The effect of lipoprotein-associated phospholipase A2 deficiency on pulmonary allergic responses in Aspergillus fumigatus sensitized mice.

BackgroundLipoprotein-associated phospholipase A2 (Lp-PLA2)/platelet-activating factor acetylhydrolase (PAF-AH) has been implicated in the pathogenesis of cardiovascular disease. A therapeutic targeting of this enzyme was challenged by the concern that increased circulating platelet activating factor (PAF) may predispose to or increase the severity of the allergic airway response. The aim of this study was to investigate whether Lp-PLA2 gene deficiency increases the risk of PAF and IgE-mediated inflammatory responses in vitro and in vivo using mouse models.MethodsLp-PLA2-/- mice were generated and back crossed to the C57BL/6 background. PAF-AH activity was measured using a hydrolysis assay in serum and bronchoalveolar lavage (BAL) samples obtained from mice. Aspergillus fumigatus (Af)-specific serum was prepared for passive allergic sensitization of mice in vivo and mast cells in vitro. β- hexosaminidase release was studied in bone marrow derived mast cells sensitized with Af-specific serum or DNP-IgE and challenged with Af or DNP, respectively. Mice were treated with lipopolysaccharide (LPS) and PAF intratracheally and studied 24 hours later. Mice were sensitized either passively or actively against Af and were studied 48 hours after a single intranasal Af challenge. Airway responsiveness to methacholine, inflammatory cell influx in the lung tissue and BAL, immunoglobulin (ELISA) and cytokine (Luminex) profiles were compared between the wild type (WT) and Lp-PLA2-/- mice.ResultsPAF-AH activity was reduced but not completely abolished in Lp-PLA2-/- serum or by in vitro treatment of serum samples with a high saturating concentration of the selective Lp-PLA2 inhibitor, SB-435495. PAF inhalation significantly enhanced airway inflammation of LPS treated WT and Lp-PLA2-/- mice to a similar extent. Sensitized WT and Lp-PLA2-/- bone-marrow derived mast cells released β-hexosaminidase following stimulation by allergen or IgE crosslinking to equivalent levels. Wild type and Lp-PLA2-/- mice responded to passive or active allergic sensitization by significant IgE production, airway inflammation and hyperresponsiveness after Af challenge. BAL cell influx was not different between these strains while IL-4, IL-5, IL-6 and eotaxin release was attenuated in Lp-PLA2-/- mice. There were no differences in the amount of total IgE levels in the Af sensitized WT and Lp-PLA2-/- mice.ConclusionsWe conclude that Lp-PLA2 deficiency in C57BL/6 mice did not result in a heightened airway inflammation or hyperresponsiveness after PAF/LPS treatment or passive or active allergic sensitization and challenge

Quercetin prevents progression of disease in elastase/LPS-exposed mice by negatively regulating MMP expression

Abstract Background Chronic obstructive pulmonary disease (COPD) is characterized by chronic bronchitis, emphysema and irreversible airflow limitation. These changes are thought to be due to oxidative stress and an imbalance of proteases and antiproteases. Quercetin, a plant flavonoid, is a potent antioxidant and anti-inflammatory agent. We hypothesized that quercetin reduces lung inflammation and improves lung function in elastase/lipopolysaccharide (LPS)-exposed mice which show typical features of COPD, including airways inflammation, goblet cell metaplasia, and emphysema. Methods Mice treated with elastase and LPS once a week for 4 weeks were subsequently administered 0.5 mg of quercetin dihydrate or 50% propylene glycol (vehicle) by gavage for 10 days. Lungs were examined for elastance, oxidative stress, inflammation, and matrix metalloproteinase (MMP) activity. Effects of quercetin on MMP transcription and activity were examined in LPS-exposed murine macrophages. Results Quercetin-treated, elastase/LPS-exposed mice showed improved elastic recoil and decreased alveolar chord length compared to vehicle-treated controls. Quercetin-treated mice showed decreased levels of thiobarbituric acid reactive substances, a measure of lipid peroxidation caused by oxidative stress. Quercetin also reduced lung inflammation, goblet cell metaplasia, and mRNA expression of pro-inflammatory cytokines and muc5AC. Quercetin treatment decreased the expression and activity of MMP9 and MMP12 in vivo and in vitro, while increasing expression of the histone deacetylase Sirt-1 and suppressing MMP promoter H4 acetylation. Finally, co-treatment with the Sirt-1 inhibitor sirtinol blocked the effects of quercetin on the lung phenotype. Conclusions Quercetin prevents progression of emphysema in elastase/LPS-treated mice by reducing oxidative stress, lung inflammation and expression of MMP9 and MMP12.http://deepblue.lib.umich.edu/bitstream/2027.42/78260/1/1465-9921-11-131.xmlhttp://deepblue.lib.umich.edu/bitstream/2027.42/78260/2/1465-9921-11-131.pdfPeer Reviewe

Self-assembling nanoparticles containing dexamethasone as a novel therapy in allergic airways inflammation.

Nanocarriers can deliver a wide variety of drugs, target them to sites of interest, and protect them from degradation and inactivation by the body. They have the capacity to improve drug action and decrease undesirable systemic effects. We have previously developed a well-defined non-toxic PEG-dendritic block telodendrimer for successful delivery of chemotherapeutics agents and, in these studies, we apply this technology for therapeutic development in asthma. In these proof-of-concept experiments, we hypothesized that dexamethasone contained in self-assembling nanoparticles (Dex-NP) and delivered systemically would target the lung and decrease allergic lung inflammation and airways hyper-responsiveness to a greater degree than equivalent doses of dexamethasone (Dex) alone. We found that ovalbumin (Ova)-exposed mice treated with Dex-NP had significantly fewer total cells (2.78 ± 0.44 × 10(5) (n = 18) vs. 5.98 ± 1.3 × 10(5) (n = 13), P<0.05) and eosinophils (1.09 ± 0.28 × 10(5) (n = 18) vs. 2.94 ± 0.6 × 10(5) (n = 12), p<0.05) in the lung lavage than Ova-exposed mice alone. Also, lower levels of the inflammatory cytokines IL-4 (3.43 ± 1.2 (n = 11) vs. 8.56 ± 2.1 (n = 8) pg/ml, p<0.05) and MCP-1 (13.1 ± 3.6 (n = 8) vs. 28.8 ± 8.7 (n = 10) pg/ml, p<0.05) were found in lungs of the Dex-NP compared to control, and they were not lower in the Dex alone group. In addition, respiratory system resistance was lower in the Dex-NP compared to the other Ova-exposed groups suggesting a better therapeutic effect on airways hyperresponsiveness. Taken together, these findings from early-stage drug development studies suggest that the encapsulation and protection of anti-inflammatory agents such as corticosteroids in nanoparticle formulations can improve efficacy. Further development of novel drugs in nanoparticles is warranted to explore potential treatments for chronic inflammatory diseases such as asthma

The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues

Inhaled Interleukin-10 before and after induction of experimental endotoxemia in the rat : effects on the inflammatory response

To determine the effects of inhaled IL-10 at different doses and different time points on the pulmonary and systemic inflammatory response during endotoxemia, 48 ventilated, anaesthetized rats (mean body weight ± standard deviation, 500 ± 33g) were randomly assigned to six groups (n = 8, each). Interleukin-10 was nebulised either prior to or following the intravenous injection of LPS (5mg/kg) at two doses (5.0 mycro-g or 0.5 mycro-g) in our groups. Eight rats received the same insult with no further treatment (LPS-only group). Another eight rats served as controls without endotoxemia but with aerosolized phosphate-buffered saline, the solvent of IL-10 (Sham group). Concentrations of TNF-alpha, IL-1beta, IL-6, and IFN-gamma were analyzed in plasma and bronchoalveolar lavage fluid (BALF). In addition, the nitrite release from ex-vivo cultured alveolar macrophages was determined. As compared to the LPS-only group, the concentrations of the proinflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IFN-gamma in plasma were significantly reduced in the group, which inhaled 5 mycro-g IL-10 before LPS injection (p< 0.0125). Spontaneous nitrite release from exvivo cultured alveolar macrophages was suppressed in this group (p< 0.0125). Inhalation of 0.5 mycro-g IL-10 before LPS injection and both dosages of IL-10 inhalation (5 mycro-g or 0.5 mycro-g) after LPS injection did not significantly influence either inflammatory cytokine concentrations in BALF, in plasma or the nitrite release from ex-vivo cultured alveolar macrophages. In this study, inhaled IL-10 only demonstrated anti-inflammatory effects when it was administered at 5 mycro-g prior to the induction of experimental endotoxemia. Interleukin-10 aerosol had no effect when it was given either following induction of endotoxemia or given at a lower dosage (which here was 0.5 mycro-g) either before or following injection of lipopolysaccharide.Das "Acute Respiratory Distress Syndrome" (ARDS) ist eine akut auftretende, überwiegend Sepsis-induzierte, inflammatorische Erkrankung der Lunge mit hoher Letalität. Ein komplexes Netzwerk aus Zytokinen und anderen proinflammatorischen Mediatoren unterhält die pulmonale Entzündungreaktion. Dem Zytokin Interleukin-10 (IL-10) könnte in diesem Zusammenhang aufgrund seines spezifisch antiinflammatorischen und immunmodulierenden Wirkspektrums eine therapeutische Bedeutung zukommen. In tierexperimentellen Untersuchungen konnten die protektiven Wirkungen von systemisch appliziertem Interleukin-10 bezüglich verringerter Wirkspiegel proinflammatorischer Mediatoren sowie des Überlebens der Versuchstiere bei Sepsis belegt werden. In einer Untersuchung an ARDS-Erkrankten wiesen Patienten, deren bronchoalveoläre Lavage (BAL) hohe Konzentrationen an Interleukin-10 enthielt, eine signifikant niedrigere Letalität auf als Patienten mit niedriger IL-10-Konzentration. Die Inhalation von IL-10 über den Luftweg könnte lokal in der Lunge die Freisetzung von Entzündungsmediatoren verringern und so den Verlauf eines ARDS positiv beeinflussen. Im Rahmen einer bereits durchgeführten Studie der eigenen Arbeitsgruppe konnte gezeigt werden, dass die Inhalation von IL-10 (0.19 mycro-g/Tier) vor Induktion einer experimentellen Endotoxinämie (Beobachtungszeitraum 6h) zur signifikanten Reduktion proinflammatorischer Zytokine im Plasma sowie der BAL führte. Daneben bewirkte IL-10 Aerosol eine signifikante Verringerung der Nitritproduktion aus ex vivo kultivierten Alveolarmakrophagen (AM). Mit der vorliegenden Studie sollte untersucht werden, ob vernebeltes IL-10 auch in einer geringeren Dosierung als in der ersten Studie angewandt antiinflammatorisch wirksam ist. Daneben sollte geklärt werden, ob der Zeitpunkt der Applikation des IL-10 Aerosol relevant ist. Konkret sollte untersucht werden, ob die Inhalation von IL-10 erst nach Induktion der experimentellen Endotoxinämie ebenfalls antinflammatorisches Potential besitzt. Die Generierung und Verneblung des IL-10 Aerosols erfolgte in der vorliegenden Untersuchung mittels eines speziell für diesen Einsatz von der eigenen Arbeitsgruppe entwickelten und charakterisierten Verneblersystems. Der Vernebler produziert stabil Aerosopartikel, deren Größenverteilung (rund 2 mycro-m) mit hoher Wahrscheinlichkeit in alvelären Bereichen deponiert. Die alveoläre Depositionsfraktion des Verneblers beträgt rund 4% und liegt damit in einem Bereich, der aus der Humanmedizin für die Verneblung bei intubierten Patienten bekannt ist. An 48 narkotisierten, kontrolliert beatmeten Ratten wurde die antiinflammatorische Wirkung eines IL-10-Aerosols untersucht. Die Induktion des experimentellen Lungenschadens erfolgte durch intravenöse Injektion von Endotoxin (Lipopolysaccharid; LPS, in einer Dosierung von 5mg/kg). Als löslicher Bestandteil der Membran gram-negativer Bakterien führt LPS über die Freisetzung proinflammatorischer Substanzen zu entzündlichen Reaktionen, die lokal beschränkt oder auch systemisch auftreten können. Bei Versuchstieren unterschiedlicher Spezies bewirkt die systemische Applikation von LPS pulmonale Veränderungen, die denen des septisch bedingten ARDS des Menschen qualitativ entsprechen. Die Tiere wurden zufällig in eine der sechs Versuchsgruppen eingeteilt (je n=8): Die LPS-Gruppe erhielt eine LPS-Injektion (5 mg/kg/KG) ohne anschließende therapeutische Intervention. Die mit IL-10 behandelten Tiere erhielten das IL-10-Aerosol entweder vor oder nach Induktion der experimentellen Endotoxinämie nach unten genanntem Protokoll. In einer Kontroll (Sham)-Gruppe wurde die Auswirkung von Narkose, chirurgischer Präparation, Beatmung und Aerosolapplikation (IL-10-Trägerlösung; phosphatgepufferte Kochsalzlösung) evaluiert. Neben der in vivo Beobachtung von Hämodynamik (Herzfrequenz, mittlerer arterieller Blutdruck), Lungenmechanik, arteriellen Blutgasen und Blutbild, untersuchten wir anhand von Blutproben und einer Bronchoalveolären Lavage (BAL) die systemische und pulmonale Inflammation (inflammatorische Zytokine TNFalpha, IL-1beta, IL-6 und IFNgamma in Plasma und BAL). Die Verneblung von IL-10 erfolgte in zwei Dosierungen und zu zwei Zeitpunkten: in einer Gruppe wurde IL-10 in einer Dosierung von 5.0 mycro-g (0.19 mycro-g/Tier) vor, und in einer weiteren Gruppe nach Induktion der experimentellen Endotoxinämie vernebelt. In zwei weiteren Versuchgruppen wurde IL-10 in einer Dosierung von 0.5 mycro-g (0.019 mycro-g/Tier) ebenfalls vor sowie nach Injektion von LPS vernebelt. Die Endotoxinämie führte nur zu geringen Verschlechterungen der klinischen Parameter, aber sowohl pulmonal (BAL) als auch systemisch (Plasma) zeigte sich ein Anstieg proinflammatorischer Mediatoren. Gegenüber den Tieren, deren Endotoxinämie unbehandelt blieb, bewirkte nur die Inhalation von IL-10 in der höheren Dosierung (5 mycro-g) das signifikante Abfallen der proinflammatorischen Zytokine TNFalpha, IL-1beta, IL-6 und IFNgamma im Plasma sowie der Freisetzung von Nitrit aus kultivierten AM. IL-10 Aerosol hatte in keiner Dosierung respektive zu keinem Applikationszeitpunkt antiinflammatorische Effekte auf die Zytokinkonzentrationen in der BAL. Die präemptive Vernebelung von IL-10 in einer Dosierung von 5 mycro-g (0.19 mycro-g/Tier) vor Induktion einer experimentellen Endotoxinämie zeigte sowohl auf die AM Kultur als auch systemisch (Plasma) antinflammatorisches Wirkprofil. Demgegenüber zeigte IL-10 keine antinflammatorische Effekte, wenn es erst nach Injektion von LPS oder aber in geringerer Dosierung vernebelt wurde. Zur antiinflammatorischen Therapie der experimentellen Endotoxinämie durch LPS Injektion in der Spezies Ratte erscheint IL-10 Aerosol nur wirksam, wenn es in ausreichender Dosierung (5 mycro-g) sowie vor Injektion von LPS appliziert wird

Perinatal nicotine with or without early life influenza infection leads to lung dysfunction with age.

Obstructive Airway Diseases (OADs) affect millions of people worldwide, and are characterized by chronic inflammation and tissue remodeling in the lung. It has been proposed that the development of OAD is greatly influenced (and perhaps pre-determined) by early life events, such as maternal smoking or a viral infection. However, direct evidence of this is limited and the mechanisms involved remain unclear. Our laboratory previously developed a murine model of perinatal nicotine exposure, and reported that nicotine leads to airway remodeling and decreased pulmonary function in the offspring. We discovered these effects were mediated through the α7 nicotinic acetylcholine receptor (nAChR). Similarly, respiratory viral infections during childhood, such as Influenza A Virus (IAV), have been implicated in the development of OADs in epidemiological and murine studies. We set out to investigate if perinatal nicotine and/or early life IAV infection could promote lung remodeling and long-term pulmonary dysfunction. We employed a previously established murine model for nicotine exposure and a newly developed model for early life IAV infection. Importantly, lung dysfunction was tested at 7-months of age, in which all other studies look at lung dysfunction at earlier times. v We show that both chronic nicotine exposure starting during embryogenesis and continuing until adulthood (but not transient exposure limited to the perinatal period) and early life IAV infection separately are capable of driving lung dysfunction with age. Interestingly, limited differences in lung dysfunction were discovered with chronic exposure and early life IAV infection alone, whereas major lung dysfunctional differences were discovered for transient perinatal nicotine exposure followed by early life IAV infection suggesting a potentiation effect. Abnormalities in lung function were accredited to increased peri-airway collagen deposition and enlarged alveolar structures; the latter appeared due to alveolar simplification during development but also perhaps destruction during aging. We discovered that α7 nAChRs partially mediated these changes. All together, we found that indeed these early life exposures resulted in abnormalities in lung structure and function that persisted into adulthood. This model of nicotine exposure and early life IAV infection in young mice provides a novel tool for studying the impact of these exposures in lung

Local lung hypoxia determines epithelial fate decisions during alveolar regeneration.

After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5 (Krt5) remodelling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, through hypoxia-inducible factor (HIF1α), drives Notch signalling and Krt5pos basal-like cell expansion. Single-cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and transdifferentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2pos LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair

Pathophysiological correlations in maedi: a chronic lymphoid interstitial pneumonia of sheep

Maedi is a chronic lymphoid interstitial pneumonia (LIP) of sheep caused by the lentivirus, maedi-visna virus (MW). Following a long prepatent phase maedi progresses to clinical disease characterised by exercise intolerance and dyspnoea. Although maedi is well characterised at a pathological level, physiological studies of the disease have not been undertaken. As a consequence, the nature of the functional abnormality that underlies the clinical disease is unknown, whether there exists measurable functional deficit in the preclinical phase of the disease is unknown and the relationship between lung pathology and functional deficit is unknown. These questions are fundamental to understanding the way in which pathological events ultimately conspire to bring about organ dysfunction and clinical disease. Further, knowledge of the way in which pathology relates to measurable lung dysfunction offers a potential means of assessing the progress and prognosis of this disease. This thesis describes an investigation into the pathophysiological mechanisms responsible for inducing lung functional deficits in maedi.As a prelude to establishing the nature of the functional deficit in maedi, repeated measurements of static lung compliance (Cst), lung distensibility (K), effective alveolar volume (VA,eff) and transfer factor for carbon monoxide (TL,CO,'sb') were made in anaesthetized control sheep, seronegative for MW, over a period of 5 months. This study furnished regression equations and prediction intervals for lung function indices in normal sheep using bodyweight as the independent variable. By comparison with predicted normal values sheep naturally infected with MW had reduced lung volumes and gas diffusing capabilities and increased lung elastic recoil.A pathophysiological study was instigated to identify structural correlates of lung functional deficits. Preliminary investigation involved the quantitative morphometric characterisation of the normal sheep lung. Data from this study indicated that the ratio of fixed to physiological lung volume ranged from 0.49 to 0.59 and that this ratio was positively correlated with the time between euthanasia and inflation fixation of the lungs. Values for tissue volume fraction within the lung parenchyma (Fvt) ranged from 0.18 to 0.25 and values for alveolar surface density (Svt) ranged from 592 to 716 cm2/cm3. Pathophysiological correlations in MW-infected sheep indicated that lung volume and transfer factor measurements were more sensitive indices of pathology than measurements of Cst or K. Transfer factor was reduced even in sheep with minimal histopathology suggesting this index as a sensitive means of assessing this condition. The density of surface forces could not account for variation in K seen in vivo, however tissue factors such as the quantity and functional tone of contractile tissue in the parenchyma, airways or blood vessels may contribute. Given that parenchymal smooth muscle hyperplasia is a pathological feature of maedi, it was hypothesized that this tissue element is responsible for the observed reduction in K.In order to further investigate this relationship, the distribution and morphometric quantitation of a-smooth muscle actin (ASMA) in lung parenchyma from normal and MW-infected 4 sheep was determined and related to in vivo functional measurements. The volume density of ASMA (IVASMA1) was negatively correlated with K and Cst, however partial correlation coefficients indicated that IVASMA' and Fvt were strongly interdependent thus complicating interpretation of the link between Fv'ASMA' and K. In order to separate the influence of dynamic and passive tissue elements, histamine and clenbuterol were administered to normal and MWinfected sheep in an attempt to cause relaxation and contraction of parenchymal contractile tissue. The functional response of the cardiopulmonary system to intravenous infusion of these agents was measured and correlated with Fv'ASMA'. K and Cst were significantly increased following clenbuterol injection, however only the increase in K was correlated with the quantity of Fv'ASMA', and this correlation was negative. These results could be explained if the site of action of clenbuterol was not the contractile tissue at the level of the alveolar ducts, but rather that which surrounds conducting airways.The dose of histamine required to lower dynamic compliance to 65% of baseline values was negatively correlated with Fv'ASMA' and the attendant percentage change in K was positively correlated with Fv'ASMA'. These findings support the contention that parenchymal contractile tissue is of functional relevance and capable of regulating overall lung elastic properties.Maedi is a naturally occurring disease in sheep in which the aetiologic agent and target cell is known and in which the pathology is well characterised. As such it has potential as a model for LIP associated with human immunodeficiency virus infection in children and adults. The present study has both served to establish the functional characteristics of this disease and indicate structural correlates of observed functional deficits. Moreover, evidence is presented to suggest that the observed reduction in lung distensibility in maedi is a consequence of increased tissue forces associated with the parenchymal smooth muscle hyperplasia that is a feature of this disease

Inflammatory Response as a Mechanism of Perinatal Programming: Long-term Impact on Pulmonary and Renal Function?

RATIONALE: Temporal changes in the fetal environment, such as malnutrition and placental insufficiency induce intrauterine growth restriction (IUGR) and lead to a permanent changes of physiological processes later in life. Interestingly, epidemiological studies demonstrated an impairment of lung and renal function in young infants subsequent to IUGR. Complementary, experimental studies showed that IUGR induces a perinatal programming of the developing lung with persisting impairment of pulmonary structure and function. Besides IUGR, early postnatal hyperalimentation (pHA) is discussed as a crucial factor of IUGR-associated diseases. Both extracellular matrix (ECM) and inflammatory processes have been shown to be dysregulated following IUGR and early pHA. However, the underlying molecular mechanisms of IUGR-associated diseases and the potential linkage of ECM and inflammation have not been addressed so far. Therefore, the ultimate goal of this project was to elucidate the role of regulation of ECM and inflammatory cytokines subsequent to IUGR and early pHA. AIMS: There are three specific aims: (1) to analyze the role of the TGF-β signaling in lung development subsequent to IUGR, (2) to determine the regulation of inflammatory cytokines and ECM-molecules in lungs subsequent to IUGR, and (3) to characterize the pathomechanistic role of early pHA using the example of the kidney. METHODS: Two simultaneous sets of animal experiments were used. One animal model addressed pre- and postnatal nutritional intervention, the other was restricted to postnatal interventions only: (A) IUGR was induced in Wister rats by isocaloric low protein diet (8% casein; IUGR) during gestation. The control group received normal protein diet (17% casein; Co). At birth the litter size was reduced to 6 male pups to induce early pHA. During lactation the mothers of both groups were fed standard chow. (B) Early pHA was induced by litter size reduction to 6 (LSR6) or 10 (LSR10) male neonates. Home-cage control (HCC; mean litter size of 16) animals without any postnatal manipulation during lactation were included. At postnatal day (P) 28 as well as P70 animals underwent whole body plethysmography and in addition metabolic cages at P70. Serum and samples of lungs and kidney were obtained at P1, P12, P21, P42, and P70 for mRNA extraction, protein extraction as well as histological analyses. RESULTS: Both respiratory system resistance and compliance were impaired subsequent to IUGR at P28; this impairment was even more significant at P70. (1) These changes were accompanied by persistent attenuated activity of the TGF-β signaling, assessed by phosphorylation of Smad2 and Smad3. Expression analysis of TGF-β-regulated ECM components in the lungs of IUGR animals at P1, such as collagen I, elastin, and tenascin N, revealed a significant deregulation. Consistently, 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. Interestingly, however, not just a deregulation of ECM components was detected at P1, but also attenuated apoptotic processes, e.g. decreased cleavage of PARP. (2) Since the TGF-β signaling has potent anti-inflammatory effects, we next determined the dynamic expression of pro-inflammatory and pro-fibrotic markers as well of ECM components in the lung subsequent to IUGR at P1, P42 and P70. The expression of ECM components and metabolizing enzymes was markedly deregulated and the deposition of collagen I was strikingly increased at P70. Concomitantly to the pro-fibrotic processes in the lung subsequent to IUGR, the expression of inflammatory cytokines and both the activity and the expression of target genes of Stat3 signaling were dynamically regulated, with unaltered or decreased expression at P1 and significantly increased expression art P70. (3) Assessment of renal function at postnatal day 70 revealed decreased glomerular filtration rate, proteinuria, and increased fractional sodium and potassium secretion following early pHA (LSR6). Moreover, the deposition of ECM molecules, such as collagen I, was increased. Interestingly, despite the elevated expression of pro-inflammatory leptin and IL-6 expression the phosphorylation of Stat3 and ERK1/2 in the kidney, however, was decreased after LSR6. In accordance, neuropeptide Y (NPY) gene expression – sympathetic co-neurotransmitter regulated by Stat3 signaling – was down-regulated. In accordance, suppressor of cytokine signaling (SOCS)3 protein expression, an inhibitor of Stat3 and Erk1/2 signaling, was strongly elevated and colocalized with NPY. Interestingly, NPY is co- localized with SOCS3 in the distal tubules in the cortex and outer medulla, and in the proximal tubules, but no expression of SOCS3 was detectable in glomeruli. CONCLUSION: Taken together, IUGR has a direct and strong negative impact on respiratory resistance and compliance of the lung. There are two major underlying mechanisms linking IUGR and deregulation of ECM: first, the attenuated TGF-β signaling during late lung development; and second, the increased expression of inflammatory cytokines subsequent to IUGR. In addition, the missing anti-inflammatory effect of TGF-β signaling could contribute to the increased inflammatory response following IUGR. Furthermore we demonstrated that early pHA leads to organ-intrinsic increased expression of NPY via a postreceptor-leptin-receptor leptin resistance. This leptin resistance could contribute to the observed profibrotic processes, and ultimately to a long-term impairment of renal function. Thus, both IUGR and early postnatal hyperalimentation have a strong impact on perinatal programming of multi-organ inflammatory response