The Role And Mechanism Of Spectrin βⅡ And TRPV6 In Regulating Atherosclerosis

Cardiovascular diseases（CVD）is the leading cause of death due to disease in the world,causing an increasingly burden and threat to public health and people’s life,and as the pathological basis of a variety of CVD,the study of pathogenesis of atherosclerosis（AS）is particularly important.A major feature of AS is that subendothelial differentiated mature macrophages engulf oxidized low-density lipoprotein（ox-LDL）through the B scavenger receptor differentiation antigen cluster 36（CD36）,and differentiate into macrophage-derived foam cells.Its formation marks the initiation of AS,and also promotes the occurrence and development of AS.At the same time,the polarization of macrophages into different subtypes in response to different stimuli in the microenvironment affects plaque formation and stability,and influences the pathological process of AS,with classically activated macrophages（M1）exacerbating the inflammatory response,tissue damage,plaque instability,and accelerating disease progression,while alternative activated macrophages（M2）promote tissue repair,enhance plaque stability,and improving disease prognosis.We therefore decided to investigate the mechanisms of macrophage polarization and foam formation and their effects on AS.SpectrinβⅡ,encoded by the SPTBN1 gene,is the most common non-erythrocytic haematopoietic protein and is widely found in many human tissues.It acts as an intracellular backbone protein cross-linked to actin and plays a key role in maintaining cell shape,regulating cytokinesis,protein sorting and epithelial cell development.However,whether it affects the development of atherosclerosis has not been reported.It has been shown that actin filaments（F-actin）,which are SpectrinβⅡ crosslinking proteins,regulates Piezo1-mediated Ca²⁺influx to affect the process of macrophages polarization and regulate the inflammatory phenotypes of macrophages,and that SpectrinβⅡ is closely associated with inflammatory responses in liver tumour cells.We therefore hypothesize that SpectrinβⅡ may play a role in the development of atherosclerosis by regulating macrophage polarization.TRPV6 channel is a member of the TRP cation channels superfamily with high calcium selectivity.TRPV6 is widely expressed in human tissues and its dysfunction is associated with a variety of diseases,such as neonatal skeletal dysplasia,inflammatory bowel disease and cancer,suggesting that TRPV6 plays a key role in controlling Ca²⁺influx and maintaining Ca²⁺homeostasis.Previous studies have shown that hyperlipidaemia induces Ca²⁺influx of macrophages to promote foam cell formation and atherogenesis.While some other TRP superfamily members,such as TRPM2 and TRPV4,have been shown to be closely associated with atherosclerosis.In view of this,we speculate that TRPV6 may also play an important role in macrophage-derived foam cell formation and atherosclerosis development.Aims1.The role and mechanism of SpectrinβⅡ in regulating macrophages polarization and influencing atherosclerosis.1)To identify changes in the expression of SpectrinβⅡ mRNA in peripheral blood mononuclear cells（PBMCs）from patients with coronary heart disease（CHD）.2)To identify changes in the expression of SpectrinβⅡ in arterial tissue and bone marrow derived macrophages（BMDMs）in mice with atherosclerosis.3)To elucidate the role of SpectrinβⅡ in macrophage polarization.4)To elucidate the molecular mechanisms by which SpectrinβⅡ regulates macrophage polarization.2.The role and mechanism of TRPV6 in regulating macrophage-derived foam cell formation and affecting atherosclerosis.1)To clarify the changes in TRPV6 expression in plaque tissue of atherosclerotic mice.2)To clarify the role of TRPV6 in macrophage-derived foam cell formation.3)To elucidate the molecular mechanisms by which TRPV6 regulates macrophage-derived foam cell formation.Methods1.The role and mechanism of SpectrinβⅡon mediating macrophages polarization and affecting atherosclerosis.1)Twenty patients with coronary artery disease and 20 non-coronary controls were consecutively included in the cardiology department of our hospital.General baseline data were collected and PBMCs were isolated and extracted.qPCR was performed to detect the expression level of SpectrinβⅡ mRNA in PBMCs.2)Apolipoprotein E knockout(Apo E^-/-)mice were fed a high cholesterol diet（HCD）for 16 weeks to establish a mouse model of atherosclerosis.Western blot and qPCR were performed to detect the expression of SpectrinβⅡ in BMDMs and aorta of C57/BL6 mice on Normal Chow Diet（NCD）,Apo E^-/-mice on NCD and Apo E^-/-mice on HCD.3)BMDMs and RAW264.7 macrophage cell lines were stimulated with 50 ng/ml LPS+100 ng/ml IFN-γto polarize them to type M1 and 20 ng/ml IL-4+IL-13 to induce polarization to type M2,respectively.Protein and RNA were extracted,and Western blot and qPCR were performed to detect SpectrinβⅡ protein and mRNA levels.4)Sptbn1^flox/flox mice were crossed with Lyz2^cre-/+mice to obtain macrophage-specific knockout Sptbn1 gene mice(Sptbn1^flox/flox Lyz2^cre/+,Sptbn1M-KO),and the same litter of Sptbn1^flox/flox mice（Sptbn1 WT）was used as control.Two groups of mouse BMDMs were extracted separately and stimulated with 50 ng/ml LPS+100 ng/ml IFN-γto polarize to type M1 and 20 ng/ml IL-4+IL-13 to induce polarization to type M2,then protein and RNA were extracted,and the effect of knockout of SpectrinβⅡ on macrophage polarization was examined by Western blot and qPCR.5)The RAW264.7 cell line was infected with the control lentivirus sh-Scramble and knockdown sptbn1 sh-Sptbn1-1 and sh-Sptbn1-2,respectively,to establish stable transgenic cell lines with knockdown of the Sptbn1 gene,which was induced to polarize to M1 and M2,respectively,and Western blot and qPCR were performed to detect the effect of knockdown of SpectrinβⅡ on macrophage polarization.6)BMDMs from Sptbn1 WT and Sptbn1 M-KO groups were extracted and divided into Sptbn1 WT unstimulated group,Sptbn1 WT polarized group,Sptbn1 M-KO unstimulated group and Sptbn1 M-KO polarized group according to different treatments.Western blot measured the changes in the expression of NF-κB p65 phosphorylation and its inhibitor IκBα,a molecule related to M1 polarization pathway,and STAT6 phosphorylation,a molecule related to M2 polarization pathway,respectively.And qPCR measured the changes of mRNA expression levels of M1 polarization-related pro-inflammatory factor IL-1β,TNF-αand M2 polarization-related anti-inflammatory factor IL-10 to see whether knockout of SpectrinβⅡ affects the activity of downstream signaling pathways of polarization in BMDMs.2.The role and mechanism of TRPV6 in regulating macrophage-derived foam cell formation and affecting atherosclerosis.1)Apo E^-/-mice were fed with HCD for 16 weeks to establish a mouse model of atherosclerosis,and plaque tissue was partially sectioned.Western blot,qPCR and immunofluorescence assays were performed to detect TRPV6 expression in BMDMs and aorta of C57/BL6 mice in the NCD group,Apo E^-/-mice in the NCD group and Apo E^-/-mice in the HCD group.2)BMDMs were treated with 50μg/m L ox-LDL for 24 h to induce macrophages foam cell formation,and changes in TRPV6 expression were detected by Western blot and qPCR after 0 h and 24 h of treatment.3)Small interfering RNA（si RNA）knocking down TRPV6 and Adenovirus（Ad）overexpressing TRPV6 were constructed respectively.The BMDMs were divided into TRPV6 knockdown group（Knockdown,KD）and overexpression group（OE）groups.KD groups BMDMs were transfected with control si-Scramble and knockdown TRPV6 si-TRPV6-1,si-TRPV6-2 and si-TRPV6-3,respectively;OE groups BMDMs were infected with control Ad-Control and overexpression Ad-TRPV6,respectively.After successful transfection,the knockdown or overexpression efficiency was verified by Western blot and qPCR.Then the control,KD and OE groups were treated with 50μg/m L ox-LDL and stained with Oil Red O or with 50μg/m L Dil-ox LDL.The effect of knockdown or overexpression of TRPV6 on the uptake of ox-LDL by macrophages was observed by Oil Red O,fluorescence microscopy and flow cytometry.4)The highest knockdown efficiency of si-TRPV6-1 was selected and BMDMs were divided into BMDMs group,BMDMs+ox-LDL group,BMDMs+si-TRPV6-1 group and BMDMs+si-TRPV6-1+ox-LDL group according to the different treatment methods.Macrophage-derived foam cell was induced by treatment with 50μg/m L ox-LDL for 24 h.Western blot detected the changes in CD36 and its downstream signaling pathway,including the expression of NF-κB p65 and its inhibitory protein IκBαexpression which are associated with inflammation,the phosphorylation level of fat-related Fyn,JNK and p38,and the expression level of macrophage chemokines MCP-1 and MIF,and qPCR detected changes in the mRNA levels of inflammatory factors IL-1β,IL-6,TNF-α,MCP-1 and MMP9.To elucidate whether knockdown of TRPV6 affects macrophage CD36 expression and activation of downstream signaling.5)The BMDMs were divided into control BMDMs group and BMDMs+ox-LDL group,KD group BMDMs+ox-LDL+si-TRPV6-1 group and BMDMs+ox-LDL+si-TRPV6-2 group,and OE group BMDMs+ox-LDL+Ad-TRPV6 group.The changes in intracellular Ca²⁺levels of BMDMs were detected by flow cytometry to clarify whether knockdown or overexpression of TRPV6 affected Ca²⁺influx in BMDMs-derived foam cells.6)BMDMs were divided into two groups,one group was divided into the control groups BMDMs group and BMDMs+BAY K8644(BAY K8644,an L-type calcium channel activator that increases intracellular Ca²⁺levels)group,the KD groups BMDMs+si-TRPV6-1 group and BMDMs+si-TRPV6-1+BAY K8644 group according to the different treatments.The other group was the control groups BMDMs group and BMDMs+BAPTA-AM(BAPTA-AM,an intracellular calcium chelator that reduces intracellular Ca²⁺levels)group,and the OE groups BMDMs+Ad-TRPV6 group and BMDMs+Ad-TRPV6+BAPTA-AM group.Then macrophages were treated with 50μg/m L Dil-ox-LDL for 24 h to induce foam cell formation,and flow cytometry was used to detect changes in Dil-ox-LDL uptake by BMDMs to study whether knockdown or overexpression of TRPV6affected ox-LDL uptake by BMDMs.7)BMDMs were divided into two groups,one group was divided into control groups BMDMs group,BMDMs+ox-LDL group and BMDMs+ox-LDL+BAY K8644 group,and KD groups BMDMs+ox-LDL+si-TRPV6-1 group and BMDMs+ox-LDL+si-TRPV6-1+BAY K8644 group according to different treatments.The other group was the control groups BMDMs group,BMDMs+ox-LDL group and BMDMs+ox-LDL+BAPTA-AM group,and the OE groups BMDMS+ox-LDL+Ad-TRPV6-1 group and BMDMs+ox-LDL+Ad-TRPV6+BAPTA-AM group according to different treatments.The changes of CD36 protein expression levels in BMDMs from different treatments were examined by Western blot to explore whether knocking down or overexpressing TRPV6affected CD36 expression in BMDMs-derived foam cells,and whether exogenously increasing or decreasing intracellular Ca²⁺levels would affect this effect.8)Apo E-/-at 6 weeks of age were randomly divided into 2 groups:high HCD+tail vein saline（control group,n=6）,and HCD+tail vein SOR-C13（TPRV6 antagonist,n=12,using saline to prepare a 50 mg/m L stock solution）,and fed continuously for 16 weeks,and two groups were treated with saline or SOR-C13 in 0.1 m L/10 g body weight by tail vein injection twice a week.At the end of feeding,the aorta and aortic roots of the mice were extracted and sectioned for oil red O staining,Hematoxylin-Eosin（HE）staining,Masson staining,and Elisa to detect serum levels of pro-inflammatory factors IL-1β,TNF-α,MCP-1 and anti-inflammatory factor IL-10,and then to detect the effect of blocking TRPV6 for the treatment of atherosclerosis.Results1.The role and mechanism of SpectrinβⅡ in regulating macrophages polarization and affecting atherosclerosis.1)The levels of SpectrinβⅡ mRNA in PBMCs were significantly higher in CHD patients than it in controls.2)Western blot and qPCR results showed that the protein expression and mRNA levels of SpectrinβⅡ were significantly higher in BMDMs and aorta of mice in the Apo E^-/-mice HCD group compared to the C57/BL6 mice NCD group and the Apo E^-/-mice NCD group.3)After the induction of macrophage polarization,Western blot and qPCR results showed that both protein and mRNA levels of SpectrinβⅡ were increased when macrophages were polarized to M1,and decreased when polarized to M2.4)Western blot and qPCR results showed that compared to the control sh-Scramble group,the Sptbn1 knockdown groups sh-Sptbn1-1,and sh-Sptbn1-2 RAW264.7macrophages showed a significant decrease in the expression of SpectrinβⅡ protein and mRNA in the stable transfer cell lines,and the knockdown efficiencies were in decreasing order.5)Compared to the control sh-Scramble group,the expression of M1 polarization markers i NOS and MCP-1 in the Sptbn1 knockdown group sh-Sptbn1-1 was significantly reduced,while the expression level of M2 polarization marker Arg1 was increased.6)After mouse tail identification,Western blot and qPCR results showed significantly lower expression of BMDMs SpectrinβⅡ protein and mRNA in the Sptbn1 M-KO group compared to the Sptbn1 WT group,indicating that the Sptbn1 M-KO mice were successfully constructed.7)Compared to the Sptbn1 WT group,the expression of the M1 macrophage markers CD86 and i NOS was significantly lower in the Sptbn1 M-KO group of mice when BMDMs were polarized to M1 type,and the expression levels of their markers CD206 and Arg1increased after polarization to M2,indicating that knockout of SpectrinβⅡ by macrophages inhibited M1 polarization of macrophages and promoted M2 polarization.8)Western blot results showed that the phosphorylation level of M1 polarization-related pathway molecule NF-B p65 and the degradation level of IκBαwere significantly reduced in the Sptbn1 M-KO M1 polarization group compared to the Sptbn1-WT polarization group;the phosphorylation level of M2 polarization-related pathway molecule STAT6 was significantly increased in the Sptbn1 M-KO M2 polarization group compared to the Sptbn1-WT polarization group.Meanwhile,qPCR results showed that the levels of M1 pro-inflammatory cytokines IL-1βand TNF-αmRNA were significantly reduced,and the mRNA expression level of M2 anti-inflammatory cytokine IL-10 was increased.It suggests that knockdout of SpectrinβⅡ in macrophages regulates macrophage polarization by inhibiting NF-κB pathway activity and enhancing STAT6 pathway activity.2.The role and mechanism of TRPV6 in regulating macrophages foam cell formation and influencing atherosclerosis.1)Western blot and qPCR results showed that the protein expression and mRNA levels of TRPV6 were significantly higher in the aorta of Apo E^-/-mice on HCD compared to the NCD group of C57/BL6 mice and Apo E^-/-mice on a NCD group.The immunofluorescence results showed that TRPV6 was highly expressed in macrophages in plaques in aortic root sections from Apo E^-/-mice on HCD compared to C57/BL6 mice on NCD and Apo E^-/-mice on NCD.2)Macrophage-derived foam cell formation was induced in vitro via ox-LDL.Western blot and qPCR results showed that both protein and mRNA levels of TRPV6 were elevated during foam cell formation.3)Western blot and qPCR results showed that TRPV6 protein expression and mRNA levels were reduced in the si-TRPV6-1,si-TRPV6-2 and si-TRPV6-3 groups compared to the si-Scramble group;while compared to the Ad-Control group,The TRPV6 protein expression and mRNA levels were all increased in the Ad-TRPV6 group.4)Compared to the si-Scramble group,in the KD TRPV6 group,oil red O staining suggested that uptake of ox-LDL by macrophages was reduced,and fluorescence microscopy and flow cytometry showed significantly reduced fluorescence intensity of Dil-ox-LDL uptaking by macrophages;while compared to the Ad-Control group,in the OE TRPV6 group,oil red O staining suggested macrophage uptake of ox-LDL was increased,and fluorescence microscopy and flow cytometry showed significantly enhanced fluorescence intensity of macrophage uptake of Dil-ox-LDL.These suggest that knockdown or overexpression of TRPV6 affects the BMDMs ability in taking up ox-LDL.5)Western blot,qPCR and Elisa results showed that the expression levels of CD36and the phosphorylation levels of macrophage CD36-related signalling pathway molecules NF-κB p65,Fyn,JNK and p38 and the degradation levels of IκBαwere significantly lower in the BMDMs+si-TRPV6-1+ox-LDL group compared to the BMDMs+ox-LDL group.Also the expression of chemokines MCP-1 and MIF was significantly reduced;the levels of pro-inflammatory factors IL-1β,IL-6,TNF-α,MCP-1 and M,P9 mRNA were significantly reduced,and the secretion level of IL-1βin cell culture supernatant was reduced.It was shown that knockdown of TRPV6 reduced the expression of CD36 and the activation of its downstream pathway during macrophages foam cell formation.6)The flow cytometry results showed that compared to the BMDMs+ox-LDL group,the intracellular Ca²⁺levels of macrophages in the BMDMs+ox-LDL+si-TRPV6-1 and BMDMs+ox-LDL+si-TRPV6-2 groups were significantly reduced,whereas the intracellular Ca²⁺levels were significantly increased in the OE group BMDMs+ox-LDL+Ad-TRPV6 group.This indicates that knockdown of TRPV6 inhibited Ca²⁺influx during foam cell formation in BMDMs,while overexpression of TRPV6 enhanced Ca²⁺influx in BMDMs.7)Compared with the BMDMs group,the intracellular Dil fluorescence signal of macrophages in the BMDMs+si-TRPV6-1 group was reduced,while the intracellular Dil fluorescence signal of macrophages in the BMDMs+si-TRPV6-1+BAY k8644 group was enhanced after treatment with BAY K8644,indicating that knockdown of TRPV6 inhibited the uptake of ox-LDL by BMDMs and increased intracellular Ca²⁺level partially restored the ability of BMDMs to uptake ox-LDL.In contrast,the intracellular Dil fluorescence signal of macrophages in the BMDMs+Ad-TRPV6 group was increased compared with the BMDMs group.And the intracellular Dil fluorescence signal was significantly reduced in the BMDMs+Ad-TRPV6+BAPTA-AM group after administration of BAPTA-AM,indicating that overexpression of TRPV6 enhanced ox-LDL uptake by BMDMs and complete antagonism of intracellular Ca²⁺significantly attenuated the ability of BMDMs to take up ox-LDL.This suggests that TRPV6 regulates the ability of lipid uptake in BMDMs by mediating Ca²⁺influx.8)Western blot results showed that the expression level of CD36 was increased in the BMDMs+ox-LDL group compared to the BMDMs group,and the level of CD36 was significantly reduced in the BMDMs+ox-LDL+si-TRPV6-1 group,while after treatment with BAY K8644,the expression level of CD36 was partially restored in the BMDMs+ox-LDL+si-TRPV6-1+BAY K8644 group.Compared to the BMDMs+ox-LDL group,CD36expression levels in the BMDMs+ox-LDL+Ad-TRPV6-1 group were significantly increased,whereas after treatment with BAPTA-AM,CD36 levels in the BMDMs+ox-LDL+Ad-TRPV6-1+BAPTA-AM group were significantly reduced.This suggests that knockdown or overexpression of TRPV6 regulates CD36 expression during foam cell formation by mediating Ca²⁺influx in BMDMs.9)Compared to the control group,oil red O staining showed a significant reduction in plaque area in the whole aorta and aortic root sections;HE staining showed a significant reduction in necrosis in the plaque area;Masson staining showed a significant increase in collagen content in the plaque area;the serum ELISA results of mice showed a decrease in the secretion of pro-inflammatory cytokines IL-1β,TNF-α,MCP-1 and an increase in the secretion of anti-inflammatory cytokine IL-10;suggesting that in vivo antagonism of TRPV6 could play a role in the treatment of AS.Conclusions1.SpectrinβⅡ promotes macrophage M1 polarization,inhibit M2 polarization,and then affect the AS process,the mechanism is that SpectrinβⅡ activates NF-κB signaling pathway,inhibits STAT6 signaling pathway,then regulates polarization phenotypes and cytokines release.2.TRPV6 promotes macrophages foam cell formation process by the mechanism that TRPV6 mediates macrophage Ca²⁺influx,promotes CD36 expression and activation of its downstream pathways Fyn and NF-κB signaling pathways,and promotes macrophage lipid uptake,retention and inflammatory activation.