We have investigated the temporal relationship between the hemodynamic and histological/morphological progression in a rat model of pulmonary arterial hypertension that develops pulmonary arterial lesions morphologically indistinguishable from those in human pulmonary arterial hypertension. Adult male rats were injected with Sugen5416 and exposed to hypoxia for 3 wk followed by a return to normoxia for various additional weeks. At 1, 3, 5, 8, and 13 wk after the Sugen5416 injection, hemodynamic and histological examinations were performed. Right ventricular systolic pressure reached its maximum 5 wk after Sugen5416 injection and plateaued thereafter. Cardiac index decreased at the 3∼5-wk time point, and tended to further decline at later time points. Reflecting these changes, calculated total pulmonary resistance showed a pattern of progressive worsening. Acute intravenous fasudil markedly reduced the elevated pressure and resistance at all time points tested. The percentage of severely occluded small pulmonary arteries showed a similar pattern of progression to that of right ventricular systolic pressure. These small vessels were occluded predominantly with nonplexiform-type neointimal formation except for the 13-wk time point. There was no severe occlusion in larger arteries until the 13-wk time point, when significant numbers of vessels were occluded with plexiform-type neointima. The Sugen5416/hypoxia/normoxia-exposed rat shows a pattern of chronic hemodynamic progression similar to that observed in pulmonary arterial hypertension patients. In addition to vasoconstriction, nonplexiform-type neointimal occlusion of small arteries appears to contribute significantly to the early phase of pulmonary arterial hypertension development, and plexiform-type larger vessel occlusion may play a role in the late deterioration.

A major limitation in the pharmacological treatment of pulmonary arterial hypertension (PAH) is the lack of pulmonary vascular selectivity. Recent studies have identified a tissue-penetrating homing peptide, CARSKNKDC (CAR), which specifically homes to hypertensive pulmonary arteries but not to normal pulmonary vessels or other tissues. Some tissue-penetrating vascular homing peptides have a unique ability to facilitate transport of co-administered drugs into the targeted cells/tissues without requiring physical conjugation of the drug to the peptide (bystander effect). We tested the hypothesis that co-administered CAR would selectively enhance the pulmonary vascular effects of i.v. vasodilators in Sugen5416/hypoxia/normoxia-exposed PAH rats. Systemically administered CAR was predominantly detected in cells of remodeled pulmonary arteries. Intravenously co-administered CAR enhanced pulmonary, but not systemic, effects of the vasodilators, fasudil and imatinib, in PAH rats. CAR increased lung tissue imatinib concentration in isolated PAH lungs without increasing pulmonary vascular permeability. Sublingual CAR was also effective in selectively enhancing the pulmonary vasodilation by imatinib and sildenafil. Our results suggest a new paradigm in the treatment of PAH, using an i.v./sublingual tissue-penetrating homing peptide to selectively augment pulmonary vascular effects of nonselective drugs without the potentially problematic conjugation process. CAR may be particularly useful as an add-on therapy to selectively enhance the pulmonary vascular efficacy of any ongoing drug treatment in patients with PAH.

Current therapy of pulmonary arterial hypertension (PAH) is inadequate. Dehydroepiandrosterone (DHEA) effectively treats experimental pulmonary hypertension in chronically hypoxic and monocrotaline-injected rats. Contrary to these animal models, SU5416/hypoxia/normoxia-exposed rats develop a more severe form of occlusive pulmonary arteriopathy and right ventricular (RV) dysfunction that is indistinguishable from the human disorder. Thus, we tested the effects of DHEA treatment on PAH and RV structure and function in this model. Chronic (5 wk) DHEA treatment significantly, but moderately, reduced the severely elevated RV systolic pressure. In contrast, it restored the impaired cardiac index to normal levels, resulting in an improved cardiac function, as assessed by echocardiography. Moreover, DHEA treatment inhibited RV capillary rarefaction, apoptosis, fibrosis, and oxidative stress. The steroid decreased NADPH levels in the RV. As a result, the reduced reactive oxygen species production in the RV of these rats was reversed by NADPH supplementation. Mechanistically, DHEA reduced the expression and activity of Rho kinases in the RV, which was associated with the inhibition of cardiac remodeling-related transcription factors STAT3 and NFATc3. These results show that DHEA treatment slowed the progression of severe PAH in SU5416/hypoxia/normoxia-exposed rats and protected the RV against apoptosis and fibrosis, thus preserving its contractile function. The antioxidant activity of DHEA, by depleting NADPH, plays a central role in these cardioprotective effects.

Pulmonary arterial hypertension (PAH) is characterized by elevated pulmonary arterial pressure with lumen-occluding neointimal and plexiform lesions. Activation of store-operated calcium entry channels promotes contraction and proliferation of lung vascular cells. TRPC4 is a ubiquitously expressed store-operated calcium entry channel, but its role in PAH is unknown. We tested the hypothesis that TRPC4 promotes pulmonary arterial constriction and occlusive remodeling, leading to right ventricular failure in severe PAH. Severe PAH was induced in Sprague-Dawley rats and in wild-type and TRPC4-knockout Fischer 344 rats by a single subcutaneous injection of SU5416 [SU (semaxanib)], followed by hypoxia exposure (Hx; 10% O2) for 3 weeks and then a return to normoxia (Nx; 21% O2) for 3 to 10 additional weeks (SU/Hx/Nx). Although rats of both backgrounds exhibited indistinguishable pulmonary hypertensive responses to SU/Hx/Nx, Fischer 344 rats died within 6 to 8 weeks. Normoxic and hypertensive TRPC4-knockout rats recorded hemodynamic parameters similar to those of their wild-type littermates. However, TRPC4 inactivation conferred a striking survival benefit, due in part to preservation of cardiac output. Histological grading of vascular lesions revealed a reduction in the density of severely occluded small pulmonary arteries and in the number of plexiform lesions in TRPC4-knockout rats. TRPC4 inactivation therefore provides a survival benefit in severe PAH, associated with a decrease in the magnitude of occlusive remodeling.

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Circulating Microparticles from Pulmonary Arterial Hypertension stimulate ICAM‐1 and E‐selectin expression in Pulmonary Artery Endothelial CellsHargett L1, O’Neill K, Yarbrough T1, and Bauer N1,2. the Center for Lung Biology1 and Department of Pharmacology 2, University of South Alabama, College of Medicine, Mobile AL 36688. Microparticles (MPs) are submicron vesicles released by various cells that serve as mediators of inflammation. MPs harbor adhesion molecules and can stimulate the expression n of adhesion molecules in target cells. MPs are increased in pulmonary arterial hypertension (PAH). The hallmark PAH arterial vasculopathy involves inflammatory cell infiltration. Rho kinase (ROCK) is implicated in the expression of adhesion molecules in the pulmonary endothelium. We propose that MPs, generated as a result of PAH, activate ROCK and increasing the expression of adhesion molecules in pulmonary endothelium. MPs were isolated from the blood of normal and PAH rats (Su/Nx/Hx model). Pulmonary artery endothelial cells (PAECs) were treated with media, MPs from normal rats, or MPs from PAH rats. ICAM‐1 and E‐selectin, as assessed by western and flow cytometry, were increased in the PAECs. ROCK activity, as indicated by phosphorylated MYPT1696 western, was increased in PAH‐MP treated PAECs. Our results indicate that MPs from 8 week PAH rats increase the expression of ICAM‐1 and E‐selectin selectively in PAECs 6 hours after treatment. Also, preliminary data show increased phosphorylated MYPT1 in response to MPs from 8 week PAH rats. In conclusion, MPs from PAH rats increase adhesion molecule expression and may enhance ROCK activity in PAECs in vitro. As our studies progress, an inflammatory role for MPs in PAH could be revealed. This research is funded by AHA 11 (SDG7390037) Grant and NHLBI T32 (HL076125) Training Grant.

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