An increasing volume of experimental data indicates that the adventitial fibroblast, in both the pulmonary and systemic circulations, is a critical regulator of vascular wall function in health and disease. A rapidly emerging concept is that the vascular adventitia acts as biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. In response to stress or injury, resident adventitial cells can be activated and reprogrammed to exhibit different functional and structural behaviors. In fact, under certain conditions, the adventitial compartment may be considered the principal injury-sensing tissue of the vessel wall. In response to vascular stresses such as overdistension and hypoxia, the adventitial fibroblast is activated and undergoes phenotypic changes, which include proliferation, differentiation, upregulation of contractile and extracellular matrix proteins, and release of factors that directly affect medial smooth muscle cell tone and growth and that stimulate recruitment of inflammatory and progenitor cells to the vessel wall. Each of these changes in fibroblast phenotype modulates either directly or indirectly changes in overall vascular function and structure. The purpose of this review is to present the current evidence demonstrating that the adventitial fibroblast acts as a key regulator of pulmonary vascular function and structure from the “outside-in.”

Arterial walls are heterogeneous three-layered structures composed of an intima, media, and adventitia. Each layer exhibits specific histological, biochemical, and functional characteristics, and, as such, each contributes in unique ways to maintaining vascular homeostasis and to regulating the vascular response to stress or injury. Endothelial cells and smooth muscle cells (SMC), the principal cellular constituents of the intima and media, respectively, have received much attention from vascular biologists, whereas the adventitial fibroblast, the principal cell type in the adventitia, has been largely overlooked. However, an increasing volume of experimental data indicates that the adventitial compartment of blood vessels, in both the pulmonary and systemic circulations, is a critical regulator of vessel wall function in health and disease. A rapidly emerging concept is that the vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. Indeed, the adventitial compartment may be considered the principal “injury-sensing tissue” of the vessel wall. In response to environmental stresses such as hypoxia or vascular distension, resident fibroblasts are activated and undergo a variety of functional changes. Increases in cell proliferation, the expression of contractile and extracellular matrix (ECM) proteins, as well as in the secretion of factors capable of directly affecting medial SMC tone and growth and vascular inflammation occur in the fibroblasts in a manner that influences overall vascular tone and wall structure. Thus the adventitia should be viewed as capable of regulating vascular function and structure from the “outside-in.”

The purpose of this review is to provide evidence that, in response to injury, resident adventitial fibroblasts can be activated to exhibit different functional characteristics that contribute significantly to pulmonary vascular remodeling. Data will be presented supporting the concept that fibroblasts (in some cases only specific subpopulations of fibroblasts) within the adventitial compartment are able to 1) proliferate with greater propensity than SMCs in response to injury or stress, 2) differentiate into SM-like cells (i.e., myofibroblasts), which can accumulate in the adventitia and/or migrate to the medial and intimal layers of the vessel wall, 3) increase and alter their profile of ECM deposition, 4) synthesize and release molecules that have potent paracrine effects on neighboring SMC and endothelial cells, 5) express molecules/receptors that facilitate the recruitment of circulating leukocytes and progenitor cells to the adventitial compartment, and 6) synthesize and release angiogenic chemokines and molecules that support neovascular growth of the vasa vasorum and perpetuate the inflammatory response. In addition, the emerging role of resident adventitial progenitor cells in vascular remodeling will be discussed.

Adventitial Fibroblast Heterogeneity

There is significant diversity in the fibroblast populations comprising the pulmonary artery (PA) adventitia (17). This is not surprising because heterogeneity of fibroblast populations within tissues, including the lung, is well documented (25, 43, 49, 72, 75, 104). One hypothesis receiving increasing attention is that only certain fibroblast subsets within a tissue respond to injury or stress with heightened proliferative or dysregulated fibrogenic responses (34, 43, 49, 72, 76). Differences among subsets of normal fibroblasts have been identified on the basis of surface markers, cytoskeletal composition, lipid content, and cytokine profile (34, 72, 75). The most extensively characterized marker that has been utilized to differentiate specific lung fibroblast sub-populations is the glycosyl phosphatidylinositol (GPI)-anchored protein Thy-1. Thy-1 is localized to lipid rafts and signals to Src family kinases to regulate cell adhesion and cytoskeletal organization (4). Thy-1(–) and Thy-1(+) mouse lung fibroblasts differ morphologically and have differing secretory profiles (76). Thy-1(–) cells, at least within the lung, appear to be a more consistent fibrogenic subtype (33, 34, 125). Thy-1(–) lung fibroblasts exhibit greater proliferative responses to PDGF-AA and CTGF (33). Thy-1(–) cells secrete twice as much latent TGF-β as Thy-1(+) cells, show increased shedding of syndecan-2, and can express five times more α-SM-actin, a characteristic of the myofibroblast phenotype, than Thy-1(+) fibroblasts (125). Recent data show that Thy-1(–/–) mice exhibit more severe lung fibrosis (increased collagen accumulation) and increased Smad 2/3 phosphorylation (indicating higher levels of active TGF-β)in response to intratracheal bleomycin than do Thy-1(+)-expressing control mice (34). That selective expansion of select fibroblast subsets can occur in response to injury is supported by studies demonstrating that fibroblasts from within a fibrogenic milieu differ from those in normal tissues. Fibroblasts isolated from lungs with active fibrotic disease have increased proliferative capacity, are capable of anchorage-independent growth, and are morphologically distinct (75, 80, 111). Interestingly, the myofibroblasts in fibroblastic foci in lung tissues from individuals with IPF/UIP are Thy-1(–), despite the fact that the majority of fibroblasts from normal lungs are Thy-1(+) (34). Collectively, these findings suggest the existence within the lung of fibroblast subsets with an increased propensity to contribute to fibrotic responses.

Although it is clear that fibroblast subsets contribute significantly to tissue fibrosis, it should also be noted that precise identification of fibroblasts in vivo (and even in vitro) is difficult with current tools because markers frequently utilized to identify fibroblasts, such as vimentin and α-SM-actin, lack specificity or sensitivity (86). Vimentin, a frequently used marker for fibroblasts, unfortunately does not distinguish fibroblasts from other cells of mesenchymal origin. α-SM-actin has been utilized to identify myofibroblasts and is not generally observed in resting fibroblasts. It is, however, obviously observed in SMC and in circulating SM progenitors. Fibroblast-specific protein-1 (FSP1), also identified as S100A4, is an 11-kDa protein that belongs to the S100 superfamily of intracellular binding proteins. It has been suggested to be a specific marker of fibroblasts in a variety of conditions including renal and pulmonary fibrosis (42, 53). However, the specificity of FSP1 as a marker for fibroblasts remains unclear at the present time (54, 59). Identification of fibroblast-specific markers is a crucial future goal in fibroblast biology.

Studies of fibroblasts derived from the adventitia of pulmonary arteries also suggest a tremendous heterogeneity in their functional characteristics. Fibroblast clones have been generated from the PA adventitia with markedly different proliferative capabilities (17). Of interest and relevance to hypoxia-induced pulmonary vascular remodeling, clones that consistently proliferated in response to hypoxia, as well as those that did not, were identified. A selective contribution of this subset of fibroblasts to hypoxia-induced pulmonary vascular remodeling was suggested by the selective expansion of the “hypoxia-proliferative” clones generated from the remodeled adventitia of chronically hypoxic animals (17, 107). These observations are consistent with selective expansion of specific fibroblast populations in lung fibrosis described above. Further studies are needed to characterize the various populations of fibroblasts present in the adventitia of different vessels and to specifically evaluate their role in vascular remodeling.

At present, the vast majority of work contained in the literature related to intact animals describes general responses of the “adventitial fibroblast” to stress or injury. Experiments in cell culture most often describe the responses of “aggregate populations” of adventitial fibroblasts. The remainder of this review will focus on work that describes generalized responses of the adventitial fibroblast, keeping in mind the caveat that not all adventitial fibroblasts may be equally exhibiting the responses described.

Stress/Injury Induces Early Proliferative Changes in the Adventitia

One of the most consistent findings in experimental models of systemic vascular injury and hypertension is early and often dramatic adventitial remodeling (87). Early evidence of adventitial cell activation and phenotypic change have been reported following balloon injury to the vessel wall (71, 101). More recent experiments in models of hypercholesterolemia and hypertension have also demonstrated that adventitial changes precede intimal and medial remodeling (37). Increases in adventitial fibroblast proliferation, which precede and exceed endothelial and SMC proliferation, are a common denominator in these studies. In fact, when quantitative analysis of cell numbers and density is applied to animal models of hypertensive remodeling, the most consistent, yet unexpected, findings that emerge are increases in adventitial cell density and decreases in SMC density (1, 50, 61). Studies evaluating the mechanical behavior of the adventitia support the idea that, under conditions of elevated blood pressure, the adventitia becomes the predominant wall component due to its pronounced stiffening behavior (90, 100). Thus the adventitia has been suggested to be the most appropriate arterial layer for “sensing” hypertensive states (60, 61, 90). These observations of unique adventitial mechanical properties and early increases in fibroblast proliferation have stimulated the hypothesis that the adventitia plays an essential role in the regulatory systems that control vascular remodeling and vascular tone at least under conditions of high wall stress.

Interestingly, similar findings have emerged from experimental studies in the pulmonary circulation (FIGURE 1). In hypoxia-induced pulmonary hypertension, there is early and often dramatic evidence of adventitial remodeling (62, 107, 108). Less pronounced but still significant adventitial changes are noted in high-flow and monocrotaline models of pulmonary hypertension as well as in idiopathic forms of human pulmonary hypertension (12, 52, 63, 77). In hypoxic models of pulmonary hypertension, adventitial fibroblasts have been noted to undergo the earliest and most significant increases in proliferation of all vascular wall cell types (FIGURE 1) (5, 62, 68). Thus, like the systemic arterial adventitial fibroblast, the pulmonary arterial fibroblast seems poised to undergo early changes in proliferation in response to a variety of injurious stimuli.


In hypoxia-induced pulmonary hypertension, the PA adventitia is the primary site of cell activation and tissue remodeling
Compared with control calf (Normoxia), pulmonary arteries of chronically (2 wk) hypoxic neonatal calf (Hypoxia) demonstrate marked adventitial thickening (A), excessive deposition of extracellular matrix proteins [type I collagen (B), cellular (ED-A) fibronectin (C), and tenascin-C (TN-C; D)], augmented cell proliferation (BrdU; E), myofibroblast accumulation/differentiation (α-SM-actin expression; F), cytokine production (active isoform of TGF-β1; G), and recruitment of circulating leukocytes and fibrocytes (CD11b; H). H&E, hematoxylin and eosin; A, adventitia; M, media. Arrows define adventitia.

Due to the early dramatic increases in proliferation of the pulmonary adventitial fibroblast in hypoxic models of pulmonary hypertension, observations again suggesting a distinct environmental “stimulus-sensing” capability of the fibroblast, many investigators have sought to understand the molecular basis of this process. It has been established that even heterogeneous populations of PA adventitial fibroblasts proliferate in response to hypoxic conditions, a response not observed in PA SMC when cultured and tested under identical experimental conditions (15, 16, 117119). Some have even suggested that this is a unique (compared to systemic) response of PA fibroblasts (119). Activation of Gαiand Gq family members, perhaps in a ligand-independent fashion, with subsequent stimulation of protein kinase C and mitogen-activated protein (MAP) kinase family members are important regulators of hypoxia-induced adventitial fibroblast proliferation (15, 16, 107). Activation of phosphatidylinositol 3-kinase (PI3K), as well as synergistic interaction with Akt, mammalian target of rapamycin (mTOR), and p70 ribosomal protein S6 kinase, has also recently been demonstrated to be necessary for the proliferative responses of PA adventitial fibroblasts in response to hypoxia (31, 51). Therefore, a complex network of signaling pathways initiated by G-protein-mediated signaling is responsible for the stimulation and proliferation of PA adventitial fibroblasts in response to hypoxia, a response that is distinct among vascular wall cells (FIGURE 2). It is currently unclear as to why hypoxia fails to initiate the same set of proliferative signals in pulmonary arterial SMC or for that matter even in certain subsets of adventitial fibroblasts.


Intercellular pathways through which hypoxia stimulates fibroblast activation
Hypoxia activates G proteins (Gαior Gq), which initiate PI3K/Akt-, PLC/PKC-, mTOR/p70S6K-, and ERK1/2-dependent signaling pathways that mediate hypoxic proliferative responses in adventitial fibroblasts. PI3K stimulates Akt, which in turn contributes to the cross talk between PI3K and mTOR/p70S6K pathways. Because hypoxia induces a dramatic increase in p70S6K activity, additional signaling mechanisms are likely involved in control of the translational machinery. The cumulative effects of PI3K, Akt, mTOR, p70S6K, and ERK1/2 integrate transcriptional and translational pathways that ultimately lead to fibroblast proliferation, differentiation, and the release of mediators under hypoxic conditions.

Downstream of the aforementioned signaling pathways lie transcription factors, which are involved in the proliferative response. Hypoxia is known to activate a diverse array of transcription factors and thus has a profound impact on the cellular transcriptome (14). However, it is likely that the degree and nature of the global transcriptional response to hypoxia in vivo is both cell-type and cell-state specific. Although the transcription factor hypoxia-inducible factor-1α(HIF-1α) plays a major role in controlling the ubiquitous transcriptional response to hypoxia, it is clear that a number of other transcriptional activators and repressors are also activated either directly or indirectly under hypoxic conditions. Activation of HIF-1α appears to be an important regulator of replication of human PA adventitial fibroblasts under hypoxic conditions (51). Hypoxia also induces a significant increase in the expression and activity of early growth response-1 (Egr-1) in adventitial fibroblasts (30). Attenuation of Egr-1 protein with antisense oligonucleotides reduces the hypoxia-induced proliferation of pulmonary arterial fibroblasts (3). Egr-1 contributes to the proliferative phenotype induced by hypoxia, at least in part, by regulating expression of cyclin D and epidermal growth factor receptor (3). Hypoxia-induced upregulation of Egr-1 has been demonstrated to be an important contributor to the pathogenesis of pulmonary vascular remodeling (121). Thus both HIF-1α and Egr-1 are important transcription factors participating in hypoxia-induced adventitial fibroblast proliferation. They are also likely involved in regulating other phenotypic changes induced by hypoxia in adventitial fibroblasts (described below).

Hypoxia or other stressors may also affect local increases in adventitial fibroblast proliferation by inducing the secretion of various autocrine/paracrine factors (FIGURE 2) (51, 107). For example, increased release of ATP from PA adventitial fibroblasts has been demonstrated under hypoxic conditions (30). Extracellular ATP can stimulate adventitial fibroblast proliferation by itself and can also act synergistically with other growth factors released by fibroblasts, endothelial cells, or macrophages, including IGF-1 and PDGF (30). The proliferative effects of ATP appear to be mediated largely through G-protein-coupled P2Y receptors and downstream signaling intermediaries strikingly similar to those included by hypoxia. Angiotensin II is another autocrine/paracrine factor released by hypoxia, under the control of HIF-1α, which has also recently been demonstrated to act in a positive feedback loop and to facilitate PA adventitial fibroblast proliferation under hypoxic conditions (51, 64). Specific increases in angiotensin-converting enzyme (ACE) expression are observed in the adventitia of hypoxic pulmonary hypertensive animals (65). This system could operate to control adventitial fibroblast proliferation in response to a variety of stresses and probably deserves the same attention that it has received with regard to fibroblasts in the systemic circulation (70, 83).

Excessive proliferation of fibroblasts to brief or unsustained stimuli such as hypoxia or stretch could lead to unwanted changes in vascular structure. Therefore, it is likely that many stimuli (i.e., hypoxia, overdistension) could also activate replication repressor signals in fibroblasts to limit or control replication. The existence of growth-limiting signaling pathways has been described in many cell types in response to other growth-promoting stimuli (110, 124). Thus, since the fibroblast seems capable of early and often dramatic growth increases in response to a number of environmental stimuli, it seems likely that it would have also developed mechanisms to limit uncontrolled growth. Protein kinase C-ζ (PKC-ζ) and MAP kinase phosphatase-1 (MKP-1) have been identified as proliferative suppressors in other cell types. We tested the hypothesis that hypoxia would also activate PKC-ζ and MKP-1 to repress proliferative signals in normal adventitial fibroblasts and thereby limit acute hypoxia-induced proliferation of fibroblasts. Using multiple molecular and pharmacological strategies, we have shown that hypoxia-induced proliferation of PA adventitial fibroblasts is indeed negatively regulated by the atypical PKC-ζ isozyme (96). We have also demonstrated that PKC-ζ attenuates the phosphorylation of extracellular regulated kinase (ERK) 1/2 through the regulation of MKP-1 in hypoxic fibroblasts exposed over 24–48 h to hypoxia. Therefore, we suggest that PKC-ζ and MKP-1 can act as signal terminators of ERK1/2 activation and thus limit hypoxia-induced proliferation of PA adventitial fibroblasts derived from normal animals.

As noted above, fibroblasts derived from fibrogenic foci in the lung exhibit a different phenotype than the majority of fibroblasts from the normal lung. Similarly, a significant modulation in the phenotype of PA adventitial fibroblasts derived from chronically (≥ 2 wk) hypoxic animals has been observed. PA adventitial fibroblasts derived from chronically hypoxic animals exhibit far greater growth responses to a number of growth-promoting stimuli, including hypoxia, than fibroblasts from normoxic animals (16, 17, 117, 119). Changes in the signaling pathways used to elicit proliferation are observed in these cells compared to controls (16, 96, 97, 117). Interestingly, we have found that the functional role of the atypical PKC-ζ isozyme in the proliferative responses is significantly altered in fibroblasts from chronically hypoxic animals. In these cells, PKC-ζ acts as proproliferative kinase for adventitial fibroblasts as opposed to its anti-proliferative actions in fibroblasts derived from normoxic control animals described above (97). These observations raise the possibility that chronic hypoxia leads to the emergence of fibroblasts in the adventitia that have lost their ability to limit stimulus-induced proliferation. Whether chronic hypoxia, or any stimulus for that matter, modulates intracellular signaling patterns to change fibroblast phenotype or causes expansion of unique fibroblast subsets or both in ways that might contribute to structural remodeling remains unclear.

Adventitial Fibroblasts Can Differentiate into Myofibroblasts

Activation of resident fibroblasts by a variety of stimuli can result in their differentiation into a myofibroblast phenotype, a process shown to be critical to a variety of fibrotic diseases, including those in the lung (19, 27, 73). Myofibroblasts express α-SM-actin, the most frequently used marker for myofibroblast identification, which allows monitoring of this cell type during experimental and clinical conditions (20, 21). Early and dramatic increases in the appearance of α-SM-actin-expressing myofibroblasts in the adventitia are observed in hypoxia-induced pulmonary hypertension as well as in numerous other vasculopathies (103, 105, 120). Myofibroblasts are implicated as key participants in tissue remodeling because of their ability to perform multiple physiological functions in response to change in the local environment (27). Myofibroblasts are the principal producers of collagen and other ECM proteins including fibronectin, tenascin, and elastin in the fibrotic tissues, including the remodeled PA adventitia (27, 92, 107, 108). Accumulation of myofibroblasts could contribute to changes in vascular tone under both basal and pathophysiological situations. In addition, its responses to vasodilating stimuli differ from those of SMC and thus could contribute to the abnormalities of vasorelaxation observed in the setting of chronic pulmonary hypertension. The myofibroblast can exert significant changes in the function and structure of the vessel wall under pathophysiological conditions because of its contractile properties and enhanced production of ECM proteins (95).

Based on work in systemic models of hypertension and vascular injury, the myofibroblast appears capable of migrating from the adventitia to the media or even the intima and thus contributing to the thickening of these components, which is observed in response to injury (92, 93, 101, 115). Accumulation of myofibroblasts in the intima of patients with pulmonary hypertension has been well documented and consistently observed (102, 123). However, whether these intimal cells originate from the adventitia is unclear.

The differentiation of fibroblasts into myofibroblasts is regulated by a complex microenvironment consisting of growth factors, cytokines, adhesion molecules, and ECM molecules. TGF-β is a well-known cytokine capable of inducing transition of a fibroblast into a myofibroblast phenotype by stimulating α-SM-actin expression and collagen production (28). Thrombin and endothelin (ET-1) have also been reported to induce differentiation of normal lung fibroblasts into a myofibroblast phenotype (6, 95). All of these factors are upregulated by hypoxia and have been observed to be present in the PA adventitia of chronically hypoxic animals (107). Furthermore, we have recently demonstrated that hypoxia alone can stimulate an adventitial fibroblast-myofibroblast transition (98). Interestingly, we found that hypoxia induces myofibroblast differentiation (α-SM-actin protein expression) along with the induction of myofibroblast proliferation of PA adventitial fibroblasts (98). However, these two cellular responses to the hypoxic challenge are regulated by different intracellular signaling modules. Hypoxia-induced proliferative responses in fibroblasts utilize a Gαi-initiated ERK1/2-dependent signaling pathway. In contrast, hypoxia-induced α-SM-actin expression, although again dependent on Gαi activation, utilizes JNK rather than ERK1/2 signaling to achieve the response. It remains unclear as to whether fibroblasts can differentiate into SMC, although this intriguing possibility has been raised and would have significant implications for vascular pathophysiology (11).

Adventitia: A Depot for Vascular Progenitor Cells

In addition to the fact that resident adventitial fibroblasts can differentiate into myofibroblasts or SM-like cells that can remain in the adventitia or migrate to medial or intimal compartments of the vessel wall, Hu et al. have recently raised the possibility that the adventitia contains progenitor cells capable of differentiating into SM or endothelial cells (40, 112). These cells could migrate to the media and neointima, contributing to lesion progression (FIGURE 3). Specifically, they demonstrated that the adventitia harbors cells expressing stem cell markers including Sca-1, cKit, CD34, and Flk-1. Explanted cultures of adventitial tissues using stem cell medium resulted in the outgrowth of heterogeneous cell populations, some of which were shown to differentiate into SMC in response to PDGF-BB stimulation. When these same Sca-1+ cells, carrying the lacZ gene, were transferred to the adventitial side of vein grafts in Apo-E-deficient mice, β-Gal+ cells were found in atherosclerotic lesions of the intima in these vessels. Evidence was presented suggesting that these progenitor cells are adventitial residents and are not of bone marrow origin. One possibility to explain the presence of progenitor cells in the adventitia is that these cells are remnants of earlier developmental stages. It is possible that mesenchymal precursors remain located in the adventitia and that these local mesenchymal cells are capable of differentiating into different types of vascular cells under the appropriate circumstances.


Schematic interpretation of the hypothesis that the cells contributing to the medial and/or neointimal thickening may originate from different cell types as well as from different vessel wall compartments
The adventitia may serve as a rich source of various cell types (resident fibroblast, myofibroblast, and local and circulating progenitor cells) that contribute to pathophysiological changes in vascular structure. VSMC, vascular smooth muscle cell.

The adventitia may also serve as a repository for circulating progenitor cells following vascular injury (FIGURES 1 AND 3). Circulating progenitor cells have been implicated in the pathophysiology of a number of systemic and pulmonary vascular diseases (35, 38, 56, 88, 106). Caplice et al. (10) demonstrated in sex-mismatched bone marrow transplant patients that SMCs in the atherosclerotic plaque can originate from cells administered at bone marrow transplantation. Interestingly, these cells appear to cluster not only in the intima but also in and around adventitial microvessels. Others have demonstrated that circulating or bone marrow-derived progenitor cells participate in the neovascularization of the adventitia and intima and may be essential for atherosclerotic disease progression (39, 66). We have recently demonstrated the appearance of cells expressing stem-cell antigens in the PA adventitia of chronically hypoxic animals (18). Many of these cells were in the rapidly expanding adventitial vasa vasorum blood vessels. Expansion of the adventitial vasa vasorum appears important in the progression of many vascular diseases. They appear to provide a conduit for delivery of inflammatory and progenitor cells to the vascular wall (66, 106).

It has become increasingly clear that inflammation is an important component of systemic and pulmonary vascular disease (41, 106). Inflammatory cells, specifically monocytes but not neutrophils, are observed in the adventitia early in the development of hypoxic pulmonary hypertension (FIGURE 1 ;Ref. 26). Contained within this inflammatory cell influx is a subset of monocytic cells that have been termed fibrocytes. Fibrocytes are characterized by the dual expression of both leukocytic markers (CD45, CD34, CD11b, CD14) and mesenchymal markers (α1-procollagen) (79). These cells are rapidly recruited to sites of tissue injury and have been shown to differentiate into collagen-producing fibroblasts and/or myofibroblasts (67, 74, 79, 89, 122). We have observed a rapid and dramatic influx of fibrocytes into the adventitia of animals (rats and calves) exposed to chronic hypoxia. These cells contribute significantly to the cell populations expressing type I collagen and α-SM-actin, supporting the idea that non-resident circulating cells recruited to the adventitia also contribute to vascular remodeling. Perhaps most importantly, when these cells were depleted in the circulation, hypoxia-induced pulmonary vascular remodeling was inhibited (26).

Collectively, these observations support the idea that adventitia can act as a source for cells involved in vascular repair, such as myofibroblasts, through differentiation of resident fibroblasts and/or progenitor cells, as well as through the recruitment of circulating mesenchymal progenitors.

Autocrine and Paracrine Roles of the Adventitial Fibroblast in the Control of Vascular Tone and Structure

Adventitial fibroblasts are activated rapidly in response to a variety of pathophysiological stimuli (FIGURE 4). One early marker of activation, observed in response to a number of stressors, is an increase in the production of reactive oxygen species (ROS) through a distinct (among vascular wall cells) NADPH oxidase system (2, 69, 70, 91). This early and often dramatic change in both extracellular and intracellular concentrations of ROS can have profound acute and chronic effects on vascular function (57, 113). Increased concentrations of ROS, particularly superoxide (O2·−), in the adventitial compartment are capable of scavenging endothelial-derived nitric oxide (NO). In fact, high adventitial (O2· −) concentrations can act as a “sink” for NO and lower its bioactive concentrations over a fairly wide diffusion radius, contributing to increases in vascular tone even in the presence of normal NO production by the vascular endothelium (82). In addition, the release of ROS by the adventitial fibroblast can have direct effects on neighboring SMC to increase their contraction (113). O2·− has been demonstrated to directly increase Erk activity, ultimately leading to increases in intracellular calcium and PA SMC contraction (32). Another ROS produced in the extracellular space via extracellular- O2· − dismutase (EC-SOD) is H2O2, which is capable of diffusing into cells, e.g., fibroblasts or SMC, and of initiating signaling pathways resulting in contraction, growth, or migration (113). In addition, adventitial fibroblasts, in response to ROS and other microenvironmental stimuli, are capable of releasing a number of mediators that could affect vascular tone (FIGURE 4). These include ET-1, PDGF, EGF, FGF-2, PGH-2, HSP90α, and cyclophilins (45, 55).


Advential fibroblasts regulate tone and structure of the vessel wall
Adventitial fibroblasts are rapidly activated in response to a variety of pathophysiological stimuli and release a number of potent mediators that directly affect vascular tone, structure, and functions of resident vessel wall cells. Fibroblasts may also release chemokines leading to the recruitment of inflammatory and progenitor cells and thus affect vascular structure and function indirectly.

It has also been demonstrated that, following a wide variety of vascular injuries, there is an almost immediate influx of leukocytes into the adventitial compartment. Fibroblasts, in response to stress or injury (including hypoxia), are known to be capable of producing a wide array of chemokines, which facilitate recruitment of leukocytes from the vasculature, a response that is probably regulated by NAD(P)H oxidase (9). Chemotactic factors documented to increase in the adventitia during the development of hypoxia-induced pulmonary hypertension include MCP-1, VEGF, ET-1, and TGF-β (18, 106). Importantly, there are likely numerous chemokines that fibroblasts release in a stimulus-specific manner that are capable of regulating influx of specific leukocyte subtypes. Importantly, the newly recruited leukocytes produce ROS, which in turn have effects on adventitial fibroblasts and/or the underlying SMC (FIGURE 4). In addition, recruited monocytes and macrophages, as well as activated resident fibroblasts, release cytokines and growth factors capable of directly affecting SMC phenotype and function (106, 116). Factors released include ET-1, Ang-II, TGF-β , IGF-I, bFGF, and PDGF- β, all of which have significant effects on growth and matrix production of fibroblasts and on the underlying SMC. Specifically, PA adventitial fibroblasts, in response to hypoxia, produce paracrine factors through HIF-dependent mechanisms, which have potent stimulatory effects on SMC proliferation (84).

In vivo evidence that activated adventitial fibroblasts play an important paracrine role in regulating vascular remodeling was recently provided by experiments showing that targeted perivascular delivery of a novel NADPH oxidase inhibitor effectively attenuated angioplasty-induced neointimal formation of the rat carotid artery and angiotensin II-induced medial thickening (22, 56, 57). Similarly, Liu et al. (58) showed that hypoxia-induced pulmonary hypertension and vascular remodeling were completely abolished in NADPH (GP91phox) knockout mice.

Thus the activated adventitial fibroblast appears to play a significant role in influencing the tone and structure of the vascular wall following a variety of injuries or stresses both directly, through the secretion of vasoactive and growth-promoting molecules, and indirectly by producing chemokines, which promote accumulation of leukocytes and progenitor cells.

Cell-Matrix Interactions in the Adventitial Compartment

In response to stress or injury, the fibroblast dramatically changes its production of ECM molecules, the accumulation of which can have a profound effect on vascular structure and function. The composition of the adventitial ECM is principally regulated by fibroblasts. The fibrillar collagens are a major component of the adventitial matrix and the most abundant types I and III collagens are the principal matrix proteins produced by adventitial fibroblasts (78). ECM-cell interactions affect the physical coupling of cells via the regulation of cell-surface adhesion molecules and may also regulate, via a positive feedback loop, the deposition of ECM (8). Thus, under normal conditions, there must be a homeostatic relationship between resident fibroblasts and this collagen matrix to maintain fibroblasts in a quiescent, undifferentiated state (99). Activation of the fibroblast leads to alterations in the production and relative composition of matrix components, which in turn ultimately contribute to further changes in cell growth, behavior, and differentiation.

Recent findings have revealed that adventitial matrix composition is altered during the progression of diseases such as atherosclerosis and restenosis (81). Excessive and progressive deposition of ECM proteins in the adventitia is common in remodeled vessels, including those in pulmonary arterial hypertension (24, 46, 105, 108). Marked increases in the production and accumulation of collagen and elastin are observed in the adventitia during the development of pulmonary hypertension (24). Although not well studied, the accumulation of collagen is likely to affect stiffness of the vessel wall, which can have profound effects on flow dynamics in the vessel and ultimately on right ventricular function (23, 85). In addition, marked increases in the accumulation of fibronectin, tenascin-C (TN-C), and elastin in the adventitial compartment of models of hypoxia-induced pulmonary hypertension are observed (24, 108). Fibronectin appears to play a critical role in facilitating proliferation of fibroblasts as well as in their differentiation into myofibroblasts (19). TN-C expression has been shown to be upregulated in pulmonary hypertensive vessels (47). TN-C, like fibronectin, is associated with fibroblast and SMC proliferation and has also been shown to contribute to differentiation into a myofibroblast (48, 109). In addition, fibronectin and TN-C deposition coincides with the expression and activity of matrix metalloproteinases (MMPs), a family of zinc enzymes responsible for degradation of the ECM components, including basement membrane collagen, interstitial collagen, fibronectin, and various proteoglycans (29, 44). Excessive or inappropriate expression of MMPs may contribute to the pathogenesis of tissue-destructive processes in a wide variety of diseases, including pulmonary hypertension (7). MMPs are produced by a number of adventitial cells, including fibroblasts and macrophages. Indeed, upregulated expression of these MMPs may be necessary for the fibroblast to move through the adventitial matrix into the media and even intima (94). MMP activity is upregulated in adventitial fibroblasts from animals with hypoxia-induced pulmonary hypertension. The potent proteolytic activities of MMPs are regulated by specific tissue inhibitors of metalloproteinases (TIMP), the activity of which have been reported to be decreased in various vasculopathies, thus creating an environment in the adventitia conducive to cell migration (114). Inhibition of MMP activity attenuates monocrotaline and hypoxia-induced pulmonary hypertension (36, 114). It is thus clear that adventitial fibroblasts dramatically change their production of ECM proteins in response to vascular stress. These changes facilitate fibroblast proliferation, migration, and differentiation. Each of these changes, in turn, affects vascular function and structure. Thus cell-matrix interactions may provide novel therapeutic targets in the prevention of remodeling that characterizes PA hypertension.

In summary, it is becoming increasingly clear that the adventitia should no longer be considered a nearly inert support structure for the vessel wall. Rather, accumulating evidence now supports the idea that the adventitia is a biological processing center for a wide variety of pathological stimuli. Resident adventitial fibroblasts, because of their distinct biochemical properties and interactions with a unique matrix protein environment, are poised to be activated in response to a variety of potentially injurious stimuli. The activated fibroblast, a process that appears to occur largely through NAD(P)H oxidase-dependent signaling, is capable of a wide array of responses, including proliferation, differentiation, migration, and secretion of paracrine factors, which have profound influences on vascular wall structure and function. Thus the adventitia, and specifically the adventitial fibroblast, under many conditions should be considered the conductor of a symphony of events that occur in the vessel wall to impart changes in function and structure. Work in the future must be directed at understanding how to control adventitial fibroblast activation to reduce unwanted changes in vascular structure. In addition, we must consider the adventitia as a potential platform for the delivery of drugs and agents that will have profound effects on overall vascular wall structure and function.


This study was supported by National Heart, Lung, and Blood Institute Grants HL-57144-09, HL-14985-33 (K. R. Stenmark), HL-64917-05 (M. Das), and American Heart Association Grants 055056Z (M. Frid) and 0560061Z (N. Davie).


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