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1.
Int J Biol Sci ; 20(8): 2943-2964, 2024.
Article in English | MEDLINE | ID: mdl-38904030

ABSTRACT

Background: Shear stress-induced Dickkopf-1 (DKK1) secretion by endothelial cells (ECs) promotes EC dysfunction and accelerates atherosclerosis (AS). However, the paracrine role of endothelial DKK1 in modulating adjacent smooth muscle cells (SMCs) in atherosclerosis remains unclear. This study investigated the role of EC-secreted DKK1 in SMC-derived foam cell formation under shear stress, in vitro and in vivo. Methods: Parallel-plate co-culture flow system was used to explore the cellular communication between ECs and SMCs under shear stress in vitro. Endothelium-specific knockout of DKK1 (DKK1ECKO/APOE-/-) and endothelium-specific overexpression of DKK1 (DKK1ECTg) mice were constructed to investigate the role of endothelial DKK1 in atherosclerosis and SMC-derived foam cell formation in vivo. RNA sequencing (RNA-seq) was used to identify the downstream targets of DKK1. Reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot, coimmunoprecipitation (Co-IP) assays and chromatin immunoprecipitation (ChIP) experiments were conducted to explore the underlying regulatory mechanisms. Results: DKK1 is transcriptionally upregulated in ECs under conditions of low shear stress, but not in co-cultured SMCs. However, DKK1 protein in co-cultured SMCs is increased via uptake of low shear stress-induced endothelial DKK1, thereby promoting lipid uptake and foam cell formation in co-cultured SMCs via the post-translational upregulation of scavenger receptor-A (SR-A) verified in parallel-plate co-culture flow system, DKK1ECKO and DKK1ECTg mice. RNA sequencing revealed that DKK1-induced SR-A upregulation in SMCs is dependent on Ubiquitin-specific Protease 53 (USP53), which bound to SR-A via its USP domain and cysteine at position 41, exerting deubiquitination to maintain the stability of the SR-A protein by removing the K48 ubiquitin chain and preventing proteasomal pathway degradation, thereby mediating the effect of DKK1 on lipid uptake in SMCs. Moreover, DKK1 regulates the transcription of USP53 by facilitating the binding of transcription factor CREB to the USP53 promoter. SMC-specific overexpression of USP53 via adeno-associated virus serotype 2 vectors in DKK1ECKO/APOE-/- mice reversed the alleviation of atherosclerotic plaque burden, SR-A expression and lipid accumulation in SMCs within plaques resulting from DKK1 deficiency. Conclusions: Our findings demonstrate that, endothelial DKK1, induced by pathological low shear stress, acts as an intercellular mediator, promoted the foam cell formation of SMCs. These results suggest that targeted intervention with endothelial DKK1 may confer beneficial effects on atherosclerosis.


Subject(s)
Atherosclerosis , Foam Cells , Intercellular Signaling Peptides and Proteins , Myocytes, Smooth Muscle , Animals , Atherosclerosis/metabolism , Mice , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Foam Cells/metabolism , Myocytes, Smooth Muscle/metabolism , Endothelial Cells/metabolism , Humans , Ubiquitination , Male , Coculture Techniques , Mice, Knockout , Ubiquitin-Specific Proteases/metabolism , Ubiquitin-Specific Proteases/genetics , Mice, Inbred C57BL
2.
Metabolism ; 148: 155690, 2023 Nov.
Article in English | MEDLINE | ID: mdl-37717724

ABSTRACT

BACKGROUND: The liver regulates metabolic balance during fasting-feeding cycle. Hepatic adaptation to fasting is precisely modulated on multiple levels. Tumor necrosis factor-α-induced protein 8-like 2 (TIPE2) is a negative regulator of immunity that reduces several liver pathologies, but its physiological roles in hepatic metabolism are largely unknown. METHODS: TIPE2 expression was examined in mouse liver during fasting-feeding cycle. TIPE2-knockout mice, liver-specific TIPE2-knockout mice, liver-specific TIPE2-overexpressed mice were examined for fasting blood glucose and pyruvate tolerance test. Primary hepatocytes or liver tissues from these mice were evaluated for glucose production, lipid accumulation, gene expression and regulatory pathways. TIPE2 interaction with Raf-1 and TIPE2 transcription regulated by PPAR-α were examined using gene overexpression or knockdown, co-immunoprecipitation, western blot, luciferase reporter assay and DNA-protein binding assay. RESULTS: TIPE2 expression was upregulated in fasted mouse liver and starved hepatocytes, which was positively correlated with gluconeogenic genes. Liver-specific TIPE2 deficiency impaired blood glucose homeostasis and gluconeogenic capacity in mice upon fasting, while liver-specific TIPE2 overexpression elevated fasting blood glucose and hepatic gluconeogenesis in mice. In primary hepatocytes upon starvation, TIPE2 interacted with Raf-1 to accelerate its ubiquitination and degradation, resulting in ERK deactivation and FOXO1 maintenance to sustain gluconeogenesis. During prolonged fasting, hepatic TIPE2 deficiency caused aberrant activation of ERK-mTORC1 axis that increased hepatic lipid accumulation via lipogenesis. In hepatocytes upon starvation, PPAR-α bound with TIPE2 promoter and triggered its transcriptional expression. CONCLUSIONS: Hepatocyte TIPE2 is a PPAR-α-induced Raf-1 inactivator that sustains hepatic gluconeogenesis and prevents excessive hepatic lipid accumulation, playing beneficial roles in hepatocyte adaptation to fasting.

3.
FASEB J ; 37(8): e23048, 2023 08.
Article in English | MEDLINE | ID: mdl-37389895

ABSTRACT

Vascular smooth muscle cells (VSMCs) are considered to be a crucial source of foam cells in atherosclerosis due to their low expression level of cholesterol exporter ATP-binding cassette transporter A1 (ABCA1) intrinsically. While the definite regulatory mechanisms are complicated and have not yet been fully elucidated, we previously reported that Dickkopf-1 (DKK1) mediates endothelial cell (EC) dysfunction, thereby aggravating atherosclerosis. However, the role of smooth muscle cell (SMC) DKK1 in atherosclerosis and foam cell formation remains unknown. In this study, we established SMC-specific DKK1-knockout (DKK1SMKO ) mice by crossbreeding DKK1flox/flox mice with TAGLN-Cre mice. Then, DKK1SMKO mice were crossed with APOE-/- mice to generate DKK1SMKO /APOE-/- mice, which exhibited milder atherosclerotic burden and fewer SMC foam cells. In vitro loss- and gain-of-function studies of DKK1 in primary human aortic smooth muscle cells (HASMCs) have proven that DKK1 prevented oxidized lipid-induced ABCA1 upregulation and cholesterol efflux and promoted SMC foam cell formation. Mechanistically, RNA-sequencing (RNA-seq) analysis of HASMCs as well as chromatin immunoprecipitation (ChIP) experiments showed that DKK1 mediates the binding of transcription factor CCAAT/enhancer-binding protein delta (C/EBPδ) to the promoter of cytochrome P450 epoxygenase 4A11 (CYP4A11) to regulate its expression. In addition, CYP4A11 as well as its metabolite 20-HETE-promoted activation of transcription factor sterol regulatory element-binding protein 2 (SREBP2) mediated the DKK1 regulation of ABCA1 in SMC. Furthermore, HET0016, the antagonist of CYP4A11, has also shown an alleviating effect on atherosclerosis. In conclusion, our results demonstrate that DKK1 promotes SMC foam cell formation during atherosclerosis via a reduction in CYP4A11-20-HETE/SREBP2-mediated ABCA1 expression.


Subject(s)
Atherosclerosis , Foam Cells , Humans , Animals , Mice , Muscle, Smooth, Vascular , Cytochrome P-450 Enzyme System , Transcription Factors , Atherosclerosis/genetics , Apolipoproteins E/genetics , Cytochrome P-450 CYP4A , ATP Binding Cassette Transporter 1/genetics
4.
Exp Biol Med (Maywood) ; 248(2): 117-129, 2023 01.
Article in English | MEDLINE | ID: mdl-36426712

ABSTRACT

Immune-mediated hepatitis is marked by liver inflammation characterized by immune cell infiltration, chemokine/cytokine production, and hepatocyte injury. C-X3C motif receptor 1 (CX3CR1), as the receptor of chemokine C-X3C motif ligand 1 (CX3CL1)/fractalkine, is mainly expressed on immune cells including monocytes and T cells. Previous studies have shown that CX3CR1 protects against liver fibrosis, but the exact role of CX3CL1/CX3CR1 in acute immune-mediated hepatitis remains unknown. Here, we investigate the role of the CX3CL1/CX3CR1 axis in immune-mediated hepatitis using concanavalin A (ConA)-induced liver injury model in CX3CR1-deficient (Cx3cr1-/-) mice. We observed that Cx3cr1-/- mice had severe liver injury and increased pro-inflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interferon-gamma [IFN-γ], interleukin-1 beta [IL-1ß], and IL-6) in serum and liver compared to wild-type (Cx3cr1+/+) mice after ConA injection. The deficiency of CX3CR1 did not affect ConA-induced immune cell infiltration in liver but led to elevated production of TNF-α in macrophages as well as IFN-γ in T cells after ConA treatment. On the contrary, exogenous CX3CL1 attenuated ConA-induced cytokine production in wild type, but not CX3CR1-deficient macrophages and T cells. Furthermore, in vitro results showed that CX3CR1 deficiency promoted the pro-inflammatory cytokine expression by increasing the phosphorylation of nuclear factor kappa B (NF-κB) p65 (p-NF-κB p65). Finally, pre-treatment of p-NF-κB p65 inhibitor, resveratrol, attenuated ConA-induced liver injury and inflammatory responses, especially in Cx3cr1-/- mice. In conclusion, our data show that the deficiency of CX3CR1 promotes pro-inflammatory cytokine production in macrophages and T cells by enhancing the phosphorylation of NF-κB p65, which exacerbates liver injury in ConA-induced hepatitis.


Subject(s)
Chemical and Drug Induced Liver Injury, Chronic , Hepatitis , Mice , Animals , NF-kappa B/metabolism , Tumor Necrosis Factor-alpha/metabolism , T-Lymphocytes/metabolism , Cytokines/metabolism , Hepatitis/pathology , Macrophages/metabolism , Interferon-gamma/metabolism , Mice, Inbred C57BL , CX3C Chemokine Receptor 1
5.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-957552

ABSTRACT

Objective:To evaluate the effect of high-concentration hydrogen inhalation on sepsis-associated encephalopathy (SAE) in mice.Methods:Healthy male ICR mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=50 each) using the random number table method: sham operation group (Sham group), SAE group, sham operation plus high-concentration hydrogen group (Sham+ H 2 group), and SAE plus high-concentration hydrogen group (SAE+ H 2 group). SAE model was prepared by cecal ligation and puncture (CLP) in anesthetized animals.At 1 and 6 h after operation, Sham+ H 2 and SAE+ H 2 groups inhaled the mixture of hydrogen and oxygen (67% hydrogen-33% oxygen) for 1 h, and Sham and SAE groups inhaled the mixture of nitrogen and oxygen (67% nitrogen-33% oxygen) for 1 h. The postoperative 7-day survival rate was recorded.Cognitive function was assessed by Y maze test at days 3, 5 and 7 after operation.The mice were sacrificed at 24 h after operation, and hippocampal tissues were obtained for microscopic examination of the pathological changes of neurons in hippocampal CA1 region (with a light microscope) and for determination of normal neuron count, contents of tumor necrosis factor-ɑ (TNF-α) and high mobility group box-1 (HMGB1) (by enzyme-linked immunosorbent assay), mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry) and content of mitochondrial ATP (by fluorescein-fluorescent enzyme luminescence method). Results:Compared with Sham group, the 7-day survival rate after operation, percentage of spontaneous alternation at each time point after operation, and the number of normal neurons were significantly decreased, the contents of TNF-ɑ and HMGB1 were increased, and the contents of ATP and MMP were decreased in SAE and SAE+ H 2 groups ( P<0.05), and no significant change was found in Sham+ H 2 group ( P>0.05). Compared with SAE group, the 7-day survival rate after operation, percentage of spontaneous alternation at each time point after operation, and the number of normal neurons were significantly increased, the contents of TNF-ɑ and HMGB1 were decreased, and the contents of ATP and MMP were increased in SAE+ H 2 group ( P<0.05). Conclusions:High-concentration hydrogen inhalation can reduce SAE, and the mechanism may be related to reduction of hippocampal inflammatory responses and improvement in mitochondrial function in mice.

6.
Front Cardiovasc Med ; 8: 731315, 2021.
Article in English | MEDLINE | ID: mdl-34651027

ABSTRACT

Rationale: Previous studies have indicated an important role for complement in atherosclerosis, a lipid-driven chronic inflammatory disease associated to oxidative stress in the vessel wall. However, it remains unclear how complement is activated in the process of atherogenesis. An accepted general model for complement activation in the context of ischemia reperfusion injury is that ischemia induces the exposure of neoepitopes that are recognized by natural self-reactive IgM antibodies, and that in turn activate complement. Objective: We investigated whether a similar phenomenon may be involved in the pathogenesis of atherosclerosis, and whether interfering with this activation event, together with inhibition of subsequent amplification of the cascade at the C3 activation step, can provide protection against atherogenesis. Methods and Results: We utilized C2scFv-Crry, a novel construct consisting of a single chain antibody (scFv) linked to Crry, a complement inhibitor that functions at C3 activation. The scFv moiety was derived from C2 IgM mAb that specifically recognizes phospholipid neoepitopes known to be expressed after ischemia. C2scFv-Crry targeted to the atherosclerotic plaque of Apoe -/- mice, demonstrating expression of the C2 neoepitope. C2scFv-Crry administered twice per week significantly attenuated atherosclerotic plaque in the aorta and aortic root of Apoe -/- mice fed with a high-fat diet (HFD) for either 2 or 4 months, and treatment reduced C3 deposition and membrane attack complex formation as compared to vehicle treated mice. C2scFv-Crry also inhibited the uptake of oxidized low-density-lipoprotein (oxLDL) by peritoneal macrophages, which has been shown to play a role in pathogenesis, and C2scFv-Crry-treated mice had decreased lipid content in the lesion with reduced oxLDL levels in serum compared to vehicle-treated mice. Furthermore, C2scFv-Crry reduced the deposition of endogenous total IgM in the plaque, although it did not alter serum IgM levels, further indicating a role for natural IgM in initiating complement activation. Conclusion: Neoepitope targeted complement inhibitors represent a novel therapeutic approach for atherosclerosis.

7.
Foods ; 10(6)2021 Jun 17.
Article in English | MEDLINE | ID: mdl-34204441

ABSTRACT

Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea. Our previous report showed that induced hormesis was a critical determinant for the promotion of a healthy lifespan in Caenorhabditis elegans. In the present study, we investigated the anti-aging effects of the main active ingredients in green tea. We found that galloylated catechins (EGCG and epicatechin gallate) could extend the lifespan of C. elegans, while their metabolites (gallic acid, epicatechin, and epigallocatechin) could not. Interestingly, the combination with theanine, not caffeine, could alleviate the adverse effects induced by high-dose EGCG, including the promotion of lifespan and locomotor ability. This was due to the attenuation of the excess production of reactive oxygen species and the activation of DAF-16. These findings will facilitate further studies on the health benefits of tea active components and their interactions.

8.
J Clin Invest ; 131(9)2021 05 03.
Article in English | MEDLINE | ID: mdl-33724957

ABSTRACT

Cholangiopathies caused by biliary epithelial cell (BEC) injury represent a leading cause of liver failure. No effective pharmacologic therapies exist, and the underlying mechanisms remain obscure. We aimed to explore the mechanisms of bile duct repair after targeted BEC injury. Injection of intermedilysin into BEC-specific human CD59 (hCD59) transgenic mice induced acute and specific BEC death, representing a model to study the early signals that drive bile duct repair. Acute BEC injury induced cholestasis followed by CCR2+ monocyte recruitment and BEC proliferation. Using microdissection and next-generation RNA-Seq, we identified 5 genes, including Mapk8ip2, Cdkn1a, Itgb6, Rgs4, and Ccl2, that were most upregulated in proliferating BECs after acute injury. Immunohistochemical analyses confirmed robust upregulation of integrin αvß6 (ITGß6) expression in this BEC injury model, after bile duct ligation, and in patients with chronic cholangiopathies. Deletion of the Itgb6 gene attenuated BEC proliferation after acute bile duct injury. Macrophage depletion or Ccr2 deficiency impaired ITGß6 expression and BEC proliferation. In vitro experiments revealed that bile acid-activated monocytes promoted BEC proliferation through ITGß6. Our data suggest that BEC injury induces cholestasis, monocyte recruitment, and induction of ITGß6, which work together to promote BEC proliferation and therefore represent potential therapeutic targets for cholangiopathies.


Subject(s)
Antigens, Neoplasm/biosynthesis , Bile Acids and Salts/metabolism , Biliary Tract/metabolism , Cell Proliferation , Epithelial Cells/metabolism , Integrins/biosynthesis , Macrophage Activation , Macrophages/metabolism , Up-Regulation , Animals , Antigens, Neoplasm/genetics , Bile Acids and Salts/genetics , Female , Humans , Integrins/genetics , Male , Mice , Mice, Transgenic , RNA-Seq
9.
Theranostics ; 10(16): 7163-7177, 2020.
Article in English | MEDLINE | ID: mdl-32641985

ABSTRACT

Rationale: Kupffer cells (KCs) play a crucial role in liver immune homeostasis through interacting with other immune cells and liver sinusoidal endothelial cells (LSECs). However, how KCs exactly interact with these cells for maintaining the homeostasis still require the further investigation. CXCL10 is a chemokine that has been implicated in chemoattraction of monocytes, T cells, NK cells, and dendritic cells, and promotion of T cell adhesion to endothelial cells. Although CXCL10 is also known to participate in the pathogenesis of hepatic inflammation, the degree to which it is functionally involved in the crosstalk between immune cells and regulation of immune response is still unclear. Methods: To dynamically investigate the function of KCs, we used our recently developed rapid cell ablation model, intermedilysin (ILY)/human CD59 (hCD59)-mediated cell ablation tool, to selectively ablate KC pool under normal condition or concanavalin A (Con A)- induced hepatitis. At certain time points after KCs ablation, we performed flow cytometry to monitor the amount of hepatic infiltrating immune cells. mRNA array was used to detect the change of hepatic cytokines and chemokines levels. Cytokines and chemokines in the serum were further measured by LEGENDplexTM mouse proinflammatory chemokine panel and inflammation panel. Evans blue staining and transmission electron microscopy were used to investigate the interaction between KCs and LSECs in steady condition. CXCL10 neutralizing antibody and CXCL10 deficient mouse were used to study the role of CXCL10 in immune cell migration and pathogenesis of Con A-induced hepatitis. Results: At steady state, elimination of KCs results in a reduction of hepatic infiltrating monocytes, T, B, and NK cells and a list of cytokines and chemokines at transcriptional level. In the meantime, the depletion of KCs resulted in increased sinusoidal vascular permeability. In the pathological condition, the KCs elimination rescues Con A-induced acute hepatitis through suppressing proinflammatory immune responses by down-regulation of hepatitis-associated cytokines/chemokines in serum such as CXCL10, and recruitment of infiltrating immune cells (monocytes, T, B, and NK cells). We further documented that deficiency or blockade of CXCL10 attenuated the development of Con A-induced hepatitis associated with reduction of the infiltrating monocytes, especially inflammatory Ly6Chi monocytes. Conclusions: This study supports the notion that KCs actively interact with immune cells and LSECs for maintaining immune response and liver homeostasis. Our data indicate that the interplay between KCs and infiltrated monocytes via CXCL10 contribute to Con A-induced hepatitis.


Subject(s)
Chemokine CXCL10/metabolism , Hepatitis C/immunology , Hepatitis/immunology , Kupffer Cells/immunology , T-Lymphocytes/immunology , Animals , Capillary Permeability/immunology , Cell Communication/immunology , Chemokine CXCL10/analysis , Chemokine CXCL10/genetics , Concanavalin A/administration & dosage , Concanavalin A/immunology , Disease Models, Animal , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Endothelium, Vascular/pathology , Hepatitis/pathology , Hepatitis C/pathology , Hepatitis C/surgery , Hepatitis C/virology , Humans , Kupffer Cells/metabolism , Liver/blood supply , Liver/immunology , Liver/pathology , Liver/surgery , Liver Cirrhosis/immunology , Liver Cirrhosis/pathology , Liver Cirrhosis/surgery , Liver Cirrhosis/virology , Liver Transplantation , Mice , Mice, Knockout , Microvessels/cytology , Microvessels/pathology
10.
Nat Commun ; 11(1): 2280, 2020 05 08.
Article in English | MEDLINE | ID: mdl-32385245

ABSTRACT

Renal macrophages (RMs) participate in tissue homeostasis, inflammation and repair. RMs consist of embryo-derived (EMRMs) and bone marrow-derived RMs (BMRMs), but the fate, dynamics, replenishment, functions and metabolic states of these two RM populations remain unclear. Here we investigate and characterize RMs at different ages by conditionally labeling and ablating RMs populations in several transgenic lines. We find that RMs expand and mature in parallel with renal growth after birth, and are mainly derived from fetal liver monocytes before birth, but self-maintain through adulthood with contribution from peripheral monocytes. Moreover, after the RMs niche is emptied, peripheral monocytes rapidly differentiate into BMRMs, with the CX3CR1/CX3CL1 signaling axis being essential for the maintenance and regeneration of both EMRMs and BMRMs. Lastly, we show that EMRMs have a higher capacity for scavenging immune complex, and are more sensitive to immune challenge than BMRMs, with this difference associated with their distinct glycolytic capacities.


Subject(s)
Bone Marrow Cells/cytology , Cell Lineage , Kidney/embryology , Macrophages/cytology , Animals , CX3C Chemokine Receptor 1/metabolism , Chemokine CX3CL1/blood , Chemokine CX3CL1/metabolism , Female , Fetus/cytology , Liver/embryology , Male , Mice , Monocytes/cytology
11.
ACS Appl Bio Mater ; 3(5): 3123-3136, 2020 May 18.
Article in English | MEDLINE | ID: mdl-35025356

ABSTRACT

The controllable release is necessary for ideal drug delivery technologies. Because of their high specific surface area and high porosity, titanium dioxide nanotubes (TNTs) have been widely used as drug carriers in medical devices. By loading copper as the catalyst, nitric oxide (NO) generation was facilitated by catalyzing the decomposition of renewable endogenous NO donors in vivo. Herein, the long-term controllable release profile of NO is highlighted owing to the multilayer polydopamine (PDA) cap structure. Different layers of PDA are used to adjust the NO release behavior, and the results show that three layers of PDA can not only effectively prevent the burst release of NO but also maintain long-term stable release of copper ion and NO. The bioactivity of the NO generated from three-layer PDA-modified copper-loaded TNTs (PDA-3L-NTCu2) and unmodified copper-loaded TNTs (NTCu2) are verified by our work, indicating effective inhibition of platelet activation, thrombosis, inflammation, and intimal hyperplasia. Importantly, the PDA-3L-NTCu2 show selectively promote the growth of endothelial cells in vitro and outstanding re-endothelialization for 4 weeks in vivo, as compared to NTCu2, TNTs, and 316L stain steel. This study suggests that copper-loaded with PDA modification helps us achieve controlled long-term stable local NO release with well-retained bioactivity and enhanced re-endothelialization.

12.
Arterioscler Thromb Vasc Biol ; 39(9): 1762-1775, 2019 09.
Article in English | MEDLINE | ID: mdl-31315440

ABSTRACT

OBJECTIVE: Atherosclerotic cardiovascular disease (ASCVD) is an increasing cause of morbidity and mortality in people with HIV since the introduction of combination antiretroviral therapy. Despite recent advances in our understanding of HIV ASCVD, controversy still exists on whether this increased risk of ASCVD is due to chronic HIV infection or other risk factors. Mounting biomarker studies indicate a role of monocyte/macrophage activation in HIV ASCVD; however, little is known about the mechanisms through which HIV infection mediates monocyte/macrophage activation in such a way as to engender accelerated atherogenesis. Here, we experimentally investigated whether HIV expression is sufficient to accelerate atherosclerosis and evaluated the role of caspase-1 activation in monocytes/macrophages in HIV ASCVD. Approach and Results: We crossed a well-characterized HIV mouse model, Tg26 mice, which transgenically expresses HIV-1, with ApoE-/- mice to promote atherogenic conditions (Tg26+/-/ApoE-/-). Tg26+/-/ApoE-/- have accelerated atherosclerosis with increased caspase-1 pathway activation in inflammatory monocytes and atherosclerotic vasculature compared with ApoE-/-. Using a well-characterized cohort of people with HIV and tissue-banked aortic plaques, we documented that serum IL (interleukin)-18 was higher in people with HIV compared with non-HIV-infected controls, and in patients with plaques, IL-18 levels correlated with monocyte/macrophage activation markers and noncalcified inflammatory plaques. In autopsy-derived aortic plaques, caspase-1+ cells and CD (clusters of differentiation) 163+ macrophages correlated. CONCLUSIONS: These data demonstrate that expression of HIV is sufficient to accelerate atherogenesis. Further, it highlights the importance of caspase-1 and monocyte/macrophage activation in HIV atherogenesis and the potential of Tg26+/-/ApoE-/- as a tool for mechanistic studies of HIV ASCVD.


Subject(s)
Atherosclerosis/etiology , Caspase 1/physiology , HIV Infections/complications , Animals , Antigens, CD/analysis , Antigens, Differentiation, Myelomonocytic/analysis , Apolipoproteins E/physiology , Cohort Studies , Disease Models, Animal , Enzyme Activation , Female , Interleukin-18/blood , Male , Mice , Mice, Transgenic , Receptors, Cell Surface/analysis
13.
AIDS ; 33(10): 1557-1564, 2019 08 01.
Article in English | MEDLINE | ID: mdl-31306164

ABSTRACT

OBJECTIVE: HIV atherosclerosis and cardiovascular disease (CVD) represent a significant human health burden in the era of combination antiretroviral therapy (cART). The pathogenesis of HIV atherosclerosis is still poorly understood, due, in part, to the lack of a suitable small animal model. Indoleamine-2,3-dioxygenase (IDO) enzyme activity is the first and rate-limiting step in tryptophan catabolism and is measured by the kynurenine to tryptophan ratio (KTR). The serum KTR is a biomarker of inflammation and has recently been implicated as an important risk factor for CVD in patients living with HIV (PLWH) who are virologically suppressed under cART. However, IDO activity in HIV-associated CVD has not been studied in mouse model before. DESIGN: A novel mouse model of HIV atherosclerosis (Tg26/ApoE) was generated and examined for IDO activity and atherogenesis throughout 8 weeks on a high-fat diet. Tg26/ApoE mice were compared with Tg26 and ApoE single transgenic mice, before and during a high-fat diet. METHOD: Serum kynurenine, tryptophan and percentage of aortic plaque formation were measured. Additionally, levels of relevant cytokines were investigated in Tg26/ApoE and ApoE. RESULTS: Tg26/ApoE developed an accelerated atherosclerosis with increasing levels of KTR that were associated with plaque progression. This accelerated plaque was potentially driven by elevated levels of circulating IL-6. CONCLUSION: These results indicate that Tg26/ApoE serve as a new mouse model for HIV-induced atherogenesis, and aid in understanding the role of tryptophan catabolism in the pathogenesis of HIV atherosclerosis/CVD.


Subject(s)
Atherosclerosis/pathology , HIV Infections/complications , Indoleamine-Pyrrole 2,3,-Dioxygenase/analysis , Animals , Aorta/pathology , Aortic Diseases/pathology , Cytokines/blood , Diet, High-Fat , Disease Models, Animal , Humans , Kynurenine/blood , Mice, Transgenic , Tryptophan/blood
14.
Cell Mol Life Sci ; 76(23): 4725-4743, 2019 Dec.
Article in English | MEDLINE | ID: mdl-31359086

ABSTRACT

Targeted cell ablation is a powerful approach for studying the role of specific cell populations in a variety of organotypic functions, including cell differentiation, and organ generation and regeneration. Emerging tools for permanently or conditionally ablating targeted cell populations and transiently inhibiting neuronal activities exhibit a diversity of application and utility. Each tool has distinct features, and none can be universally applied to study different cell types in various tissue compartments. Although these tools have been developed for over 30 years, they require additional improvement. Currently, there is no consensus on how to select the tools to answer the specific scientific questions of interest. Selecting the appropriate cell ablation technique to study the function of a targeted cell population is less straightforward than selecting the method to study a gene's functions. In this review, we discuss the features of the various tools for targeted cell ablation and provide recommendations for optimal application of specific approaches.


Subject(s)
Bacteriocins/metabolism , Clodronic Acid/chemistry , Diphtheria Toxin/genetics , Optogenetics/methods , Simplexvirus/physiology , Animals , Clodronic Acid/toxicity , Diphtheria Toxin/metabolism , Humans , MPTP Poisoning/metabolism , MPTP Poisoning/pathology , Neurons/physiology , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Simplexvirus/enzymology
15.
Hepatology ; 69(5): 1965-1982, 2019 05.
Article in English | MEDLINE | ID: mdl-30681731

ABSTRACT

Adipocyte death occurs under various physiopathological conditions, including obesity and alcohol drinking, and can trigger organ damage particularly in the liver, but the underlying mechanisms remain obscure. To explore these mechanisms, we developed a mouse model of inducible adipocyte death by overexpressing the human CD59 (hCD59) on adipocytes (adipocyte-specific hCD59 transgenic mice). Injection of these mice with intermedilysin (ILY), which rapidly lyses hCD59 expressing cells exclusively by binding to the hCD59 but not mouse CD59, resulted in the acute selective death of adipocytes, adipose macrophage infiltration, and elevation of serum free fatty acid (FFA) levels. ILY injection also resulted in the secondary damage to multiple organs with the strongest injury observed in the liver, with inflammation and hepatic macrophage activation. Mechanistically, acute adipocyte death elevated epinephrine and norepinephrine levels and activated lipolysis pathways in adipose tissue in a chemokine (C-C motif) receptor 2-positive (CCR2+ ) macrophage-dependent manner, which was followed by FFA release and lipotoxicity in the liver. Additionally, acute adipocyte death caused hepatic CCR2+ macrophage activation and infiltration, further exacerbating liver injury. Conclusion: Adipocyte death predominantly induces liver injury and inflammation, which is probably due to the superior sensitivity of hepatocytes to lipotoxicity and the abundance of macrophages in the liver.


Subject(s)
Adipocytes/physiology , Adipose Tissue/enzymology , Liver Diseases/etiology , Macrophages/physiology , Receptors, CCR2/metabolism , Animals , Bacteriocins , Cell Death , Disease Models, Animal , Epinephrine/blood , Fatty Acids, Nonesterified/blood , Female , Inflammation/etiology , Isoproterenol , Lipolysis , Liver Diseases/blood , Male , Mice, Transgenic , Norepinephrine/blood , Receptors, CCR2/genetics
16.
Mol Ther ; 25(5): 1168-1186, 2017 05 03.
Article in English | MEDLINE | ID: mdl-28366764

ABSTRACT

CRISPR-associated protein 9 (Cas9)-mediated genome editing provides a promising cure for HIV-1/AIDS; however, gene delivery efficiency in vivo remains an obstacle to overcome. Here, we demonstrate the feasibility and efficiency of excising the HIV-1 provirus in three different animal models using an all-in-one adeno-associated virus (AAV) vector to deliver multiplex single-guide RNAs (sgRNAs) plus Staphylococcus aureus Cas9 (saCas9). The quadruplex sgRNAs/saCas9 vector outperformed the duplex vector in excising the integrated HIV-1 genome in cultured neural stem/progenitor cells from HIV-1 Tg26 transgenic mice. Intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 excised HIV-1 proviral DNA and significantly reduced viral RNA expression in several organs/tissues of Tg26 mice. In EcoHIV acutely infected mice, intravenously injected quadruplex sgRNAs/saCas9 AAV-DJ/8 reduced systemic EcoHIV infection, as determined by live bioluminescence imaging. Additionally, this quadruplex vector induced efficient proviral excision, as determined by PCR genotyping in the liver, lungs, brain, and spleen. Finally, in humanized bone marrow/liver/thymus (BLT) mice with chronic HIV-1 infection, successful proviral excision was detected by PCR genotyping in the spleen, lungs, heart, colon, and brain after a single intravenous injection of quadruplex sgRNAs/saCas9 AAV-DJ/8. In conclusion, in vivo excision of HIV-1 proviral DNA by sgRNAs/saCas9 in solid tissues/organs can be achieved via AAV delivery, a significant step toward human clinical trials.


Subject(s)
Endonucleases/genetics , Genetic Therapy/methods , Genome, Viral , HIV Infections/therapy , HIV-1/genetics , Proviruses/genetics , RNA, Guide, Kinetoplastida/genetics , Animals , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Base Sequence , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , Dependovirus/genetics , Dependovirus/metabolism , Disease Models, Animal , Endonucleases/metabolism , Gene Editing/methods , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HIV Infections/pathology , HIV Infections/virology , HIV Long Terminal Repeat , HIV-1/metabolism , Humans , Mice , Mice, Transgenic , Oligonucleotides/genetics , Oligonucleotides/metabolism , Proviruses/metabolism , RNA, Guide, Kinetoplastida/metabolism , Staphylococcus aureus/chemistry , Staphylococcus aureus/enzymology , gag Gene Products, Human Immunodeficiency Virus/genetics , gag Gene Products, Human Immunodeficiency Virus/metabolism , pol Gene Products, Human Immunodeficiency Virus/genetics , pol Gene Products, Human Immunodeficiency Virus/metabolism
17.
Article in Chinese | WPRIM (Western Pacific) | ID: wpr-508168

ABSTRACT

Objective To observe the feasibility of phase-rectified signal averaging applied for the evaluation of automatic nervous system activity in general anesthesia.Methods Forty-seven fe-males and 63 males aged 18-65 years,ASA falling into category Ⅰ or Ⅱ,scheduled for oral maxillo-facial surgery under general anesthesia were selected.The heart rate variability(HRV)signal was re-corded by Powerlab throughout the operation,and then it was sectioned into three segments with 5 minutes:pre-anesthesia(T0 ),means the waking state before the operation;intra-anesthesia(T1 ),the period of operation in 25-30 min;post-anesthesia(T2 ),the consciousness recovery state in the end of the operation.All indicators of HRV analysis was calculated in the time domain.SpO 2 ,MAP and BIS were recorded synchronously in the three periods.Results Compared with T0 ,DC, RMSSD, SDNN,SD1,SD2,logTP,logLF,logHF were significantly lower at T1 and T2 (P <0.05).In con-trast to T1 ,HR,MAP and BIS were significantly higher at T2 (P <0.05 ),DC,RMSSD,SDNN, SD1,SD2,logTP,logLF,logHF were significantly increased at T2 (P <0.05 ).There was positive relation between DC and RMSSD,SD1,logHF (r =0.905,0.909,0.932,respectively,P <0.01). Conclusion As 5 min-DC could indicate the character of PRSA,especially its specificity in showing vagus nerves activities,it is feasible that DC is used to observe the autonomic nerve activity intra op-eration.

18.
Cell Death Dis ; 7(12): e2498, 2016 12 01.
Article in English | MEDLINE | ID: mdl-27906187

ABSTRACT

A growing body of evidence demonstrates that autophagy, an evolutionarily conserved intracellular degradation process, is involved in the pathogenesis of atherosclerosis and has become a potential therapeutic target. Here we tested the effect of two inhibitors of phosphatidylinositol 3-kinase, 3-methyladenine (3-MA) and 2-(4-morpholinyl)-8-phenyl-chromone (LY294002), commonly used as inhibitors of autophagy, in atherosclerosis in apolipoprotein E-/- mice. Systemic application of 3-MA but not LY294002 markedly reduced the size of atherosclerotic plaque and increased the stability of lesions in high-fat diet-fed mice as compared with controls. Furthermore, 3-MA had multiple atheroprotective effects, including modulating macrophage autophagy and foam cell formation and altering the immune microenvironment. Long-term treatment with 3-MA promoted oxidized low-density lipoprotein (oxLDL)-induced macrophage autophagy and suppressed foam cell formation and cell viability in vitro. Furthermore, systemic application of 3-MA promoted lipid droplet breakdown and decreased apoptosis, most likely associated with autophagy. 3-MA treatment strikingly enhanced the expression of immune-negative molecules such as interleukin 10 (IL-10), transforming growth factor ß and IL-35, as well as forkhead box P3 (Foxp3), the specific transcriptional factor for regulatory T cells, but did not affect the level of proinflammatory cytokines in the arterial wall. We provide strong evidence for the potential therapeutic benefit of 3-MA in inhibiting atherosclerosis development and improving plaque stability.


Subject(s)
Adenine/analogs & derivatives , Apolipoproteins E/deficiency , Atherosclerosis/immunology , Atherosclerosis/pathology , Autophagy/drug effects , Foam Cells/pathology , Adenine/pharmacology , Animals , Apolipoproteins E/metabolism , Apoptosis/drug effects , Cell Count , Cell Survival/drug effects , Cellular Microenvironment/drug effects , Diet, High-Fat , Foam Cells/drug effects , Foam Cells/metabolism , Lipid Droplets/drug effects , Lipid Droplets/metabolism , Lipoproteins, LDL/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Macrophages/pathology , Male , Mice, Inbred C57BL , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/metabolism , Plaque, Atherosclerotic/metabolism , Plaque, Atherosclerotic/pathology
19.
Physiol Genomics ; 48(8): 626-32, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27368711

ABSTRACT

Effective methods for cell ablation are important tools for examining the anatomical, functional, and behavioral consequences of selective loss of specific cell types in animal models. We have developed an ablation system based on creating genetically modified animals that express human CD59 (hCD59), a membrane receptor, and administering intermedilysin (ILY), a toxin produced by Streptococcus intermedius, which binds specifically to hCD59 to induce cell lysis. As proof-of-concept in the rat, we generated an anemia model, SD-Tg(CD59-HBA1)Bryd, which expresses hCD59 on erythrocytes. Hemolysis is a common complication of inherited or acquired blood disorders, which can result in cardiovascular compromise and death. A rat model that can replicate hemolysis through specific ablation of erythrocytes would allow further study of disease and novel treatments. In vitro, complete lysis of erythrocytes expressing hCD59 was observed at and above 250 pM ILY, while no lysis was observed in wild-type erythrocytes at any ILY concentration (8-1,000 pM). In vivo, ILY intravenous injection (100 ng/g body wt) dramatically reduced the hematocrit within 10 min, with a mean hematocrit reduction of 43% compared with 1.4% in the saline control group. Rats injected with ILY at 500 ng/g intraperitoneally developed gross signs of anemia. Histopathology confirmed anemia and revealed hepatic necrosis, with microthrombi present. These studies validate the hCD59-ILY cell ablation technology in the rat and provide the scientific community with a new rapid conditional targeted ablation model for hemolytic anemia and hemolysis-associated sequelae.


Subject(s)
Anemia, Hemolytic/genetics , Animals, Genetically Modified/genetics , Anemia, Hemolytic/metabolism , Animals , Animals, Genetically Modified/metabolism , CD59 Antigens/genetics , CD59 Antigens/metabolism , DNA, Complementary/genetics , Erythrocytes/metabolism , Female , Hemolysis/genetics , Humans , Male , Rats
20.
J Clin Invest ; 126(6): 2321-33, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27159394

ABSTRACT

Cell ablation is a powerful tool for studying cell lineage and/or function; however, current cell-ablation models have limitations. Intermedilysin (ILY), a cytolytic pore-forming toxin that is secreted by Streptococcus intermedius, lyses human cells exclusively by binding to the human complement regulator CD59 (hCD59), but does not react with CD59 from nonprimates. Here, we took advantage of this feature of ILY and developed a model of conditional and targeted cell ablation by generating floxed STOP-CD59 knockin mice (ihCD59), in which expression of human CD59 only occurs after Cre-mediated recombination. The administration of ILY to ihCD59+ mice crossed with various Cre-driver lines resulted in the rapid and specific ablation of immune, epithelial, or neural cells without off-target effects. ILY had a large pharmacological window, which allowed us to perform dose-dependent studies. Finally, the ILY/ihCD59-mediated cell-ablation method was tested in several disease models to study immune cell functionalities, hepatocyte and/or biliary epithelial damage and regeneration, and neural cell damage. Together, the results of this study demonstrate the utility of the ihCD59 mouse model for studying the effects of cell ablation in specific organ systems in a variety of developmental and disease states.


Subject(s)
Bacteriocins/administration & dosage , CD59 Antigens/physiology , Animals , Astrocytes/drug effects , Astrocytes/immunology , Astrocytes/pathology , Bacteriocins/toxicity , CD59 Antigens/genetics , Gene Knock-In Techniques , Hemolysis/genetics , Hemolysis/physiology , Hepatitis, Animal/etiology , Hepatitis, Animal/genetics , Hepatitis, Animal/immunology , Hepatocytes/drug effects , Hepatocytes/immunology , Hepatocytes/pathology , Humans , Integrases , Lymphocyte Depletion , Mice , Mice, Inbred C57BL , Mice, Transgenic , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/pathology
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