Inhibition of TGF-β Signaling and Decreased Apoptosis in IUGR-Associated Lung Disease in Rats

Intrauterine growth restriction is associated with impaired lung function in adulthood. It is unknown whether such impairment of lung function is linked to the transforming growth factor (TGF)-β system in the lung. Therefore, we investigated the effects of IUGR on lung function, expression of extracellular matrix (ECM) components and TGF-β signaling in rats. IUGR was induced in rats by isocaloric protein restriction during gestation. Lung function was assessed with direct plethysmography at postnatal day (P) 70. Pulmonary activity of the TGF-β system was determined at P1 and P70. TGF-β signaling was blocked in vitro using adenovirus-delivered Smad7. At P70, respiratory airway compliance was significantly impaired after IUGR. These changes were accompanied by decreased expression of TGF-β1 at P1 and P70 and a consistently dampened phosphorylation of Smad2 and Smad3. Furthermore, the mRNA expression levels of inhibitors of TGF-β signaling (Smad7 and Smurf2) were reduced, and the expression of TGF-β-regulated ECM components (e.g. collagen I) was decreased in the lungs of IUGR animals at P1; whereas elastin and tenascin N expression was significantly upregulated. In vitro inhibition of TGF-β signaling in NIH/3T3, MLE 12 and endothelial cells by adenovirus-delivered Smad7 demonstrated a direct effect on the expression of ECM components. Taken together, these data demonstrate a significant impact of IUGR on lung development and function and suggest that attenuated TGF-β signaling may contribute to the pathological processes of IUGR-associated lung disease

Changes in 11 beta-hydroxysteroid dehydrogenase type 2 expression in a low-protein rat model of intrauterine growth restriction

Methods. IUGR rats after maternal low-protein diet (n = 17) were compared with controls (n = 18). At 70 and 120 days of age, in situ distribution of 11 beta-HSD2 gene and protein expression was investigated by RT-PCR of microdissected tubules and immunohistochemistry. For in situ localization studies, double staining for 11 beta-HSD2 and calbindin was used. Serum levels of corticosterone and dehydrocorticosterone were measured by tandem mass spectrometry. Results. In IUGR rats, intra-arterial blood pressure significantly increased at Day 120 of life. Serum corticosterone/dehydrocorticosterone ratios and 11 beta-HSD2 mRNA in total kidney were not altered in IUGR animals. However, 11 beta-HSD2 mRNA concentration was significantly lower in microdissected tubuli of IUGR animals (Day 120: 0.18 +/- 0.14 vs 1.00 +/- 0.32 rel. units in controls; P < 0.05). In IUGR animals, immunostaining scores for 11 beta-HSD2 were significantly lower than in controls (P < 0.05). Double staining with calbindin showed lower expression of 11 beta-HSD2 in distal segments of the distal tubule. Conclusions. Our data indicate lower gene and protein expression of the pre-receptor enzyme 11 beta-HSD2 in IUGR animals when looking at specific renal compartments, but not in total kidney extracts. Thus, lower 11 beta-HSD2 as a mechanism for hypertension later in life might be missed without methods for in situ detection

Prevention of Early Postnatal Hyperalimentation Protects against Activation of Transforming Growth Factor-beta/Bone Morphogenetic Protein and Interleukin-6 Signaling in Rat Lungs after Intrauterine Growth Restriction

Background: Intrauterine growth restriction (IUGR) is intimately linked with postnatal catch-up growth, leading to impaired lung structure and function. However, the impact of catch-up growth induced by early postnatal hyperalimentation (HA) on the lung has not been addressed to date. Objective: The aim of this study was to investigate whether prevention of HA subsequent to IUGR protects the lung from 1) deregulation of the transforming growth factor-beta(TGF-beta)/bone morphogenetic protein (BMP) pathway, 2) activation of interleukin (IL)-6 signaling, and 3) profibrotic processes. Methods: IUGR was induced in Wistar rats by isocaloric protein restriction during gestation by feeding a control (Co) or a low-protein diet with 17% or 8% casein, respectively. On postnatal day 1 (P1), litters from both groups were randomly reduced to 6 pups per dam to induce HA or adjusted to 10 pups and fed with standard diet: Co, Co with HA (Co-HA), IUGR, and IUGR with HA (IUGR-HA). Results: Birth weights in rats after IUGR were lower than in Co rats (P < 0.05). HA during lactation led to accelerated body weight gain from P1 to P23 (Co vs. Co-HA, IUGR vs. IUGR-HA; P < 0.05). At P70, prevention of HA after IUGR protected against the following: 1) activation of both TGF-beta [phosphorylated SMAD (pSMAD) 2; plasminogen activator inhibitor 1 (Pai1)] and BMP signaling [pSMAD1; inhibitor of differentiation (Id1)] compared with Co (P < 0.05) and Co or IUGR (P < 0.05) rats, respectively; 2) greater mRNA expression of interleukin (Il) 6 and Il13 (P < 0.05) as well as activation of signal transducer and activator of transcription 3 (STAT3) signaling (P < 0.05) after IUGR-HA; and 3) greater gene expression of collagen I alpha 1 and osteopontin (P < 0.05) and increased deposition of bronchial subepithelial connective tissue in IUGR-HA compared with Co and IUGR rats. Moreover, HA had a significant additive effect (P < 0.05) on the increased enhanced pause (indicator of airway resistance) in the IUGR group (P < 0.05). at P70. Conclusions: This study demonstrates a dual mechanism in IUGR-associated lung disease that is 1) IUGR-dependent and 2) HA-mediated and thereby offers new avenues to develop innovative preventive strategies for perinatal programming of adult lung diseases

Expression of the TGF-β signaling machinery in lungs of neonatal and adult rats.

<p>Changes in the expression of genes encoding components of the TGF-β signaling machinery as assessed by real-time RT-PCR at days P1 and P70; n = 8–15 for each bar. The significance for each bar is indicated by p values, IUGR vs. CO; two-tailed Mann-Whitney test. A: Expression of genes encoding the TGF-β receptors <i>tgfbr1</i>, <i>tgfbr2</i> and <i>tgfbr3</i> at P1 (white bar) and P70 (striped bar). B: Expression of genes encoding the regulatory <i>smad2</i>, <i>smad3</i> and <i>smad4</i> at days P1 (white bar) and P70 (striped bar). C: Expression of genes encoding the inhibitory <i>smad7</i>, <i>smurf2</i> and smad anchor for receptor activation (<i>sara</i>) at days P1 (white bar) and P70 (striped bar).</p

Effect of IUGR on the expression of extracellular matrix (ECM) proteins and modulators of the ECM in neonatal rat lungs.

<p>Expression of TGF-β-regulated genes encoding elastin (Eln), tenascin N (Ten), collagens (Coll) I, Coll III, and Fibrillin (A), and genes of ECM modulators including matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitor of metalloproteinases (TIMP)-1, and TIMP-2 (B) in lungs extracted at day P1 from neonatal rats after IUGR and control rats. The mRNA expression, illustrated as relative fold induction, was assessed by real-time PCR. The control group was normalized to 1 as indicated by a scattered line; n = 15 for each group. The significance for each bar is indicated by p values, IUGR vs. Co; two-tailed Mann-Whitney test.</p

Body weight, respiration and physiological lung parameters of IUGR rats.

<p>A: Body weight (g) and relative lung weight (lung weight/body weight) of rats after IUGR (white bars) and control rats (black bars) at days P1 and P70; n = 8–15 for each bar. B: Architectural changes in lung structure were evident in hematoxylin and eosin-stained lung sections from IUGR rats and control rats at day P70. Measurement of septal thickness (µm) and mean linear intercept (MLI; µm) in IUGR rats and control rats (CO) at day P70; n = 6–10 for each bar. C: Assessment of respiratory system compliance by whole body plethysmography in IUGR rats (white bars) and in the control group (CO; black bars) at P70. n = 15–17 for each bar. D: Expression pattern of genes encoding surfactant protein A (SP-A), SP-C, and SPD. IUGR rats (white bars) and control group (black bars). n = 6–15 for each bar. The significance for each bar is indicated by p values, IUGR vs. Co, n.s. = not significant; two-tailed Mann-Whitney test.</p

Effect of IUGR on apoptosis in lungs of rats at days P1 and P70.

<p>Apoptosis is assessed by cleaved caspase-3 and cleaved fragment of Poly (ADP-ribose) polymerase (PARP). A: Representative immunoblots illustrating the expression of cleaved and total caspase-3, fragments of PARP and total PARP in lung homogenates of rats with IUGR and without IUGR (Co) at <i>day</i> P1 (A) and P70 (B). The β-actin served as loading control; n = 4–6 for each bar.</p