Hypoxia-induced pulmonary hypertension (PH) observed in chronic obstructive pulmonary diseases such as emphysema and chronic bronchitis, and sleep-related alveolar hypoventilation disorders (1
) is a major cause of morbidity and mortality. There has been a doubling of PH and increased death rate from this set of diseases in the past two decades. Despite major advances in the treatment of severe cardiopulmonary conditions, PH remains a deadly disease that is largely unresponsive to current treatment regimens.
Hypoxia-induced PH is characterized by profound structural remodeling of the PA wall due in large part to the proliferation, migration, and hypertrophy of PA SMCs and adventitial fibroblasts. The most important changes involve the remodeling of thin-walled intermediate size arteries and small arterioles to thickened walled “resistance vessels” which are believed to have the greatest impact on pulmonary vascular resistance (2
). Remodeling of these arteries is characterized by medial thickening as the result of SMC proliferation, hypertrophy, and extracellular matrix (ECM) deposition. In response to injury, normally quiescent SMCs switch to a proliferative and synthetic phenotype as demonstrated by an increase in DNA synthesis and production of ECM components (3
Changes in SMC phenotype reflect the action of extracellular signals and intracellular signaling systems that regulate biochemical processes and the expression of genes that regulate growth. Hypoxia elicits changes in SMC phenotype via a variety of mechanisms including induction of Ca2+ influx (4
), and the production of reactive oxygen species by SMCs (6
). Importantly, hypoxia, like other vascular insults, stimulates the expression of growth factors including PDGF-BB and IGF-1 (11
), VEGF (12
), TGF-β, endothelin-1 (11
), and thrombos-pondin-1 (12
) that modulate SMC phenotype.
These remodeling pathways are normally restrained in healthy arteries due to the low level of growth factor and cytokine production in healthy arterial walls. In addition, vasodilatory agents such as prostacyclins and NO exert antiproliferative effects on SMCs by increasing intracellular levels of cyclic nucleotides (13
), which stimulate the activity of protein kinases A and G (PKA and PKG). There is now substantial evidence that cAMP/PKA signaling acts as a molecular gate to block MAP kinase induced proliferation in response to mitogens like PDGF (14
). Cyclic AMP signaling in SMCs has also been shown to decrease the expression of cyclin D1 and Cdk2 (16
), and increase the expression of antiproliferative molecules like p53 and p21 (17
The transcription factor CREB is a target for cAMP/PKA signaling and the primary regulator of gene transcription in response to elevated cAMP levels. Therefore, we hypothesized that CREB might regulate PA SMC phenotype. In testing this hypothesis we found that phosphorylated (P-CREB) and total CREB levels were reduced in SMCs treated with PDGF-BB or exposed to hypoxia, but elevated in quiescent cells (18
). These data were confirmed in studies of lung and PA tissue samples from adult rats and neonatal cows raised under hypoxic conditions to produce PH. Total CREB content was reduced in actin positive SMCs surrounding remodeled, hypertensive vessels. Subsequent studies revealed that ectopic expression of wild type and constitutively active CREB isoforms in isolated PA SMCs inhibited their proliferation under basal conditions or in response to PDGF-BB (18
). These CREB isoforms also attenuated PDGF-stimulated migration, but dominant negative CREBs enhanced PDGF-stimulated migration (18
In more recent studies we found that siRNA-mediated depletion of CREB inhibited the expression of several SMC markers including SM-myosin, calponin, and fibronectin (19
). Cyclin D1 expression, and DNA synthesis were upregulated by CREB depletion. CREB siRNA also increased intracellular elastin production and deposition of extracellular elastin fibers, a hallmark of dedifferentiated or immature SMCs. The changes induced by loss of CREB are consistent with the changes in SMC phenotype noted in PH and other vascular pathologies including decreased expression of SMC markers, elevated SMC cell cycle entry and DNA synthesis, and enhanced extracellular matrix deposition. These data implicate CREB in the regulation of SMC phenotypic regulation, and link CREB to PA remodeling in hypoxia-induced PH.
In a separate series of experiments we investigated whether thiazolidinediones (TZDs), agonists of the transcription factor peroxisome proliferator-activated receptor γ (PPARγ), would prevent or reverse PA remodeling induced by chronic hypoxia. These studies were based on the ability of TZDs to prevent arterial remodeling and vasoconstriction in the systemic vasculature (20
). We reported that PA remodeling was reduced in rats exposed to hypoxia and simultaneously treated with the TZD rosiglitazone (ROSI). ROSI treatment also blocked muscularization of distal pulmonary arterioles, and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia.
Here we test the hypothesis that TZDs suppress PA remodeling and PA SMC phenotypic modulation, in part, by preventing the loss of CREB induced by hypoxia or PDGF. The data demonstrate that ROSI prevents CREB depletion and PA remodeling in rats exposed to chronic hypoxia. Likewise, ROSI blocks CREB loss and PA SMC proliferation induced with PDGF-BB in culture. Loss of CREB in response to PDGF is due to increased expression of CK2 catalytic subunit, which is increased in the PA media of animals exposed to chronic hypoxia. ROSI prevents upregulation of CK2 catalytic subunit and thereby prevents CREB depletion by hypoxia or PDGF.