Won Hee Lee

Irradiation induces regionally specific alterations in pro- inflammatory environments in rat brain

Purpose: Pro-inflammatory environments in the brain have been implicated in the onset and progression of neurological disorders. In the present study, we investigate the hypothesis that brain irradiation induces regionally specific alterations in cytokine gene and protein expression. Materials and methods: Four month old F344 × BN rats received either whole brain irradiation with a single dose of 10 Gy γ-rays or sham-irradiation, and were maintained for 4, 8, and 24 h following irradiation. The mRNA and protein expression levels of pro-inflammatory mediators were analysed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining. To elucidate the molecular mechanisms of irradiation-induced brain inflammation, effects of irradiation on the DNA-binding activity of pro-inflammatory transcription factors were also examined. Results: A significant and marked up-regulation of mRNA and protein expression of pro-inflammatory mediators, including tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1), was observed in hippocampal and cortical regions isolated from irradiated brain. Cytokine expression was regionally specific since TNF-α levels were significantly elevated in cortex compared to hippocampus (57% greater) and IL-1β levels were elevated in hippocampus compared to cortical samples (126% greater). Increases in cytokine levels also were observed after irradiation of mouse BV-2 microglial cells. A series of electrophoretic mobility shift assays (EMSA) demonstrated that irradiation significantly increased activation of activator protein-1 (AP-1), nuclear factor-κB (NF-κB), and cAMP response element-binding protein (CREB). Conclusion: The present study demonstrated that whole brain irradiation induces regionally specific pro-inflammatory environments through activation of AP-1, NF-κB, and CREB and overexpression of TNF-α, IL-1β, and MCP-1 in rat brain and may contribute to unique pathways for the radiation-induced impairments in tissue function.

Cross Talk of Combined Gene and Cell Therapy in Ischemic Heart Disease Role of Exosomal MicroRNA Transfer

Background— Despite the promise shown by stem cells for restoration of cardiac function after myocardial infarction, the poor survival of transplanted cells has been a major issue. Hypoxia-inducible factor-1 (HIF1) is a transcription factor that mediates adaptive responses to ischemia. Here, we hypothesize that codelivery of cardiac progenitor cells (CPCs) with a nonviral minicircle plasmid carrying HIF1 (MC-HIF1) into the ischemic myocardium can improve the survival of transplanted CPCs. Methods and Results— After myocardial infarction, CPCs were codelivered intramyocardially into adult NOD/SCID mice with saline, MC-green fluorescent protein, or MC-HIF1 versus MC-HIF1 alone (n=10 per group). Bioluminescence imaging demonstrated better survival when CPCs were codelivered with MC-HIF1. Importantly, echocardiography showed mice injected with CPCs+MC-HIF1 had the highest ejection fraction 6 weeks after myocardial infarction (57.1±2.6%; P=0.002) followed by MC-HIF1 alone (48.5±2.6%; P=0.04), with no significant protection for CPCs+MC-green fluorescent protein (44.8±3.3%; P=NS) when compared with saline control (38.7±3.2%). In vitro mechanistic studies confirmed that cardiac endothelial cells produced exosomes that were actively internalized by recipient CPCs. Exosomes purified from endothelial cells overexpressing HIF1 had higher contents of miR-126 and miR-210. These microRNAs activated prosurvival kinases and induced a glycolytic switch in recipient CPCs, giving them increased tolerance when subjected to in vitro hypoxic stress. Inhibiting both of these miRs blocked the protective effects of the exosomes. Conclusions— In summary, HIF1 can be used to modulate the host microenvironment for improving survival of transplanted cells. The exosomal transfer of miRs from host cells to transplanted cells represents a unique mechanism that can be potentially targeted for improving survival of transplanted cells.

Microfluidic Single-Cell Analysis Shows That Porcine Induced Pluripotent Stem Cell–Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

Rationale: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. Objective: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. Methods and Results: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. Conclusions: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

HIF-1 reduces ischaemia–reperfusion injury in the heart by targeting the mitochondrial permeability transition pore

AIMS Hypoxia-inducible factor-1 (HIF-1) has been reported to promote tolerance against acute myocardial ischaemia-reperfusion injury (IRI). However, the mechanism through which HIF-1 stabilization actually confers this cardioprotection is not clear. We investigated whether HIF-1α stabilization protects the heart against acute IRI by preventing the opening of the mitochondrial permeability transition pore (MPTP) and the potential mechanisms involved. METHODS AND RESULTS Stabilization of myocardial HIF-1 was achieved by pharmacological inhibition of prolyl hydroxylase (PHD) domain-containing enzyme using GSK360A or using cardiac-specific ablation of von Hippel-Lindau protein (VHL(fl/fl)) in mice. Treatment of HL-1 cardiac cells with GSK360A stabilized HIF-1, increased the expression of HIF-1 target genes pyruvate dehydrogenase kinase-1 (PDK1) and hexokinase II (HKII), and reprogrammed cell metabolism to aerobic glycolysis, thereby resulting in the production of less mitochondrial oxidative stress during IRI, and less MPTP opening, effects which were shown to be dependent on HKII. These findings were further confirmed when HIF-1 stabilization in the rat and murine heart resulted in smaller myocardial infarct sizes (both in vivo and ex vivo), decreased mitochondrial oxidative stress, and inhibited MPTP opening following IRI, effects which were also found to be dependent on HKII. CONCLUSIONS We have demonstrated that acute HIF-1α stabilization using either a pharmacological or genetic approach protected the heart against acute IRI by promoting aerobic glycolysis, decreasing mitochondrial oxidative stress, activating HKII, and inhibiting MPTP opening.

Microfluidic Single Cell Analysis Show Porcine Induced Pluripotent Stem Cell–Derived Endothelial Cells Improve Myocardial Function by Paracrine Activation

Rationale: Induced pluripotent stem cells (iPSCs) hold great promise for the development of patient-specific therapies for cardiovascular disease. However, clinical translation will require preclinical optimization and validation of large-animal iPSC models. Objective: To successfully derive endothelial cells from porcine iPSCs and demonstrate their potential utility for the treatment of myocardial ischemia. Methods and Results: Porcine adipose stromal cells were reprogrammed to generate porcine iPSCs (piPSCs). Immunohistochemistry, quantitative PCR, microarray hybridization, and angiogenic assays confirmed that piPSC-derived endothelial cells (piPSC-ECs) shared similar morphological and functional properties as endothelial cells isolated from the autologous pig aorta. To demonstrate their therapeutic potential, piPSC-ECs were transplanted into mice with myocardial infarction. Compared with control, animals transplanted with piPSC-ECs showed significant functional improvement measured by echocardiography (fractional shortening at week 4: 27.2±1.3% versus 22.3±1.1%; P<0.001) and MRI (ejection fraction at week 4: 45.8±1.3% versus 42.3±0.9%; P<0.05). Quantitative protein assays and microfluidic single-cell PCR profiling showed that piPSC-ECs released proangiogenic and antiapoptotic factors in the ischemic microenvironment, which promoted neovascularization and cardiomyocyte survival, respectively. Release of paracrine factors varied significantly among subpopulations of transplanted cells, suggesting that transplantation of specific cell populations may result in greater functional recovery. Conclusions: In summary, this is the first study to successfully differentiate piPSCs-ECs from piPSCs and demonstrate that transplantation of piPSC-ECs improved cardiac function after myocardial infarction via paracrine activation. Further development of these large animal iPSC models will yield significant insights into their therapeutic potential and accelerate the clinical translation of autologous iPSC-based therapy.

Protective effects of genistein on proinflammatory pathways in human brain microvascular endothelial cells.

Proinflammatory cerebromicrovascular environment has been implicated in the critical early pathologic events in a variety of neurodegenerative diseases. Recent studies also have demonstrated the potential beneficial effects of soy isoflavones. However, cellular and molecular mechanisms underlying these processes are not fully understood. The present study was designed to examine the hypothesis that soy isoflavone genistein may attenuate cytokine-induced proinflammatory pathways in human brain microvascular endothelial cells. The quantitative real-time reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay showed that pretreatment of HBMEC with increasing concentrations of genistein significantly and dose-dependently inhibited cytokine-induced up-regulation of mRNA and protein expression of proinflammatory mediators such as tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, interleukin-8, and intercellular adhesion molecule-1. In addition, genistein pretreatment significantly reduced cytokine-mediated up-regulation of transmigration of blood leukocytes in a dose-dependent manner. Our results suggest that genistein may attenuate proinflammatory pathways through inhibition of cytokine-induced overexpression of proinflammatory mediators and inflammatory reactions in human brain microvascular endothelial cells.

Irradiation Alters MMP-2/TIMP-2 System and Collagen Type IV Degradation in Brain

PURPOSE Blood-brain barrier (BBB) disruption is one of the major consequences of radiation-induced normal tissue injury in the central nervous system. We examined the effects of whole-brain irradiation on matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) and extracellular matrix (ECM) degradation in the brain. METHODS AND MATERIALS Animals received either whole-brain irradiation (a single dose of 10 Gy γ-rays or a fractionated dose of 40 Gy γ-rays, total) or sham-irradiation and were maintained for 4, 8, and 24 h following irradiation. mRNA expression levels of MMPs and TIMPs in the brain were analyzed by real-time reverse transcriptase-polymerase chain reaction (PCR). The functional activity of MMPs was measured by in situ zymography, and degradation of ECM was visualized by collagen type IV immunofluorescent staining. RESULTS A significant increase in mRNA expression levels of MMP-2, MMP-9, and TIMP-1 was observed in irradiated brains compared to that in sham-irradiated controls. In situ zymography revealed a strong gelatinolytic activity in the brain 24 h postirradiation, and the enhanced gelatinolytic activity mediated by irradiation was significantly attenuated in the presence of anti-MMP-2 antibody. A significant reduction in collagen type IV immunoreactivity was also detected in the brain at 24 h after irradiation. In contrast, the levels of collagen type IV were not significantly changed at 4 and 8 h after irradiation compared with the sham-irradiated controls. CONCLUSIONS The present study demonstrates for the first time that radiation induces an imbalance between MMP-2 and TIMP-2 levels and suggests that degradation of collagen type IV, a major ECM component of BBB basement membrane, may have a role in the pathogenesis of brain injury.

Microfluidic Single-Cell Analysis of Transplanted Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes After Acute Myocardial Infarction

Background— Human induced pluripotent stem cells (iPSCs) are attractive candidates for therapeutic use, with the potential to replace deficient cells and to improve functional recovery in injury or disease settings. Here, we test the hypothesis that human iPSC-derived cardiomyocytes (iPSC-CMs) can secrete cytokines as a molecular basis to attenuate adverse cardiac remodeling after myocardial infarction. Methods and Results— Human iPSCs were generated from skin fibroblasts and differentiated in vitro with a small molecule–based protocol. Troponin+ iPSC-CMs were confirmed by immunohistochemistry, quantitative polymerase chain reaction, fluorescence-activated cell sorting, and electrophysiological measurements. Afterward, 2×106 iPSC-CMs derived from a cell line transduced with a vector expressing firefly luciferase and green fluorescent protein were transplanted into adult NOD/SCID mice with acute left anterior descending artery ligation. Control animals received PBS injection. Bioluminescence imaging showed limited engraftment on transplantation into ischemic myocardium. However, magnetic resonance imaging of animals transplanted with iPSC-CMs showed significant functional improvement and attenuated cardiac remodeling compared with PBS-treated control animals. To understand the underlying molecular mechanism, microfluidic single-cell profiling of harvested iPSC-CMs, laser capture microdissection of host myocardium, and in vitro ischemia stimulation were used to demonstrate that the iPSC-CMs could release significant levels of proangiogenic and antiapoptotic factors in the ischemic microenvironment. Conclusions— Transplantation of human iPSC-CMs into an acute mouse myocardial infarction model can improve left ventricular function and attenuate cardiac remodeling. Because of limited engraftment, most of the effects are possibly explained by paracrine activity of these cells.

Oxidative mechanisms of IL-4-induced IL-6 expression in vascular endothelium

The present study is designed to investigate the effects of interleukin-4 (IL-4) on expression of interleukin-6 (IL-6), as well as to examine the role of distinct sources of reactive oxygen species (ROS) in this process. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA) showed that IL-4 significantly up-regulated the mRNA and protein expression of IL-6 in human aortic endothelial cells (HAEC) and C57BL/6 mice. Dihydroethidium (DHE) and dichlorofluorescein (DCF) fluorescence staining demonstrated that IL-4 significantly increased ROS generation in HAEC. A significant and dose-dependent inhibition of IL-4-induced IL-6 expression was observed in HAEC pre-treated with antioxidants, such as pyrrolidine dithiocarbamate (PDTC) and epigallocatechin gallate (EGCG), indicating that IL-4-induced IL-6 expression is mediated via an ROS-dependent mechanism. Additionally, pharmacological inhibitor of NADPH oxidase (NOX) significantly attenuated IL-4-induced ROS generation and IL-6 expression in HAEC. Furthermore, the disruption of NOX gene dramatically and significantly reduced IL-4-induced IL-6 expression in NOX knockout mice (B6.129S6-Cybb(tm1Din)/J). In contrast, overexpression of IL-6 in IL-4-activated HAEC was not affected by inhibiting other ROS generating pathways, such as xanthine oxidase, arachidonic acid metabolism, and the mitochondrial electron transport chain. These results demonstrate that IL-4 up-regulates IL-6 expression in vascular endothelium through NOX-mediated ROS generation.

Whole Brain Radiation-Induced Cognitive Impairment: Pathophysiological Mechanisms and Therapeutic Targets

Radiation therapy, the most commonly used for the treatment of brain tumors, has been shown to be of major significance in tu-mor control and survival rate of brain tumor patients. About 200,000 patients with brain tumor are treated with either partial large field or whole brain radiation every year in the United States. The use of radiation therapy for treatment of brain tumors, however, may lead to devastating functional deficits in brain several months to years after treatment. In particular, whole brain radiation therapy results in a significant reduction in learning and memory in brain tumor patients as long-term consequences of treatment. Although a number of in vitro and in vivo studies have demonstrated the pathogenesis of radiation-mediated brain injury, the cel-lular and molecular mechanisms by which radiation induces damage to normal tissue in brain remain largely unknown. Therefore, this review focuses on the pathophysiological mechanisms of whole brain radiation-induced cognitive impairment and the iden-tification of novel therapeutic targets. Specifically, we review the current knowledge about the effects of whole brain radiation on pro-oxidative and pro-inflammatory pathways, matrix metalloproteinases (MMPs)/tissue inhibitors of metalloproteinases (TIMPs) system and extracellular matrix (ECM), and physiological angiogenesis in brain. These studies may provide a foundation for defin-ing a new cellular and molecular basis related to the etiology of cognitive impairment that occurs among patients in response to whole brain radiation therapy. It may also lead to new opportunities for therapeutic interventions for brain tumor patients who are undergoing whole brain radiation therapy.