Fibrosis In Aged Mice Research Articles

IL-10 deficiency aggravates cell senescence and accelerates BLM-induced pulmonary fibrosis in aged mice via PTEN/AKT/ERK pathway

BackgroundPulmonary fibrosis (PF) is an aging-related progressive lung disorder. The aged lung undergoes functional and structural changes termed immunosenescence and inflammaging, which facilitate the occurrence of fibrosis. Interleukin-10 (IL-10) is a potent anti-inflammatory and immunoregulatory cytokine, yet it remains unclear how IL-10 deficiency-induced immunosenescence participates in the development of PF.MethodsFirstly we evaluated the susceptibility to fibrosis and IL-10 expression in aged mice. Then 13-month-old wild-type (WT) and IL-10 knockout (KO) mice were subjected to bleomycin(BLM) and analyzed senescence-related markers by PCR, western blot and immunohistochemistry staining of p16, p21, p53, as well as DHE and SA-β-gal staining. We further compared 18-month-old WT mice with 13-month-old IL-10KO mice to assess aging-associated cell senescence and inflamation infiltration in both lung and BALF. Moreover, proliferation and apoptosis of alveolar type 2 cells(AT2) were evaluated by FCM, immunofluorescence, TUNEL staining, and TEM analysis. Recombinant IL-10 (rIL-10) was also administered intratracheally to evaluate its therapeutic potential and related mechanism. For the in vitro experiments, 10-week-old naïve pramily lung fibroblasts(PLFs) were treated with the culture medium of 13-month PLFs derived from WT, IL-10KO, or IL-10KO + rIL-10 respectively, and examined the secretion of senescence-associated secretory phenotype (SASP) factors and related pathways.ResultsThe aged mice displayed increased susceptibility to fibrosis and decreased IL-10 expression. The 13-month-old IL-10KO mice exhibited significant exacerbation of cell senescence compared to their contemporary WT mice, and even more severe epithelial-mesenchymal transition (EMT) than that of 18 month WT mice. These IL-10 deficient mice showed heightened inflammatory responses and accelerated PF progression. Intratracheal administration of rIL-10 reduced lung CD45 + cell infiltration by 15%, including a 6% reduction in granulocytes and a 10% reduction in macrophages, and increased the proportion of AT2 cells by approximately 8%. Additionally, rIL-10 significantly decreased α-SMA and collagen deposition, and reduced the expression of senescence proteins p16 and p21 by 50% in these mice. In vitro analysis revealed that conditioned media from IL-10 deficient mice promoted SASP secretion and upregulated senescence genes in naïve lung fibroblasts, which was mitigated by rIL-10 treatment. Mechanistically, rIL-10 inhibited TGF-β-Smad2/3 and PTEN/PI3K/AKT/ERK pathways, thereby suppressing senescence and fibrosis-related proteins.ConclusionsIL-10 deficiency in aged mice leads to accelerated cell senescence and exacerbated fibrosis, with IL-10KO-PLFs displaying increased SASP secretion. Recombinant IL-10 treatment effectively mitigates these effects, suggesting its potential as a therapeutic target for PF.Graphical

Lysosomal-Associated Protein Transmembrane 5, Tubular Senescence, and Progression of CKD.

Tubular senescence is a major determinant of chronic kidney disease (CKD) and identification of potential therapeutic targets involved in senescent tubular epithelial cells has clinical importance. Lysosomal-associated protein transmembrane 5 (LAPTM5) is a key molecule related to T and B cell receptor expression and inflammation. However, the expression pattern of LAPTM5 in the kidney and the contribution of LAPTM5 to the development of CKD keep unknown. LAPTM5-/- mice and tubule specific-LAPTM5 knockout mice were used to examine the role of LAPTM5 in tubular senescence by establishing different experimental mouse CKD models. LAPTM5 expression was significantly induced in the kidney, especially in proximal tubules and distal convoluted tubules, from mice with aristolochic acid nephropathy, bilateral ischemia/reperfusion injury (IRI)-induced CKD or unilateral ureter obstruction (UUO). Tubule-specific deletion of LAPTM5 inhibited senescence of tubular epithelial cells and alleviated tubulointerstitial fibrosis in aged mice. Moreover, LAPTM5 deficiency ameliorated kidney injury and tubular senescence in mice with CKD. Mechanistically, LAPTM5 inhibited ubiquitination of NICD1 by mediating WWP2 lysosomal degradation, then leading to cellular senescence in tubular epithelial cells. Notably, we also observed a higher expression of LAPTM5 in tubules from individuals with CKD and the level of LAPTM5 was correlated with kidney fibrosis and tubular senescence in people with CKD. LAPTM5 contributed to tubular senescence by regulating WWP2/NICD1 signaling pathway and exacerbated kidney injury during the progression of CKD.

Resolvin D1 suppresses macrophage senescence and splenic fibrosis in aged mice

Aging is associated with systemic, non-resolving inflammation and the accumulation of senescent cells. The resolution of inflammation (or inflammation-resolution) is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory leukotrienes (LTs). Aged mice (i.e. 2 years of age) exhibit a significant decrease in the SPM:LT ratio in specific organs including the spleen, which suggests that this organ may exhibit heightened inflammation and may be particularly amenable to SPM therapy. Previous studies have shown that resolvin D1 (RvD1) is decreased in spleens of aged mice compared with young controls. Therefore, we asked whether treatment of RvD1 in aged mice would impact markers of cellular senescence in splenic macrophages, and downstream effects on splenic fibrosis, a hallmark of splenic aging. We found that in aged mice, both zymosan-elicited and splenic macrophages showed an increase in mRNA expression of inflammatory and eicosanoid biosynthesis genes and a dysregulation of genes involved in the cell cycle. Injections with RvD1 reversed these changes. Importantly, RvD1 also decreased splenic fibrosis, a hallmark of splenic aging. Our findings suggest that RvD1 treatment may limit several features of aging, including senescence and fibrosis in spleens from aged mice.SummaryAging is associated with systemic, low grade, non-resolving inflammation. The resolution of inflammation is in part mediated by the balance between specialized pro-resolving mediators (SPMs) and pro-inflammatory lipid mediators, like leukotrienes (LTs). A hallmark of aging is the accumulation of senescent cells that promote low grade inflammation by secreting pro-inflammatory cytokines and lipid mediators. Splenic macrophages contribute to systemic aging, and spleens of aged mice demonstrate decreased levels of the SPM called resolvin D1 (RvD1). Whether addition of RvD1 is protective in spleens of aged mice is unknown and is focus of this study. RvD1 treatment to aged mice led to decreased mRNA expression of markers of cellular senescence and inflammation in splenic macrophages compared with age-matched vehicle controls. Moreover, RvD1 decreased splenic fibrosis, which occurs due to persistent low-grade inflammation in aging. Promoting inflammation resolution with RvD1 thus limits macrophage senescence, pro-inflammatory signals and established splenic fibrosis in aging.

Protective Effect of Heparan Sulphate Derivative Against Glycocalyx Damage-Induced Renal Fibrosis in Aging Mice

Protective Effect of Heparan Sulphate Derivative Against Glycocalyx Damage-Induced Renal Fibrosis in Aging Mice

Excessive sodium chloride ingestion promotes inflammation and kidney fibrosis in aging mice.

In aging kidneys, a decline of function resulting from extracellular matrix (ECM) deposition and organ fibrosis is regarded as "physiological." Whether a direct link between high salt intake and fibrosis in aging kidney exists autonomously from arterial hypertension is unclear. This study explores kidney intrinsic changes (inflammation, ECM derangement) induced by a high-salt diet (HSD) in a murine model lacking arterial hypertension. The contribution of cold shock Y-box binding protein (YB-1) as a key orchestrator of organ fibrosis to the observed differences is determined by comparison with a knockout strain (Ybx1ΔRosaERT+TX). Comparisons of tissue from mice fed with normal-salt diet (NSD, standard chow) or high-salt diet (HSD, 4% NaCl in chow; 1% NaCl in water) for up to 16 mo revealed that with HSD tubular cell numbers decrease and tubulointerstitial scarring [periodic acid-Schiff (PAS), Masson's trichrome, Sirius red staining] prevails. In Ybx1ΔRosaERT+TX animals tubular cell damage, a loss of cell contacts with profound tubulointerstitial alterations, and tubular cell senescence was seen. A distinct tubulointerstitial distribution of fibrinogen, collagen type VI, and tenascin-C was detected under HSD, transcriptome analyses determined patterns of matrisome regulation. Temporal increase of immune cell infiltration was seen under HSD of wild type, but not Ybx1ΔRosaERT+TX animals. In vitro Ybx1ΔRosaERT+TX bone marrow-derived macrophages exhibited a defect in polarization (IL-4/IL-13) and abrogated response to sodium chloride. Taken together, HSD promotes progressive kidney fibrosis with premature cell aging, ECM deposition, and immune cell recruitment that is exacerbated in Ybx1ΔRosaERT+TX animals.NEW & NOTEWORTHY Short-term experimental studies link excessive sodium ingestion with extracellular matrix accumulation and inflammatory cell recruitment, yet long-term data are scarce. Our findings with a high-salt diet over 16 mo in aging mice pinpoints to a decisive tipping point after 12 mo with tubular stress response, skewed matrisome transcriptome, and immune cell infiltration. Cell senescence was aggravated in knockout animals for cold shock Y-box binding protein (YB-1), suggesting a novel protective protein function.

Angiotensin II type-1 receptor-associated protein interacts with transferrin receptor-1 and promotes its internalization

Kidney fibrosis is a common pathway that leads to chronic kidney disease. Angiotensin II type-1 receptor (AT1R)-associated protein (ATRAP) was originally identified as an AT1R-binding protein. Previously, we reported that systemic knockout of ATRAP exacerbates kidney fibrosis in aged mice. Although these effects of ATRAP appeared to be AT1R-independent actions, the molecular mechanism remains poorly understood. To elucidate the molecular mechanism of ATRAP independent of AT1R, we explored novel ATRAP-interacting proteins. Mass spectrometric analysis of the immunoprecipitants of a Flag-tagged ATRAP complex revealed 376 candidate proteins that potentially interact with ATRAP. Gene ontology analysis revealed that proteins related to vesicle trafficking, membrane transport, and many membrane proteins, including transferrin receptor 1 (TfR1), were enriched. Because TfR1 promotes cellular iron uptake and iron is a key factor involved in kidney fibrosis, we focused on TfR1 and confirmed that it interacts with ATRAP. In addition, our findings revealed that enhanced ATRAP expression decreased cell-surface TfR1 expression without altering the overall cellular TfR1 expression levels. Furthermore, enhanced ATRAP expression attenuated cellular iron levels. Together, our results highlight the role of ATRAP as a suppressor of TfR1 that functions by facilitating TfR1 internalization, which affects iron metabolism and oxidative stress signaling.

Lactoferrin ameliorates myocardial fibrosis by inhibiting inflammatory response via the AMPK/NF-κB pathway in aged mice

Graphical abstract demonstrating lactoferrin inhibits NF-κB - related inflammation by activating AMPK, thereby improving cardiac fibrosis and protecting the aging heart. Abbreviations AMPK, 5′-AMP-activated protein kinase, NF-κB, nuclear factor kappa-B. • Lactoferrin ameliorated cardiac aging and myocardial fibrosis in aged mice. • Lactoferrin inhibited TGF-β1-induced cardiac fibroblast activation and collagen synthesis. • NF-κB -related inflammation was inhibited by lactoferrin via AMPK activation. • Lactoferrin has potential use in alleviating age-related myocardial fibrosis. Cardiac fibrosis is more likely to promote the development of heart failure in the elderly. However, effective prevention and improvement of cardiac fibrosis are lacking. Here, we investigated the ameliorative effect of lactoferrin (LF) on aging cardiac fibrosis and its possible mechanism. We treated aged mice with LF and measured its effects on cardiac function, cardiac aging, cardiac fibrosis, and inflammatory response. LF effects on proliferation, transformation, collagen synthesis, and related signalling pathways of fibroblasts were observed in vitro, indicating that LF could delay heart aging, improve heart function, reduce fibrosis, and inhibit inflammation. LF inhibited proliferation, transformation, collagen synthesis, and inflammation associated with nuclear factor kappa-B (NF-κB) via 5′-AMP-activated protein kinase (AMPK) activation in myocardial fibroblasts, suggesting that LF ameliorates cardiac fibrosis and protects the heart in aged mice via AMPK activation. Thus, LF is a functional food for preventing and improving heart disease associated with cardiac fibrosis.

Modulation of H4K16Ac levels reduces pro-fibrotic gene expression and mitigates lung fibrosis in aged mice.

Histone H4 lysine16 acetylation (H4K16Ac) modulates chromatin structure by serving as a switch from a repressive to a transcriptionally active state. This euchromatin mark is associated with active transcription. In this study, we investigated the effects of H4K16Ac on the expression of pro-fibrotic genes in lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and in an aging murine model of lung fibrosis.Methods: The lung tissues and fibroblasts from human IPF/non-IPF donors and from aged mice with/without bleomycin induced lung fibrosis were used in this study. The H4K16Ac levels were examined by immunohistochemistry or western blots. RNA silencing of H4K16Ac acetyltransferase Mof was used to reduce H4K16Ac levels in IPF fibroblasts. The effects of reduced H4K16Ac on pro-fibrotic gene expression were examined by western blots and real-time PCR. The association of H4K16Ac with these genes' promoter region were evaluated by ChIP assays. The gene expression profile in siRNA Mof transfected IPF cells were determined by RNA-Seq. The impact of H4K16Ac levels on lung fibrosis was evaluated in an aging murine model.Results: Aged mice with bleomycin induced lung fibrosis showed increased H4K16Ac levels. Human lung fibroblasts with siRNA Mof silencing demonstrated reduced H4K16Ac, and significantly down-regulated profibrotic genes, such as α-smooth muscle actin (α-SMA), collagen I, Nox4, and survivin. ChIP assays confirmed the associations of these pro-fibrotic genes' promoter region with H4K16Ac, while in siRNA Mof transfected cells the promoter/H4K16Ac associations were depleted. RNA-seq data demonstrated that Mof knockdown altered gene expression and cellular pathways, including cell damage and repair. In the aging mice model of persistent lung fibrosis, 18-month old mice given intra-nasal siRNA Mof from week 3 to 6 following bleomycin injury showed improved lung architecture, decreased total hydroxyproline content and lower levels of H4K16Ac.Conclusions: These results indicate a critical epigenetic regulatory role for histone H4K16Ac in the pathogenesis of pulmonary fibrosis, which will aid in the development of novel therapeutic strategies for age-related diseases such as IPF.

The cell membrane repair protein MG53 modulates transcription factor NF-κB signaling to control kidney fibrosis

Kidney fibrosis is associated with the progression of acute kidney injury to chronic kidney disease. MG53, a cell membrane repair protein, has been shown to protect against injury to kidney epithelial cells and acute kidney injury. Here, we evaluated the role of MG53 in modulation of kidney fibrosis in aging mice and in mice with unilateral ureteral obstruction (UUO) a known model of progressive kidney fibrosis. Mice with ablation of MG53 developed more interstitial fibrosis with age than MG53-intact mice of the same age. Similarly, in the absence of MG53, kidney fibrosis was exaggerated compared to mice with intact MG53 in the obstructed kidney compared to the contralateral unobstructed kidney or the kidneys of sham operated mice. The ureteral obstructed kidneys from MG53 deficient mice also showed significantly more inflammation than ureteral obstructed kidneys from MG53 intact mice. Invitro experiments demonstrated that MG53 could enter the nuclei of proximal tubular epithelial cells and directly interact with the p65 component of transcription factor NF-κB, providing a possible explanation of enhanced inflammation in the absence of MG53. To test this, enhanced MG53 expression through engineered cells or direct recombinant protein delivery was given to mice subject to UUO. This reduced NF-κB activation and inflammation and attenuated kidney fibrosis. Thus, MG53 may have a therapeutic role in treating chronic kidney inflammation and thereby provide protection against fibrosis that leads to the chronic kidney disease phenotype.

Regulation of connective tissue growth factor expression by miR-133b for the treatment of renal interstitial fibrosis in aged mice with unilateral ureteral obstruction

IntroductionRenal interstitial fibrosis, an important pathological feature of kidney aging and chronic renal failure, is regulated by mesenchymal stem cells (MSCs). We have previously demonstrated low expression of miR-133b in MSC-derived extracellular vesicles (MSC-EVs) in aged rats. However, miR-133b can mediate the inhibition of epithelial-mesenchymal transition (EMT) of renal tubules induced by transforming growth factor-β1 (TGF-β1). We investigated the effect of miR-133b for the treatment of geriatric renal interstitial fibrosis and evaluated its target genes.MethodsWe performed real-time polymerase chain reaction to detect miR-133b expression induced during EMT of HK2 cells by TGF-β1 at different concentrations (0, 6, 8, and 10 ng/mL) and at different time points (0, 24, 48, and 72 h). The target genes of miR-133b were validated using the dual-luciferase reporter assay. In vitro experiments were performed to evaluate mRNA and protein expression of miR-133b targets, E-cadherin, α-smooth muscle actin (SMA), fibronectin, and collagen 3A1 (Col3A1), in HK2 cells transfected with miR-133b under TGF-β1 stimulation. A 24-month-old unilateral ureteral obstruction (UUO) mouse model was established and injected with transfection reagent and miR-133b into the caudal vein. The target gene of miR-133b and other parameters mentioned above such as mRNA and protein expression levels and renal interstitial fibrosis were detected at 7 and 14 days.ResultsmiR-133b expression gradually decreased with an increase in TGF-β1 concentration and treatment time, and the miR-133b mimic downregulated connective tissue growth factor (CTGF) expression. The dual-luciferase reporter assay confirmed CTGF as a direct target of miR-133b. Transfection of the miR-133b mimic inhibited TGF-β1-induced EMT of HK2 cells; this effect was reversed by CTGF overexpression. miRNA-133b expression significantly increased (approximately 70–100 times) in mouse kidney tissues after injection of the miRNA-133b overexpression complex, which significantly alleviated renal interstitial fibrosis in mice with UUO.ConclusionmiR-133b exerted targeted inhibitory effects on CTGF expression, which consequently reduced TGF-β1-induced EMT of HK2 cells and renal interstitial fibrosis in aged mice with UUO.

Targeting mechanosensitive MDM4 promotes lung fibrosis resolution in aged mice.

Aging is a strong risk factor and an independent prognostic factor for progressive human idiopathic pulmonary fibrosis (IPF). Aged mice develop nonresolving pulmonary fibrosis following lung injury. In this study, we found that mouse double minute 4 homolog (MDM4) is highly expressed in the fibrotic lesions of human IPF and experimental pulmonary fibrosis in aged mice. We identified MDM4 as a matrix stiffness-regulated endogenous inhibitor of p53. Reducing matrix stiffness down-regulates MDM4 expression, resulting in p53 activation in primary lung myofibroblasts isolated from IPF patients. Gain of p53 function activates a gene program that sensitizes lung myofibroblasts to apoptosis and promotes the clearance of apoptotic myofibroblasts by macrophages. Destiffening of the fibrotic lung matrix by targeting nonenzymatic cross-linking or genetic ablation of Mdm4 in lung (myo)fibroblasts activates the Mdm4-p53 pathway and promotes lung fibrosis resolution in aged mice. These findings suggest that mechanosensitive MDM4 is a molecular target with promising therapeutic potential against persistent lung fibrosis associated with aging.

Vascular dysfunction in aged mice contributes to persistent lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease thought to result from impaired lung repair following injury and is strongly associated with aging. While vascular alterations have been associated with IPF previously, the contribution of lung vasculature during injury resolution and fibrosis is not well understood. To compare the role of endothelial cells (ECs) in resolving and non‐resolving models of lung fibrosis, we applied bleomycin intratracheally to young and aged mice. We found that injury in aged mice elicited capillary rarefaction, while injury in young mice resulted in increased capillary density. ECs from the lungs of injured aged mice relative to young mice demonstrated elevated pro‐fibrotic and reduced vascular homeostasis gene expression. Among the latter, Nos3 (encoding the enzyme endothelial nitric oxide synthase, eNOS) was transiently upregulated in lung ECs from young but not aged mice following injury. Young mice deficient in eNOS recapitulated the non‐resolving lung fibrosis observed in aged animals following injury, suggesting that eNOS directly participates in lung fibrosis resolution. Activation of the NO receptor soluble guanylate cyclase in human lung fibroblasts reduced TGFβ‐induced pro‐fibrotic gene and protein expression. Additionally, loss of eNOS in human lung ECs reduced the suppression of TGFβ‐induced lung fibroblast activation in 2D and 3D co‐cultures. Altogether, our results demonstrate that persistent lung fibrosis in aged mice is accompanied by capillary rarefaction, loss of EC identity, and impaired eNOS expression. Targeting vascular function may thus be critical to promote lung repair and fibrosis resolution in aging and IPF.

Mitochondrial Uncoupling Protein‐2 Drives Fibroblast Senescence in Age‐Related Lung Fibrosis by Altering Bioenergetics and Reactive Oxygen Species

Background Mitochondrial dysfunction is a potential link between aging and age‐related pathology, including lung fibrosis (idiopathic pulmonary fibrosis, IPF). (Myo)Fibroblasts are the effector cells in fibrosis. Transcriptomic analyses revealed high expression of mitochondrial uncoupling protein‐2 (UCP2) in lung fibroblasts isolated from patients with idiopathic pulmonary fibrosis, compared to healthy controls. Our objective was to examine the role of UCP2 in mediating mitochondrial dysfunction, fibroblast senescence and myofibroblast differentiation in age‐related pathologic fibrosis.MethodsWe studied tissues and fibroblasts obtained from lung explants of IPF patients and utilized the murine model of lung fibrosis and delayed resolution following bleomycin‐induced injury in aged mice. We silenced UCP2 expression using siRNA in vitro and in vivo. Specific endpoints were analyzed by RT‐PCR, SDS‐PAGE western blotting, immunofluorescence, flow cytometry, mass spectrometry and extracellular flux analysis.ResultsUCP2 gene expression was increased in lungs (whole lung lysates as well as fibroblasts) of IPF patients compared to those without IPF; in particular, mesenchymal stromal cells derived from bronchoalveolar lavage of patients with rapidly progressive clinical course had higher UCP2 expression compared to those with a slower course. Lung fibroblasts from aged mice expressed higher UCP2 than younger counterparts, more pronounced after bleomycin‐induced lung injury. The lungs as well as fibroblasts isolated from IPF patients in addition, demonstrated lower steady state ATP levels than those from non‐IPF controls. UCP2 silencing in IPF fibroblasts restored ATP levels, decreased pro‐fibrotic markers such as α‐SMA and collagen; this was associated with enhanced mitochondrial biogenesis, decreased mitochondrial ROS (superoxide and hydrogen peroxide), increased ATP‐linked oxygen consumption and decreased aerobic glycolysis. In addition, UCP2 silencing induced a decrease in senescence markers and decreased expression of senescence‐associated secretory phenotype (SASP) genes. In aged mice after bleomycin injury, therapeutic oro‐tracheal administration of UCP2 siRNA resulted in decreased lung collagen and fibrotic remodeling, indicating enhanced resolution capacity.ConclusionOur results suggest that expression of mitochondrial UCP2 in lung fibroblasts increases with age. UCP2 is highly expressed in lung fibroblasts of patients with IPF, more so in those with rapidly progressive clinical course; UCP2 drives a senescent fibroblast phenotype involving lower ATP levels and impairs resolution of bleomycin injury‐induced fibrosis in aged mice. Further studies are warranted to study the link between cellular aging and UCP2, and the therapeutic potential of targeting UCP2 in age‐related fibrotic disorders such as IPF.Support or Funding InformationNIH grants K08 HL135399 (SR); P01 HL114470 and R01 AG046210 (VJT), VA Merit Award I01BX003056 (VJT)Schematic depicting the putative role played by UCP2 in aging fibroblast phenotypes.Figure 1

New Trends in Regenerative Medicine: Reprogramming and Reconditioning.

New Trends in Regenerative Medicine: Reprogramming and Reconditioning.

Incremental load training improves renal fibrosis by regulating the TGF‑β1/TAK1/MKK3/p38MAPK signaling pathway and inducing the activation of autophagy in aged mice.

Recent studies have confirmed that kidney tissue fibrosis is closely linked to the natural aging of organs. One of its major characteristics is the reduction of autophagic activity. However, to date, few studies have assessed whether incremental load training is able to improve the occurrence of renal fibrosis caused by natural aging and the underlying mechanisms. In the present study involving male C57/BL mice, an elderly exercise group (OY group) was subjected to progressive load-increasing rotary-bar training (5 days/week, lasting for 6 weeks), with an elderly control group (OC group) and a young control group (YC group) used as controls. Renal fibrosis and autophagy-associated indicators were assessed by Masson's staining, reverse transcription-quantitative PCR analysis, western blotting, immunofluorescence and transmission electron microscopy. The results suggested that collagen deposition in the basal part of the renal tubular epithelium and glomeruli in the OY group was significantly lower than that in the OC group. In the OC group, the protein expression levels of E-cadherin, Beclin 1 and light chain 3 were significantly decreased, and increases in α-smooth muscle actin-positive signals were observed in the glomerular matrix and renal capsule wall. Furthermore, the expression of transforming growth factor (TGF)-β1 and its downstream signaling molecules TGF-β-activated kinase 1 (TAK1), mitogen-activated protein kinase (MAPK) kinase (MKK3) and p38MAPK were downregulated following training. The present study confirmed that incremental load training may improve renal fibrosis in aged mice by regulating the TGF-β1/TAK1/MMK3/p38MAPK signaling pathway and inducing the activation of autophagy to reduce the synthesis of extracellular matrix and delay the epithelial-mesenchymal transition. The present study provides a novel experimental basis for the intervention of incremental load training to prevent senile renal fibrosis.

Dysregulation of IL-33/ST2 signaling and myocardial periarteriolar fibrosis

Microvascular dysfunction in the heart and its association with periarteriolar fibrosis may contribute to the diastolic dysfunction seen in heart failure with preserved ejection fraction. Interleukin-33 (IL-33) prevents global myocardial fibrosis in a pressure overloaded left ventricle by acting via its receptor, ST2 (encoded by the gene, Il1rl1); however, whether this cytokine can also modulate periarteriolar fibrosis remains unclear. We utilized two approaches to explore the role of IL-33/ST2 in periarteriolar fibrosis. First, we studied young and old wild type mice to test the hypothesis that IL-33 and ST2 expression change with age. Second, we produced pressure overload in mice deficient in IL-33 or ST2 by transverse aortic constriction (TAC). With age, IL-33 expression increased and ST2 expression decreased. These alterations accompanied increased periarteriolar fibrosis in aged mice. Mice deficient in ST2 but not IL-33 had a significant increase in periarteriolar fibrosis following TAC compared to wild type mice. Thus, loss of ST2 signaling rather than changes in IL-33 expression may contribute to periarteriolar fibrosis during aging or pressure overload, but manipulating this pathway alone may not prevent or reverse fibrosis.

Quercetin Enhances Ligand-induced Apoptosis in Senescent Idiopathic Pulmonary Fibrosis Fibroblasts and Reduces Lung Fibrosis In Vivo

Although cellular senescence may be a protective mechanism in modulating proliferative capacity, fibroblast senescence is now recognized as a key pathogenic mechanism in idiopathic pulmonary fibrosis (IPF). In aged mice, abundance and persistence of apoptosis-resistant senescent fibroblasts play a central role in nonresolving lung fibrosis after bleomycin challenge. Therefore, we investigated whether quercetin can restore the susceptibility of senescent IPF fibroblasts to proapoptotic stimuli and mitigate bleomycin-induced pulmonary fibrosis in aged mice. Unlike senescent normal lung fibroblasts, IPF lung fibroblasts from patients with stable and rapidly progressing disease were highly resistant to Fas ligand (FasL)-induced and TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Senescent IPF fibroblasts exhibited decreased expression of FasL and TRAIL receptors and caveolin-1, as well as increased AKT activation, compared with senescent normal lung fibroblasts. Although quercetin alone was not proapoptotic, it abolished the resistance to FasL- or TRAIL-induced apoptosis in IPF fibroblasts. Mechanistically, quercetin upregulated FasL receptor and caveolin-1 expression and modulated AKT activation. In vivo quercetin reversed bleomycin-induced pulmonary fibrosis and attenuated lethality, weight loss, and the expression of pulmonary senescence markers p21 and p19-ARF and senescence-associated secretory phenotype in aged mice. Collectively, these data indicate that quercetin reverses the resistance to death ligand-induced apoptosis by promoting FasL receptor and caveolin-1 expression and inhibiting AKT activation, thus mitigating the progression of established pulmonary fibrosis in aged mice. Therefore, quercetin may be a viable therapeutic option for IPF and other age-related diseases that progress with the accumulation of senescent fibroblasts.

308 - Redox control of liver fibrosis in aged mice

Glutaredoxin-1 (Glrx) removes glutathione (GSH) adducts of protein thiols (S-glutathionylated protein) and controls cellular signaling and gene transcription. Human lung fibrosis decreases Glrx protein expression, and conversely, Glrx administration attenuates bleomycin-induced lung fibrosis in mice. We previously showed that Glrx knockout (KO) mice develop liver steatosis and diet-induced liver fibrosis. Aging is another factor which promotes liver fibrosis. Objective We investigated whether Glrx expression can affect age-induced liver fibrosis in mice and provoke functional changes in isolated hepatocytes and hepatic stellate cells. Methods Aged liver samples of 4, 12, and 18 months old mice were obtained from NIA. Liver samples from global Glrx transgenic (TG) and wild-type (WT) mice were collected at age 9-24 months. Liver sections were stained with Picrosirius red to assess collagen deposition and fibrotic areas. Hepatocytes and hepatic stellate cells were isolated from mouse liver to examine the effects of Glrx overexpression or ablation on hepatocyte apoptosis and collagen synthesis in hepatic stellate cells. Results Liver fibrosis significantly increased during aging, while Glrx expression levels tended to decrease. Overexpressed Glrx attenuated collagen accumulation in Glrx TG mice compared to aged WT mice. In vitro studies showed that Glrx KO enhanced Fas-mediated apoptosis in hepatocytes. Also, Glrx KO increased TGFb-induced fibrotic markers (collagen, aSM-actin) in hepatic stellate cells. Conclusion Aging increased liver fibrosis which is likely related to a decline in Glrx expression. In contrast, Glrx overexpression significantly attenuated the age-induced changes in liver fibrosis. Glrx protected hepatocytes from apoptosis and hepatic stellate cells from fibrotic activation. Thus, upregulated Glrx may ameliorate liver fibrosis in aged animals. (Supported by R01HL133013, R03AG051857, R01DK103750).

A9871 Insulin regulated aminopeptidase (irap) inhibition completely reverses age-induced cardiac fibrosis and improves cardiac function

Objectives: There is an urgent need to discover targets that can reverse established fibrosis that occurs with advanced ageing and in many disease states thus improving organ function. The aim of this study was to determine whether inhibition or deficiency of IRAP would reverse age-induced cardiac fibrosis and improve left ventricular function in 2 year old mice. Methods: Cardiovascular fibrosis and function (using echocardiography) was assessed in young (∼4–5 months old) and aged (∼22–24 months old) WT (C57BL/6J) or IRAP KO mice. Aged WT mice were chronically treated with either the synthetic IRAP inhibitor (HFI-419; 500 ng/kg/min) or vehicle for 4 or 8 weeks. Results: Ageing resulted in marked interstitial cardiac fibrosis and increased IRAP expression that was co-localised with myofibroblasts. Global IRAP gene deletion protected against age-induced fibrosis and resulted in improved left ventricular function. Importantly chronic treatment with 3 structurally-unrelated specific IRAP inhibitors for 4 weeks reversed established cardiac fibrosis in aged mice. Mechanisms involved down-regulation of collagen synthesis, increased collagen degradation and reduced tissue inflammation. In separate animals, we examined if HFI-419 treatment for 8 weeks improved left ventricular function assessed by serial echocardiography at weeks 0, 4 and 8. Our preliminary data suggest improvement of left ventricular function within 4 weeks of HFI-419 treatment and was associated with reduced cardiac fibrosis in these mice. Conclusion: Our data provides compelling evidence that inhibition of IRAP represents a novel therapeutic strategy for treatment of established cardiac fibrosis leading to improved organ function.

GW29-e1913 Urotensin II gene knockout reduces blood pressure, glucose and alleviates cardiac and vascular fibrosis in aged mice

GW29-e1913 Urotensin II gene knockout reduces blood pressure, glucose and alleviates cardiac and vascular fibrosis in aged mice