Single Nucleotide Polymorphisms Mutation of Tropoelastin Gene Affects Tropoelastin mRNA and Elastin Expressions in Human Aortic Smooth Muscle Cells.

We aimed to explore the effects of single nucleotide polymorphisms (SNPs) in tropoelastin gene on tropoelastin mRNA and elastin expressions in human aortic smooth muscle cells (HASMCs). Two SNP loci, rs2071307 (G/A) and rs1785598 (G/C), were selected to construct recombinant lentivirus vectors carrying wild-type and mutant tropoelastin gene. Recombinant plasmids including pWSLV-02-ELN, pWSLV-02-ELN-mut1, and pWSLV-02-ELN-mut2 were constructed, before being amplified by polymerase chain reaction (PCR) and sequenced. The prepared plasmids and the packaging plasmids (pVSV-G and psPAX2) were cotransfected into HEK293T cells to obtain recombinant lentiviruses carrying tropoelastin gene. Afterward, HASMCs were infected with recombinant lentiviruses, and the positive cells sorted by flow cytometry were amplified. Four stable HASMCs cell lines including pWSLV-02-ELN, pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02 vector were constructed. The expressions of tropoelastin mRNA and elastin in HASMCs were detected by real-time quantitative reverse transcription-PCR and western blot, respectively. Recombinant plasmids including pWSLV-02-ELN-mut1, pWSLV-02-ELN-mut2, and pWSLV-02-ELN were successfully constructed. Recombinant lentiviruses carrying tropoelastin gene were obtained via lentivirus packaging. After infection for 24 h, 3 days and 5 days in HASMCs, tropoelastin mRNA expressions in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that of pWSLV-02-ELN group. Besides, after infection for 24 h, 3 days, and 5 days, elastin levels in pWSLV-02-ELN-mut1 and pWSLV-02-ELN-mut2 groups were significantly lower than that in pWSLV-02-ELN group. In conclusion, SNPs mutation of tropoelastin gene affected the expression of tropoelastin mRNA and elastin, suggesting that the polymorphisms of rs2071307 and rs17855988 in tropoelastin gene might be important factors for AD development.

CircHIPK3 relieves vascular calcification via mediating SIRT1/PGC-1α/MFN2 pathway by interacting with FUS.

BACKGROUND: Circular RNAs (circRNAs) have been reported to regulate the biological processes of human diseases. CircHIPK3 has been implicated in vascular calcification, but the downstream regulatory mechanisms remain unclear. Our study aimed to understand the regulatory function of circHIPK3 in vascular calcification. METHODS: CircHIPK3 expression in atherosclerosis (AS) serum samples and vascular smooth muscle cells (VSMCs) calcification model was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The binding relationships between fused in sarcoma (FUS) and circHIPK3 or sirtuin 1 (SIRT1) were verified by RNA immunoprecipitation (RIP) assay and RNA pull-down assays. Alkaline phosphatase (ALP) activity and alizarin red staining assays were performed to evaluate the biological effect of ß-glycerophosphate (ß-GP) and circHIPK3 on calcium deposition. qRT-PCR and western blot assays were used to examine the effect of ß-GP, circHIPK3, SIRT1, mitofusin 2 (MFN2), and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) on VSMCs calcification and the expression of calcification-related proteins. RESULTS: In AS serum samples and VSMCs calcification model, the expression of circHIPK3 was significantly reduced. CircHIPK3 overexpression inhibited ALP activity and calcium deposition in ß-GP-induced VSMCs. Moreover, circHIPK3 could recruit FUS to further stabilize SIRT1 mRNA. CircHIPK3 promoted MFN2 expression to alleviate VSMCs calcification via activating SIRT1/PGC-1α signaling. CONCLUSION: The positive regulation of circHIPK3/FUS/SIRT1/PGC-1α/MFN2 signaling pathway contributed to the alleviate VSMCs calcification, revealing a novel regulatory axis for vascular calcification.

Down-regulating of MFN2 promotes vascular calcification via regulating RAS-RAF-ERK1/2 pathway.

BACKGROUND: Vascular calcification (VC), as a prevalent feature of atherosclerosis (AS), is a life-threatening pathological change. Mitofusin 2 (MFN2) has been reported to be down-regulated and participate in the pathogenesis of AS. Here, we explored the feasible impacts of MFN2 on VC in AS. METHODS: Atherosclerotic lesion was evaluated by Oil Red O staining. The VC was detected by Alizarin Red S staining, ALP staining, and calcium content in vascular smooth muscle cells (VSMCs) or atherosclerotic mice. The chondrocyte differentiation of VSMCs was measured by Alcian blue staining. Western blotting and qRT-PCR were used to determine the protein and mRNA expression of associated molecules. Intermolecular interaction was measured by ChIP and dual luciferase assays. RESULTS: The expression of MFN2 and E2F1 was reduced in the aorta tissues of AS patients and mice. Silencing of MFN2 drove calcification in VSMCs and aortas of atherosclerotic mice as confirmed by up-regulating RUNX2, OPG levels, and down-regulating SM22α, α-SMA levels. The chondrocyte differentiation of VSMCs was accelerated by MFN2 knockdown through inducing the expression of Aggrecan, Collagen II, and SOX9. In addition, E2F1 promoted the transcription and expression of MFN2 in VSMCs. Overexpression of MFN2 or E2F1 suppressed ox-LDL-induced VSMC calcification. Finally, MFN2 depletion enhanced VSMC calcification via activating RAS-RAF-ERK1/2 pathway. CONCLUSION: Our results suggest that silencing of MFN2 drives VC via activating RAS-RAF-ERK1/2 pathway in the progression of AS, thus MFN2 may be a therapeutic target for AS.

CircHIPK3 Regulates Vascular Smooth Muscle Cell Calcification Via the miR-106a-5p/MFN2 Axis.

Atherosclerosis is the most common arterial disease and is closely related to vascular calcification. CircHIPK3 has been implicated in atherosclerosis development, but the possible downstream regulatory mechanisms remain unclear. The levels of circHIPK3, miR-106a and MFN2 in tissues and blood samples of patients with atherosclerosis were detected by RT-qPCR. The levels of circHIPK3, miR-106a and MFN2 were detected by RT-qPCR and the expression levels of MFN2, osteogenic and cartilage differentiation marker proteins were detected by western blot in vitro. ALP staining, Alizarin Red staining, and calcium content detection evaluated the degree of osteogenic differentiation of cells. Alcian blue staining detected the level of cell cartilage differentiation. Luciferase detected the targeting relationship between circHIPK3 and miR-106a-5p, as well as miR-106a-5p and MFN2. CircHIPK3 and MFN2 were low expressed and miR-106a-5p was highly expressed in tissues and blood samples of patients with atherosclerosis, as well as vascular smooth muscle cell (VSMC) with osteogenic and cartilage differentiation. Overexpression of circHIPK3 reduced the cell mineralization and calcium content. Overexpression of circHIPK3 inhibited osteogenic differentiation by decreasing ALP activity, RUNX2, and OPG expression, and increasing SM22α and SMA level. What's more, overexpression of circHIPK3 decreased the chondrogenic differentiation by inhibiting the protein level of SOX9, aggrecan, and collagen II. CircHIPK3 targeted miR-106a-5p and miR-106a-5p targeted MFN2. MiR-106a-5p overexpression or MFN2 depletion repressed the effect of circHIPK3 overexpression on VSMC calcification. CircHIPK3 regulated osteogenic and cartilage differentiation of VSMC via miR-106a-5p/MFN2 axis, indicating a target for treating vascular calcification.

Association between single nucleotide polymorphisms of tropoelastin gene and aortic dissection. / å¼¹åŠ›è›‹ç™½åŽŸåŸºå› å•æ ¸è‹·é ¸å¤šæ€æ€§ä¸Žä¸»åŠ¨è„‰å¤¹å±‚çš„å ³ç³».

OBJECTIVES: To evaluate the relation between single nucleotide polymorphisms (SNPs) of tropoelastin gene and aortic dissection (AD) via identifying SNPs in the tropoelastin gene, and to detect the level of tropoelastin mRNA, elastin and elastic fibers. METHODS: The specimens of the AD group (n=96) and the control group (n=95), including their blood and aortic wall tissues, were collected. DNA was extracted from the blood samples in the 2 groups, and the SNPs in the tropoelastin gene were examined by the MassARRAY genotyping technique, and their haplotypes were constructed by PHASE software. The expression of tropoelastin mRNA and elastin in the aortic tunica media was respectively detected by real-time PCR or Western blotting. Elastin Van Gieson (EVG) staining was used to observe the shape of aortic tunica media and clarify the distribution of elastic fibers. The frequency of genotypes and haplotypes of SNP loci in the tropoelastin gene was analyzed and compared between the 2 groups, and the expression of tropoelastin mRNA, elastin and elastic fibers were also compared. RESULTS: Seven SNP loci of the tropoelastin gene were detected in these samples. Among them, 5 SNP loci were polymorphic. The frequency of 3 SNP loci[rs2071307 (G/A), rs34945509 (C/T) and rs17855988 (G/C)] was significantly different between the AD group and the control group (all P<0.05). There were significantly different in the haplotypes frequency of rs2071307 (G/A), rs34945509 (C/T) and rs17855988 (G/C) between the 2 groups (all P<0.01). Real-time PCR and Western blotting showed that the relative expression of tropoelastin mRNA and elastin in the aortic tunica media in the AD group was significantly lower than that in the control group (P<0.05). EVG staining showed that the aortic tunica media was torn, the morphology and structure of elastic fibers were broken, cracked, and disordered in the AD group, while the aortic tunica media was in complete structure and well arrangement.The elastic fibers were presented closely and orderly in the control group. CONCLUSIONS: The polymorphisms of rs2071307 (G/A), rs34945509 (C/T), and rs17855988(G/C) in the tropoelastin gene may eventually affect the synthesis of elastic fibers and they may play an important role in the occurrence of AD.

The protective effect of puerarin on angiotensin II-induced aortic aneurysm formation by the inhibition of NADPH oxidase activation and oxidative stress-triggered AP-1 signaling pathways.

Puerarin, an active ingredient of Pueraria lobata, has a range of pharmacological effects and excellent pharmacodynamic properties. In the present study, the effect of puerarin on angiotensin II-induced aortic aneurysm formation and the potential underlying molecular mechanisms were examined. The results revealed that puerarin significantly suppressed the viability, and induced the apoptosis, of aneurysm-inducing cells in a time- and dose-dependent manner. Furthermore, treatment with puerarin significantly suppressed the production of reactive oxygen species (ROS) and the expression of matrix metalloproteinase-2 (MMP-2) protein in aneurysm cells. Puerarin treatment significantly increased caspase-9 and -3 activity, induced the protein expression of phosphorylated (p)-Jun and inhibited the protein expression of activator protein 1 (AP-1) in aneurysm cells. It was also demonstrated that Puerarin significantly suppressed the reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity in aneurysm cells. Therefore, it was demonstrated that puerarin on suppressed the cell growth of angiotensin II-induced aortic aneurysm formation by affecting the rate of apoptosis, the generation of ROS, MMP-2, AP-1 and p-Jun protein expression and NADPH oxidase.

Post-Transcriptional Control of Tropoelastin in Aortic Smooth Muscle Cells Affects Aortic Dissection Onset.

Aortic dissection (AD) is a catastrophic disease with high mortality and morbidity, characterized with fragmentation of elastin and loss of smooth muscle cells. Although AD has been largely attributable to polymorphisms defect in the elastin-coding gene, tropoelastin (TE), other undermined factors also appear to play roles in AD onset. Here, we investigated the effects of post-transcriptional control of TE by microRNAs (miRNAs) on elastin levels in aortic smooth muscle cells (ASMC). We found that miR-144-3p is a miRNA that targets TE mRNA in both human and mouse. Bioinformatics analyses and dual luciferase reporter assay showed that miR-144-3p inhibited protein translation of TE, through binding to the 3'-UTR of the TE mRNA. Interestingly, higher miR-144-3p levels and lower TE were detected in the ASMC obtained from AD patients, compared to those from non-AD controls. In a mouse model for human AD, infusion of adeno-associated viruses (serotype 6) carrying antisense for miR-144-3p (as-miR-144-3p) under CAG promoter significantly reduced the incidence and severity of AD, seemingly through enhancement of TE levels in ASMC. Thus, our data suggest an essential role of miR-144-3p on the pathogenesis of AD.

Infected Abdominal Aortic Aneurysms Treated with Extra-anatomic Prosthesis Bypass in the Retroperitoneum.

BACKGROUND: Infected abdominal aortic aneurysms (iAAAs) are rare but life-threatening diseases. The purpose of the present study was to report our experience of extra-anatomic prosthesis bypass in the retroperitoneum as a treatment for iAAAs. METHODS: Data of 8 consecutive patients diagnosed with iAAAs and treated by an extra-anatomic prosthesis bypass in the retroperitoneum were retrospectively collected. Operative details were as follows: one side of the retroperitoneal space was selected to build a track, and a bifurcated expanded polytetrafluoroethylene prosthesis was placed through the track. The proximal end of the prosthesis was sutured with the normal segment of abdominal aorta proximal to the infected aneurysm by end-to-end anostomosis. The 2 distal ends of the prosthesis were, respectively, sutured with the external iliac artery distal to the aneurysm. The anastomoses were then consolidated with the nearby connective tissue. After the closure of the retroperitoneum, the infected aneurysm was incised, and the infected tissue was debrided. Drainage tubes were placed in the aneurysm sac, which was packed with an omentum flap. All patients received perioperative antibiotic therapy for a period of time. All 8 patients were regularly followed up by outpatient observation. RESULTS: Eight patients with iAAAs underwent an extra-anatomic prosthesis bypass in the retroperitoneum and debridement of the infected aneurysm. An emergency operation was performed for 1 patient who underwent concomitant gastrointestinal procedures for aortoduodenal fistula. All 8 patients were definitively diagnosed by one or more sequential computed tomography scans combined with other methods. The blood or tissue cultures of all cases were positive in the perioperative period, with Salmonella (5 cases) being the most common pathogens. Other pathogens included Burkholderia pseudomallei (2 cases) and Escherichia coli (1 case). All patients survived and were discharged in 4-5 weeks after their operations. All patients were free from graft infection during the follow-up period. CONCLUSIONS: The extra-anatomic prosthesis bypass in the retroperitoneum for treating iAAAs was safe and effective. Our experience with the procedure may provide a new approach for the treatment of this disease.

Endoscopic variceal ligation combined with Hassab's procedure in preventing the recurrence of esophageal varices.

OBJECTIVE: To assess the efficacy of endoscopic variceal ligation (EVL) combined with Hassab's procedure in the prevention of variceal recurrence. METHODS: One hundred and thirty-five patients with esophageal varices were randomized to receive EVL alone, Hassab's procedure alone or a combination of EVL and Hassab's procedure for variceal eradication. Ultrasonographic venous network images were recorded by an esophageal microprobe before and after the EVL or Hassab's procedure. The clinical outcome and vascular network images of the 3 groups were analyzed. RESULTS: Esophageal varices were obliterated immediately after EVL alone, while both perforating veins and periesophageal collaterals remained patent, and 83% had recurrence of esophageal varices during an initial 3-year follow-up. Esophageal varices were reduced in size, periesophageal collaterals were obliterated after Hassab's procedure alone, and 30% experienced rebleeding and 95% with variceal recurrence. EVL combined with Hassab's procedure obliterated all esophageal varices, perforating veins and periesophageal collaterals, and only 3 patients (8%) recurred. CONCLUSION: The existence of patent perforating veins and periesophageal collaterals is the reason of esophageal variceal recurrence after EVL alone. EVL combined with Hassab's procedure can effectively prevent the recurrence, even if the cirrhojtic portal hypertension persisted.