Differential Proliferative Responses to Hypoxia of Proximal Pulmonary Artery SMC Subpopulations In Vitro Analysis

Of great interest is the possibility that an in vitro cell culture system could be utilized as a model system to investigate the mechanisms responsible for the marked differences in the response to hypoxia observed in vivo. Thus, we evaluated the proliferative responses of cell subpopulations isolated from distinct media regions of the main pulmonary artery to hypoxia (Table 2, Fig. 4). We found that in the presence of serum, DNA synthesis was increased in nonmuscle-like cells (subendothelial L1 and outer media "L3-R") in response to hypoxia, whereas it was decreased in SMC (middle media L2 and outer media "L3-S"). We also observed that certain nonmuscle-like cell populations could proliferate in response to hypoxia even in the absence of exogenously added serum, a unique response that was never observed in any of the SMC subpopulations even after prolonged periods in culture.

Phenotype Smc

Figure 4. Progressive increase in the phenotypic uniformity of SMC comprising the mature bovine arterial media along the proximal-to-distal axis of the pulmonary vascular tree. The phenotype of SMC was defined based on expression of several muscle-specific markers (a-SM-actin, SM-myosin, calponin, desmin, metavinculin). Only immunostaining for smooth muscle myosin heavy chains (SM-MHC) is shown here (right). Phenotypic heterogeneity of SMC, identified in intralobar pulmonary arteries (iLPA), is not observed in distal arteries of approximate diameter <1500 |im. Rather, all SMC in small size distal arteries display a similar, uniform phenotype. H&E, hematoxyllin-eosin staining (left).

Figure 4. Progressive increase in the phenotypic uniformity of SMC comprising the mature bovine arterial media along the proximal-to-distal axis of the pulmonary vascular tree. The phenotype of SMC was defined based on expression of several muscle-specific markers (a-SM-actin, SM-myosin, calponin, desmin, metavinculin). Only immunostaining for smooth muscle myosin heavy chains (SM-MHC) is shown here (right). Phenotypic heterogeneity of SMC, identified in intralobar pulmonary arteries (iLPA), is not observed in distal arteries of approximate diameter <1500 |im. Rather, all SMC in small size distal arteries display a similar, uniform phenotype. H&E, hematoxyllin-eosin staining (left).

Given the differential proliferative responses exhibited by the cell populations to hypoxic conditions, we initiated an evaluation of the molecular mechanisms that contribute to the hypoxia-induced proliferative responses observed in distinct cell subpopulations (nonmuscle SMC). We found that non-muscle-like cells which consistently demonstrated augmented growth capabilities under hypoxic conditions, were characterized by exuberant responses to G-protein coupled receptor (GPCR) agonists compared to the SMC that did not exhibit proliferative response to hypoxia (11). These findings suggest that there may be differences in receptor expression and/or susceptibility to activation in hypoxia-proliferative versus hypoxia-nonproliferative cells that contribute to the sensitivity to reduction in oxygen concentrations.

We also found that the nonmuscle-like cell populations, which responded with increased proliferation to hypoxia, had augmented responses to stimulation of the protein kinase C (PKC) pathway. Since we previously demonstrated that activation of PKC is a requisite step for SMC to proliferate under hypoxic conditions (8), we evaluated the specific isozymes of PKC, which might be linked to this specific cell function. We found that nonmuscle-like cells had increased levels of immunodetectable PKCa compared with the middle media SMC (8). This pattern of isozyme expression was paralleled by increased PKC catalytic activity in nonmuscle-like subendothelial L1-cells compared with middle media L2-SMC. These observations raise the possibility that hypoxia-proliferative cells have membrane-bound receptors that are sensitive to hypoxic activation, as well as the ability to engage specific intracellular signaling pathways, which confer unique proliferative responses to cells.

Since G-protein coupled receptors (GPCR) are also known to have potent effects on cAMP, and because cAMP has been shown to be an important modulator of cell proliferation, we tested the hypothesis that differences in cAMP response element binding protein (CREB) expression can function as a molecular determinant of SMC proliferative capability (17). We found that in the large pulmonary conduit vessels, CREB content was high in the "proliferation-resistant" subpopulations of medial SMC (i.e. in the outer media L3-S and middle media L2-SMC) and low in proliferation-prone regions (especially the subendothelial L1 medial region). We found in general that CREB content was decreased and SMC proliferation was accelerated in vessels from neonatal calves exposed to chronic hypoxia. In culture, we found that serum deprivation of "traditional" middle media L2-SMC led to increased CREB content and a quiescent growth state. In contrast, a highly proliferative population of subendothelial nonmuscle-like cells had low CREB content even under serum-deprived conditions. A correlation between CREB content and proliferation was further demonstrated by the observation that over-expression of wild type or constituently active CREB arrested cell cycle progression. Additionally, expression of constituently active CREB decreased both proliferation and chemokinesis. Consistent with these functional properties, active CREB decreased the expression of multiple cell cycle regulatory genes as well as genes encoding growth factors, growth factor receptors, and cytokines. These data suggest that CREB, at least in vascular SMC, could act as a unique modulator of cellular phenotype determination (17).

Collectively, our in vivo and in vitro findings regarding the cells which comprise the large conducting portion of the pulmonary circulation as well as the findings of others, support the concept that there exists heterogeneity in growth, ion channel expression, and in matrix-producing capabilities of different SMC populations, and that these differences are intrinsic to the cell type. The data also strongly supports the idea that the differential proliferative and matrix-producing capabilities of distinct SMC populations contained within the large conducting vessels govern, at least in part, the pattern of abnormal cell proliferation and matrix protein synthesis that characterize chronic hypoxic forms of pulmonary hypertension. The observation that the isolated phenotypically distinct medial SMC subpopulations exhibit differential proliferative responses to hypoxia demonstrates that these cells can be used as a model system to evaluate the mechanisms that regulate selective responsiveness of medial SMC to low oxygen concentration. Finally, these data support the idea that specific PKC isozymes and mitogen-activated protein kinases are uniquely coupled to upstream membrane-bound receptors (including GPCR), which are activated under hypoxic conditions. These signaling pathways control expression of specific transcription factors, including CREB, that likely are important determinants of the differential growth responses to hypoxia exhibited by distinct SMCs.

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Your heart pumps blood throughout your body using a network of tubing called arteries and capillaries which return the blood back to your heart via your veins. Blood pressure is the force of the blood pushing against the walls of your arteries as your heart beats.Learn more...

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