Norbert Weissmann

Reversal of experimental pulmonary hypertension by PDGF inhibition

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 antire-modeling therapy in progressed pulmonary hypertension.

Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial

BACKGROUND Lung fibrosis can be complicated by pulmonary hypertension, limiting exercise tolerance and life expectancy. Furthermore, vasodilators might cause deterioration in gas exchange. Our aim was to compare acute effects of sildenafil, nitric oxide, and epoprostenol in individuals with pulmonary hypertension secondary to lung fibrosis. METHODS We did a randomised controlled, open-label trial, in 16 individuals admitted to our hospital with pulmonary hypertension secondary to lung fibrosis. After inhalation of nitric oxide (10-20 ppm), we assigned patients to either maximum tolerated dose of intravenous epoprostenol (mean 8.0 ng/kg per min; n=8) or oral sildenafil (50 mg; n=8). Our primary objective was to assess pulmonary vasodilative potency (decrease in pulmonary vascular resistance index) of sildenafil by comparison with inhaled nitric oxide and infused epoprostenol. Analyses were by intention to treat. FINDINGS Pulmonary vascular resistance index was reduced by nitric oxide (-21.9%, 95% CI -14.1 to -36.2), epoprostenol (-36.9%, -24.4 to -59.6), and sildenafil (-32.5%, -10.2 to -54.1). However, ratio of pulmonary to systemic vascular resistance decreased only in individuals who received nitric oxide and sildenafil. Baseline measurement of multiple-inert-gas elimination showed right-to-left shunt flow (4.8%, 0.0-28.2) and little perfusion of low ventilation(V)/perfusion(Q) areas (0.1%, 0.0-13.0). Prostacyclin increased V/Q mismatch (shunt 16.8%, 10.8-35.9; low V/Q 3.8%, 0.0-13.0) and decreased arterial oxygenation. By contrast, nitric oxide (4.5%, 0.0-18.0; 0.0%, 0.0-17.3) and sildenafil (3.3%, 0.0-11.3; 0.0%, 0.0-12.4) maintained V/Q matching, with raised arterial partial pressure of oxygen (14.3 mm Hg, -1.7 to 31.3) noted for sildenafil. We recorded no adverse events. INTERPRETATION Sildenafil causes preferential pulmonary vasodilation and improves gas exchange in patients with severe lung fibrosis and secondary pulmonary hypertension.

Cellular and molecular basis of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is caused by functional and structural changes in the pulmonary vasculature, leading to increased pulmonary vascular resistance. The process of pulmonary vascular remodeling is accompanied by endothelial dysfunction, activation of fibroblasts and smooth muscle cells, crosstalk between cells within the vascular wall, and recruitment of circulating progenitor cells. Recent findings have reestablished the role of chronic vasoconstriction in the remodeling process. Although the pathology of PAH in the lung is well known, this article is concerned with the cellular and molecular processes involved. In particular, we focus on the role of the Rho family guanosine triphosphatases in endothelial function and vasoconstriction. The crosstalk between endothelium and vascular smooth muscle is explored in the context of mutations in the bone morphogenetic protein type II receptor, alterations in angiopoietin-1/TIE2 signaling, and the serotonin pathway. We also review the role of voltage-gated K(+) channels and transient receptor potential channels in the regulation of cytosolic [Ca(2+)] and [K(+)], vasoconstriction, proliferation, and cell survival. We highlight the importance of the extracellular matrix as an active regulator of cell behavior and phenotype and evaluate the contribution of the glycoprotein tenascin-c as a key mediator of smooth muscle cell growth and survival. Finally, we discuss the origins of a cell type critical to the process of pulmonary vascular remodeling, the myofibroblast, and review the evidence supporting a contribution for the involvement of endothelial-mesenchymal transition and recruitment of circulating mesenchymal progenitor cells.

Combination Therapy with Oral Sildenafil and Inhaled Iloprost for Severe Pulmonary Hypertension

Context Common therapies for pulmonary hypertension have disadvantages: Continuous intravenous epoprostenol may cause sepsis, hypotension, and tachyphylaxis; nitric oxide requires continuous inhalation; and inhaled iloprost requires up to 12 doses per day. Phosphodiesterase in lung tissue (PDE-5) inhibits the action of these therapies by inactivating the second messengers of prostacyclin and nitric oxide (cyclic adenosine monophosphate and cyclic guanosine monophosphate). Because sildenafil blocks the action of PDE-5, thereby causing vascular dilatation, it could be useful in treating pulmonary hypertension. Contribution This randomized, controlled trial of low- or high-dose sildenafil, with or without inhaled iloprost, showed dose-dependent improvement in mean pulmonary artery pressure and hemodynamics with sildenafil alone. Iloprost amplified the effects. Implications Sildenafil may enhance the management of pulmonary hypertension. The Editors Severe pulmonary hypertension is a debilitating disease with short life expectancy that often affects young people. Continuous intravenous administration of epoprostenol has been used for pulmonary vasodilatation and was shown to improve exercise capacity and survival in patients with primary pulmonary hypertension (1). However, this therapy is limited by serious infectious complications of the intravenous line, systemic side effects due to the nonselectivity of the vasodilator response, and very high costs incurred because of tachyphylaxis with long-term administration. The vasodilatory effects of nitric oxide administered by inhalation are restricted to the pulmonary vasculature. Nitric oxide has a very short half-life and is used as a screening agent for lung vasoreactivity (2). Inhalation of aerosolized iloprost, a long-acting prostacyclin analogue, causes strong preferential pulmonary vasodilatation in both primary and secondary pulmonary hypertension (3-6). Long-term use of nebulized iloprost was shown to be beneficial in severe primary pulmonary hypertension and overt right-heart failure (7, 8), but because the drug wears off in about 60 minutes, patients must take 6 to 12 inhalations daily to achieve sustained relief of pulmonary hypertension. Phosphodiesterases are a superfamily of enzymes that inactivate cyclic adenosine monophosphate and cyclic guanosine monophosphate, the second messengers of prostacyclin and nitric oxide. The phosphodiesterases have different tissue distributions and substrate affinities (9); in particular, phosphodiesterase-5 is abundantly expressed in lung tissue (10). Inhibition of phosphodiesterase may augment and prolong prostanoid- and nitric oxiderelated vascular effects. The novel selective phosphodiesterase-5 inhibitor sildenafil has been approved for treatment of erectile dysfunction. Sildenafil causes only very minor systemic hemodynamic effects in healthy humans (11). Data from an experimental model of pulmonary hypertension (12) and two recent case reports (13, 14) suggested that sildenafil might be an effective pulmonary vasodilator. We compared the pulmonary vasodilatory effect of sildenafil with that of inhaled nitric oxide and aerosolized iloprost in 30 patients with severe pulmonary hypertension. We evaluated doseresponse characteristics of sildenafil alone and in combination with inhaled prostanoid during right-heart catheterization. Methods Patients Thirty patients (23 women and 7 men) with severe pulmonary hypertension (mean pulmonary arterial pressure > 40 mm Hg) were included. Sixteen patients had pulmonary arterial hypertension, as defined by the World Health Organization World Symposium on Primary Pulmonary Hypertension (15); of these patients, 10 had primary pulmonary hypertension and 6 had calcinosis, the Raynaud phenomenon, esophageal dysfunction, sclerodactyly, and telangiectasia (the CREST syndrome). One patient had aplasia of the left pulmonary artery. Thirteen patients had chronic thromboembolic pulmonary hypertension and were not candidates for surgery. All patients were admitted for testing of pulmonary vasoreactivity and evaluation of therapeutic options. Seventeen patients were tested for the first time and had not been previously treated with inhaled iloprost. Thirteen patients had been tested at least 3 months after previous vasodilator testing; 11 of these patients were receiving long-term therapy with inhaled iloprost. Exclusion criteria were pulmonary hypertension secondary to chronic obstructive pulmonary disease, pulmonary venous congestion, congenital heart disease, acute or chronic inflammatory lung disease, pregnancy or insufficient contraceptive measures, or previous treatment with phosphodiesterase inhibitors, including theophylline preparations. The individual response to vasodilators, including inhaled nitric oxide, was neither an inclusion nor an exclusion criterion. Treatment The study protocol was approved by the Justus-Liebig-University Ethics Committee, and each patient gave written informed consent. A 7.5-French SwanGanz fiberoptic thermodilution pulmonary artery catheter (93A-754H, Baxter Healthcare, Irvine, California) was inserted via the right jugular vein by using standard techniques. Each patient received short-term inhaled nitric oxide; the maximum vasodilator response to this agent required nitric oxide, 20 to 40 parts per million. If necessary, patients received nasal oxygen throughout the test to achieve arterial oxygen saturation greater than 88%. After hemodynamic values returned to baseline, inhaled iloprost was delivered by using an ultrasonic nebulizer (Multisonic compact [Otto Schill GmbH, Probstzella, Germany]; mass median aerodynamic diameter, 3.9 m; duration of inhalation, 4 minutes; total inhaled dose, 2.8 g) (16). Hemodynamic and gas exchange variables were measured at 5, 15, 30, 60, and 90 minutes after iloprost inhalation. At the end of a 2-hour observation period, when hemodynamics had returned to baseline values, patients were randomly assigned to one of four treatment groups: 12.5 mg of oral sildenafil alone (n = 7); 12.5 mg of oral sildenafil, followed by 2.8 g of inhaled iloprost 1 hour later (n = 7); 50 mg of oral sildenafil alone (n = 8); or 50 mg of oral sildenafil, followed by 2.8 g of inhaled iloprost 1 hour later (n = 8). Patients were assigned to the therapeutic regimens by using computerized randomization in groups of four; no more than two patients in a row were assigned to one group. Hemodynamic measurements and blood sampling were performed at 15, 30, 60, 90, and 120 minutes (and at 150 and 180 minutes in the group receiving 12.5 mg of sildenafil plus iloprost and in the group receiving 50 mg of sildenafil plus iloprost) after administration of oral sildenafil. To assess the overall vasodilator response, the area under the curve (AUC) of reduction in pulmonary vascular resistance was calculated as the integral of the difference between preintervention baseline values until pulmonary vascular resistance again reached 95% of baseline values or 120 minutes. Statistical Analysis Data are given as the mean and 95% CIs. For every variable, the response to vasodilator treatment was considered significant if the 95% (P < 0.05), 99% (P < 0.01), or 99.9% (P < 0.001) CI of the difference between pretreatment and post-treatment values did not overlap with zero. Two-way analysis of variance (ANOVA) was performed to test for significant differences and possible interaction of vasoreactivity to different vasodilators with underlying disease. One-way ANOVA with the Scheff post-test was used to determine hemodynamic responsiveness to the vasodilator regimens when two-way ANOVA indicated that underlying disease was a significant factor. Results Baseline Hemodynamics The Table shows baseline hemodynamics in all treatment groups. All patients had severe pulmonary hypertension and low cardiac index values. No pulmonary venous congestion was observed, as indicated by low pulmonary arterial occlusion pressure (8.7 0.6 mm Hg). On ANOVA, the groups did not differ significantly in any baseline characteristic. Table. Baseline Hemodynamic Data Inhaled Nitric Oxide Inhalation of nitric oxide decreased mean pulmonary arterial pressure (change, 7.0% [95% CI, 3.8% to 10.2%]), increased the cardiac index (change, 7.9% [CI, 3.4% to 12.5%]), and decreased pulmonary vascular resistance (change, 14.1% [CI, 19.1% to 9.2%]) (Figure 1). The pulmonary selectivity of the vasodilatory effect was indicated by a significant reduction in the ratio of pulmonary to systemic vascular resistance. The decrease in pulmonary vascular resistance in response to inhaled nitric oxide did not differ significantly among groups (Figure 2). The effects of nitric oxide wore off within 15 minutes. Figure 1. Hemodynamic responses to vasodilators. P P P P Figure 2. Time to decrease in pulmonary vascular resistance in response to vasodilator challenge. NO Inhaled Iloprost Aerosolized iloprost decreased the pulmonary vascular resistance by 27.1% (CI, 22.2% to 32.1%) and increased the cardiac index by 22.8% (CI, 17.6% to 27.9%) (Figure 1). The decrease in the ratio of pulmonary to systemic vascular resistance indicated preferential pulmonary vasodilatation. No significant differences among groups in the vasodilator response to inhaled iloprost were observed (Figure 2). The effects of iloprost wore off within 60 to 90 minutes. Oral Sildenafil Administration of 12.5 mg or 50 mg of sildenafil decreased mean pulmonary arterial pressure in a dose-dependent manner (change, 8.5% [CI, 15.1% to 1.9%] and 13.5% [CI, 23.8% to 3.2%], respectively) (Figure 1). The cardiac index increased by 5.0% (CI, 0.5% to 10.5%) and 13.2% (CI, 4.3% to 22.2%), and pulmonary vascular resistance decreased by 14.7% (CI, 6.6% to 22.7%) and 24.3% (CI, 16.7% to 31.8%). The decrease in the ratio of pulmonary to systemic vascular resistance indicated preferential pulmonary vasodilatation. Arterial oxygen did not decrease significantly (data not sh

Nox4 Is a Protective Reactive Oxygen Species Generating Vascular NADPH Oxidase

Rationale: The function of Nox4, a source of vascular H2O2, 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 H2O2 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 H2O2, 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.

Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension

OBJECTIVES We sought to investigate the impact of adjunct sildenafil on exercise capacity and hemodynamic parameters in patients with pulmonary arterial hypertension (PAH) who fulfilled predefined criteria of deterioration despite ongoing treatment with inhaled iloprost. BACKGROUND Inhaled iloprost is an effective therapy in PAH. The phosphodiesterase-5 inhibitor sildenafil exerts pulmonary vasodilation and may amplify prostanoid efficacy. METHODS Of 73 PAH patients receiving long-term inhaled iloprost treatment, 14 fulfilled criteria of deterioration unresponsive to conventional treatment. These patients received adjunct oral sildenafil over a period of nine to 12 months, leaving the inhalative iloprost regimen unchanged. RESULTS Before iloprost therapy, the baseline 6-min walking distance was 217 +/- 31 m (mean +/- SEM), with an improvement to 305 +/- 28 m within the first three months of iloprost treatment and a subsequent decline to 256 +/- 30 m after 18 +/- 4 months. Adjunct therapy with sildenafil reversed the deterioration and increased the 6-min walk distance to 346 +/- 26 m (p = 0.002, Wilcoxon test) at three months of combined therapy, with a sustained efficacy up to 12 months (349 +/- 32 m, p = 0.002). The distribution of New York Heart Association functional classes (IV/III/II) improved from September 9, 2000, before sildenafil, to January 8, 2003, after nine to 12 months with sildenafil. All hemodynamic variables changed favorably: pulmonary vascular resistance decreased from 2,494 +/- 256 before sildenafil to 1,950 +/- 128 dynes.s.cm(-5).m(2) after three months of adjunct sildenafil (p = 0.036). Two patients died of severe pneumonia during the period of combined therapy. No further serious adverse events occurred. CONCLUSIONS; In patients with severe PAH deteriorating despite ongoing prostanoid treatment, long-term adjunct oral sildenafil improves exercise capacity and pulmonary hemodynamics. A combination of prostanoids and sildenafil is an appealing concept for future treatment of pulmonary hypertension.

Post-Stroke Inhibition of Induced NADPH Oxidase Type 4 Prevents Oxidative Stress and Neurodegeneration

The identification of NOX4 as a major source of oxidative stress in stroke and demonstration of dramatic protection after stroke in mice by NOX4 deletion or NOX inhibition, opens up new avenues for treatment.

Hypoxia-Dependent Regulation of Nonphagocytic NADPH Oxidase Subunit NOX4 in the Pulmonary Vasculature

Nonphagocytic NADPH oxidases have recently been suggested to play a major role in the regulation of physiological and pathophysiological processes, in particular, hypertrophy, remodeling, and angiogenesis in the systemic circulation. Moreover, NADPH oxidases have been suggested to serve as oxygen sensors in the lung. Chronic hypoxia induces vascular remodeling with medial hypertrophy leading to the development of pulmonary hypertension. We screened lung tissue for the expression of NADPH oxidase subunits. NOX1, NOXA1, NOXO1, p22phox, p47phox, p40phox, p67phox, NOX2, and NOX4 were present in mouse lung tissue. Comparing mice maintained for 21 days under hypoxic (10% O2) or normoxic (21% O2) conditions, an upregulation exclusively of NOX4 mRNA was observed under hypoxia in homogenized lung tissue, concomitant with increased levels in microdissected pulmonary arterial vessels. In situ hybridization and immunohistological staining for NOX4 in mouse lungs revealed a localization of NOX4 mRNA and protein predominantly in the media of small pulmonary arteries, with increased labeling intensities after chronic exposure to hypoxia. In isolated pulmonary arterial smooth muscle cells (PASMCs), NOX4 was localized primarily to the perinuclear space and its expression levels were increased after exposure to hypoxia. Treatment of PASMCs with siRNA directed against NOX4 decreased NOX4 mRNA levels and reduced PASMC proliferation as well as generation of reactive oxygen species. In lungs from patients with idiopathic pulmonary arterial hypertension (IPAH), expression levels of NOX4, which was localized in the vessel media, were 2.5-fold upregulated. These results support an important role for NOX4 in the vascular remodeling associated with development of pulmonary hypertension.

Classical transient receptor potential channel 6 (TRPC6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange

Regional alveolar hypoxia causes local vasoconstriction in the lung, shifting blood flow from hypoxic to normoxic areas, thereby maintaining gas exchange. This mechanism is known as hypoxic pulmonary vasoconstriction (HPV). Disturbances in HPV can cause life-threatening hypoxemia whereas chronic hypoxia triggers lung vascular remodeling and pulmonary hypertension. The signaling cascade of this vitally important mechanism is still unresolved. Using transient receptor potential channel 6 (TRPC6)-deficient mice, we show that this channel is a key regulator of acute HPV as this regulatory mechanism was absent in TRPC6−/− mice whereas the pulmonary vasoconstrictor response to the thromboxane mimetic U46619 was unchanged. Accordingly, induction of regional hypoventilation resulted in severe arterial hypoxemia in TRPC6−/− but not in WT mice. This effect was mirrored by a lack of hypoxia-induced cation influx and currents in smooth-muscle cells from precapillary pulmonary arteries (PASMC) of TRPC6−/− mice. In both WT and TRPC6−/− PASMC hypoxia caused diacylglycerol (DAG) accumulation. DAG seems to exert its action via TRPC6, as DAG kinase inhibition provoked a cation influx only in WT but not in TRPC6−/− PASMC. Notably, chronic hypoxia-induced pulmonary hypertension was independent of TRPC6 activity. We conclude that TRPC6 plays a unique and indispensable role in acute hypoxic pulmonary vasoconstriction. Manipulation of TRPC6 function may thus offer a therapeutic strategy for the control of pulmonary hemodynamics and gas exchange.

Activation of Soluble Guanylate Cyclase Reverses Experimental Pulmonary Hypertension and Vascular Remodeling

Background— Severe pulmonary hypertension is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. Using wild-type and homozygous endothelial nitric oxide synthase (NOS3−/−) knockout mice with pulmonary hypertension induced by chronic hypoxia and rats with monocrotaline-induced pulmonary hypertension, we examined whether the soluble guanylate cyclase (sGC) stimulator Bay41-2272 or the sGC activator Bay58-2667 could reverse pulmonary vascular remodeling. Methods and Results— Both Bay41-2272 and Bay58-2667 dose-dependently inhibited the pressor response of acute hypoxia in the isolated perfused lung system. When wild-type (NOS3+/+) or NOS3−/− mice were housed under 10% oxygen conditions for 21 or 35 days, both strains developed pulmonary hypertension, right heart hypertrophy, and pulmonary vascular remodeling, demonstrated by an increase in fully muscularized peripheral pulmonary arteries. Treatment of wild-type mice with the activator of sGC, Bay58-2667 (10 mg/kg per day), or the stimulator of sGC, Bay41-2272 (10 mg/kg per day), after full establishment of pulmonary hypertension from day 21 to day 35 significantly reduced pulmonary hypertension, right ventricular hypertrophy, and structural remodeling of the lung vasculature. In contrast, only minor efficacy of chronic sGC activator therapies was noted in NOS3−/− mice. In monocrotaline-injected rats with established severe pulmonary hypertension, both compounds significantly reversed hemodynamic and structural changes. Conclusions— Activation of sGC reverses hemodynamic and structural changes associated with monocrotaline- and chronic hypoxia-induced experimental pulmonary hypertension. This effect is partially dependent on endogenous nitric oxide generated by NOS3.