Astrocytic PAR1 and mGluR2/3 control synaptic glutamate time course at hippocampal CA1 synapses.

Astrocytes play an essential role in regulating synaptic transmission. This study describes a novel form of modulation of excitatory synaptic transmission in the mouse hippocampus by astrocytic G-protein-coupled receptors (GPCRs). We have previously described astrocytic glutamate release via protease-activated receptor-1 (PAR1) activation, although the regulatory mechanisms for this are complex. Through electrophysiological analysis and modeling, we discovered that PAR1 activation consistently increases the concentration and duration of glutamate in the synaptic cleft. This effect was not due to changes in the presynaptic glutamate release or alteration in glutamate transporter expression. However, blocking group II metabotropic glutamate receptors (mGluR2/3) abolished PAR1-mediated regulation of synaptic glutamate concentration, suggesting a role for this GPCR in mediating the effects of PAR1 activation on glutamate release. Furthermore, activation of mGluR2/3 causes glutamate release through the TREK-1 channel in hippocampal astrocytes. These data show that astrocytic GPCRs engage in a novel regulatory mechanism to shape the time course of synaptically-released glutamate in excitatory synapses of the hippocampus.

Visualizing Cancer-Originating Acetate Uptake Through MCT1 in Reactive Astrocytes in the Glioblastoma Tumor Microenvironment.

BACKGROUND: Reactive astrogliosis is a hallmark of various brain pathologies, including neurodegenerative diseases and glioblastomas. However, the specific intermediate metabolites contributing to reactive astrogliosis remain unknown. This study investigated how glioblastomas induce reactive astrogliosis in the neighboring microenvironment and explores 11C-acetate PET as an imaging technique for detecting reactive astrogliosis. METHODS: Through in vitro, mouse models, and human tissue experiments, we examined the association between elevated 11C-acetate uptake and reactive astrogliosis in gliomas. We explored acetate from glioblastoma cells, which triggers reactive astrogliosis in neighboring astrocytes by upregulating MAO-B and MCT1 expression. We evaluated the presence of cancer stem cells in the reactive astrogliosis region of glioblastomas and assessed the correlation between the volume of 11C-acetate uptake beyond MRI and prognosis. RESULTS: Elevated 11C-acetate uptake is associated with reactive astrogliosis and astrocytic MCT1 in the periphery of glioblastomas in human tissues and mouse models. Glioblastoma cells exhibit increased acetate production as a result of glucose metabolism, with subsequent secretion of acetate. Acetate derived from glioblastoma cells induces reactive astrogliosis in neighboring astrocytes by increasing the expression of MAO-B and MCT1. We found cancer stem cells within the reactive astrogliosis at the tumor periphery. Consequently, a larger volume of 11C-acetate uptake beyond contrast-enhanced MRI was associated with worse prognosis. CONCLUSION: Our results highlight the role of acetate derived from glioblastoma cells in inducing reactive astrogliosis and underscore the potential value of 11C-acetate PET as an imaging technique for detecting reactive astrogliosis, offering important implications for the diagnosis and treatment of glioblastomas.

4-EA-NBOMe, an amphetamine derivative, alters glutamatergic synaptic transmission through 5-HT1A receptors on cortical neurons from SpragueDawley rat and pyramidal neurons from C57BL/6 mouse.

New psychoactive substances (NPSs) are compounds designed to mimic illegal recreational drugs. In particular, there are difficulties in legal restrictions because there is no fast NPS detection method to suppress the initial spread of NPS with criminal records; thus, they expose the public to serious health threats, including toxicity and dependence. However, the effects of NPSs on the brain and the related cellular mechanisms are well unknown. One of the recently emerging drugs is 4-ethylamphetamine-NBOMe (4-EA-NBOMe), a member of the 2 C phenylalanine family with a similar structure to methamphetamine (methA). In this study, we tested the effect of methA analogs on the glutamatergic synaptic transmission on primary cultured cortical neurons of SpragueDawley (SD) rats and C57BL/6 mice, and also layer 2/3 pyramidal neurons of the medial prefrontal cortex (mPFC) of C57BL/6 mice. We found that acute treatment with 4-EA-NBOMe inhibits spontaneous excitatory postsynaptic currents (EPSCs) and that withdrawal after chronic inhibition by 4-EA-NBOMe augments glutamatergic synaptic transmission. These modifications of synaptic responses are mediated by 5-HT1A receptors. These findings suggest that 4-EA-NBOMe directly affects the central nervous system by changing the efficacy of glutamatergic synaptic transmission.

Mechanisms of Invasion in Glioblastoma: Extracellular Matrix, Ca2+ Signaling, and Glutamate.

Glioblastoma (GBM) is the most common and malignant form of primary brain tumor with a median survival time of 14-16 months in GBM patients. Surgical treatment with chemotherapy and radiotherapy may help increase survival by removing GBM from the brain. However, complete surgical resection to eliminate GBM is almost impossible due to its high invasiveness. When GBM cells migrate to the brain, they interact with various cells, including astrocytes, neurons, endothelial cells, and the extracellular matrix (ECM). They can also make their cell body shrink to infiltrate into narrow spaces in the brain; thereby, they can invade regions of the brain and escape from surgery. Brain tumor cells create an appropriate microenvironment for migration and invasion by modifying and degrading the ECM. During those processes, the Ca2+ signaling pathway and other signaling cascades mediated by various ion channels contribute mainly to gene expression, motility, and invasion of GBM cells. Furthermore, GBM cells release glutamate, affecting migration via activation of ionotropic glutamate receptors in an autocrine manner. This review focuses on the cellular mechanisms of glioblastoma invasion and motility related to ECM, Ca2+ signaling, and glutamate. Finally, we discuss possible therapeutic interventions to inhibit invasion by GBM cells.

Tumor Spheroids of an Aggressive Form of Central Neurocytoma Have Transit-Amplifying Progenitor Characteristics with Enhanced EGFR and Tumor Stem Cell Signaling.

Central neurocytoma (CN) has been known as a benign neuronal tumor. In rare cases, CN undergoes malignant transformation to glioblastomas (GBM). Here we examined its cellular origin by characterizing differentiation potential and gene expression of CN-spheroids. First, we demonstrate that both CN tissue and cultured primary cells recapitulate the hierarchal cellular composition of subventricular zone (SVZ), which is comprised of neural stem cells (NSCs), transit amplifying progenitors (TAPs), and neuroblasts. We then derived spheroids from CN which displayed EGFR+/ MASH+ TAP and BLBP+ radial glial cell (RGC) characteristic, and mitotic neurogenesis and gliogenesis by single spheroids were observed with cycling multipotential cells. CN-spheroids expressed increased levels of pluripotency and tumor stem cell genes such as KLF4 and TPD5L1, when compared to their differentiated cells and human NSCs. Importantly, Gene Set Enrichment Analysis showed that gene sets of GBM-Spheroids, EGFR Signaling, and Packaging of Telomere Ends are enriched in CN-spheroids in comparison with their differentiated cells. We speculate that CN tumor stem cells have TAP and RGC characteristics, and upregulation of EGFR signaling as well as downregulation of eph-ephrin signaling have critical roles in tumorigenesis of CN. And their ephemeral nature of TAPs destined to neuroblasts, might reflect benign nature of CN.

Signaling mechanisms of µ-opioid receptor (MOR) in the hippocampus: disinhibition versus astrocytic glutamate regulation.

µ-opioid receptor (MOR) is a class of opioid receptors that is critical for analgesia, reward, and euphoria. MOR is distributed in various brain regions, including the hippocampus, where traditionally, it is believed to be localized mainly at the presynaptic terminals of the GABAergic inhibitory interneurons to exert a strong disinhibitory effect on excitatory pyramidal neurons. However, recent intensive research has uncovered the existence of MOR in hippocampal astrocytes, shedding light on how astrocytic MOR participates in opioid signaling via glia-neuron interaction in the hippocampus. Activation of astrocytic MOR has shown to cause glutamate release from hippocampal astrocytes and increase the excitability of presynaptic axon fibers to enhance the release of glutamate at the Schaffer Collateral-CA1 synapses, thereby, intensifying the synaptic strength and plasticity. This novel mechanism involving astrocytic MOR has been shown to participate in hippocampus-dependent conditioned place preference. Furthermore, the signaling of hippocampal MOR, whose action is sexually dimorphic, is engaged in adult neurogenesis, seizure, and stress-induced memory impairment. In this review, we focus on the two profoundly different hippocampal opioid signaling pathways through either GABAergic interneuronal or astrocytic MOR. We further compare and contrast their molecular and cellular mechanisms and their possible roles in opioid-associated conditioned place preference and other hippocampus-dependent behaviors.

Climbing fiber synapses rapidly and transiently inhibit neighboring Purkinje cells via ephaptic coupling.

Climbing fibers from the inferior olive make strong excitatory synapses onto cerebellar Purkinje cell (PC) dendrites and trigger distinctive responses known as complex spikes. We found that, in awake mice, a complex spike in one PC suppressed conventional simple spikes in neighboring PCs for several milliseconds. This involved a new ephaptic coupling, in which an excitatory synapse generated large negative extracellular signals that nonsynaptically inhibited neighboring PCs. The distance dependence of complex spike-simple spike ephaptic signaling, combined with the known CF divergence, allowed a single inferior olive neuron to influence the output of the cerebellum by synchronously suppressing the firing of potentially over 100 PCs. Optogenetic studies in vivo and dynamic clamp studies in slice indicated that such brief PC suppression, as a result of either ephaptic signaling or other mechanisms, could effectively promote firing in neurons in the deep cerebellar nuclei with remarkable speed and precision.

Polybenzimidazole-Benzophenone Composite Nanofiber Window Air Filter with Superb UV Resistance and High Chemical and Thermal Durability.

There is a growing interest in window air filters to protect indoor air quality from ultrafine particulate matter (PM) in outdoor air. The filters for this purpose must achieve high filtering efficiency without compromising the original functions of the window, such as high air permeability and visibility. Several filters meeting these requirements have been developed and demonstrate a high PM2.5 filtering efficiency. However, these filters are installed outside the window or on the window screen guard, thereby requiring high levels of ultraviolet (UV), chemical, and thermal resistance. These requirements have been overlooked so far. In this study, we examine the fabrication and performance of a polybenzimidazole-benzophenone (PBI-BP) composite nanofiber air filter that demonstrates superb UV resistance and chemical and thermal durability. Because of the UV absorbance of the BP in the nanofibers, the filter membrane is robust even under prolonged UV exposure, which is essential for filters for this purpose. The filter membrane is not damaged even after treatment in strong acids or annealing at high temperature up to 400 °C. Thus, the PBI-BP composite filter is suitable for practical application in window air filters and can be adapted to develop filters used under other harsh environments.

Activation of Astrocytic µ-Opioid Receptor Causes Conditioned Place Preference.

The underlying mechanisms of how positive emotional valence (e.g., pleasure) causes preference of an associated context is poorly understood. Here, we show that activation of astrocytic µ-opioid receptor (MOR) drives conditioned place preference (CPP) by means of specific modulation of astrocytic MOR, an exemplar endogenous Gi protein-coupled receptor (Gi-GPCR), in the CA1 hippocampus. Long-term potentiation (LTP) induced by a subthreshold stimulation with the activation of astrocytic MOR at the Schaffer collateral pathway accounts for the memory acquisition to induce CPP. This astrocytic MOR-mediated LTP induction is dependent on astrocytic glutamate released upon activation of the astrocytic MOR and the consequent activation of the presynaptic mGluR1. The astrocytic MOR-dependent LTP and CPP were recapitulated by a chemogenetic activation of astrocyte-specifically expressed Gi-DREADD hM4Di. Our study reveals that the transduction of inhibitory Gi-signaling into augmented excitatory synaptic transmission through astrocytic glutamate is critical for the acquisition of contextual memory for CPP.

Reusable Polybenzimidazole Nanofiber Membrane Filter for Highly Breathable PM2.5 Dust Proof Mask.

Ultrafine particulate matters (PMs) are an imminent threat to the human respiratory system, as their sizes are comparable to and even smaller than human tissues. To cope with this situation, researchers have developed and commercialized various personal dust proof masks. However, because of the relatively thick filter membrane to guarantee filtering efficiency, a huge pressure drop across the active filter layer is inevitable and breathing through it becomes uncomfortable. In this work, we investigated the performance of electrospun polybenzimidazole (PBI) nanofiber membrane filters that can potentially be used for dust proof masks or other high-performance filters. Thanks to its high dipole moment (6.12) as confirmed by density functional theory (DFT) calculation, the surface potential of the PBI nanofiber air filter, measured by KPFM, was higher than that of other commercially available mask filters. The filter developed in this work provides high PM filtering efficiency of ∼98.5% at much reduced pressure drop (130 Pa) in comparison to those used in commercially available masks (386 Pa) with similar filtering efficiencies. Consequently, an approximately 3-fold higher quality factor (∼0.032), evaluated for PM2.5, in comparison to that of commercial ones (∼0.011) was achieved by using PBI nanofiber. Furthermore, we developed a cleaning method effective for the filter contaminated by both inorganic and organic PMs. Even after several cycles of cleaning, the PBI filter membrane demonstrated negligible damage and retained its original performance because of its mechanical, thermal, and chemical durability.

Ephaptic Coupling Promotes Synchronous Firing of Cerebellar Purkinje Cells.

Correlated neuronal activity at various timescales plays an important role in information transfer and processing. We find that in awake-behaving mice, an unexpectedly large fraction of neighboring Purkinje cells (PCs) exhibit sub-millisecond synchrony. Correlated firing usually arises from chemical or electrical synapses, but, surprisingly, neither is required to generate PC synchrony. We therefore assessed ephaptic coupling, a mechanism in which neurons communicate via extracellular electrical signals. In the neocortex, ephaptic signals from many neurons summate to entrain spiking on slow timescales, but extracellular signals from individual cells are thought to be too small to synchronize firing. Here we find that a single PC generates sufficiently large extracellular potentials to open sodium channels in nearby PC axons. Rapid synchronization is made possible because ephaptic signals generated by PCs peak during the rising phase of action potentials. These findings show that ephaptic coupling contributes to the prevalent synchronization of nearby PCs.

Activation of Astrocytic µ-opioid Receptor Elicits Fast Glutamate Release Through TREK-1-Containing K2P Channel in Hippocampal Astrocytes.

Recently, µ-opioid receptor (MOR), one of the well-known Gi-protein coupled receptors (Gi-GPCR), was reported to be highly expressed in the hippocampal astrocytes. However, the role of astrocytic MOR has not been investigated. Here we report that activation of astrocytic MOR by [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO), a selective MOR agonist, causes a fast glutamate release using sniffer patch technique. We also found that the DAMGO-induced glutamate release was not observed in the astrocytes from MOR-deficient mice and MOR-short hairpin RNA (shRNA)-expressed astrocytes. In addition, the glutamate release was significantly reduced by gene silencing of the TREK-1-containing two-pore potassium (K2P) channel, which mediates passive conductance in astrocytes. Our findings were consistent with the previous study demonstrating that activation of Gi-GPCR such as cannabinoid receptor CB1 and adenosine receptor A1 causes a glutamate release through TREK-1-containing K2P channel from hippocampal astrocytes. We also demonstrated that MOR and TREK-1 are significantly co-localized in the hippocampal astrocytes. Furthermore, we found that both MOR and TREK-1-containing K2P channels are localized in the same subcellular compartments, soma and processes, of astrocytes. Our study raises a novel possibility that astrocytic MOR may participate in several physiological and pathological actions of opioids, including analgesia and addiction, through astrocytically released glutamate and its signaling pathway.

SAP102 regulates synaptic AMPAR function through a CNIH-2-dependent mechanism.

The postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD/DLG-MAGUK) family of proteins scaffold α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) complexes to the postsynaptic compartment and are postulated to orchestrate activity-dependent modulation of synaptic AMPAR functions. SAP102 is a key member of this family, present from early development, before PSD-95 and PSD-93, and throughout life. Here we investigate the role of SAP102 in synaptic transmission using a cell-restricted molecular replacement strategy, where SAP102 is expressed against the background of acute knockdown of endogenous PSD-95. We show that SAP102 rescues the decrease of AMPAR-mediated evoked excitatory postsynaptic currents (AMPAR eEPSCs) and AMPAR miniature EPSC (AMPAR mEPSC) frequency caused by acute knockdown of PSD-95. Further analysis of the mini events revealed that PSD-95-to-SAP102 replacement but not direct manipulation of PSD-95 increases the AMPAR mEPSC decay time. SAP102-mediated rescue of AMPAR eEPSCs requires AMPAR auxiliary subunit cornichon-2, whereas cornichon-2 knockdown did not affect PSD-95-mediated regulation of AMPAR eEPSC. Combining these observations, our data elucidate that PSD-95 and SAP102 differentially influence basic synaptic properties and synaptic current kinetics potentially via different AMPAR auxiliary subunits. NEW & NOTEWORTHY Synaptic scaffold proteins postsynaptic density (PSD)-95-like, disk-large (DLG) membrane-associated guanylate kinase (PSD-MAGUKs) regulate synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) function. However, the functional diversity among different PSD-MAGUKs remains to be categorized. We show that distinct from PSD-95, SAP102 increase the AMPAR synaptic current decay time, and the effect of SAP102 on synaptic AMPAR function requires the AMPAR auxiliary subunit cornichon-2. Our data suggest that PSD-MAGUKs target and modulate different AMPAR complexes to exert specific experience-dependent modification of the excitatory circuit.

The role of netrin-1 in metastatic renal cell carcinoma treated with sunitinib.

INTRODUCTION: Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies like sunitinib, target the formation of new vessels allowing nutrient deprivation and cell death. However, recent studies have shown that patients develop resistance after approximately 1 year of treatment and show disease progression while on therapy. Therefore, we propose to identify the protein(s) responsible for increased migration with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease. MATERIAL AND METHODS: Human renal cancer cell lines (Caki-1, Caki-2, ACHN) were treated with increasing doses of sunitinib to develop a sunitinib-conditioned renal cell carcinoma cell line. mRNA microarray and qPCR were performed to compare the differences in gene expression between Caki-1 sunitinib-conditioned and non-conditioned cells. NTN1 was assessed in our in vivo sunitinib-conditioned mouse model using immunostaining. xCELLigence and scratch assays were used to evaluate migration and MTS was used to evaluate cell viability. RESULTS: Human renal cell carcinoma sunitinib-conditioned cell lines showed upregulation of netrin-1 in microarray and q-PCR. Increased migration was demonstrated in Caki-1 sunitinib-conditioned cells when compared to the non-treated ones as well as, increased endothelial cell migration. Silencing of netrin-1 in sunitinib-conditioned Caki-1 cells did not demonstrate a significant reduction in cell migration. CONCLUSION: Netrin-1 is highly upregulated in renal cell carcinoma treated with sunitinib, but has no influence on cell viability or cell migration in metastatic RCC.

Expression of µ-Opioid Receptor in CA1 Hippocampal Astrocytes.

µ-opioid receptor (MOR) is a class of opioid receptors with a high affinity for enkephalins and beta-endorphin. In hippocampus, activation of MOR is known to enhance the neuronal excitability of pyramidal neurons, which has been mainly attributed to a disinhibition of pyramidal neurons via activating Gαi subunit to suppress the presynaptic release of GABA in hippocampal interneurons. In contrast, the potential role of MOR in hippocampal astrocytes, the most abundant cell type in the brain, has remained unexplored. Here, we determine the cellular and subcellular distribution of MOR in different cell types of the hippocampus by utilizing MOR-mCherry mice and two different antibodies against MOR. Consistent with previous findings, we demonstrate that MOR expression in the CA1 pyramidal layer is co-localized with axon terminals from GABAergic inhibitory neurons but not with soma of pyramidal neurons. More importantly, we demonstrate that MOR is highly expressed in CA1 hippocampal astrocytes. The ultrastructural analysis further demonstrates that the astrocytic MOR is localized in soma and processes, but not in microdomains near synapses. Lastly, we demonstrate that astrocytes in ventral tegmental area and nucleus accumbens also express MOR. Our results provide the unprecedented evidence for the presence of MOR in astrocytes, implicating potential roles of astrocytic MOR in addictive behaviors.

Experience-Dependent Equilibration of AMPAR-Mediated Synaptic Transmission during the Critical Period.

Experience-dependent synapse refinement is essential for functional optimization of neural circuits. However, how sensory experience sculpts excitatory synaptic transmission is poorly understood. Here, we show that despite substantial remodeling of synaptic connectivity, AMPAR-mediated synaptic transmission remains at equilibrium during the critical period in the mouse primary visual cortex. The maintenance of this equilibrium requires neurogranin (Ng), a postsynaptic calmodulin-binding protein important for synaptic plasticity. With normal visual experience, loss of Ng decreased AMPAR-positive synapse numbers, prevented AMPAR-silent synapse maturation, and increased spine elimination. Importantly, visual deprivation halted synapse loss caused by loss of Ng, revealing that Ng coordinates experience-dependent AMPAR-silent synapse conversion to AMPAR-active synapses and synapse elimination. Loss of Ng also led to sensitized long-term synaptic depression (LTD) and impaired visually guided behavior. Our synaptic interrogation reveals that experience-dependent coordination of AMPAR-silent synapse conversion and synapse elimination hinges upon Ng-dependent mechanisms for constructive synaptic refinement during the critical period.

FOXC2 and CLIP4 : a potential biomarker for synchronous metastasis of ≤7-cm clear cell renal cell carcinomas.

Renal cell carcinomas (RCC) smaller than 7-cm are heterogeneous and exhibit metastatic potential in approximately 15% of cases. Although large-scale characterization of mutations in clear cell RCC (ccRCC), the most common RCC subtype, has been established, the genetic alterations related to ≤7-cm ccRCCs undergoing synchronous metastasis are poorly understood. To discover biomarkers that can be used to estimate the risk of synchronous metastasis in these ccRCC patients, we performed whole exome sequencing on the formalin-fixed paraffin-embedded (FFPE) samples of 10 ccRCC patients with ≤7-cm tumors and synchronous metastasis and expanded our study using The Cancer Genome Atlas (TCGA) ccRCC dataset (n = 201). Recurrent mutations were selected according to functional prediction and statistical significance. Mutations in three candidate genes, RELN (1 out of 10), FOXC2 (1 out of 10), and CLIP4 (2 out of 10) were found in expanded analysis using a TCGA cohort. Furthermore, siRNA-mediated target gene knockdown (FOXC2 and CLIP4) and overexpression (RELN) assays showed that FOXC2 and CLIP4 significantly increased cell migration and viability in ccRCCs. Our study demonstrated that FOXC2 and CLIP4 activity correlates to the presence of ≤7-cm ccRCCs with synchronous metastasis and may be potential molecular predictors of synchronous metastasis of ≤7-cm ccRCCs.

A Case of von Hippel-Lindau Disease with Colorectal Adenocarcinoma, Renal Cell Carcinoma and Hemangioblastomas.

von Hippel-Lindau (VHL) disease is an autosomal dominant inherited tumor syndrome associated with mutations of the VHL tumor suppressor gene located on chromosome 3p25. The loss of functional VHL protein contributes to tumorigenesis. This condition is characterized by development of benign and malignant tumors in the central nervous system (CNS) and the internal organs, including kidney, adrenal gland, and pancreas. We herein describe the case of a 74-year-old man carrying the VHL gene mutation who was affected by simultaneous colorectal adenocarcinoma, renal clear cell carcinoma, and hemangioblastomas of CNS.

Cellular Adaptation to VEGF-Targeted Antiangiogenic Therapy Induces Evasive Resistance by Overproduction of Alternative Endothelial Cell Growth Factors in Renal Cell Carcinoma.

Vascular endothelial growth factor (VEGF)-targeted antiangiogenic therapy significantly inhibits the growth of clear cell renal cell carcinoma (RCC). Eventually, therapy resistance develops in even the most responsive cases, but the mechanisms of resistance remain unclear. Herein, we developed two tumor models derived from an RCC cell line by conditioning the parental cells to two different stresses caused by VEGF-targeted therapy (sunitinib exposure and hypoxia) to investigate the mechanism of resistance to such therapy in RCC. Sunitinib-conditioned Caki-1 cells in vitro did not show resistance to sunitinib compared with parental cells, but when tested in vivo, these cells appeared to be highly resistant to sunitinib treatment. Hypoxia-conditioned Caki-1 cells are more resistant to hypoxia and have increased vascularity due to the upregulation of VEGF production; however, they did not develop sunitinib resistance either in vitro or in vivo. Human endothelial cells were more proliferative and showed increased tube formation in conditioned media from sunitinib-conditioned Caki-1 cells compared with parental cells. Gene expression profiling using RNA microarrays revealed that several genes related to tissue development and remodeling, including the development and migration of endothelial cells, were upregulated in sunitinib-conditioned Caki-1 cells compared with parental and hypoxia-conditioned cells. These findings suggest that evasive resistance to VEGF-targeted therapy is acquired by activation of VEGF-independent angiogenesis pathways induced through interactions with VEGF-targeted drugs, but not by hypoxia. These results emphasize that increased inhibition of tumor angiogenesis is required to delay the development of resistance to antiangiogenic therapy and maintain the therapeutic response in RCC.