Effect And Mechanism Of CD38 On Angiotensin Ⅱ-induced Abdominal Aortic Aneurysms In Mice

Background and objective:Abdominal aortic aneurysm(AAA)is a serious cardiovascular disease to threaten human health,the incidence of the people over 65 age is about 8%.Expansion of aortic vessels,vascular membrane thickening and remodeling are its pathological features,in which the abdominal aortic balloon bulge appears and its artic vessel diameter is increased by more than 50%compared with normal vessel.The patients usually have no any symptoms,once the affected aorta is ruptured,the mortality rate of the patients is up to 80%.Studies have shown that AAA is closely associated with the phenotypic transformation of vascular smooth muscle cells(VSMCs).CD38 is the major nicotinamide adenine dinucleotide(NAD+)degrading enzyme in mammalian cells,and deletion of CD38 gene increased intracellular NAD+levels.As NAD+is an intracellular electron transmitter and coenzymes or substrates of many metabolic enzymes such as Sirtuins family,etc.,NAD+participates in the regulation of many cellular biological functions,and plays an important role in cell metabolism,inhibiting senescence and maintaining cardiovascular functional homeostasis.Previous studies showed that SIRT1 was involved in the development of mouse aortic dissection.We analyzed the transcriptome of mouse aorta and aortic aneurysm with different ages and found that CD38 expression was increased in old and affected vessels.These studies suggest that CD38 may play an important role in the development of the aortic aneurysm in mice.Based on the importance of vascular media in the development of aortic aneurysm,this project aimed to explore the effects and mechanisms of CD38 on the occurrence and development of AAA and the phenotypic change of vascular smooth muscle cells during AAA formation using vascular smooth muscle cell-specific CD38 knockout mice.Methods1.Preparation and identification of CD38flx/flxCreSMAApoE-/-mice:the mice which the exon2 and exon3 of CD38 gene were flanked with LoxP were mated with mice expressing Cre recombinase driven by the smooth muscle-specific promoter-SMA with a genetic background of C57BL/6 to obtain the CD38flx/flxCresMA mice which were subsequently crossed with C57BL/6 mice with ApoE-/-background to further obtain the CD38flx/flxCreSMAApoE-/-mice.The mice were used for subsequent experiments.2.Establishment of the angiotensin Ⅱ-induced the mouse models of the abdominal aortic aneurysm:the angiotensin Ⅱ(Ang Ⅱ)-induced abdominal aortic aneurysm model was prepared by following steps:specifically,3-monthold CD38flx/flxApoE-/-and CD38flx/flxCreSMAApoE-/-male mice were selected for continuous injection of Ang Ⅱ(1000 ng/kg/min)for 28 days with a small animal minipump.After the models were established,the whole aorta was isolated to observe the presence of the balloon bulge on the aorta,and then the maximum diameter of the aorta was measured by a vernier caliper.If it exceeded 50%of the normal diameter and had a high incidence,the model construction was judged to be successful.3.Effect and phenotypic analysis of vascular smooth muscle cells-specific CD38 deficiency on Ang Ⅱ-induced AAA in mice:Prior to the Ang Ⅱ perfusion,the blood pressures were measured with Tail-cuff method and the diameters of abdominal aorta were determined by Ultrasonography as basic characterization.During the Ang Ⅱ perfusion,the measurements were conducted weekly till the perfusion was terminated.At the end of the infusion,the abdominal aortas were isolated and AAA were examined,and the aortas were sectioned for histochemical studies,HE-,Masson-,and EVG-staining were performed to determine vascular remodeling,including outer/inner vascular diameter,media thickness,and fibrosis.The prevalence of AAA was calculated in Ang Ⅱ-perfused mice based on the observation.4.Effect of NMN supplement on Ang Ⅱ-induced AAA in mice:male mice(CD38flx/flxApoE-/-)with 3-months old were divided into two groups(NMN+AngⅡ group and saline+AngⅡ group)for preparing AngⅡ-induced AAA mice as described above.NMN was given to the mice by intragastrical administration at a dosage of 400 mg/kg/day for 7 days before Ang Ⅱ perfusion and then the drug with same dosage was continuously administrated one time each day for 28 days during the AngⅡ infusion.The aortas were isolated for evaluating the therapeutic effect of NMN on AngⅡ-induced AAA model in mice.5.Effect of CD38 on Ang Ⅱ-induced abdominal aortic aneurysm and the mechanism:The phenotype transformation,oxidative stress,mitochondrial homeostasis and inflammatory cell infiltration were investigated in the vascular smooth muscle cells-specific CD38 knockout mice in vivo and in vitro.1)The smooth muscle cell markers(α-SMA,MYH11),transcription factor for phenotype transformation(KLF4),mitochondrial fusion/fission-related proteins(MFN1,Drp1)and inflammatory cell infiltration(CD68,F4/80)were examined by immunofluorescence staining in the frozen sections of mouse abdominal aortic tissue;2)The primary aortic smooth muscle cells were isolated and cultured by two-type collagenase digestion and tissue adhering method in vitro,and the cells within 5 generations were selected for subsequent experiments;3)the effect of CD38 knockdown on smooth muscle cell migration was detected by cell scratch experiments;4)The effect of CD38 deficiency on smooth muscle cell marker aSMA,transcription factor KLF4,matrix metalloproteinases(MMPs)MMP2 and Sirtuins families were assayed by Western blot;5)The restoration of smooth muscle cell phenotypic transformation was analyzed by SIRT1 inhibitor.Results:1.CD38flx/flxApoE-/-and CD38flx/flxCreSMAApoE-/-mice were successfully generated:By PCR and gel electrophoresis assays,the target bands with 313bp for CD38flx/flx gene,350 bp for CreSMA gene,and 245 bp for ApoE-/-gene were identified in the mice.CD38 protein level of vascular smooth muscle tissues in CD38flx/flxCreSMAApoE-/-mice was reduced to 26±8.08%of that in control CD38flx/flxApoE-/-mice.These results demonstrated that the SMC double knockout(CD38flx/flxCreSMAApoE-/-)mice and its control(CD38flx/flxApoE-/-)mice were successfully generated and the required mouse models with were generated.2.Generation of Ang Ⅱ-induced mouse AAA model:Following perfusion of AngⅡ for 28 days,about 80%of mice(CD38flx/flxApoE-/-)were developed to AAA which was located nearly to renal arteries,and was consistent with the reports from literature.The maximum vascular diameters in AAA and control(sham group)were 2.05±0.59 mm and 0.93±0.27 mm,respectively.These results indicated that the AAA model was successfully generated and was able to be used in the consequent experiments.3.Smooth muscle cells-specific CD38 deficiency significantly protected mice from AngⅡ-induced AAA:1)Tissue immunofluorescence results showed that the expression of vascular smooth muscle cell marker α-SMA was decreased,whereas CD38 expression was increased in abdominal aortic aneurysm tissues.;2)After 28day Ang Ⅱ perfusion,the incidence of CD38flx/flxApoE-/-mice was 80%,while none of CD38flx/flxCreSMAApoE-/-mice had the AAA;3)CD38 deficiency suppressed blood pressure including systolic and diastolic blood pressure caused by Ang Ⅱ;4)The histological staining results showed that CD38 knockout significantly reduced aortic vessel enlargement and vessel wall thickening induced by AngⅡ.The results indicated that vascular smooth muscle cell-specific CD38 knockout inhibited AngⅡ-induced abdominal aortic aneurysm in mice and alleviated AngⅡinduced the vascular remodeling.4.NMN supplementation has no significant effect on AngⅡ-induced the incidence of abdominal aortic aneurysm,but might aggravate its severity in CD38flx/flxApoE-/-mice:The results showed that although NMN supplementation reduced the incidence of Ang Ⅱ-induced abdominal aortic aneurysm from 67%in control group to 57%in mice treated with NMN,the protection of NMN on AngⅡinduced AAA need to be further confirmed by increasing the experimental numbers.It is worth noting that we also observed that oral NMN supplementation might aggravate AngⅡ-induced mouse abdominal aortic aneurysm because AngⅡinduced abdominal aortic aneurysm was limited to the abdominal aorta with a single aneurysm without NMN supplementation,however,the aneurysm could occur in the thoracic aorta and abdominal aorta with multiple aneurysms when NMN supplementation was applied.Moreover,oral NMN also failed to reduce the maximum diameter of the aneurysmal body.5.Protectory roles and molecular mechanism of vascular smooth muscle cellspecific CD38 deficiency on AngⅡ-induced AAA in mice:underlying:1)The aortic tissue immunofluorescence showed that CD38 deficiency inhibited AngⅡinduced decreased expressions of smooth muscle cell markers α-SMA,MYH11 and increased expression of the key transcription factor KLF4 for phenotype transformation;2)tissue reactive oxygen staining and immunofluorescence results showed that CD38 knockdown inhibited increased aortic wall reactive oxygen species and macrophage infiltration caused by AngⅡ,and increased the expression of mitochondrial fusion protein MFN1 and dynamic-related protein Drpl;3)Cell experiments showed that the α-SMA expression was successfully downregulated by about 50%in vascular smooth muscle cells with 24 h of AngⅡ(10-6mol/L)stimulation;4)Consistent with the animal experiments,the Western blot results showed that CD38 knockdown inhibited the downregulation of-SMA,upregulation of KLF4,and MMP2 caused by Ang Ⅱ;5)Cell scratch experiments showed that CD38 knockdown inhibited smooth muscle cell migration;6)CD38 knockdown significantly upregulated SIRT1 expression in vascular smooth muscle cells;7)The Sirt 1 inhibitor Ex527 attenuated the inhibitory effect of CD38 on smooth muscle cell migration.Conclusions:1.VSMC specific CD38 deficiency inhibits Ang Ⅱ-induced AAA and the elevation of blood pressure,in which the underlying mechanisms may be related to suppression of VSMC phenotypic transformation and ROS production in VSMCs.2.CD38 regulates VSMC phenotypic transformation through inhibiting SIRT1KLF4 signaling axis,promoting the occurrence and development of Ang IIinduced AAA in VSMCs.3.NMN supplementation may not prevent,but aggravate the AngⅡ-induced AAA in mice.