Figure 1 Continued.
sess specialized functions. It is not surprising then that pulmonary artery endothelial cells flow-align in vivo, but microvascular endothelial cells do not flow-align. Macro-and microvascular endothelial cells in vivo each fulfill a barrier function, but the microvascular barrier is more restrictive. To more rigorously divulge the environment-independent features of macro- and microvascular endothe-lial cells, we sought approaches to isolate and culture cells from these different vascular segments.
Ryan and colleagues  approached the problem of isolating lung capillary (microvascular) endothelial cells in the 1970s. They (and other groups) developed methods for enriching endothelial cells from capillaries using minimal vascular protease digestion and perfusion of small beads. As an alternative, peripheral lung segments were cut (pleural cut technique), minced, exposed to minimal protease digestion, and expanded from cell colonies. Both approaches were successful, and have been adapted to further improve the isolation success rate. One major advance was recognition that endothelial cells from different vascular segments uniquely interact with plant lectins. Griffonia simplicifolia and Glycine max have been shown by many different labo ratories to recognize pulmonary microvascular endothelial cells and not pulmonary artery endothelial cells, whereas Helixpomatia (in rat) recognizes pulmonary artery endothelial cells . Thus, by incubating cells isolated using the pleural cut technique with Griffonia simplicifolia coated beads, microvascular endothelial cells can be efficiently ascertained (Figure 1C, top panel) and isolated (Figure 1C, bottom panel).
Use of purified pulmonary artery and microvascular endothelial cells has greatly contributed to our understanding of heterogeneity along the vascular tree. As in the intact circulation, pulmonary artery endothelial cells in vitro flow-align in response to shear stress, whereas microvascular endothelial cells do not flow-align (Al-Mehdi, unpublished). Similarly, cultured pulmonary artery endothelial cells align tangential to the axis of mechanical perturbation, whereas microvascular endothelial cells do not (Parker, unpublished). These findings are consistent with conclusions from the intact circulation, suggesting that these cell types possess unique mechanotransduction signaling programs.
Because oxidant-induced lung injury occurred principally in postcapillary (venule/vein) segments, Gillespie and others  examined potential mechanisms that may account for such site-specific oxidant sensitivity. Using endothelial cells isolated from each of the three vascular segments, their results indicated that pulmonary vein endothelial cells were most susceptible to xanthine oxidase and hypoxanthine-induced DNA damage. This DNA damage corresponded with DNA repair rates in the respective cell types, where vein endothelium exhibited the lowest rate of DNA repair. These findings therefore support the results from the intact circulation, suggesting that the propensity for oxidants to increase permeability in postcapillary segments could result from the inability of these cells to mitigate the oxidant signal.
Since thapsigargin initiated site-specific responses in situ, our group examined its effect in vitro. Activation of store-operated calcium entry triggered reorganization of the actin cytoskeleton into stress fibers, increased myosin light chain phosphorylation, decreased adhesive forces, and induced intercellular gap formation in pulmonary artery endothelial cells. Gap formation corresponded to increased macromolecular permeability. Although thapsigargin increased myosin light chain phosphorylation in pulmonary microvascular endothelial cells, it did not induce intercellular gaps and was not sufficient to increase micromolecular permeability. Thus, these findings are also consistent with results from the intact circulation.
Mechanisms accounting for the enhanced barrier function of pulmonary microvascular endothelial cells are not well understood, but microarray analysis comparing the two cell types has begun to reveal some insight. Microvascular endothelial cells express a number of cell-cell adhesion molecules not typically observed in pulmonary artery endothelial cells, including activated leukocyte cell adhesion molecule (ALCAM/CD166; immunoglobulin family), N-cadherin (cadherin family), and ZO-2 (ZO/MAGUK fam ily). We have confirmed our results from microarray studies using either quantitative PCR or Western blot analysis, but the functional contribution of any of these molecules to barrier enhancement has not been established. Nonetheless, these findings provide a step toward understanding the distinct molecular anatomy of the macro- and microvascular endothelial cell barrier.
A similar but more comprehensive approach has been undertaken by Brown and colleagues , when they performed global expression profiling experiments using mRNAfrom 53 different endothelial cell phenotypes. More than 2.4 million gene expression measurements were made. Using stable gene profiles in culture, they were able to discriminate macrovascular from microvascular endothelial cells and, further, artery from vein endothelial cells. Relevance of these unique expression profiles in vitro has not been confirmed in the intact circulation. Nonetheless, their work makes the important point that endothelial cells in culture are able to retain a stable memory of their origin, even under otherwise similar environmental conditions.
Whereas the wealth of in vivo, in situ, and in vitro evidence supports the notion of distinct endothelial phenotypes among precapillary, capillary, and postcapillary segments, an even more puzzling diversity exists within highly confined vascular segments. The idea of pacemaker cells was introduced in cell culture experiments to acknowledge that single cells control in synchrony the signal transduction events of near neighbors. Bhattarcharya and colleagues  have recently illustrated this concept in the pulmonary circulation in situ. They showed that calcium oscillations originate first in pacemaker endothelial cells that control the calcium response of their surrounding neighbors, indicating the presence of heterogeneity within the microvascular cells themselves in the pulmonary circulation. More recently, the same group has shown that endothelial cells at branch points of lung venular capillaries possess twofold more mitochondrial content by MitoTracker Green staining than their midsegment counterparts. The spatial pattern of the TNF-a-induced cytosolic Ca2+ transition and oxygen radical generation corresponded to the mitochondrial distribution pattern . A major difference in the environment of midsegment and branch point cells is the magnitude and pattern of shear stress. In midsegment, flow is laminar and shear stress is thought to be of consistent magnitude; in the branch points flow is turbulent and shear stress is thought to be highly variable. This suggests that shear stress regulates the endothelial cell phenotype, perhaps partly by altering mitochondrial mass and function in individual cells.
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