| Background:Atherosclerosis is a macrophage-driven chronic inflammatory disease of the arterial wall.Macrophages build up in atherosclerotic plaques throughout the progression of atherosclerosis,as the consequence of multiple pathological changes including increased monocyte influx,impaired emigration,defective efferocytosis and enhanced local proliferation,etc..Plaque macrophages are capable to ingest modified lipoproteins(such as oxidized-LDL:OxLDL),export cholesterol,efferocytose debris and initiate tissue repair,and thus can mediate the restoration of the homeostasis of the vascular wall.However,continuous pathological stimulations,such as hypercholesterolemia and tissue injury,can induce a malfunctioned phenotype among the macrophages,leading to their abnormal inflammation activation,which can be detrimental and fuel atherogenesis.Uncontrolled inflammation hampers macrophages cholesterol efflux and efferocytosis directly,leading to the accumulation of debris and enlargement of necrotic cores.Moreover,inflammatory mediators released by plaque macrophages aggravate local tissue damage,which in turn arouses more inflammation,forming a vicious circle.As shown by numerous studies,blocking critical inflammation pathways in macrophages,such as NF?B or MAPKs pathways,can alter plaque phenotype profoundly and suppress atherosclerosis.Therefore,macrophage inflammation plays a critical role in atherogenesis.However,the functions of plaque macrophages are highly complicated,and the influences of many risk factors on plaque macrophages remain unclear,which are the major challenges for the improvement of treatments for atherosclerosis.Hypothyroidism has been well recognized to be accompanied by hypercholesterolemia and cardiovascular diseases.Traditionally,it is attributed to the decreased thyroid hormone levels in these patients.However,increased hypercholesterolemia and cardiovascular diseases including atherosclerosis are also found among subclinical hypothyroid(SH)patients,whose thyroid hormone levels remain normal and only thyroid-stimulating hormone(TSH)levels are increased.This suggests that TSH may also play roles in atherosclerosis independent from its regulation of thyroid hormones.TSH,also named thyrotropin,is a glycoprotein hormone synthesized and secreted by the pituitary,well known for its classic role in hypothalamus-pituitary-thyroid axis to bind to TSH receptor(TSHR)expressed by the thyrocytes and stimulate thyroid hormone synthesis and secretion.TSHR belongs to G protein-coupled receptors which transduce signals mainly by the trimeric G proteins coupled to it.Each G proteins trimeric is composed of three subunits,?,?,and y,and the its corresponding downstream pathway is mainly determined by the G? subunit(includes 4 subclasses:G?s,G?i,G?q/11/15,G?12/13)that it contains.The expression of TSHR is not confined to thyrocytes,and a variety of tissues and cell types,such as hepatocytes,adipocyte,and osteoclasts,etc.have been found to express TSHR.A series of studies have shown that TSH is able to act on these extra-thyroidal TSHR,and regulates cholesterol metabolism in the liver,promotes adipocyte triglyceride synthesis and suppresses osteoclast differentiation.Of noted,TSHR has also been reported to be expressed in macrophages,endothelial cells,and smooth muscle cells,three cell types critically involved in atherosclerosis.Therefore,TSH is likely to act on these TSHR-expressed cells in the aortic wall and contribute to atherogenesis directly.To test the potential roles of TSH in atherogenesis,a former project of our lab generated ApoE-/-Tshr-/-mice by crossing ApoE-/-,the well-known atherosclerosis model mice,with Tshr+/-mice,and compared their severity of atherosclerosis with ApoE-/-Tshr+/+ littermate controls.The thyroid function of ApoE-/-Tshr-/-mice were maintained by thyroid powder supplementation in the diet.While no significant change was observed in either their thyroid function or serum lipid profile,ApoE-/-Tshr-/-mice showed a significant reduction in atherosclerosis size compared to their littermates.This suggests TSH to be directly involved in atherogenesis,likely by acting on cells in the atherosclerotic lesions.Further analysis of the composition of the plaques showed that the smooth muscle content and collagen in the plaque was not significantly affected in the plaque of ApoE-/-Tshr-/-mice,but there was a significant decrease in macrophage content and inflammatory cytokines expression.Moreover,treating macrophages with TSH in vitro led to dose-dependent production of IL-6 and TNF-?.Based on the above observations,we hypothesize:TSH is very likely to act on macrophages directly and promote atherosclerosis.To test this hypothesis,the present study examined the presence of TSH in atherosclerotic plaque,and the expression of TSHR in plaque macrophages in the first place.Secondly,we explored,in vitro,the potential mechanisms by which TSH regulates plaque macrophage burden and vascular inflammation,and the molecular signaling pathways that mediate the effects of TSH on macrophages.The in vivo pathophysiological significance of the pro-inflammatory effect of TSH on the macrophages was further tested in a myeloid-specific Tshr KO ApoE-/-mouse model.The present study thus provides mechanistic insights into the predisposition to atherosclerosis in hypothyroidism,and may help improve current prevention and treatment of thyroid and cardiovascular diseases.Objectives:1.Examined the presence of TSH in atherosclerotic plaque and the expression of TSHR in plaque macrophages2.Explored the regulatory effects of TSH on macrophages3.Verify in vivo the significance of the action of TSH on the macrophages in atherosclerosis4.Unravel the molecular mechanisms by which TSH promote macrophage inflammationMethods:1.Observe the distribution of TSH in plaques by immunohistochemistry(IHC)of aortic root cryosections of ApoE-/-mice fed 12 weeks of western diet(WD).Compare TSH abundance in the aortas of wildtype C57,chow fed ApoE-/-mice and WD fed ApoE-/-mice to study whether TSH build up in the aorta with the progression of disease.2.Detect TSHR expression in plaque macrophages by co-staining TSHR and macrophage marker MOMA-2 by immunofluorescence(IF)of aortic root sections.3.Murine bone marrow derived macrophages(BMDMs)were obtained by differentiation of bone marrow cells 7 days in vitro,and were used for in vitro experiments studying the effect of TSH on OxLDL-induced macrophage inflammation markers expression,and apoptosis,and the culture medium of which was used as source of chemoattractant in the transwell assay.Murine bone marrow monocytes and peripheral blood monocytes were isolated using a negative magnetic sorting kit.Mouse peritoneal macrophages were obtained by peritoneal lavage of 3%starch solution intraperitoneally injected mice,72 hr after the injection,and seeded to cell slides for IF.RAW 264.7 monocyte-macrophages were recovered,maintained and passaged as instructed on the ATCC website,used for signaling pathway investigation.4.The influence of TSH on the expression of a series of inflammatory markers(including Tnf-?,Il-6,Nos2,Ccl2,Arg1,Pparg,Abca1)of macrophages towards OxLDL was studied by qPCR:BMDMs were preconditioned with 0.5 ng/ml TSH(or equal volume PBS as the control)before incubating in 25 ?g/ml OxLDL for another 24 hours,and then RNA was extracted from cells and retro-transcribed and analyzed with qPCR.5.The effect of TSH treatment on the attractivity of the macrophage conditioned medium was studied by transwell assay.BMDMs were treated with TSH(or equal volume PBS as the control)and 25 ?g/ml OxLDL for 12 hours,and the culture medium were collected and placed to the lower chamber.The upper chambers were then added with freshly isolated bone marrow monocytes and after incubation in cell incubator for 3 hr,the inlets were removed and transmigrated monocytes in the lower chamber were photographed and counted by software.6.The effect of Tshr KO on macrophage differentiation was studied by qPCR analysis of the expression of macrophage markers(F4/80,Lyz1/2,MerTK,Nr4a1)in Tshr+/+ and Tshr-/BMDMs differentiated at the 0,1st,3rd,5th,7th day of differentiation.Effect of TSH on monocyte maturation was studied by flow cytometry(FCM)analysis of F4/80 levels of monocytes cultured in mediums with or without 0.5 ng/ml TSH for 24 or 48 hr in vitro.7.Effect of TSH on OxLDL induced macrophage apoptosis was examined by FCM.BMDMs preconditioned with 0.5 ng/ml TSH(or equal volume of PBS as the control)for 24 hours were further treated with 80?g/ml OxLDL for another 24 hours and then stained with a PI/Annexin V apoptosis detection kit before loaded for FCM analysis;8.The pathophysiological significance of macrophage TSHR in atherogenesis was examined in myeloid-specific Tshr KO;ApoE-/-mice.By crossing Tshrflox/flox mice with LyzCre+mice(myeloid-specific CRE expressed mice),myeloid-specific Tshr KO mice(LyzCre+ Tshrflox/flox)were obtained and then were crossed with ApoE-/-mice,and after subsequence mating and screening,the LyzM-cre+ Tshrflox/flox ApoE-/-(TSHRMKO)mice and their littermate controls LyzM-cre+ Tshrwt/wt ApoE-/-(LC)were obtained finally.TSHRMKO mice and their littermates were given WD after 6 weeks of age and executed 12 or 16 weeks later,and their anti-coagulant blood(50 ?l),serum,whole aorta,aortic root were harvested for experiments.9.Serum IL-6 and CCL2 levels of TSHRMKO or LC mice were quantified by ELISA.10.Serum levels of total T3,total T4 were quantified by radioimmunoassay.11.Serum total cholesterol,high-density lipoprotein-and low-density lipoprotein-cholesterol,and triglyceride were quantified by enzymatic methods on Olympus AU5400 system.12.The relative count of leukocyte populations in peripheral blood was determined by immunostaining and FCM analysis.13.The size of atherosclerosis was analyzed by Oil Red O staining of whole aorta en face,and the cryosections of aortic root.Expression levels of F4/80 and CCL2,IL-6 were examined by IHC of the cryosections of aortic root.14.The relative count of CD45+leukocytes,macrophages and monocytes were determined by FCM of immunostained aortic cells.15.The rate of monocyte entry into atherosclerotic plaque was monitored by doing FCM analysis of whole aorta 24 hr after the adoptive transfer of CFSE labelled monocytes into WD fed TSHRMKO or LC mice.16.Monocyte recruitment to the plaques was also studied by labeling blood monocytes in vivo by fluorescent bead injection 5 days before executing the mice and harvesting the aortic roots,which were cryosectioned and the beads recovered in the lesions were counted.Blood was also sampled on execution and the monocyte marker CD115 was stained to evaluate the efficiency and specificity of the labelling method.17.The rate of apoptosis and proliferation in atherosclerotic plaques were determined by IF co-staining F4/80 and TdT-mediated dUTP nick end labelling(TUNEL),and Ki67 IHC,respectively,of the aortic cryosections.18.Mouse peritoneal macrophages were treated with 1 ng/ml TSH(or equal volume of PBS as the negative control or 50 ng/ml LPS as the positive control)for 30 minutes,and then cells were fixed and stained NF?B p65 by IF to study the effect of TSH treatment on p65 nuclear translocation.19.RAW 264.7 cells were treated with escalating dose of TSH for different time and their whole protein or nuclear/cytoplasmic protein was extracted for WB analysis of the levels of the total and phosphorylated I?B? and MAPKs to study the activating effect of TSH on these pathways.20.RAW 264.7 cells were treated with 1 ng/ml TSH for 30 minutes after critical molecules of respective pathways(Tshr,G?s,G?i,G?q,G?11,G?15,G?12,G?13)were silenced by siRNA or blocked by the corresponding inhibitor,and then the cell lysates were obtained for WB analysis of the activation states of I?B/NF?B and MAPKs pathways to explore the critical G proteins and the corresponding downstream molecular pathways that mediate the pro-inflammatory effect of TSH in macrophages.Results:1.TSH exists in the plaque and plaque macrophages express TSHRTSH IHC of the aortic root section of ApoE-/-mice fed Western diet(WD)observed the presence of TSH in plaques of different stages.Comparing the levels of TSH in the aortic tissues of mouse aortas with different grades of atherosclerosis by Western Blotting(WB),we found that TSH levels increases along with the progression of atherosclerosis.Co-staining TSHR and MOMA-2(a macrophage-monocyte marker)in the plaque by immunofluorescence(IF)showed overlapping stained area,confirming the expression of TSHR in plaque macrophages.2.TSH preconditioning in vitro boosts the OxLDL induced inflammatory cytokines expression in macrophages,and thus promotes monocyte chemotaxis towards macrophages.qPCR analysis showed that,preconditioning macrophages with 0.5 ng/ml TSH for 24 hours can significantly boost the expression of a series of acute inflammation markers(Tnf-?,Il-6,Nos2,Ccl2),and suppress markers related to inflammation resolution(Arg1,Pparg,Abca1),in the face of OxLDL(vs control:P<0.05).Transwell assay showed that,compared to the control,0.5 ng/ml TSH overnight treatment rendered the macrophage-conditioned medium much more attractive to monocytes(P<0.01).The above effects can all be blocked by Tshr KO of the macrophages.3.TSH treatment in vitro has little effects on macrophage differentiation and OxLDLinduced apoptosisAs shown by qPCR,Tshr+/+ and Tshr-/-bone marrow cells have similar expression level of differentiation markers(F4/80,Lyz1/2,MerTK,Nr4a1)during their differentiation(P>0.05).Moreover,no significant effect on F4/80 level was observed by FCM in incubating monocytes with 0.5 ng/ml TSH during their maturation to macrophages(vs control:P>0.05).On the other hand,FCM also showed no significant effect of TSH treatment on the apoptosis rate of 80?g/ml OxLDL treated macrophages(vs control:P>0.05).These suggest that changes in the rate of differentiation or apoptosis may not be the main mechanisms by which TSH alters macrophage burden in atherosclerosis.4.Myeloid-specific Tshr KO reduced plaque macrophage burden,suppressed vascular inflammation and delayed atherosclerosisCompared to their littermate controls(LC),myeloid-specific Tshr KO(TSHRMKO)mice had similar body weight,thyroid function,serum lipid profile,and leukocytes counts in peripheral blood(vs LC:P>0.05).However,TSHRMKO mice displayed decreased serum IL-6 and CCL2 levels(vs LC:P<0.05).Oil Red O staining of en face whole aorta and aortic root sections both revealed a significant reduction in plaque size in TSHRMKO mice aorta when compared to litter mate controls(vs LC:P<0.01).Moreover,IHC of aortic root section showed a significant reduction of F4/80,CCL2,IL-6 levels in the plaque(vs LC:P<0.05),suggesting myeloid Tshr KO can reduce macrophage burden alleviate inflammation in the plaque.5.Myeloid-specific Tshr KO reduce macrophage burden in plaques by suppressing monocyte recruitment in atherosclerosisWhole aorta FCM analysis revealed significantly fewer CD45+cells,F4/80+macrophages and freshly recruited CD11b+F4/80lo monocytes were detected in the TSHRMKO aorta,compared to littermates(for CD45+cells and macrophages:P<0.05;for monocytes:P<0.01).In addition,adoptive transfer of CFSE labelled monocytes showed reduced entry of monocytes into the aorta(vs LC:P<0.01).Moreover,despite the fact that monocytes were labelled with similar efficiency and specificity by in vivo bead labelling in TSHRMKO and littermate mice,significantly fewer beads were recovered in atherosclerotic lesions of TSHRMKO mice(vs LC:P<0.001).These above results consistently suggest that monocyte recruitment to the plaque is significantly reduced by myeloid-specific Tshr KO.On the other hand,TUNEL staining in aortic root lesions revealed no significantly alteration in the rate of apoptosis between the two groups of mice(vs LC:P>0.05);Ki67+IHC revealed a mild increase in proliferative cells in TSHRMKO plaque(vs LC:12w:P<0.01;16w:P<0.05),suggesting that neither macrophage proliferation nor apoptosis was the main mechanism by which TSH increases macrophage burden in the plaques.6.G?13/RhoGTPase/ERK&p38 and G?15/PLC/PKC/IKB/NF-?B transduced TSH signals in macrophagesI?B/NF?B and MAPKs(classical subclasses include ERKS,JNKs,and p38s)pathways play critical roles in the activation of macrophage inflammation.IF observed the nuclear translocation of the critical inflammation transcriptional factor NF?B p65 within 30 minutes after treating RAW 264.7 cells with 1 ng/ml TSH.WB also showed that,TSH stimulation can promote the phosphorylation and degradation of I?B?(and thus the activation of NF?B)and induce ERK1/2,JNKs(p46/p54)and p38? phosphorylation,in a dose-(0.2,0.5,1 ng/ml)and time-(15,30,60 min)dependent manner.The above effects can all be blocked by Tshr siRNA interference.Further WB analysis showed that neither pertussis toxin(PTX,G?i inhibitor)nor NF-449(Gas inhibitor)inhibited TSH-induced I?B,ERK1/2,p38 or JNK phosphorylation,while U73122(inhibitor of PLC,act downstream of G?q/11/15)suppressed TSH-induced phosphorylation of I?B.G?15 siRNA,but not G?q or G?11 siRNA,significantly suppressed TSH-induced I?B phosphorylation.On the other hand,G?13,but not G?l2,interference markedly decreased the phosphorylation of ERK and p38 induced by TSH.Moreover,the inhibition of PKCs or Rho GTPases(RhoA,Rac,and cdc42),the respective canonical downstream effectors of Gq and G?12/13 class of G proteins,blocked the activation of I?B or ERK1/2,p38 phosphorylation,similar to G?15 or G?13 interference.The above results suggest,two pathways,G?13/RhoGTPase/ERK&p38 and G?15/PLC/PKC/I?B/NF-?B,transduce TSH signals in macrophages.We then studied their relative importance by silencing G?13 or G?15 by siRNA before treating cells with TSH.Both G?15 and G?13 silencing led to a reduction in TSH-induced Ccl2 expression and in macrophage-conditioned medium attracted monocyte chemotaxis,which suggests the activation of both pathways are responsible for the proinflammatory effect of TSH.Conclusions:By acting on macrophage TSHR in the plaque,TSH activates G?13/RhoGTPase/ERK&p38 and G?15/PLC/PKC/I?B/NF-?B pathways,leading to activation of macrophage inflammation in the plaque,promoting their production of chemokines,and result in higher macrophage burden and increased atherosclerosis. |
PDF Full Text Request