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

The existence of phenotypically distinct SMC populations raises the possibility that these cells play unique roles in the adaptation of the vessel wall to stress and/or injury. Increasing evidence suggests that phenotypically distinct cells may exhibit differential responses to stress or injury and thus participate selectively in the subsequent vascular remodeling process (9-12,18,25). This work challenges conventional wisdom that medial and/or intimal thickening following injury is simply the result of a dedifferentiation and subsequent proliferation and migration of a single population of "well-differentiated" SMC. We thus tested the hypothesis that cells exhibiting distinct phenotypes would exhibit selective proliferative responses to chronic hypoxia (36). We evaluated the proliferative behavior of distinct medial SMC subpopulations, which were identified by immunobiochemical staining characteristics. Two distinct SMC subpopulations residing contiguous to each other in the outer media were selectively identified based on their expression of metavinculin or lack thereof (Fig. 3A). The proliferative response of these distinct SMC subpopulations to hypoxia was concurrently evaluated by performing immunofluorescent staining of the nuclear proliferation-associated antigen, Ki-67. Quantitative analysis of double-labeled tissue sections demonstrated that, at every post-hypoxic time point studied, >95% of the overall cell proliferation occurred within only one cell population, the metavinculin-negative SMC population (Fig. 3B). In contrast, the metavinculin-positive SMC population (labeled green in Fig. 3A) remained quiescent. These data provides compelling evidence that distinct SMC populations within the neonatal pulmonary artery media exhibit markedly different proliferative responses to hypoxic exposure (36). The data also raise the possibility that highly differentiated SMC are less susceptible to hypoxia-induced proliferation than cells exhibiting a less differentiated SM phenotype.

Hypoxia Response Curve

Figure 3. Phenotypically distinct medial SMC populations exhibit markedly different growth capabilities under identical culture conditions. A: Growth curves demonstrating heterogeneic proliferative capabilities of cell populations isolated from defined medial regions in response to stimulation with 10% serum (left) or 10% plasma (right). B: 3H-thymidine incorporation in response to hypoxia (under serum stimulation) differs significantly in distinct cell subtypes obtained from different medial regions of the main pulmonary artery. Cell populations were isolated from the following medial regions: Ll-cells from subendothelial media, L2-SMC from the middle media, L3-R cells and L3-S SMC from the outer media.

Figure 3. Phenotypically distinct medial SMC populations exhibit markedly different growth capabilities under identical culture conditions. A: Growth curves demonstrating heterogeneic proliferative capabilities of cell populations isolated from defined medial regions in response to stimulation with 10% serum (left) or 10% plasma (right). B: 3H-thymidine incorporation in response to hypoxia (under serum stimulation) differs significantly in distinct cell subtypes obtained from different medial regions of the main pulmonary artery. Cell populations were isolated from the following medial regions: Ll-cells from subendothelial media, L2-SMC from the middle media, L3-R cells and L3-S SMC from the outer media.

In an effort to provide further support to the idea that distinct subpopulations of medial cells respond differentially to the stimuli imposed by chronic hypoxic exposure, we evaluated the expression pattern of tropoelastin (9). We chose to evaluate tropoelastin because in its mature form, elastin, it plays a crucial role in determining the structure and function of large conduit arteries. We found that, in response to hypoxic exposure, tropoelastin expression was induced at high levels in metavinculin-negative SMC populations but not in metavinculin-positive ones. This data supported the existence of distinct SMC populations within the vascular media that differ in their functional responses to the stresses induced by hypoxia. Collectively, these observations lend further support to the idea that distinct SMC subpopulations contribute selectively to the pathogenesis of vascular disease.

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