PERSPECTIVE
Adventitial Fibroblasts
Defining a Role in Vessel Wall Remodeling

Bradley H. Strauss and Marlene Rabinovitch

St. Michael's Hospital and Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada

In arterial wall pathology, we have traditionally focused our attention on the development of medial hypertrophy and the neointimal lesion. These lesions are a feature of pulmonary hypertension and restenosis after balloon angioplasty, atherosclerosis, and transplant arteriopathy. Intimal hyperplasia is characterized pathologically by smooth-muscle cell (SMC) proliferation and migration into the developing neointima, and synthesis and secretion of extracellular matrix (ECM) proteins, including collagen, elastin, and proteoglycans (1). Investigators have also implicated a second process termed vascular remodeling to refer to alterations in the entire vessel architecture that occur in response to hemodynamic changes or various forms of vascular injury. In pulmonary hypertension, this term has been used to describe the medial and adventitial thickening that occurs particularly in response to hypoxia. In systemic arterial wall pathophysiology, a somewhat different definition of remodeling has been used to describe changes in overall vessel dimensions, either enlargement or constriction (5). In this context, remodeling can be either an adaptive response, resulting in increased overall luminal diameter (positive remodeling) or a maladaptive response, resulting in a smaller overall lumen size (negative remodeling). Recent insights into arterial remodeling have implicated the adventitial layer as an important modulator of remodeling through its interactions with the media and intima. In pulmonary hypertension, adventitial thickening with increased cellularity and ECM deposition are most prominent in the small, muscular pulmonary arteries (10, 11), which typically share a number of structural features in common with systemic muscular arteries. This includes a vascular supply, the vasa vasorum, which provides nutrients to the adventitia and the outer part of the media, and which also acts as an entry site for circulating inflammatory cells stimulated to migrate from the outer vessel layers of the adventitia and media toward the intima (12). The adventitia is also normally populated by fibroblasts, which seem to play a significant role in arterial repair.

A wide variety of vessel wall injuries contribute to significant structural changes in both the adventitia and media. These include transition of fibroblasts to myofibroblasts, myofibroblast proliferation, myofibroblast apoptosis, myofibroblast migration into the intima, adventitial fibrosis, and expression of matrix metalloproteinases (MMPs). It is important to assess evidence for these activities in the pathogenesis of remodeling in the pulmonary and systemic circulation. Although many properties are shared between adventitial fibroblasts in the muscular arteries of the pulmonary and systemic circulation, differences also exist that need further elucidation, particularly in the response to hypoxia.

Fibroblast Phenotypic Transition

In response to pulmonary hypertension, pulmonary adventitial fibroblasts express SMC contractile proteins, in particular -SM actin. These "myofibroblasts" have contractile properties and demonstrate a marked increase in proliferative and synthetic activities (10). In arterial balloon injury, a similar phenotypic alteration in adventitial fibroblasts toward expression of SMC markers, -SM actin and vimentin, has been described, beginning at Day 3 and reaching a maximum at Days 7 to 14 (13, 14). In contrast, medial SMCs show a loss of -SM actin during this time. Platelet-derived growth factor (PDGF)-A chain and PDGF -receptor expression are also strongly expressed in the adventitia three days after injury concurrent with these changes in phenotype (13). The presence of myofibroblasts in the adventitial layer after balloon injury and the subsequent vessel retraction that results in a decrease in the arterial cross-section have been compared to healing skin wounds that demonstrate scar retraction secondary to dermal myofibroblasts. The remodeling properties of myofibroblasts have been attributed to the presence of -SM actin (15). However, the relative absence of myofibroblasts in the adventitia at three months, which corresponds to the time of peak arterial constrictive remodeling, suggests that other fibroblast mechanisms such as collagen deposition may be contributing (see subsequent discussion). It should also be stressed that the identification of phenotypic transition of fibroblasts currently relies predominantly on -SM actin, a rather imprecise and limited marker for characterizing these adventitial cells. Better cytoskeletal and molecular markers are required.

Fibroblast Proliferation

Fibroblast proliferation is more prominent in the neonate with pulmonary hypertension than in the adult with pulmonary hypertention. In this issue Das and coworkers have studied the proliferation of adventitial fibroblasts isolated from neonatal pulmonary arteries during a number of experimental conditions (16). Adventitial fibroblasts demonstrate augmented growth responses under hypoxic conditions that are additive to the effects of standard growth factors PDGF, basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF)-I. Moreover, hypoxia induces proliferation in pulmonary artery adventitial fibroblasts even in the absence of exogenous growth factors, which does not occur in neighboring medial SMCs (17, 18) or in systemic arterial adventitial fibroblasts (19). This pattern of pulmonary fibroblast proliferation is accompanied by changes in specific protein kinase C (PKC) isozymes, suggesting novel signal transduction pathways unique to the adventitial fibroblast (10). There also appear to be developmental differences in the PKC signaling pathways, which may explain the differential rates of fibroblast proliferation in adult and neonatal forms of pulmonary hypertension (20).

In the swine coronary angioplasty model that causes deep medial injury, time-course studies have shown intense adventitial proliferation at Days 2 to 3 followed by a decline at Day 7 (13, 14, 21). This adventitial proliferation is accompanied by inflammatory cell infiltration into the adventitia in the initial 24 h after balloon injury (22). There are disagreements in the literature about the nature of the proliferating adventitial fibroblast because -SM actin positivity is noted in these proliferating cells in some studies (13), but not in others (14, 22). This early burst of adventitial proliferation is much greater than the levels of proliferation measured in the medial SMC.

Fibroblast Apoptosis

Tissue remodeling also involves apoptosis, or programmed cell death, to counteract the effects of proliferation. Apoptosis is a ubiquitous physiologic process that regulates cell mass and structure. In contrast to cell necrosis, apoptosis is a tightly regulated process that requires energy (adenosine triphosphate [ATP]), gene transcription, and protein synthesis. The complex signaling pathways leading to the activation of proteases (e.g., interleukin-1 converting enzyme [ICE]) and endonucleases that mediate apoptosis continue to be an intense area of research. We were unable to detect apoptotic cells in lung biopsy specimens from a small number of patients with pulmonary hypertension (23). In monocrotaline-injured pulmonary hypertension, apoptosis was only evident in the adult rat pulmonary artery endothelial cell layer, with no evidence of apoptosis in the adventitial layer (24). In the porcine coronary balloon injury model, a maximal number of apoptotic cells was identified in the adventitia at six hours with a return to basal levels at Day 14 (22). This temporal pattern differed from proliferation, which peaked at a later time (at Day 3) and back to basal levels at Day 28. Apoptotic cells within the adventitial layer did not colocalize with either SMC markers or inflammatory cell markers, suggesting these cells were fibroblasts. The rates of cell proliferation exceed the apoptotic rates in the adventitia, which would account for a net increase in overall cell number.

Fibroblast Migration

There is no data available for pulmonary hypertension. In systemic arterial injury, there is some evidence to support migration of fibroblasts from the adventitia to the intimal layer and the development of the neointimal lesion, although tracking migrating fibroblasts remains a technically challenging task (13, 14, 21). The cells that are purported to be migrating display -SM actin positivity (i.e., the myofibroblast phenotype).

Adventitial Fibrosis

In response to hypoxia, fibroblasts, unlike SMCs, show increased type 1 collagen messenger RNA (mRNA) expression (25). Pulmonary arterial fibroblasts also demonstate enhanced procollagen production in response to mechanical loads and serum growth factors such as PDGF (34). In the porcine coronary balloon injury model, procollagen mRNA and protein levels were increased in the adventitia within two days of injury (26). Intracellular type I procollagen was detected in adventitial fibroblasts with enhanced expression for the first month after injury, leading to collagen accumulation in the adventitia up to three months. Moreover, double-labeling studies indicated that collagen expression and proliferation were colocalized in adventitial fibroblasts and later in myofibroblasts. Tropoelastin mRNA expression was not upregulated in the adventitia.

Protease Expression

MMPs are a family of degradative enzymes that have been implicated in two important processes in vessel wall repair, cellular migration (27), and regulating the extracellular matrix composition and content (28). Serine elastases also appear to play a causative role in the development of pulmonary hypertension (29, 30) and in restenosis after balloon angioplasty (31). Several MMPs (including MMP-1, -2, -3, and -9) are produced in the vessel wall by vascular SMCs. Only limited data are available for pulmonary hypertension. In a rat pulmonary hypertension model, MMP expression is increased in the posthypoxic period when there is active resorption of collagen (32). Stromelysin (MMP-3) was localized to the luminal surface of the pulmonary artery rather than the adventitia. More extensive data are available for adventitial fibroblasts in systemic arteries. Coronary adventitial explants in culture demonstrate increased gelatinolytic (MMP-2 and -9) activity and lower levels of the endogenous MMP inhibitor, TIMP-1, compared with SMCs that grow out of explants of the arterial medial layer (33). The enhanced protease expression should favor cell migration, which was confirmed by the increased outgrowth of adventitial cells compared with medial SMCs from the explants (33).

Future Directions

Important questions to be addressed include the identification of the link between inciting factors and the early remodeling response (e.g., the role of inflammation) as well as the contribution of the vaso vasorum vasculature in the initiation and propagation (maintenance) of the remodeling process. The interactions between locally generated growth factors and cytokines and the various adventitial events affected by these vasoactive substancescell proliferation, cell death, extracellular matrix synthesis, and degradationneed to be elucidated for both the pulmonary and systemic circulation. We also need to understand the consistent but unexplained differences in the hypoxic response of adventitial fibroblasts in the pulmonary and systemic arteries. A critical issue that also must be addressed is the relationship between the adventitial fibroblast and the constrictive arterial remodeling that is the predominant cause of restenosis after balloon angioplasty. Effective therapy for the large number of diseases characterized by adverse arterial remodeling will depend on our ability to understand and modify the role of the adventitial fibroblast in arterial repair.

	Footnotes

Abbreviations: alpha smooth muscle, -SM; matrix metalloproteinases, MMPs; platelet-derived growth factor, PDGF; smooth-muscle cell(s), SMC(s).

(Received in original form October 11, 1999).

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