ADAMTS16 activates latent TGF-β, accentuating fibrosis and dysfunction of the pressure-overloaded heart

AIMS: Cardiac fibrosis is a major cause of heart failure (HF), and mediated by the differentiation of cardiac fibroblasts into myofibroblasts. However, limited tools are available to block cardiac fibrosis. ADAMTS16 is a member of the ADAMTS superfamily of extracellular protease enzymes involved in extracellular matrix (ECM) degradation and remodelling. In this study, we aimed to establish ADAMTS16 as a key regulator of cardiac fibrosis. METHODS AND RESULTS: Western blot and qRT-PCR analyses demonstrated that ADAMTS16 was significantly up-regulated in mice with transverse aortic constriction (TAC) associated with left ventricular hypertrophy and HF, which was correlated with increased expression of Mmp2, Mmp9, Col1a1, and Col3a1. Overexpression of ADAMTS16 accelerated the AngII-induced activation of cardiac fibroblasts into myofibroblasts. Protein structural analysis and co-immunoprecipitation revealed that ADAMTS16 interacted with the latency-associated peptide (LAP)-transforming growth factor (TGF)-β via a RRFR motif. Overexpression of ADAMTS16 induced the activation of TGF-β in cardiac fibroblasts; however, the effects were blocked by a mutation of the RRFR motif to IIFI, knockdown of Adamts16 expression, or a TGF-β-neutralizing antibody (ΝAb). The RRFR tetrapeptide, but not control IIFI peptide, blocked the interaction between ADAMTS16 and LAP-TGF-β, and accelerated the activation of TGF-β in cardiac fibroblasts. In TAC mice, the RRFR tetrapeptide aggravated cardiac fibrosis and hypertrophy by up-regulation of ECM proteins, activation of TGF-β, and increased SMAD2/SMAD3 signalling, however, the effects were blocked by TGF-β-NAb. CONCLUSION: ADAMTS16 promotes cardiac fibrosis, cardiac hypertrophy, and HF by facilitating cardiac fibroblasts activation via interacting with and activating LAP-TGF-β signalling. The RRFR motif of ADAMTS16 disrupts the interaction between ADAMTS16 and LAP-TGF-β, activates TGF-β, and aggravated cardiac fibrosis and hypertrophy. This study identifies a novel regulator of TGF-β signalling and cardiac fibrosis, and provides a new target for the development of therapeutic treatment of cardiac fibrosis and HF

Effects of Vegetation Restoration Age on Soil C: N: P Stoichiometry in Yellow River Delta Coastal Wetland of China

Vegetation restoration can alter carbon (C), nitrogen (N), and phosphorus (P) cycles in coastal wetlands affecting C: N: P stoichiometry. However, the effects of restoration age on soil C: N: P stoichiometry are unclear. In this study, we examined the responses of soil C, N, and P contents and their stoichiometric ratios to vegetation restoration age, focusing on below-ground processes and their relationships to aboveground vegetation community characteristics. We conducted an analysis of temporal gradients based on the 'space for time' method to synthesize the effects of restoration age on soil C: N: P stoichiometry in the Yellow River Delta wetland of China. The findings suggest that the combined effects of restoration age and soil depth create complex patterns of shifting soil C: N: P stoichiometry. Specifically, restoration age significantly increased all topsoil C: N: P stoichiometries, except for soil total phosphorus (TP) and the C: N ratio, and slightly affected subsoil C: N: P stoichiometry. The effects of restoration age on the soil C: N ratio was well constrained owing to the coupled relationship between soil organic carbon (SOC) and total nitrogen (TN) contents, while soil TP content was closely related to changes in plant species diversity. Importantly, we found that the topsoil C: N: P stoichiometry was significantly affected by plant species diversity, whereas the subsoil C: N: P stoichiometry was more easily regulated by pH and electric conductivity (EC). Overall, this study shows that vegetation restoration age elevated SOC and N contents and alleviated N limitation, which is useful for further assessing soil C: N: P stoichiometry in coastal restoration wetlands

Precipitation and plant community-weighted mean traits determine total transpirable soil water in a desert grassland

The desert grassland has low precipitation, high evaporation and a limited soil water supply. Thus the tension between water shortage and sustainable vegetation restoration is increasingly evident. Change in total transpirable soil water (TTSW) reflects the water utilization and extraction capacity of plant communities, which are the basis for maintaining productivity and stability in communities, as well as the key ecological processes alleviating the above tension. Change in TTSW is influenced by precipitation, vegetation and soil. Quantifying the relative contribution of these factors to change in TTSW is key to the sustainable restoration of desert grassland. However, the current understanding of such effects remains limited. This study examined four typical plant communities in the Ningxia desert grassland, monitoring soil water content and evaluating plant community characteristics and diversity over three consecutive years. Using redundancy analysis and hierarchical partitioning methods, the influence of annual precipitation, vegetation (functional and species diversity, community characteristics) and soil physical properties on TTSW variation were investigated. Findings revealed: (1) The factors contributing to TTSW variation were annual precipitation (41 %-68 %,), vegetation (29 %-45 %), and soil physical properties (0 %-6.7 %). Specifically, community-weighted mean traits (CWM) and functional diversity (FD) explained TTSW variation by 24–37 % and 0.25–11 % respectively. In Gramineae communities, the influence of annual precipitation and vegetation on TTSW variation was consistent across soil depths. However, in degraded Sophora alopecuroides + Artemisia scoparia (SA) communities, the influence of vegetation (CWM, FD, community composition and community function) on TTSW variation was more pronounced in shallow soils (45 %) than in deeper soils (33 %), while annual precipitation effects were more substantial in the deep soil layers (66 %) than in the shallow soil layers (42 %). (2) Annual precipitation and CWM were the main biological and abiotic factors affecting TTSW. Interestingly, increased annual precipitation was negatively correlated with TTSW. Similarly, the primary controlling factor, CWM, also showed a significant negative relationship with TTSW. This relationship varied across community types and could be linear or quadratic, highlighting the need to understand the selection effects mediated by CWM in ensuring sustainable vegetation restoration. This research offers insights into the relationship between plant functional diversity and soil water retention function

Inundation depth controls leaf photosynthetic capacity by regulating leaf area and N content in an estuarine wetland

Background and AimsEstuarine wetlands are important carbon sinks, with plant photosynthesis being a vital component of this process. Changes in the inundation depth of wetlands could alter leaf photosynthesis and thus ecosystem carbon uptake capacity, ultimately determining the size of carbon sink. However, the relationship between inundation depth and photosynthetic capacity has yet to be determined, especially in estuarine wetlands with complex hydrological conditions. In addition, there is also conflicting evidence regarding the effect of inundation depth on photosynthetic capacity.MethodsTo better understand the mechanisms of photosynthetic capacity responding to inundation depth, we performed a field experiment with a gradient of inundation depths (0, 5, 10, 20, 30 and 40 cm) in estuarine wetland dominated by Phragmites australis in the Yellow River Delta, China.ResultsOur results showed that inundation depth significantly altered leaf morphological traits, elements and photosynthetic capacity. In particular, leaf photosynthetic capacity was obviously increased with increasing inundation depth. The increased leaf area enhanced light harvesting ability of leaves, and then increased Pn at different inundation depths. Besides, higher leaf N content promoted leaf photosynthetic capacity at different inundation depths.ConclusionOverall, the findings demonstrated that inundation depth significantly enhanced the photosynthetic capacity of P. australis, which was correlated with altered leaf functional traits in wetlands. Our results hold important implications for more accurately predicting the relationship between photosynthetic capacity and inundation depth in wetland ecosystems under climate change and more accurately estimating of the carbon sink capacity of wetland ecosystems in the future

Protein therapy of skeletal muscle atrophy and mechanism by angiogenic factor AGGF1

Abstract Background Skeletal muscle atrophy is a common condition without a pharmacologic therapy. AGGF1 encodes an angiogenic factor that regulates cell differentiation, proliferation, migration, apoptosis, autophagy and endoplasmic reticulum stress, promotes vasculogenesis and angiogenesis and successfully treats cardiovascular diseases. Here, we report the important role of AGGF1 in the pathogenesis of skeletal muscle atrophy and attenuation of muscle atrophy by AGGF1. Methods In vivo studies were carried out in impaired leg muscles from patients with lumbar disc herniation, two mouse models for skeletal muscle atrophy (denervation and cancer cachexia) and heterozygous Aggf1+/− mice. Mouse muscle atrophy phenotypes were characterized by body weight and myotube cross‐sectional areas (CSA) using H&E staining and immunostaining for dystrophin. Molecular mechanistic studies include co‐immunoprecipitation (Co‐IP), western blotting, quantitative real‐time PCR analysis and immunostaining analysis. Results Heterozygous Aggf1+/− mice showed exacerbated phenotypes of reduced muscle mass, myotube CSA, MyHC (myosin heavy chain) and α‐actin, increased inflammation (macrophage infiltration), apoptosis and fibrosis after denervation and cachexia. Intramuscular and intraperitoneal injection of recombinant AGGF1 protein attenuates atrophy phenotypes in mice with denervation (gastrocnemius weight 81.3 ± 5.7 mg vs. 67.3 ± 5.1 mg for AGGF1 vs. buffer; P < 0.05) and cachexia (133.7 ± 4.7 vs. 124.3 ± 3.2; P < 0.05). AGGF1 expression undergoes remodelling and is up‐regulated in gastrocnemius and soleus muscles from atrophy mice and impaired leg muscles from patients with lumbar disc herniation by 50–60% (P < 0.01). Mechanistically, AGGF1 interacts with TWEAK (tumour necrosis factor‐like weak inducer of apoptosis), which reduces interaction between TWEAK and its receptor Fn14 (fibroblast growth factor‐inducing protein 14). This leads to inhibition of Fn14‐induced NF‐kappa B (NF‐κB) p65 phosphorylation, which reduces expression of muscle‐specific E3 ubiquitin ligase MuRF1 (muscle RING finger 1), resulting in increased MyHC and α‐actin and partial reversal of atrophy phenotypes. Autophagy is reduced in Aggf1+/− mice due to inhibition of JNK (c‐Jun N‐terminal kinase) activation in denervated and cachectic muscles, and AGGF1 treatment enhances autophagy in two atrophy models by activating JNK. In impaired leg muscles of patients with lumbar disc herniation, MuRF1 is up‐regulated and MyHC and α‐actin are down‐regulated; these effects are reversed by AGGF1 by 50% (P < 0.01). Conclusions These results indicate that AGGF1 is a novel regulator for the pathogenesis of skeletal muscle atrophy and attenuates skeletal muscle atrophy by promoting autophagy and inhibiting MuRF1 expression through a molecular signalling pathway of AGGF1‐TWEAK/Fn14‐NF‐κB. More importantly, the results indicate that AGGF1 protein therapy may be a novel approach to treat patients with skeletal muscle atrophy

Angiogenic Factor AGGF1 Activates Autophagy with an Essential Role in Therapeutic Angiogenesis for Heart Disease.

AGGF1 is an angiogenic factor with therapeutic potential to treat coronary artery disease (CAD) and myocardial infarction (MI). However, the underlying mechanism for AGGF1-mediated therapeutic angiogenesis is unknown. Here, we show for the first time that AGGF1 activates autophagy, a housekeeping catabolic cellular process, in endothelial cells (ECs), HL1, H9C2, and vascular smooth muscle cells. Studies with Atg5 small interfering RNA (siRNA) and the autophagy inhibitors bafilomycin A1 (Baf) and chloroquine demonstrate that autophagy is required for AGGF1-mediated EC proliferation, migration, capillary tube formation, and aortic ring-based angiogenesis. Aggf1+/- knockout (KO) mice show reduced autophagy, which was associated with inhibition of angiogenesis, larger infarct areas, and contractile dysfunction after MI. Protein therapy with AGGF1 leads to robust recovery of myocardial function and contraction with increased survival, increased ejection fraction, reduction of infarct areas, and inhibition of cardiac apoptosis and fibrosis by promoting therapeutic angiogenesis in mice with MI. Inhibition of autophagy in mice by bafilomycin A1 or in Becn1+/- and Atg5 KO mice eliminates AGGF1-mediated angiogenesis and therapeutic actions, indicating that autophagy acts upstream of and is essential for angiogenesis. Mechanistically, AGGF1 initiates autophagy by activating JNK, which leads to activation of Vps34 lipid kinase and the assembly of Becn1-Vps34-Atg14 complex involved in the initiation of autophagy. Our data demonstrate that (1) autophagy is essential for effective therapeutic angiogenesis to treat CAD and MI; (2) AGGF1 is critical to induction of autophagy; and (3) AGGF1 is a novel agent for treatment of CAD and MI. Our data suggest that maintaining or increasing autophagy is a highly innovative strategy to robustly boost the efficacy of therapeutic angiogenesis

Up-regulation of AGGF1 in response to acute MI and robust therapeutic effects of AGGF1 protein therapy on MI.

<p><b>(A)</b> Real-time reverse transcription (RT)-PCR analysis for <i>AGGF1</i> mRNA expression in mice with LAD ligation or sham operation (Sham) (<i>n</i> = 6/group). Pre, before surgery. <b>(B)</b> Western blot analysis for AGGF1 protein expression (<i>n</i> = 6 mice/group). <b>(C)</b> Immunostaining analysis for AGGF1 protein expression in cross-sections of hearts (<i>n</i> = 5). Scale bar = 50 μm. <b>(D)</b> AGGF1 (500 ng/ml) induces autophagy in HUVECs under a hypoxic condition for different time points (<i>n</i> = 3/group). <b>(E)</b> AGGF1-specific siRNA inhibited autophagy under normoxia or hypoxia. <b>(F)</b> Post-MI survival of mice with AGGF1 treatment (<i>n</i> = 20), mice with IgG treatment (<i>n</i> = 30), and sham mice (<i>n</i> = 14). <b>(G)</b> Representative M-mode echocardiograms. <b>(H)</b> Effects of AGGF1 protein therapy on myocardial function and contraction. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

AGGF1-induced angiogenesis is suppressed by autophagic flux inhibition.

<p><b>(A)</b> HUVEC proliferation induced by AGGF1 (500 ng/ml) was blocked by pretreatment with bafilomycin A1 (Baf) or chloroquine (CQ) for 2 h before treatment with IgG (control) or AGGF1 (<i>n</i> = 4/group). <b>(B)</b> Baf or CQ inhibited AGGF1-induced migration of HUVECs using scratch assays (<i>n</i> = 4/group). <b>(C)</b> Baf or CQ inhibited AGGF1-induced migration of HUVECs using Transwell migration assays (<i>n</i> = 4/group). <b>(D)</b> Baf or CQ inhibited AGGF1-induced endothelial tube formation (<i>n</i> = 4/group). Scale bar = 50 μm. <b>(E)</b> Baf or CQ inhibited AGGF1-induced sprout angiogenesis in an e<i>x vivo</i> aortic ring angiogenesis assay (<i>n</i> = 4/group). Scale bar = 125 μm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p

<i>Atg5</i> KO blunted AGGF1-induced therapeutic angiogenesis, survival, and recovery of myocardial functions and contraction of MI mice.

<p><b>(A)</b> AGGF1-induced angiogenesis (CD31 immunostaining) in heart sections 28 d after MI in AAV9-GFP mice. <i>Atg5</i> KO blocks AGGF1-induced angiogenesis (CD31 immunostaining signal) in heart sections 28 d after treatment (<i>n</i> = 5/group, AAV9-CMV-Cre <i>Atg5</i> KO mice). Scale bar = 50 μm. (<b>B</b>) Four-week survival of MI mice under different treatments (Sham + AAV9-GFP + IgG, n = 14; MI + AAV9-GFP + IgG, n = 20; MI + AAV9-GFP + AGGF1, n = 20; Sham + AAV9-Cre + IgG, n = 15; MI + AAV9-Cre + IgG, n = 25; MI + AAV9-Cre + AGGF1, n = 25). (<b>C</b>) Effects of AGGF1 protein therapy under different treatments on the function of the left ventricle (Sham + AAV9-GFP + IgG, n = 13; MI + AAV9-GFP + IgG, n = 10; MI + AAV9-GFP + AGGF1, n = 14; Sham + AAV9-Cre + IgG, n = 13; MI + AAV9-Cre + IgG, n = 7; MI + AAV9-Cre + AGGF1, n = 8). (<b>D</b>) Effects of AGGF1 protein therapy under different treatments on cardiac fibrosis (Masson trichrome staining of cross-sections) and LV wall thickness 4 wk after treatment (<i>n</i> = 5/group). Scale bar = 1 mm. Underlying data are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1002529#pbio.1002529.s001" target="_blank">S1 Data</a>.</p