Cluster analysis identifies novel real-world lung disease-pulmonary hypertension subphenotypes: implications for treatment response.

Background: Clinical trials repurposing pulmonary arterial hypertension (PAH) therapies to patients with lung disease- or hypoxia-pulmonary hypertension (PH) (classified as World Health Organization Group 3 PH) have failed to show a consistent benefit. However, Group 3 PH clinical heterogeneity suggests robust phenotyping may inform detection of treatment-responsive subgroups. We hypothesised that cluster analysis would identify subphenotypes with differential responses to oral PAH therapy. Methods: Two k-means analyses were performed on a national cohort of US veterans with Group 3 PH; an inclusive model (I) of all treated patients (n=196) and a haemodynamic model (H) limited to patients with right heart catheterisations (n=112). The primary outcome was organ failure or all-cause mortality by cluster. An exploratory analysis evaluated within-cluster treatment effects. Results: Three distinct clusters of Group 3 PH patients were identified. In the inclusive model (C1I n=43, 21.9%; C2I n=102, 52.0%; C3I n=51, 26.0%), lung disease and spirometry drove cluster assignment. By contrast, in the haemodynamic model (C1H n=44, 39.3%; C2H n=43, 38.4%; C3H n=25, 22.3%), right heart catheterisation data surpassed the importance of lung disease and spirometry. In the haemodynamic model, compared to C3H, C1H experienced the greatest hazard for respiratory failure or death (HR 6.1, 95% CI 3.2-11.8). In an exploratory analysis, cluster determined treatment response (p=0.006). Conclusions regarding within-cluster treatment responses were limited by significant differences between select variables in the treated and untreated groups. Conclusions: Cluster analysis identifies novel real-world subphenotypes of Group 3 PH patients with distinct clinical trajectories. Future studies may consider this methodological approach to identify subgroups of heterogeneous patients that may be responsive to existing pulmonary vasodilatory therapies.

Real-world use of inhaled treprostinil for lung disease-pulmonary hypertension: A protocol for patient evaluation and prescribing.

Inhaled treprostinil was approved recently for interstitial lung disease-pulmonary hypertension; however, efficacy in "real-world" populations is not known. We designed a protocol and report our experience evaluating 10 patients referred for therapy. Misdiagnosis at presentation was common; ultimately, three patients (30%) were prescribed drug. This protocol offers an opportunity to standardize longitudinal assessment of inhaled treprostinil in clinical practice.

Tadalafil for veterans with chronic obstructive pulmonary disease-pulmonary hypertension: A multicenter, placebo-controlled randomized trial.

Treating Veterans with chronic obstructive pulmonary disease complicated by pulmonary hypertension (COPD-PH) using phosphodiesterase type-5 inhibitor pharmacotherapy is common, but efficacy data are lacking. To address this further, patients with COPD-PH from five Department of Veterans Affairs hospitals were randomized (1∶1) to receive placebo or oral tadalafil (40 mg/day) for 12 months. The primary endpoint was changed from baseline in 6-min walk distance at 12 months. Secondary endpoints included change from baseline in pulmonary vascular resistance, mean pulmonary artery pressure, and symptom burden by the University of California San Diego shortness of breath questionnaire scale at 6 months. A total of 42 subjects (all male; 68 ± 7.6 years old) were randomized to placebo (N = 14) or tadalafil (N = 28). The group imbalance was related to under-enrollment. Compared to placebo, no significant difference was observed in the tadalafil group for change from the primary endpoint or change in mean pulmonary artery pressure or pulmonary vascular resistance from baseline at 6 months. A clinically meaningful improvement was observed in the secondary endpoint of shortness of breath questionnaire score in the tadalafil versus placebo group at 6 months. There was no significant difference in major adverse events between treatment groups, and tadalafil was well tolerated overall. For Veterans with COPD-PH enrolled in this study, once-daily treatment with tadalafil did not improve 6-min walk distance or cardiopulmonary hemodynamics although a decrease in shortness of breath was observed. Under-enrollment and imbalanced randomization confound interpreting conclusions from this clinical trial and limit the generalization of our findings.

Pulmonary vascular resistance and clinical outcomes in patients with pulmonary hypertension: a retrospective cohort study.

BACKGROUND: In pulmonary hypertension subgroups, elevated pulmonary vascular resistance (PVR) of 3·0 Wood units or more is associated with poor prognosis. However, the spectrum of PVR risk in pulmonary hypertension is not known. To address this area of uncertainty, we aimed to analyse the relationship between PVR and adverse clinical outcomes in pulmonary hypertension. METHODS: We did a retrospective cohort study of all patients undergoing right heart catheterisation (RHC) in the US Veterans Affairs health-care system (Oct 1, 2007-Sep 30, 2016). Patients were included in the analyses if data from a complete RHC and at least 1 year of follow-up were available. Both inpatients and outpatients were included, but individuals with missing mean pulmonary artery pressure (mPAP), pulmonary artery wedge pressure, or cardiac output were excluded. The primary outcome measure was time to all-cause mortality assessed by the Veteran Affairs vital status file. Cox proportional hazards models were used to assess the association between PVR and outcomes, and the mortality hazard ratio was validated in a RHC cohort from Vanderbilt University Medical Center (Sept 24, 1998-June 1, 2016). FINDINGS: The primary cohort (N=40 082; 38 751 [96·7%] male; median age 66·5 years [IQR 61·1-73·5]; median follow-up 1153 days [IQR 570-1971]), included patients with a history of heart failure (23 201 [57·9%]) and chronic obstructive pulmonary disease (13 348 [33·3%]). We focused on patients at risk for pulmonary hypertension based on a mPAP of at least 19 mm Hg (32 725 [81·6%] of 40 082). When modelled as a continuous variable, the all-cause mortality hazard for PVR was increased at around 2·2 Wood units compared with PVR of 1·0 Wood unit. Among patients with a mPAP of at least 19 mm Hg and pulmonary artery wedge pressure of 15 mm Hg or less, the adjusted hazard ratio (HR) for mortality was 1·71 (95% CI 1·59-1·84; p<0·0001) and for heart failure hospitalisation was 1·27 (1·13-1·43; p=0·0001), when comparing PVR of 2·2 Wood units or more to less than 2·2 Wood units. The validation cohort (N=3699, 1860 [50·3%] male, median age 60·4 years [49·5-69·2]; median follow-up 1752 days [IQR 1281-2999]) included 2870 patients [77·6%] with mPAP of at least 19 mm Hg (1418 [49·4%] male). The adjusted mortality HR for patients in the mPAP of 19 mm Hg or more group and with PVR of 2·2 Wood units or more and pulmonary artery wedge pressure of 15 mm or less Hg (1221 [42·5%] of 2870) was 1·81 (95% CI 1·33-2·47; p=0·0002). INTERPRETATION: These data widen the continuum of clinical risk for mortality and heart failure in patients referred for RHC with elevated pulmonary artery pressure to include PVR of around 2.2 Wood units and higher. Testing the generalisability of these findings in at-risk populations with fewer cardiopulmonary comorbidities is warranted. FUNDING: None.

Association of Borderline Pulmonary Hypertension With Mortality and Hospitalization in a Large Patient Cohort: Insights From the Veterans Affairs Clinical Assessment, Reporting, and Tracking Program.

BACKGROUND: Pulmonary hypertension (PH) is associated with increased morbidity across the cardiopulmonary disease spectrum. Based primarily on expert consensus opinion, PH is defined by a mean pulmonary artery pressure (mPAP) ≥25 mm Hg. Although mPAP levels below this threshold are common among populations at risk for PH, the relevance of mPAP <25 mm Hg to clinical outcome is unknown. METHODS AND RESULTS: We analyzed retrospectively all US veterans undergoing right heart catheterization (2007-2012) in the Veterans Affairs healthcare system (n=21,727; 908-day median follow-up). Cox proportional hazards models were used to evaluate the association between mPAP and outcomes of all-cause mortality and hospitalization, adjusted for clinical covariates. When treating mPAP as a continuous variable, the mortality hazard increased beginning at 19 mm Hg (hazard ratio [HR]=1.183; 95% confidence interval [CI], 1.004-1.393) relative to 10 mm Hg. Therefore, patients were stratified into 3 groups: (1) referent (≤18 mm Hg; n=4,207); (2) borderline PH (19-24 mm Hg; n=5,030); and (3) PH (≥25 mm Hg; n=12,490). The adjusted mortality hazard was increased for borderline PH (HR=1.23; 95% CI, 1.12-1.36; P<0.0001) and PH (HR=2.16; 95% CI, 1.96-2.38; P<0.0001) compared with the referent group. The adjusted hazard for hospitalization was also increased in borderline PH (HR=1.07; 95% CI, 1.01-1.12; P=0.0149) and PH (HR=1.15; 95% CI, 1.09-1.22; P<0.0001). The borderline PH cohort remained at increased risk for mortality after excluding the following high-risk subgroups: (1) patients with pulmonary artery wedge pressure >15 mm Hg; (2) pulmonary vascular resistance ≥3.0 Wood units; or (3) inpatient status at the time of right heart catheterization. CONCLUSIONS: These data illustrate a continuum of risk according to mPAP level and that borderline PH is associated with increased mortality and hospitalization. Future investigations are needed to test the generalizability of our findings to other populations and study the effect of treatment on outcome in borderline PH.

Protocol for exercise hemodynamic assessment: performing an invasive cardiopulmonary exercise test in clinical practice.

Invasive cardiopulmonary exercise testing (iCPET) combines full central hemodynamic assessment with continuous measurements of pulmonary gas exchange and ventilation to help in understanding the pathophysiology underpinning unexplained exertional intolerance. There is increasing evidence to support the use of iCPET as a key methodology for diagnosing heart failure with preserved ejection fraction and exercise-induced pulmonary hypertension as occult causes of exercise limitation, but there is little information available outlining the methodology to use this diagnostic test in clinical practice. To bridge this knowledge gap, the operational protocol for iCPET at our institution is discussed in detail. In turn, a standardized iCPET protocol may provide a common framework to describe the evolving understanding of mechanism(s) that limit exercise capacity and to facilitate research efforts to define novel treatments in these patients.

Study design and rationale for investigating phosphodiesterase type 5 inhibition for the treatment of pulmonary hypertension due to chronic obstructive lung disease: the TADA-PHiLD (TADAlafil for Pulmonary Hypertension associated with chronic obstructive Lung Disease) trial.

In patients with chronic obstructive pulmonary disease (COPD), moderate or severe pulmonary hypertension (COPD-PH) is associated with increased rates of morbidity and mortality. Despite this, approaches to treatment and the efficacy of phosphodiesterase type 5 inhibition (PDE-5i) in COPD-PH are unresolved. We present the clinical rationale and study design to assess the effect of oral tadalafil on exercise capacity, cardiopulmonary hemodynamics, and clinical outcome measures in COPD-PH patients. Male and female patients 40-85 years old with GOLD stage 2 COPD or higher and pulmonary hypertension diagnosed on the basis of invasive cardiac hemodynamic assessment (mean pulmonary artery pressure [mPAP] >30 mmHg, pulmonary vascular resistance [PVR] >2.5 Wood units, and pulmonary capillary wedge pressure ≤18 mmHg at rest) will be randomized at a 1∶1 ratio to receive placebo or oral PDE-5i with tadalafil (40 mg daily for 12 months). The primary end point is change from baseline in 6-minute walk distance at 12 months. The secondary end points are change from baseline in PVR and mPAP at 6 months and change from baseline in peak volume of oxygen consumption ([Formula: see text]) during exercise at 12 months. Changes in systemic blood pressure and/or oxyhemoglobin saturation (Sao2) at rest and during exercise will function as safety outcome measures. TADA-PHiLD (TADAlafil for Pulmonary Hypertension assocIated with chronic obstructive Lung Disease) is the first sufficiently powered randomized clinical trial testing the effect of PDE-5i on key clinical and drug safety outcome measures in patients with at least moderate PH due to COPD.

Cellular FLICE-like inhibitory protein deviates myofibroblast fas-induced apoptosis toward proliferation during lung fibrosis.

A prominent feature of fibrotic tissue in general and of lungs in particular is fibroblast proliferation and accumulation. In patients overcoming fibrosis, apoptosis limits this excessive cell growth. We have previously shown resistance to Fas-induced apoptosis of primary lung fibroblasts from mice with bleomycin-induced lung fibrosis, their escape from immune surveillance, and continued accumulation in spite of overexpression of the Fas death receptor. Cellular FLICE-like inhibitory protein (c-FLIP) is a regulator of cell death receptor-induced apoptosis in many cell types. We aimed to determine c-FLIP levels in myofibroblasts from fibrotic lungs and to directly assess c-FLIP's role in apoptosis and proliferation of primary lung myofibroblasts. c-FLIP levels were determined by apoptosis gene array, flow cytometry, Western blot, and immunofluorescence before and after down-regulation with a specific small interfering RNA. Apoptosis was assessed by caspase cleavage in Western blot and by Annexin V affinity labeling after FACS and tissue immunofluorescence. Proliferation was assessed by BrdU uptake, also using FACS and immunofluorescence. We show that myofibroblasts from lungs of humans with idiopathic pulmonary fibrosis and from bleomycin-treated versus normal saline-treated mice up-regulate c-FLIP levels. Using the animal model, we show that fibrotic lung myofibroblasts divert Fas signaling from apoptosis to proliferation and that this requires signaling by TNF receptor-associated factor (TRAF) and NF-κB. c-FLIP down-regulation reverses the effect of Fas activation, causing increased apoptosis, decreased proliferation, and diminished recruitment of TRAF to the DISC complex. This indicates that c-FLIP is essential for myofibroblast accumulation and may serve as a potential target to manipulate tissue fibrosis.

Transcriptional changes associated with recovery from heart failure in the SHR.

To identify biological pathways associated with myocardial recovery from heart failure (HF), gene profiling and gene set enrichment analysis (GSEA) were examined in left ventricle of spontaneously hypertensive rats with HF (SHR-F) with no treatment, following treatment with the angiotensin converting enzyme inhibitor captopril, and treatment with captopril combined with the short chain fatty acid derivative phenylbutyrate. Failing hearts demonstrated depressed left ventricular ejection fraction, while ventricular volume and mass increased. Captopril treatment alone prevented further deterioration but did not improve myocardial function; relatively few transcripts were differentially expressed relative to untreated SHR-F. Gene sets identified by GSEA as downregulated with captopril treatment compared to SHR-F group included those related to hypoxia and reactive oxygen species, while upregulated gene sets included G protein signaling. Treatment with phenylbutyrate alone did not improve survival (no animals in this group survived the 30 day treatment period), while phenylbutyrate combined with captopril increased survival and significantly improved cardiac function in vivo and in vitro. Normalized microarray data identified 780 genes that demonstrated a combined treatment effect of which 258 genes were modified with HF. Fatty acid metabolism and ion transport were among the most significantly upregulated pathways in the combined treatment group compared to untreated SHR with HF, whereas those related to oxidative stress, growth, inflammation, protein degradation, and TGF-beta signaling were downregulated. These findings demonstrate improved myocardial function and regression of cardiac hypertrophy, and identify many HF related gene sets altered with phenylbutyrate and captopril treatment, but not captopril alone. These results characterize gene sets associated with recovery from HF, and suggest that phenylbutyrate may be a potentially effective adjunctive treatment, together with captopril, by synergistically modulating pathways that contribute to restoration of contractile function of the failing SHR heart.

Transition from compensated hypertrophy to systolic heart failure in the spontaneously hypertensive rat: Structure, function, and transcript analysis.

Gene expression, determined by micro-array analysis, and left ventricular (LV) remodeling associated with the transition to systolic heart failure (HF) were examined in the spontaneously hypertensive rat (SHR). By combining transcript and gene set enrichment analysis (GSEA) of the LV with assessment of function and structure in age-matched SHR with and without HF, we aimed to better understand the molecular events underlying the onset of hypertensive HF. Failing hearts demonstrated depressed LV ejection fraction, systolic blood pressure, and LV papillary muscle force while LV end-diastolic and systolic volume and ventricular mass increased. 1431 transcripts were differentially expressed between failing and non-failing animals. GSEA identified multiple enriched gene sets, including those involving inflammation, oxidative stress, cell degradation and cell death, as well as TGF-beta and insulin signaling pathways. Our findings support the concept that these pathways and mechanisms may contribute to deterioration of cardiac function and remodeling associated with hypertensive HF.

Activation of elastin transcription by transforming growth factor-beta in human lung fibroblasts.

Elastin synthesis is essential for lung development and postnatal maturation as well as for repair following injury. Using human embryonic lung fibroblasts that express undetectable levels of elastin as assessed by Northern analyses, we found that treatment with exogenous transforming growth factor-beta (TGF-beta) induced rapid and transient increases in levels of elastin heterogeneous nuclear RNA (hnRNA) followed by increases of elastin mRNA and protein expression. In fibroblasts derived from transgenic mice, TGF-beta induced increases in the expression of a human elastin gene promoter fragment driving a chloramphenicol acetyl transferase reporter gene. The induction of elastin hnRNA and mRNA expression by TGF-beta was abolished by pretreatments with TGF-beta receptor I inhibitor, global transcription inhibitor actinomycin D, and partially blocked by addition of protein synthesis inhibitor cycloheximide, but was not affected by the p44/42 MAPK inhibitor U0126. Pretreatment with the p38 MAPK inhibitor SB-203580 also partially attenuated the levels of TGF-beta-induced elastin mRNA but not its hnRNA. Western analysis indicated that TGF-beta stimulated Akt phosphorylation. Inhibition of phosphatidylinositol 3-kinase and Akt phosphorylation by LY-294002 abolished TGF-beta-induced increases in elastin hnRNA and mRNA expression. Treatment of lung fibroblasts with interleukin-1beta or the histone deacetylase inhibitor trichostatin A inhibited TGF-beta-induced elastin mRNA and hnRNA expression by a mechanism that involved inhibition of Akt phosphorylation. Downregulation of Akt2 but not Akt1 expression employing small interfering RNA duplexes blocked TGF-beta-induced increases of elastin hnRNA and mRNA levels. Together, our results demonstrated that TGF-beta activates elastin transcription that is dependent on phosphatidylinositol 3-kinase/Akt activity.

Fibulin-5 gene expression in human lung fibroblasts is regulated by TGF-beta and phosphatidylinositol 3-kinase activity.

Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor-beta (TGF-beta) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steady-state level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-beta markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF-beta induction. Electrophoretic gel mobility shift assay revealed that TGF-beta strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3-dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF-beta was blocked by pretreatment with TGF-beta receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002, and actinomycin D. Basal and TGF-beta-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-beta-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-beta-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-beta/Smad signaling and requires the PI3-kinase/Akt pathway.

Modulation of amino acid uptake by TGF-beta in lung myofibroblasts.

Hormones such as insulin, growth factors, and cell stress stimulate system A amino acid transporter. Transforming growth factor-beta (TGF-beta) stimulates amino acid uptake thereby inducing cell proliferation, cellular hypertrophy, and matrix synthesis. Insulin appears to activate amino acid in smooth muscle cells via a phosphatidylinositol 3-kinase (PI3-kinase)-dependent pathway. We examine the effect and interaction of TGF-beta, insulin, and PI3-kinase activity on amino acid uptake in human lung myofibroblasts. TGF-beta treatment induced large increases in system A activity and a small delayed increase in the phosphorylation of protein kinase B, also termed phospho-Akt. In contrast, insulin induced small increases in system A activity and large increases in phospho-Akt levels. LY294002, a PI3-kinase inhibitor, blocked the TGF-beta-induced amino acid uptake only partially, but completely blocked TGF-beta-induced Akt phosphorylation. Moreover, the level of phospho-Smad3 was found to be high even when LY294002 blocked TGF-beta-induced phospho-Akt levels. Inhibition of PI3-kinase activity resulted in increase in Km, consistent with a major change in transporter activity without change in transporter number. The PI3-kinase inhibitor also did not change the amino acid transporter 2 (ATA2) mRNA levels. Taken together, these results suggest that TGF-beta induced Smad-3 and amino acid uptake through a PI3-kinase independent pathway.

Interleukin-1beta induces osteopontin expression in pulmonary fibroblasts.

Osteopontin is a multifunctional matricellular protein identified as one of the most upregulated genes in pulmonary fibrosis. Experimental animal models have identified early pro-fibrotic cytokines as essential to the pathogenesis of inflammation-induced pulmonary fibrosis. However, the principal sources of osteopontin in the fibroproliferative lung, and the factors responsible for its induction, have not been fully defined. We isolated primary rat lung fibroblasts in culture to examine the expression and regulation of lung fibroblast-derived osteopontin. Our results demonstrate a potent and dramatic increase in osteopontin expression induced by interleukin-1beta (IL-1beta), whereas tumor necrosis factor-alpha, transforming growth factor-beta, and angiotensin II had minimal effect. Stimulation with IL-1beta resulted in the secretion of soluble osteopontin protein. We found that osteopontin expression by IL-1beta was regulated via signaling primarily through the mitogen-activated protein kinase member ERK1/2, partially by p38 MAPK, but not at all by JNK. Finally, the mechanism of IL-1beta increase in osteopontin mRNA requires de novo transcription and translation. In conclusion, we find that osteopontin is expressed by primary lung fibroblasts and is potently upregulated by the early inflammatory and pro-fibrotic cytokine IL-1beta. Activated fibroblasts may be a significant source of osteopontin production during lung fibrogenesis.

Induction of the myofibroblast phenotype following elastolytic injury to mouse lung.

The repair of alveolar structures following endotracheal administration of porcine pancreatic elastase (PPE) to mice involves the coordinated deposition of new matrix elements. We determined the induction of the myofibroblast phenotype following elastolytic injury to mouse lung by examining the expression of alpha-smooth muscle actin (alpha-SMA) by immunohistochemistry. We also examined elastin and alpha1(I) collagen mRNA expression by in situ hybridization. Changes in airspace dimensions were assessed by determining mean linear intercept. In untreated mice, alpha-SMA was localized to vascular structures and large airways, with no detectable expression in alveolar units. PPE induced alpha-SMA expression in damaged areas surrounding large vessels, in septal remnants, and in the opening ring of alveolar ducts. Elastin and alpha1(I) collagen mRNA expression were up-regulated in residual alveolar structures and septal walls. PPE dose-response studies indicated that alpha1(I) collagen and elastin mRNA expression were not induced in areas of normal lung adjacent to damaged lung. The administration of low dose PPE resulted in increased alpha-SMA protein and elastin mRNA expression in the cells comprising the opening ring of alveolar ducts. Our data suggest that repair mechanisms following elastolytic injury are confined to overtly damaged alveolar structures and involve the induction of the myofibroblast phenotype.

Formation of lung alveolar-like structures in collagen-glycosaminoglycan scaffolds in vitro.

The objective of this study was to investigate the histology of tissue formed when fetal rat lung cells were grown in a collagen-glycosaminoglycan (GAG) tissue-engineering scaffold. The goal was the formation of lung histotypic structures in the tissue-engineering scaffolds in vitro. Achieving this goal would facilitate future investigations of the effects of selected scaffold design parameters on processes that may underlie aspects of lung regeneration in vivo. Lung cells were obtained from Sprague-Dawley rats after 16 and 19 days of gestation. These dissociated cells were seeded into type I collagen-chondroitin 6-sulfate matrices, 8 mm in diameter by 2 mm in thickness, cross-linked and sterilized by dehydrothermal treatment. Approximately 28 million cells were seeded into each spongelike sample. Histological and immunohistochemical studies were performed at termination periods of 2 days and 1, 2, and 3 weeks. The enzymatically dissociated 19-day gestation fetal rat lung cells formed and maintained alveolar-like structures, 50-60 microm in diameter, in the collagen- GAG scaffold. A novel finding was that all of the cell-seeded scaffolds underwent cell-mediated contraction that appeared to be associated with the finding by immunohistochemistry of expression of alpha-smooth muscle actin in some cells. These results demonstrate the capability of dissociated lung cells to form lung histotypic structures in collagen-GAG tissue-engineering scaffolds in vitro. This culture system may be of value in facilitating exploration of strategies for preparing such scaffolds for the regeneration of lung tissue in vivo.

Hypoxia suppresses elastin repair by rat lung fibroblasts.

Macrophage and neutrophil proteinases damage lung elastin, disrupting alveolar epithelium and filling alveoli with inflammatory exudate. Alveolar collapse and regional hypoxia occur. Whether low oxygen tension alters fibroblast-mediated lung repair is unknown. To determine the effect of chronic hypoxia on repair of enzyme-induced elastin disruption, primary rat lung fibroblasts produced elastin matrix for 5 wk before treatment with porcine pancreatic elastase (PPE). After exposure to PPE or saline, cultures recovered for 2 wk in normoxia (21% O(2)) or hypoxia (3% O(2)). Hypoxia suppressed regeneration of hot alkali-resistant elastin, achieving only 49% of the repair achieved in normoxic cultures. Vascular smooth muscle cells and lung fibroblasts repair elastin by two pathways: de novo synthesis and salvage repair. Although both pathways were affected, hypoxia predominantly inhibited de novo synthesis, decreasing formation of new elastin matrix by 63% while inhibiting salvage repair by only 36%. Prolonged hypoxia alone downregulated steady-state levels of elastin mRNA by 45%, whereas PPE had no significant effect on elastin gene expression. Electron microscopy documented preservation of intracellular organelles and intact nuclei. Together, these data suggest that regional hypoxia limits lung elastin repair following protease injury at least in part by inhibiting elastin gene expression.

Methods for measuring type I collagen synthesis in vitro.

The excess accumulation of type I collagen within tissues leads to organ dysfunction and occurs as a result of an imbalance between synthesis and degradation. This chapter outlines several methods to assess the in vitro production of type I collagen that are employed in our laboratory. We describe Western immunoblotting of intact alpha1(I) collagen using antibodies directed to alpha1(I) collagen amino and carboxyl propeptides. The measurement of alpha1(I) collagen mRNA levels using real-time polymerase chain reaction is then outlined. Finally, methods to determine the transcriptional regulation of alpha1(I) collagen using a nuclear run-on assay are described.

Engraftment of neonatal lung fibroblasts into the normal and elastase-injured lung.

Interstitial fibroblasts are an integral component of the alveolar wall. These cells produce matrix proteins that maintain the extracellular scaffold of alveolar structures. Emphysema is characterized by airspace enlargement resulting from the loss of alveolar cellularity and matrix. In this study, we explored the endotracheal delivery of fibroblasts to the lung parenchyma as a means of repairing damaged alveolar structures directly or indirectly for the delivery of transgenes. Fibroblasts were isolated from the lungs of neonatal transgenic mice expressing GFP during the period of rapid alveolarization. These GFP+ cells maintained their myofibroblast phenotype in culture and expressed elastin and alpha-smooth muscle actin mRNA. We administered GFP+ fibroblasts to saline- and elastase-treated mice by endotracheal instillation. We detected more GFP+ fibroblasts in the alveolar walls and in the interstitial areas of elastase-injured lungs than in normal lungs as assessed by immunohistochemistry and fluorescent imaging. The presence of GFP+ fibroblasts in the interstitium demonstrated transepithelial migration of these cells. Expression of GFP+ fibroblasts in recipient lungs was maintained for at least 20 d after endotracheal administration. These cells synthesize matrix components including elastin in vitro and could contribute to restoring the structural integrity of the alveolar wall.

Regulation of elastin gene transcription by proteasome dysfunction.

Elastin, a major extracellular matrix protein and the core component of elastic fiber, is essential to maintain lung structural integrity and normal physiological function. We previously found that the downregulation of elastin gene transcription by IL-1beta is mediated via activation of NF-kappaB and CCAAT/enhancer binding protein (C/EBP)beta, both targets of the ubiquitin-proteasome pathway. To further investigate the molecular mechanisms that underlie the control of elastin gene expression, we disrupted the ubiquitin-proteasome pathway with specific proteasome inhibitors. We found that specific proteasome inhibitors decreased the steady-state level of elastin mRNA in a dose-responsive manner. Run-on assay and promoter reporter study indicated that the proteasome inhibitor MG-132 repressed the rate of elastin transcription. MG-132 did not affect mRNA levels of NF-kappaB and C/EBPbeta, or the nuclear presence of NF-kappaB, but markedly increased C/EBPbeta isoforms, including liver-enriched transcriptional activating protein and liver-enriched transcriptional inhibitory protein. Addition of cycloheximide blocked these increases and the downregulation of elastin mRNA by MG-132. The MG-132-induced downregulation of elastin transcription was dependent on C/EBPbeta expression as assessed with small interfering RNA. These results indicate that the ubiquitin-proteasome pathway plays an essential role in maintaining elastin gene expression in lung fibroblasts. Disruption of this pathway results in the downregulation of tropoelastin transcription via posttranscriptionally induced C/EBPbeta isoforms.