Dynamin-related protein 1 mediates the therapeutic effect of isoliquiritigenin in diabetic intimal hyperplasia via regulation of mitochondrial fission.

Phenotypic shift of vascular smooth muscle cells (VSMCs) plays a key role in intimal hyperplasia, especially in patients with diabetes mellitus (DM). This study aimed to investigate the role of dynamin-related protein 1 (DRP1) in mitochondrial fission-mediated VSMC phenotypic shift and to clarify whether DRP1 is the therapeutic target of isoliquiritigenin (ISL). Wire injury of carotid artery or platelet-derived growth factor treatment was performed in DM mice or high-glucose cultured human aortic smooth muscle cells (HASMCs), respectively. The effects of DRP1 silencing on DM-induced intimal hyperplasia were investigated both in vivo and in vitro. Phenotypic shift of HASMCs was evaluated by detection of reactive oxygen species (ROS) generation, cell viability, and related protein expressions. The effects of ISL on DM-induced intimal hyperplasia were evaluated both in vivo and in vitro. DRP1 silencing and ISL treatment attenuated DM-induced intimal hyperplasia with reduced ROS generation, cell viability, and VSMC dedifferentiation. The GTPase domain of DRP1 protein played a critical role in mitochondrial fission in DM-induced VSMC phenotypic shift. Cellular experiments showed that ISL inhibited mitochondrial fission and reduced the GTPase activity of DRP1, which was achieved by the directly binding to K216 of the DRP1 GTPase domain. ISL attenuated mouse intimal hyperplasia by reducing GTPase activity of DRP1 and inhibiting mitochondrial fission in vivo. In conclusion, increased GTPase activity of DRP1 aggregated DM-induced intimal hyperplasia by increasing mitochondrial fission-mediated VSMC phenotypic shift. ISL attenuated mouse intimal hyperplasia by reducing DRP1 GTPase activity and inhibiting mitochondrial fission of VSMCs.

Glucagon-like peptide-1 receptor agonist exendin 4 ameliorates diabetes-associated vascular calcification by regulating mitophagy through the AMPK signaling pathway.

BACKGROUND: Vascular calcification (VC) is a complication in diabetes mellitus (DM) patients. Osteogenic phenotype switching of vascular smooth muscle cells (VSMCs) plays a critical role in diabetes-related VC. Mitophagy can inhibit phenotype switching in VSMCs. This study aimed to investigate the role of the glucagon-like peptide-1 receptor (GLP-1R) agonist exendin 4 (EX4) in mitophagy-induced phenotype switching. MATERIALS AND METHODS: The status of VC in T2DM mice was monitored using Von Kossa and Alizarin Red S (ARS) staining in mouse aortic tissue. Human aortic smooth muscle cells were cultured in high glucose (HG) and ß-glycerophosphate (ß-GP) conditioned medium. Accumulation of LC3B and p62 was detected in the mitochondrial fraction. The effect of EX4 in vitro and in vivo was investigated by knocking down AMPKα1. RESULTS: In diabetic VC mice, EX4 decreased the percentage of von Kossa/ARS positive area. EX4 inhibited osteogenic differentiation of HG/ß-GP-induced VSMCs. In HG/ß-GP-induced VSMCs, the number of mitophagosomes was increased, whereas the addition of EX4 restored mitochondrial function, increased the number of mitophagosome-lysosome fusions, and reduced p62 in mitochondrial frictions. EX4 increased the phosphorylation of AMPKα (Thr172) and ULK1 (Ser555) in HG/ß-GP-induced VSMCs. After knockdown of AMPKα1, ULK1 could not be activated by EX4. The accumulation of LC3B and p62 could not be reduced after AMPKα1 knockdown. Knockdown of AMPKα1 negated the therapeutic effects of EX4 on VC of diabetic mice. CONCLUSION: EX4 could promote mitophagy by activating the AMPK signaling pathway, attenuate insufficient mitophagy, and thus inhibit the osteogenic phenotype switching of VSMCs.

CDKN2B-AS1 mediates proliferation and migration of vascular smooth muscle cells induced by insulin.

Excessive proliferation and migration of vascular smooth muscle cells (VSMCs) contribute to the intimal hyperplasia in type 2 diabetes mellitus (T2DM) patients after percutaneous coronary intervention. We aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in VSMC proliferation and migration, as well as the underlying mechanism. T2DM model mice with carotid balloon injury were used in vivo and mouse aortic vascular smooth muscle cells (MOVAS) stimulated by insulin were used in vitro to assess the role of CDKN2B-AS1 in VSMC proliferation and migration following vascular injury in T2DM state. To investigate cell viability and migration, MTT assay and Transwell assay were conducted. To elucidate the underlying molecular mechanisms, the methylation-specific polymerase chain reaction, RNA immunoprecipitation, RNA-pull down, co-immunoprecipitation, and chromatin immunoprecipitation were performed. In vivo, CDKN2B-AS1 was up-regulated in common carotid artery tissues. In vitro, insulin treatment increased CDKN2B-AS1 level, enhanced MOVAS cell proliferation and migration, while the promoting effect was reversed by CDKN2B-AS1 knockdown. CDKN2B-AS1 forms a complex with enhancer of zeste homolog 2 (EZH2) and DNA methyltransferase (cytosine-5) 1 (DNMT1) to regulate smooth muscle 22 alpha (SM22α) methylation levels. In insulin-stimulated cells, SM22α knockdown abrogated the inhibitory effect of CDKN2B-AS1 knockdown on cell viability and migration. Injection of lentivirus-sh-CDKN2B-AS1 relieved intimal hyperplasia in T2DM mice with carotid balloon injury. Up-regulation of CDKN2B-AS1 induced by insulin promotes cell proliferation and migration by targeting SM22α through forming a complex with EZH2 and DNMT1, thereby aggravating the intimal hyperplasia after vascular injury in T2DM.

In vivo MRI tracking of iron oxide nanoparticle-labeled human mesenchymal stem cells in limb ischemia.

BACKGROUND: Stem cell transplantation has been investigated for repairing damaged tissues in various injury models. Monitoring the safety and fate of transplanted cells using noninvasive methods is important to advance this technique into clinical applications. METHODS: In this study, lower-limb ischemia models were generated in nude mice by femoral artery ligation. As negative-contrast agents, positively charged magnetic iron oxide nanoparticles (aminopropyltriethoxysilane-coated Fe2O3) were investigated in terms of in vitro labeling efficiency, effects on human mesenchymal stromal cell (hMSC) proliferation, and in vivo magnetic resonance imaging (MRI) visualization. Ultimately, the mice were sacrificed for histological analysis three weeks after transplantation. RESULTS: With efficient labeling, aminopropyltriethoxysilane-modified magnetic iron oxide nanoparticles (APTS-MNPs) did not significantly affect hMSC proliferation. In vivo, APTS-MNP-labeled hMSCs could be monitored by clinical 3 Tesla MRI for at least three weeks. Histological examination detected numerous migrated Prussian blue-positive cells, which was consistent with the magnetic resonance images. Some migrated Prussian blue-positive cells were positive for mature endothelial cell markers of von Willebrand factor and anti-human proliferating cell nuclear antigen. In the test groups, Prussian blue-positive nanoparticles, which could not be found in other organs, were detected in the spleen. CONCLUSION: APTS-MNPs could efficiently label hMSCs, and clinical 3 Tesla MRI could monitor the labeled stem cells in vivo, which may provide a new approach for the in vivo monitoring of implanted cells.

Endovascular interventions for TransAtlantic InterSociety Consensus II C and D femoropopliteal lesions.

BACKGROUND: Peripheral artery disease accounts for more than 400 000 hospitalizations in the USA and results in symptoms ranging from claudication to gangrene. Recent advances in endovascular techniques have led to a more aggressive approach for treating peripheral artery disease. The aim of this retrospective study was to evaluate the outcomes of endovascular interventions on TransAtlantic InterSociety Consensus (TASC) II C and D femoropopliteal occlusive disease. METHODS: Data for all patients undergoing endovascular interventions for femoropopliteal occlusive disease from December 2007 through December 2010 were reviewed. Demographic data, risk factor data, preprocedural and postprocedural ankle-brachial indices, technical success rates, and complication rates were obtained. Primary, assisted primary, and secondary patency were determined by Kaplan-Meier survival analysis. Univariate and multivariate analyses were performed to identify factors adversely affecting primary patency. RESULTS: The study group included 52 TASC II C and 106 TASC II D limbs in 126 patients (mean age, (68.0 ± 18.0) years). The technical success rate was 91.1%. Complications occurred in 19 limbs (12.0%), including 8 (5.1%) major complications. The mean follow-up period was (17.6 ± 5.1) months (range, 12.0 - 48.0 months). Primary patency rates at 1, 2, 3, and 4 years were 95%, 78%, 74%, and 74% in TASC II C lesions and 89%, 62%, 52%, and 52% in TASC II D lesions, respectively. Secondary patency rates at 1, 2, 3, and 4 years were 97%, 94%, 94%, and 94% in TASC II C lesions and 97%, 95%, 83%, and 83% in TASC II D lesions, respectively. It is significantly different between primary patency rates (P < 0.05) but not secondary patency rates of TASC II C and D groups (P > 0.05). Predictors of restenosis/occlusion included hyperlipidemia, lesion length, and popliteal artery involvement. CONCLUSIONS: Endovascular treatment of TASC II C and D femoropopliteal artery occlusion has a high technical success rate with favorable mid-term secondary patency rate. Hyperlipidemia, lesion length, and popliteal artery involvement were independent risk factors for in-stent restenosis.

Long-term MRI tracking of dual-labeled adipose-derived stem cells homing into mouse carotid artery injury.

BACKGROUND: Stem cell therapy has shown great promise for regenerative repair of injured or diseased tissues. Adipose-derived stem cells (ADSCs) have become increasingly attractive candidates for cellular therapy. Magnetic resonance imaging has been proven to be effective in tracking magnetic-labeled cells and evaluating their clinical relevance after cell transplantation. This study investigated the feasibility of imaging green fluorescent protein-expressing ADSCs (GFP-ADSCs) labeled with superparamagnetic iron oxide particles, and tracked them in vivo with noninvasive magnetic resonance imaging after cell transplantation in a model of mouse carotid artery injury. METHODS: GFP-ADSCs were isolated from the adipose tissues of GFP mice and labeled with superparamagnetic iron oxide particles. Intracellular stability, proliferation, and viability of the labeled cells were evaluated in vitro. Next, the cells were transplanted into a mouse carotid artery injury model. Clinical 3 T magnetic resonance imaging was performed immediately before and 1, 3, 7, 14, 21, and 30 days after cell transplantation. Prussian blue staining and histological analysis were performed 7 and 30 days after transplantation. RESULTS: GFP-ADSCs were found to be efficiently labeled with superparamagnetic iron oxide particles, with no effect on viability and proliferation. Homing of the labeled cells into the injured carotid artery tissue could be monitored by magnetic resonance imaging. CONCLUSION: Magnetically labeled ADSCs with expression of GFP can home into sites of vascular injury, and may provide new insights into understanding of cell-based therapy for cardiovascular lesions.

[Subintimal angioplasty for chronic arterial occlusive disease: a systematic review].

OBJECTIVE: To evaluate the clinical effectiveness of subintimal angioplasty in treating chronic arterial occlusive disease of lower extremity. METHODS: Eligible studies concerning treatment by subintimal angioplasty in patients with arterial occlusive disease of lower extremity were identified from electronic database, cross-reference search and relative articles. The study quality and data extraction of all relevant articles were assessed by three independent reviewers. The study endpoints were technical success, primary patency, limb salvage, and complications. RESULTS: A total of 352 studies were selected for comprehensive review. Fourteen studies including a total of 2350 patients matched the selection criteria. According to whether selective using of re-entry catheter were used or not, the technical success rates were 96.4% and 87% (P < 0.01). Limb salvage rate was 90.5% and 81.5% at 6 and 12 months respectively. Depending on whether preoperative use of anti-platelet drug, primary patency rate was 91% and 68.5% at 6 months respectively (P < 0.01). The complication rate was 8.25% without serious complications. CONCLUSION: This Meta-analysis suggests that subintimal angioplasty is a safe and effective method in treating chronic arterial occlusive disease of lower extremity with high technical success rate and limb salvage, and low serious complications. Selective using of re-entry devices and preoperative anti-platelet drug can improve the technical success rate and primary patency rate significantly. Despite the high rate of technical and clinical success of the procedure, randomized contrast trials and long-term follow-up results are required to confirm the efficacy of these results.

[Proteomics analysis of distinct proteins in human atherosclerosis obliterans: identification and verification].

OBJECTIVE: To identify distinct proteins involved in human atherosclerosis obliterans (ASO) by a differential proteomic approach. METHODS: Eight atherosclerotic femoral arteries with a mean age of 68.6 years (6 male and 2 female) and 5 normal femoral arteries with a mean age of 44.2 years (3 male and 2 female) were obtained from high amputation patients. Then the first 2-dimensional maps of the proteome of human femoral arteries was plotted to compare ASO and control specimens. Proteomic profiling was to differentiate and identify histological proteins that were associated with ASO. The differentially expressed proteins were sequenced by matrix assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). The result was verified by immunohistochemistry (IHC) and Western blot. RESULTS: ASO was associated with distinct patterns of protein expression in the femoral arteries. A total of 25 distinct spots corresponding to 13 different proteins were identified by MALDI-TOF-MS using the NCBI and IPI databases. These proteins were mainly involved in the pathogenetic mechanisms such as inflammation, oxidative stress, proliferation and transformation of SMCs. The low level of heat shock protein 27 (HSP27) in ASO was verified by IHC and western-blot in accord with the result of MS. CONCLUSION: Proteomic analysis can be used to investigate differentially expressed proteins, which may provide new insights into ASO pathogenesis, such as HSP27.

[Endovascular angioplasty for the treatment of long iliac artery chronic total occlusions].

OBJECTIVE: To evaluate the effectiveness, safety and feasibility of endovascular angioplasty in treating long iliac artery chronic total occlusions (CTO). METHODS: The clinical data from a consecutive series of patients with long (> 5 cm) iliac artery CTO who treated by endovascular angioplasty from January 2006 to December 2010 was retrospectively analyzed. There were 139 patients (157 limbs) with long iliac artery CTO treated by endovascular angioplasty in this study [male 93 and female 46, mean age (77 ± 10) years]. According to TASC II classification, there were 18 patients in type B, 89 patients in type C and 32 patients in type D. Recanalization of the occluded lesions was attempted with the left brachial and/or femoral access. RESULTS: The ankle brachial index increased from 0.42 ± 0.19 before treatment to 0.81 ± 0.26 after treatment. The rate of technical success was 96.2% (151/157) and the patency rate of iliac artery was 94.1% (111/118) during the follow-up. Significant restenosis or reocclusion was found in 7 iliac lesions and there were no major interventional complications, such as iliac artery rupture, stent displacement, pseudoaneurysms, and arteriovenous fistula. CONCLUSIONS: Endovascular angioplasty is an effective, safe and feasible method in treating long iliac CTO with high patency rate. Combined left brachial and femoral access can increase the technical success rate significantly.

Optimization of the model of abdominal aortic aneurysm by co-incubation of calcium chloride and collagenase in rats.

OBJECTIVE: To optimize the model of abdominal aortic aneurysm (AAA) in rats using calcium chloride (CaCl2) and collagenase together. METHODS: This study was performed at the 9th People's Hospital, Institute of Traumatic Medicine, Shanghai Jiao Tong University, School of Medicine, Shanghai, China from July 2008 to February 2009. Aortas of 55 adult male Sprague-Dawley rats were exposed and incubated for 20 minutes with fresh normal saline solutions supplemented with CaCl2 (0.4 M) and collagenase (4%, w/v) (group A), CaCl2 alone (group B), collagenase alone (group C), or normal saline alone (group D). After 4 weeks, the treated aortas were evaluated by digital measurement, angiography, and histological examination. RESULTS: In group A, there was a mean increase in diameter of 87.86% +/- 69.49% (range, 35.33-299.29%) weeks after surgery. The frequency of AAA in this group was 83.3% (10/12). One (1/13) AAA occurred in group C and none in other groups. Partial endothelial loss, elastin disruption, and abnormal collagen deposition were noted in the AAA tissues in group A, corresponded well to native aneurysms in human. CONCLUSION: The use of collagenase optimized the established CaCl2-induced rat model, giving a high frequency of AAA in a short period of time.

[The prevention and management of subintimal angioplasty complications].

OBJECTIVE: The purpose of this study was to assess the prevention and management of subintimal angioplasty (SIA) to treat lower extremity arterial occlusions. METHODS: From December 2003 to May 2008, 106 lower extremities with arterial occlusions (median length of 10.8 cm, range from 4.5 to 28.0 cm) were treated on an intention-to-treat basis with SIA. Twenty-one lower extremities had disabling claudication and 85 had limb-threatening ischemia. Main outcome measures included the occurrences of SIA complications and their prevention and management. In order to prevent and cure perforation of a vessel and the important collaterals being compromised, the recanalisation of SIA was performed in the "roadmap" of DSA, the guide wire was advanced with top loop through the subintimal plane until the occlusion was passed, the position of the catheter which was confirmed by injection of a small amount of contrast media during the manipulations. In order to prevent and cure acute reocclusion and embolisation, subintimal angioplasty of long occlusion was performed by long suitable diameter balloon catheters, with prolonged (2 to 3 min) inflation, stents were only placed with residual stenoses and intimal flaps. Anticoagulation was administered for 3 to 5 d and then antiplatelet treatment was given for 6 months. RESULTS: The subcutaneous light gore of arterial access sites was seen in five legs, no arterial embolisation occurred. The perforation rate was 6% (7 legs), but no serious outcomes occurred. One patient had been amputated because of important collaterals being compromised. Three legs had acute occlusion in subintimal recanalisation and one patient converted what would have been an above-knee bypass, into a below-knee bypass. CONCLUSIONS: The complications of subintimal angioplasty include perforation, embolisation, acute recanalisation occlusion and important collaterals being compromised. Most of these complications can be prevented and cured, few serious outcomes occur.

[Surgical treatment of visceral artery aneurysms].

OBJECTIVE: To analyze the experience with diagnosis and surgical treatment of visceral artery aneurysms (VAAs). METHODS: From June 2003 to December 2008, 8 patients (2 male and 6 female) with 9 VAAs underwent surgical treatment. Mean age was 49 years (ranged from 30 to 72 years). The site of aneurysmal disease was splenic artery in 4 cases, superior mesenteric artery in 2 cases, renal artery in 2 cases (3 aneurysms). In 1 patient of splenic artery aneurysm, portal vein hypertension coexisted. All the VAAs of preoperative diagnostic workup consisted of a ultrasound, computed tomography (CT) scan, and digital subtraction angiography. Six patients were operated on and two patients was treated with endovascular procedures. Only one small VAAs was treated with follow-up. RESULTS: No deaths or major complications occurred in the perioperative period. All the patients remained symptom free during a follow-up of 26.5 months (ranged from 2 to 60 months). Follow-up consisted of clinical and ultrasound scan examinations or CT scan at 1 and 6 months, and yearly thereafter. CONCLUSIONS: Aggressive approach to the treatment of VAAs is essential. Elective open surgical treatment and an endovascular procedure of visceral artery aneurysms are both safe and effective, and offers satisfactory early and long term results. There is some evidence that small (< 2 cm) and asymptomatic VAAs may be safely observed.

[Effect of transarterial embolization on persistent type II endoleaks after endovascular aneurysm repair: a meta-analysis].

OBJECTIVE: To evaluate the clinical effectiveness of transarterial embolization in persistent type II endoleak after endovascular aneurysm repair (EVAR). METHODS: Eligible studies concerning treatment by transarterial embolization in patients with persistent type II endoleak after EVAR were identified from electronic database, cross-reference search and pertinent articles. All relevant studies were systematically reviewed and the data were extracted by two independent reviewers. The study endpoints were technical success, clinical success, mortality, and complications. RESULTS: A total of 56 studies were selected for comprehensive review. No randomized controlled trials were identified. Seven patient series, comprising 129 type II endoleaks, matched the selection criteria. The rate of technical success ranged from 69.7% to 100.0% (95% CI: 78.3% +/- 7.3%). During the follow-up, the clinical success rate was 100.0% in three studies using the criterion without increase of the aneurysm diameter by the end of followup; and in other 3 studies the disappearance rates of type II endoleak were 61.5%, 20.0%, and 78.9% respectively. No incidence of serious complications, rupture of aneurysmal sac, and intra-operational death were reported. CONCLUSION: Transarterial embolization is a main option in treating type II endoleak without serious complications. Despite the high rate of technical and clinical success of the procedure, long term follow-up and randomized contrast trials are needed to confirm the efficacy.

[The clinical values of subintimal angioplasty for treatment lower extremity arterial occlusions].

OBJECTIVE: The purpose of this study was to assess the technical feasibility and early outcomes of subintimal angioplasty to treat lower extremity arterial occlusions. METHODS: During a 2-year period (from December 2003 to December 2005), 36 lower extremities with arterial occlusions (median length, 11.5 cm; range, 4.5 to 28.0 cm) were treated on an intention-to-treat basis with SIA. Thirteen lower extremities had disabling claudication and twenty-three had limb-threatening ischemia. MAIN OUTCOME MEASURES: technical success, cumulative patency, clinical results and complications. RESULTS: The technical success rate was 80% and four out of seven failures were treated by conventional surgery. After 13.2 months follow-up, recanalization vessel patency rate was 70%, and clinical effectiveness rate was 81%, no serious complications occurred. CONCLUSION: In a selected group of patients, SIA is feasible with a high initial technical success rate and the short-term results are satisfied, SIA is a good treatment alternative in patients who are lower extremity arterial occlusions. The durability of this method of therapy is unknown, and our length of follow-up is not sufficient to answer this question.