Pro-ferroptotic signaling promotes arterial aging via vascular smooth muscle cell senescence.

Senescence of vascular smooth muscle cells (VSMCs) contributes to aging-related cardiovascular diseases by promoting arterial remodelling and stiffness. Ferroptosis is a novel type of regulated cell death associated with lipid oxidation. Here, we show that pro-ferroptosis signaling drives VSMCs senescence to accelerate vascular NAD+ loss, remodelling and aging. Pro-ferroptotic signaling is triggered in senescent VSMCs and arteries of aged mice. Furthermore, the activation of pro-ferroptotic signaling in VSMCs not only induces NAD+ loss and senescence but also promotes the release of a pro-senescent secretome. Pharmacological or genetic inhibition of pro-ferroptosis signaling, ameliorates VSMCs senescence, reduces vascular stiffness and retards the progression of abdominal aortic aneurysm in mice. Mechanistically, we revealed that inhibition of pro-ferroptotic signaling facilitates the nuclear-cytoplasmic shuttling of proliferator-activated receptor-γ and, thereby impeding nuclear receptor coactivator 4-ferrtin complex-centric ferritinophagy. Finally, the activated pro-ferroptotic signaling correlates with arterial stiffness in a human proof-of-concept study. These findings have significant implications for future therapeutic strategies aiming to eliminate vascular ferroptosis in senescence- or aging-associated cardiovascular diseases.

LncRNA FGF7-5 and lncRNA GLRX3 together inhibits the formation of carotid plaque via regulating the miR-2681-5p/ERCC4 axis in atherosclerosis.

Atherosclerotic plaques belong to the common vascular disease in the aged, which rupture will lead to acute thromboembolic diseases, the leading cause of fatal cardiovascular events. Accumulating evidence indicates that the lncRNAs-miRNAs-mRNA regulatory network plays a critical role in atherosclerosis. Based on RNA sequencing (GSE207252), we constructed expression profiles of lncRNAs, microRNAs, and mRNA in the carotid plaque of atherosclerosis patients and analyzed differentially expressed genes (DEGs). We identified three candidate lncRNAs using two algorithms (LASSO and SVM-RFE): lnc_GLRX3, lnc_FGF7-5, and DISC1FP1). LNCipedia, TargetScan, and miRDB databases were used to predict target miRNAs of lncRNAs and target genes of miRNAs. Gene ontology (GO) functional annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and Gene Set Enrichment Analysis (GSEA) analysis of DEGs was carried out using the R package clusterProfiler. A PPI network was constructed using the STRING website and visualized by Cytoscape. According to the "MCC" method of the plug-in cytoHubba in Cytoscape, ERCC4 was the top hub gene of the PPI network. We constructed a lncRNA_FGF7-5/lncRNA_GLRX3-miR-2681-5p-ERCC4 regulatory network for carotid plaque using lncRNA-miRNA and miRNA-mRNA pairs. Next, lncRNA_FGF7-5 and lncRNA_GLRX3 targeted miR-2681-5p directly to upregulate ERCC4 expression. Silencing of lncRNA_FGF7-5 and lncRNA_GLRX3 promoted apoptosis and TP53 expression in HUVECs treated with ox-LDL; however, these effects were reversed by ERCC4-overexpression. Taken together, these findings indicated that lncRNA_FGF7-5 and lncRNA_GLRX3 together reduced atherosclerosis-induced apoptosis of HUVECs via targeting miR-2681-5p to increase ERCC4 expression, thereby preventing the formation of carotid plaque and finally inhibiting atherosclerosis progression.

LSD1 downregulates p21 expression in vascular smooth muscle cells and promotes neointima formation.

Neointima formation is characterized by the proliferation of vascular smooth muscle cells (VSMC). Although lysine-specific demethylase 1 (LSD1) has critical functions in several diseases, its role in neointima formation remains to be clarified. In this study, we aimed to explore the crucial role of LSD1 on neointima formation using a carotid artery injury model in mice. We observed that aberrant LSD1 expression was increased in human and mouse stenotic arteries and platelet-derived growth factor-BB (PDGF-BB)-treated VSMC. Furthermore, LSD1 knockdown significantly mitigated neointima formation in vivo and inhibited PDGF-BB-induced VSMC proliferation in vitro. We further uncovered that LSD1 overexpression exhibited opposite phenotypes in vivo and in vitro. Finally, LSD1 knockdown inhibited VSMC proliferation by increasing p21 expression, which is associated with LSD1 mediated di-methylated histone H3 on lysine 4 (H3K4me2) modification. Taken together, our data suggest that LSD1 may be a potential therapeutic target for the treatment of neointima formation.

Sorting Nexin 10 Mediates Metabolic Reprogramming of Macrophages in Atherosclerosis Through the Lyn-Dependent TFEB Signaling Pathway.

RATIONALE: SNX10 (sorting nexin 10) has been reported to play a critical role in regulating macrophage function and lipid metabolism. OBJECTIVE: To investigate the precise role of SNX10 in atherosclerotic diseases and the underlying mechanisms. METHODS AND RESULTS: SNX10 expression was compared between human healthy vessels and carotid atherosclerotic plaques. Myeloid cell-specific SNX10 knockdown mice were crossed onto the APOE-/- (apolipoprotein E) background and atherogenesis (high-cholesterol diet-induced) was monitored for 16 weeks. We found that SNX10 expression was increased in atherosclerotic lesions of aortic specimens from humans and APOE-/- mice. Myeloid cell-specific SNX10 deficiency (Δ knockout [KO]) attenuated atherosclerosis progression in APOE-/- mice. The population of anti-inflammatory monocytes/macrophages was increased in the peripheral blood and atherosclerotic lesions of ΔKO mice. In vitro experiments showed that SNX10 deficiency-inhibited foam cell formation through interrupting the internalization of CD36, which requires the interaction of SNX10 and Lyn-AKT (protein kinase B). The reduced Lyn-AKT activation by SNX10 deficiency promoted the nuclear translocation of TFEB (transcription factor EB), thereby enhanced lysosomal biogenesis and LAL (lysosomal acid lipase) activity, resulting in an increase of free fatty acids to fuel mitochondrial fatty acid oxidation. This further promoted the reprogramming of macrophages and shifted toward the anti-inflammatory phenotype. CONCLUSIONS: Our data demonstrate for the first time that SNX10 plays a crucial role in diet-induced atherogenesis via the previously unknown link between the Lyn-Akt-TFEB signaling pathway and macrophage reprogramming, suggest that SNX10 may be a potentially promising therapeutic target for atherosclerosis treatment.

Cholinergic anti-inflammatory pathway inhibits neointimal hyperplasia by suppressing inflammation and oxidative stress.

Neointimal hyperplasia as a consequence of vascular injury is aggravated by inflammatory reaction and oxidative stress. The α7 nicotinic acetylcholine receptor (α7nAChR) is a orchestrator of cholinergic anti-inflammatory pathway (CAP), which refers to a physiological neuro-immune mechanism that restricts inflammation. Here, we investigated the potential role of CAP in neointimal hyperplasia using α7nAChR knockout (KO) mice. Male α7nAChR-KO mice and their wild-type control mice (WT) were subjected to wire injury in left common carotid artery. At 4 weeks post injury, the injured aortae were isolated for examination. The neointimal hyperplasia after wire injury was significantly aggravated in α7nAChR-KO mice compared with WT mice. The α7nAChR-KO mice had increased collagen contents and vascular smooth muscle cells (VSMCs) amount. Moreover, the inflammation was significantly enhanced in the neointima of α7nAChR-KO mice relative to WT mice, evidenced by the increased expression of tumor necrosis factor-α/interleukin-1ß, and macrophage infiltration. Meanwhile, the chemokines chemokine (C-C motif) ligand 2 and chemokine (CXC motif) ligand 2 expression was also augmented in the neointima of α7nAChR-KO mice compared with WT mice. Additionally, the depletion of superoxide dismutase (SOD) and reduced glutathione (GSH), and the upregulation of 3-nitrotyrosine, malondialdehyde and myeloperoxidase were more pronounced in neointima of α7nAChR-KO mice compared with WT mice. Accordingly, the protein expression of NADPH oxidase 1 (Nox1), Nox2 and Nox4, was also higher in neointima of α7nAChR-KO mice compared with WT mice. Finally, pharmacologically activation of CAP with a selective α7nAChR agonist PNU-282987, significantly reduced neointima formation, arterial inflammation and oxidative stress after vascular injury in C57BL/6 mice. In conclusion, our results demonstrate that α7nAChR-mediated CAP is a neuro-physiological mechanism that inhibits neointima formation after vascular injury via suppressing arterial inflammation and oxidative stress. Further, these results imply that targeting α7nAChR may be a promising interventional strategy for in-stent stenosis.

The novel exercise-induced hormone irisin protects against neuronal injury via activation of the Akt and ERK1/2 signaling pathways and contributes to the neuroprotection of physical exercise in cerebral ischemia.

BACKGROUND: Irisin is a novel exercise-induced myokine involved in the regulation of adipose browning and thermogenesis. In this study, we investigated the potential role of irisin in cerebral ischemia and determined whether irisin is involved in the neuroprotective effect of physical exercise in mice. MATERIALS AND METHODS: The middle cerebral artery occlusion (MCAO) model was used to produce cerebral ischemia in mice. First, the plasma irisin levels and changes in expression of the irisin precursor protein FNDC5 in skeletal muscle were determined post ischemic stroke. Second, the association between plasma irisin levels and the neurological deficit score, brain infarct volume, or plasma concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1ß in mice with MCAO were evaluated. Third, the therapeutic effect of irisin on ischemic brain injury was evaluated in vivo and in vitro. Recombinant irisin was injected directly into the tail vein 30min after the MCAO operation, and then the effects of irisin treatment on brain infarct volume, neurological deficit, neuroinflammation, microglia activation, monocyte infiltration, oxidative stress and intracellular signaling pathway activation (Akt and ERK1/2) were measured. Irisin was also administered in cultured PC12 neuronal cells with oxygen and glucose deprivation (OGD). Finally, to assess the potential involvement of irisin in the neuroprotection of physical exercise, mice were exercised for 2weeks and an irisin neutralizing antibody was injected into these mice to block irisin 1h before the MCAO operation. RESULTS: The plasma irisin concentration and intramuscular FNDC5 protein expression decreased after ischemic stroke. Plasma irisin levels were negatively associated with brain infarct volume, the neurological deficit score, plasma TNF-α and plasma IL-6 concentrations. In OGD neuronal cells, irisin protected against cell injury. In mice with MCAO, irisin treatment reduced the brain infarct volume, neurological deficits, brain edema and the decline in body weight. Irisin treatment inhibited activation of Iba-1+ microglia, infiltration of MPO-1+ monocytes and expression of both TNF-α and IL-6 mRNA. Irisin significantly suppressed the levels of nitrotyrosine, superoxide anion and 4-hydroxynonenal (4-HNE) in peri-infarct brain tissues. Irisin treatment increased Akt and ERK1/2 phosphorylation, while blockade of Akt and ERK1/2 by specific inhibitors reduced the neuroprotective effects of irisin. Finally, the exercised mice injected with irisin neutralizing antibody displayed more severe neuronal injury than the exercised mice injected with control IgG. CONCLUSION: Irisin reduces ischemia-induced neuronal injury via activation of the Akt and ERK1/2 signaling pathways and contributes to the neuroprotective effect of physical exercise against cerebral ischemia, suggesting that irisin may be a factor linking metabolism and cardio-cerebrovascular diseases.

NADPH oxidase 4 contributes to connective tissue growth factor expression through Smad3-dependent signaling pathway.

Transforming growth factor-ß (TGF-ß)/Smad signaling has been implicated in connective tissue growth factor (CTGF) expression in vascular smooth muscle cells (VSMC). Reactive oxygen species (ROS) are involved in activation of TGF-ß/Smad signaling. However, detailed mechanisms underlying the process remain unclear. In present study, we demonstrated TGF-ß1 strongly induced CTGF expression, Smad3 activation, NADPH oxidase 4 (Nox4) expression and increased ROS production in primary rat VSMC in vitro. NADPH oxidases inhibitor diphenylene iodonium (DPI) eliminated TGF-ß1-induced CTGF expression and ROS generation. In addition, small-interfering RNA (siRNA) silencing of Smad3 or Nox4 significantly suppressed TGF-ß1-mediated CTGF expression in VSMC. Furthermore, Nox4 silencing or inhibition eliminated TGF-ß1-induced Smad3 activation and interaction between Nox4 and Smad3. In vivo studies further identified a positive correlation of Nox4 levels with Smad3 activation and CTGF expression in atherosclerotic arteries of patients and animal models. These data established that a novel mechanistic link of Nox4-dependent activation of Smad3 to increased TGF-ß1-induced CTGF in the process of vascular remodeling, which suggested a new potential pathway for therapeutic interventions.

Multiple overlapping bare stents for endovascular visceral aneurysm repair: a potential alternative endovascular strategy to multilayer stents.

BACKGROUND: Multilayer stent has become a new endovascular strategy for visceral artery aneurysm repair. However, its use was not allowed in some areas, such as China. This study evaluates an alternative method: multiple overlapping bare stents for repairing visceral artery aneurysms. METHODS: Twenty-four patients with celiac artery aneurysm (n = 2), splenic artery aneurysm (n = 8), hepatic artery aneurysm (n = 3), superior mesenteric artery aneurysm (n = 6), and renal artery aneurysm (n = 5) were treated with 2 to 4 overlapping bare stents. Long-term results, including clinical achievement ratio and target artery patency, were followed up with computed tomographic angiography. RESULTS: Insertion of overlapping bare stents was successful in all patients. Five aneurysms (21%) were totally excluded 3 months after operation, increasing to 12 (50%) and 20 (83%) aneurysms with total isolation at 6 and 12 months' follow-up, respectively. The clinical achievement ratios of multiple overlapping bare stents on splenic artery aneurysms, hepatic artery aneurysms, renal artery aneurysms, celiac artery aneurysms, and superior mesenteric artery aneurysms were 75%, 100%, 80%, 50%, and 100%, respectively. All cases combined had 100% target artery patency. CONCLUSIONS: Preliminary experience showed that repair using multiple overlapping bare stents seemed to be a potential alternative strategy for treating visceral artery aneurysm, resulting in target artery patency. However, the exact mechanism requires further study and more cases should be involved.

[Aortorenal bypass with autologous saphenous vein in Takayasu arteritis-induced renal artery stenosis: an analysis of 33 cases].

OBJECTIVE: To clarify the outcome of surgical reconstruction for renal artery in Takayasu arteritis-induced renal artery stenosis (TARAS). METHODS: A retrospective chart review was conducted on 33 consecutive patients with TARAS, who underwent aortorenal bypass (ARB) with autologous saphenous vein graft. There were 9 male and 24 female patients, with a mean age of (25 ± 11) years. The effects on blood pressure and renal function were analyzed. Primary, primary assisted, and secondary patency rates were determined. The effects of various factors on primary patency rate were analyzed. All patients showed hypertension. The mean blood pressure was (175 ± 26)/(100 ± 19) mmHg (1 mmHg = 0.133 kPa). The mean antihypertensive dosage was (2.1 ± 0.6) defined daily dose (DDD). Seventeen patients showed intractable hypertension. Mean estimated glomerular filtration rate was (78 ± 5) ml/min. One patient was dialysis-dependent, and 3 patients were combined with congestive heart failure. RESULTS: ARB was performed for the 39 renal arteries, including 27 unilateral and 6 bilateral bypasses. Postoperative morbidity was 15.2%. All patients survived. During follow-up of mean (56 ± 18) months, two graft occlusions and four graft restenoses occurred. All graft restenoses were eliminated successfully with percutaneous angioplasty, but one patient experienced restenosis again six months later. At 1, 3, and 5 years of follow-up, primary patency was 92%, 89%, and 79%, respectively, primary assisted patency was 95%, 95%, and 91%, respectively, and secondary patency was 95%, 95%, and 91%, respectively. ARB resulted in a decrease in mean blood pressure to 139/85 mmHg (one month post-ARB, P = 0.000) and 136/80 mmHg (last follow-up, P = 0.000), and a reduction in mean antihypertensive dosage to 1.4 DDD (one month post-ARB, P = 0.084) and 0.6 DDD (last follow-up, P = 0.000). Mean estimated glomerular filtration rate increased to 82 ml/min (P = 0.458) one month post-ARB, and 91 ml/min (P = 0.044) at last follow-up, respectively. The dialysis-dependent patient no longer required hemodialysis, and left ventricular dysfunction resolved in all of the three patients. CONCLUSION: ARB using the autologous saphenous vein graft is safe, effective and durable for treating TARAS.

[Effect of the size of abdominal aortic aneurysm on endovascular exclusion and its results].

OBJECTIVE: To evaluate the effect of the diameter of abdominal aortic aneurysm (AAA) on endovascular exclusion (EVE) and its results. METHODS: From March 1997 to June 2007, 429 AAA patients were treated with endovascular stent-graft exclusion. According to the maximal diameter of abdominal aortic aneurysm, the patients were divided into two groups: group A (diameter < 55 mm, n = 274) and group B (diameter > or = 55 mm, n = 155). The diameter of AAA, involvement of iliac artery, length, diameter and distortion of aneurismal neck in the two groups were recorded and compared retrospectively. RESULTS: Patients in group B were significantly older than group A (73.7 vs 71.1 years, P < 0.05). More patients in group B was complicated with coronary artery disease than those in group A (P < 0.05). The mean diameter of AAA in group A was (46.6 +/- 6.8) mm, and (66.8 +/- 11.2) mm in group B (P < 0.05). Proximal aneurysmal necks were shorter, wider and more tortuous in group B than those in group A (P < 0.05). Extraperitoneal approach, embolism of inner iliac artery and reconstruction of another inner iliac artery and stretch technique were more applied in group B. There were more endoleak during operation in group B and more stent-grafts were used. There was significant difference in morbidity rate between the two groups, while no statistic difference in mortality. And in group B, there were a high rate of endoleak and secondary intervention post operation. CONCLUSIONS: The diameter of AAA affects EVE and its results. In small aneurysms, EVE carries better outcome than in big aneurysms.

Gene expression pattern in apoptotic QGY-7703 cells induced by homoharringtonine.

AIM: To classify the genes responsible for apoptosis in QGY-7703 cells induced by homoharringtonine (HHT). METHODS: Apoptosis in QGY-7703 cells induced by HHT was demonstrated by DNA fragmentation and morphological observation. cDNA microarray technology was used to detect gene transcription, and the result of microarrays for genes was confirmed by RT-PCR. RESULTS: Seventy-eight individual mRNA were identified and their transcription levels changed significantly. Those genes, of which 68% were upregulated and 32% were downregulated, were partially related to apoptosis. They were mostly oncogenes, tumor suppressors, enzymes, and kinases. CONCLUSION: HHT is a potential drug in the treatment of liver cancer. TGF-beta, TNF, FAS, p38MAPK, and p53 apoptosis signaling pathways were activated during apoptosis in QGY-7703 cells. Such inducible genes may play important roles in apoptosis and deserve to be further studied.

Identification of genes responsive to apoptosis in HL-60 cells.

AIM: To identify genes responsive to apoptosis in HL-60 cells treated by homoharringtonine. METHODS: cDNA microarray technology was used to detect gene expression and the result of microarrays for genes (TIEG and VDUP1) was confirmed by Northern analysis. RESULTS: Seventy-five individual mRNAs whose mass changed significantly were identified. Among these genes (25 were up-regulated and 50 were down-regulated), most are known related to oncogenes and tumor suppressor. Some genes were involved in apoptosis signaling pathways. CONCLUSION: TGF beta and TNF apoptosis signaling pathways were initiated during apoptosis in HL-60 cells. TIEG and VDUP1 play important roles in mediating apoptosis.

[Endovascular stent-graft exclusion for Stanford B type aortic dissections: a report of 146 patients].

OBJECTIVE: To assess the operation indications, preoperative evaluation, technique essentials and clinical prospect of endovascular stent-graft exclusion for aortic dissection. METHODS: From September 1998 to April 2003, endovascular stent-graft exclusion for aortic dissection (Stanford B) was preformed in 146 patients. CTA or MRA were used as preoperative evaluation methods. Graft was constructed from self-expanding Z-stents covered with a woven Dacron polyester fabric graft (Talent). The stent-grafts were inserted from the femoral or iliac artery to exclude the tear of dissection, and all operations were performed under DSA guidance. RESULTS: The grafts were installed successfully in 145 patients. In 119 patients only proximal tears were excluded, and 26 patients who had both the proximal and distal tears were excluded. The mean follow-up period was 16 months (1 - 54 months). Six patients died within the perioperative period, 2 patients died during the follow-up, 2 patients had recurrence of aortic dissection (Stanford A) and cured by Bentall operation. The others were in good state. No accidents related to the dissection and operation occurred. CONCLUSIONS: Endovascular graft exclusion may be a safe and effective treatment for selected patients with thoracic aortic dissection. Endoleak may lead to aneurysmal expansion and rupture. Further follow-up is necessary to evaluate its long-term effect.