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Acid sphingomyelinase (ASM) has been reported to increase tissue ceramide and thereby mediate hyperhomocysteinemia (hHcy)-induced glomerular nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation, inflammation, and sclerosis. In the present study, we tested whether somatic podocyte-specific silencing of Smpd1 gene (mouse ASM gene code) attenuates hHcy-induced NLRP3 inflammasome activation and associated extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. In vivo, somatic podocyte-specific Smpd1 gene silencing almost blocked hHcy-induced glomerular NLRP3 inflammasome activation in Podocre (podocyte-specific expression of cre recombinase) mice compared with control littermates. By nanoparticle tracking analysis (NTA), floxed Smpd1 shRNA transfection was found to abrogate hHcy-induced elevation of urinary EV excretion in Podocre mice. In addition, Smpd1 gene silencing in podocytes prevented hHcy-induced immune cell infiltration into glomeruli, proteinuria, and glomerular sclerosis in Podocre mice. Such protective effects of podocyte-specific Smpd1 gene silencing were mimicked by global knockout of Smpd1 gene in Smpd1−/− mice. On the contrary, podocyte-specific Smpd1 gene overexpression exaggerated hHcy-induced glomerular pathological changes in Smpd1trg/Podocre (podocyte-specific Smpd1 gene overexpression) mice, which were significantly attenuated by transfection of floxed Smpd1 shRNA. In cell studies, we also confirmed that Smpd1 gene knockout or silencing prevented homocysteine (Hcy)-induced elevation of EV release in the primary cultures of podocyte isolated from Smpd1−/− mice or podocytes of Podocre mice transfected with floxed Smpd1 shRNA compared with WT/WT podocytes. Smpd1 gene overexpression amplified Hcy-induced EV secretion from podocytes of Smpd1trg/Podocre mice, which was remarkably attenuated by transfection of floxed Smpd1 shRNA. Mechanistically, Hcy-induced elevation of EV release from podocytes was blocked by ASM inhibitor (amitriptyline, AMI), but not by NLRP3 inflammasome inhibitors (MCC950 and glycyrrhizin, GLY). Super-resolution microscopy also showed that ASM inhibitor, but not NLRP3 inflammasome inhibitors, prevented the inhibition of lysosome-multivesicular body interaction by Hcy in podocytes. Moreover, we found that podocyte-derived inflammatory EVs (released from podocytes treated with Hcy) induced podocyte injury, which was exaggerated by T cell coculture. Interstitial infusion of inflammatory EVs into renal cortex induced glomerular injury and immune cell infiltration. In conclusion, our findings suggest that ASM in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy and that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effect. NEW & NOTEWORTHY: In the present study, we tested whether podocyte-specific silencing of Smpd1 gene attenuates hyperhomocysteinemia (hHcy)-induced nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome activation and associated inflammatory extracellular vesicle (EV) release in podocytes and thereby suppresses glomerular inflammatory response and injury. Our findings suggest that acid sphingomyelinase (ASM) in podocytes plays a crucial role in the control of NLRP3 inflammasome activation and inflammatory EV release during hHcy. Based on our findings, it is anticipated that the development of podocyte-specific ASM inhibition or Smpd1 gene silencing may be a novel therapeutic strategy for treatment of hHcy-induced glomerular disease with minimized side effects. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. Methods and Results: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1β production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. Conclusion: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response. [ABSTRACT FROM AUTHOR]

The activation of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been reported to importantly contribute to glomerular inflammation and injury under different pathological conditions such as obesity. However, the mechanism mediating NLRP3 inflammasome activation in podocytes and subsequent glomerular injury remains poorly understood. Given that the ceramide signaling pathway has been reported to be implicated in obesity-related glomerulopathy (ORG), the present study was designed to test whether the ceramide-producing enzyme, acid sphingomyelinase (ASM), determines NLRP3 inflammasome activation and inflammatory exosome release in podocytes leading to glomerular inflammation and injury during ORG. In Smpd1trg/Podocre mice, podocyte-specific overexpression of Smpd1 gene which encodes ASM significantly exaggerated high-fat diet (HFD)-induced NLRP3 inflammasome activation in podocytes and immune cell infiltration in glomeruli compared to WT/WT mice. Smpd1 gene deletion, however, blocked these pathological changes induced by HFD in Smpd1−/− mice. Accompanied with NLRP3 inflammasome activation and glomerular inflammation, urinary excretion of exosomes containing podocyte marker and NLRP3 inflammasome products (IL-1β and IL-18) in Smpd1trg/Podocre mice on the HFD was much higher than that in WT/WT mice. In contrast, Smpd1−/− mice on the HDF had significantly lower urinary exosome excretion than WT/WT mice. Correspondingly, HFD-induced podocyte injury, glomerular sclerosis, and proteinuria were more severe in Smpd1trg/Podocre mice, but milder in Smpd1−/− mice compared to WT/WT mice. Using podocytes isolated from these mice, we demonstrated that visfatin, a prototype pro-inflammatory adipokine, induced NLRP3 inflammasome activation and enrichment of multivesicular bodies (MVBs) containing IL-1β in podocytes, which was much stronger in podocytes from Smpd1trg/Podocre mice, but weaker in those from Smpd1−/− mice than WT/WT podocytes. By quantitative analysis of exosomes, it was found that upon visfatin stimulation, podocytes from Smpd1trg/Podocre mice released much more exosomes containing NLRP3 inflammasome products, but podocytes from Smpd1−/− mice released much less exosomes compared to WT/WT podocytes. Super-resolution microscopy demonstrated that visfatin inhibited lysosome-MVB interaction in podocytes, indicating impaired MVB degradation by lysosome. The inhibition of lysosome-MVB interaction by visfatin was amplified by Smpd1 gene overexpression but attenuated by Smpd1 gene deletion. Taken together, our results suggest that ASM in podocytes is a crucial regulator of NLRP3 inflammasome activation and inflammatory exosome release that instigate glomerular inflammation and injury during obesity. [ABSTRACT FROM AUTHOR]

BACKGROUND Our previous studies showed that renal medullary sphingosine-1-phosphate receptor 1 (S1PR1) mediated sodium excretion, high salt intake increased S1PR1 level, deoxycorticosterone acetate (DOCA) blocked high salt-induced S1PR1 in the renal medulla, and that conditional knockout of S1PR1 in the collecting duct aggravated DOCA-salt hypertension. The present study tested the hypothesis that overexpression of S1PR1 transgene in the renal medulla attenuates the sodium retention and hypertension in DOCA-salt mouse model. METHODS Male C57BL/6J mice received renal medullary transfection of control or S1PR1-expressing plasmids and then DOCA-salt treatment. Renal sodium excretion and arterial pressure were compared between control and S1PR1-overexpressed mice in response to high salt loading or pressure natriuresis. RESULTS S1PR1-transfected mice showed significantly enhanced urinary sodium excretion in response to acute sodium loading (0.93 ± 0.27 in control vs. 4.72 ± 1.12 µmol/min/gKW in S1PR1-overexpressed mice, P < 0.05) and the pressure natriuresis (3.58 ± 1.77 vs. 9.52 ± 1.38, P < 0.05), less positive sodium balance in response to chronic high-salt intake (3.05 ± 0.39 vs. 1.65 ± 0.39 mmol/72 hr, P < 0.05), and consequently, the attenuation of DOCA-salt hypertension (134.2 ± 6.79 vs. 109.8 ± 3.54 mm Hg, P < 0.05). The αENaC protein amount in the renal medulla was not changed, however, the βENaC was significantly decreased and the γENaC was significantly increased in S1PR1-overexpressed mice. The immunostaining showed apical membrane translocation of γENaC, while no change of αENaC and βENaC in control mice, and that the apical membrane translocation of γENaC was blocked in S1PR1-treasffected mice. CONCLUSIONS These results suggested that activation of S1PR1 in the renal medulla attenuates DOCA-induced sodium retention and salt-sensitive hypertension associated with inhibition of ENaC. [ABSTRACT FROM AUTHOR]

Background We have previously shown that high salt stimulates the expression of miR-429 in the renal medulla, which induces mRNA decay of HIF prolyl-hydroxylase 2 (PHD2), an enzyme to promote the degradation of hypoxia-inducible factor (HIF)-1α, and increases the HIF-1α-mediated activation of antihypertensive genes in the renal medulla, consequently promoting extra sodium excretion. Our preliminary results showed that high salt-induced increase of miR-429 was not observed in Dahl S rats. This present study determined whether correction of this impairment in miR-429 would reduce PHD2 levels, increase antihypertensive gene expression in the renal medulla and attenuate salt-sensitive hypertension in Dahl S rats. Methods Lentiviruses encoding rat miR-429 were transfected into the renal medulla in uninephrectomized Dahl S rats. Sodium excretion and blood pressure were then measured. Results Transduction of lentiviruses expressing miR-429 into the renal medulla increased miR-429 levels, decreased PHD2 levels, and upregulated HIF-1α target gene NOS-2, which restored the adaptive mechanism to increase the antihypertensive gene after high-salt intake in Dahl S rats. Functionally, overexpression of miR-429 transgene in the renal medulla significantly improved pressure natriuretic response, enhanced urinary sodium excretion, and reduced sodium retention upon extra sodium loading, and consequently, attenuated the salt-sensitive hypertension in Dahl S rats. Conclusions Our results suggest that the impaired miR-429-mediated PHD2 inhibition in response to high salt in the renal medulla may represent a novel mechanism for salt-sensitive hypertension in Dahl S rats and that correction of this impairment in miR-429 pathway could be a therapeutic approach for salt-sensitive hypertension. [ABSTRACT FROM AUTHOR]

Background: Exosomes have been reported to mediate activation of the inflammatory response by secretion of inflammasome products such as IL-1β or IL-18 and that changes in exosomes production or secretion may be a therapeutic target for treatment of a variety of different chronic diseases. The present study tested the hypothesis that exosome-mediated release of NLRP3 inflammasome products instigates the inflammatory response in the lung during emphysema, a type of chronic obstructive pulmonary disease (COPD) and that electroacupuncture (EA) may attenuate emphysema by inhibition of NLRP3 inflammasome activation and consequent inflammation. Methods: The COPD mice model was developed by injecting porcine pancreatic elastase (PPE) via puncture tracheotomy and instillation. EA (4 Hz/20 Hz, 1 to 3 mA) was applied to the bilateral BL13 and ST36 for 30 min, once every other day for 2 weeks. Micro computed tomography (micro-CT) was performed to measure lung function. Histopathological changes in the lungs were displayed by HE staining. Results: In a mouse model of porcine pancreatic elastase (PPE)-induced emphysema, the lung tissue was found to display several key features of emphysema, including alveolar septal thickening, enlarged alveoli, interstitial edema, and inflammatory cells infiltration. Lungs of mice receiving PPE exhibited substantially increased low attenuation area (LAA) in micro-CT images. The colocalization of NLRP3 vs ASC or caspase-1 detected by confocal microscopy was shown to increase in both bronchial and alveolar walls, indicating the increased formation of NLRP3 inflammasomes. IL-1β, a prototype NLRP3 inflammasome activating product, was also found to have increased in the lung during emphysema, which was colocalized with CD63 (an exosome marker), an indicative of inflammatory exosome formation. By nanoparticle tracking analysis (NTA), IL-1β-containing exosomes were shown to significantly increase in the bronchoalveolar lavage (BAL) from mice with emphysema. Therapeutically, IL-1β production in the lung during emphysema was significantly reduced by EA at the acupoint Feishu (BL13) and Zusanli (ST36), accompanied by decreased colocalization of NLRP3 vs ASC or caspase-1. Increased exosome release into BAL during emphysema was shown to be significantly attenuated in EA-treated mice compared to their controls. However, EA of non-specific BL23 together with ST36 acupoint had no effects on NLRP3 inflammasome activation, exosome release and associated lung pathology during emphysema. Conclusion: NLRP3 inflammasome activation in concert with increased release of exosomes containing IL-1β or other inflammasome products contributes to the development of lung inflammation and injury during PPE-induced emphysema and that EA of lung-specific acupoints attenuates inflammasome activation and exosome release, thereby reducing inflammatory response in the lung of mice with emphysema. [ABSTRACT FROM AUTHOR]

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum -infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo , pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources. [ABSTRACT FROM AUTHOR]

Arterial medial calcification (AMC) involves an increased small extracellular vesicle (sEV) secretion and apatite calcium precipitation in the arterial wall. The mechanisms mediating AMC remain poorly understood. In the present study, smooth muscle-specific acid ceramidase (Ac) gene knockout mice (Asah1fl/fl/SMCre) were used to demonstrate the role of lysosomal ceramide signaling pathway in AMC. Asah1fl/fl/SMCre mice were found to have more severe AMC in both aorta and coronary arteries compared to their littermates (Asah1fl/fl/SMwt and WT/WT mice) after receiving a high dose vitamin D. These mice also had pronounced upregulation of osteopontin and RUNX2 (osteogenic markers), CD63, AnX2 (sEV markers) and ALP expression (mineralization marker) in the arterial media. In cultured coronary arterial smooth muscle cells (CASMCs) from Asah1fl/fl/SMCre mice, high dose of Pi led to a significantly increased calcium deposition, phenotypic change and sEV secretion compared to WT CASMCs, which was associated with reduced lysosome-multivesicular body (MVB) interaction. Also, GW4869, sEV release inhibitor decreased sEV secretion and calcification in these cells. Lysosomal transient receptor potential mucolipin 1 (TRPML1) channels regulating lysosome interaction with MVBs were found remarkably inhibited in Asah1fl/fl/SMCre CASMCs as shown by GCaMP3 Ca2+ imaging and Port-a-Patch patch clamping of lysosomes. Lysosomal Ac in SMCs controls sEV release by regulating lysosomal TRPML1 channel activity and lysosome-MVB interaction, which importantly contributes to phenotypic transition and AMC. [ABSTRACT FROM AUTHOR]

Arterial medial calcification (AMC) is associated with crystallization of hydroxyapatite in the extracellular matrix and arterial smooth muscle cells (SMCs) leading to reduced arterial compliance. The study was performed to test whether lysosomal acid sphingomyelinase (murine gene code: Smpd1)‐derived ceramide contributes to the small extracellular vesicle (sEV) secretion from SMCs and consequently leads to AMC. In Smpd1trg/SMcre mice with SMC‐specific overexpression of Smpd1 gene, a high dose of Vit D (500 000 IU/kg/d) resulted in increased aortic and coronary AMC, associated with augmented expression of RUNX2 and osteopontin in the coronary and aortic media compared with their littermates (Smpd1trg/SMwt and WT/WT mice), indicating phenotypic switch. However, amitriptyline, an acid sphingomyelinase (ASM) inhibitor, reduced calcification and reversed phenotypic switch. Smpd1trg/SMcre mice showed increased CD63, AnX2 and ALP levels in the arterial wall, accompanied by reduced co‐localization of lysosome marker (Lamp‐1) with multivesicular body (MVB) marker (VPS16), a parameter for lysosome‐MVB interaction. All these changes related to lysosome fusion and sEV release were substantially attenuated by amitriptyline. Increased arterial stiffness and elastin disorganization were found in Smpd1trg/SMcre mice as compared to their littermates. In cultured coronary arterial SMCs (CASMCs) from Smpd1trg/SMcre mice, increased Pi concentrations led to markedly increased calcium deposition, phenotypic change and sEV secretion compared with WT CASMCs, accompanied by reduced lysosome‐MVB interaction. However, amitriptyline prevented these changes in Pi‐treated CASMCs. These data indicate that lysosomal ceramide plays a critical role in phenotype change and sEV release in SMCs, which may contribute to the arterial stiffness during the development of AMC. [ABSTRACT FROM AUTHOR]

The glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone secreted by intestinal enteroendocrine L-cells, which plays a crucial role in glucose control, regulation, and protection from different pathological conditions such as diabetes mellitus. The present study sought to test whether GLP-1 release increases gut injury with a high-fat diet (HFD) and whether this GLP-1 release is associated with NLRP3 inflammasome activation. Our results showed that the NLRP3 inflammasome is activated in the intestinal tissue of wild-type mice on a HFD, accompanied by GLP-1 overexpression. The number of intestinal L-cells and the GLP-1 level in serum are increased in WT mice with HFD. However, in the Asc–/– and Nlrp3–/– mice, these HFD-induced intestinal and serum GLP-1 changes were suppressed. Using confocal microscopy, the colocalization of GLP-1 and FLICA that labels activated caspase-1 in intestine was decreased in the Asc–/– and Nlrp3–/– mice compared to WT mice. Mechanistically, the inhibitor of caspase-1 or HMGB1 blocker is used to demonstrate the regulatory action of NRLP3 inflammasome in GLP-1 release. It was found that the level of GLP-1 and its colocalization with IL-1β were reduced by inhibition of the caspase-1 activity, but not altered by blockade of HMGB1 action. Our results suggest that NLRP3 inflammasome activation triggers GLP-1 production from the intestine, which is associated with IL-1β, but not with HMGB1. These findings for the first time provide evidence that the activation of NLRP3 inflammasome in the intestine increases GLP-1 release in mice, which may serve as an adaptive response to intestinal inflammation. [ABSTRACT FROM AUTHOR]

Elevated homocysteine (Hcy) levels have been shown to activate nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome leading to podocyte dysfunction and glomerular injury. However, it remains unclear how this inflammasome activation in podocytes is a therapeutic target for reversal of glomerular injury and ultimate sclerosis. The present study tested whether inhibition of Rac1 GTPase activity suppresses NLRP3 inflammation activation and thereby blocks podocyte injury induced by elevated Hcy. In cultured podocytes, we found that L-Hcy (the active Hcy form) stimulated the NLRP3 inflammasome formation, as shown by increased colocalization of NLRP3 with apoptosis-associated speck-like protein (ASC) or caspase-1, which was accompanied by increased interleukin-1β production and caspase-1 activity, indicating NLRP3 inflammasome activation. Rac1 activator, uridine triphosphate (UTP), mimicked L-Hcy-induced NLRP3 inflammasome activation, while Rac1 inhibitor NSC23766 blocked it. This Rac1 inhibition also prevented L-Hcy-induced podocyte dysfunction. All these effects were shown to be mediated via lipid raft redox signaling platforms with nicotinamide adenine dinucleotide phosphate oxidase subunits and consequent O2– production. In animal studies, hyperhomocysteinemia (hHcy) induced by folate-free diet was shown to induce NLRP3 inflammasome formation and activation in glomeruli, which was also mimicked by UTP and inhibited by NSC23766 to a comparable level seen in Nlrp3 gene knockout mice. These results together suggest that Rac1 inhibition protects the kidney from hHcy-induced podocyte injury and glomerular sclerosis due to its action to suppress NLRP3 inflammasome activation in podocytes. [ABSTRACT FROM AUTHOR]

Amitriptyline is a tricyclic antidepressant and an inhibitor of lysosomal acid sphingomyelinase (ASM). Amitriptyline is well known for its cardiovascular side effects and toxicity in psychiatric patients. However, the mechanisms underlying the cardiovascular side effects of amitriptyline remain largely undefined. This study aimed to determine the effects of amitriptyline on angiogenic capability of vascular endothelial cells in physiological settings and identify its mechanism of action. The ex vivo aortic ring angiogenesis and in vitro‐cultured endothelial cell tube formation assay were used to assess the effects of amitriptyline on endothelial angiogenic capability. It was demonstrated that amitriptyline impaired the angiogenesis of aortic rings, which was similar to that found in aortic rings with haploinsufficiency of the ASM gene. In cultured mouse microvascular endothelial cells (MVECs), amitriptyline impaired the proliferation and tube formation under basal condition, which were accompanied by attenuated angiogenic signalling pathways such as endothelial nitric oxide synthase, Akt and Erk1/2 pathways. Mechanistically, amitriptyline inhibited autophagic flux without affecting autophagosome biogenesis at basal condition. ASM gene silencing or autophagy inhibition mimics the inhibitory effects of amitriptyline on endothelial cell proliferation and tube formation. Collectively, our data suggest that amitriptyline inhibits endothelial cell proliferation and angiogenesis via blockade of ASM‐autophagic flux axis. It is implicated that the cardiovascular side effects of amitriptyline may be associated with its inhibitory action on physiological angiogenesis. [ABSTRACT FROM AUTHOR]

Background/Aim: Plasma trimethylamine-N-oxide (TMAO), a product of intestinal microbial metabolism of dietary phosphatidylcholine has been recently associated with atherosclerosis and increased risk of cardiovascular diseases (CVD) in rodents and humans. However, the molecular mechanisms of how TMAO induces atherosclerosis and CVD progression are still unclear. The present study tested whether TMAO induces NLRP3 inflammasome formation and activation and thereby contributes to endothelial injury initiating atherogenesis. Methods: Inflammasome formation and activation was determined by confocal microscopy, caspase-1 activity was measured by colorimetric assay, IL-1ß production was measured using ELISA, cell permeability was determined by microplate reader and ZO-1 expression was determined by western blot analysis and confocal microscopy. In in vivo experiments, TMAO was infused by osmotic pump implantation. Results: TMAO treatment significantly increased the colocalization of NLRP3 with Asc or NLRP3 with caspase-1, caspase-1 activity, IL-1ß production, cell permeability in carotid artery endothelial cells (CAECs) compared to control cells. Pretreatment with caspase-1 inhibitor, WEHD or Nlrp3 siRNA abolished the TMAO-induced inflammasome formation, activation and cell permeability in these cells. In addition, we explored the mechanisms by which TMAO activates NLRP3 inflammasomes. TMAO-induced the activation of NLRP3 inflammasomes was associated with both redox regulation and lysosomal dysfunction. In animal experiments, direct infusion of TMAO in mice with partially ligated carotid artery were found to have increased NLRP3 inflammasome formation and IL-1ß production in the intima of wild type mice. Conclusion: The formation and activation of NLRP3 inflammasomes by TMAO may be an important initiating mechanism to turn on the endothelial inflammatory response leading to endothelial dysfunction. [ABSTRACT FROM AUTHOR]

Background: Our previous study has detected a stem cell deficiency in the renal medulla in Dahl salt-sensitive (S) rats. This study determined whether infusion of valproic acid (VA), an agent known to stimulate the stem cell function, attenuated salt-sensitive hypertension in Dahl S rats. Methods: Uninephrectomized Dahl S rats were infused with vehicle or VA (50mg/kg/d) into the renal medulla and fed with a low (LS) or high salt diet (HS). Stem cell marker and number were analyzed by immunohistochemistry, Real-time RTPCR and Western blot. Sodium excretion and blood pressure were measured. Results: VA significantly increased the mRNA and protein levels of FGF2, a stem cell niche factor, and CD133, a stem cell marker. The number of CD133+ cells was significantly increased in the renal medulla in VA-treated rats. Meanwhile, high salt-induced increases in the mRNA level of proinflammatory factors interleukin-1β and interleukin-6 were blocked in VA-treated rats. Functionally, sodium excretion in response to the blood pressure increase and acute sodium loading was significantly enhanced, sodium retention attenuated, high salt-induced increase of blood pressure reduced in VA-treated rats. Conclusion: Activation of stem cell function by VA inhibits the activation of proinflammatory factors and attenuates salt-sensitive hypertension in Dahl S rats. [ABSTRACT FROM AUTHOR]

Background: Autophagy disorder contributes to dedifferentiation of arterial smooth muscle cells, but the mechanisms are poorly understood. Here, we sought to investigate the role of scaffolding adaptor p62/SQSTM1 (p62) in phenotype switching of mouse coronary arterial myocytes (CAMs) induced by CD38 gene deficiency or lysosomal dysfunction which blocks autophagic flux in the cells. Methods: Protein expression was measured by western blot analysis and immunofluorescent staining. Cell cycle and proliferation rate were analyzed by flow cytometry and MTS assay respectively. mRNA abundance was tested by qRT-PCR. Results: CD38 gene deficiency or bafilomycin A1 (baf), a selective lysosomal inhibitor treatment increased proliferation rate and vimentin expression in CAMs which was prevented by p62 gene silencing. Cell percentage in G2/M and G0/G1 phase was decreased and increased by CD38 deficiency or baf treatment, respectively which was accompanied by accrual of cyclin-dependent kinase 1 (CDK1) protein. Although free ubiquitin content was increased, the colocalization of it to CDK1 was markedly decreased in CD38-/- or baf treated CAMs. Furthermore, the changes in both cell cycle and CDK1 ubiquitinylation could be restored by p62 gene silencing. Conclusion: The results suggest in CD38-/- or baf treated CAMs, p62 accumulation promotes phenotype transition and proliferation by accelerating cell cycle progress through G2/M which might relate to the compromised ubiquitinylation and degradation of CDK1. [ABSTRACT FROM AUTHOR]

Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 ( NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis. [ABSTRACT FROM AUTHOR]

Recent studies indicate that inflammasomes serve as intracellular machinery to initiate classical cytokine-mediated inflammatory responses and play a crucial role in the pathogenesis of cardiovascular diseases. However, whether or not the activation of endothelial inflammasomes directly causes cell dysfunction or tissue injury without recruitment of inflammatory cells is unknown. We explored the role of endothelial cell inflammasome activation in mediating tight junction disruption, a hallmark event of endothelial barrier dysfunction leading to endothelial hyperpermeability in diabetes. We used confocal microscopy to study the formation and activation of NOD-like receptor family pyrin domain containing-3 (Nlrp3) inflammasomes and expression of tight junction proteins in coronary arteries of streptozotocin-treated diabetic wild type and Nlrp3 gene-deleted mice. We found that Nlrp3 ablation prevented inflammasome activation and tight junction disassembly in the coronary arterial endothelium of diabetic mice. Similarly, Nlrp3 gene silencing prevented high glucose-induced down-regulation of tight junction proteins in cultured mouse vascular endothelial cells (MVECs). The high glucose-induced tight junction disruption and consequent endothelial permeability were attributed to increased release of the high mobility group box protein-1 (HMGB1), which is dependent on enhanced Nlrp3 inflammasome activity. Mechanistically, reducing reactive oxygen species (ROS) production abolished high glucose-induced inflammasome activation, tight junction disruption, and endothelial hyperpermeability in MVECs. Collectively, the ROS-dependent activation of endothelial Nlrp3 inflammasomes by hyperglycemia may be an important initiating mechanism to cause endothelial dysfunction. These effects could contribute to the early onset of endothelial injury in diabetes. Key message: [ABSTRACT FROM AUTHOR]

The disruption in transportation of ox LDL-derived cholesterol and the subsequent lipid accumulation in macrophages are the hallmark events in atherogenesis. Our recent studies demonstrated that lysosomal Ca2+ messenger of nicotinic acid adenine dinucleotide phosphate ( NAADP), an enzymatic product of CD38 ADP-ribosylcyclase ( CD38), promoted lipid endocytic trafficking in human fibroblast cells. The current studies are designed to examine the functional role of CD38/ NAADP pathway in the regulation of lysosomal cholesterol efflux in atherosclerosis. Oil red O staining showed that ox LDL concentration-dependently increased lipid buildup in bone marrow-derived macrophages from both wild type and CD38−/−, but to a significant higher extent with CD38 gene deletion. Bodipy 493/503 fluorescence staining found that the deposited lipid in macrophages was mainly enclosed in lysosomal organelles and largely enhanced with the blockade of CD38/ NAADP pathway. Filipin staining and direct measurement of lysosome fraction further revealed that the free cholesterol constituted a major portion of the total cholesterol segregated in lysosomes. Moreover, in situ assay disclosed that both lysosomal lumen acidity and the acid lipase activity were reduced upon cholesterol buildup in lysosomes. In CD38−/− mice, treatment with Western diet (12 weeks) produced atherosclerotic damage in coronary artery with striking lysosomal cholesterol sequestration in macrophages. These data provide the first experimental evidence that the proper function of CD38/ NAADP pathway plays an essential role in promoting free cholesterol efflux from lysosomes and that a defection of this signalling leads to lysosomal cholesterol accumulation in macrophages and results in coronary atherosclerosis in CD38−/− mice. [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that local inflammatory mediators are importantly involved in the regulation of renal function. However, it remains unknown how such local inflammation is triggered intracellularly in the kidney. The present study was designed to characterize the inflammasome centered by Nlrp3 in the kidney and also test the effect of its activation in the renal medulla. Methods and Results: By immunohistochemistry analysis, we found that inflammasome components, Nlrp3, Asc and caspase-1, were ubiquitously distributed in different kidney areas. The caspase-1 activity and IL-1β production were particularly high in the renal outer medulla compared to other kidney regions. Further confocal microscopy and RT-PCR analysis showed that Nlrp3, Asc and caspase-1 were particularly enriched in the thick ascending limb of Henle's loop. In anesthetized mice, medullary infusion of Nlrp3 inflammasome activator, monosodium urate (MSU), induced significant decreases in sodium excretion and medullary blood flow without changes in mean arterial blood pressure and renal cortical blood flow. Caspase-1 inhibitor, Ac-YVAD-CMK and deletion of Nlrp3 or Asc gene abolished MSU-induced decreases in renal sodium excretion and MBF. Conclusion: Our results indicate that renal medullary Nlrp3 inflammasomes represent a new regulatory mechanism of renal MBF and sodium excretion which may not depend on classical inflammatory response. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that the inflammasome plays a critical role in the pathogenesis of vascular diseases. However, the pathological relevance of this inflammasome activation, particularly in vascular cells, remains largely unknown. Here, we investigated the role of endothelial (Nucleotide-binding Oligomerization Domain) NOD-like receptor family pyrin domain containing three (Nlrp3) inflammasomes in modulating inter-endothelial junction proteins, which are associated with endothelial barrier dysfunction, an early onset of obesity- associated endothelial injury. Our findings demonstrate that the activation of Nlrp3 inflammasome by visfatin markedly decreased the expression of inter-endothelial junction proteins including tight junction proteins ZO-1, ZO-2 and occludin, and adherens junction protein VE-cadherin in cultured mouse vascular endothelial (VE) cell monolayers. Such visfatin-induced down-regulation of junction proteins in endothelial cells was attributed to high mobility group box protein 1 (HMGB1) release derived from endothelial inflammasomedependent caspase-1 activity. Similarly, in the coronary arteries of wild-type mice, high-fat diet (HFD) treatment caused a down-regulation of inter-endothelial junction proteins ZO-1, ZO-2, occludin and VE-cadherin, which was accompanied with enhanced inflammasome activation and HMGB1 expression in the endothelium as well as transmigration of CD43+ T cells into the coronary arterial wall. In contrast, all these HFD-induced alterations in coronary arteries were prevented in mice with Nlrp3 gene deletion. Taken together, these data strongly suggest that the activation of endothelial Nlrp3 inflammasomes as a result of the increased actions of injurious adipokines such as visfatin produces HMGB1, which act in paracrine or autocrine fashion to disrupt inter-endothelial junctions and increase paracellular permeability of the endothelium contributing to the early onset of endothelial injury during metabolic disorders such as obesity or highfat/ cholesterol diet. [ABSTRACT FROM AUTHOR]

Background/Aims: The accumulation of free cholesterol in atherosclerotic lesions has been well documented in both animals and humans. In studying the relevance of free cholesterol buildup in atherosclerosis, contradictory results have been generated, indicating that free cholesterol produces both pro- and anti-atherosclerosis effects in macrophages. This inconsistency might stem from the examination of only select concentrations of free cholesterol. In the present study, we sought to investigate the implication of excess free cholesterol loading in the pathophysiology of atherosclerosis across a broad concentration range from (in µg/ml) 0 to 60. Methods: Macrophage viability was determined by measuring formazan formation and flow cytometry viable cell counting. The polarization of M1 and M2 macrophages was differentiated by FACS (Fluorescence-Activated Cell Sorting) assay. The secretion of IL-1β in macrophage culture medium was measured by ELISA kit. Macrophage apoptosis was detected by flow cytometry using a TUNEL kit. Results: Macrophage viability was increased at the treatment of lower concentrations of free cholesterol from (in µg/ml) 0 to 20, but gradually decreased at higher concentrations from 20 to 60. Lower free cholesterol loading induced anti-inflammatory M2 macrophage polarization. The activation of the PPARγ (Peroxisome Proliferator-Activated Receptor gamma) nuclear factor underscored the stimulation of this M2 phenotype. Nevertheless, higher levels of free cholesterol resulted in pro-inflammatory M1 activation. Moreover, with the application of higher free cholesterol concentrations, macrophage apoptosis and secretion of the inflammatory cytokine IL-1β increased significantly. Conclusion: These results for the first time demonstrate that free cholesterol could render concentration-dependent diversification effects on macrophage viability, polarization, apoptosis and inflammatory cytokine secretions, thereby reconciling the pros and cons of free cholesterol buildup in macrophages to the pathophysiology of atherosclerosis. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background/Aims: Recent studies have indicated that CD38 gene deficiency results in dedifferentiation or transdifferentiation of arterial smooth muscle cells upon atherogenic stimulations. However, the molecular mechanisms mediating this vascular smooth muscle (SMC) phenotypic switching remain unknown. Methods & Results: In the present study, we first characterized the phenotypic change in the primary cultures of coronary arterial myocytes (CAMs) from CD38-/- mice. It was shown that CD38 deficiency decreased the expression of contractile marker calponin, SM22α and α-SMA but increased the expression of SMC dedifferentiation marker, vimentin, which was accompanied by enhanced cell proliferation. This phenotypic change in CD38-/- CAMs was enhanced by 7-ketocholesterol (7-Ket), an atherogenic stimulus. We further found that the CD38 deficiency decreased the expression and activity of nuclear factor E2-related factor 2 (Nrf2), a basic leucine zipper (bZIP) transcription factor sensitive to redox regulation. Similar to CD38 deletion, Nrf2 gene silencing increased CAM dedifferentiation upon 7-Ket stimulation. In contrast, the overexpression of Nrf2 gene abolished 7-Ket-induced dedifferentiation in CD38-/- CAMs. Given the sensitivity of Nrf2 to oxidative stress, we determined the role of redox signaling in the regulation of Nrf2 expression and activity associated with CD38 effect in CAM phenotype changes. It was demonstrated that in CD38-/- CAMs, 7-Ket failed to stimulate the production of O2-., while in CD38+/+ CAMs 7-Ket induced marked O2-. production and enhancement of Nrf2 activity, which was substantially attenuated by NOX4 gene silencing. Finally, we demonstrated that 7-Ket-induced and NOX4-dependent O2-. production was inhibited by 8-Br-cADPR, an antagonist of cADPR or NED-19, an antagonist of NAADP as product of CD38 ADP-ribosylcyclase, which significantly inhibited the level of cytosolic Ca2+ and the activation of Nrf2 under 7-Ket. Conclusion: Taken together, these results suggest that CD38 activity is required for 7-Ket-induced Ca2+ and consequently O2-. production in CAMs, which increases Nrf2 activity to maintain their differentiated status. When CD38 gene expression and function are deficient, the Nrf2 activity is suppressed, thereby leading to phenotypic switching of CAMs. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pulmonary edema associated with increased vascular permeability is a severe complication of Staphylococcus aureus-induced sepsis and an important cause of human pathology and death. We investigated the role of the mammalian acid sphingomyelinase (Asm)/ceramide system in the development of lung edema caused by S. aureus. Our findings demonstrate that genetic deficiency or pharmacologic inhibition of Asm reduced lung edema in mice infected with S. aureus. The Asm/ceramide system triggered the formation of superoxide, resulting in degradation of tight junction proteins followed by lung edema. Treatment of infected mice with amitriptyline, a potent inhibitor of Asm, protected mice from lung edema caused by S. aureus, but did not reduce systemic bacterial numbers. In turn, treatment with antibiotics reduced bacterial numbers but did not protect mice from lung edema. In contrast, only the combination of antibiotics and amitriptyline inhibited both pulmonary edema and bacteremia protecting mice from lethal sepsis and lung dysfunction suggesting the combination of both drugs as novel treatment option for sepsis. Key messages: [ABSTRACT FROM AUTHOR]

Background: Proteinuria-induced epithelial-mesenchymal transition (EMT) plays an important role in progressive renal tubulointerstitial fibrosis in chronic renal disease. Stem cell therapy has been used for different diseases. Stem cell conditioned culture media (SCM) exhibits similar beneficial effects as stem cell therapy. The present study tested the hypothesis that SCM inhibits albumin-induced EMT in cultured renal tubular cells. Methods: Rat renal tubular cells were treated with/without albumin (20 µmg/ml) plus SCM or control cell media (CCM). EMT markers and inflammatory factors were measured by Western blot and fluorescent images. Results: Albumin induced EMT as shown by significant decreases in levels of epithelial marker E-cadherin, increases in mesenchymal markers fibroblast-specific protein 1 and α-smooth muscle actin, and elevations in collagen I. SCM inhibited all these changes. Meanwhile, albumin induced NF-κB translocation from cytosol into nucleus and that SCM blocked the nuclear translocation of NF-κB. Albumin also increased the levels of pro-inflammatory factor monocyte chemoattractant protein-1 (MCP)-1 by nearly 30 fold compared with control. SCM almost abolished albumin-induced increase of MCP-1. Conclusion: These results suggest that SCM attenuated albumin-induced EMT in renal tubular cells via inhibiting activation of inflammatory factors, which may serve as a new therapeutic approach for chronic kidney diseases. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Autophagy is of importance in the regulation of cell differentiation and senescence in podocytes. It is possible that derangement of autophagy under different pathological conditions activates or enhances Epithelial-to-Mesenchymal Transition (EMT) in podocytes, resulting in glomerular sclerosis. To test this hypothesis, the present study produced lysosome dysfunction by inhibition of the vacuolar H+-ATPase (V-ATPase) to test whether deficiency of autophagic flux leads to enhancement of EMT in podocytes. Methods and Results: By Western blot and confocal analysis, lysosome inhibition using a V-ATPase inhibitor or its siRNA was found to markedly decreases the epithelial markers (P-cadherin and ZO-1) and increases the mesenchymal markers (FSP-1 and α-SMA). This enhancement was accompanied by deficient autophagic flux, as demonstrated by marked increases in LC3B-II and p62/Sequestosome 1. However, inhibition of autophagosome formation using spaudin-1 significantly attenuated both enhancement of EMT and deficiency of autophagic flux. To explore the mechanisms by which deficient autophagic flux enhances EMT, we tested the role of accumulated p62 as a signal hub in this process. Neither the nuclear factor erythroid 2-related factor 2 (Nrf2) and nuclear kappa-light-chain-enhancer pathways of p62 contributed to enhanced EMT. However, inhibition of cyclin-dependent kinase 1 (CDK1) activity reduced the phosphorylation of p62 and enhanced EMT in podocytes similar to lysosome dysfunction. Conclusion: The lack of phosphorylated p62 leads to a faster exit from cell mitosis, enhanced EMT associated with lysosome dysfunction may be attributed to accumulation of p62 and associated reduction of p62 phosphorylation. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Adult stem cell deficiency has been implicated in the pathogenic mechanism for various diseases. Renal medullary dysfunction is one of the major mechanisms for the development of hypertension in Dahl salt-sensitive (S) rats. The present study first detected a stem cell deficiency in the renal medulla in Dahl S rats and then tested the hypothesis that transplantation of mesenchymal stem cells (MSCs) into the renal medulla improves salt-sensitive hypertension in Dahl S rats. Immunohistochemistry and flowcytometry analyses showed a significantly reduced number of stem cell marker CD133+ cells in the renal medulla from Dahl S rats compared with controls, suggesting a stem cell deficiency. Rat MSCs or control cells were transplanted into the renal medulla in uninephrectomized Dahl S rats, which were then treated with a low- or high-salt diet for 20 days. High-salt-induced sodium retention and hypertension was significantly attenuated in MSC-treated rats compared with control cell-treated rats. Meanwhile, high-salt-induced increases of proinflammatory factors, monocyte chemoattractant protein-1, and interleukin-1β, in the renal medulla were blocked by MSC treatment. Furthermore, immunostaining showed that high-salt-induced immune cell infiltration into the renal medulla was substantially inhibited by MSC treatment. These results suggested that stem cell defect in the renal medulla may contribute to the hypertension in Dahl S rats and that correction of this stem cell defect by MSCs attenuated hypertension in Dahl S rats through anti-inflammation. Key message: [ABSTRACT FROM AUTHOR]

Background/Aims: Hyperhomocysteinemia (hHcys) has been reported to initiate Nod-like receptor protein 3 (NLRP3) inflammasome formation and activation in podocytes, leading to glomerular dysfunction and sclerosis. However, it remains unknown whether Nlrp3 gene is critical for the formation and activation of inflammasomes in glomeruli of hHcys mice. Methods: Plasma homocysteine concentration was estimated utilizing HPLC, inflammasome formation and immunofluorescence expression from confocal microscopy, IL-1β production from ELISA. Results: Uninephrectomized Nlrp3 knockout (Nlrp3-/-) and wild type (Nlrp3+/+) and intra renal Nlrp3 shRNA-transfected wild type mice (Nlrp3 shRNA) were fed a folate free (FF) diet or normal chow (ND) for 4 weeks to produce hHcys. The plasma Hcys levels were significantly elevated in both Nlrp3-/- and Nlrp3+/+ mice fed a FF diet compared to ND fed mice. The FF diet significantly increased the colocalization of Nlrp3 with apoptosis-associated speck-like protein (ASC) or caspase-1, caspase-1 activity and IL-1β production in glomeruli of Nlrp3+/+, but not in Nlrp3-/- mice and local Nlrp3 shRNA transfected mice. Correspondingly, the glomerular damage index (GDI) and urinary protein excretion were significantly higher in Nlrp3+/+ mice compared to ND fed mice. However, the hHcys-induced increase in GDI and proteinuria were significantly lower in Nlrp3-/- and local Nlrp3 shRNA transfected mice than in Nlrp3+/+ mice. Immunocytochemical analysis showed that hHcys decreased expression of podocin and nephrin, but increased desmin expression in glomeruli of Nlrp3+/+ mice compared to Nlrp3-/- mice. Conclusion: Nlrp3 gene is an essential component of Nlrp3 inflammasomes and that targeting Nlrp3 may be important therapeutic strategy to prevent inflammasome activation and thereby protect podocytes and glomeruli from hHcys-induced injury. © 2014 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Recent studies have indicated a protective role of autophagy in regulating vascular smooth muscle cells homeostasis in atherogenesis, but the mechanisms controlling autophagy, particularly autophagy maturation, are poorly understood. Here, we investigated whether acid sphingomyelinase (ASM)-regulated lysosome function is involved in autophagy maturation in coronary arterial smooth muscle cells (CASMCs) in the pathogenesis of atherosclerosis. In coronary arterial wall of ASM-deficient ( Smpd1) mice on Western diet, there were high expression levels of both LC3B, a robust marker of autophagosomes (APs), and p62, a selective autophagy substrate, compared with those in wild-type ( Smpd1) mice. By Western blotting and flow cytometry, atherogenic stimulation of Smpd1 CASMCs with 7-ketocholesterol was found to significantly enhance LC3B expression and increase the content of both APs and autophagolysosomes (APLs). In Smpd1 CASMCs, such 7-ketocholesterol-induced increases in LC3B and p62 expression and APs were further augmented, but APLs formation was abolished. Analysis of fluorescence resonance energy transfer between fluorescence-labeled LC3B and Lamp1 (lysosome marker) showed that 7-ketocholesterol markedly induced fusion of APs with lysosomes in Smpd1 CASMCs, which was abolished in Smpd1 CASMCs. Moreover, 7-ketocholesterol-induced expression of cell dedifferentiation marker vimentin and proliferation was enhanced in Smpd1 CASMCs compared with those in Smpd1 CASMCs. Lastly, overexpression of ASM further increased APLs formation in Smpd1 CASMCs and restored APLs formation in Smpd1 CASMCs indicating that increased ASM expression is highly correlated with enhanced APLs formation. Taken together, our data suggest that the control of lysosome trafficking and fusion by ASM is essential to a normal autophagic flux in CASMCs, which implicates that the deficiency of ASM-mediated regulation of autophagy maturation may result in imbalance of arterial smooth muscle cell homeostasis and thus serve as an important atherogenic mechanism in coronary arteries. Key messages: • Acid sphingomyelinase (ASM) controls autophagy maturation in smooth muscle cells. • ASM maintains smooth muscle cell homeostasis and its contractile phenotype. • ASM plays a protective role in smooth muscle dysfunction and atherosclerosis. [ABSTRACT FROM AUTHOR]

Aim Autophagic flux is an important process during autophagy maturation in smooth muscle cells. However, the molecular mechanisms underlying autophagic flux in these cells are largely unknown. Here, we revealed a previously undefined role of CD38, an enzyme that metabolizes NADP+ into NAADP, in the regulation of autophagic flux in coronary arterial myocytes (CAMs). Methods and results In vivo CD38 gene knockout mice (CD38−/−) fed the high-fat Western diet showed increased accumulation of autophagosomes in coronary arterial media compared with that in wild-type (CD38+/+) mice, suggesting that CD38 gene deletion results in a defective autophagic process in CAMs of coronary arteries. In primary cultured CAMs, CD38 gene deletion markedly enhanced 7-ketocholesterol (7-Ket, an atherogenic stimulus and autophagy inducer)-induced accumulation of autophagosomes and increased expression of an autophagic marker, LC3B. However, no difference in autophagosome formation was observed between CD38+/+ and CD38−/− CAMs when autophagic flux was blocked, which indicates that CD38 regulates autophagic flux rather than induction of autophagosome formation. Further, 7-Ket-induced formation of autophagolysosomes was markedly attenuated in CD38−/− CAMs compared with CD38+/+ CAMs. Mechanistically, CD38 gene deletion markedly inhibited 7-Ket-induced dynein activation and autophagosome trafficking, which were associated with attenuated lysosomal Ca2+ release. Importantly, coronary arterial smooth muscle from CD38−/− mice fed the Western diet exhibited phenotypic changes towards a more dedifferentiated state with abnormal extracellular matrix metabolism. Conclusion Taken together, these results suggest that CD38 plays a critical role in autophagosome trafficking and fusion with lysosomes, thus controlling autophagic flux in CAMs under atherogenic stimulation. [ABSTRACT FROM PUBLISHER]

Although Nlrp3 inflammasome activation in macrophages has been shown to be critical for the development of atherosclerosis upon atherogenic stimuli, it remains unknown whether activated Nlrp3 inflammasomes by other non-atherogenic stimuli induce alterations in macrophages that may contribute in the concert with other factors to atherogenesis. Thus, the present study tested the hypothesis that activation of Nlrp3 inflammasomes by ATP, which is a classical non-lipid danger stimulus, enhances the migration of macrophage and increases lipids deposition in macrophages accelerating foam cell formation. We first demonstrated that extracellular ATP (2.5 mM) markedly increased the formation and activation of Nlrp3 inflammasomes in bone marrow macrophages (BMMs) from wild type (Asc+/+) mice resulting in activation of caspase-1 and IL-1β production. In these Asc+/+ macrophages, such stimulation of inflammasomes by non-lipid ATP was similar to those induced by atherogenic stimuli such as cholesterol crystals or 7-ketocholesterol. Both non-lipid and lipid forms of stimuli induced formation and activation of Nlrp3 inflammasomes, which were prevented by Asc gene deletion. Interestingly, Asc+/+ BMMs had dramatic lipids accumulation after stimulation with ATP. Further, we demonstrated that large amount of cholesterol was accumulated in lysosomes of Asc+/+ BMMs when inflammasomes were activated by ATP. Such intracellular and lysosomal lipids deposition was not observed in Asc−/− BMMs and also prevented by caspase-1 inhibitor WEHD. In addition, in vitro and in vivo experiments revealed that migration of Asc+/+ BMMs increased due to stimulation of Nlrp3 inflammasomes, which was markedly attenuated in Asc−/− BMMs. Together, these results suggest that activation of Nlrp3 inflammasomes remarkably increases the susceptibility of macrophages to lipid deposition and their migration ability. Such novel action of inflammasomes may facilitate entry or retention of macrophages into the arterial wall, where they form foam cells and ultimately induce atherosclerosis. [ABSTRACT FROM AUTHOR]

BACKGROUND In response to high salt intake, transcription factor hypoxia-inducible factor (HIF) 1α activates many antihypertensive genes, such as heme oxygenase 1 (HO-1) 1 and cyclooxygenase 2 (COX-2) in the renal medulla, which is an important molecular adaptation to promote extra sodium excretion. We recently showed that high salt inhibited the expression of HIF prolyl-hydroxylase 2 (PHD2), an enzyme that promotes the degradation of HIF-1α, thereby upregulating HIF-1α, and that high salt–induced inhibition in PHD2 and subsequent activation of HIF-1α in the renal medulla was blunted in Dahl salt-sensitive hypertensive rats. This study tested the hypothesis that silencing the PHD2 gene to increase HIF-1α levels in the renal medulla attenuates salt-sensitive hypertension in Dahl S rats. METHODS PHD2 short hairpin RNA (shRNA) plasmids were transfected into the renal medulla in uninephrectomized Dahl S rats. Renal function and blood pressure were then measured. RESULTS PHD2 shRNA reduced PHD2 levels by >60% and significantly increased HIF-1α protein levels and the expression of HIF-1α target genes HO-1 and COX-2 by >3-fold in the renal medulla. Functionally, pressure natriuresis was remarkably enhanced, urinary sodium excretion was doubled after acute intravenous sodium loading, and chronic high salt-induced sodium retention was remarkably decreased, and as a result, salt-sensitive hypertension was significantly attenuated in PHD2 shRNA rats compared with control rats. CONCLUSIONS Impaired PHD2 response to high salt intake in the renal medulla may represent a novel mechanism for hypertension in Dahl S rats, and inhibition of PHD2 in the renal medulla could be a therapeutic approach for salt-sensitive hypertension. [ABSTRACT FROM AUTHOR]

Podocytes are highly differentiated glomerular epithelial cells that contribute to the glomerular barrier function of kidney. A role for autophagy has been proposed in maintenance of their cellular integrity, but the mechanisms controlling autophagy in podocytes are not clear. The present study tested whether CD38-mediated regulation of lysosome function contributes to autophagic flux or autophagy maturation in podocytes. Podocytes were found to exhibit a high constitutive level of LC3-II, a robust marker of autophagosomes ( APs), suggesting a high basal level of autophagic activity. Treatment with the m TOR inhibitor, rapamycin, increased LC3-II and the content of both APs detected by Cyto-ID Green staining and autophagolysosomes ( APLs) measured by acridine orange staining and colocalization of LC3 and Lamp1. Lysosome function inhibitor bafilomycin A1 increased APs, but decreased APLs content under both basal and rapamycin-induced conditions. Inhibition of CD38 activity by nicotinamide or silencing of CD38 gene produced the similar effects to that bafilomycin A1 did in podocytes. To explore the possibility that CD38 may control podocyte autophagy through its regulation of lysosome function, the fusion of APs with lysosomes in living podocytes was observed by co-transfection of GFP- LC3B and RFP-Lamp1 expression vectors. A colocalization of GFP- LC3B and RFP-Lamp1 upon stimulation of rapamycin became obvious in transfected podocytes, which could be substantially blocked by nicotinamide, CD38 sh RNA, and bafilomycin. Moreover, blockade of the CD38-mediated regulation by PPADS completely abolished rapamycin-induced fusion of APs with lysosomes. These results indicate that CD38 importantly control lysosomal function and influence autophagy at the maturation step in podocytes. [ABSTRACT FROM AUTHOR]