Progression of pulmonary hypertension is associated with increased proliferation and migration of pulmonary vascular smooth muscle cells. PDGF is a potent mitogen and involved in this process. We now report that the PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension. In rats with monocrotaline-induced pulmonary hypertension, therapy with daily administration of STI571 was started 28 days after induction of the disease. A 2-week treatment resulted in 100% survival, compared with only 50% in sham-treated rats. The changes in RV pressure, measured continuously by telemetry, and right heart hypertrophy were reversed to near-normal levels. STI571 prevented phosphorylation of the PDGF receptor and suppressed activation of downstream signaling pathways. Similar results were obtained in chronically hypoxic mice, which were treated with STI571 after full establishment of pulmonary hypertension. Moreover, expression of the PDGF receptor was found to be significantly increased in lung tissue from pulmonary arterial hypertension patients compared with healthy donor lung tissue. We conclude that STI571 reverses vascular remodeling and cor pulmonale in severe experimental pulmonary hypertension regardless of the initiating stimulus. This regimen offers a unique novel approach for antiremodeling therapy in progressed pulmonary hypertension.

Rationale:: The function of Nox4, a source of vascular H ₂ O ₂ , is unknown. Other Nox proteins were identified as mediators of endothelial dysfunction. Objective:: We determined the function of Nox4 in situations of increased stress induced by ischemia or angiotensin II with global and tamoxifen-inducible Nox4 ^−/− mice. Methods and Results:: Nox4 was highly expressed in the endothelium and contributed to H ₂ O ₂ formation. Nox4 ^−/− mice exhibited attenuated angiogenesis (femoral artery ligation) and PEG-catalase treatment in control mice had a similar effect. Tube formation in cultured Nox4 ^−/− lung endothelial cells (LECs) was attenuated and restored by low concentrations of H ₂ O _2, whereas PEG-catalase attenuated tube formation in control LECs. Angiotensin II infusion was used as a model of oxidative stress. Compared to wild-type, aortas from inducible Nox4-deficient animals had development of increased inflammation, media hypertrophy, and endothelial dysfunction. Mechanistically, loss of Nox4 resulted in reduction of endothelial nitric oxide synthase expression, nitric oxide production, and heme oxygenase-1 (HO-1) expression, which was associated with apoptosis and inflammatory activation. HO-1 expression is controlled by Nrf-2. Accordingly, Nox4-deficient LECs exhibited reduced Nrf-2 protein level and deletion of Nox4 reduced Nrf-2 reporter gene activity. In vivo treatment with hemin, an inducer of HO-1, blocked the vascular hypertrophy induced by Nox4 deletion in the angiotensin II infusion model and carbon monoxide, the product of HO-1, blocked the Nox4-deletion-induced apoptosis in LECs. Conclusion:: Endogenous Nox4 protects the vasculature during ischemic or inflammatory stress. Different from Nox1 and Nox2, this particular NADPH oxidase therefore may have a protective vascular function.

Tetrahydrobiopterin (BH₄) serves as a critical co-factor for the endothelial nitric-oxide synthase (eNOS). A deficiency of BH₄ results in eNOS uncoupling, which is associated with increased superoxide and decreased NO^• production. BH₄ has been suggested to be a target for oxidation by peroxynitrite (ONOO^–), and ascorbate has been shown to preserve BH₄ levels and enhance endothelial NO^• production; however, the mechanisms underlying these processes remain poorly defined. To gain further insight into these interactions, the reaction of ONOO^– with BH₄ was studied using electron spin resonance and the spin probe 1-hydroxy-3-carboxy-2,2,5-tetramethyl-pyrrolidine. ONOO^– reacted with BH₄ 6–10 times faster than with ascorbate or thiols. The immediate product of the reaction between ONOO^– and BH₄ was the trihydrobiopterin radical ( ), which was reduced back to BH₄ by ascorbate, whereas thiols were not efficient in recycling of BH₄. Uncoupling of eNOS caused by peroxynitrite was investigated in cultured bovine aortic endothelial cells (BAECs) by measuring superoxide and NO^• using spin probe 1-hydroxy-3-methoxycarbonyl-2,2,5,5-tetramethyl-pyrrolidine and the NO^•-spin trap iron-diethyldithiocarbamate. Bolus ONOO^–, the ONOO^– donor 3-morpholinosydnonimine, and an inhibitor of BH₄ synthesis (2,4-diamino-6-hydroxypyrimidine) uncoupled eNOS, increasing superoxide and decreasing NO^• production. Exogenous BH₄ supplementation restored endothelial NO^• production. Treatment of BAECs with both BH₄ and ascorbate prior to ONOO^– prevented uncoupling of eNOS by ONOO^–. This study demonstrates that endothelial BH₄ is a crucial target for oxidation by ONOO^– and that the BH₄ reaction rate constant exceeds those of thiols or ascorbate. We confirmed that ONOO^– uncouples eNOS by oxidation of tetrahydrobiopterin and that ascorbate does not fully protect BH₄ from oxidation but recycles radical back to BH₄.