The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo.

We here examined the potential biological function of phosphoenolpyruvate carboxykinase 1 (PCK1) in angiogenesis. shRNA- or CRISPR-Cas9-induced PCK1 depletion potently inhibited endothelial cell proliferation, migration, sprouting, and tube formation, whereas ectopic PCK1 overexpression exerted opposite activity. In HUVECs, Gαi3 expression and Akt activation were decreased following PCK1 depletion, but were augmented by ectopic PCK1 overexpression. In vivo, retinal expression of PCK1 gradually increased from postnatal day 1 (P1) to P5. The intravitreous injection of endothelial-specific PCK1 shRNA adenovirus at P1 potently inhibited the radial extension of vascular plexus at P5. Conditional endothelial knockdown of PCK1 in adult mouse retina increased vascular leakage and the number of acellular capillaries while decreasing the number of RGCs in murine retinas. In diabetic retinopathy patients, PCK1 mRNA and protein levels were up-regulated in retinal tissues. Together, PCK1 is essential for angiogenesis possibly by mediating Gαi3 expression and Akt activation.

High alcohol-producing Klebsiella pneumoniae causes fatty liver disease through 2,3-butanediol fermentation pathway in vivo.

High alcohol-producing Klebsiella pneumoniae (HiAlc Kpn) in the gut microbiota had been demonstrated to be the causative agent of fatty liver disease (FLD). However, the catabolic pathways for alcohol production in vivo remain unclear. Here, we characterized the genome of HiAlc and medium alcohol-producing (MedAlc) Kpn and constructed an adh (an essential gene encoding alcohol dehydrogenase) knock-out HiAlc Kpn W14 strain (W14Δadh) using CRISPR-Cas9 system. Subsequently, we established the mouse model via gavage administration of HiAlc Kpn W14 and W14 Δadh strains, respectively. Proteome and metabolome analysis showed that 10 proteins and six major metabolites involved in the 2,3-butanediol fermentation pathway exhibited at least a three-fold change or greater during intestinal growth. Compared with HiAlc Kpn W14-fed mice, W14Δadh-fed mice with weak alcohol-producing ability did not show apparent pathological changes at 4 weeks, although some steatotic hepatocytes were observed at 12 weeks. Our data demonstrated that carbohydrate substances are catabolized to produce alcohol and 2,3-butanediol via the 2,3-butanediol fermentation pathway in HiAlc Kpn, which could be a promising clinical diagnostic marker. The production of high amounts of endogenous alcohol is responsible for the observed steatosis effects in hepatocytes in vivo.

Requirement of Gαi1 and Gαi3 in interleukin-4-induced signaling, macrophage M2 polarization and allergic asthma response.

IL-4 induces Akt activation in macrophages, required for full M2 (alternative) polarization. We examined the roles of Gαi1 and Gαi3 in M2 polarization using multiple genetic methods. Methods and Results: In MEFs and primary murine BMDMs, Gαi1/3 shRNA, knockout or dominant negative mutations attenuated IL-4-induced IL4Rα endocytosis, Gab1 recruitment as well as Akt activation, leaving STAT6 signaling unaffected. Following IL-4 stimulation, Gαi1/3 proteins associated with the intracellular domain of IL-4Rα and the APPL1 adaptor, to mediate IL-4Rα endosomal traffic and Gab1-Akt activation in BMDMs. In contrast, gene silencing of Gαi1/3 with shRNA or knockout resulted in BMDMs that were refractory to IL-4-induced M2 polarization. Conversely, Gαi1/3-overexpressed BMDMs displayed preferred M2 response with IL-4 stimulation. In primary human macrophages IL-4-induced Akt activation and Th2 genes expression were inhibited with Gαi1/3 silencing, but augmented with Gαi1/3 overexpression. In Gαi1/3 double knockout (DKO) mice, M2 polarization, by injection of IL-4 complex or chitin, was potently inhibited. Moreover, in a murine model of asthma, ovalbumin-induced airway inflammation and hyperresponsiveness were largely impaired in Gαi1/3 DKO mice. Conclusion: These findings highlight novel and essential roles for Gαi1/3 in regulating IL-4-induced signaling, macrophage M2 polarization and allergic asthma response.

Loss of GRB2 associated binding protein 1 in arteriosclerosis obliterans promotes host autophagy.

BACKGROUND: Arteriosclerosis obliterans (ASO) is a major cause of adult limb loss worldwide. Autophagy of vascular endothelial cell (VEC) contributes to the ASO progression. However, the molecular mechanism that controls VEC autophagy remains unclear. In this study, we aimed to explore the role of the GRB2 associated binding protein 1 (GAB1) in regulating VEC autophagy. METHODS: In vivo and in vitro studies were applied to determine the loss of adapt protein GAB1 in association with ASO progression. Histological GAB1 expression was measured in sclerotic vascular intima and normal vascular intima. Gain- and loss-of-function of GAB1 were applied in VEC to determine the effect and potential downstream signaling of GAB1. RESULTS: The autophagy repressor p62 was significantly downregulated in ASO intima as compared to that in healthy donor (0.80 vs. 0.20, t = 6.43, P < 0.05). The expression level of GAB1 mRNA (1.00 vs. 0.24, t = 7.41, P < 0.05) and protein (0.72 vs. 0.21, t = 5.97, P < 0.05) was significantly decreased in ASO group as compared with the control group. Loss of GAB1 led to a remarkable decrease in LC3II (1.19 vs. 0.68, t = 5.99, P < 0.05), whereas overexpression of GAB1 significantly led to a decrease in LC3II level (0.41 vs. 0.93, t = 7.12, P < 0.05). Phosphorylation levels of JNK and p38 were significantly associated with gain- and loss-of-function of GAB1 protein. CONCLUSION: Loss of GAB1 promotes VEC autophagy which is associated with ASO. GAB1 and its downstream signaling might be potential therapeutic targets for ASO treatment.

Open surgical treatment for rare epithelioid hemangioendothelioma of the common femoral vein.

Epithelioid hemangioendothelioma is a very rare disease affecting big blood vessels, especially veins. There have been very few articles describing the disease. We hereby present the case of a 56-year-old woman presenting with lower limb edema, who was initially being treated for residual thrombus in the common femoral vein but was eventually diagnosed with epithelioid hemangioendothelioma (EHE). The common femoral vein was resected and reconstructed using the external jugular vein. No additional therapy was administered. In this article, previous literature about EHE has been reviewed and oncologic principles have been discussed.

Therapeutic ultrasound plus pulsed electromagnetic field improves recovery from peripheral arterial disease in hypertension.

The objective of this investigation was to evaluate the therapy effect of combined therapeutic ultrasound (TUS) treatment and pulsed electromagnetic field (PEMF) therapy on angiogenesis in hypertension-related hindlimb ischemia. After subjecting excision of the left femoral artery, spontaneously hypertensive rats (SHRs) were randomly distributed to one of four groups: SHR; TUS treated SHR (SHR-TUS); PEMF treated SHR (PEMF-TUS); and TUS plus PEMF treated SHR (SHR-TUS-PEMF). Wistar-Kyoto rats (WKYs) with femoral artery excision were regarded as a control group. At day 14 after surgery, the TUS plus PEMF united administration had the greatest blood perfusion accompanied by elevated capillary density and the lowest TUNEL index. Interestingly, the united administration up-regulated the angiogenic factors expression of phosphorylated Akt (p-Akt), phosphorylated endothelial nitric oxide synthase (p-eNOS), vascular endothelial growth factor (VEGF), anti-apoptotic protein of Bcl-2 and down-regulated pro-apoptotic protein levels of Bax and Cleaved caspase-3 in vivo. Our results demonstrated that the united administration could significantly rescue hypertension-related inhibition of ischemia-induced neovascularization partly by promoting angiogenesis and inhibiting apoptosis.

Catheter-directed thrombolysis and stenting in the treatment of iliac vein compression syndrome with acute iliofemoral deep vein thrombosis: outcome and follow-up.

BACKGROUND: The purpose of this study was to evaluate the safety and effectiveness of catheter-directed thrombolysis (CDT) and stenting in the treatment of iliac vein compression syndrome (IVCS) with acute iliofemoral deep vein thrombosis (DVT). METHODS: A retrospective analysis was conducted in 61 patients (36 women, 25 men, age range 32-90 years, mean 64 years) who had IVCS with acute iliofemoral thrmobosis (≤10 days) and were treated by CDT and stenting between June 2006 and August 2011. All patients presented with IVCS with a median duration of 4.1 days and were treated with CDT (urokinase: initial dose of 125,000-250,000 U followed by 20,000-60,000 U/hr) followed by stent placement. Filters were implanted in those patients with existing pulmonary embolism (PE), inferior caval vein thrombosis, or in accordance with the patients' request. The patency, the pressure gradient crossing the stenosis of the iliac vein, both thigh and calf limb circumferences, and complications were assessed before and after CDT and stenting. A Duplex ultrasound was used to perform follow-up examinations at 1 month, 6 months, 1 year, 2 years, 3 years, 4 years, and 5 years after the operation. RESULTS: Three patients had PE before CDT as assessed by the computed tomography angiography. A total of 28 patients had a filter implanted (25 patients had a Cordis permanent filter and 3 patients had a Braun temporary filter). A total of 68 stents were implanted in 61 patients. Overall, the 1-month, 6-month, 1-year, 2-year, 3-year, and 5-year primary patency rates were 96.7%, 95.1%, 91.8%, 90.2%, 88.5%, and 85.2%, respectively. The pressure gradient crossing the stenosis of the iliac vein decreased significantly after CDT and stenting (7.22 ± 4.64 vs. 1.82 ± 2.78 cm H2O, P < 0.001). The reductions of thigh and calf circumferences were 66.7% (6.19 ± 2.67 vs. 1.98 ± 1.43 cm) and 61.6% (4.36 ± 2.10 vs. 1.46 ± 1.10 cm), respectively. Reocclusion occurred in 7 patients within 1-27 months. Four patients (7%) experienced minor bleeding and were treated successfully with sandbag compression. One patient felt light pain on the left waist after 3 months of stenting. No large hematoma, stent migration, or acute thrombosis complications occurred during the procedure. Two patients died from nonvascular causes during a follow-up of 2-62 months (mean, 31.0 months). Four patients were found with limb swelling and three patients felt heaviness. The incidence rate of postthrombotic syndrome was 11.5% (7/61). CONCLUSIONS: Treatment with CDT for IVCS with acute DVT achieves good patency and vein function after 5 years of follow-up in this study. However, further evidence is required to establish longer term benefits.

[Retrograde tibial approach for endovascular recanalization of popliteal and tibioperoneal artery occlusion].

OBJECTIVES: To describe a procedure of the retrograde approach for endovascular treatment of complex popliteal and/or infrapopliteal occlusions and to determine its safety and efficacy in minimizing failure rates. METHODS: Between January 2010 and March 2012, 28 patients (16 male and 12 female patients) received retrograde tibial approach after failure of antegrade intervention. There were 3 patients with severe claudication (Rutherford category 3) and 25 patients with critical limb ischemia (Rutherford category 4 to 6). From this group, two techniques were employed. Twenty-four patients were treated via a retrograde transpedal access site and 4 patients via a transcollateral loop technique. The clinical and follow-up data of these patients were analyzed retrospectively. RESULTS: The technique success rates were 92.8% (26/28). No major complications and 3 (10.7%) minor sequelaes were documented in this study. Twenty-three patients were followed up for 3 to 29 months, with a mean of (14 ± 9) months. Overall patency was 73.9% (17/23) and 47.8% (11/23) at 6 and 12 months. Overall survival and limb salvage was 95.7% (22/23), ulcer were healed in 9/10 patients. CONCLUSION: The use of retrograde tibial or pedal approach seems feasible and safety in case of failure in antegrade revascularization of popliteal and/or infrapopliteal occlusions.

[Molecular mechanisms of antithrombin deficiency caused by novel double heterozygous mutations].

OBJECTIVE: To explore the molecular mechanisms of antithrombin (AT) deficiency caused by novel double heterozygous mutations. METHODS: Wild-type and mutant AT cDNA expression plasmids (ATwt, AT-c.134G > A, AT-c.342T > G, AT-c.134G > A and AT-c.342T > G) were transfected into HEK293T cells. Western blot was used to detect the AT:Ag in cell lysates. Homology was used to reestablish 3-D spatial structure of AT. Laser confocal assay was utilized to analyze the distribution of AT in endoplasmic reticulum (ER). RESULTS: Compared to the wild-types, the AT expression of HEK293T cells sharply increased when they were transfected by AT-c.342T > G or AT-c.134G > A and c.342T > G. Homology modeling showed that the mutation (AT-c.342T > G) caused a decreased distance between Arg and surrounding bases as Arg's side chain was significantly longer than Ser's. The mutation of 13th base pair decreases the distance between AT and heparin. Laser confocal assay showed that the AT protein concreted in HEK293T cells when they were transfected by mutant plasmids (AT-c.134G > A and c.342T > G) and aggregated in ER. CONCLUSIONS: The main molecular mechanism of AT deficiency in this pedigree is the disturbed AT secretion as a result of the mutation of AT-c.342T > G.