Ronald H. Goldstein

Severity of Elastase-Induced Emphysema Is Decreased in Tumor Necrosis Factor-α and Interleukin-1β Receptor-Deficient Mice

A single intratracheal dose of porcine pancreatic elastase, which is cleared from the lung by 24 hours, was administered to wild-type, IL-1β type 1 receptor-deficient, double TNF-α (type 1 and type 2) receptor-deficient, and combined TNF-α (type 1 receptor) plus IL-1β receptor-deficient mice. The mean linear intercept (Lm) of saline-treated mice was 32(3) μm [mean(SE)]. For wild-type elastase-treated mice, Lm was 81(6) μm at 21 days versus 52(5) μm at 5 days after treatment, indicating that alveolar wall remodeling occurs long after the elastase injury. At 21 days, Lm values were 67(10), 62(3), and 39(5) μm in elastase-treated mice deficient in the IL-1β receptor, double TNF-α receptors, and combined receptors, respectively. The level of apoptosis assessed by a terminal deoxynucleotidyl transferase-catalyzed in situ nick end-labeling assay was increased at 5 days after elastase treatment and was markedly and similarly attenuated in the IL-1β, the double TNF-α, and the combined receptor-deficient mice. Our results indicate that inflammatory mediators exacerbate elastase-induced emphysema. We estimate that in the combined TNF-α + IL-1β receptor-deficient mice, inflammation accounts for about 80% of the emphysema that develops after elastase treatment; decreased apoptosis of lung cells likely contributes to decreased severity of emphysema.

Regulation of connective tissue growth factor expression by prostaglandin E2

Transforming growth factor-beta (TGF-beta) stimulates alpha(1)(I) collagen mRNA synthesis in human lung fibroblasts through a mechanism that is partially sensitive to cycloheximide and that may involve synthesis of connective tissue growth factor (CTGF). Northern blot analyses indicate that TGF-beta stimulates time- and dose-dependent increases in CTGF mRNA. In TGF-beta-stimulated fibroblasts, maximal levels of CTGF mRNA (3.7-fold above baseline) occur at 6 h. The TGF-beta-stimulated increase in CTGF mRNA was not blocked by cycloheximide. Nuclear run-on analysis indicates that TGF-beta increases the CTGF transcription rate. The TGF-beta-stimulated increases in CTGF transcription and steady-state levels of CTGF mRNA are attenuated in prostaglandin E(2) (PGE(2))-treated fibroblasts. PGE(2) fails to attenuate luciferase activity induced by TGF-beta in fibroblasts transfected with the TGF-beta-responsive luciferase reporter construct p3TP-LUX. In amino acid-deprived fibroblasts, PGE(2) and insulin regulate alpha(1)(I) collagen mRNA levels without affecting CTGF mRNA levels. The data suggest that the regulation of alpha(1)(I) collagen mRNA levels by TGF-beta and PGE(2) may function through both CTGF-dependent and CTGF-independent mechanisms.

Regulation of lysyl oxidase and cyclooxygenase expression in human lung fibroblasts: interactions among TGF‐β, IL‐1β, and prostaglandin E

Prostaglandin E2, transforming growth factor‐β and interleukin‐1β variably regulate the expression of cyclooxygenase 1, cyclooxygenase 2, and lysyl oxidase in IMR90, human embryo lung fibroblasts. Prostaglandin E2 at 100 nM upregulates cyclooxygenase 1 mRNA by approximately three‐fold while it downregulates lysyl oxidase mRNA levels. Notably, prostaglandin E2 suppresses the enhancing effect of TGF‐β on basal levels of lysyl oxidase mRNA. These changes in steady state mRNA levels reflect transcriptional level control, at least in part. Corresponding changes are seen in the protein levels of lysyl oxidase, cyclooxygenase 1 and cyclooxygenase 2 and in catalytic activities of these enzymes, including net prostaglandin E2 synthesis. Cyclooxygenase 2 mRNA(t1 2, 30 min) is considerably less stable than that of cyclooxygenase 1 (t1 2, 4h) while lysyl oxidase mRNA is unusually stable (t1 2 > 14h). Taken together with the differing kinetics with which these genes respond to perturbation by these cytokines, the present results suggest a coordinated, autocrine‐like mechanism of regulation of cyclooxygenase 1 and cyclooxygenase 2 and further point to the potential of their metabolic product, prostaglandin E2, to suppress the expression of lysyl oxidase in the inflammatory response to injury.

Retinoic acid does not affect alveolar septation in adult FVB mice with elastase-induced emphysema.

Background: Administration of all-trans retinoic acid (ATRA) to adult Sprague-Dawley rats with emphysema induced by porcine pancreatic elastase (PPE) reversed the emphysema perhaps by inducing new alveolar formation. Objective: A study was conducted to determine whether ATRA can induce new alveolar septa and reverse the airspace enlargement caused in adult mice by PPE treatment. Methods: 48 FVB mice were divided into 6 groups. Three groups received 15 µg of PPE in 0.1 ml of 0.9% saline and 3 groups received 0.1 ml of saline, intratracheally. Starting at day 22, the mice received 12 daily intraperitoneal injections of cottonseed oil, with or without ATRA (12.5 µg or 50 µg). The mice were killed for study 1 day after the last injection. Results: Measurements of plasma and lung tissue ATRA levels showed statistically significant elevated levels after the 50-µg but not after the 12.5-µg doses of ATRA. In situ hybridization studies of elastin and α1(I) collagen mRNA expression in pulmonary parenchyma as well as in airways and blood vessels showed no effect of ATRA. Airspace size was determined by the mean linear intercept (Lm) method. The Lm of the groups receiving PPE and ATRA (46.2 ± 4.1 µm, mean ± SD) was not significantly different from the group receiving PPE and oil (47.8 ± 6.0 µm). The Lm for groups receiving saline and ATRA (40.6 ± 2.5 µm) were not significantly different from the group receiving saline and oil (41.0 ± 2.7 µm). Comparison of the fixed lung volume data and calculated internal surface area also showed no differences between the control and ATRA-treated groups. Conclusion: ATRA treatment does not affect airspace size or expression of elastin or α1(I) collagen mRNA in adult FVB mice with PPE-induced emphysema.

Structure and expression of the promoter for the R4/ALK5 human type I transforming growth factor-β receptor : regulation by TGF-β

Abstract The type I transforming growth factor-β (TGF-β) receptors are serine/threonine kinases that are essential for the action of TGF-β. In this paper, we describe the molecular cloning and expression of the R4/ALK5 human type I TGF-β receptor promoter. DNA sequence analysis indicates that the promoter lacks a TATA and CAAT box but is highly GC-rich and contains putative Spl binding sites. The transcriptional start site is approx. 232 base pairs upstream of the AUG start codon. In human lung fibroblasts, TGF-β induced a 3-fold increase in steady-state level for type I receptor mRNA. Exposure of cells transfected with a 618 by promoter fragment to TGF-β1 up-regulated transcriptional activity indicating that a TGF-β response element is contained within this region.

The effect of PGE2 on the activation of quiescent lung fibroblasts

The effect of prostaglandin E2 (PGE2) on fibroblast proliferation was examined. The presence of PGE2 for 24 h inhibited the growth of quiescent cells stimulated with serum, platelet-derived growth factor and macrophage-derived factors. Maximal inhibition of nuclear labeling with [3H]thymidine occurred at concentrations greater than 10(-7) M. The inhibitory effect of PGE2 was less potent in exponentially growing cells and was not the result of conversion of PGE2 to PGA2 during incubation in growth medium. The G1 phase was determined to be 12-14 h in untreated cultures. The extent of growth inhibition by PGE2 was similar with addition of PGE2 at 0, 3, 6, or 9 h following restimulation of quiescent cell cultures. Approximately 25% of the cells that enter S phase are refractory to PGE2-induced growth inhibition. Short-term exposure to PGE2 (5 min and 30 min) caused substantial growth inhibition. The serum-induced proliferation was also inhibited by the cAMP analogue, dibutyrl cAMP. Our results suggest that PGE2 affects a distinct subpopulation of cells. Restimulation of quiescent cells treated with PGE2 for 24 h, indicated that release from PGE2 exposure is associated with prolongation of the G1 phase of the cell cycle.

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.

Phenylbutyrate decreases type I collagen production in human lung fibroblasts

Fibrotic lung diseases are characterized by excess extracellular matrix production, in particular type I collagen. Phenylbutyrate (PB) is a non‐toxic pharmacological compound that functions as a weak histone deacetylase inhibitor. In hepatic stellate cells, the synthesis of type I collagen expression is decreased by inhibiting histone acetylation. Our studies examined the regulation of type I collagen by PB in human lung fibroblasts. We found that PB decreases basal and transforming growth factor‐β‐stimulated α1(I) collagen mRNA and protein levels. Northern blot analyses demonstrated that PB decreases steady‐state α1(I) collagen mRNA levels by 78% without significantly changing the stability of the mRNA transcript. PB stimulates cAMP production and increases the acetylation of histone H4, but does not affect the activity of two transforming growth factor‐β (TGF‐β)‐responsive luciferase reporter constructs. These data suggest that PB regulates type I collagen expression in human lung fibroblasts by mechanisms that include cAMP production and histone acetylation. PB may have therapeutic use in fibrotic lung diseases.

Interleukin-1β 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‐1β (IL‐1β), whereas tumor necrosis factor‐α, transforming growth factor‐β, and angiotensin II had minimal effect. Stimulation with IL‐1β resulted in the secretion of soluble osteopontin protein. We found that osteopontin expression by IL‐1β 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‐1β 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‐1β. Activated fibroblasts may be a significant source of osteopontin production during lung fibrogenesis. J. Cell. Biochem.

The Accumulation of Type I Collagen Mrnas in Human Embryonic Lung Fibroblasts Stimulated by Transforming Growth Factor-β

We examined the expression of type I collagen mRNAs (alpha 1(I) and alpha 2 (I)) by embryonic lung fibroblasts in cultures treated with transforming growth factor-beta (TGF-beta). TGF-beta caused a concentration dependent increase in the expression of alpha 1(I) mRNA for type I collagen. TGF-beta at low concentration (0.1 ng/ml) slightly increased the level of alpha 1(I) mRNA. Higher concentrations of TGF-beta (1.0 and 5.0 ng/ml) further increased the amount of alpha 1(I) mRNA. The increase in alpha 1(I) mRNA was associated with a marked increase in production of intact type I collagen molecules. TGF-beta did not increase expression of alpha 2(I) mRNA. The alpha 2(I) mRNA levels in human lung fibroblast cultures were not affected by varying the duration of exposure to TGF-beta nor the concentration of TGF-beta. In contrast, TGF-beta increased the amount of both alpha 1(I) and alpha 2(I) mRNA in NIH3T3 cells. These data suggest that the amount of alpha 2(I) mRNA is not rate limiting with respect to type I collagen production during TGF-beta stimulation in human lung fibroblast cultures.