Frank Rose

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.

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

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.

WNT1-inducible signaling protein–1 mediates pulmonary fibrosis in mice and is upregulated in humans with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is characterized by distorted lung architecture and loss of respiratory function. Enhanced (myo)fibroblast activation, ECM deposition, and alveolar epithelial type II (ATII) cell dysfunction contribute to IPF pathogenesis. However, the molecular pathways linking ATII cell dysfunction with the development of fibrosis are poorly understood. Here, we demonstrate, in a mouse model of pulmonary fibrosis, increased proliferation and altered expression of components of the WNT/beta-catenin signaling pathway in ATII cells. Further analysis revealed that expression of WNT1-inducible signaling protein-1 (WISP1), which is encoded by a WNT target gene, was increased in ATII cells in both a mouse model of pulmonary fibrosis and patients with IPF. Treatment of mouse primary ATII cells with recombinant WISP1 led to increased proliferation and epithelial-mesenchymal transition (EMT), while treatment of mouse and human lung fibroblasts with recombinant WISP1 enhanced deposition of ECM components. In the mouse model of pulmonary fibrosis, neutralizing mAbs specific for WISP1 reduced the expression of genes characteristic of fibrosis and reversed the expression of genes associated with EMT. More importantly, these changes in gene expression were associated with marked attenuation of lung fibrosis, including decreased collagen deposition and improved lung function and survival. Our study thus identifies WISP1 as a key regulator of ATII cell hyperplasia and plasticity as well as a potential therapeutic target for attenuation of pulmonary fibrosis.

Ultrasonic versus jet nebulization of iloprost in severe pulmonary hypertension

Inhalation of iloprost, a stable prostacyclin analogue, is a promising perspective in the treatment of pulmonary hypertension. In initial clinical studies, a conventional jet nebulizer system was successfully used to decrease pulmonary vascular resistance and pressure, requiring however, up to twelve inhalations of 12-15 min per day. The aim of this study was to investigate if the application of an equal dose of iloprost at a drastically reduced duration of inhalation with the use of a more efficient ultrasonic nebulizer, leads to comparable haemodynamic effects, without escalation of side effects. The physical features of the jet nebulizer system (Ilo-Neb) and the ultrasonic nebulizer (Multisonic Compact) were characterized by laser diffractometry and a Tc99m-tracer technique. Mass median aerodynamic diameters were 3.2 microm for the jet and 3.9 microm for the ultrasonic nebulizer. Total output (mean+/-SD) was 60+/-7 microL.min(-1) (jet) and 163+/-15 microL.min(-1)(ultrasonic), and efficiency of the devices was 39+/-3% (jet) and 86+/-5% (ultrasonic). Based on these data, a total inhalative dose of 2.8 microg iloprost was delivered by jet nebulization within 12 min and by ultrasonic nebulization within 4 min, in 18 patients with severe primary and secondary pulmonary hypertension (New York Heart Association class III and IV), in a randomized crossover design. Haemodynamics were assessed by right heart catheterization. Inhalation with the ultrasonic device and jet nebulizer, reduced mean+/-SEM pulmonary artery pressure from 54.3+/-2.1 to 47.1+/-2.0 and from 53.5+/-2.2 to 47.0+/-2.2 mmHg, respectively, and mean+/-SEM pulmonary vascular resistance from 1,073+/-109 to 804+/-87 and from 1,069+/-125 to 810+/-83 dyn.s.cm(-5), respectively. Both modes of aerosolization were well tolerated. In conclusion, due to the markedly higher efficiency and output of the ultrasonic device, wastage of drug is largely avoided and the duration of inhalation can be shortened to one-third, with comparable haemodynamic effects and without enforcing side effects.

Metastasis: the seed and soil theory gains identity

The metastatic spread of tumor cells to distant sites represents the major cause of cancer-related deaths. Cancer metastasis involves a series of complex interactions between tumor cells and microenvironment that influence its biological effectiveness and facilitate tumor cell arrest to distant organs. More than a century since Paget developed the theory of seed and soil, the enigma of tissue specificity observed in metastatic colonization of tumor cells begins to unfold itself. The advent of new technologies has led to the discovery of novel molecules and pathways that confer metastasis-associated properties to the cancer cells, mediating organ specificity and unique genetic signatures have been developed using microarray studies. Future clinical studies and new antimetastatic compounds aiming to improve survival of patients with metastasis will most probably be based on these signatures. This review summarizes the plethora of old and new molecules that are strongly correlated with organ-specific metastases and which provide now an identity to the theory of seed and soil.

NOX4 regulates ROS levels under normoxic and hypoxic conditions, triggers proliferation, and inhibits apoptosis in pulmonary artery adventitial fibroblasts.

The NADPH oxidases are involved in vascular remodeling processes and oxygen sensing. Hypoxia-induced pulmonary arterial remodeling results in thickening of the vessel wall and reduction of the area of vessel lumen, leading to pulmonary hypertension and cor pulmonale. The proliferation of pulmonary artery adventitial fibroblasts (PAFB) is critically involved in this process. In this study, we analyzed the role of the non-phagocytic NADPH oxidase subunits NOX1 and NOX4 in PAFB. NOX4 was predominantly expressed in comparison to NOX1 at mRNA levels. Under hypoxic conditions, NOX4 was significantly upregulated at mRNA and protein levels. Silencing of NOX4 by siRNA caused reduction of ROS levels under both normoxic and hypoxic (24 h) conditions and suppressed the significant hypoxic-induced ROS increase. PAFB proliferation was significantly decreased in cells transfected with NOX4 siRNA, whereas apoptosis was enhanced. Also, the expression of NOX4 was studied in PAFB isolated from the lungs of patients with idiopathic pulmonary arterial hypertension (IPAH). Interestingly, a significant increase of NOX4 mRNA expression was observed under hypoxic conditions in PAFB from the lungs with IPAH compared to healthy donors. In conclusion, NOX4 maintains ROS levels under normoxic and hypoxic conditions and enhances proliferation and inhibits apoptosis of PAFB.

Hypoxic pulmonary artery fibroblasts trigger proliferation of vascular smooth muscle cells: role of hypoxia-inducible transcription factors

Chronic lung hypoxia causes vascular remodeling with pulmonary artery smooth muscle cell (SMCPA) hyperplasia, resulting in pulmonary hypertension and cor pulmonale. We investigated SMCPA and pulmonary artery adventitial fibroblasts (FBPA) for their proliferative response to hypoxia. Strong SMCPA growth occurred under hypoxic conditions in SMCPA/FBPA co‐cultures, but not in SMCPA monocultures. SMCPA growth was fully reproduced by transferring serum‐free supernatant from hypoxic cultured FBPA to normoxic SMCPA. Hypoxia‐inducible‐transcriptionfactor subtypes (HIF‐1α, HIF‐2α, HIF‐3α) and its dependent target genes, carrying the hypoxiaresponsive‐element as regulatory component, were strongly activated in both hypoxic FBPA and SMCPA. HIF‐transcription‐factor decoy technique, employed to FBPA during hypoxic culturing, blocked the mitogenic activity of FBPA conditioned medium on SMCPA. The data suggest that hypoxia‐driven gene regulation in pulmonary artery fibroblasts results in a mitogenic stimulus on adjacent pulmonary artery smooth muscle cells, and HIF‐transcription‐decoy may offer a new therapeutic approach to suppress these events.

cDNA array hybridization after laser-assisted microdissection from nonneoplastic tissue.

Differential gene expression can be investigated effectively by cDNA arrays. Because tissue homogenates result inevitably in an average expression of a bulk of different cells, we aimed to combine mRNA profiling with cell-type-specific microdissection. Using a polymerase chain reaction (PCR)-based preamplification technique, the expression profile was shown to be preserved. We modified the existing protocol enabling to apply the total amount of extracted RNA from microdissected cells. A mean amplification factor of nearly 1000 allowed to reduce the demand of initial RNA to approximately 10 ng. This technique was used to investigate intrapulmonary arteries from mouse lungs ( approximately 500 cell equivalents). Using filters with 1176 spots, three independent experiments showed a high consistency of expression for the preamplified cDNAs. These profiles differed primarily from those of total lung homogenates. Additionally, in experimental hypoxia-induced pulmonary hypertension, amplified cDNA from intrapulmonary vessels of these lungs was compared to cDNA from vessels dissected from normoxic lungs. Validation by an alternative method was obtained by linking microdissection with real-time polymerase chain reaction (PCR). As suggested by the array data, nine selected genes with different factors of up-regulation were fully confirmed by the PCR technique. Thus, a rapid protocol is presented combining microdissection and array profiling that demands low quantities of initial RNA to assess reliably cell-type-specific gene regulation even within nonneoplastic complex tissues.

Hypoxia-driven proliferation of human pulmonary artery fibroblasts: cross-talk between HIF-1α and an autocrine angiotensin system

Pulmonary artery adventitial fibroblasts (FBPA) may play a central role in lung vascular remodeling under conditions of hypoxia and inflammation, the result being pulmonary hypertension and cor pulmonale. In cultured human FBPA, both angiotensin II (Ang II) and hypoxia promoted cell cycle progression and cell proliferation and suppressed apoptosis. These effects were further enhanced when both stimuli were applied simultaneously. Hypoxia elevated the expression of hypoxia‐inducible factor 1α (HIF‐1α) and increased the expression of genes regulated by the hypoxia‐responsive element (HRE). Up‐regulation of both angiotensin‐converting enzyme (ACE) and Ang II receptor type 1 (AT1) was also observed. Exogenous Ang II further increased HIF/HRE‐dependent signaling in FBPA, whereas suppression of the autocrine ACE‐Ang II‐AT1 loop with inhibitors of ACE, AT1, and phosphatidylinositol 3‐kinase (PI3K) reduced the proliferative response to both hypoxia and exogenous Ang II. Overexpression of HIF‐1α by transient transfection caused the same proliferative effect and up‐regulation of AT1 expression that were observed under hypoxic conditions. In contrast, small interfering RNA targeting HIF‐1α inhibited hypoxia‐induced ACE and AT1 expression. Our studies indicate that the ACE‐Ang II‐AT1 system serves as a positive feedback loop and fosters FBPA proliferation under hypoxic conditions, with the PI3K‐HIF‐HRE axis as the central effector pathway. This pathway may thus facilitate vascular remodeling under hypoxic conditions.