PAH exists in both an idiopathic form and in association with a wide variety of diseases such as HIV, connective tissue diseases such as scleroderma, liver disease, and exposure to certain drugs and toxins (24
). Despite the wide variety of underlying causes, patients with PAH tend to develop common histologic findings of the lung vasculature. These findings include a proliferation of smooth muscle cells with medial hypertrophy and the muscularization of arterioles, in situ
thrombosis, the proliferation of ECs with the formation of neointima and plexogenic lesions, and the infiltration of vascular lesions with inflammatory cells (macrophages and lymphocytes) (6
). Vascular injury with a secondary inflammatory response appears to be a common finding that may help explain the shared histologic endpoint for the diverse variety of diseases causing pulmonary hypertension.
Inflammation plays a key role in vascular biology. The role of inflammatory cells (and particularly T cells) in peripheral vascular disease and atherosclerosis is well known. Markers of chronic inflammation, such as C-reactive protein and macrophage colony–stimulating factor, were shown to be predictors of cardiovascular disease, including PAH in humans (2
). T and B cells were shown to be key players in the development of atherosclerotic lesions, with immunodeficient mice showing a 40–80% decrease in the formation of lesions (26
). When apoprotein E–deficient mice, which are hypercholesterolemic and prone to the formation of atherosclerosis, are crossed with severe combined immunodeficiency mice, which lack T and B cells, the formation of atherosclerotic lesions drops by 70%. Interestingly, when CD4+
T cells are transferred into these mice, the development of atherosclerosis is similar to that in immunocompetent control mice (28
). Similarly, in a wire injury model of vascular disease, the importance of T cells was demonstrated by the observation that Rag1−/−
mice were protected from vascular proliferation after injury, as were mice that had undergone a thymectomy (17
). This shows that an inflammatory response (including CD4+
T cells) to a vascular injury seems to play a key role in the remodeling process of the vessel wall.
The role of inflammatory cells in pulmonary vascular disease is less clear. Although they were described as present in lesions of PAH, whether they play a role in the pathogenesis and progression of the disease or are incidental findings is unclear. Evidence suggests that the immune system may play an active role in disease progression. PAH, a rare disease with an incidence of about six cases per million in the idiopathic form, has an increased prevalence in patients with autoimmune disorders such as systemic sclerosis and lupus (4
). Patients with PAH were found to have elevated concentrations of autoantibodies (30
). Patients with idiopathic PAH have increased concentrations of FoxP3+
T cells or T-regulatory cells in peripheral blood compared with control subjects (32
). These findings suggest that the immune system, and particularly T cells, may play a role in the pathogenesis of pulmonary hypertension.
Here we describe a murine model of monocrotaline-induced pulmonary vascular injury and remodeling. Monocrotaline-induced pulmonary hypertension in rats is a well-established animal model of the disease (33
). It is characterized by monocrotaline-induced EC injury, followed by an inflammatory response and vascular remodeling (34
). Few publications have described this model in mice (35
). Interspecies differences in the amount of liver cytochrome P450 3A enzyme required to convert monocrotaline to its toxic pyrrole metabolite may explain the difficulty in establishing a murine model of monocrotaline-induced pulmonary hypertension. Mice require a much higher exposure to this toxin than do rats to induce pulmonary vascular injury (35
Within 1 week of injection with monocrotaline, we began to see histologic evidence of EC injury. Serum ACE concentrations, previously shown to correlate with lung injury, were significantly upregulated (39
). Furthermore, VEGF concentrations were decreased, which was shown to occur in the rat monocrotaline model as well as in a rat model of pulmonary fibrosis with pulmonary hypertension. This decrease in VEGF concentrations is thought to be related to EC injury and apoptosis (40
We found that serial injections with monocrotaline led to a perivascular CD4+
T-cell inflammatory response. The inflammation was characterized by elevations in the T-cell inflammatory cytokine IL-6. This response began as early as 1 week after the initial injection with monocrotaline, and lasted as long as the exposure to monocrotaline. The chemokine MCP-1 appeared to play a role in the trafficking of CD4+
T cells. MCP-1 was observed to be trending upward by 2 weeks of injections with monocrotaline, and was significantly elevated by 4 weeks, which correlated with the infiltration of CD3+
T cells. This chemokine was previously shown to play a role in experimental animal models of pulmonary hypertension and in human disease (42
). Specifically, blocking the MCP-1 signaling pathway was shown to attenuate the development of pulmonary hypertension in the rat monocrotaline model of pulmonary hypertension (43
). MCP-1 concentrations were shown to be significantly elevated in the serum of patients with idiopathic PAH, compared with control subjects (42
The inflammatory reaction induced by monocrotaline led to a remodeling of the pulmonary vasculature. This was characterized by medial thickening and smooth muscle proliferation, with increased muscularization (as assessed by smooth muscle actin staining) of the small (<30 μm) pulmonary arterioles. MMP-2 concentrations were elevated and likely play a role in remodeling of the vessel wall. MMP-2 participates in the degradation of basement membrane, facilitates smooth muscle proliferation, and was previously shown to play a role in rat models of pulmonary hypertension (21
). These vascular changes coincide with an increase in RV pressures and RV hypertrophy. The remodeling of vessels persisted even after 4 weeks off monocrotaline therapy, despite a drop in the number of perivascular inflammatory cells. RV pressure and the Fulton index remained significantly elevated above control levels. This suggests that inflammatory remodeling appears to be fixed, and that it may lead to a progressive rise in pressures and right heart failure, even after the removal of the inflammatory insult.
We found that CD4+ T cells are necessary for the induction of pulmonary hypertension by monocrotaline in mice. When Rag1−/− mice are exposed to monocrotaline, they have an attenuated inflammatory response, and markers of vascular remodeling such as MMP-2 are not significantly elevated. Furthermore, they do not manifest significant elevations in RV pressures or increased RV hypertrophy. When CD 4+ cells are transplanted from normal control mice into Rag1−/− mice and are then exposed to serial injections of monocrotaline, they experience exuberant perivascular inflammation, a threefold rise in MMP-2 concentrations, and a significant rise in RV pressures and the Fulton index.
Our findings highlight the important role of inflammation, and specifically of CD4+
T cells, in the propagation of vascular remodeling in a monocrotaline model of vascular injury. These findings seem to be in conflict with the findings of Taraseviciene-Stewart and colleagues, who showed a protective role for T cells in the development of a proliferative vasculopathy in athymic nude rats exposed to vascular endothelial growth factor receptor 2 inhibitor (44
). Several key differences between our studies may help explain these seemingly disparate findings. First, the athymic nude rat is known to be deficient in T cells, but with normal B-cell function and increased natural killer and macrophage cell populations. In contrast, wild-type mice obviously have an intact immune system, and Rag1−/−
mice are characterized by an absence of mature T or B cells. These three different animal lines have very different immunologic backgrounds, and thus likely different immunologic responses to vascular injury. Furthermore, the vascular injury caused by the VEGF inhibitor is likely different from that caused by serial monocrotaline injections. The end result of the injury in each model is also quite different. The model of Taraseviciene-Stewart and colleagues (44
) attempted to model PAH by inducing an EC proliferative response, whereas the monocrotaline model involves inflammatory injury that is not primarily characterized by EC proliferation. The presence of T cells appears to play an important role in the response of athymic nude rats to exposure to SU5416. The absence of T cells likely alters the immune response and allows for EC proliferation under normoxic conditions. The introduction of T cells into the athymic rat presumably normalizes the immune response and confers protection from SU5416 during normoxia. These two studies are linked insofar as they both highlight the importance of inflammation and immune response to vascular injury and vascular remodeling. However, we feel that the specific role of T cells differs because of the differences in animal models used and the mode of injury in the separate experiments.
We found that monocrotaline induces EC damage in the pulmonary vasculature of mice. Vascular remodeling in response to this EC injury was characterized by CD4+ T cell–dominated inflammation and smooth muscle cell proliferation. This CD4+ T cell–dependent remodeling of the pulmonary vasculature leads to a rise in RV pressure and RV hypertrophy. This model of monocrotaline-induced pulmonary artery injury and remodeling allows for insights into the role of inflammation in the remodeling of the pulmonary vasculature, and further facilitates the use of genetic murine models in pulmonary vascular research. This, in turn, may help us understand the role of inflammation in vascular lesions in human pulmonary hypertension.