Browsing by Author "Xie, Jiahua"

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    Asialo-rhuEPO as a Potential Neuroprotectant for Ischemic Stroke Treatment
    Kittur, Farooqahmed S.; Hung, Chiu-Yueh; Li, P. Andy; Sane, David C.; Xie, Jiahua (MDPI, 2023-04-18)
    Neuroprotective drugs to protect the brain against cerebral ischemia and reperfusion (I/R) injury are urgently needed. Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been demonstrated to have excellent neuroprotective functions in preclinical studies, but its neuroprotective properties could not be consistently translated in clinical trials. The clinical failure of rhuEPOM was thought to be mainly due to its erythropoietic activity-associated side effects. To exploit its tissue-protective property, various EPO derivatives with tissue-protective function only have been developed. Among them, asialo-rhuEPO, lacking terminal sialic acid residues, was shown to be neuroprotective but non-erythropoietic. Asialo-rhuEPO can be prepared by enzymatic removal of sialic acid residues from rhuEPOM (asialo-rhuEPOE) or by expressing human EPO gene in glycoengineered transgenic plants (asialo-rhuEPOP). Both types of asialo-rhuEPO, like rhuEPOM, displayed excellent neuroprotective effects by regulating multiple cellular pathways in cerebral I/R animal models. In this review, we describe the structure and properties of EPO and asialo-rhuEPO, summarize the progress on neuroprotective studies of asialo-rhuEPO and rhuEPOM, discuss potential reasons for the clinical failure of rhuEPOM with acute ischemic stroke patients, and advocate future studies needed to develop asialo-rhuEPO as a multimodal neuroprotectant for ischemic stroke treatment.
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    Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants
    Kittur, Farooqahmed S.; Bah, Mamudou; Archer-Hartmann, Stephanie; Hung, Chiu-Yueh; Azadi, Parastoo; Ishihara, Mayumi; Sane, David C.; Xie, Jiahua (PLOS, 2013-10-04)
    Asialo-erythropoietin, a desialylated form of human erythropoietin (EPO) lacking hematopoietic activity, is receiving increased attention because of its broader protective effects in preclinical models of tissue injury. However, attempts to translate its protective effects into clinical practice is hampered by unavailability of suitable expression system and its costly and limit production from expensive mammalian cell-made EPO (rhuEPOM) by enzymatic desialylation. In the current study, we took advantage of a plant-based expression system lacking sialylating capacity but possessing an ability to synthesize complex N-glycans to produce cytoprotective recombinant human asialo-rhuEPO. Transgenic tobacco plants expressing asialo-rhuEPO were generated by stably co-expressing human EPO and β1,4-galactosyltransferase (GalT) genes under the control of double CaMV 35S and glyceraldehyde-3-phosphate gene (GapC) promoters, respectively. Plant-produced asialo-rhuEPO (asialo-rhuEPOP) was purified by immunoaffinity chromatography. Detailed N-glycan analysis using NSI-FTMS and MS/MS revealed that asialo-rhuEPOP bears paucimannosidic, high mannose-type and complex N-glycans. In vitro cytoprotection assays showed that the asialo-rhuEPOP (20 U/ml) provides 2-fold better cytoprotection (44%) to neuronal-like mouse neuroblastoma cells from staurosporine-induced cell death than rhuEPOM (21%). The cytoprotective effect of the asialo-rhuEPOP was found to be mediated by receptor-initiated phosphorylation of Janus kinase 2 (JAK2) and suppression of caspase 3 activation. Altogether, these findings demonstrate that plants are a suitable host for producing cytoprotective rhuEPO derivative. In addition, the general advantages of plant-based expression system can be exploited to address the cost and scalability issues related to its production.
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    Plant-Produced Asialo-Erythropoietin Restores Pancreatic Beta-Cell Function by Suppressing Mammalian Sterile-20-like Kinase (MST1) and Caspase-3 Activation
    Arthur, Elena; Kittur, Farooqahmed S.; Lin, Yuan; Hung, Chiu-Yueh; Sane, David C.; Xie, Jiahua (Frontiers, 2017-04-19)
    Pancreatic beta-cell death adversely contributes to the progression of both type I and II diabetes by undermining beta-cell mass and subsequently diminishing endogenous insulin production. Therapeutics to impede or even reverse the apoptosis and dysfunction of beta-cells are urgently needed. Asialo-rhuEPO, an enzymatically desialylated form of recombinant human erythropoietin (rhuEPO), has been shown to have cardioprotective and neuroprotective functions but with no adverse effects like that of sialylated rhuEPO. Heretofore, the anti-apoptotic effect of asialo-rhuEPO on pancreatic beta-cells has not been reported. In the current study, we investigated the cytoprotective properties of plant-produced asialo-rhuEPO (asialo-rhuEPO(P)) against staurosporine-induced cell death in the pancreatic beta-cell line RIN-m5F. Our results showed that 60 IU/ml asialo-rhuEPO(P) provided 41% cytoprotection while 60 IU/ml rhuEPO yielded no effect. Western blotting results showed that asialo-rhuEPO(P) treatment inhibited both MST1 and caspase-3 activation with the retention of PDX1 and insulin levels close to untreated control cells. Our study provides the first evidence indicating that asialo-rhuEPO(P)-mediated protection involves the reduction of MST1 activation, which is considered a key mediator of apoptotic signaling in beta-cells. Considering the many advantages its plant-based expression, asialo-rhuEPO(P) could be potentially developed as a novel and inexpensive agent to treat or prevent diabetes after further performing studies in cell-based and animal models of diabetes.
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    Quantitative Proteomics Reveals the Beneficial Effects of Low Glucose on Neuronal Cell Survival in an in vitro Ischemic Penumbral Model
    Li, Hua; Kittur, Farooqahmed S.; Hung, Chiu-Yueh; Li, P. Andy; Ge, Xinghong; Sane, David C.; Xie, Jiahua (2020-09-01)
    Understanding proteomic changes in the ischemic penumbra are crucial to rescue those salvageable cells and reduce the damage of an ischemic stroke. Since the penumbra region is dynamic with heterogeneous cells/tissues, tissue sampling from animal models of stroke for the molecular study is a challenge. In this study, cultured hippocampal HT22 cells under hypoxia treatment for 17.5 h with 0.69 mM low glucose (H+LG) could mimic ischemic penumbral cells since they had much higher cell viability and viable cell number compared to hypoxia without glucose (H-G) treatment. To validate established cell-based ischemic penumbral model and understand the beneficial effects of low glucose (LG), quantitative proteomics analysis was performed on H+LG, H-G, and normoxia with normal 22 mM glucose (N+G) treated cells. We identified 427 differentially abundant proteins (DAPs) between H-G and N+G and further identified 105 DAPs between H+LG and H-G. Analysis of 105 DAPs revealed that LG promotes cell survival by activating HIF1 alpha to enhance glycolysis; preventing the dysregulations of extracellular matrix remodeling, cell cycle and division, and antioxidant and detoxification; as well as attenuating inflammatory reaction response, protein synthesis and neurotransmission activity. Our results demonstrated that this established cell-based system could mimic penumbral conditions and can be used for molecular studies.