Background-The plexiform lesion is the hallmark of severe pulmonary arterial hypertension. However, its genesis and hemodynamic effects are largely unknown because of the limited availability of lung tissue samples from patients with pulmonary arterial hypertension and the lack of appropriate animal models. This study investigated whether rats with severe progressive pulmonary hypertension developed plexiform lesions. Methods and Results-After a single subcutaneous injection of the vascular endothelial growth factor receptor blocker Sugen 5416, rats were exposed to hypoxia for 3 weeks. They were then returned to normoxia for an additional 10 to 11 weeks. Hemodynamic and histological examinations were performed at 13 to 14 weeks after the Sugen 5416 injection. All rats developed pulmonary hypertension (right ventricular systolic pressure Ϸ100 mm Hg) and severe pulmonary arteriopathy, including concentric neointimal and complex plexiform-like lesions. There were 2 patterns of complex lesion formation: a lesion forming within the vessel lumen (stalk-like) and another that projected outside the vessel (aneurysm-like). Immunohistochemical analyses showed that these structures had cellular and molecular features closely resembling human plexiform lesions. Conclusions-Severe, sustained pulmonary hypertension in a very late stage of the Sugen 5416/hypoxia/normoxiaexposed rat is accompanied by the formation of lesions that are indistinguishable from the pulmonary arteriopathy of human pulmonary arterial hypertension. This unique model provides a new and rigorous approach for investigating the genesis, hemodynamic effects, and reversibility of plexiform and other occlusive lesions in pulmonary arterial hypertension. (Circulation. 2010;121:2747-2754.)

Abstract-Vascular remodeling, rather than vasoconstriction, is believed to account for high vascular resistance in severe pulmonary arterial hypertension (PAH). We have found previously that acute Rho kinase inhibition nearly normalizes PAH in chronically hypoxic rats that have no occlusive neointimal lesions. Here we examined whether Rho kinase-mediated vasoconstriction was also important in a rat model of severe occlusive PAH. Adult rats were exposed to chronic hypoxia (Ϸ10% O 2 ) after subcutaneous injection of the vascular endothelial growth factor receptor inhibitor SUGEN 5416. Hemodynamic measurements were made in anesthetized rats after 2 weeks of hypoxia (early group) and 3 weeks of hypoxia plus 2 weeks of normoxia (late group). Both groups developed PAH, with greater severity in the late group. In the early group, intravenous fasudil was more effective than intravenous bradykinin, inhaled NO, or intravenous iloprost in reducing right ventricular systolic pressure. Despite more occlusive vascular lesions, fasudil also markedly reduced right ventricular systolic pressure in late-stage rats. Blood-perfused lungs from late-stage rats showed spontaneous vasoconstriction, which was reversed partially by the endothelin A receptor blocker BQ123 and completely by fasudil or Y-27632. Phosphorylation of MYPT1, a downstream target of Rho kinase, was increased in lungs from both groups of rats, and fasudil (intravenous) reversed the increased phosphorylation in the late group. Thus, in addition to structural occlusion, Rho kinase-mediated vasoconstriction is an important component of severe PAH in SUGEN 5416/hypoxia-exposed rats, and PAH can be significantly reduced in the setting of a severely remodeled lung circulation if an unconventional vasodilator is used. S evere pulmonary arterial hypertension (PAH) in adult patients is characterized by progressive narrowing/occlusion of small pulmonary arteries, which frequently leads to right heart failure and death. 1,2 Factors thought to contribute to the formation of pulmonary vascular lesions include sustained vasoconstriction, vascular remodeling, and in situ thrombosis. However, it is now widely believed that fixed obstruction resulting from vascular remodeling is the major cause of the elevated vascular resistance in severe, progressive PAH. 3,4 Large clinical studies show that only Ϸ13% of adult PAH patients have a significant decrease in pulmonary artery pressure during acute vasodilator testing at the time of diagnosis, 5 suggesting a major fixed structural but minor reversible vasoconstrictor component in this group of pulmonary vascular diseases.Reeves et al proposed in 1986 that over time in PAH, the hypertensive component attributable to vasoconstriction decreases, whereas that attributable to fixed obstruction increases. 6 This concept cannot be tested clinically, because it is essentially impossible to obtain serial hemodynamic data and matched lung tissue samples for thorough assessment of the lung vascular morphology. At best, a single lung specimen becom...

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.

Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 287: L665-L672, 2004. First published September 5, 2003 10.1152/ ajplung.00050.2003.-Recent evidence suggests that Rho/Rho kinase signaling plays an important role in the sustained vasoconstriction induced by many agonists and is involved in the pathogenesis of systemic vascular diseases. However, little is known about its role in increased vascular tone in hypoxic pulmonary hypertension (PH). The purpose of this study was to examine whether Rho/Rho kinasemediated Ca 2ϩ sensitization contributed to sustained vasoconstriction and increased vasoreactivity in hypoxic PH in rats. Acute intravenous administration of Y-27632, a Rho kinase inhibitor, nearly normalized the high pulmonary arterial blood pressure and total pulmonary resistance in chronically hypoxic rats. In contrast to nifedipine, Y-27632 also markedly decreased elevated basal vascular tone in hypertensive blood-perfused lungs and isolated pulmonary arteries. Y-27632 and another Rho kinase inhibitor, HA-1077, completely reversed nitro-L-arginine-induced vasoconstriction in physiological salt solution-perfused hypertensive lungs, whereas inhibitors of myosin light chain kinase (ML-9), protein kinase C (GF-109203X), phosphatidylinositol 3-kinase (LY-294002), and tyrosine kinase (tyrphostin A23) caused only partial or no reversal of the vasoconstriction. Vasoconstrictor responses to KCl were augmented in hypertensive physiological salt solution-perfused lungs and pulmonary arteries, and the augmentation was eliminated by Y-27632. These results suggest that Rho/Rho kinase-mediated Ca 2ϩ sensitization plays a central role in mediating sustained vasoconstriction and increased vasoreactivity in hypoxic PH.hypoxic pulmonary hypertension; Y-27632; calcium sensitization; RhoA SUSTAINED ABNORMAL VASOCONSTRICTION is one of the major causes of many cardiovascular diseases, including pulmonary hypertension (PH). ]. This is referred to as Ca 2ϩ sensitization (9, 43). Recent evidence indicates that although Ca 2ϩ /calmodulindependent MLCK-mediated MLC phosphorylation is the key factor for triggering VSMC contraction, Ca 2ϩ sensitization is important for the sustained phase of contraction (9, 43).The small GTPase RhoA, a member of the Rho family of small GTP-binding proteins, and its downstream effector Rho kinase (Rho/Rho kinase signaling) play a major role in the regulation of MLCP activity and, thus, Ca 2ϩ sensitization (9, 30, 43). RhoA is activated by various vasoconstrictors, including thromboxane, endothelin-1 (ET-1), and serotonin, the receptors of which are coupled to G proteins. Thus Rho/Rho kinase-mediated Ca 2ϩ sensitization is thought to be a major component in the sustained vasoconstriction induced by G protein-coupled receptor agonists. Selective Rho kinase inhibitors, such as Y-27632 (45) and fasudil (HA-1077) (2, 41, 45), effectively reverse the sustained vasoconstriction induced by many agonists (3,25,34).The pathogenesi...

We have found in chronically hypoxic rats that acute intravenous administration of the Rho kinase inhibitor Y-27632 nearly normalizes the pulmonary hypertension (PH) but has no pulmonary vascular selectivity. In this study, we tested if oral or inhaled Y-27632 would be an effective and selective pulmonary vasodilator in hypoxic PH. Although acute oral Y-27632 caused a marked and sustained decrease in mean pulmonary arterial pressure (MPAP), it also decreased mean systemic arterial pressure (MSAP). In contrast, 5 minutes of inhaled Y-27632 decreased MPAP without reducing MSAP. The hypotensive effect of inhaled Y-27632 on hypoxic PH was greater than that of inhaled nitric oxide, and the effect lasted for at least 5 hours. Inhaled fasudil, another Rho kinase inhibitor, caused selective MPAP reductions in monocrotaline-induced PH and in spontaneous PH in fawn-hooded rats, as well as in chronically hypoxic rats. These results suggested that inhaled Y-27632 was more effective than inhaled nitric oxide as a selective pulmonary vasodilator in hypoxic PH, and that Rho kinase-mediated vasoconstriction was also involved in the other models of PH. Inhaled Rho kinase inhibitors might be useful for acute vasodilator testing in patients with PH, and future work should evaluate their efficacy in the long-term treatment of PH.

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.

Current pharmacological interventions for pulmonary arterial hypertension (PAH) require continuous infusions, multiple inhalations, or oral administration of drugs that act on various pathways involved in the pathogenesis of PAH. However, invasive methods of administration, short duration of action, and lack of pulmonary selectivity result in noncompliance and poor patient outcomes. In this study, we tested the hypothesis that encapsulation of an investigational anti-PAH molecule fasudil (HA-1077), a Rho-kinase inhibitor, into liposomal vesicles results in prolonged vasodilation in distal pulmonary arterioles. Liposomes were prepared by hydration and extrusion method and fasudil was loaded by ammonium sulfate-induced transmembrane electrochemical gradient. Liposomes were then characterized for various physicochemical properties. Optimized formulations were tested for pulmonary absorption and their pharmacological efficacy in a monocrotaline (MCT) induced rat model of PAH. The entrapment efficiency of optimized liposomal fasudil formulations was between 68.1±0.8% and 73.6±2.3%, and the cumulative release at 37°C was 98–99% over a period of 5 days. Compared to intravenous (IV) fasudil, a ~10 fold increase in the terminal plasma half-life was observed when liposomal fasudil was administered as aerosols. The t1/2 of IV fasudil was 0.39±0.12 h. and when given as liposomes via pulmonary route, the t1/2 extended to 4.71±0.72 h. One h after intratracheal instillation of liposomal fasudil, mean pulmonary arterial pressure (MPAP) was reduced by 37.6±5.7% and continued to decrease for about 3 h, suggesting that liposomal formulations produced pulmonary preferential vasodilation in MCT induced PAH rats. Overall, this study established the proof-of-principle that aerosolized liposomal fasudil is a feasible option for a non-invasive, controlled release and pulmonary preferential treatment of PAH.

A burgeoning body of evidence suggests that RhoA/Rho kinase (ROCK) signalling plays an important role in the pathogenesis of various experimental models of pulmonary hypertension (PH), including chronic hypoxia-, monocrotaline-, bleomycin-, shunt-and vascular endothelial growth factor receptor inhibition plus chronic hypoxia-induced PH. ROCK has been incriminated in pathophysiologic events ranging from mediation of sustained abnormal vasoconstriction to promotion of vascular inflammation and remodelling. In addition, the 3-hydoxy-3-methylglutaryl CoA reductase inhibitors, statins, which inhibit activation of RhoA by preventing post-translational isoprenylation of the protein and its translocation to the plasma membrane ameliorate PH in several different rat models, and may also be effective in PH patients. Also, phosphorylation of RhoA and prevention of its translocation to the plasma membrane are involved in the protective effect of the type 5-PDE inhibitor, sildenafil, against hypoxia-and bleomycin-induced PH. Collectively, these and other observations indicate that independent of the cause of PH, activation of the RhoA/ROCK pathway serves as a point of convergence of various signalling cascades in the pathogenesis of the disease. We propose that ROCK inhibitors and other drugs that inhibit this pathway might be useful in the treatment of various forms of PH.