1.Abnormalities In Diabetic Perivascular Adipose Tissue And The Roles And Mechanisms Of Its Cfd+ Macrophages In Vasculopathy 2.The Roles And Mechanisms Of Changed MiRNA Mitochondrial-cytoplasmic Distribution In Abnormal Hepatic Lipid Metabolism Induced By

Part 1:Abnormalities in diabetic perivascular adipose tissue and the roles and mechanisms of its Cfd+macrophages in vasculopathyBackgroundWith the development of social economy,the incidence of type 2 diabetes mellitus(T2DM)has increased rapidly,with the number of diabetics in China reaching 92.4 million.Patients with T2DM are more likely to suffer from various cardio-cerebrovascular complications,which are the main cause of death in patients.Numerous studies have shown that abnormalities in the cellular composition and function of adipose tissue play a central role in the development of vascular complications in T2DM.However,most studies have focused on visceral adipose,whereas few studies have been conducted on perivascular adipose tissue(PVAT),which is strongly related to vasculopathy.PVAT can secrete a variety of adipokines and cytokines to regulate vascular function by paracrine or endocrine.PVAT consists of adipocytes and the stromal vascular fraction(SVF).The SVF include a variety of cellular components such as adipose-derived stem cells(ADSC),endothelial cells and immune cells,which is the major contributor to the complexity of PVAT cellular composition and function.Abnormalities in cellular composition and function of PVAT play key roles in the development of atherosclerosis(AS).For example,immune cells(macrophages and T cells)infiltrate in aortic PVAT in AS,promoting adipose tissue inflammation and impairing vascular function.However,the cellular composition and function of PVAT have not been fully elucidated,as well as its abnormalities and pathological significance in diabetes.Macrophages are one of the most vital immune cells in PVAT.In recent years,a growing number of studies have demonstrated that the complexity of macrophage types and functions profoundly exceeds the dichotomy described by M1 and M2 phenotypes.Macrophages in adipose tissue are highly heterogeneous and plastic.However,the cell types and function of macrophages,particularly the macrophages in PVAT,and the abnormalities and significance of intercellular communication in PVAT in T2DM vasculopathy remain incompletely understood to date.Adipocytes are the most abundant cell type in PVAT and are the main source of PVATderived adipokines/cytokines,which play a major role in the regulation of vascular function and diabetic vasculopathy.Adipocytes are also the main target cells for the actions of other cells in PVAT,and many cells in PVAT can indirectly regulate vascular function through adipocytes.However,the interaction between other cells in PVAT and adipocytes has not yet been elucidated in detail.In recent years,the development of single cell RNA sequencing(scRNA-seq)technology has provided an effective tool to address the above questions.However,to date,studies on PVAT using scRNA-seq technology have mainly focused on non-immune cells such as ADSC.The whole picture of the cellular composition and functional characteristics of PVAT,as well as their changes in T2DM and their significance,remains poorly understood.This study intends to characterize the heterogeneity of cellular composition and function in the S VF of PVAT and their characteristic alterations in T2DM by scRNA-seq,and to verify the mechanism of Cfd+macrophages in vasculopathy by in vitro experiments,providing new ideas for exploring the pathogenesis and treatment of diabetic vasculopathy.Objectives1.To reveal the cellular composition/types and function in PVAT,and their characteristic changes in diabetes.2.To reveal the regulatory network among cell populations in the SVF of PVAT,and their characteristic changes in diabetes.3.To elucidating the mechanism of protective effects on vascular function through macrophage-adipocyte communication with a focus on the Cfd+macrophage subpopulation.Methods1.Cellular composition,function,and cellular communication in the SVF of PVAT,and their characteristic changes in T2DM were revealed using analysis of scRNA-seq data,and experiments were performed to verify specific macrophage subsets.(1)T2DM rat model was constructed by combination of high fat and high sugar diet and low dose streptozotocin(STZ);(2)Isolation of the SVF of PVAT from normal and T2DM rats,preparation of single cell suspensions,construction of cDNA library for single cell sequencing;(3)Study on the cellular composition and function,and cell communication in the SVF of PVAT,and their characteristic changes in diabetes using various bioinformatics tools;(4)Altered proportion of macrophage subpopulations identified by scRNA-seq was confirmed by immunofluorescence co-localization.2.Establishment of a co-culture system for adipocytes and macrophages to study the regulation of Cfd+macrophages on adipocyte functions.(1)PVAT-derived adipocyte was isolated from rats,and the PVAT-derived Cfd+macrophages was sorted by flow cytometry;(2)Isolated PVAT-derived adipocytes or 3T3-L1 adipocytes were cultured in the lower layer of the Transwell system,and the macrophages sorted by flow cytometry were cultured in the upper layer of the Transwell system;(3)The effect of Cfd+macrophages on adipocytes was confirmed using Western Blot and qRT-PCR to detect the expression levels of molecules in insulin signaling pathway,as well as adipokines/cytokine levels in adipocytes.3.Establishment ternary(aorta,adipocytes and macrophages)co-culture system to study the regulation of Cfd+ macrophages on vascular functions.(1)Aortic vessels and PVAT-derived adipocyte was isolated from rats;(2)PVAT-derived Cfd+macrophages was sorted by flow cytometry;(3)Isolated aorta with or without PVAT were cultured in the lower layer of the Transwell system,and the isolated adipocytes and macrophages were co-cultured in the upper layer of the Transwell system using the contact system;(4)The effect of Cfd+macrophages on vascular function in vitro was confirmed using Western Blot,immunohistochemistry and qRT-PCR to detect indicators related to vascular endothelial and smooth muscle function,as well as cytokine levels in aorta.4.The expression of C5L2 receptor on adipocyte surface was inhibited using siRNA,and then co-cultured with Cfd+ macrophages,to explore the mechanism of Cfd+macrophages effecting adipocyte functions by Western Blot and ELISA.Results1.Construction of T2DM rat modelT2DM was induced in SD rats by high fat and high sugar diet combined with low dose STZ intraperitoneally,and the T2DM rats showed increased blood glucose and weight loss,which indicated that the model was successfully constructed.2.Heterogeneity of cellular composition and function in the SVF of PVATThe SVF of PVAT in normal and diabetic rats was composed of 9 cell types,including nonimmune and immune cells.There were 4 types of nonimmune cells:ADSCs,fibroblasts,smooth muscle cells and endothelial cells.There were 5 types of immune cells in the SVF of PVAT:macrophages,T cells,NK cells,B cells and dendritic cells.Macrophages were divided into 4 subpopulations,which were involved in processes such as inflammatory response,insulin sensitivity,cytokine secretion and lymphocyte chemotaxis.NK cells could also be divided into 4 subpopulations related to functions such as immunity,metabolic reprogramming and cytotoxicity.T cells were divided into 6 subpopulations involved in T cell proliferation,apoptosis and activation,and involved in cellular immune response.ADSC and smooth muscle cells could both be further subdivided into 2 subpopulations involved in processes such as vascular remodeling,inflammatory response and regulation of lipid metabolism.3.The cellular composition and function in the SVF of PVAT were changed significantly in T2DM ratsThere was no change in the cell types in the SVF of PVAT in rats with T2DM,but the proportion of macrophages and NK cells increased significantly,and the number of several macrophage and NK cell subpopulations was significantly changed.The functions related to inflammatory and angiogenesis were generally enhanced in all types of cells in SVF.4.Macrophages were the hub of cell communication in the SVF of PVATAnalysis of cellular communication showed that there were complex interactions/communications between cell populations in the SVF and that these cell communications were increased and enhanced in the T2DM.In addition,we found that the number and strength of communications between macrophages and other cell types(both immune and non-immune cells)in PVAT was the greatest,both as ligands and as receptors,and significantly increased in T2DM,suggesting that macrophages played an important bridging and central role in cell communication in the SVF of PVAT.5.The proportion of Cfd+macrophages was decreased in PVAT from T2DM ratsThere were four functionally distinct subpopulations in PVAT macrophages,of which the number of Cfd+ macrophage subpopulations was reduced in T2DM rats.We also confirmed the finding of scRNA-seq using immunofluorescence that the proportion of Cfd+macrophages was significantly reduced in PVAT of T2DM rats.In addition,we found that Cfd+macrophage subpopulations were mainly found in PVAT using immunofluorescence,with little distribution in visceral and subcutaneous adipose tissue,indicating a distinct tissue specificity.6.Cfd+macrophage significantly improved vasculopathy in T2DM rats(1)Cfd+macrophages performed significantly protective effects on aortaTo reveal the function of Cfd+macrophages,which were significantly reduced in PVAT of T2DM,we did further studies using co-culture systems of aortic vessels with or without PVAT and Cfd+macrophages.We found that Cfd+macrophages significantly increased vascular NO levels,promoted the conversion of smooth muscle to a contractile phenotype,reduced levels of pro-inflammatory cytokines,upregulated levels of the antiinflammatory cytokine IL-10,and exerted vascular protective effects in vitro dependent on PVAT.(2)The vascular protective effects of Cfd+macrophages were dependent on adipocytesUsing co-culture system of PVAT-free aortic vessels isolated from T2DM rats with PVAT-derived adipocytes and flow-sorted Cfd+ macrophages,we found that when the three were co-cultured,they significantly reduced ROS levels in the vessels,attenuated oxidative stress,increased vascular NO levels,promoted smooth muscle conversion to a contractile phenotype,and reduced vascular inflammation.However,when adipocytes were absent from the co-culture system,Cfd+macrophages were unable to exert the above vasoprotective effects.These results suggested that the vascular protective effects of Cfd+macrophages were dependent on adipocytes.7.Cfd+macrophages regulated the response to insulin and the level of active factors in adipocytes(1)Cfd+macrophages significantly improved insulin resistance in adipocytesCo-cultured flow-sorted PVAT-derived Cfd+macrophages with 3T3-L1 adipocytes(or PVAT-derived primary adipocytes),we found that Cfd+macrophages promoted glucose consumption in adipocytes,enhanced insulin signaling pathways and improved adipocyte insulin resistance.(2)Cfd+macrophages effected the levels of adipokines/cytokines in adipocytesCo-cultured flow-sorted PVAT-derived Cfd+macrophages with 3T3-L1 adipocytes(or PVAT-derived primary adipocytes),we found that Cfd+macrophages decreased the levels of proinflammatory cytokines(TNF-α,IL-6 and MCP-1)and vasoconstriction factors(Resistin and AngⅡ),and increased the levels of anti-inflammatory cytokines(IL1RN and IL-10)and vasorelaxation factors(Adiponectin and Omentin).8.Cfd+macrophages modulated the function of adipocytes through Cfd-C3-ASP-C5L2 axisWhen co-cultured with 3T3-L1 adipocytes,Cfd+macrophages reduced the levels of complement C3 and increased the levels of acylation stimulating protein(ASP)in cocultured supernatant.ASP could bind to C5L2 receptors on the surface of adipocytes,increased the phosphorylation of Akt in 3T3-L1 adipocytes,and facilitated GLUT4 expression and membrane translocation.However,using siRNA to knock down the expression of the C5L2 receptor in adipocytes,we found that the effects of Cfd+macrophages on adipocytes were significantly attenuated.Conclusion1.There is strong heterogeneity in cellular composition and function of the SVF of PVAT,which were changed significantly in T2DM.Particularly,the proportion of macrophages,NK cells,and their subpopulations changed significantly,and the inflammatory and angiogenesis function was enhanced in T2DM.2.Communication between cell populations in the SVF of PVAT is complex and enhanced in T2DM,and macrophages,as key nodes,play an important role in the above cell communication.3.Cfd+macrophages significantly ameliorate insulin resistance and modulate adipokines/cytokines expression in adipocytes through Cfd-C3-ASP-C5L2 axis,thus inhibiting vascular inflammation,improving endothelial functions,promoting smooth muscle conversion to a contractile phenotype,exerting vascular protective effects.Part 2:The roles and mechanisms of changed miRNA mitochondrialcytoplasmic distribution in abnormal hepatic lipid metabolism induced by TSHBackgroundIn recent years,the significantly accelerated pace of life and the rapid increase in various stress led to a continuous increase in the incidence of various autoimmune diseases,represented by hypothyroidism.About 90%of them are subclinical hypothyroidism(SH),characterized by elevated serum levels of thyroid stimulating hormone(TSH)and normal levels of FT3 and FT4.To date,many epidemiological studies have demonstrated that the elevated TSH levels are one of the independent risk factors for cardiovascular diseases such as atherosclerosis,and metabolic syndrome.However,the molecular mechanisms are still not fully elucidated.The liver is an important organ and critical regulator in the lipid metabolism.Some studies have found significant disorders of hepatic lipid metabolism in SH patients.However,the correlation between SH and hepatic steatosis and non-alcoholic fatty liver disease(NAFLD)is still controversial.Some studies have suggested that SH are an important risk factor for the development and progression of NAFLD;others have suggested that SH is not associated with NAFLD or only in men.Previous studies have shown that functional TSH receptors(TSHR)exist on the surface of hepatocytes.TSH binds to TSHR on the surface of hepatocytes to regulate the expression of key molecules in downstream lipid metabolism pathways,promoting hepatic triglyceride(TG)accumulation,leading to hypercholesterolemia,and also regulating the synthesis of bile acids,ultimately causing disorders of hepatic lipid metabolism.MiRNAs are a group of small noncoding RNAs,are involved in almost all life process,and play key roles in hepatic lipid metabolism.Many miRNAs can regulate hepatic lipid metabolism directly by targeting key enzymes in lipid metabolism;or indirectly by targeting molecules in lipid metabolism pathways.Mitochondria,one of the most abundant organelles in hepatocytes,is a key node in hepatic lipid metabolism.There is a large number of miRNAs in mitochondria,most of which are encoded by the nucleus and enter the cytoplasm to regulate the expression of target genes and be involved in the hepatic lipid metabolism.The distribution of miRNA between mitochondria and cytoplasm is closely related to cellular and mitochondrial functions.There are also miRNAs distributed in mitochondria that exert post-transcriptional regulation by altering their distribution in the mitochondrial-cytoplasmic compartment even if their total expression in the cell remains unchanged,thereby regulating cellular and/or mitochondrial functions.However,very little is known about the pathological and physiological significance of this regulatory mode and its mechanism.The roles and mechanisms of changed miRNA mitochondrialcytoplasmic distribution in abnormal hepatic lipid metabolism induced by TSH have not been reported.This study aims to screen miRNAs with significantly changed mitochondrial-cytoplasmic distribution,and further verify the roles and mechanisms of these miRNAs in TSH-induced hepatic lipid accumulation,providing new insights into studying the molecular mechanisms on lipid metabolism disorders in SH patients.Objectives1.To reveal the characteristic changes of miRNA mitochondrial-cytoplasmic distribution in TSH-induced abnormal lipid metabolism in hepatocytes.2.To elucidate the roles and mechanisms of changed miRNA mitochondrial-cytoplasmic distribution in TSH-induced abnormal lipid metabolism by focusing on the miRNAs with the most significant changes in mitochondrial-cytoplasmic distribution.Methods1.The changes of miRNA mitochondrial-cytoplasmic distribution were studied using Small RNA sequencing(1)The mitochondria were isolated and purified from HepG2 cells treated or untreated with TSH,RNA in cell lysate and mitochondria was extracted for Small RNA sequencing;(2)The sequencing data were analyzed to identify the miRNAs with unchanged levels in total RNA but significantly altered levels in mitochondrial RNA compared to control HepG2 cells using multiple bioinformatics tools;(3)The changes in the expression of the screened differential miRNAs in total RNA,mitochondrial RNA and cytoplasmic RNA were further confirmed using absolute quantitative PCR;(4)The distribution changes in mitochondria and cytoplasm of the key miRNAs screened above were confirmed using in situ hybridization.2.Prediction and validation of miRNA targets(1)The target genes associated with lipid metabolism for these miRNAs with unchanged total levels but significantly altered mitochondrial levels were predicted using multiple target gene prediction software,and then performed functional enrichment analysis;(2)The HepG2 cells were transfected with miRNA mimics or miRNA inhibitors,and the target genes of each miRNA were confirmed using qRT-PCR,Western Blot and dualluciferase reporter assay.3.Detection the role of key miRNAs and their target genes in lipid metabolism in HepG2 cells(1)Transfection of miRNA mimics,miRNA inhibitors and co-transfection of miRNA mimics and overexpression vectors of corresponding target genes in HepG2 cells;(2)The accumulation of TG in HepG2 cells was evaluated using oil red staining;(3)The expression levels of key downstream molecules in HepG2 cells were detected using Western Blot and qRT-PCR;(4)The level of very long chain fatty acids(VLCFAs)in HepG2 cells was detected by GCMS.4.Detection the role of key miRNAs and PPARα in lipid metabolism in HepG2 cells(1)Transfection of miRNA mimics and/or overexpression vectors of PPARa in HepG2 cells;(2)The expression levels of key enzymes of fatty acid β-oxidation in HepG2 cells were detected using Western Blot and qRT-PCR;(3)Detection the level of O2 consumption and O2 consumption rate,and the activity of ACOX1 in hepatocytes of each group;(4)Detection the level of fatty acid in hepatocytes of each group.5.Detection the role of key miRNAs in TSH-induced triglyceride accumulation in HepG2 cells(1)HepG2 cells were treated with TSH,and then transfected of miRNA inhibitors;(2)Detection the level of key enzymes involved in fatty acid β-oxidation and the levels of fatty acid in hepatocytes of each group;(3)Detection the level of O2 consumption and O2 consumption rate,and the activity of ACOX1 in hepatocytes of each group.Results1.TSH promoted the accumulation of fatty acids and triglycerides in HepG2 cells in a dose-and time-dependent manner,and also inhibited the fatty acid oxidation in HepG2 cells.2.TSH significantly changed the distribution of many miRNAs in mitochondria of hepatocytes,without affecting their total expression in cells.There were 84 of these miRNAs,69 miRNAs of which were reduced in mitochondria.The distribution changes of representative miRNAs(miR-449a,miR-449b-5p,miR-5194)in mitochondria were confirmed by absolute quantification PCR and in situ hybridization experiments,which were transport from mitochondria to cytoplasm.3.The potential target genes of miR-449a,miR-449b-5p and miR-5194 was closely related to multiple lipid metabolism functions and pathways.For example,fatty acid oxidation,regulation of lipid metabolism by PPARα,NAFLD,and adipokines signaling pathway.According to a comprehensive assessment of the binding energy of the target genes and sequence conservativeness,PGC1B,ABCD1 and ADIPOR1 were the most potential target genes for miR-449a,miR-449b-5p and miR-5194,respectively.The results were confirmed by the dual luciferase reporter gene assays.Also,transfection of these three miRNA mimics respectively in HepG2 cells inhibited the expression of the target genes.4.miR-449a、miR-449b-5p、miR-5194 significantly promoted the accumulation of triglyceride in HepG2 cellsWe found that respectively transfection of above three miRNA mimics significantly increased TG accumulation in HepG2 cells.However,when co-transfection with miRNA mimics and overexpression vector of corresponding target genes,TG accumulation caused by miRNA mimics was attenuated.Transfection of miRNA inhibitors had little effect on TG accumulation in HepG2 cells.5.miR-449b-5p could significantly increase the level of VLCFA in HepG2 cells.However,when miR-449b-5p and ABCD1 were co-transfected,the effects of miR-449b-5p were inhibited.6.miR-449a and miR-5194 synergistically inhibited the expression of PPARα,a key molecule in metabolic regulation,and inhibited FA oxidation in hepatocyte(1)Transfection of the two above-mentioned miRNAs in HepG2 cells could significantly suppressed the expression of target genes and PPARa at the mRNA and protein levels,respectively.When miRNA mimics and target gene overexpression vectors were cotransfected,the inhibition of miR-449a and miR-5194 on the expression levels of PPARa were lost.Co-transfection of miR-449a mimic and miR-5194 mimics in HepG2 cells inhibited PPARa expression more significantly than single transfection of either miRNA mimic.(2)Both miR-449a mimic and miR-5194 mimic reduced the expression of key enzymes for fatty acid β-oxidation(CPT1A and ACOX1),inhibited FA oxidation and increased the level of fatty acid in HepG2 cells.Overexpression of PPARa attenuated the above effects of miR-449a and miR-5194.7.miR-449a and/or miR-5194 inhibitors could attenuate the inhibition of fatty acid oxidation in HepG2 cells induced by TSHTransfection of miR-449a and/or miR-5194 inhibitors increased the levels of key enzymes for fatty acid β-oxidation(CPT1A and ACOX1),enhanced mitochondrial and peroxisomal fatty acid oxidation,and reduced FA levels,thereby,attenuating the inhibition of TSH on fatty acid oxidation in HepG2 cells.Conclusion1.TSH significantly change miRNAs mitochondrial-cytoplasmic distribution in HepG2 cells,without affecting the total expression of these miRNAs.2.As the representative of the above-mentioned miRNAs,the distribution changes of miR449a/449b-5p/5194 in mitochondria and cytoplasm play key roles in TSH-induce TG accumulation,which were independent on their total expression in cells.3.miR-449b-5p increases VLCFA accumulation in HepG2 cells through ABCD1.4.miR-449a and miR-5194 inhibit fatty acid oxidation,promote the accumulation of triglyceride in HepG2 cells through the regulatory network of their target genes(PGC1B and ADIPOR1)respectively and downstream molecule(PPARα).