Age-related Fibrosis Research Articles

The naturally occurring peptide GHK reverses age-related fibrosis by modulating myofibroblast functio

The naturally occurring peptide GHK reverses age-related fibrosis by modulating myofibroblast functio

Age-related changes in the fiber structure around adipocytes in the subcutaneous fat layer and their association with skin viscoelasticity.

Age-related changes in the fiber structure around adipocytes were investigated via scanning electron microscopy (SEM) of excised skin tissues. In addition, the viscoelasticity of the subcutaneous fat layer was evaluated via elastography, and the association between the fiber structure and the viscoelastic properties was assessed. Skin tissues excised from the facial cheek area were used. Then, SEM images of these tissues were obtained. The thickness and quantity of the fibers around adipocytes were assessed using a 5-point scale. The score was used to grade 18 tissue samples. Moreover, the viscoelasticity of the subcutaneous fat layer in the same samples was evaluated via ultrasound elastography. Based on the SEM image score, an association was observed between the fiber status score and age, thereby indicating a tendency toward age-related fibrosis. Fiber structures with high scores, which indicate fibrosis, had a significantly lower viscoelasticity based on ultrasound elastography. The thickness and quantity of fibrous structures around adipocytes in the subcutaneous fat layer increase with age, and these changes can be associated with decreased viscoelasticity in the subcutaneous fat layer.

Chk2 Modulates Bmi1-Deficiency-Induced Renal Aging and Fibrosis via Oxidative Stress, DNA Damage, and p53/TGFβ1-Induced Epithelial-Mesenchymal Transition.

Renal aging may lead to fibrosis and dysfunction, yet underlying mechanisms remain unclear. We explored whether deficiency of the Polycomb protein Bmi1 causes renal aging via DNA damage response (DDR) activation, inducing renal tubular epithelial cell (RTEC) senescence and epithelial-mesenchymal transition (EMT). Bmi1 knockout mice exhibited oxidative stress, DDR activation, RTEC senescence, senescence-associated secretory phenotype (SASP), and age-related fibrosis in kidneys. Bmi1 deficiency impaired renal structure and function, increasing serum creatinine/urea, reducing creatinine clearance, and decreasing cortical thickness and glomerular number. However, knockout of the serine-threonine kinase Chk2 alleviated these aging phenotypes. Transcriptomics identified transforming growth factor beta 1 (TGFβ1) upregulation in Bmi1-deficient RTECs, but TGFβ1 was downregulated upon Chk2 knockout. The tumor suppressor protein p53 transcriptionally activated TGFβ1, promoting EMT in RTECs. Bmi1 knockout or oxidative stress (induced with H2O2) increased TGFβ1 expression, and EMT in RTECs and was partly reversed by p53 inhibition. Together, Bmi1 deficiency causes oxidative stress and DDR-mediated RTEC senescence/SASP, thus activating p53 and TGFβ1 to induce EMT and age-related fibrosis. However, blocking DDR (via Chk2 knockout) or p53 ameliorates these changes. Our study reveals mechanisms whereby Bmi1 preserves renal structure and function during aging by suppressing DDR and p53/TGFβ1-mediated EMT. These pathways represent potential targets for detecting and attenuating age-related renal decline.

TGF-β as A Master Regulator of Aging-Associated Tissue Fibrosis.

Fibrosis is the abnormal accumulation of extracellular matrix proteins such as collagen and fibronectin. Aging, injury, infections, and inflammation can cause different types of tissue fibrosis. Numerous clinical investigations have shown a correlation between the degree of liver and pulmonary fibrosis in patients and telomere length and mitochondrial DNA content, both of which are signs of aging. Aging involves the gradual loss of tissue function over time, which results in the loss of homeostasis and, ultimately, an organism's fitness. A major feature of aging is the accumulation of senescent cells. Senescent cells abnormally and continuously accumulate in the late stages of life, contributing to age-related fibrosis and tissue deterioration, among other aging characteristics. Furthermore, aging generates chronic inflammation, which results in fibrosis and decreases organ function. This finding suggests that fibrosis and aging are closely related. The transforming growth factor-beta (TGF-β) superfamily plays a crucial role in the physiological and pathological processes of aging, immune regulation, atherosclerosis, and tissue fibrosis. In this review, the functions of TGF-β in normal organs, aging, and fibrotic tissues is discussed: TGF-β signalling is altered with age and is an indicator of pathology associated with tissue fibrosis. In addition, this review discusses the potential targeting of noncoding.

160-LB: Age-Related Adipose Tissue Fibrosis and Neuropathy in the Genetically Diverse HET3 Mouse

Changes in adipose tissue's extracellular matrix, as with fibrosis, inhibits the tissue's ability to shrink and expand as needed, resulting in sheer stress on adipocytes and chronic inflammation, as well as decreased insulin sensitivity. With aging, a state of metabolic dysregulation and increased incidence of insulin resistance, we have observed a reduction in adipose innervation. This ‘adipose neuropathy' likely poses a physiological challenge for tissue metabolic function and glucose homeostasis, as has been observed in adipose denervation studies. Using second-harmonic generation label-free imaging of adipose collagen and advanced image co-localization techniques, we found that obese adipose tissue exhibits higher co-localization of nerves with collagen fibers, indicating that changes in tissue fibrosis may tie mechanistically with tissue neuropathy. We compared C57BL/6J mice with the more genetically diverse HET3 mouse model that is used by the NIA's intervention testing program, to investigate age-related neuropathy and adipose fibrosis. We found that HET3 mice displayed mitigated neuropathy phenotypes in skin, muscle and adipose, compared to inbred C56BL/6J mice. Tissue fibrosis was significantly increased with age in subcutaneous and visceral white adipose depots of both strains of mice. Using birefringence imaging, we found that the relative distribution of type I / thick filament collagen increased with age while type III / thin filament collagen decreased. Gene expression by qPCR revealed significant increases in collagen genes Col1a1, Col3a1, and Col5a1 in adipose with age. Despite its success as a longevity treatment, the mTOR inhibitor rapamycin had little to no effect on reducing or preventing the onset of neuropathy in HET3 aged mice, and importantly it worsened the fibrotic phenotype with increased type 1 collagen in adipose. Together, we found that rapamycin's longevity effects are uncoupled from healthspan producing effects, with detrimental impacts on adipose fibrosis that may impact age-related insulin resistance. Disclosure J. Willows: None. G. Mishra: None. M. Blaszkiewicz: None. K. L. Townsend: Other Relationship; Neuright, Inc. Funding American Heart Association (AHA) Collaborative Sciences Award (18CSA34090028)

Long Non-coding RNA MALAT1 Is Depleted With Age in Skeletal Muscle in vivo and MALAT1 Silencing Increases Expression of TGF-β1 in vitro

Long non-coding RNAs (lncRNAs) are thought to function as “sponges” for microRNAs, but a role for such competing endogenous RNAs (ceRNAs) in muscle aging is not well understood. We therefore examined in skeletal muscles of young (4–6 months) and aged (22–24) male and female mice the expression of lncRNA MALAT1, which is predicted in silico to bind the senescence-associated microRNA miR-34a-5p. Results indicate a significant decrease in lncRNA MALAT1 expression in mouse skeletal muscle with age that coincides with an age-related increase in miR-34a-5p expression. In vitro studies using mouse C2C12 myoblasts demonstrate that MALAT1 silencing using siRNA increases miR-34a expression, consistent with a role for MALAT1 as an inhibitor of miR-34a-5p activity. Levels of reactive oxygen species (ROS) are known to increase in muscle with age, and so we treated C2C12 cells with hydrogen peroxide (10 and 100 μM) to examine changes in MALAT1 expression. MALAT1 expression decreased significantly with H2O2 treatment, but this effect was attenuated with p53 siRNA. Finally, miR-34a-5p is implicated in tissue fibrosis, and so we assessed the expression of TGF-β1 after MALAT1 silencing. MALAT1 siRNA significantly increased the expression of TGF-β1 in C2C12 cells. These findings suggest that age-related fibrosis and muscle atrophy mediated by ROS may result at least in part from an increase in miR-34a bioavailability resulting from a decline in miR-34a “sponging” due to ceRNA MALAT1 depletion. Crosstalk between MALAT1 and miR-34a may therefore represent a therapeutic target for improving muscle function with aging.

Neutrophil PAD4 negatively influences cardiac function and remodeling with increasing age

Abstract Background Aging can be viewed as a status of chronic inflammation, in which neutrophils have a lower threshold for activation. The enzyme peptidylarginine deiminase 4 (PAD4), which catalyzes the conversion of arginine to citrulline, will be activated in a certain population of neutrophils. When this conversion takes place on the histones, neutrophils can form neutrophil extracellular traps (NETs), which are both prothrombotic and proinflammatory. Mice lacking this enzyme systemically were previously reported to be protected from age-related fibrosis. Purpose We aimed to study the long-term effect of neutrophils on cardiac health during the process of natural aging. We hypothesized that neutrophil PAD4, and in consequence NETs, are involved in cardiac fibrosis development, which in turn will result in impaired cardiac function. Methods We generated a mouse model of impaired NET release capability via deletion of PAD4, a NET-essential gene, under the neutrophil-specific promoter (PAD4fl/flMRP8Cre+). In order to study heart failure (HF) development, these specific deletion mice and their littermate controls were aged for a period of two years (coinciding with approximately 70 years of age in the human population; the age at which HF is the number one cause of hospitalization), after which cardiac function and remodeling were evaluated by echocardiography and histology, respectively. A separate set of young mice (12 weeks) were evaluated in parallel. Results We performed a comprehensive echocardiography analysis including both structural and functional parameters. As for systolic function, we could see that in old wild type (WT) mice, ejection fraction (EF) significantly decreased as compared to EF in young and healthy (YH) mice (YH - 67±6%, WT - 53±10%; p<0.0001) (Figure 1B). However, this decrease in systolic function was absent in the old PAD4fl/flMRP8Cre+ mice, with EF being comparable to the YH group (PAD4fl/flMRP8Cre+ - 67±7%; p=0.9169) (figure 1 A,B). As for diastolic function, again we could see a marked decrease in E/A ratio in the WT as compared to the YH population (YH- 1.50±0.23, WT – 1.21±0.17; p<0.0001), while this functional deterioration was absent in aged PAD4fl/flMRP8Cre+ animals (PAD4fl/flMRP8Cre+ - 1.38±0.21; p=0.0837) (Figure 1 C,D). To link this decline in heart function to tissue remodeling, we quantified collagen deposition in the heart. We saw that natural aging resulted in an increase in cardiac collagen deposition in the WT population as compared to YH mice (YH – 0.86±0.63%, WT – 4.02±1.71%). This increased collagen deposition was absent in the neutrophil deletion mice (PAD4fl/flMRP8Cre+ - 1.7±0.76%). Additionally, when comparing WT to PAD4 deletion-mice, we saw that the increase in collagen deposition is significantly higher in the WT mice (p<0.0001). Conclusion Our data confirms neutrophil PAD4 involvement in heart failure progression by promoting cardiac fibrosis, resulting in cardiac dysfunction. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Fonds Wetenschappelijk Onderzoek (FWO) - Vlaanderen

Abstract P139: Genetic Signature Of Accelerated Cardiac Fibrosis Due To Trpa1 Ablation

While our previous study demonstrated that loss of transient receptor potential ankyrin 1 ( Trpa1 ) accelerates age-related cardiac fibrosis in mice, the underlying mechanism of potential anti-fibrotic property of TRPA1 remains largely unknown. TRPA1 is a sensor of oxidative stress and may play a protective role in age-related diseases. In this study, we performed quantitative polymerase chain reaction array analyses of the mRNA expression of 84 fibrosis-related genes in the myocardial tissue of 12-month-old Trpa1 -/- mice with significant cardiac fibrosis and age-matched wild-type mice without cardiac fibrosis. The mRNA levels of Col1a2 and Col3a1 in the myocardial tissue were similar between Trpa1 -/- and wild-type mice, suggesting comparable cardiac collagen synthesis in the two strains. Matrix metalloproteinases are major enzymes responsible for degradation of collagen fibers. The results show that the mRNA levels of matrix metalloproteinases, including Mmp1a , Mmp2 , Mmp3 , Mmp8 , Mmp9 , Mmp13 , and Mmp14 , in the heart were similar between Trpa1 -/- and wild-type mice. Nevertheless, we identified 7 significantly changed genes in the heart between the two strains. The expression levels of Acta2 , Inhbe , Ifng , and Ccl11 were significantly increased with fold changes of 3.1, 1.9, 1.9, and 1.5 (all P < 0.05), respectively, while Timp3 , Stat6 , and Ilk were significantly decreased with fold changes of 0.3, 0.5, and 0.7 (all P < 0.05), respectively, in the heart of Trpa1 -/- mice compared with wild-type mice. Acta2 , the most upregulated gene in Trpa1 -/- hearts, is a marker of myofibroblasts. Its upregulation indicates increased differentiation from fibroblasts into myofibroblasts in Trpa1 -/- hearts compared with wild-type hearts. Timp3 , the most downregulated gene in Trpa1 -/- hearts, codes an extracellular matrix protein TIMP3, which not only inhibits matrix metalloproteinases but also regulate post-translational modification of collagen fibers. Taken together, these findings suggest that upregulation of Acta2 and downregulation of Timp3 may serve as genetic signature or play a role in accelerated age-related cardiac fibrosis due to TRPA1 ablation.

SIRT6 enhances telomerase activity to protect against DNA damage and senescence in hypertrophic ligamentum flavum cells from lumbar spinal stenosis patients.

Lumbar spinal stenosis (LSS) is a condition wherein patients exhibit age-related fibrosis, elastin-to-collagen ratio reductions, and ligamentum flavum hypertrophy. This study was designed to assess the relationship between SIRT6 and telomerase activity in hypertrophic ligamentum flavum (LFH) cells from LSS patients. We observed significant reductions in SIRT6, TPP1, and POT1 protein levels as well as increases in telomerase reverse transcriptase (TERT) levels and telomerase activity in LFH tissues relative to non- hypertrophic ligamentum flavum (LFN) tissues. When SIRT6 was overexpressed in these LFH cells, this was associated with significant increases in telomerase activity and a significant reduction in fibrosis-related protein expression. These effects were reversed, however, when telomerase activity was inactivated by hTERT knockdown in these same cells. SIRT6 overexpression was further found to reduce the frequency of senescence-associated β-galactosidase (SA-β-Gal)-positive LFH cells and to decrease p16, MMP3, and L1 mRNA levels and telomere dysfunction-induced foci (TIFs) in LFH cells. In contrast, hTERT knockdown-induced telomerase inactivation eliminated these SIRT6-dependent effects. Overall, our results indicate that SIRT6 functions as a key protective factor that prevents cellular senescence and telomere dysfunction in ligamentum flavum cells, with this effect being at least partially attributable to SIRT6-dependent telomerase activation.

Age-dependent regulation of cell-mediated collagen turnover.

Although aging represents the most important epidemiologic risk factor for fibrotic disease, the reasons for this are incompletely understood. Excess collagen deposition in tissues is the sine qua non of tissue fibrosis and can be viewed as an imbalance between collagen production and collagen degradation. Yet we still lack a detailed understanding of the changes that take place during development, maturation, and aging in extracellular matrix (ECM) dynamics. Resolution of fibrosis is impaired in aging, and this impairment may explain why age is the most important risk factor for fibrotic diseases, such as idiopathic pulmonary fibrosis. However, ECM dynamics and impaired resolution of fibrosis in aging remain understudied. Here we show that cell-mediated collagen uptake and degradation are diminished in aged animals and this finding correlates with downregulation of the collagen endocytic receptor mannose receptor, C-type 2 (Mrc2). We identify myeloid zinc finger-1 as a potentially novel transcriptional regulator of Mrc2, and both this transcription factor and Mrc2 are downregulated in multiple tissues and organisms in an age-dependent manner. Thus, cell-mediated degradation of collagen is an essential process that promotes resolution of fibrosis, and impairment in this process contributes to age-related fibrosis.

P5982Transcatheter aortic valve replacement in elderly patients with severe symptomatic aortic stenosis

Abstract Introduction TAVR has proven to be a safe technique for high-risk patients. The objective of our work is to know the results of TAVR in patients older than 90 years; which are the riskiest and had more complications during the admission. Methods Observational retrospective analysis based on all a single-center registry of all consecutive TAVR (N=518) during the last 10 years (2008–2018). Patients older than 90 years old represent 5.8 of the procedures (N=30). We performed a descriptive analysis of the baseline characteristics of our patient cohort, and the development of events during follow-up. Results Our population, as reflected in TABLE 1, had a median age of 91,32 years old, and the majority were women (57.58%). The procedures were mostly elective, unless in 4 patients (12.12%) that were urgent. None valvuloplasty was done before TAVR in this cohort, neither Valve-in-valve. Stratification scores had a wide range, from 1.45 to 28.8% for EurSCORE II and from 4.19 to 17.19% for STS, with a median of 5.53 and 8.49% respectively. Mortality in the first 30 days and in the first year was 0.00%. Mortality after the first year was 71.40% due to non-cardiovascular events (median time of follow-up of 34.34 months). Finally, we would also like to remark that the incidence of heart failure during the first year was 14.29%, taking into account all the follow-up, the incidence was 37.04%. Despite these data, in our registry, the need for pacemaker after the valve implantation was higher than in younger populations (40.62%), probably due to the age-related fibrosis of the cardiac conduction system. Conclusions TAVR is a very safe procedure with very successful results in elderly patients, nevertheless, due to the frailty of this population, individualization is mandatory in order to optimize the resources. Acknowledgement/Funding None

Toward a unified theory of aging and regeneration.

Growing evidence supports the antagonistic pleiotropy theory of mammalian aging. Accordingly, changes in gene expression following the pluripotency transition, and subsequent transitions such as the embryonic-fetal transition, while providing tumor suppressive and antiviral survival benefits also result in a loss of regenerative potential leading to age-related fibrosis and degenerative diseases. However, reprogramming somatic cells to pluripotency demonstrates the possibility of restoring telomerase and embryonic regeneration pathways and thus reversing the age-related decline in regenerative capacity. A unified model of aging and loss of regenerative potential is emerging that may ultimately be translated into new therapeutic approaches for establishing induced tissue regeneration and modulation of the embryo-onco phenotype of cancer.

1769-P: Development of Adipose Fibrosis and Dysfunction in Aged Rxfp1-/- Mice

The hormone relaxin has roles in insulin sensitivity and age-related fibrosis in several organs, but the effect on adipose is unknown. Given the association of adipose fibrosis to dysfunction and progression of metabolic disease, we analyzed adipose tissue in aged mice lacking the relaxin receptor RXFP1. Male and female Rxfp1-/- or age-matched wild type (WT) mice were used at full adulthood (5-7 months) or when aged (12-18 months). Total fat and lean mass was determined, then gonadal white adipose tissue (gWAT) or inguinal white adipose tissue (iWAT) was collected. Sections were stained with Sirius red to visualize collagen. Gene expression was determined by qPCR, and serum adiponectin measured by ELISA. There were no significant differences in total body fat or lean mass between WT and Rxfp1-null mice at either age. All mice had similar adipose collagen content at age 5-7 months. In aged male mice, there was a remarkable increase in collagen in the iWAT of Rxfp1-/- mice, including pericellular deposits and thick collagen fibrils, and modestly elevated gWAT collagen compared to WT. In aged female mice, Rxfp1-null mice had modestly increased pericellular collagen in iWAT, but no apparent difference in gWAT. In aged male iWAT, the expression of the collagen types I, III, and VI genes, and the lysyl oxidase genes Lox, Loxl1, and Loxl2 were all significantly elevated. Serum adiponectin was significantly reduced in Rxfp1-/- mice compared to WT (12.6±1.6 vs. 23.9±2.8 ug/ml respectively, p<.02). In summary, Rxfp1-/- mice developed age-related adipose fibrosis, particularly in the iWAT. The effect was sex-specific, in that the fibrosis was much more extensive in male mice. The Rxfp1-/- mice had increased expression of fibrotic collagen and lysyl oxidase genes, and decreased serum adiponectin, suggesting adipose dysfunction. These results suggest that disruption of relaxin signaling plays a role in preventing age-related adipose fibrosis, which could contribute to the development and progression of metabolic dysfunction. Disclosure R. Bennett: None. F. Hamel: None. R.L. Simpson: None. A.I. Agoulnik: None. Funding U.S. Department of Veterans Affairs

Chronic aerobic exercise training alleviates myocardial fibrosis in aged rats through restoring bioavailability of hydrogen sulfide.

Age-related fibrosis is attenuated by aerobic exercise; however, little is known concerning the underlying molecular mechanism. To address this question, aged rats were given moderate-intensity exercise for 12 weeks. After exercise in aged rats, hydrogen sulfide levels in plasma and heart increased 39.8% and 90.9%, respectively. Exercise upregulated expression of cystathionine γ-lyase and 3-mercaptopyruvate sulfurtransferase in heart of aged rats. Furthermore, aged rats were given moderate-intensity exercise for 12 weeks or treated with NaHS (intraperitoneal injection of 0.1 mL/kg per day of 0.28 mol/L NaHS). After exercise in aged rats, Masson-trichrome staining area decreased 34.8% and myocardial hydroxyproline levels decreased 29.6%. Exercise downregulated expression of collagen-I and α- smooth muscle actin in heart of aged rats. Exercise in aged rats reduced malondialdehyde levels in plasma and heart and 3-nitrotyrosine in heart. Exercise in aged rats reduced mRNA and protein expression of C/EBP homologous protein, glucose regulated protein 78, and X-box protein 1. Exercise also reduced mRNA and protein expression of interleukin 6 and monocyte chemotactic protein 1and suppressed activation of c-Jun N-terminal kinase in aging heart. Similar effects were demonstrated in aged rats treated with NaHS. Collectively, exercise restored bioavailability of hydrogen sulfide in the heart of aged rats, which partly explained the benefits of exercise against myocardial fibrosis of aged population.

Reduced collagen accumulation and augmented MMP-2 activity in left ventricle of old rats submitted to high-intensity resistance training.

Progressive fibrosis is a hallmark of the aging heart. Age-related fibrosis is modulated by endurance exercise training; however, little is known concerning the influence of resistance training (RT). Therefore we investigated the chronic effects of high-intensity RT on age-associated alterations of left ventricle (LV) structure, collagen content, matrix metalloproteinase-2 (MMP-2), and extracellular matrix-related gene expression, including transforming growth factor-β (TGF-β). Young adult (3 mo) and aged (21 mo) male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), three times a week for 12 wk. Forty-eight hours posttraining, arterial systolic and diastolic pressure, LV end-diastolic pressure (LVEDP) and dP/dt were recorded. LV morphology, collagen deposition, and gene expression of type I (COL-I) and type III (COL-III) collagen, MMP-2, tissue inhibitor of metalloproteinases-1 (TIMP-1), and TGF-β1 were analyzed by quantitative reverse transcriptase-PCR. MMP-2 content was assessed by zymography. Increased collagen deposition was observed in LV from aged rats. These parameters were modulated by RT and were associated with increased MMP-2 activity and decreased COL-I, TGF-β1, and TIMP-1 mRNA content. Despite the effect of RT on collagen accumulation, there was no improvement on LVEDP and maximal negative LV dP/dt of aged rats. Cardiomyocyte diameter was preserved in all experimental conditions. In conclusion, RT attenuated age-associated collagen accumulation, concomitant to the increase in MMP-2 activity and decreased expression of COL-I, TGF-β1, and TIMP-1 in LV, illustrating a cardioprotective effect of RT on ventricular structure and function.NEW & NOTEWORTHY We demonstrated the beneficial resistance-training effect against age-related left ventricle collagen accumulation in the left ventricle, which was associated with decreased type I collagen (COL-I), transforming growth factor-β1 (TGF-β1), and tissue inhibitor of metalloproteinases-1 (TIMP-1) gene expression and matrix metalloproteinase-2 (MMP-2) activity. Our findings suggest for the first time the potential effects of resistance training in modulating collagen accumulation and possibly fibrosis in the aging heart.

Aging, Cellular Senescence, and Kidney Fibrosis

Fibrosis is a hallmark of chronic kidney disease, and a primary risk factor for chronic kidney disease is age. Senescent cells accumulate in the kidney during natural aging and after injury. Understanding the effect of senescence on kidney maintenance and how senescence is linked to fibrosis may reveal new therapeutic opportunities. The load of senescent cells in the kidney is predictive of biological age and correlates with renal disease and allograft outcome. Senescent cells do not proliferate to repair injury. Instead, they secrete proteins that enhance inflammation and modulate fibrosis. Pharmaceutical blockade of these effects or elimination of senescent cells can counteract age-related disease. This review explores the relationships among aging, cellular senescence, inflammation, and kidney fibrosis. Drawing from data from various disciplines, we discuss senescence-associated secretory phenotype, klotho, and senotherapy, analyzing the research and looking for new solutions to age-related kidney fibrosis.

Dissecting the role of myeloid and mesenchymal fibroblasts in age-dependent cardiac fibrosis.

Aging is associated with increased cardiac interstitial fibrosis and diastolic dysfunction. Our previous study has shown that mesenchymal fibroblasts in the C57BL/6J (B6J) aging mouse heart acquire an inflammatory phenotype and produce higher levels of chemokines. Monocyte chemoattractant protein-1 (MCP-1) secreted by these aged fibroblasts promotes leukocyte uptake into the heart. Some of the monocytes that migrate into the heart polarize into M2a macrophages/myeloid fibroblasts. The number of activated mesenchymal fibroblasts also increases with age, and consequently, both sources of fibroblasts contribute to fibrosis. Here, we further investigate mechanisms by which inflammation influences activation of myeloid and mesenchymal fibroblasts and their collagen synthesis. We examined cardiac fibrosis and heart function in three aged mouse strains; we compared C57BL/6J (B6J) with two other strains that have reduced inflammation via different mechanisms. Aged C57BL/6N (B6N) hearts are protected from oxidative stress and fibroblasts derived from them do not develop an inflammatory phenotype. Likewise, these mice have preserved diastolic function. Aged MCP-1 null mice on the B6J background (MCP-1KO) are protected from elevated leukocyte infiltration; they develop moderate but reduced fibrosis and diastolic dysfunction. Based on these studies, we further delineated the role of resident versus monocyte-derived M2a macrophages in myeloid-dependent fibrosis and found that the number of monocyte-derived M2a (but not resident) macrophages correlates with age-related fibrosis and diastolic dysfunction. In conclusion, we have found that ROS and inflammatory mediators are necessary for activation of fibroblasts of both developmental origins, and prevention of either led to better functional outcomes.

The impact of aging on cardiac extracellular matrix.

Age-related changes in cardiac homeostasis can be observed at the cellular, extracellular, and tissue levels. Progressive cardiomyocyte hypertrophy, inflammation, and the gradual development of cardiac fibrosis are hallmarks of cardiac aging. In the absence of a secondary insult such as hypertension, these changes are subtle and result in slight to moderate impaired myocardial function, particularly diastolic function. While collagen deposition and cross-linking increase during aging, extracellular matrix (ECM) degradation capacity also increases due to increased expression of matrix metalloproteinases (MMPs). Of the MMPs elevated with cardiac aging, MMP-9 has been extensively evaluated and its roles are reviewed here. In addition to proteolytic activity on ECM components, MMPs oversee cell signaling during the aging process by modulating cytokine, chemokine, growth factor, hormone, and angiogenic factor expression and activity. In association with elevated MMP-9, macrophage numbers increase in an age-dependent manner to regulate the ECM and angiogenic responses. Understanding the complexity of the molecular interactions between MMPs and the ECM in the context of aging may provide novel diagnostic indicators for the early detection of age-related fibrosis and cardiac dysfunction.

Abstract 567: Transcription Factor Runx2 is Induced in Vascular Aging and May Promote Age-related Arterial Stiffness

Background: Arterial stiffening is a hallmark of vascular aging and may constitute a critical mechanism linking age to increased cardiovascular risk. However, up to now there is no therapy available to efficiently and specifically target age-related arterial stiffening. We recently identified the osteogenic transcription factor Runx2 as an inducer of diabetic arterial stiffness. Objective: The present study investigated the role of Runx2 in the setting of age-related arterial stiffness. Methods and Results: Aortic stiffness – quantified by ex vivo mechanical testing (pressure myography) – was markedly increased in 1-year old male C57Bl/6 mice compared to young (10 week-old) controls. At the same time, Runx2 was aberrantly upregulated in the medial layer of aged aortae, coming along with significant medial fibrosis. Additionally, we detected increased aortic expression of interleukin 6 ( Il6 ) – a key cytokine involved in vascular “inflammaging”. Mechanistically, we found IL-6-induced RUNX2 expression in aortic smooth muscle cells (SMCs) via a NFkB-dependent pathway. Conclusion: In conclusion this study suggests Runx2 as a potential mediator of age-related arterial fibrosis and stiffness warranting further interventional/therapeutic studies.

Abstract 15628: Prevalence of Non-Pulmonary Vein Triggers in Patients With Previous History Of Coronary Artery Bypass Grafting Undergoing Catheter Ablation for Atrial Fibrillation

Introduction: Post-operative atrial fibrillation (AF) is reported in up to 5-40% of patients following coronary artery bypass graft (CABG) surgery. Age-related atrial dilatation and fibrosis, electrical remodeling, autonomic imbalance and post-operative inflammation are few of the many pathophysiological factors that play an important role in the genesis and persistence of post-CABG AF. Recent data on fibroblast-myocyte interaction indicates the central role of cardiac fibrosis in the origin of non-pulmonary vein (non-PV) drivers that serve as a source for maintenance of AF. Therefore, we sought to examine the prevalence of non-PV triggers and their impact on ablation outcome in post-CABG AF patients. Methods: Two-hundred sixty seven patients with history of CABG undergoing catheter ablation for atrial fibrillation were included in this analysis (61±11 years, 69% male, LVEF 50±10%, LA size 41.0±6.8mm, non-PAF 72%). All patients underwent pulmonary vein antrum (PVAI) and posterior wall isolation. Non-PV triggers were defined as ectopic triggers originating from sites other than pulmonary veins such as interatrial septum, superior vena cava, left atrial appendage, coronary sinus, ligament of Marshall, crista terminalis. Those were identified by isoproterenol challenge and were ablated based on operator choice. Results: During ablation, non-PV triggers were detected in 243 (91%) patients. The triggers were most commonly mapped to CS (62%) and LAA (70%). Additional sites of non-PV triggers were LA septum, superior vena cave, crista terminalis and mitral valve annulus. Based on operator choice, non-PV triggers were ablated in 118 of the 243 patients and not targeted in 125 patients. At the end of 32±7 month follow-up after a single procedure, overall success rate was 130 (53.5%). Of the 118 patients undergoing non-PV trigger ablation, 81(68.6%) patients remained arrhythmia-free off anti-arrhythmic drugs compared to 49 of 125 (39.2%) where non-PV ablation was not performed (log rank p <0.001, hazard ratio 3.76 [95% CI 2-6.43]). Conclusion: Our results for the first time demonstrate that non-PV foci are highly prevalent in post-CABG AF patients and ablation of those triggers results in significantly better long-term outcome.