Mingyang Deng

Deregulation of Mitochondrial ATPsyn-β in Acute Myeloid Leukemia Cells and with Increased Drug Resistance

The mechanisms underlying the development of multidrug resistance in acute myeloid leukemia are not fully understood. Here we analyzed the expressions of mitochondrial ATPsyn-β in adriamycin-resistant cell line HL-60/ADM and its parental cell line HL-60. Meanwhile we compared the differences of mitochondrial ATPsyn-β expression and ATP synthase activity in 110 acute myeloid leukemia (AML, non-M3) patients between relapsed/refractory and those in remission. Our results showed that down-regulation of ATPsyn-β expression by siRNA in HL-60 cells increased cell viability and apoptotic resistance to adriamycin, while up-regulation of mitochondrial ATPsyn-β in HL-60/ADM cells enhanced cell sensitivity to adriamycin and promoted apoptosis. Mitochondrial ATPsyn-β expression and ATP synthase activity in relapsed/refractory acute myeloid leukemia patients were downregulated. This downregulated ATPsyn-β expression exhibited a positive correlation with the response to adriamycin of primary cells. A lower expression of ATPsyn-β in newly diagnosed or relapsed/refractory patients was associated with a shorter first remission duration or overall survival. Our findings show mitochondrial ATPsyn-β plays an important role in the mechanism of multidrug resistance in AML thus may present both a new marker for prognosis assessment and a new target for reversing drug resistance.

An intron mutation in the ACVRL1 may be associated with a transcriptional regulation defect in a Chinese family with hereditary hemorrhagic telangiectasia.

Purpose To identify a novel pathogenic gene mutation present in a Chinese family with hereditary hemorrhagic telangiectasia (HHT) and to determine if an intron mutation may influence the transcriptional activity of the ACVRL1 gene. Methods HHT family members were ascertained following the presentation of proband and involved subjects. All family members (n = 5) and 113 healthy individuals were genotyped for the variant in intron 6 c.772+27G>C of ACVRL1 gene. The genomic structure of ACVRL1 in affected HHT patients and healthy individuals was determined by long range PCR and sequencing. The expression of ACVRL1 mRNA and protein in patients with HHT was evaluated using real-time polymerase chain reaction and immunoblot analysis. Luciferase activity assay and electrophoretic mobility shift assay (EMSA) were performed to uncover the mechanism of intron-related transcriptional regulation. Results Only one novel mutation in intron 6 (c.772+27G>C) of ACVRL1 gene, no other mutation, abnormal splice, gross genomic deletion or rearrangement was found in this HHT2 family. Compared with healthy individuals, ACVRL1 mRNA and protein were significantly decreased in affected HHT2 individuals. Luciferase activity assay demonstrated that the transcriptional activity of the mutated ACVRL1 was significantly lower than that of the wild-type of intron 6; EMSA results showed that intron 6 c.772+27G>C mutation was able to inhibit the binding of transcriptional factor Sp1. Conclusions A novel intron mutation in ACVRL1 gene is associated with familial HHT2. The mechanisms may be involved in the down-regulation of ACVRL1 gene transcription.

Thrombotic thrombocytopenic purpura complicating Graves disease: dramatic response to plasma exchange and infusion.

Dear Sir, Thrombotic thrombocytopenic purpura (TTP) is an uncommon but fatal disease if untreated. It is associated with microangiopathic hemolytic anaemia, thrombocytopenia and microvascular thrombosis that results in variable injury of the central nervous system, kidney and other organs. Most cases are caused by autoantibodies to ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and inhibits VWF-dependent platelet aggregation (Kenneth et al., 2010). TTP can complicate autoimmune diseases such as systemic lupus erythematosus, Sjögren’s syndrome and polymyositis (Noda et al., 1990). TTP as major presentation of Graves disease is rare. There is only one documented case in the literature, searched via Pubmed, associating these two disorders (Chaar et al., 2007). Here we report a second case of Graves disease presenting with TTP having dramatic response to plasma exchange and infusion. A 51-year-old female was admitted to our hospital in November 2008 for a 10-day history of fatigue, dizziness and confusion. Ten days before admission, she had a sudden onset of fatigue, dizziness and palpitations but no fever, diarrhoea or oliguria. On examination she had jaundice, petechiae, goitre and proptosis. Further inquiry revealed a 2 year history of weight loss with increased appetite. Laboratory tests showed hemoglobin (Hb) of 57 g/L, platelet count of 11 × 109/L, reticulocytes of 12%, total bilirubin (TBIL) of 88·3 μmol/L (reference 5·0–16·7 μmol/L) and direct bilirubin (DBIL) of 11·8 μmol/L (0·0–5·7 μmol/L). Thyroid function tests showed FT3 of 11·3 pg/mL (1·45–3·48 pg/mL), FT4 of 3·7 ng/dL (0·71–1·85 ng/dL) and TSH of 0·001 μIU/mL (0·47–4·64 μIU/mL). Ultrasonic scan of the thyroid revealed diffuse enlargement. Hyperthyroidism was diagnosed. Therefore, propylthiouracil was commenced a week before together with treatment of transfusion with packed RBC of 3 Units and platelet suspension of 6 Units to prevent cerebral bleeding. However, after transfusion she developed slight fever around 38 ◦C and episodes of confusion persisting for 30 min to

Pretreatment with 17β-Estradiol Attenuates Cerebral Ischemia-Induced Blood-Brain Barrier Disruption in Aged Rats: Involvement of Antioxidant Signaling

Background/Aims: Blood-brain barrier (BBB) disruption is often induced in brain injury and particularly associated with cerebral ischemia in stroke. Estradiol (17β-estradiol 3-benzoate, E2), an endogenous steroid hormone, has been reported to play a protective role against cerebrovascular pathologies. Here we aimed to determine the potential effects of E2 on the integrity of BBB and brain damage following middle cerebral artery occlusion (MCAO). Methods: Aged (24- month-old) ovariectomized female rats were administered E2 for 2 months through daily intraperitoneal injections prior to induction of MCAO. Results: Compared to vehicle controls, E2 had a dose-dependent effect in reducing the severity of brain injuries while maintaining the integrity of the BBB in aged rats. The neuroprotective effects of E2 were associated with the rescued tight junction loss, decreased oxidative stress, and attenuated ERK activation in the ischemic brains. Conclusion: Our data suggest a beneficial role of E2 in aged stroke patients, associated with a protective effect of E2 against BBB disruption in aging-related brain ischemia.

Role of glycogen synthase kinase 3 in ischemia-induced blood–brain barrier disruption in aged female rats

Estrogen receptors have protective effects against ischemic brain injury. However, the molecular mechanisms underlying this phenomenon have yet to be well studied. Given that inhibition of glycogen synthase kinase (GSK3) can reduce cerebral ischemia/reperfusion injury, we hypothesized that estrogen receptors‐mediated protective effects against ischemia‐induced blood–brain barrier (BBB) disruption involve inhibition of GSK3. Thus, we evaluated GSK3 expression in the brain of ovariectomized female rats, and examined the effects of intracerebroventricular pre‐treatments of SB216763, GSK3 inhibitor, on BBB permeability following middle cerebral artery occlusion (MCAO). We also examined the role of specific estrogen receptor subtype in regulation of GSK3 expression and BBB permeability after MCAO. We found that ovariectomized female rats exhibited increased mRNA levels of estrogen receptor α (ERα) and estrogen receptor β (ERβ), and increased protein levels of GSK3β but not GSK3α in brain cortical areas. Furthermore, intracerebroventricular pre‐treatments of SB216763 dose‐dependently attenuated brain infarction volume, brain water contents, neurological deficits, and BBB disruption, and increased tight junction protein ZO‐1 and occludin expression at 24 h following MCAO. Finally, activation of ERβ but not ERα dose‐dependently decreased GSK3β expression at 24 h following MCAO. This was associated with increased tight junction protein expression and improved neurological scores. Thus, our study suggested that activation of ERβ may protect against brain ischemia‐induced BBB disruption by inhibiting GSK3β‐mediated signaling.

Mesenchymal Stem Cell-Derived Extracellular Vesicles Ameliorates Hippocampal Synaptic Impairment after Transient Global Ischemia

Recent studies have found that administration of stem cells or extracellular vehicles (EVs) derived from stem cells exert neuroprotective effects after transient global ischemia. However, the underlying mechanisms of this effect remain unclear, especially at the level of synaptic functions. In this study, we compared the suppressive effects on cyclooxygenase-2 (COX-2) upregulation by EVs derived from bone marrow mesenchymal stem cells (BMSC-EV), adipose tissue MSC (AdMSC-EV) and serum (serum-EV). Then we examined whether BMSC-EVs could restore functional integrity of synaptic transmission and plasticity. Mice were randomly assigned to four groups: sham, sham with EV treatment, ischemia and ischemia with EV treatment. EVs were administered by intracerebroventricular injection (ICVI). We examined the consequence of transient global ischemia on pre- and post-synaptic functions of the hippocampal CA3-CA1 synapses at basal level, and long-term potentiation (LTP), an activity-dependent form of synaptic plasticity. Then we tested the therapeutic effects of EVs on these synaptic deficits. Meanwhile, Morris water maze (MWM) test was performed to examine the efficacy of EVs in rescuing ischemia-induced impairments in spatial learning and memory. EV treatment significantly restored impaired basal synaptic transmission and synaptic plasticity, and improved spatial learning and memory compared with the control group. In addition, EVs significantly inhibited ischemia-induced pathogenic expression of COX-2 in the hippocampus. EVs exert ameliorating effects on synaptic functions against transient global cerebral ischemia, which may be partly attributed to suppression of COX-2 pathogenic expression.

Hypermethylation of CpG sites at the promoter region is associated with deregulation of mitochondrial ATPsyn-β and chemoresistance in acute myeloid leukemia

BACKGROUND Aberrant DNA methylation status of some genes has been shown to be involved in chemoresistance of acute myeloid leukemia (AML). We have recently found that down-regulation of the β subunit of mitochondrial ATP synthase (ATPsyn-β) leads to adriamycin resistance in acute and chronic myeloid leukemia cells, and hypermethylation of the ATPsyn-β gene promoter is associated with chemoresistance in chronic myeloid leukemia. OBJECTIVE To further investigate the relationship between methylation of ATPsyn-β gene, mRNA expression as well as chemoresistance in AML. METHODS Quantitative RT-PCR and methylation specific PCR were performed to assess mRNA expression and methylation status of ATPsyn-β gene on primary bone marrow nuclear cells (BMMCs), and cell proliferation assay was used to determine the sensitivity of BMMCs to adriamycin. RESULTS Hypermethylation status of ATPsyn-β gene promoter existed in those relapsed/refractory AML patients, and this hypermethylation of the gene was associated with a suppressed mRNA expression levels. Four patients at diagnosis and relapse underwent gene methylation status shift from hypermethylation to hypomethylation, which was accompanied by reduced mRNA expression of the gene. 5-azacitidine(5-Aza)- a demethylating agent, could restore ATPsyn-β mRNA expression and increase the adriamycin sensitivity of primary leukemic cells from seven relapsed/ refractory AML patients. CONCLUSIONS Hypermethylation of ATPsyn-β gene promoter is associated with a down-regulated mRNA expression and chemoresistance in AML patients.

Analysis of clinical and laboratory characteristics in 42 patients with thrombotic thrombocytopenic purpura from a single center in China.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by microvascular platelet deposition and thrombus formation with resulting microangiopathic hemolytic anemia. Deficiency of the von Willebrand factor cleavage protease, also known as ADAMTS 13, has been implicated as an important etiological factor in TTP. Little studies were obtained on Chinese patients with TTP until now. Our aim was to analyze the clinical features, outcome and laboratory characteristics of Chinese TTP patients, and determine whether plasma ADAMTS 13 activity is decreased in TTP and its diagnostic value for TTP. Forty-two TTP patients (29 females; 13 males) admitted to our hospital from 1998 to 2010 were analyzed. There were 34 patients (81%) with the triad of TTP, including hemolytic anemia, thrombocytopenia and neurologic abnormalities; 7 (16.7%) had the classical pentad of TTP. Major etiologic factors were acquired autoimmunological abnormalities (31%); no familial TTP was identified in this series. The schistocytes of peripheral blood smears were present in all cases with a mean frequency of 4.6% (range from 0.3% to 13.4%). Plasma ADAMTS 13 activity was determined in 22 patients with the FRET-vWF86 assay. Only 4 idiopathic TTP patients (18.2%) had severe ADAMTS 13 deficiency (activity<10%); 9 (40.9%) had moderate decrease of ADAMTS 13 activity (activity: 10-40%); another 9 (40.91%) had normal ADAMTS 13 activity (>40%). T lymphocyte subpopulation was measured in 23 TTP patients with FACS Calibur; 14 of the 23 (60.9%) had significantly decreased CD4 cells count and CD4/CD8 ratio, suggesting cellular immune dysfunction may be involved in the pathogenesis of TTP. In the studies, plasmapheresis is the main therapeutic method. 26 of 31 patients (83.9%) accepting plasmapheresis achieved complete remission; those patients who only underwent plasma infusion had low remission rate (18.2%) and high mortality (9/11; 81.8%). Four patients with packed RBC infusion manifested transient exacerbation of neurologic or psychiatric symptoms. In conclusion, the diagnosis of TTP in China is still based on clinical features including evidence of microangiopathic hemolysis. Severe ADAMTS 13 activity deficiency might be a valuable indicator for idiopathic TTP diagnosis. Further studies are needed to determine the real value of ADAMTS 13 activity for TTP diagnosis and whether T lymphocytes subset dysregulation plays important role in TTP pathogenesis.

Preservation of neuronal functions by exosomes derived from different human neural cell types under ischemic conditions

Stem cell‐based therapies have been reported in protecting cerebral infarction‐induced neuronal dysfunction and death. However, most studies used rat/mouse neuron as model cell when treated with stem cell or exosomes. Whether these findings can be translated from rodent to humans has been in doubt. Here, we used human embryonic stem cell‐derived neurons to detect the protective potential of exosomes against ischemia. Neurons were treated with in vitro oxygen–glucose deprivation (OGD) for 1 h. For treatment group, different exosomes were derived from neuron, embryonic stem cell, neural progenitor cell and astrocyte differentiated from H9 human embryonic stem cell and added to culture medium 30 min after OGD (100 μg/mL). Western blotting was performed 12 h after OGD, while cell counting and electrophysiological recording were performed 48 h after OGD. We found that these exosomes attenuated OGD‐induced neuronal death, Mammalian target of rapamycin (mTOR), pro‐inflammatory and apoptotic signaling pathway changes, as well as basal spontaneous synaptic transmission inhibition in varying degrees. The results implicate the protective effect of exosomes on OGD‐induced neuronal death and dysfunction in human embryonic stem cell‐derived neurons, potentially through their modulation on mTOR, pro‐inflammatory and apoptotic signaling pathways.

Antithrombin Cambridge II(A384S) mutation frequency and antithrombin activity levels in 120 of deep venous thrombosis and 150 of cerebral infarction patients in a single center in Southern China

Antithrombin Cambridge II(A384S) mutation shows a relatively high frequency in western population. Some studies suggest that the mutation is an independent genetic risk factor both for deep vein thrombosis (DVT) and for arterial thrombosis, but whether the mutation has racial difference or has a general significance for thrombophilia remains unclear. In this study we performed an analysis of the prevalence of the mutation in Chinese southern population; Also, the antithrombin activity levels were evaluated in each investigated individual. The studies included 120 patients with DVT, 150 patients with cerebral infarction, and 110 controls. The mutation was detected using polymerase chain reaction/PvuII restrictive fragment length polymorphism procedures. Antithrombin activity assay was done using chromogenic substrate method. The results showed that no antithrombin Cambridge II mutation was detected in all three groups (DVT, cerebral infarction and controls), the incidence was 0/380. Plasma antithrombin activity was 91.37% ± 16.15% in the DVT patients and 102.68% ± 13.10% in the controls; the antithrombin activity was significantly reduced in the DVT group (P < 0.0001). In DVT patients, eight cases were identified as primary antithrombin deficiency, accounting for an incidence of 6.7%. No significant difference was found for antithrombin activity between cerebral infarction group and controls. These results suggest that antithrombin Cambridge II mutation has a racial difference, and may not be a valuable risk factor of thrombophilia in Asian population, and antithrombin deficiency remains a major genetic risk factor for DVT patients in China.