Activation of CXCR7 promotes endothelial repair and reduces the carotid atherosclerotic lesions through inhibition of pyroptosis signaling pathways

Endothelial injuries, including cell pyroptosis, are ongoing inflammatory processes with key roles in atherosclerosis development. Our previous report showed that the chemokine CXCL12 and its receptor CXCR7 are associated with the proliferation and angiogenesis of endothelial cells. Nevertheless, the mechanism underlying these effects on atherosclerotic lesions, especially on endothelial dysfunction, remains unknown. Here, we demonstrated that CXCR7 was upregulated in human carotid atherosclerotic plaques, apolipoprotein E knockout (ApoE^?/?) mice fed with a high‐fat diet (HFD), and oxidized lipopolysaccharide‐treated (ox‐LDL) human umbilical vein endothelial cells (HUVECs). Further, the activation of CXCR7 reversed ox‐LDL‐induced HUVEC dysfunction, such as migration, tube formation, and cell pyroptosis; all of these protective effects were alleviated by inhibition of CXCR7. The NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasomes were also elevated in human carotid atherosclerotic plaques, ApoE^?/? mice fed with HFD, and ox‐LDL‐injured HUVECs by regulation of caspase‐1 and interleukin (IL)‐1β expression. The activation of CXCR7 by TC14012 led to a decrease in atherosclerotic lesions in ApoE^?/? mice fed with HFD. TC14012 also inhibited the expression of the NLRP3 inflammasome signaling pathway in vivo. In conclusion, our study suggests that CXCR7 plays an important role in regulating NLRP3 inflammasome‐modulated pyroptosis in HUVECs, providing a potential novel therapy for atherosclerosis.     

Autophagy in FOXD1 stroma-derived cells regulates renal fibrosis through TGF-β and NLRP3 inflammasome pathway

Renal fibrosis is the final common pathway of various renal injuries and it leads to chronic kidney disease. Recent studies reported that FOXD1-lineage pericyte plays a critical role in tubulointerstitial fibrosis (TIF). However the regulatory mechanisms remain unclear. Autophagy is a cellular process of degradation of damaged cytoplasmic components that regulates cell death and proliferation. To investigate the role of autophagy in FOXD1-lineage pericytes on renal TIF, we generated the FOXD1-lineage stromal cell-specific Atg7 deletion (Atg7^△FOXD1) mice. FOXD1-lineage stromal cell-specific Atg7 deletion enhanced renal TIF through Smad-dependent transforming growth factor (TGF)-β signaling after unilateral ureteral obstruction (UUO). FOXD1-lineage stromal cell-specific Atg7 deletion increased the accumulation of interstitial myofibroblasts and enhanced the differentiation of pericytes into myofibroblasts after UUO. Peritubular capillary rarefaction was accelerated in Atg7^△FOXD1 mice after UUO. Atg7^△FOXD1 mice increased the accumulation of SQSTM1/p62-positive aggregates in the obstructed kidney and resulted in increased expression of NLRP3 inflammasome, interleukin (IL) 1-β and caspase-1 signaling pathway, which enhanced apoptosis of interstitial cells after UUO. In summary, our data showed that autophagy in FOXD1-lineage stromal cells plays a protective role in renal TIF through regulating the Smad4 dependent TGF-β an NLRP3 inflammasome signaling pathway.     

SQSTM1/p62 and PPARGC1A/PGC-1alpha at the interface of autophagy and vascular senescence

ABSTRACT

Defective macroautophagy/autophagy and mitochondrial dysfunction are known to stimulate senescence. The mitochondrial regulator PPARGC1A (peroxisome proliferator activated receptor gamma, coactivator 1 alpha) regulates mitochondrial biogenesis, reducing senescence of vascular smooth muscle cells (VSMCs); however, it is unknown whether autophagy mediates PPARGC1A-protective effects on senescence. Using ppargc1a^?/- VSMCs, we identified the autophagy receptor SQSTM1/p62 (sequestosome 1) as a major regulator of autophagy and senescence of VSMCs. Abnormal autophagosomes were observed in VSMCs in aortas of ppargc1a^?/- mice. ppargc1a^?/- VSMCs in culture presented reductions in LC3-II levels; in autophagosome number; and in the expression of SQSTM1 (protein and mRNA), LAMP2 (lysosomal-associated membrane protein 2), CTSD (cathepsin D), and TFRC (transferrin receptor). Reduced SQSTM1 protein expression was also observed in aortas of ppargc1a^?/- mice and was upregulated by PPARGC1A overexpression, suggesting that SQSTM1 is a direct target of PPARGC1A. Inhibition of autophagy by 3-MA (3 methyladenine), spautin-1 or Atg5 (autophagy related 5) siRNA stimulated senescence. Rapamycin rescued the effect of Atg5 siRNA in Ppargc1a^+/+ , but not in ppargc1a^?/- VSMCs, suggesting that other targets of MTOR (mechanistic target of rapamycin kinase), in addition to autophagy, also contribute to senescence. Sqstm1 siRNA increased senescence basally and in response to AGT II (angiotensin II) and zinc overload, two known inducers of senescence. Furthermore, Sqstm1 gene deficiency mimicked the phenotype of Ppargc1a depletion by presenting reduced autophagy and increased senescence in vitro and in vivo. Thus, PPARGC1A upregulates autophagy reducing senescence by a SQSTM1-dependent mechanism. We propose SQSTM1 as a novel target in therapeutic interventions reducing senescence.     

Clostridium difficile toxin B induces autophagic cell death in colonocytes

Toxin B (TcdB) is a major pathogenic factor of Clostridum difficile. However, the mechanism by which TcdB exerts its cytotoxic action in host cells is still not completely known. Herein, we report for the first time that TcdB induced autophagic cell death in cultured human colonocytes. The induction of autophagy was demonstrated by the increased levels of LC3‐II, formation of LC3⁺ autophagosomes, accumulation of acidic vesicular organelles and reduced levels of the autophagic substrate p62/SQSTM1. TcdB‐induced autophagy was also accompanied by the repression of phosphoinositide 3‐kinase (PI3K)/Akt/mechanistic target of rapamycin (mTOR) complex 1 activity. Functionally, pharmacological inhibition of autophagy by wortmannin or chloroquine or knockdown of autophagy‐related genes Beclin 1, Atg5 and Atg7 attenuated TcdB‐induced cell death in colonocytes. Genetic ablation of Atg5, a gene required for autophagosome formation, also mitigated the cytotoxic effect of TcdB. In conclusion, our study demonstrated that autophagy serves as a pro‐death mechanism mediating the cytotoxic action of TcdB in colonocytes. This discovery suggested that blockade of autophagy might be a novel therapeutic strategy for C. difficile infection.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Macrophage autophagy regulates mitochondria‐mediated apoptosis and inhibits necrotic core formation in vulnerable plaques

The vulnerable plaque is a key distinguishing feature of atherosclerotic lesions that can cause acute atherothrombotic vascular disease. This study was designed to explore the effect of autophagy on mitochondria‐mediated macrophage apoptosis and vulnerable plaques. Here, we generated the mouse model of vulnerable carotid plaque in ApoE^?/? mice. Application of ApoE^?/? mice with rapamycin (an autophagy inducer) inhibited necrotic core formation in vulnerable plaques by decreasing macrophage apoptosis. However, 3‐methyladenine (an autophagy inhibitor) promoted plaque vulnerability through deteriorating these indexes. To further explore the mechanism of autophagy on macrophage apoptosis, we used macrophage apoptosis model in vitro and found that 7‐ketocholesterol (7‐KC, one of the primary oxysterols in oxLDL) caused macrophage apoptosis with concomitant impairment of mitochondria, characterized by the impairment of mitochondrial ultrastructure, cytochrome c release, mitochondrial potential dissipation, mitochondrial fragmentation, excessive ROS generation and both caspase‐9 and caspase‐3 activation. Interestingly, such mitochondrial apoptotic responses were ameliorated by autophagy activator, but exacerbated by autophagy inhibitor. Finally, we found that MAPK‐NF‐κB signalling pathway was involved in autophagy modulation of 7‐KC–induced macrophage apoptosis. So, we provide strong evidence for the potential therapeutic benefit of macrophage autophagy in regulating mitochondria‐mediated apoptosis and inhibiting necrotic core formation in vulnerable plaques.     

FGF21 induces autophagy‐mediated cholesterol efflux to inhibit atherogenesis via RACK1 up‐regulation

Fibroblast growth factor 21 (FGF21) acts as an anti‐atherosclerotic agent. However, the specific mechanisms governing this regulatory activity are unclear. Autophagy is a highly conserved cell stress response which regulates atherosclerosis (AS) by reducing lipid droplet degradation in foam cells. We sought to assess whether FGF21 could inhibit AS by regulating cholesterol metabolism in foam cells via autophagy and to elucidate the underlying molecular mechanisms. In this study, ApoE^?/? mice were fed a high‐fat diet (HFD) with or without FGF21 and FGF21 + 3‐Methyladenine (3MA) for 12 weeks. Our results showed that FGF21 inhibited AS in HFD‐fed ApoE^?/? mice, which was reversed by 3MA treatment. Moreover, FGF21 increased plaque RACK1 and autophagy‐related protein (LC3 and beclin‐1) expression in ApoE^?/? mice, thus preventing AS. However, these proteins were inhibited by LV‐RACK1 shRNA injection. Foam cell development is a crucial determinant of AS, and cholesterol efflux from foam cells represents an important defensive measure of AS. In this study, foam cells were treated with FGF21 for 24 hours after a pre‐treatment with 3MA, ATG5 siRNA or RACK1 siRNA. Our results indicated that FGF21‐induced autophagy promoted cholesterol efflux to reduce cholesterol accumulation in foam cells by up‐regulating RACK1 expression. Interestingly, immunoprecipitation results showed that RACK1 was able to activate AMPK and interact with ATG5. Taken together, our results indicated that FGF21 induces autophagy to promote cholesterol efflux and reduce cholesterol accumulation in foam cells through RACK1‐mediated AMPK activation and ATG5 interaction. These results provided new insights into the molecular mechanisms of FGF21 in the treatment of AS.     

Smooth muscle-specific HuR knockout induces defective autophagy and atherosclerosis

Human antigen R (HuR) is a widespread RNA-binding protein involved in homeostatic regulation and pathological processes in many diseases. Atherosclerosis is the leading cause of cardiovascular disease and acute cardiovascular events. However, the role of HuR in atherosclerosis remains unknown. In this study, mice with smooth muscle-specific HuR knockout (HuR^SMKO) were generated to investigate the role of HuR in atherosclerosis. HuR expression was reduced in atherosclerotic plaques. As compared with controls, HuR^SMKO mice showed increased plaque burden in the atherosclerotic model. Mechanically, HuR could bind to the mRNAs of adenosine 5′-monophosphate-activated protein kinase (AMPK) α1 and AMPKα2, thus increasing their stability and translation. HuR deficiency reduced p-AMPK and LC3II levels and increased p62 level, thereby resulting in defective autophagy. Finally, pharmacological AMPK activation induced autophagy and suppressed atherosclerosis in HuR^SMKO mice. Our findings suggest that smooth muscle HuR has a protective effect against atherosclerosis by increasing AMPK-mediated autophagy.Subject terms: Cardiovascular diseases, Pathogenesis     

Dual Benefit of Reduced Cx43 on Atherosclerosis in LDL Receptor-Deficient Mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR^?/?) mice were intercrossed with mice heterozygous for Cx43 (Cx43^+/?mice). Male mice with normal (Cx43^+/+LDLR^?/?) or reduced (Cx43^+/?LDLR^?/?) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43^+/?LDLR^?/?mice compared with Cx43^+/+LDLR^?/?littermate controls. In addition, atheroma in Cx43^+/?LDLR^?/?mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Interleukin-17 deficiency reduced vascular inflammation and development of atherosclerosis in Western diet-induced apoE-deficient mice

ObjectiveSeveral reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE^?/?IL-17^?/? mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis.Methods and resultsAtherosclerotic plaque areas in apoE^?/?IL-17^?/? mice fed a Western diet (WD) were significantly reduced compared with those in apoE^?/? mice. No significant differences in plasma lipid profiles were observed between apoE^?/? and apoE^?/?IL-17^?/? mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE^?/?IL-17^?/? mice compared with WD-fed apoE^?/? mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1β, IL-6, IFN-γ, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE^?/?IL-17^?/? mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE^?/?IL-17^?/? mice.ConclusionOur results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis.     

Macrophage autophagy plays a protective role in advanced atherosclerosis

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Highlights? Proapoptotic oxidative stress induces autophagy in macrophages (M?s) ? Inhibition of autophagy enhances oxidative stress and apoptosis in M?s ? Atheromata of M?-Atg5^?/?Ldlr^?/? mice have increased apoptosis and plaque necrosis ? Apoptotic Atg5^?/? M?s are poorly recognized by phagocytes in vitro and in atheromata     

Age‐associated vascular inflammation promotes monocytosis during atherogenesis

Aging leads to a proinflammatory state within the vasculature without disease, yet whether this inflammatory state occurs during atherogenesis remains unclear. Here, we examined how aging impacts atherosclerosis using Ldlr^?/? mice, an established murine model of atherosclerosis. We found that aged atherosclerotic Ldlr^?/? mice exhibited enhanced atherogenesis within the aorta. Aging also led to increased LDL levels, elevated blood pressure on a low‐fat diet, and insulin resistance after a high‐fat diet (HFD). On a HFD, aging increased a monocytosis in the peripheral blood and enhanced macrophage accumulation within the aorta. When we conducted bone marrow transplant experiments, we found that stromal factors contributed to age‐enhanced atherosclerosis. To delineate these stromal factors, we determined that the vasculature exhibited an age‐enhanced inflammatory response consisting of elevated production of CCL‐2, osteopontin, and IL‐6 during atherogenesis. In addition, in vitro cultures showed that aging enhanced the production of osteopontin by vascular smooth muscle cells. Functionally, aged atherosclerotic aortas displayed higher monocyte chemotaxis than young aortas. Hence, our study has revealed that aging induces metabolic dysfunction and enhances vascular inflammation to promote a peripheral monocytosis and macrophage accumulation within the atherosclerotic aorta.     

Role of autophagy,SQSTM1, SH3GLB1, and TRIM63 in the turnover of nicotinic acetylcholine receptors

Removal of ubiquitinated targets by autophagosomes can be mediated by receptor molecules, like SQSTM1, in a mechanism referred to as selective autophagy. While cytoplasmic protein aggregates, mitochondria, and bacteria are the best-known targets of selective autophagy, their role in the turnover of membrane receptors is scarce. We here showed that fasting-induced wasting of skeletal muscle involves remodeling of the neuromuscular junction (NMJ) by increasing the turnover of muscle-type CHRN (cholinergic receptor, nicotinic/nicotinic acetylcholine receptor) in a TRIM63-dependent manner. Notably, this process implied enhanced production of endo/lysosomal carriers of CHRN, which also contained the membrane remodeler SH3GLB1, the E3 ubiquitin ligase, TRIM63, and the selective autophagy receptor SQSTM1. Furthermore, these vesicles were surrounded by the autophagic marker MAP1LC3A in an ATG7-dependent fashion, and some of them were also positive for the lysosomal marker, LAMP1. While the amount of vesicles containing endocytosed CHRN strongly augmented in the absence of ATG7 as well as upon denervation as a model for long-term atrophy, denervation-induced increase in autophagic CHRN vesicles was completely blunted in the absence of TRIM63. On a similar note, in trim63^?/? mice denervation-induced upregulation of SQSTM1 and LC3-II was abolished and endogenous SQSTM1 did not colocalize with CHRN vesicles as it did in the wild type. SQSTM1 and LC3-II coprecipitated with surface-labeled/endocytosed CHRN and SQSTM1 overexpression significantly induced CHRN vesicle formation. Taken together, our data suggested that selective autophagy regulates the basal and atrophy-induced turnover of the pentameric transmembrane protein, CHRN, and that TRIM63, together with SH3GLB1 and SQSTM1 regulate this process.     

Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis

Antisense oligonucleotides (ASOs) against Ldl receptor (Ldlr-ASO) represent a promising strategy to promote hypercholesterolemic atherosclerosis in animal models without the need for complex breeding strategies. Here, we sought to characterize and contrast atherosclerosis in mice given Ldlr-ASO with those bearing genetic Ldlr deficiency. To promote atherosclerosis, male and female C57Bl6/J mice were either given weekly injections of Ldlr-ASO (5 mg/kg once per week) or genetically deficient in Ldlr (Ldlr^?/?). Mice consumed either standard rodent chow or a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. While both models of Ldlr deficiency promoted hypercholesterolemia, Ldlr^?/? mice exhibited nearly 2-fold higher cholesterol levels than Ldlr-ASO mice, reflected by increased VLDL and LDL levels. Consistent with this, the en face atherosclerotic lesion area was 3-fold and 3.6-fold greater in male and female mice with genetic Ldlr deficiency, respectively, as compared with the modest atherosclerosis observed following Ldlr-ASO treatment. Aortic sinus lesion sizes, fibrosis, smooth muscle actin, and necrotic core areas were also larger in Ldlr^?/? mice, suggesting a more advanced phenotype. Despite a more modest effect on hypercholesterolemia, Ldlr-ASO induced greater hepatic inflammatory gene expression, macrophage accumulation, and histological lobular inflammation than was observed in Ldlr^?/? mice. We conclude Ldlr-ASO is a promising tool for the generation of complex rodent models with which to study atherosclerosis but does not promote comparable levels of hypercholesterolemia or atherosclerosis as Ldlr^?/? mice and increases hepatic inflammation. Thus, genetic Ldlr deficiency may be a superior model, depending on the proposed use.     

Spreading of pathological TDP-43 along corticospinal tract axons induces ALS-like phenotypes in Atg5+/- mice

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by phosphorylated TDP-43 (pTDP-43)-positive inclusions in neurons and glial cells. However, the pathogenic mechanism that underlies ALS remains largely unknown. To investigate the effects of autophagy deficiency in the formation and spreading of pathological TDP-43 along corticospinal tract axons, TDP-43 preformed fibrils (PFFs) were prepared and unilaterally injected into the fifth layer of the left primary motor cortex (M1) or the left anterior horn of the seventh cervical spinal cord segment (C7) of Atg5^+/- mice. After the injection of TDP-43 PFFs, the elevated levels of pTDP-43 were present in several pyramidal tract-associated regions of Atg5^+/- mice. Additionally, the occurrence of spontaneous potentials detected by electromyography demonstrates evidence of lower motor neuron dysfunction in M1-TDP-43 PFFs-injected Atg5^+/- mice, and prolonged central motor conduction time detected by motor evoked potentials provides evidence of upper motor neuron dysfunction in C7-TDP-43 PFFs-injected Atg5^+/- mice. These results show that injection of TDP-43 PFFs into the M1 or C7 of Atg5^+/- mice induces the spreading of pathological TDP-43 along corticospinal tract axons in both an anterograde and retrograde manner. Importantly, TDP-43 PFFs-injected Atg5^+/- mice also display ALS-like motor dysfunction. Taken together, our findings provide direct evidence that TDP-43 PFFs-injected Atg5^+/- mice exhibited ALS-like neuropathology and motor phenotypes, suggesting that autophagy deficiency promotes the formation and spreading of pathological TDP-43 in vivo.     

Diminished Autophagy Limits Cardiac Injury in Mouse Models of Type 1 Diabetes

Cardiac autophagy is inhibited in type 1 diabetes. However, it remains unknown if the reduced autophagy contributes to the pathogenesis of diabetic cardiomyopathy. We addressed this question using mouse models with gain- and loss-of-autophagy. Autophagic flux was inhibited in diabetic hearts when measured at multiple time points after diabetes induction by streptozotocin as assessed by protein levels of microtubule-associated protein light chain 3 form 2 (LC3-II) or GFP-LC3 puncta in the absence and presence of the lysosome inhibitor bafilomycin A1. Autophagy in diabetic hearts was further reduced in beclin 1- or Atg16-deficient mice but was restored partially or completely by overexpression of beclin 1 to different levels. Surprisingly, diabetes-induced cardiac damage was substantially attenuated in beclin 1- and Atg16-deficient mice as shown by improved cardiac function as well as reduced levels of oxidative stress, interstitial fibrosis, and myocyte apoptosis. In contrast, diabetic cardiac damage was dose-dependently exacerbated by beclin 1 overexpression. The cardioprotective effects of autophagy deficiency were reproduced in OVE26 diabetic mice. These effects were associated with partially restored mitophagy and increased expression and mitochondrial localization of Rab9, an essential regulator of a non-canonical alternative autophagic pathway. Together, these findings demonstrate that the diminished autophagy is an adaptive response that limits cardiac dysfunction in type 1 diabetes, presumably through up-regulation of alternative autophagy and mitophagy.     

MDA‐5 activation by cytoplasmic double‐stranded RNA impairs endothelial function and aggravates atherosclerosis

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA‐5) belongs to the intracellular retinoic acid inducible gene‐I like receptors and its activation promotes pro‐inflammatory mechanisms. Here, we studied the effect of MDA‐5 stimulation in vascular biology. To gain insights into MDA‐5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA‐5 agonist polyIC (long double‐stranded RNA). Human coronary endothelial cell expressed MDA‐5 and reacted with receptor up‐regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro‐inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA‐5 stimulation. To test these effects in vivo, wild‐type mice were transfected with 32.5 μg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC‐treated wild‐type mice, endothelium‐dependent vasodilation and re‐endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA‐5 deficiency in vivo. Finally, chronic MDA‐5 stimulation in Apolipoprotein E/toll‐like receptor 3 (TLR3) double^‐deficient (ApoE^?/?/TLR3^?/?) mice‐enhanced atherosclerotic plaque formation. This study demonstrates that MDA‐5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA‐5.