal lymphatics and also is the site implicated in many forms of pulmonary vascular remodeling, including plexigenic arteriopathy. Thus, in addition to environmental stimuli that regulate differentiation, endothelial phenotype may partly be regulated though the cell's genetic preprogram. Efforts now have been made to understand signaling events that regulate site-specific endothelial cell function in the developing and adult lung circulation.

As indicated previously, cell-cell interaction is a critical determinant of endothelial differentiation. In the microcirculation of the lung, endothelium arises from blood islands to form a mature alveolar-capillary barrier. However, signals responsible for terminal differentiation are poorly understood. Akeson et al. (3) developed and characterized immortalized cell lines obtained from the lung mesenchyme, so-called MFLM cells. MFLM cells stain positive for CD34, VEGF-R1/-R2, Tie-1, and Tie-2 and are capable of vasculogenesis and angiogenesis. Moreover, introduction of these cells into mouse blastocysts results in formation of cardiopulmonary vascular structures, indicating that MFLM cells function as endothelial precursors. When cocultured with airway type II-like epithelial cells, MLE-12, the two cell types align to form parallel tubular structures that are analogous to the alveolar-capillary membrane in vivo. This model of developmental interaction between branching airways and vascular plexus of lung mesenchyme was combined with microarray differential expression to establish the molecular anatomy of a developing lung microcirculation. The resulting molecular fingerprint identified differential expression of SLIT and ROBO, a protein first messenger and its receptor, respectively, in the developing mesenchyme. SLIT and ROBO signaling pathways promote axonal guidance in neural development, indicating that they may also guide association of the developing vasculature and airway. Future studies are required to establish the functional molecular anatomy of a developing microcirculation, particularly in comparison with a developing macrocirculation, to better understand the signals that guide terminal differentiation of the vascular circuit of the lung.

If genetically programmed determinants contribute to endothelial cell differentiation, then cells isolated from macro- and microvascular segments should retain a phenotype in culture when their environments are the same. Indeed, similar to their in vivo phenotype, microvascular cells express more vascular endothelial (VE)-cadherin and less eNOS than do pulmonary artery endothelial cells, and these phenotypes can be further discriminated on the basis of lectin binding. Several functional studies also have demonstrated that pulmonary artery and microvascular endothelial cells exhibit unique functional characteristics both in vitro and in vivo. For example, microvascular endothelial cells possess enhanced barrier function compared with their macrovascular counterparts and do not similarly change shape in response to inflammatory calcium agonists (40). The disparate responses to inflammatory calcium agonists has led to integrated studies examining which specific segment of the lung circulation is responsive to calcium agonists. Structure-function analysis has revealed that the activation of a store-operated calcium entry pathway produced visible intercellular gaps in only macrovascular (arteriolar and venular, >= 100 µm) and not microvascular (<= 100 µm) lung segments (9). Similarly, activation of store-operated calcium entry disrupted the pulmonary artery endothelial cell barrier, although it did not increase microvascular permeability in vitro (33). This site-specific control of endothelial function partly results from phenotypically distinct signal transduction cascades. In macrovascular endothelial cells, inflammatory calcium agonists produce large store-operated calcium entry responses that decrease cAMP concentrations important for disruption of cell adhesion (55). However, in microvascular endothelial cells, the magnitude of calcium entry is substantially reduced, and these cells possess an intrinsic capacity to preserve global cAMP concentrations (54). Indeed, preliminary studies designed to specifically decrease cAMP in microvascular cells have revealed that these cells are capable of producing intercellular gaps. However, these studies also have demonstrated that lung microvascular endothelium possesses an intrinsic capacity for repair that is not seen in its macrovascular counterpart. Thus genetically preprogrammed determinants control the function of fully differentiated adult lung endothelial cells.

In addition to its barrier function, endothelium actively delivers circulating substances to tissue through transcellular trafficking of vesicles. Albumin and other molecules recognize specific binding epitopes on the apical membrane of microvascular endothelial cells. In particular, a 60-kDa glycoprotein (gp60) has been implicated in the albumin binding (24) and transcellular trafficking of albumin (49). Gp60 interacts with caveolin-1 enriched in the caveolar membranes of lipid raft domains. Caveolin-1 associates with the heterotrimeric GTP binding proteins Galpha q and Galpha i and with the signaling molecules Src kinase and Fyn (39, 58). The activation of gp60 induces endocytosis of albumin and other molecules in the fluid phase (39), which may be dependent on dynamin-regulated fission of caveoli and the vectorial transfer of vesicles to the basolateral membrane (43). Although considerable progress has been made in identifying certain components of the signaling pathway(s) activated upon albumin binding to endothelium, the essential signaling events remain to be described. The signals that initiate endocytosis and induce vesicular traffic remain important future challenges. It is interesting that not all endothelia possess the machinery to activate vesicle formation; for example, gp60 is not present in brain microvascular endothelial cells, perhaps reflecting macromolecular impermeability of the blood-brain barrier. Thus transport of albumin (via binding its receptor, e.g., gp60) and other macromolecules (via fluid phase endocytosis) after budding of vesicles may represent an important mode of permeation across site- or organ-specific microvascular endothelia.

Endothelial cell gap formation, transcytosis of vesicles, angiogenesis, and motility associated with vascular repair all require locomotion. Molecular motors utilize the cytoskeleton to initiate and sustain locomotion. Perhaps the best described molecular motor is the actin- and myosin-based motor that in nonexcitable cells proceeds in response to phosphorylation of a 20-kDa myosin light chain. Such phosphorylation initiates actomyosin interaction to increase centripetally directed tension. However, endothelial cells were not historically considered to be "contractile," and only more recently has significant emphasis been placed on gaining insight into the role of molecular motors in nonexcitable cell function. The cloning of multiple endothelial cell-specific myosin light chain kinase isoforms and splice variants by Garcia and colleagues (34, 62) has advanced the understanding of molecular motors in nonexcitable cells. Myosin light chain kinase 1 possesses a 922-amino acid NH2-terminal sequence not present in smooth muscle myosin light chain kinase. This isoform possesses a tyrosine residue (Tyr-464 and Tyr-471) that, when phosphorylated, is sufficient to activate kinase activity without a change in calcium-calmodulin. Indeed, stimulation of tyrosine kinase activity results in activation of a "contractile complex" that includes myosin light chain kinase, Src, and cortactin (22). Future studies are required to address the role of the myosin light chain kinase-dependent motor in site-specific endothelial cell function, including control of endothelial cell shape, vesicular trafficking, angiogenesis, and repair.

The presence of distinct endothelial cell phenotypes throughout the lung vasculature has important therapeutic implications, particularly with consideration of evidence that macro- and microvascular endothelial cells possess different surface antigens. Conjugation of effector compounds with carrier antibodies directed against endothelial surface antigens is a promising new strategy for site-specific delivery of drugs, called vascular immunotargeting (42). The lung represents an especially attractive target for immunotargeting because the pulmonary vasculature contains approximately one-third of the body's endothelium and receives all the cardiac output, and site-specific endothelial cell antigens have been identified. The feasibility of this approach has been demonstrated by conjugating active enzymes to monoclonal antibodies against a variety of endothelial cell antigens, including angiotensin-converting enzyme, thrombomodulin, intercellular adhesion molecule (ICAM)-1 and platelet endothelial cell adhesion molecule (PECAM)-1 (8, 12, 41, 42, 64). Conjugates selectively accumulate in lungs of intact animals and result in delivery of active enzymes. These findings indicate that antigens expressed in discrete endothelial cell locations, angiogenic vessels, vessels undergoing repair, or tumor vasculature could be selectively targeted to deliver drug therapy. Future studies are required to further expand the site-specific endothelial molecular anatomy to identify viable antigens that are suitable targets for drug delivery.


    ENDOTHELIUM IN DISEASE STATES
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The role of healthy endothelium in maintaining vascular function is now widely accepted, and, similarly, the role of dysfunctional endothelium in promoting disease progression is widely acknowledged. Identification of circulating endothelial cells and a better appreciation for the phenotypic distinctions of differentiated endothelium have provided new means to use these cells in identifying disease processes and as a target for therapeutic advantage.

As indicated previously, circulating endothelial cells can be isolated and studied for both enumeration and characterization of their phenotype. Circulating endothelial cells from sickle cell disease patients possess an abnormal proadhesive and procoagulant phenotype, which includes expression of molecules such as ICAM, vascular cell adhesion molecule (VCAM), E-selectin, and tissue factor (51-53). Interestingly, circulating endothelial cells also show a decrease in apoptosis, which may be conferred by increased levels of VEGF in platelet-poor plasma. Similarly, procoagulant circulating endothelial cells are found in mouse models of sickle cell anemia, suggesting a role for activated circulating endothelial cells in the hemostatic abnormalities of sickle cell disease. Consistent with this possibility, Solovey and coworkers (53a) demonstrated that sulfazalazine, a strong inhibitor of nuclear factor-kappa B (NF-kappa B) in vitro reduced the expression of activation markers in tissue and circulating endothelial cells both in the sickle cell anemia mouse model and in patients with sickle cell disease. Thus future studies to determine the role of a procoagulant circulating endothelial cell phenotype in hemostatic abnormalities present in disease states such as sickle cell anemia are important.

Endothelial cells of the tunica intima also contribute importantly to hemostasis and inflammatory cell recruitment, at least in part, through expression of vWf and P-selectin. vWf may have a structural role in the formation of Weibel-Palade bodies, which are also the site of P-selectin storage. To examine the role of endothelial cell-derived vWf in development of vascular disease, transgenic low-density lipoprotein receptor (LDLR) negative (-/-) mice were crossed with vWf+/+ or vWf-/- mice. After being fed diets rich in saturated fat and cholesterol, both LDLR-/-vWF-/- and LDLR-/-vWf+/+ mice had preserved P-selectin-dependent leukocyte rolling in mesenteric venules. However, at both 8 and 22 wk, atherosclerotic lesions were smaller in LDLR-/-vWF-/- mice compared with LDLR-/-vWf+/+ mice. This effect was most apparent in areas of disturbed flow, such as at branch points of renal and mesenteric arteries. To clarify the role of soluble P-selectin, Andre and colleagues (4) studied a mouse engineered to express a truncated form of P-selectin without its cytoplasmic tail. This form of P-selectin is shed from endothelial cells and results in three- to fourfold higher levels of soluble P-selectin than in wild-type mice. Transgenic animals expressed an increase in fibrin deposits and a decrease in hemorrhagic lesions in the skin, with more fibrin formation at the site of platelet adhesion in an ex vivo perfusion chamber. This overall procoagulant phenotype is likely due to excess soluble P-selectin, because it could be reproduced in the wild-type mice perfused with recombinant P-selectin Ig (4). These data indicate that P-selectin is not only a marker of inflammation but also a direct activator of procoagulant activity associated with vascular and thrombotic disorders.

Because cells surrounding endothelium contribute to endothelial differentiation, Edelberg and colleagues (20) studied the cross talk of tissue environment and endothelial cell phenotype by examining the myocardial contribution to coagulability of the coronary microcirculation. Expression of vWf was followed by using a phenotypically distinct subset of cardiac endothelial cells in transgenic mice that expressed vWF in tandem with the lacZ reporter. These special cardiac endothelial cells expressed platelet-derived growth factor-alpha (PDGF-alpha ) receptor. However, the cells transcribed the PDGF-alpha ligand as an AA dimer that was unable to activate the PDGF-alpha receptor. However, cardiac muscle cells synthesize a paracrine factor that triggers endothelial cell transcription of the PDGF-beta subunit. In conjunction with constituent endothelial cell expression of the A subunit, the AB dimer was produced. This dimer was able to bind to the PDGF-alpha receptor and initiate production of components critical for regulating blood coagulation, such as vWF and tissue factor, as well as products that play a critical role in angiogenesis, such as VEGF and its receptor, KDR/FLK (20). Thus growth factor circuits originating in cardiac muscles cells can lead to endothelia heterogeneity within the heart and control the site-specific biological function of this cell type. Genetic deletions of endothelial cell natural anticoagulant mechanisms coupled with genetically induced alterations of PDGF circuits in mice are able to produce myocardial infarction in the absence of atherosclerosis (11). Alterations in the environment around blood vessels may ultimately be responsible for myocardial infarction and stroke (47). Current therapeutic approaches that decrease coagulation may be optimized by altering the function of local tissue circuits, thereby minimizing bleeding complications.

Endothelium contributes to vascular remodeling in response to injury. In severe pulmonary hypertension there is a marked elevation of pulmonary artery pressure that can achieve near-systemic levels. Such hypertension is due in part to a decrease in endothelium-dependent vasodilation (19, 25, 26) and the proliferation of endothelial cells in precapillary pulmonary arteries, which play a central role in the obliteration of these vessels (14). Endothelial cells in primary pulmonary hypertension, but not in pulmonary hypertension due to congenital heart malformations, expand in a monoclonal fashion, i.e. they arise from a single cell and therefore display certain mechanistic features in common with neoplastic cell growth (35). Yeager and colleagues (65) recently determined that endothelial cells obtained from lesions found in primary pulmonary hypertension have microsatellite instability within the hMSH2 DNA repair enzyme gene. Similarly, microsatellite instability was found within the TGF-beta receptor II (RII) and Bax, a proapoptotic member of the Bcl-2 family. Such microsatellite instability is found only in endothelial cell lesions obtained from patients with primary pulmonary hypertension and not in endothelial cell lesions obtained from patients with pulmonary hypertension due to congenital heart malformations. These somatic mutations may allow a selective growth advantage for a rare pulmonary artery endothelial cell phenotype. Interestingly, germline mutations in a TGF-beta RII-related receptor, bone morphogenetic protein receptor II, have been described in families with primary pulmonary hypertension (18, 56). The presence of mutations in lung endothelial cells suggests that pulmonary vasoconstriction is not the main disease trigger. Nevertheless, the local lung vascular environment likely allows for the growth of progenitor-like endothelial cells. Infusion of ECV304 cells, a spontaneously transformed endothelial cell line, in severe combined immunodeficiency mice generates clusters of endothelial cells in branching points of the pulmonary arteries. This pattern of cell clusters is similar to the pattern that is present in human severe pulmonary hypertension. Understanding the basic mechanisms of blood vessel formation and maturation also may impact our understanding of other endothelial cell proliferative processes, such as pediatric hemangiomas.


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Recent advances in endothelial biology have begun to unravel a high level of cell heterogeneity, established by both environmental and genetic determinants. However, these findings have raised additional questions that become of paramount importance when considering the central role of endothelium in control of vascular function and in progression of vasculopathic disease states. Thus several important and unresolved issues are presented as future challenges for endothelial cell biologists.

Understand the Developmental Program for Endothelial Cell Heterogeneity

Understand the developmental mechanisms that mediate endothelial cell heterogeneity. Endothelial cells may arise with a prespecified genetic program and contribute to a specific vascular bed on the basis of this predetermined program. Alternatively, endothelial cells may arise as a uniform population that is adapted to various functions on the basis of inductive signals that come from organ-specific cell populations. It is important to understand from where and when endothelial cells arise and what signaling pathways regulate the differentiation of this cell type from the primitive mesoderm. It is also important to identify signaling molecules that regulate the terminal differentiation of the endothelial cell at a local level.

Characterize the molecular basis of endothelial cell heterogeneity at various developmental stages. The characterization of the genetic differences between endothelial cells at different developmental stages is an important area for future study. This includes examining endothelial cells at different developmental stages in different vascular beds associated with different organ systems.

Identify and characterize the hemangioblast. The existence of a common precursor for hematopoietic and endothelial cells is still controversial. Future studies need to define whether the hemangioblast exists in murine and human embryology and whether cells with hemangioblast potential exist in the blood and/or marrow of adult humans.

Describe and Characterize Endothelial Cell Heterogeneity

Map endothelial cell phenotypes in vivo. There is an increasing appreciation that endothelial cells differ from one another in structure and function. It is important to systematically catalog endothelial cell phenotypes in specific vascular beds, such as the pulmonary and coronary arteries. These phenotypic maps will provide a powerful foundation for designing vascular bed-specific therapies.

Develop functional or physiological assays for endothelial cell heterogeneity. Functional assays need to be developed and performed, giving specific consideration to the organ-specific nature of endothelial cell heterogeneity. Functional assays also need to be developed to differentiate between circulating endothelial precursors and mature endothelial cells, including assays to determine the role of circulating endothelial cell precursors in tumor growth and wound healing.

Understand the Molecular and Cellular Basis of Endothelial Cell Heterogeneity

Identification and characterization of vascular bed-specific signaling pathways. Increasing data support the role of local circuits in mediating differential gene expression and phenotypic heterogeneity within the endothelium. An important goal is to delineate the extracellular signals, the downstream signaling pathways, and the DNA-protein interactions that mediate programmed gene expression in different sites of the vascular tree. Implicit in this goal is the development of novel in vivo model systems with which to study endothelial cells in the context of their native environment.

Identify heterotypic cell-cell interactions. There is increasing evidence that communication between endothelial cells and smooth muscle cells regulates various aspects of blood vessel development, vascular contractility, remodeling, and endothelial cell function. Furthermore, interactions between endothelial cells and epithelial cells influence pattern formation and proper organ function, especially in the lung and kidney. Additional work is necessary to define the molecular signaling pathways that regulate the communication between endothelial cells and other cell types and the genetic programs that lie downstream of these signaling pathways.

Determine the distinct characteristics of circulating endothelial cells. The relationship among circulating endothelial cells, endothelial cell outgrowth from blood, and the putative circulating angioblast needs to be determined. Markers need to be developed that discriminate between these cells as well as between endothelial or hematopoietic progenitors and mature endothelial cells. Methods for single-cell endothelial culture need to be developed to help in studies aimed at distinguishing endothelial cells and their progenitors.

Understand Endothelial Cell Heterogeneity in Disease States and for Therapeutic Uses

Understand the contribution of endothelial cell phenotypes to normal vascular function and to the susceptibility to site-specific disease. The unique interaction between genetic and environmental determinants that regulate endothelial cell behavior confers a site-specific function and also a specialized response to inflammation. Thus it is important to understand how these determinants of endothelial cell function confer protection or sensitivity to vascular disease states.

Understand endothelial cell heterogeneity in development or diagnosis of clinical phenotypes and the role in therapeutics. To further understand the contribution of endothelial cell-based differences to the clinical phenotype, it is important to establish accessible libraries of endothelial cell phenotypes from various sites. This would allow examination by state-of-the-art technologies to ascertain candidate gene mutations in different disease states as well as to develop a comprehensive, site-specific molecular anatomy of endothelium. In addition, the ability to expand endothelial progenitor cells ex vivo, as opposed to outgrowth of differentiated endothelial cells, needs to be explored for its utility of blood outgrowth endothelial cells in biomedical diagnostics or gene therapy. Furthermore, additional work is necessary to understand strategies using specific endothelial cell antigens for the site- and organ-specific delivery of therapeutics to endothelial cells.


    ACKNOWLEDGEMENTS

Participants in the June 22-23, 2000, workshop were as follows: Ruhul Abid, Ann Akeson, Steven M. Albelda, Scott Baldwin, Joyce Bischoff, Kyunghee Choi, Carlyne D. Cool, Diane Darland, Jay Edelberg, Greg Evans, Don Fischman, James Greenberg, Jeffrey M. Isner, Yi Lin, Asrar B. Malik, Neil M. Matsui, Takashi Mikawa, Natalie Norwood, Renata Pasqualini, Shahin Rafii, John Rudge, Sonia Skarlatos, James N. Topper, Jo Tsai, and Denisa Wagner.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Garfinkel, NHLBI, Division of Lung Diseases, 6701 Rockledge Drive, Suite 10018, Bethesda, MD 20892 (E-mail: garfinks{at}nih.gov).


    REFERENCES
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ABSTRACT
INTRODUCTION
ORIGIN OF ENDOTHELIUM
ENDOTHELIAL PHENOTYPES IN THE...
ENDOTHELIAL PHENOTYPES IN BLOOD
ENDOTHELIAL PHENOTYPES IN LUNG
ENDOTHELIUM IN DISEASE STATES
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REFERENCES

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