Smooth Muscle Cell Heterogeneity in Proximal Pulmonary Arteries In Vivo Analysis

Although for years investigators had suggested differences between the SMC of large and small vessels, until the 1990's there was limited information regarding the existence of site-specific heterogeneity of SMC within the vascular media. Then, studies in the systemic circulation of experimental animals began to suggest diversity in the SMC composition of large vessel media based on expression patterns of a limited number of contractile and cytoskeletal proteins. There remained, however, a paucity of data regarding the existence of SMC diversity within the pulmonary arterial circulation. A comprehensive evaluation of the adult bovine main and proximal pulmonary arteries was therefore performed to specifically address the hypothesis that the adult pulmonary arterial media was comprised of heterogeneous subpopulations of SMC expressing different biochemical markers (13). This study provided compelling data regarding the existence of numerous SMC phenotypes within the media of the main and proximal pulmonary arteries. As shown in Figure 1 and Table 1, at least 4 phenotypically distinct SMC subpopulations (based on differential expression of muscle-specific contractile and cytoskeletal proteins) could be identified within the adult bovine arterial media.

These phenotypically distinct cells were found to reside in distinct regions in the medial compartment. In the main pulmonary artery, 3 distinct regions of the media were defined based on the morphology of the cells residing in them and matrix fiber orientation patterns (Fig. 1).

■endothelium

£j A 60 140 1211 Tii Adult

Fetal Period (d) | Postnatal Period (d) Birth

£j A 60 140 1211 Tii Adult

Fetal Period (d) | Postnatal Period (d) Birth

Figure 1. Phenotypic heterogeneity of the cells comprising the proximal pulmonary artery tunica media (A and B), and schematic representation of their distinct differentiation pathways during development (C). A: Schematic diagram demonstrating the structural and cellular heterogeneity of the mature bovine main pulmonary artery media. These distinct regions of the media were identified based on cell morphology and arrangement, as well as elastic lamellae shape and orientation; LI, subendothelial region; L2, middle media region; L3, outer media region. B: Immunoperoxidase staining (brown) with specific antibodies against smooth muscle myosin heavy chain (SM-MHC) demonstrates phenotypic heterogeneity of cells within the arterial media, with intense positive staining in middle media L2-SMC and outer media "L3-S" SMC expressing SM-MHC, but the absence of immunoreactivity in subendothelial LI - and outer media "L3-R" cells. The tissue section was concurrently labeled with hematoxyllin to identify cell nuclei. C; Schematic representation of differentiation pathways based on expression of muscle-specific contractile and cytoskeletal proteins by different cell populations within the main pulmonary artery media. LI, subendothelial cells; L2, middle media SMC; "L3-S"-SMC and "L3-R"-cells, two phenotypically distinct cell populations within the outer region of the media.

A subendothelial region, arbitrarily termed L1 (layer 1), is identified and is composed of small, irregularly shaped cells interspersed among fragmented particles of elastin. As shown in Table 1, cells in the subendothelial (L1) region do not express any of the smooth muscle markers evaluated. Accordingly, the phenotype of cells in this region could be defined as non-muscle. A middle media region, arbitrarily termed L2 (layer 2), is composed of elongated, spindle-shaped cells, oriented circumferentially between well-developed and continuous elastic lamellae (Fig. 1). Immunobiochemical analysis (e.g., immunostaining and Western blotting techniques) revealed that cells within this region express a-smooth muscle actin (aSMA), SM-myosin heavy chain (SM-MHC) SM-1 isoform, calponin, and desmin (Table 1). Cells in this region, however, do not express any of the alternatively spliced muscle-specific proteins, such as SM-2 MHC isoform, metavinculin or SM-caldesmon (Table 1). An outer medial region, arbitrarily termed L3 (layer 3), is composed of two cell populations, each with distinct morphologic appearance and pattern of cell arrangement.

Table 1. Immunobiochemical Analysis of a-Actin, Myosin, SM-1 Isoform, Calponin, and Desmin

Staining for aSMA

Main Pulmonary Artery

Li L2 L3-S L3-R + +

Distal Pulmonary Artery 3000 pm 1500 pm 100-150 pm

L3-S L3-R + ~ + +

Calpoinin SM-MHC:

+

4-

+

+

+

SM-1

+

+

+

-

+

+

SM-2

-

+

ND

ND

ND

ND

SM-B

-

+

+

-

+

+

Desmin

+/-

+

+

-

+

+

Metavinculin

-

+

+

-

+

+

SM-caldesmon

-

+

ND

ND

ND

ND, not determined.

As schematically presented in Figure 1, large spindle-shaped cells (termed L3-S) arranged in compact cell clusters and oriented longitudinally within the vessel wall in areas devoid of elastic lamellae are seen in this region. A population of significantly smaller spindle-shaped cells (termed L3-R), oriented circum-ferentially is observed in interstitial areas between compact L3-S cell clusters. The L3-R cells are interspersed between well-developed, continuous elastic lamellae. Marked differences in the expression of muscle-specific markers were observed between the L3-S and L3-R cell types. L3-S cells expressed all the SM-specific proteins evaluated, including the alternatively spliced form of vinculin, metavinculin, as well as SM-2 MHC, and SM-caldesmon. Conversely, L3-R cells did not express any of the muscle-specific markers analyzed and therefore, their phenotype was defined as non-muscle.

2 Day 8 Day IS Day 2 Day 8 Day IS Day

Figure 2. Proliferation in the proximal pulmonary arterial media of neonatal calves with hypoxia-induced pulmonary hypertension occurs almost exclusively in a less differentiated SMC population. A: Double-label immunofluorescence staining of the outer medial region of the main PA of a newborn calf exposed to hypobaric hypoxia for 2 weeks. "Well-differentiated" SMC (as defined previously by expression of several SM-markers) are marked here by expression of meta-vinculin (M-VN+, green fluorescence), whereas less differentiated SMC do not express this muscle-specific protein (dark areas with no immunoreactivity). Proliferation (as defined by expression of Ki-67 nuclear proliferation-associated antigen, red fluorescence) is identified only in less differentiated (metavinculin-negative) cells. B: Quantitative analysis of the double-labeled tissue sections (as seen in A) demonstrates that more than 95% of cell proliferation observed within the media of different sized proximal pulmonary arteries occurs in less-differentiated (metavinculin-negative) cells. (TO.OOl, n=3 in each age group at each time point).

2 Day 8 Day IS Day 2 Day 8 Day IS Day

Figure 2. Proliferation in the proximal pulmonary arterial media of neonatal calves with hypoxia-induced pulmonary hypertension occurs almost exclusively in a less differentiated SMC population. A: Double-label immunofluorescence staining of the outer medial region of the main PA of a newborn calf exposed to hypobaric hypoxia for 2 weeks. "Well-differentiated" SMC (as defined previously by expression of several SM-markers) are marked here by expression of meta-vinculin (M-VN+, green fluorescence), whereas less differentiated SMC do not express this muscle-specific protein (dark areas with no immunoreactivity). Proliferation (as defined by expression of Ki-67 nuclear proliferation-associated antigen, red fluorescence) is identified only in less differentiated (metavinculin-negative) cells. B: Quantitative analysis of the double-labeled tissue sections (as seen in A) demonstrates that more than 95% of cell proliferation observed within the media of different sized proximal pulmonary arteries occurs in less-differentiated (metavinculin-negative) cells. (TO.OOl, n=3 in each age group at each time point).

To address the possibility that the differences biochemical phenotype expressed by cells in the adult arterial media were not simply the result of temporal "phenotypic" modulation of a single SMC type, experiments were performed to "track" the developmental fate of each "phenotypically unique" cell population (13). These studies, took advantage of the fact that cells with distinct phenotypes could be compared at different developmental stages because they were either localized to a specific medial region (i.e. L1- vs. L2-cells residing in the subendothelial vs. middle media, respectively) or exhibited a specific pattern of cell arrangement (e.g. "L3-S" cells forming longitudinally oriented compact cell clusters). These studies showed that the phenotypically distinct medial SMC subpopulations, observed in the adult media, progressed along distinct cyto-differentiation pathways during development, suggesting the existence of cells with unique genetic lineages within the large vessels of the lung (Fig. 2).

At the present time, the origins of these different cells remain unclear. However, the observations are consistent with the idea that cells of different origin and genetic composition contribute to the formation of the arterial media in large conduit vessels. In addition to cells arising from the local mesenchyme, mesenchymal precursor cells, neural crest cells, or bone marrow-derived progenitor cells may be recruited from distant sites to unique locations within the vessel wall and may give rise to cells with specific functional capabilities. Additional work is needed to define the precise lineages of SMC composing large pulmonary arteries.

In summary, the composition of the vascular media in large vessels is complex with multiple subpopulations of both smooth muscle and nonmuscle-like cells existing in close proximity within the arterial media. The bovine species provides, perhaps, one of the most dramatic examples of this, although similar but less marked findings have been reported in numerous species from avians to small rodents to humans (4,24,25,33).

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