An oral selective M3 cholinergic receptor antagonist in COPD.

Cholinergic antagonists have been used since the early 1900s as bronchodilators for chronic obstructive pulmonary disease (COPD). The present study investigated whether an oral muscarinic M3-selective anticholinergic agent (OrM3) would provide an improved therapeutic advantage compared with an inhaled anticholinergic agent in patients with COPD. A 6-week, multicentre, randomised, placebo- and active-controlled, parallel-group study was performed at 56 sites in the USA. In total, 412 male and female patients (aged 35-86 yrs) with a clinical history consistent with COPD were randomised to receive OrM3 0.5, 2, 3 or 4 mg orally once daily, ipratropium bromide 36 mug by inhalation four times daily or placebo. OrM3 demonstrated a significant dose-related improvement in serial forced expiratory volume in one second and a trend for dose-related improvement in patient-reported symptoms compared with placebo. However, at a dose that provided efficacy less than that of ipratropium, the incidence of dose-related, mechanism-based side-effects for OrM3 exceeded those observed for ipratropium. In patients with chronic obstructive pulmonary disease, the oral M3-selective agent did not offer a therapeutic advantage over inhaled ipratropium. These results do not support the hypothesis that high selectivity for muscarinic M3 receptors over airway neuronal M2 receptors will represent a more effective therapy than current inhaled anticholinergics in obstructive airway disease.

Effect of montelukast on lung function in asthma patients with allergic rhinitis: analysis from the COMPACT trial.

BACKGROUND: The Clinical Outcomes with Montelukast as a Partner Agent to Corticosteroid Therapy (COMPACT) trial demonstrated that montelukast added to budesonide (MNT + BD) was as efficacious as double the dose of budesonide (dBD) in improving morning peak expiratory flow (AM PEF) in adult asthmatics. Recent studies have demonstrated that montelukast is also effective in treating daytime and nighttime allergic rhinitis (AR) symptoms in asthmatic patients. This analysis was designed to examine whether asthmatic patients with comorbid AR respond differently than patients without comorbid AR in terms of asthma control (lung function). METHODS: There were 216 asthmatic patients in the MNT+BD group and 184 patients in the dBD group with AR. Treatment differences in the change from baseline in AM PEF were compared. Least square (LS) mean and 95% confidence interval (CI) were derived from an anova model adjusting for baseline and study site. RESULTS: There was a 9.2% increase in AM PEF from baseline in the MNT+BD group compared with a 6% increase in the dBD group. The LS mean difference [(MNT+BD)-dBD] was 14.2 l/min (P=0.028). Other secondary endpoints were similar between groups. CONCLUSION: In the subgroup of asthmatic patients with AR, a combined treatment approach that included montelukast and budesonide provided significantly greater efficacy in reducing airflow obstruction compared with doubling the dose of budesonide. These results support recommendations by the Allergic Rhinitis and its Impact on Asthma initiative that suggest a unified approach aimed at treating the airway inflammation common to both diseases is beneficial for the large proportion of asthmatics who also suffer from AR.

Increase in urinary leukotriene LTE4 levels in acute asthma: correlation with airflow limitation.

BACKGROUND: Leukotrienes play a key role in the pathophysiology of chronic asthma. Activation of leukotriene pathways is accompanied by rises in detectable urinary levels of leukotriene E4 (LTE4). The relationship between urinary LTE4 levels and factors associated with acute asthma has not been determined. METHODS: Adults aged 15-54 years presenting with moderate to severe acute asthma were evaluated at emergency departments in 16 US sites. Forced expiratory volume in 1 second (FEV1) was measured during the first 60 minutes after arrival and at specified times until discharge or admission. Urine samples for measurement of LTE4 levels were obtained either on arrival at the study site and/or before discharge. Patients were seen 2 weeks later for follow up, at which time repeat FEV1 measurements and urine samples for LTE4 were obtained. RESULTS: One hundred and eighty four patients were evaluated; LTE4 results from both the acute and follow up periods were available for analysis in 146. Urinary LTE4 levels were increased during asthma exacerbations compared with levels obtained 2 weeks later (geometric means 111.7 and 75.6 pg/mg creatinine, respectively, mean percentage change -32.3; 95% confidence interval (CI) for the mean percentage change -39.6 to -24.3, p<0.001). The correlation between improvement in FEV1 and decline in LTE4 over the 2 week interval was significant (p<0.001, r=0.43). CONCLUSIONS: Activation of leukotriene pathways in acute asthma is correlated with the degree of airflow obstruction, and resolution of the asthma exacerbation is associated with a reduction in leukotriene levels.

Expression of heme oxygenase in the oxygen-sensing regions of the rostral ventrolateral medulla.

Recently, unique regions in the rostral ventrolateral medulla (RVLM) have been found to be oxygen sensitive. However, the mechanism of sensing oxygen in these RVLM regions is unknown. Because heme oxygenase (HO) has been shown to be involved in the hypoxic responses of the carotid body and pulmonary artery, the aim of this study was to determine whether HO is present in the RVLM and whether expression of HO is altered by chronic hypoxia. Adult rats were exposed to hypoxia (10% O(2)) or normoxia (21% O(2)) for 10 days, and the mRNA for HO-1 and HO-2 was examined in the RVLM by using RT-PCR. Expression of HO-2 mRNA was seen in the RVLM of both control and hypoxic samples, whereas expression of HO-1 mRNA was only seen in the RVLM of hypoxic samples. HO-2 was immunocytochemically localized in brain sections (40 microm) to the C1 region and pre-Bötzinger complex of the RVLM. Together, these results indicate that HO-2 is present in the RVLM under control conditions and that HO-1 is induced in the RVLM during chronic hypoxia, consistent with a potential role for HO in the oxygen-sensing function of these cardiorespiratory RVLM regions.

Role of proteolysis and apoptosis in regression of pulmonary vascular remodeling.

Remodeled pulmonary arteries return to normal structural conditions after the increase in pulmonary artery flow resistance is reversed. We studied whether proteolysis of extracellular matrix proteins and apoptosis occur during reversal of remodeling produced by chronic hypoxia in the rat. Main pulmonary arteries were removed at different times during a 10-day period of exposure to 10% O2 and 14 days after return to air. Content and rates of degradation of collagen and elastin as well as immunoreactive collagenase in tissue and isolated mast cells were measured. Immunoblots for collagenase and tissue inhibitor of metalloproteinases (TIMP) were performed. Apoptosis was assessed by cleavage of DNA and TUNEL assay. Excess collagen and elastin present at 10 days of hypoxia decreased to near normal levels after 3-5 days of air. Transient increases in collagenolytic and elastolytic enzyme activities accompanied the rapid decrease in matrix proteins. Mast cells containing collagenase accumulated in remodeled pulmonary arteries, and the active form of collagenase appeared at the time of peak proteolytic activity. TIMP increased during remodeling. Apoptosis was maximal 3 days after return to air. Our results suggest that activation of enzymes, which degrade matrix proteins, and apoptosis play a role in resolution of vascular remodeling.

Flow cytometric determination of cell proliferation in hypertensive blood vessels.

BACKGROUND: Measurement of vascular cell proliferation in animal models of hypertension is currently accomplished by demonstrating [(3)H]-thymidine ([(3)H]-dT) incorporation into DNA using autoradiography. This method, however, is labor intensive, requires radioactivity, and is limited by the inherent difficulty in discriminating labeled and unlabeled cells. To address these limitations, a flow cytometric-based method is described utilizing incorporation of 5-bromo-2'-deoxyuridine (BrdU) into DNA of nuclei isolated from blood vessels. METHODS: Pulmonary hypertension was induced in rats by exposure to 10% O(2) (hypoxia) for varying periods of time. Pulmonary arteries and aorta from rats injected with BrdU prior to sacrifice were isolated, fixed with 10% formalin, and digested with Protease XIV. The intact nuclei liberated by this treatment were successively treated with HCl/Triton X-100 and sodium borate. Processed nuclei were probed with a BrdU-specific fluorescein-conjugated antibody, and the percentage of BrdU staining cells was determined using flow cytometry. RESULTS: An approximately 20-fold increase in BrdU-positive cells at 3 days of hypoxia in pulmonary arteries (relative to control) with no change in aorta was observed. These results were similar to previous studies using [(3)H]-dT labeling. CONCLUSIONS: Flow cytometric determination of cell proliferation in blood vessels is a simple, objective technique that may facilitate measurement of cell proliferation in animal models of vascular disease.

Remodeling of rat neonatal pulmonary artery: role of matrix metalloproteinases.

We studied whether rapid thinning of large pulmonary arteries of neonatal rats is associated with breakdown of collagen. Pulmonary artery extracts from fetal to 21 days of age were assayed for collagen content and matrix metalloproteinases. Within 3 days postpartum, no changes in collagen content, collagenolytic activity, or levels of stromelysin-l or gelatinase A were observed. After day 3, collagen content and total proteolytic activity increased with little change in matrix metalloproteinase expression. Thus, collagen was not degraded, and the late increases in collagen and total proteolytic activity were probably growth related. Unlike adult rats in which collagen is broken down after reversal of hypoxic pulmonary artery remodeling, collagen is not broken down in neonatal pulmonary arteries during adaptation to extrauterine life.

Isolation of differentially expressed genes in hypertensive pulmonary artery of rats.

Pulmonary artery remodeling is a complex biological process, and a key molecular mechanism regulating this process is selective up- and downregulation of genes. We used reverse transcriptase-polymerase chain reaction (RT-PCR) differential display in a rat model of hypoxic pulmonary hypertension to identify selectively expressed genes relevant to pulmonary artery remodeling. We characterized the pattern of gene expression in hypertensive and normal arteries. Eight differentially expressed cDNAs were selected, isolated, and characterized. Homology searches identified 4 previously identified genes and 4 novel genes that were not further characterized. The known genes were beta-glucoronidase, hemeoxygenase-2 (HO-2), glycerol-3-phosphate dehydrogenase, and cytoplasmic gamma-actin. Each of the 4 known genes was relevant to processes involved in pulmonary artery remodeling. We conclude that mRNA differential display was informative in identifying genes coding for products directly involved in pulmonary artery remodeling.

Expression of matrix-degrading enzymes in pulmonary vascular remodeling in the rat.

Exposure of rats to hypoxia causes pulmonary arterial remodeling, which is partly reversible after return to air. We hypothesized that degradation of excess collagen in remodeled pulmonary arteries in the posthypoxic period is mediated by endogenous matrix metalloproteinases (MMPs). Total proteolytic, collagenolytic, and gelatinolytic activities, levels of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1), and immunolocalization of stromelysin-1 in main pulmonary arteries were determined after exposure of rats to 10% O2 for 10 days followed by normoxia. We observed transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and expression of approximately 72-, 68-, and 60-kDa gelatinases by zymography within 3 days of cessation of hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1 level increased. Immunoreactive stromelysin-1 was localized predominantly in the luminal region of normal and hypertensive pulmonary arteries. These results are consistent with the notion that endogenous MMPs may mediate the breakdown of excess collagen in remodeled pulmonary arteries during the early posthypoxic period.

Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat.

Pulmonary vascular remodeling, produced by cell hypertrophy and extracellular matrix protein synthesis in response to hemodynamic stress, regresses after reduction of blood pressure, possibly by proteolysis of structural proteins. To test this postulate, we assessed the breakdown of extracellular matrix proteins and expression of collagenase and elastase in pulmonary arteries of rats exposed to hypoxia (10% O2 for 10 d) followed by normoxia. During hypoxia, contents of collagen and elastin increased in pulmonary arteries and latent rat interstitial collagenase was expressed without increased collagenolytic activity or mRNA levels. At 3 days after normoxia, collagen and elastin contents decreased coincident with the new appearance of activated collagenase and transient increases in collagenolytic and elastolytic activities. The amount of immunoreactive collagenase, localized predominately in connective tissue-type mast cells, was increased in the adventitia and media of hypertensive vessels. We conclude that mast cells containing latent collagenase are recruited into the outer walls of pulmonary arteries during remodeling. It is possible that mast cell-derived collagenase contributes to collagen breakdown in pulmonary arteries during early recovery from hypoxia and plays a role in restoration of vascular architecture.

Polymeric carrier of proline analogue with antifibrotic effect in pulmonary vascular remodeling.

The proline analogue cis-4-hydroxy-L-proline (cHyp) inhibits collagen accumulation but diffuses out of tissues. To prolong the antifibrotic effect, we used a copolymer of cHyp attached to a backbone of poly(ethylene glycol) (PEG) and lysine. The copolymer was encapsulated in liposomes conjugated with PEG or in liposomes coated with the polysaccharide amylopectin to improve uptake by lungs after intravenous infusion. Amylopectin-liposomes had approximately 3-fold greater uptake in cultured endothelial cells compared with PEG-liposomes and greater lung retention 1 wk after infusion (5.2 +/- 0.8% versus 2.7 +/- 0.2%, p < 0.05). Sustained antifibrotic activity, assayed by growth inhibition of smooth muscle cells and fibroblasts over 4 d, was greater for amylopectin-liposomes/copolymer than PEG-liposomes/copolymer. Inhibition of collagen accumulation in pulmonary arteries of hypoxic (10% O2) rats was used to assess antifibrotic activity. Amylopectin-liposomes/copolymer attenuated increased right ventricular pressure by approximately 50% and completely prevented excess vascular collagen 1 wk after a single intravenous injection. The copolymer in liposomes was > 1,000-fold more effective by weight than unencapsulated monomeric cHyp. Thus, the copolymer, a potent, long-acting antifibrotic agent, totally prevented collagen accumulation for 1 wk in pulmonary arteries undergoing vascular remodeling when delivered in amylopectin-liposomes.

Collagen changes in rat cervix in pregnancy--polarized light microscopic and electron microscopic studies.

The structural arrangement of collagen fibers in cervical ripening was studied in normal pregnant rats by picrosirius red staining and polarized light microscopy. The macromolecular arrangement of collagen fibers in the cervices of nonpregnant controls and in firm and rigid cervices of rats in early pregnancy (1-10 days of gestation) were optically anisotropic and had birefringence and a positive sign of elongation when examined by polarized light microscopy. The findings indicated that the structure of these collagen fibers was assembled from well-packed parallel collagen molecules. The direction of fibrous formation was arranged with regularity. In contrast, most of the collagen fibers in the soft cervices were optically isotropic. The fibers were fragmented and had a structure with discontinuous birefringence. Disarray and disorientation of the collagen fibers was found in the soft cervices. These collagen fibers changed their direction of formation. The disorganization of these collagen fibers might have a major impact on weakening the tensile strength of the cervix. Thus, we conclude that the processes of rearrangement of collagen fibers might be an important process in the cervical ripening. Electron microscopic studies suggest that in the focal hydrolytic processes of collagen and other matrix components degradation by lysosomal and phagosomal vesicles were associated with atrophic smooth muscle cells and fibroblasts of the cervices. Hydrolases released from lysosomes from these apoptotic cells may presumably be one of the processes in the remodeling of collagen structure.

Excess collagen in hypertensive pulmonary arteries decreases vascular distensibility.

Increased vascular collagen content is a major feature of pulmonary vascular remodeling. The functional role of excess collagen in decreasing pulmonary vascular compliance has not been established. We determined whether there was a correlation between hydroxyproline content of rat pulmonary artery segments and elastance (EPA) of the pulmonary artery bed during development of hypoxic pulmonary hypertension (10% O2, 10 d) and normoxic recovery. EPA was measured by air-filled pressure-volume curves. After 10 d of hypoxia, hydroxyproline content increased approximately 2-fold in large segments (1,200-250 microns in diameter) but not significantly in small segments (> 250 microns). Elastance increased from 87 +/- 6 (SEM) to 145 +/- 8 mm Hg/ml (p < 0.05) within 5 d of hypoxia and returned to control value 3 wk after recovery. There was a correlation between collagen content and EPA in large segments during development of hypertension; no correlation was found during recovery from hypoxia. The ratio of hydroxyproline to total protein was unchanged in large segments after recovery from hypoxia but was increased in small segments after recovery. We conclude that increased collagen in large pulmonary arteries directly influences EPA during the development of hypoxic pulmonary hypertension.

Effect of glucocorticoids on collagen accumulation in pulmonary vascular remodeling in the rat.

Administration of corticosteroids may attenuate the development of pulmonary hypertension by inhibiting the cell proliferation and protein synthesis that occur in early pulmonary vascular remodeling. However, in vitro studies show that corticosteroids stimulate collagen synthesis in vascular smooth muscle cells, and corticosteroid administration may be deleterious in stimulating collagen deposition. To test whether corticosteroid treatment promotes vascular collagen production in vivo, we administered triamcinolone diacetate to rats exposed to 10% O2 for 3 days and measured pro alpha 1(I) collagen mRNA and the hydroxyproline/protein ratio in the main pulmonary artery. Triamcinolone treatment (12 mg/kg intraperitoneally, once daily for 3 days) reduced mean right ventricular pressure (11 +/- 1 versus 14 +/- 1 mm Hg) and protein content of pulmonary arteries (1.8 +/- 0.1 versus 2.7 +/- 0.1 mg/vessel) (both p < 0.05). However, corticosteroid treatment produced a dose-related increase in pro alpha 1(I) mRNA levels and increased the ratio of hydroxyproline/protein (47 +/- 2 versus 38 +/- 3 micrograms/mg; p < 0.05). Thus, corticosteroid administration ameliorated the increase in pulmonary hypertension in early hypoxia, but increased the proportion of collagen in the vessel wall. Corticosteroid treatment in pulmonary vascular remodeling may be deleterious in increasing the concentration of collagen in the vessel wall.

Liposome-entrapped antifibrotic agent prevents collagen accumulation in hypertensive pulmonary arteries of rats.

We studied the therapeutic efficacy of an intravenously injected antifibrotic agent encapsulated in liposomes on inhibiting collagen accumulation in hypertensive blood vessels. cis-4-Hydroxy-L-proline (cHyp) in liposomes was injected into rats exposed to 10% O2, and drug effect was evaluated by measuring right ventricular pressure and hydroxyproline content of the pulmonary artery. Right ventricular pressure was 11 +/- 1 mm Hg (mean +/- SEM) 5 days after a single intravenous injection of 200 mg/kg cHyp in liposomes compared with 14 +/- 1 mm Hg in rats injected with empty liposomes; hydroxyproline content was also reduced by cHyp treatment (87 +/- 6 versus 107 +/- 7 micrograms per vessel) (p less than 0.05 for both, n = 6-9). Injections of cHyp in liposomes every 5 days partially prevented hypertension and vascular collagen accumulation during a 3-week exposure to hypoxia, and the dose required was one tenth the dose of unencapsulated cHyp. Therapeutic doses of cHyp in liposomes injected for 6 months affected tensile properties of main pulmonary artery and aorta, but there were no apparent histological effects on other organs. Liposomes injected intravenously were identified in pulmonary artery endothelial cells. The prolonged effect of a single injection of cHyp in liposomes may be due to uptake of the liposomes by the endothelium. Liposome delivery of drugs to the arterial wall may be useful in the study and treatment of hypertensive vascular disease.

Changes in aortic levels of tropoelastin mRNA following treatment of rats with the antihypertensive drugs captopril and hydralazine.

This manuscript describes changes in the steady state levels of aortic tropoelastin mRNA in spontaneously hypertensive rats (SHR) and normotensive controls (WKY) following treatment with two antihypertensive drugs. Three-week-old WKY and SHR rats were treated with hydralazine (15 mg/kg/day) or captopril (25 mg/kg/day). Tail artery blood pressure was monitored twice weekly. Both drugs prevented the development of hypertension in the SHR rat. At 6 weeks of age, total aortic RNA was extracted and the steady state levels of mRNAs coding for tropoelastin and pro alpha 1 (III) collagen were determined by slot blot hybridization analysis using radiolabeled tropoelastin and pro alpha 1 (III) collagen cDNA clones. Hydralazine treatment resulted in a threefold increase in tropoelastin mRNA levels in both the SHR and the WKY animals (P less than 0.01). Captopril-treated SHR animals demonstrated a similar significant increase. In contrast, no differences in pro alpha 1 (III) collagen mRNA levels were observed in the aorta of SHR or WKY rats following treatment with either captopril or hydralazine. These data suggest that antihypertensive agents can act specifically to directly induce tropoelastin mRNA levels in large arteries and thus may induce vascular remodeling independent of an increase in blood pressure.

An antifibrotic agent reduces blood pressure in established pulmonary hypertension in the rat.

We have shown that administration of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) to rats at the onset of exposure to hypoxia prevents collagen accumulation in pulmonary arteries and the rise in pulmonary blood pressure. In this experiment, we tested whether cHyp is effective when administered after hypertension was already established. Rats were exposed to hypoxia (10% O2) for 21 days. Groups were hypoxic animals treated with cHyp (200 mg/kg sc twice daily) on days 10-21 (hypoxic cHyp) and saline-injected hypoxic animals (hypoxic). On day 21, we measured mean right ventricular pressure, hematocrit, collagen content of main and intrapulmonary arteries, and wall thickness of arterioles. Treatment reduced right ventricular pressure from 21 +/- 1 to 17 +/- 1 mmHg (P less than 0.05), hematocrit from 66 +/- 1 to 56 +/- 1% (P less than 0.05), hydroxyproline content of intrapulmonary arteries from 30 +/- 3 to 11 +/- 2 micrograms/vessel (P less than 0.05), and wall thickness from 27 +/- 3 to 16 +/- 2 microns (P less than 0.05). These results show that vascular collagen content is increased in established pulmonary hypertension and that cHyp treatment is effective in partially preventing the hemodynamic, structural, and biochemical changes if started after pulmonary hypertension is established. cHyp may also affect the rheological properties of blood.

Collagen and elastin metabolism in hypertensive pulmonary arteries of rats.

We evaluated the processes controlling the accumulation of collagen and elastin in main pulmonary arteries of rats during an episode of hypoxic pulmonary hypertension. Explant cultures of main pulmonary arteries were incubated with [3H]proline to measure collagen and protein synthesis and percent collagen synthesis. Elastin synthesis was measured by [14C]valine incorporation into insoluble elastin. Relative collagen synthesis increased twofold (from 1.1 +/- 0.2 x 10(3) to 2.0 +/- 1.0 x 10(3) disintegrations per minute [14C]hydroxyproline/vessel/hr/mg protein), relative collagen synthesis doubled (from 2% to 4-5% of total protein synthesis), and elastin synthesis increased ninefold (from 0.4 +/- 0.2 x 10(4) to 3.6 +/- 0.6 x 10(4) dpm [14C]valine/vessel/hr/mg protein) in early hypertension. The level of pro alpha l(I) collagen RNA paralleled the relative collagen synthetic rate during the study period. Within 7 days of recovery from hypoxia, collagen and elastin contents were normal. We conclude that collagen and elastin in main pulmonary arteries are synthesized rapidly during an episode of hypoxic pulmonary hypertension and that collagen and elastin are rapidly removed from the hypertensive vessel during normoxic recovery.

Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium.

Physiologic stimuli of connective tissue accumulation in pulmonary vascular remodeling are poorly defined. We postulated that increased pressure within central pulmonary arteries is a stimulus for connective tissue synthesis and the response is dependent on an intact endothelium. Mechanical tension equivalent to 50 mmHg pressure was applied for 4 h to isolated rat main pulmonary arteries (endothelium intact or removed), and incorporation of [14C]proline into collagen, [14C]valine into elastin, [3H]thymidine into DNA and pro alpha 1 (I) collagen mRNA levels were measured. In intact vessels, tension induced synthesis of collagen (3.1 +/- 0.4 vs. 2.3 +/- 0.5 [SEM] dpm X 10(2) [14C]-hydroxyproline/[mg protein.h]) (n = 10) and elastin (6.1 +/- 2.4 vs. 2.9 +/- 0.4 dpm X 10(3) [14C]valine/[mg protein.h]) (n = 5) (both P less than 0.05). Steady state mRNA levels of pro alpha 1 (I) collagen were also increased by tension (46 vs. 30 X 10(2) dpm hybridized/100 ng total RNA). However, the stimulus did not increase [3H]thymidine incorporation into DNA. In denuded vessels, tension had no effect on connective tissue synthesis or mRNA level of pro alpha 1 (I) collagen. Messenger RNA levels for v-sis were induced by tension in intact but not denuded vessels. Our findings establish that induction of vascular connective tissue synthesis by mechanical tension is dependent on an intact endothelium.