Remodeling Intestinal Microbiota Alleviates Severe Combined Hyperlipidemia-Induced Nonalcoholic Steatohepatitis and Atherosclerosis in LDLR-/- Hamsters.

Combined hyperlipidemia (CHL) manifests as elevated cholesterol and triglycerides, associated with fatty liver and cardiovascular diseases. Emerging evidence underscores the crucial role of the intestinal microbiota in metabolic disorders. However, the potential therapeutic viability of remodeling the intestinal microbiota in CHL remains uncertain. In this study, CHL was induced in low-density lipoprotein receptor-deficient (LDLR-/-) hamsters through an 8-week high-fat and high-cholesterol (HFHC) diet or a 4-month high-cholesterol (HC) diet. Placebo or antibiotics were administered through separate or cohousing approaches. Analysis through 16S rDNA sequencing revealed that intermittent antibiotic treatment and the cohousing approach effectively modulated the gut microbiota community without impacting its overall abundance in LDLR-/- hamsters exhibiting severe CHL. Antibiotic treatment mitigated HFHC diet-induced obesity, hyperglycemia, and hyperlipidemia, enhancing thermogenesis and alleviating nonalcoholic steatohepatitis (NASH), concurrently reducing atherosclerotic lesions in LDLR-/- hamsters. Metabolomic analysis revealed a favorable liver lipid metabolism profile. Increased levels of microbiota-derived metabolites, notably butyrate and glycylglycine, also ameliorated NASH and atherosclerosis in HFHC diet-fed LDLR-/- hamsters. Notably, antibiotics, butyrate, and glycylglycine treatment exhibited protective effects in LDLR-/- hamsters on an HC diet, aligning with outcomes observed in the HFHC diet scenario. Our findings highlight the efficacy of remodeling gut microbiota through antibiotic treatment and cohousing in improving obesity, NASH, and atherosclerosis associated with refractory CHL. Increased levels of beneficial microbiota-derived metabolites suggest a potential avenue for microbiome-mediated therapies in addressing CHL-associated diseases.

Depletion of ApoA5 aggravates spontaneous and diet-induced nonalcoholic fatty liver disease by reducing hepatic NR1D1 in hamsters.

Background: ApoA5 mainly synthesized and secreted by liver is a key modulator of lipoprotein lipase (LPL) activity and triglyceride-rich lipoproteins (TRLs). Although the role of ApoA5 in extrahepatic triglyceride (TG) metabolism in circulation has been well documented, the relationship between ApoA5 and nonalcoholic fatty liver disease (NAFLD) remains incompletely understood and the underlying molecular mechanism still needs to be elucidated. Methods: We used CRISPR/Cas9 gene editing to delete Apoa5 gene from Syrian golden hamster, a small rodent model replicating human metabolic features. Then, the ApoA5-deficient (ApoA5-/-) hamsters were used to investigate NAFLD with or without challenging a high fat diet (HFD). Results: ApoA5-/- hamsters exhibited hypertriglyceridemia (HTG) with markedly elevated TG levels at 2300 mg/dL and hepatic steatosis on a regular chow diet, accompanied with an increase in the expression levels of genes regulating lipolysis and small adipocytes in the adipose tissue. An HFD challenge predisposed ApoA5-/- hamsters to severe HTG (sHTG) and nonalcoholic steatohepatitis (NASH). Mechanistic studies in vitro and in vivo revealed that targeting ApoA5 disrupted NR1D1 mRNA stability in the HepG2 cells and the liver to reduce both mRNA and protein levels of NR1D1, respectively. Overexpression of human NR1D1 by adeno-associated virus 8 (AAV8) in the livers of ApoA5-/- hamsters significantly ameliorated fatty liver without affecting plasma lipid levels. Moreover, restoration of hepatic ApoA5 or activation of UCP1 in brown adipose tissue (BAT) by cold exposure or CL316243 administration could significantly correct sHTG and hepatic steatosis in ApoA5-/- hamsters. Conclusions: Our data demonstrate that HTG caused by ApoA5 deficiency in hamsters is sufficient to elicit hepatic steatosis and HFD aggravates NAFLD by reducing hepatic NR1D1 mRNA and protein levels, which provides a mechanistic link between ApoA5 and NAFLD and suggests the new insights into the potential therapeutic approaches for the treatment of HTG and the related disorders due to ApoA5 deficiency in the clinical trials in future.

Vascular smooth muscle cell c-Fos is critical for foam cell formation and atherosclerosis.

BACKGROUND: Hyperlipidemia-induced vascular smooth muscle cell (VSMC)-derived foam cell formation is considered a crucial event in the development of atherosclerosis. Since c-Fos emerges as a key modulator of lipid metabolism, we investigated whether c-Fos plays a role in hyperlipidemia-induced VSMC-derived foam cell formation and atherosclerosis. APPROACH AND RESULTS: c-Fos expression was observed in VSMCs in atherosclerotic plaques from patients and western diet-fed atherosclerosis-prone LDLR-/- and ApoE-/- mice by immunofluorescence staining. To ascertain c-Fos's function in atherosclerosis development, VSMC-specific c-Fos deficient mice in ApoE-/- background were established. Western diet-fed c-FosVSMCKOApoE-/- mice exhibited a significant reduction of atherosclerotic lesion formation as measured by hematoxylin and eosin staining, accompanied by decreased lipid deposition within aortic roots as determined by Oil red O staining. Primary rat VSMCs were isolated to examine the role of c-Fos in lipid uptake and foam cell formation. oxLDL stimulation resulted in VSMC-derived foam cell formation and elevated intracellular mitochondrial reactive oxygen species (mtROS), c-Fos and LOX-1 levels, whereas specific inhibition of mtROS, c-Fos or LOX-1 lessened lipid accumulation in oxLDL-stimulated VSMCs. Mechanistically, oxLDL acts through mtROS to enhance transcription activity of c-Fos to facilitate the expression of LOX-1, exerting a feedforward mechanism with oxLDL to increase lipid uptake and propel VSMC-derived foam cell formation and atherogenesis. CONCLUSION: Our study demonstrates a fundamental role of mtROS/c-Fos/LOX-1 signaling pathway in promoting oxLDL uptake and VSMC-derived foam cell formation during atherosclerosis. c-Fos may represent a promising therapeutic target amenable to clinical translation in the future.

Chlamydia pneumoniae Infection Induces Vascular Smooth Muscle Cell Migration and Atherosclerosis Through Mitochondrial Reactive Oxygen Species-Mediated JunB-Fra-1 Activation.

Infection is closely related to atherosclerosis, which is a major pathological basis for cardiovascular diseases. Vascular smooth muscle cell (VSMC) migration is an important trigger in development of atherosclerosis that is associated with Chlamydia pneumoniae (C. pneumoniae) infection. However, the mechanism of VSMC migration remains unclear, and whether antioxidant could be a therapeutic target for C. pneumoniae infection-induced atherosclerosis also remains unknown. The results showed that C. pneumoniae infection mainly impaired mitochondrial function and increased the level of mitochondrial reactive oxygen species (mtROS). The expressions of protein JunB, Fra-1 and Matrix metalloproteinase 2 (MMP) evidently increased after C. pneumoniae infection, and the interaction between JunB and Fra-1 was also enhanced. After scavenging mtROS by antioxidant Mito-TEMPO, the increasing expressions of JunB, Fra-1, MMP2 and the capacity of VSMC migration induced by C. pneumoniae infection were all inhibited. In comparison with infected ApoE-/- mice, the level of ROS in atherosclerotic lesion in ApoE-/-TLR2-/- mice with C. pneumoniae infection decreased. Knocking out TLR2 suppressed the expressions of JunB, Fra-1 and MMP2 in VSMCs and the formation of atherosclerotic lesion after C. pneumoniae infection. Furthermore, after using small interfering RNA to inhibit the expression of TLR2, the level of mtROS and the expressions of JunB, Fra-1 and MMP2 apparently decreased. Taken together, C. pneumoniae infection may promote VSMC migration and atherosclerosis development by increasing the level of mtROS through TLR2 to activate the JunB-Fra-1/MMP2 signaling pathway. The data provide the first evidence that antioxidant could reduce C. pneumoniae infection-induced VSMC migration and atherosclerosis.

Targeting ApoC3 Paradoxically Aggravates Atherosclerosis in Hamsters With Severe Refractory Hypercholesterolemia.

RATIONALE: ApoC3 plays a central role in the hydrolysis process of triglyceride (TG)-rich lipoproteins mediated by lipoprotein lipase (LPL), which levels are positively associated with the incidence of cardiovascular disease (CVD). Although targeting ApoC3 by antisense oligonucleotide (ASO), Volanesorsen markedly reduces plasma TG level and increase high-density lipoprotein cholesterol (HDL-C) in patients with hypertriglyceridemia (HTG), the cholesterol-lowering effect of ApoC3 inhibition and then the consequential outcome of atherosclerotic cardiovascular disease (ASCVD) have not been reported in patients of familial hypercholesterolemia (FH) with severe refractory hypercholesterolemia yet. OBJECTIVE: To investigate the precise effects of depleting ApoC3 on refractory hypercholesterolemia and atherosclerosis, we crossed ApoC3-deficient hamsters with a background of LDLR deficiency to generate a double knockout (DKO) hamster model (LDLR-/-, XApoC3-/-, DKO). APPROACH AND RESULTS: On the standard laboratory diet, DKO hamsters had reduced levels of plasma TG and total cholesterol (TC) relative to LDLR-/- hamsters. However, upon high-cholesterol/high-fat (HCHF) diet feeding for 12 weeks, ApoC3 deficiency reduced TG level only in female animals without affecting refractory cholesterol in the circulation, whereas apolipoprotein A1 (ApoA1) levels were significantly increased in DKO hamsters with both genders. Unexpectedly, loss of ApoC3 paradoxically accelerated diet-induced atherosclerotic development in female and male LDLR-/- hamsters but ameliorated fatty liver in female animals. Further analysis of blood biological parameters revealed that lacking ApoC3 resulted in abnormal platelet (PLT) indices, which could potentially contribute to atherosclerosis in LDLR-/- hamsters. CONCLUSIONS: In this study, our novel findings provide new insight into the application of ApoC3 inhibition for severe refractory hypercholesterolemia and ASCVD.

TLR2/CXCR4 coassociation facilitates Chlamydia pneumoniae infection-induced atherosclerosis.

Chlamydia pneumoniae infection could play a role in atherosclerosis. Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have been both shown to be involved in atherosclerosis. However, whether and how TLR2/CXCR4 cross talk is involved in C. pneumoniae infection-induced atherosclerosis remains to be determined. Our study aims to demonstrate that C. pneumoniae infection induced the cross talk between TLR2 and CXCR4 to mediate C. pneumoniae infection-induced vascular smooth muscle cell (VSMC) migration and even accelerate atherosclerosis. We first found that C. pneumoniae infection increased the aortic lesion size (en face), cross-sectional lesion area, and lipid content in aortic root lesion, which were both significantly reduced in apolipoprotein E-null (ApoE-/-)TLR2-/- or CXCR4-blocked ApoE-/- mice and were almost reversed in CXCR4-blocked ApoE-/-TLR2-/- mice. Subsequently, our data showed that C. pneumoniae infection-induced increases in VSMC contents in the atherosclerotic lesion were remarkably suppressed in ApoE-/-TLR2-/- mice or CXCR4-blocked ApoE-/- mice, and were further decreased in CXCR4-blocked ApoE-/-TLR2-/- mice. We then demonstrated that the increase in VSMC migratory capacity caused by C. pneumoniae infection was inhibited by either TLR2 or CXCR4 depletion, and downregulating both TLR2 and CXCR4 further decreased C. pneumoniae infection-induced VSMC migration by suppressing the infection-stimulated F-actin reorganization through the inhibition of the phosphorylation of focal adhesion kinase. Taken together, our data indicate that TLR2/CXCR4 coassociation facilitates C. pneumoniae infection-induced acceleration of atherosclerosis by inducing VSMC migration via focal adhesion kinase-mediated F-actin reorganization.NEW & NOTEWORTHY Toll-like receptor 2 (TLR2) and C-X-C motif chemokine receptor 4 (CXCR4) have both been shown to be involved in atherosclerosis. We demonstrate for the first time the presence of TLR2/CXCR4 coassociation during Chlamydia pneumoniae infection-induced atherosclerosis. Amazingly, blocking of both TLR2 and CXCR4 significantly retards and even almost reverses this infection-induced atherosclerosis. Our work reveals new mechanisms about C. pneumoniae infection-induced atherosclerosis and identifies potential new therapeutic targets for the prevention and treatment of atherosclerosis.

Chlamydia pneumoniae infection promotes vascular smooth muscle cell migration via c-Fos/interleukin-17C signaling.

Chlamydia pneumoniae (C. pneumoniae) infection is associated with the initiation and progression of atherosclerosis. The migration of vascular smooth muscle cell (VSMC) from the media to the intima is a key event in the development of atherosclerosis. Interleukin-17C (IL-17C) could enhance cell migration ability. The aim of our study is to investigate the role of IL-17C in C. pneumoniae infection-promoted VSMC migration, thereby possibly accelerating atherosclerosis. We firstly demonstrated that C. pneumoniae infection significantly increased IL-17C expression in VSMCs in the atherosclerotic lesion area from ApoE deficient mice. Our in vitro study further showed that IL-17C is required for C. pneumoniae infection-promoted VSMC migration, and its expression could be regulated by c-Fos through phosphorylating extracellular signal-regulated kinase (ERK). Unexpectedly, in the present study, we also found that IL-17C is critical for C. pneumoniae infection-induced c-Fos activation. c-Fos expression and activation induced by the exposure to recombinant IL-17C were markedly suppressed in the presence of the ERK inhibitor PD98059. These results suggest a possible positive feedback between c-Fos and IL-17C after C. pneumoniae infection. Taken together, our results indicate that C. pneumoniae infection promotes VSMC migration via c-Fos/IL-17C signaling.

Chlamydia pneumoniae infection promotes monocyte transendothelial migration by increasing vascular endothelial cell permeability via the tyrosine phosphorylation of VE-cadherin.

Migration of monocytes into the subendothelial layer of the intima is one of the critical events in early atherosclerosis. Chlamydia pneumoniae (C. pneumoniae) infection has been shown to promote monocyte transendothelial migration (TEM). However, the exact mechanisms have not yet been fully clarified. In this study, we tested the hypothesis that C. pneumoniae infection increases vascular endothelial cell (VEC) permeability and subsequent monocyte TEM through stimulating the tyrosine phosphorylation of vascular endothelial-cadherin (VE-cadherin). Here, we demonstrated that C. pneumoniae infection promoted monocyte TEM in a TEM assay possibly by increasing the permeability of a VEC line EA.hy926 cell as assessed by measuring the passage of FITC-BSA across a VEC monolayer. Subsequently, Western blot analysis showed that C. pneumoniae infection induced VE-cadherin internalization. Our further data revealed that Src-mediated VE-cadherin phosphorylation at Tyr658 was involved in C. pneumoniae infection-induced internalization of VE-cadherin, VEC hyperpermeability and monocyte TEM. Taken together, our data indicate that C. pneumoniae infection promotes monocyte TEM by increasing VEC permeability via the tyrosine phosphorylation and internalization of VE-cadherin in VECs.

Mesenchymal stem cells reverse high­fat diet­induced non­alcoholic fatty liver disease through suppression of CD4+ T lymphocytes in mice.

Although the multipotency of mesenchymal stem cells (MSCs) makes them an attractive choice for clinical applications, immune modulation is an important factor affecting MSC transplantation. At present, the effect of treatment with MSCs on non­alcoholic fatty liver disease (NAFLD) has received little attention. In the present study, a compact bone­derived method was used to isolate mouse MSCs (mMSCs) and a high­fat diet was used to establish a mouse model of NAFLD. Immunophenotypic features of mMSCs were analyzed using flow cytometry. Paraffin sections were stained with hematoxylin and eosin to assess inflammation and steatosis, and with picrosirius red to assess fibrosis. Spleen leukocytes were analyzed by flow cytometry. The results demonstrated that compact bone­derived MSC transplantation decreased high­fat diet­induced weight gain, expansion of subcutaneous adipose tissue, steatosis, lobular inflammation and liver fibrogenesis. Flow cytometry analysis of spleen leukocytes demonstrated that compact bone­derived MSC transplantation suppressed the proliferation of cluster of differentiation (CD) 4+ T lymphocytes in the spleen, which had been induced by the high­fat diet. In conclusion, compact bone­derived MSCs may exhibit clinical value in the treatment of NAFLD through their capacity to suppress the activation of CD4+ T cells.

Overexpression of CHD1L is associated with poor survival and aggressive tumor biology in esophageal carcinoma.

Esophageal carcinoma (EC) is a malignancy with high metastatic potential. Chromosomal helicase/ATPase DNA binding protein 1-like (CHD1L) gene is a newly identified oncogene located at Chr1q21, and it is amplified in many solid tumors. However, the status of CHD1L protein expression in EC and its clinical significance is uncertain. This study was designed to investigate the significance of CHD1L expression in human EC and its biological function in EC cells. The expression of CHD1L was examined by immunohistochemistry in 191 surgically resected ECs. The associations between CHD1L expression and clinical pathological parameters and the prognostic value of CHD1L were analyzed. Western blot analysis showed that CHD1L was overexpressed in EC cell lines. In addition, positive CHD1L expression was strongly related to advanced clinical stage (P<0.01), and lymph node metastasis (P<0.01) of EC. The Kaplan-Meier curve indicated that high expression of CHD1L may result in poor prognosis of EC patients (P<0.01), and multivariate analysis showed that CHD1L overexpression was an independent predictor of overall survival. Furthermore, suppression of CHD1L in EC cells increased apoptosis and decreased cell proliferation invasion ability. Our results suggest that CHD1L is a target oncogene with the potential to serve as a novel prognostic biomarker in EC pathogenesis.

Plumbagin protects liver against fulminant hepatic failure and chronic liver fibrosis via inhibiting inflammation and collagen production.

Plumbagin is a quinonoid constituent extracted from Plumbago genus, and it exhibits diverse pharmacological effects. This study thoroughly investigated the effects of plumbagin on thioacetamide-induced acute and chronic liver injury. Results shown that plumbagin increased survival rate, reduced liver congestion and inflammation, and decreased macrophages and neutrophils in the fulminant hepatic failure model, and remarkably diminished liver fibrosis and inflammation in the chronic liver injury model. Furthermore, plumbagin significantly suppress the HSCs/myofibroblasts activation by reduced expression of markers α-SMA and COL-1/3, and reduced macrophage in liver. In the in vitro study, plumbagin induced apoptosis and suppressed the proliferation of LX-2 cells (human HSCs). Plumbagin treatment increased AMPK phosphorylation and attenuated NF-κB, STAT3, and Akt/mTOR signals in LX-2 cells, while SMAD2 phosphorylation was not changed. Noticeably, plumbagin promoted AMPK binding to p300 which is a cofactor of SMAD complex, this may further competitively decreases the p300/SMAD complex initiated transcription of COL-1/3 and α-SMA. Additionally, plumbagin hampered inflammation related NF-κB signal in RAW 264.7 cells. In conclusion, these findings indicate that plumbagin may be a powerful drug candidate to protect the liver from acute and chronic damage by inhibiting inflammation and collagen production.