Younggeon Jin

TTAC-0001, a human monoclonal antibody targeting VEGFR-2/KDR, blocks tumor angiogenesis

Angiogenesis is one of the most important processes for cancer cell survival, tumor growth and metastasis. Vascular endothelial growth factor (VEGF) and its receptor, particularly VEGF receptor-2 (VEGFR-2, or kinase insert domain-containing receptor, KDR), play critical roles in tumor-associated angiogenesis. We developed TTAC-0001, a human monoclonal antibody against VEGFR-2/KDR from a fully human naïve single-chain variable fragment phage library. TTAC-0001 was selected as a lead candidate based on its affinity, ligand binding inhibition and inhibition of VEGFR-2 signal in human umbilical vein endothelial cells (HUVEC). TTAC-0001 inhibited binding of VEGF-C and VEGF-D to VEGFR-2 in addition to VEGF-A. It binds on the N-terminal regions of domain 2 and domain 3 of VEGFR-2. It could inhibit the phosphorylation of VEGFR-2/KDR and ERK induced by VEGF in HUVEC. TTAC-0001 also inhibited VEGF-mediated endothelial cell proliferation, migration and tube formation in vitro, as well as ex vivo vessel sprouting from rat aortic rings and neovascularization in mouse matrigel model in vivo. Our data indicates that TTAC-0001 blocks the binding of VEGFs to VEGFR-2/KDR and inhibits VEGFR-induced signaling pathways and angiogenesis. Therefore, these data strongly support the further development of TTAC-0001 as an anti-cancer agent in the clinic.

Functional neural stem cell isolation from brains of adult mutant SOD1 (SOD1G93A) transgenic amyotrophic lateral sclerosis (ALS) mice

Abstract Objectives: The aim of present study is to investigate more functional neural stem cells (NSCs) could be isolated from brains with amyotrophic lateral sclerosis (ALS) and expanded in vitro, based on previous reports demonstrating de novo neurogenesis is enhanced to replace degenerating neural tissue. Methods: Thirteen- or eighteen-week-old mutant human Cu/Zn superoxide dismutase (SOD1G93A) transgenic ALS and wild-type SOD1 transgenic control mice were utilized. Changes in numbers of NSCs in the dentate gyrus were analyzed by immunohistochemistry against nestin and CD133. NSCs were primarily cultured from hippocampus of ALS or control mice. Expression of NSC markers, in vitro expansion capacity, and differentiating potential were compared. Results: Hippocampus of 13-week-old pre-symptomatic ALS mice harbor more cells that can be propagated for more than 12 passages in vitro, compared with same age control mice. Primarily-cultured cells formed neurospheres in the NSC culture medium, expressed NSC markers, and differentiated into cells with differentiated neural cell characteristics in the differentiation condition confirming that they are NSCs. In contrast, long-term expansible NSCs could not be derived from brains of 18-week-old symptomatic ALS mice with the same experimental techniques, although they had comparable nestin-immunoreactive cells in the dentate gyrus. Discussion: These results would suggest that increased neuroregeneration in early phase of ALS could be translated to regenerative approaches; however, long-term exposure to ALS microenvironments could abolish functional capacities of NSCs.

ClC-2 regulation of intestinal barrier function: Translation of basic science to therapeutic target.

The ClC-2 chloride channel is a member of the voltage-gated chloride channel family. ClC-2 is involved in various physiological processes, including fluid transport and secretion, regulation of cell volume and pH, maintaining the membrane potential of the cell, cell-to-cell communication, and tissue homeostasis. Recently, our laboratory has accumulated evidence indicating a critical role of ClC-2 in the regulation of intestinal barrier function by altering inter-epithelial tight junction composition. This review will detail the role of ClC-2 in intestinal barrier function during intestinal disorders, including experimental ischemia/reperfusion injury and dextran sodium sulfate (DSS)-induced inflammatory bowel disease. Details of pharmacological manipulation of ClC-2 via prostone agonists will also be provided in an effort to show the potential therapeutic relevance of ClC-2 regulation, particularly during intestinal barrier disruption.

Myosin light chain kinase mediates intestinal barrier dysfunction via occludin endocytosis during anoxia/reoxygenation injury.

Intestinal anoxia/reoxygenation (A/R) injury induces loss of barrier function followed by epithelial repair. Myosin light chain kinase (MLCK) has been shown to alter barrier function via regulation of interepithelial tight junctions, but has not been studied in intestinal A/R injury. We hypothesized that A/R injury would disrupt tight junction barrier function via MLCK activation and myosin light chain (MLC) phosphorylation. Caco-2BBe1 monolayers were subjected to anoxia for 2 h followed by reoxygenation in 21% O2, after which barrier function was determined by measuring transepithelial electrical resistance (TER) and FITC-dextran flux. Tight junction proteins and MLCK signaling were assessed by Western blotting, real-time PCR, or immunofluorescence microscopy. The role of MLCK was further investigated with select inhibitors (ML-7 and peptide 18) by using in vitro and ex vivo models. Following A/R injury, there was a significant increase in paracellular permeability compared with control cells, as determined by TER and dextran fluxes (P < 0.05). The tight junction protein occludin was internalized during A/R injury and relocalized to the region of the tight junction after 4 h of recovery. MLC phosphorylation was significantly increased by A/R injury (P < 0.05), and treatment with the MLCK inhibitor peptide 18 attenuated the increased epithelial monolayer permeability and occludin endocytosis caused by A/R injury. Application of MLCK inhibitors to ischemia-injured porcine ileal mucosa induced significant increases in TER and reduced mucosal-to-serosal fluxes of 3H-labeled mannitol. These data suggest that MLCK-induced occludin endocytosis mediates intestinal epithelial barrier dysfunction during A/R injury. Our results also indicate that MLCK-dependent occludin regulation may be a target for the therapeutic treatment of ischemia/reperfusion injury.

Pharmaceutical Activation or Genetic Absence of ClC-2 Alters Tight Junctions During Experimental Colitis.

Background:We have previously reported that the ClC-2 chloride channel has an important role in regulation of tight junction barrier function during experimental colitis, and the pharmaceutical ClC-2 activator lubiprostone initiates intestinal barrier repair in ischemic-injured intestine. Thus, we hypothesized that pharmaceutical ClC-2 activation would have a protective and therapeutic effect in murine models of colitis, which would be absent in ClC-2−/− mice. Methods:We administered lubiprostone to wild-type or ClC-2−/− mice with dextran sulfate sodium (DSS) or 2, 4, 5-trinitrobenzene sulfonic acid–induced colitis. We determined the severity of colitis and assessed intestinal permeability. Selected tight junction proteins were analyzed by Western blotting and immunofluorescence/confocal microscopy, whereas proliferative and differentiated cells were examined with special staining and immunohistochemistry. Results:Oral preventive or therapeutic administration of lubiprostone significantly reduced the severity of colitis and reduced intestinal permeability in both DSS and trinitrobenzene sulfonic acid–induced colitis. Preventive treatment with lubiprostone induced significant recovery of the expression and distribution of selected sealing tight junction proteins in mice with DSS-induced colitis. In addition, lubiprostone reduced crypt proliferation and increased the number of differentiated epithelial cells. Alternatively, when lubiprostone was administered to ClC-2−/− mice, the protective effect against DSS colitis was limited. Conclusions:This study suggests a central role for ClC-2 in restoration of barrier function and tight junction architecture in experimental murine colitis, which can be therapeutically targeted with lubiprostone.

Intestinal Ischemia–Reperfusion: Rooting for the SOCS?

Ischemia–reperfusion (IR) injury of the intestine occurs when blood flow is temporarily interrupted, which occurs during interventions such as surgery for an abdominal aortic aneurysm, small bowel transplantation, strangulating hernias, and neonatal necrotizing enterocolitis. It can also occur as a result of collapse of the systemic circulation during conditions such as hemorrhagic shock following trauma, septic shock, or heat stress [1]. At the cellular level, the initial intestinal ischemic injury reduces cellular mitochondrial ATP generation, activates hydrolases, reduces cell membrane selective permeability, and increases calcium influx into ischemic cells. Reperfusion may exacerbate the extent of injury through the activation of an intense systemic inflammatory response [2] such as marked pro-inflammatory cytokine release, production of reactive oxygen species (ROS), increased expression of nitric oxide (NO), Toll-like receptor (TLR)-4 signaling, and activation of inflammatory transcription factors, among other pro-inflammatory mechanisms [3]. IR injury is therefore difficult to manage clinically with consequent high morbidity and mortality [4]. Ischemic preconditioning is an alternative strategy to reduce IR injury among therapeutic interventions aimed at reducing IR injury by inhibiting the activation of inflammatory cells [4]. Ischemic preconditioning consists of a brief episode of ischemia preceding the major ischemic event, an intervention that activates tissue-adaptive mechanisms. Ischemic preconditioning can be induced mechanically or pharmacologically; mechanical preconditioning, in which the target organ is exposed to a brief ischemic episode by mechanically compromising its blood supply prior to prolonged ischemia, has the benefit of reducing IR injury, but it has the principal disadvantage of traumatizing major vessels and stressing the target organ. Alternatively, the identification of the signaling pathways underlying ischemic preconditioning has created the possibility of using pharmacological agents that confer protection against IR injury [5]. Ischemic preconditioning increases the generation of endogenous antioxidants such as glutathione, superoxide dismutase, and hemoxygenase-1 (HO-1). Nuclear transcription factors such as nuclear factor-kappa B (NFjB) and NO are also affected by ischemic preconditioning, reducing the generation of pro-inflammatory cytokines, and preserving blood flow, oxygenation, and mitochondrial function [3, 6]. Clinically, ischemic preconditioning may be applied to patients prior to a planned surgical procedure such as cardiac, hepatic, or pulmonary surgery, in order to reduce the potential adverse effects of IR injury in the postoperative period. In this issue of Digestive Diseases and Sciences, Liu et al. [7] report that ischemic preconditioning-induced suppressor of cytokine signaling-1 (SOCS-1) activation protects the intestine from IR injury via downregulation of the Toll-like receptor 4 (TLR4) pathway (Fig. 1). Among the molecular mechanisms integral to the pathogenesis of IR injury, the authors focused on TLR4 signaling and its downstream signaling intermediate tumor necrosis factor receptor-associated factor 6 (TRAF6), which has been understudied in IR injury. Additionally, the investigators also studied the contribution of receptor interacting protein 1 (RIP1), a key regulator of cellular apoptosis, regulated by tumor necrosis factor (TNF) signaling. SOCS-1 is a key regulator of inflammatory responses, including the TLR4 signaling pathway, thereby putatively inhibiting & Anthony T. Blikslager anthony_blikslager@ncsu.edu

Knockout of ClC-2 reveals critical functions of adherens junctions in colonic homeostasis and tumorigenicity

Adherens junctions (AJs), together with tight junctions (TJs), form an apical junctional complex that regulates intestinal epithelial cell-to-cell adherence and barrier homeostasis. Within the AJ, membrane-bound E-cadherin binds β-catenin, which functions as an essential intracellular signaling molecule. We have previously identified a novel protein in the region of the apical junction complex, chloride channel protein-2 (ClC-2), that we have used to study TJ regulation. In this study, we investigated the possible effects of ClC-2 on the regulation of AJs in intestinal mucosal epithelial homeostasis and tumorigenicity. Mucosal homeostasis and junctional proteins were examined in wild-type (WT) and ClC-2 knockout (KO) mice as well as associated colonoids. Tumorigenicity and AJ-associated signaling were evaluated in a murine colitis-associated tumor model and in a colorectal cancer cell line (HT-29). Colonic tissues from ClC-2 KO mice had altered ultrastructural morphology of intercellular junctions with reduced colonocyte differentiation, whereas jejunal tissues had minimal changes. Colonic crypts from ClC-2 KO mice had significantly higher numbers of less-differentiated forms of colonoids compared with WT. Furthermore, the absence of ClC-2 resulted in redistribution of AJ proteins and increased β-catenin activity. Downregulation of ClC-2 in colorectal cells resulted in significant increases in proliferation associated with disruption of AJs. Colitis-associated tumors in ClC-2 KO mice were significantly increased, associated with β-catenin transcription factor activation. The absence of ClC-2 results in less differentiated colonic crypts and increased tumorigenicity associated with colitis via dysregulation of AJ proteins and activation of β-catenin-associated signaling. NEW & NOTEWORTHY Disruption of adherens junctions in the absence of chloride channel protein-2 revealed critical functions of these junctional structures, including maintenance of colonic homeostasis and differentiation as well as driving tumorigenicity by regulating β-catenin signaling.