Although endothelium has historically been considered a homogeneous cell layer, there is increasing appreciation that it exhibits a rich diversity in structure and function (1
). Heterogeneity is apparent between endothelial cells in different organs, in endothelial cells along a single vascular segment within an organ, and in those between immediately adjacent cells. Endothelial cells along the arterial–capillary–venous axis display remarkable anatomic distinctions (4
). As an example, pulmonary artery endothelial cells reside on a thick basement membrane that separates the intima from underlying smooth muscle layers, whereas only a thin basement membrane separates capillary endothelial cells from interacting with nearby type I pneumonocytes. Pulmonary artery endothelia interact with as many as six adjacent endothelial cells, whereas capillary endothelial cells interact with just one neighbor. Although the former cells are aligned in the direction of blood flow, capillary endothelium exhibits no such flow alignment. Although these (and other) anatomic distinctions have been recognized for a number of years, the mechanisms responsible for maintaining such heterogeneity, and the functional consequences of heterogeneity have largely remained an enigma.
Infusion of fluorescently labeled lectins into the pulmonary circulation reveals a distinct border at approximately 25 μm, where pulmonary artery endothelial cells change their phenotype and become microvascular (capillary) endothelial cells (6
). Helix pomatia
lectin interacts prominently with pulmonary artery endothelial cells, whereas Griffonia simplicifolia
primarily interacts with pulmonary microvascular endothelial cells. Functional studies in the intact circulation have demonstrated that capillary endothelial cells possess a highly resistant barrier function when compared with pulmonary artery and vein endothelial cells (7
). Moreover, inflammatory agonists discretely target arterial, capillary, and venous endothelium to increase permeability, resulting in site-specific edema formation. A striking example of this heterogeneity is seen using two discrete calcium agonists. Thapsigargin is a plant alkaloid that directly activates calcium entry through store-operated calcium entry channels. Application of thapsigargin to the intact pulmonary circulation increases endothelial cell permeability (10
). However, the increase in permeability is due to interendothelial cell gap formation only in extraalveolar arterial and venous endothelial cells (12
). In contrast, 4 alpha-phorbol 12, 13-didecanoate (4αPDD) is a phorbol ester that directly activates the transient receptor potential 4 channel in the vanilloid family of ion channels (TRPV4). Application of 4αPDD to the intact pulmonary circulation increases endothelial cell permeability (13
). The increase in permeability is due, at least in part, to loss of cell–matrix adhesion in capillary endothelial cells, and is not due to gap formation in extraalveolar endothelial cells. Thus, whereas thapsigargin increases extraalveolar endothelial cell permeability, 4αPDD increases capillary endothelial cell permeability (14
The discrete nature of extraalveolar and alveolar vascular compartments can be further divulged using animal models of heart failure (15
). Chronic heart failure leads to hydrostatic edema. However, these animals fail to respond to thapsigargin with an increase in permeability, and the store-operated calcium entry channels that are responsible for increasing extraalveolar permeability are not expressed at normal levels (i.e., down-regulated). In contrast, the 4αPDD-induced increase in permeability is retained in animals with heart failure, and the TRPV4 channel that is responsible for increasing alveolar permeability is expressed at normal levels.
These findings exemplify how little we know about the cellular and molecular events that govern site-specific endothelial cell function, and highlight the importance of gaining a better understanding of how phenotypically distinct endothelial cell populations control the local physiology, and how regionally restricted environmental factors impact on the phenotype of endothelial cells within a given vascular location. As an example, thapsigargin induces the accumulation of large fluid cuffs around extraalveolar vessels, reminiscent of what has been reported in heart failure, high-altitude pulmonary edema, acute lung injury (which also displays alveolar edema), and asthma. Although we have known for some time that fluid collects in these vascular cuffs, the impact of such cuffs on lung mechanics has not been thoroughly studied. Recent findings indicate that perivascular cuffs decrease lung compliance, and negatively impact the functional coupling of bronchioles to lung parenchyma (14
). These new findings indicate that factors other than surfactant inactivation decrease lung compliance, and suggest that edema accumulation can decrease lung compliance without concomitant hypoxemia. In this scenario, extraalveolar endothelium, and not capillary endothelium, represents a suitable clinical target; improving extraalveolar barrier integrity would decrease permeability, potentially eradicate cuff formation, and improve lung compliance. Although endothelium is an increasingly important target cell type for therapeutics, we have, to this point, not accepted the importance or impact of phenotypic heterogeneity on therapeutic targeting.
Both pulmonary artery and microvascular endothelial cells can be isolated from the intact circulation, and cultured in vitro
. Even in in vitro
studies, pulmonary microvascular endothelial cells exhibit a more resistant barrier function than do pulmonary artery endothelial cells, and they uniquely respond to different inflammatory agonists with an increase in permeability, just as in the intact circulation (16
). These collective findings have led to an appreciation of the stable, distinct nature of extraalveolar and alveolar endothelial cell phenotypes, and have challenged the idea that environmental pressure is the only important determinant of phenotype specification. Indeed, interactions between cell specification and environmental pressure determine cell phenotype (1
SUMMARY OF THE PRINCIPAL ENDOTHELIAL CELL ATTRIBUTES
Although there is an increasing appreciation for the functional diversity of endothelium along the arterial–capillary–venous axis, heterogeneity exists within a cell population, and between adjacent cells as well. The basis for such interendothelial cell heterogeneity is poorly understood, and has partly been ascribed to environmental factors, such as production of autocrine and paracrine factors, and mechanical forces, such as shear stress and pressure (1
). In addition, pacemaker cells have been described within cell populations, in which a leading cell entrains adjacent population members to cyclically shift membrane potential or ion concentrations, as in calcium oscillations (18
). Such pacemaker activity has been described both in vitro
and in vivo
. The presence of pacemaker cells within a population suggests the presence of some intrinsic, imprinted intercellular heterogeneity.
Recently, Ingram and colleagues (20
) have addressed this possibility by adapting a single-cell cloning approach commonly used in the hematology field and applying it to the study of endothelium. In these studies, endothelial cells are seeded at single-cell density, and growth is observed over a 2-week time course. Most aortic and human umbilical cord endothelial cells that are seeded in the single-cell clonogenic assay do not divide (≈75%), and are considered to be differentiated endothelium. A smaller proportion (≈25%) divide, but to different degrees, most often growing to no more than 500–2,000 cells. Few single cells (<3%) grow to more than 10,000 cells. Interestingly, when these highly proliferative cells are reseeded at the single-cell density, they repopulate the entire hierarchy of growth potentials. The slower growing cells, in contrast, cannot repopulate the entire hierarchy of growth potentials. Thus, highly proliferative potential cells fulfill the criterion for “progenitor” cells, because they divide at high rates and are able to renew the entire cell population. Moreover, these progenitor cells are angio-vasculogenic, and thus fulfill an important, defining endothelial attribute.
Not all endothelial cell populations possess the same number of high-proliferating progenitors. Pulmonary artery and microvascular endothelial cell growth potentials were assessed using the single-cell approach (22
). Remarkably, nearly 50% of the individual microvascular endothelial cells could expand into large cell colonies, whereas only approximately 3% of pulmonary artery endothelial cells exhibited such profound proliferative capacity (22
). Reseeding the large colonies in the single-cell assay revealed that the high proliferative potential cells repopulate the entire hierarchy of endothelial cell growth potentials, consistent with the idea that the high proliferative potential cells are progenitor cells. Perhaps most interesting, pulmonary artery– and microvascular-derived progenitor cells retain both endothelial-specific and segment-specific attributes, based on functional assays and surface antigen expression. These findings therefore suggest that the microcirculation is enriched with progenitor cells that are phenotypically related to their vascular origin.
It is important to identify a molecular basis for the high proliferative potential of lung microvascular endothelium. Global expression profiling resolved higher expression of nucleosome assembly protein (NAP)-1 in microvascular endothelial cells, when compared with pulmonary artery endothelial cells. NAP-1, and the related NAP-1 family, has recently been shown to control proliferation, most notably in yeast, of Xenopus
, and Arabidopsis thaliana
). Pulmonary microvascular endothelial cells express more NAP-1 than do pulmonary artery endothelial cells (31
). Overexpressing NAP-1 in pulmonary artery endothelial cells increases their growth, and decreasing NAP-1 expression in pulmonary microvascular endothelial cells decreases their growth. Moreover, whereas microvascular endothelial cells form more blood vessels in in vivo
Matrigel vasculogenesis assays than do pulmonary artery endothelial cells, overexpressing NAP-1 in the conduit-derived cells normalizes this vasculogenic potential. Thus, there is considerable cellular diversity within extraalveolar and alveolar endothelial cell populations. Pulmonary microvascular endothelial cells, in particular, are enriched in progenitors, and NAP-1 contributes to the pro-proliferative and vasculogenic phenotype of these cells.
The study of endothelial progenitor cells has been exciting, and may offer new insights into our understanding of vascular development, homeostasis, and repair after injury. It is not presently clear whether such rapidly growing cells contribute to vascular disease, as in the plexiform lesion in pulmonary hypertension. Recent studies from Xu and colleagues and Masri and associates (32
) have illustrated that the endothelium of patients with idiopathic pulmonary hypertension displays hyperproliferative, apoptosis-resistant growth characteristics. Interestingly, these cell features are retained in culture. It is unclear whether this vascular disease originates from genetic (i.e., somatic mutation) and epigenetic causes, or whether vascular disease selects for a resident cell type, such as an endothelial progenitor cell (35
Although it is exciting to consider that resident progenitor cells exist within populations of endothelium, we still know very little about these cells. How do we identify them in vivo? What is their relationship to pacemaker cells? What is their origin, and how do they relate to stem cells and progenitor cells found in the bone marrow and elsewhere? What is their level of differentiation? How do they retain a memory? What is their role in vascular development, homeostasis, and repair after vascular injury? What role do these cells play in vascular disease? Clearly, we have only scratched the surface; the next 10 years stand to redefine our understanding of endothelial cell biology.