Effects of Drag-Reducing Polymers on Hemodynamics and Whole Blood-Endothelial Interactions in 3D-Printed Vascular Topologies.

Most in vitro models use culture medium to apply fluid shear stress to endothelial cells, which does not capture the interaction between blood and endothelial cells. Here, we describe a new system to characterize whole blood flow through a 3D-printed, endothelialized vascular topology that induces flow separation at a bifurcation. Drag-reducing polymers, which have been previously studied as a potential therapy to reduce the pressure drop across the vascular bed, are evaluated for their effect on mitigating the disturbed flow. Polymer concentrations of 1000 ppm prevented recirculation and disturbed flow at the wall. Proteomic analysis of plasma collected from whole blood recirculated through the vascularized channel with and without drag-reducing polymers provides insight into the effects of flow regimes on levels of proteins indicative of the endothelial-blood interaction. The results indicate that blood flow alters proteins associated with coagulation, inflammation, and other processes. Overall, these proof-of-concept experiments demonstrate the importance of using whole blood flow to study the endothelial response to perfusion.

Engineered Dual Antioxidant Enzyme Complexes Targeting ICAM-1 on Brain Endothelium Reduce Brain Injury-Associated Neuroinflammation.

The neuroinflammatory cascade triggered by traumatic brain injury (TBI) represents a clinically important point for therapeutic intervention. Neuroinflammation generates oxidative stress in the form of high-energy reactive oxygen and nitrogen species, which are key mediators of TBI pathology. The role of the blood-brain barrier (BBB) is essential for proper neuronal function and is vulnerable to oxidative stress. Results herein explore the notion that attenuating oxidative stress at the vasculature after TBI may result in improved BBB integrity and neuroprotection. Utilizing amino-chemistry, a biological construct (designated "dual conjugate" for short) was generated by covalently binding two antioxidant enzymes (superoxide dismutase 1 (SOD-1) and catalase (CAT)) to antibodies specific for ICAM-1. Bioengineering of the conjugate preserved its targeting and enzymatic functions, as evaluated by real-time bioenergetic measurements (via the Seahorse-XF platform), in brain endothelial cells exposed to increasing concentrations of hydrogen peroxide or a superoxide anion donor. Results showed that the dual conjugate effectively mitigated the mitochondrial stress due to oxidative damage. Furthermore, dual conjugate administration also improved BBB and endothelial protection under oxidative insult in an in vitro model of TBI utilizing a software-controlled stretching device that induces a 20% in mechanical strain on the endothelial cells. Additionally, the dual conjugate was also effective in reducing indices of neuroinflammation in a controlled cortical impact (CCI)-TBI animal model. Thus, these studies provide proof of concept that targeted dual antioxidant biologicals may offer a means to regulate oxidative stress-associated cellular damage during neurotrauma.

Activation of CB2R by synthetic CB2R agonist, PM289, improves brain endothelial barrier properties, decreases inflammatory response and enhances endothelial repair.

The Cannabinoid 2 Receptor (CB2R) has been found to provide immunological modulation in different cell types. More recently, detection of CB2R in the cerebral endothelium suggests a possible role in the resolution of inflammation at the level of the blood-brain-barrier (BBB). Here, the notion that CB2R upregulation in brain endothelial cells could be exploited to promote vascular protection and BBB integrity was evaluated. Targeting and activation of CB2R was accomplished by a novel and highly specific chromenopyrazole based CB2R agonist, PM289. This study demonstrates that CB2R upregulation is induced as early as 8 h in the cortical vasculature in an experimental mouse model of TBI. Unlike CB2R, CB1R was marginally detected and not significantly induced. In the human brain endothelial cell line, hCMEC/D3 cells, similar induction of CB2R was observed upon stimulation with TNFα. Analysis of transendothelial electrical resistance shows that PM289 markedly prevented the barrier-leakiness induced by TNFα. The BBB is also responsible for maintaining an immunological barrier. The five-fold increase in ICAM1 expression in stimulated endothelial cells was significantly diminished due to CB2R activation. Utilizing wounding assays, results showed that wound repair could be accomplished in nearly half the time when the novel CB2R agonist is present compared to the untreated control. Lastly, mechanistically, the effects of CB2R may be explained by the observed inhibition of the p65 NFκB subunit. Overall, these studies support the notion that targeting and activating CB2R in the brain vasculature could aid in BBB and vascular protection in the context of neuroinflammation.

A Next-Generation 3D Tissue-Engineered Model of the Human Brain Microvasculature to Study the Blood-Brain Barrier.

More than a billion people are affected by neurological disorders, and few have effective therapeutic options. A key challenge that has prevented promising preclinically proven strategies is the translation gap to the clinic. Humanized tissue engineering models that recreate the brain environment may aid in bridging this translational gap. Here, we showcase the methodology that allows for the practical fabrication of a comprehensive microphysicological system (MPS) of the blood-brain barrier (BBB). Compared to other existing 2D and 3D models of the BBB, this model features relevant cytoarchitecture and multicellular arrangement, with branching and network topologies of the vascular bed. This process utilizes 3D bioprinting with digital light processing to generate a vasculature lumen network surrounded by embedded human astrocytes. The lumens are then cellularized with primary human brain microvascular endothelial cells and pericytes. To initiate mechanotransduction pathways and complete maturation, vascular structures are continuously perfused for 7 days. Constructs are validated for complete endothelialization with viability dyes prior to functional assessments that include barrier integrity (permeability) and immune-endothelial interactions. This MPS has applications for the study of novel therapeutics, toxins, and elucidating mechanisms of pathophysiology.

An Engineered Adeno-Associated Virus Capsid Mediates Efficient Transduction of Pericytes and Smooth Muscle Cells of the Brain Vasculature.

Neurodegeneration and cerebrovascular disease share an underlying microvascular dysfunction that may be remedied by selective transgene delivery. To date, limited options exist in which cellular components of the brain vasculature can be effectively targeted by viral vector therapeutics. In this study, we characterize the first engineered adeno-associated virus (AAV) capsid mediating high transduction of cerebral vascular pericytes and smooth muscle cells (SMCs). We performed two rounds of in vivo selection with an AAV capsid scaffold displaying a heptamer peptide library to isolate capsids that traffic to the brain after intravenous delivery. One identified capsid, termed AAV-PR, demonstrated high transduction of the brain vasculature, in contrast to the parental capsid, AAV9, which transduces mainly neurons and astrocytes. Further analysis using tissue clearing, volumetric rendering, and colocalization revealed that AAV-PR enabled high transduction of cerebral pericytes located on small-caliber vessels and SMCs in the larger arterioles and penetrating pial arteries. Analysis of tissues in the periphery indicated that AAV-PR also transduced SMCs in large vessels associated with the systemic vasculature. AAV-PR was also able to transduce primary human brain pericytes with higher efficiency than AAV9. Compared with previously published AAV capsids tropisms, AAV-PR represents the first capsid to allow for effective transduction of brain pericytes and SMCs and offers the possibility of genetically modulating these cell types in the context of neurodegeneration and other neurological diseases.

SARS-CoV-2 Spike Protein Disrupts Blood-Brain Barrier Integrity via RhoA Activation.

The SARS-CoV-2 spike protein has been shown to disrupt blood-brain barrier (BBB) function, but its pathogenic mechanism of action is unknown. Whether angiotensin converting enzyme 2 (ACE2), the viral binding site for SARS-CoV-2, contributes to the spike protein-induced barrier disruption also remains unclear. Here, a 3D-BBB microfluidic model was used to interrogate mechanisms by which the spike protein may facilitate barrier dysfunction. The spike protein upregulated the expression of ACE2 in response to laminar shear stress. Moreover, interrogating the role of ACE2 showed that knock-down affected endothelial barrier properties. These results identify a possible role of ACE2 in barrier homeostasis. Analysis of RhoA, a key molecule in regulating endothelial cytoskeleton and tight junction complex dynamics, reveals that the spike protein triggers RhoA activation. Inhibition of RhoA with C3 transferase rescues its effect on tight junction disassembly. Overall, these results indicate a possible means by which the engagement of SARS-CoV-2 with ACE2 facilitates disruption of the BBB via RhoA activation. Understanding how SARS-CoV-2 dysregulates the BBB may lead to strategies to prevent the neurological deficits seen in COVID-19 patients.

Extracellular Microvesicles Released From Brain Endothelial Cells are Detected in Animal Models Of HIV-1 Signifying Unresolved Inflammation.

Treatment of HIV-infected patients with antiretroviral therapy (ART) has effectively suppressed viral replication; however, the central nervous system is still a major target and reservoir of the virus leading to the possible development of HIV-associated neurocognitive disorders (HAND). Furthermore, a hallmark feature of HAND is the disruption of the blood-brain barrier that leads to loss of tight junction protein (TJP) complexes. Extracellular vesicles (EVs), released by every cell type in the body, occur in greater quantities in response to cellular activation or injury. We have found that inflammatory insults activate brain endothelial cells (EC) and induce the release of EVs containing TJPs such as Occludin. We thus hypothesized that HIV infection and unresolved neuroinflammation will result in the release of brain-EC derived EVs. Herein, our results show elevated levels of brain-EC EVs in a humanized mouse model of HIV infection. Furthermore, while ART reduced brain-EC EVs, it was unable to completely resolve increased vesicles detectable in the blood. In addition to inflammatory insults, HIV-1 viral proteins (Tat and gp120) increased the release of Occludin + vesicles from human brain microvasculature ECs. This increase in vesicle release could be prevented by knock-down of the small GTPase ARF6. ARF6 has been shown to regulate EV biogenesis in other cell types, and we provide further evidence for the involvement of ARF6 in brain EC derived EVs. Overall, this study offers insight into the process of brain vascular remodeling (via EVs) in the setting of neuroinflammation and thus provides possibilities for biomarker monitoring and targeting of ARF6.

The psychoactive drug of abuse mephedrone differentially disrupts blood-brain barrier properties.

BACKGROUND: Synthetic cathinones are a category of psychostimulants belonging to the growing number of designer drugs also known as "Novel Psychoactive Substances" (NPS). In recent years, NPS have gained popularity in the recreational drug market due to their amphetamine-like stimulant effects, low cost, ease of availability, and lack of detection by conventional toxicology screening. All these factors have led to an increase in NPS substance abuse among the young adults, followed by spike of overdose-related fatalities and adverse effects, severe neurotoxicity, and cerebral vascular complications. Much remains unknown about how synthetic cathinones negatively affect the CNS and the status of the blood-brain barrier (BBB). METHODS: We used in vitro models of the BBB and primary human brain microvascular endothelial cells (hBMVEC) to investigate the effects of the synthetic cathinone, 4-methyl methcathinone (mephedrone), on BBB properties. RESULTS: We showed that mephedrone exposure resulted in the loss of barrier properties and endothelial dysfunction of primary hBMVEC. Increased permeability and decreased transendothelial electrical resistance of the endothelial barrier were attributed to changes in key proteins involved in the tight junction formation. Elevated expression of matrix metalloproteinases, angiogenic growth factors, and inflammatory cytokines can be explained by TLR-4-dependent activation of NF-κB signaling. CONCLUSIONS: In this first characterization of the effects of a synthetic cathinone on human brain endothelial cells, it appears clear that mephedrone-induced damage of the BBB is not limited by the disruption of the barrier properties but also include endothelial activation and inflammation. This may especially be important in comorbid situations of mephedrone abuse and HIV-1 infections. In this context, mephedrone could negatively affect HIV-1 neuroinvasion and NeuroAIDS progression.

ErbB3 is a critical regulator of cytoskeletal dynamics in brain microvascular endothelial cells: Implications for vascular remodeling and blood brain barrier modulation.

Neuregulin (NRG)1 - ErbB receptor signaling has been shown to play an important role in the biological function of peripheral microvascular endothelial cells. However, little is known about how NRG1/ErbB signaling impacts brain endothelial function and blood-brain barrier (BBB) properties. NRG1/ErbB pathways are affected by brain injury; when brain trauma was induced in mice in a controlled cortical impact model, endothelial ErbB3 gene expression was reduced to a greater extent than that of other NRG1 receptors. This finding suggests that ErbB3-mediated processes may be significantly compromised after injury, and that an understanding of ErbB3 function would be important in the of study of endothelial biology in the healthy and injured brain. Towards this goal, cultured brain microvascular endothelial cells were transfected with siRNA to ErbB3, resulting in alterations in F-actin organization and microtubule assembly, cell morphology, migration and angiogenic processes. Importantly, a significant increase in barrier permeability was observed when ErbB3 was downregulated, suggesting ErbB3 involvement in BBB regulation. Overall, these results indicate that neuregulin-1/ErbB3 signaling is intricately connected with the cytoskeletal processes of the brain endothelium and contributes to morphological and angiogenic changes as well as to BBB integrity.

VPS35 regulates tau phosphorylation and neuropathology in tauopathy.

The vacuolar protein sorting 35 (VPS35) is a major component of the retromer recognition core complex which regulates intracellular protein sorting and trafficking. Deficiency in VPS35 by altering APP/Aß metabolism has been linked to late-onset Alzheimer's disease. Here we report that VPS35 is significantly reduced in Progressive Supra-nuclear Palsy and Picks' disease, two distinct primary tauopathies. In vitro studies show that overexpression of VPS35 leads to a reduction of pathological tau in neuronal cells, whereas genetic silencing of VPS35 results in its accumulation. Mechanistically the availability of active cathepsin D mediates the effect of VPS35 on pathological tau accumulation. Moreover, in a relevant transgenic mouse model of tauopathy, down-regulation of VPS35 results in an exacerbation of motor and learning impairments as well as accumulation of pathological tau and loss of synaptic integrity. Taken together, our data identify VPS35 as a novel critical player in tau metabolism and neuropathology, and a new therapeutic target for human tauopathies.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in-vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system include neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-CoV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. The spike protein, which plays a key role in receptor recognition, is formed by the S1 subunit containing a receptor binding domain (RBD) and the S2 subunit. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is ubiquitously expressed throughout various vessel calibers in the frontal cortex. Moreover, ACE2 expression was upregulated in cases of hypertension and dementia. ACE2 was also detectable in primary hBMVECs maintained under cell culture conditions. Analysis of cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48 h exposure window. Introduction of spike proteins to invitro models of the blood-brain barrier (BBB) showed significant changes to barrier properties. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluidic model of the human BBB, a platform that more closely resembles the physiological conditions at this CNS interface. Evidence provided suggests that the SARS-CoV-2 spike proteins trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

HIV infects astrocytes in vivo and egresses from the brain to the periphery.

HIV invades the brain during acute infection. Yet, it is unknown whether long-lived infected brain cells release productive virus that can egress from the brain to re-seed peripheral organs. This understanding has significant implication for the brain as a reservoir for HIV and most importantly HIV interplay between the brain and peripheral organs. Given the sheer number of astrocytes in the human brain and their controversial role in HIV infection, we evaluated their infection in vivo and whether HIV infected astrocytes can support HIV egress to peripheral organs. We developed two novel models of chimeric human astrocyte/human peripheral blood mononuclear cells: NOD/scid-IL-2Rgc null (NSG) mice (huAstro/HuPBMCs) whereby we transplanted HIV (non-pseudotyped or VSVg-pseudotyped) infected or uninfected primary human fetal astrocytes (NHAs) or an astrocytoma cell line (U138MG) into the brain of neonate or adult NSG mice and reconstituted the animals with human peripheral blood mononuclear cells (PBMCs). We also transplanted uninfected astrocytes into the brain of NSG mice and reconstituted with infected PBMCs to mimic a biological infection course. As expected, the xenotransplanted astrocytes did not escape/migrate out of the brain and the blood brain barrier (BBB) was intact in this model. We demonstrate that astrocytes support HIV infection in vivo and egress to peripheral organs, at least in part, through trafficking of infected CD4+ T cells out of the brain. Astrocyte-derived HIV egress persists, albeit at low levels, under combination antiretroviral therapy (cART). Egressed HIV evolved with a pattern and rate typical of acute peripheral infection. Lastly, analysis of human cortical or hippocampal brain regions of donors under cART revealed that astrocytes harbor between 0.4-5.2% integrated HIV gag DNA and 2-7% are HIV gag mRNA positive. These studies establish a paradigm shift in the dynamic interaction between the brain and peripheral organs which can inform eradication of HIV reservoirs.

The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood-brain barrier.

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Experimental Traumatic Brain Injury during Adolescence Enhances Cocaine Rewarding Efficacy and Dysregulates Dopamine and Neuroimmune Systems in Brain Reward Substrates.

Although clinical studies identify traumatic brain injury (TBI) as a risk factor for the development of substance use disorder, much remains unknown about the possible underlying pathogenesis and age-specific effects. Thus, the aim of this study is to test the hypothesis that at an age of ongoing maturation, adolescent TBI alters elements of the reward pathway, resulting in increased sensitivity to the rewarding effects of a subthreshold dose of cocaine that does not induce significant behavioral changes in naïve, non-injured mice. Specifically, these results were derived from the combination of the controlled cortical impact model of TBI, performed on either adolescent (6 weeks) or young adult (8 weeks) mice, followed by the cocaine-induced conditioned place preference assay 2 weeks later. Using three-dimensional isosurface rendering and volumetric image analysis, TBI was found to induce neuromorphological changes such as decreased dendritic complexity and reduced spine density in brain regions essential for reward perception and processing of drug-induced euphoria. Further, we demonstrated that these neuronal changes may affect the differential expression of dopamine-associated genes. Our analysis also provided evidence for age-related differences in immune response and the distinct involvement of augmented microglial phagocytic activity in the remodeling of neuronal structures in the adolescent TBI brain. Our studies suggest that TBI during adolescence, a period associated with ongoing maturation of dopaminergic systems, may subsequently enhance the abuse liability of cocaine in adulthood.

Reward and immune responses in adolescent females following experimental traumatic brain injury.

The pathology of traumatic brain injury (TBI) adversely affects many brain regions, often resulting in the development of comorbid psychiatric disorders including substance use disorders (SUD). Although traditionally thought to be an epidemic that predominantly affects males, recent clinical studies report females have higher rates of concussions and longer recovery times than males. Yet, how neurotrauma, particularly deep within the brain, between the sexes is differentially manifested remains largely unknown. The risk of TBI peaks during adolescence when neuronal networks that regulate reward behaviors are not fully developed. Previously, using the conditioned place preference (CPP) assay, we found that adolescent TBI increased susceptibility to the rewarding effects of cocaine in male mice. Further, we observed augmented inflammatory profiles, increased microglial phagocytosis of neuronal proteins, and decreased neuronal spine density in the NAc. Notably, the extent of sex differences in SUD susceptibility following TBI has not be investigated. Thus, here we ask the central question of whether the adolescent TBI-induced neuroinflammatory profile at reward centers is divergent in a sex-dependent manner. Using the CPP assay, we found that female mice with high levels of female sex hormones at the time of adolescent TBI demonstrated neuroprotection against increased sensitivity to the rewarding effects of cocaine. These studies also provide evidence of significantly reduced microglial activation and phagocytosis of neuronal proteins within the NAc of females. Overall, our results offer crucial insight into how adolescent TBI impacts the reward pathway in a sex depending manner that could explain a vulnerability to addiction-like behavior.

Selection of an Efficient AAV Vector for Robust CNS Transgene Expression.

Adeno-associated virus (AAV) capsid libraries have generated improved transgene delivery vectors. We designed an AAV library construct, iTransduce, that combines a peptide library on the AAV9 capsid with a Cre cassette to enable sensitive detection of transgene expression. After only two selection rounds of the library delivered intravenously in transgenic mice carrying a Cre-inducible fluorescent protein, we flow sorted fluorescent cells from brain, and DNA sequencing revealed two dominant capsids. One of the capsids, termed AAV-F, mediated transgene expression in the brain cortex more than 65-fold (astrocytes) and 171-fold (neurons) higher than the parental AAV9. High transduction efficiency was sex-independent and sustained in two mouse strains (C57BL/6 and BALB/c), making it a highly useful capsid for CNS transduction of mice. Future work in large animal models will test the translation potential of AAV-F.

Endothelial Targeted Strategies to Combat Oxidative Stress: Improving Outcomes in Traumatic Brain Injury.

The endothelium is a thin monolayer of specialized cells that lines the luminal wall of blood vessels and constitutes the critical innermost portion of the physical barrier between the blood and the brain termed the blood-brain barrier (BBB). Aberrant changes in the endothelium occur in many neuropathological states, including those with high morbidity and mortality that lack targeted therapeutic interventions, such as traumatic brain injury (TBI). Utilizing ligands of surface determinants expressed on brain endothelium to target and combat injury mechanisms at damaged endothelium offers a new approach to the study of TBI and new avenues for clinical advancement. Many factors influence the targets that are expressed on endothelium. Therefore, the optimization of binding sites and ideal design features of nanocarriers are controllable factors that permit the engineering of nanotherapeutic agents with applicability that is specific to a known disease state. Following TBI, damaged endothelial cells upregulate cell adhesion molecules, including ICAM-1, and are key sites of reactive oxygen species (ROS) generation, including hydrogen peroxide. Reactive oxygen species along with pro-inflammatory mediators are known to contribute to endothelial damage and loss of BBB integrity. The use of targeted endothelial nanomedicine, with conjugates of the antioxidant enzyme catalase linked to anti-ICAM-1 antibodies, has recently been demonstrated to minimize oxidative stress at the BBB and reduce neuropathological outcomes following TBI. Here, we discuss targeted endothelial nanomedicine and its potential to provide benefits in TBI outcomes and future directions of this approach.

Characterization of cancer-associated IDH2 mutations that differ in tumorigenicity, chemosensitivity and 2-hydroxyglutarate production.

The family of isocitrate dehydrogenase (IDH) enzymes is vital for cellular metabolism, as IDH1 and IDH2 are required for the decarboxylation of isocitrate to α-ketoglutarate. Heterozygous somatic mutations in IDH1 or IDH2 genes have been detected in many cancers. They share the neomorphic production of the oncometabolite (R)-2-hydroxyglutarate [(R)-2-HG]. With respect to IDH2, it is unclear whether all IDH2 mutations display the same or differ in tumorigenic properties and degrees of chemosensitivity. Here, we evaluated the three most frequent IDH2 mutations occurring in cancer. The predicted changes to the enzyme structure introduced by these individual mutations are supported by the observed production of (R)-2-HG. However, their tumorigenic properties, response to chemotherapeutic agents, and baseline activation of STAT3 differed. Paradoxically, the varying levels of endogenous (R)-2-HG produced by each IDH2 mutant inversely correlated with their respective growth rates. Interestingly, while we found that (R)-2-HG stimulated the growth of non-transformed cells, (R)-2-HG also displayed antitumor activity by suppressing the growth of tumors harboring wild type IDH2. The mitogenic effect of (R)-2-HG in immortalized cells could be switched to antiproliferative by transformation with oncogenic RAS. Thus, our findings show that despite their shared (R)-2-HG production, IDH2 mutations are not alike and differ in shaping tumor cell behavior and response to chemotherapeutic agents. Our study also reveals that under certain conditions, (R)-2-HG has antitumor properties.