Charlie Norwood VA Medical Center

About: Charlie Norwood VA Medical Center is a healthcare organization based out in Augusta, Georgia, United States. It is known for research contribution in the topics: Autophagy & Kidney. The organization has 349 authors who have published 490 publications receiving 16360 citations. The organization is also known as: Augusta VA Medical Center.

S-Glutathionylation of LMW-PTP regulates VEGF-mediated FAK activation and endothelial cell migration

Abstract: Although promising, the ability to regulate angiogenesis through delivery of VEGF remains an unrealized goal. We have shown previously that physiological levels of peroxynitrite (1 µM) are required for a VEGF-mediated angiogenic response, yet the redox-regulated mechanisms that govern the VEGF signal remain unexplored. We assessed the impact of VEGF and peroxynitrite on modifying redox-state, the level of reduced-glutathione (GSH) and S-glutathionylation on regulation of the low molecular weight protein tyrosine phosphatase (LMW-PTP) and focal adhesion kinase (FAK), which are key mediators of VEGF-mediated cell migration. Stimulation of human microvascular endothelial (HME) cells with VEGF (20 ng/ml) or peroxynitrite (1 µM) caused an immediate and reversible negative-shift in the cellular redox-state and thiol oxidation of LMW-PTP, which culminated in cell migration. VEGF causes reversible S-glutathionylation of LMW-PTP, which inhibits its phosphorylation and activity, and causes the transient activation of FAK. Modulating the redox-state using decomposing peroxynitrite (FeTPPS, 2.5 µM) or the GSH-precursor [N-acetylcysteine (NAC), 1 mM] caused a positive-shift of the redox-state and prevented VEGF-mediated S-glutathionylation and oxidative inhibition of LMW-PTP. NAC and FeTPPS prevented the activation of FAK, its association with LMW-PTP and cell migration. Inhibiting LMW-PTP expression markedly enhanced FAK activation and cell migration. Although mild oxidative stress achieved by combining VEGF with 0.1-0.2 mM peroxynitrite augmented cell migration, an acute shift to oxidative stress achieved by combining VEGF with 0.5 mM peroxynitrite induced and sustained FAK activation, and LMW-PTP S-glutathionylation, resulting in LMW-PTP inactivation and inhibited cell migration. In conclusion, our findings demonstrate that a balanced redox-state is required for VEGF to facilitate reversible S-glutathionylation of LMW-PTP, FAK activation and endothelial cell migration. Shifting the redox-state to reductive stress or oxidative stress inhibited the VEGF-mediated angiogenic response.

Metabolic Reprogramming by MYCN Confers Dependence on the Serine-Glycine-One-Carbon Biosynthetic Pathway.

Abstract: MYCN amplification drives the development of neuronal cancers in children and adults. Given the challenge in therapeutically targeting MYCN directly, we searched for MYCN-activated metabolic pathways as potential drug targets. Here we report that neuroblastoma cells with MYCN amplification show increased transcriptional activation of the serine-glycine-one-carbon (SGOC) biosynthetic pathway and an increased dependence on this pathway for supplying glucose-derived carbon for serine and glycine synthesis. Small molecule inhibitors that block this metabolic pathway exhibit selective cytotoxicity to MYCN-amplified cell lines and xenografts by inducing metabolic stress and autophagy. Transcriptional activation of the SGOC pathway in MYCN-amplified cells requires both MYCN and ATF4, which form a positive feedback loop, with MYCN activation of ATF4 mRNA expression and ATF4 stabilization of MYCN protein by antagonizing FBXW7-mediated MYCN ubiquitination. Collectively, these findings suggest a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited as a strategy for selective cancer therapy. SIGNIFICANCE: This study identifies a MYCN-dependent metabolic vulnerability and suggests a coupled relationship between metabolic reprogramming and increased sensitivity to metabolic stress, which could be exploited for cancer therapy.See related commentary by Rodriguez Garcia and Arsenian-Henriksson, p. 3818.

Low-dose bone morphogenetic protein-2/stromal cell-derived factor-1β cotherapy induces bone regeneration in critical-size rat calvarial defects.

Abstract: Increasing evidence suggests that stromal cell-derived factor-1 (SDF-1/CXCL12) is involved in bone formation, though underlying molecular mechanisms remain to be fully elucidated. Also, contributions of SDF-1β, the second most abundant splice variant, as an osteogenic mediator remain obscure. We have shown that SDF-1β enhances osteogenesis by regulating bone morphogenetic protein-2 (BMP-2) signaling in vitro. Here we investigate the dose-dependent contribution of SDF-1β to suboptimal BMP-2-induced local bone formation; that is, a dose that alone would be too low to significantly induce bone formation. We utilized a critical-size rat calvarial defect model and tested the hypotheses that SDF-1β potentiates BMP-2 osteoinduction and that blocking SDF-1 signaling reduces the osteogenic potential of BMP-2 in vivo. In preliminary studies, radiographic analysis at 4 weeks postsurgery revealed a dose-dependent relationship in BMP-2-induced new bone formation. We then found that codelivery of SDF-1β potentiates sub...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis.

Abstract: Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.