Hurdles to effective treatments for glioblastoma multiforme (GBM), the most aggressive form of brain cancer, are resistance to chemotherapy, radiation, and immune checkpoint inhibitors. GBMs, like other “difficult to treat” tumors, often over-utilize the phosphatidyl inositol 3 kinase (PI3K) pathway for survival, cell growth, and cell division, conferring both chemotherapy and radiation resistance. Furthermore, the PI3K pathway has been shown to modulate expression of Major Histocompatibility Complex (MHC) molecules, key components of immune recognition. Based on these observations, we examined the potential of a novel, small molecule PI3K inhibitor GCT.Glio.1 (NPT520-337), which was designed to cross the blood brain barrier, to increase the sensitivity of GBM to immune based therapies and radiation. We hypothesized that targeting PI3K would result in growth arrest and increase cell surface expression of MHC molecules and the PD-L1 checkpoint target. Treatment of GL261 mouse and human U251 GBM cell lines with GCT.Glio.1 caused growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1 and MHC class II, and synergized with radiation to cause growth arrest and cell death. Based on these results we designed and performed in vivo animal studies, implanting luciferase-flagged GL261 into the caudate nucleus of C57BL6 mice. We treated the animals orally with GCT.Glio.1 followed 24 hours later by anti-PD1 therapy in repeating cycles. The treatments significantly delayed progression of the tumor when compared to no treatment or either treatment alone, with several animals exhibiting complete regression as measured by MRI and bioluminescence. In conclusion, these results indicate that GCT.Glio.1 may sensitize GBM to immune checkpoint inhibitor and/or radiation therapies. Based on our encouraging pre-clinical data and early results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using this combination therapy.

The purpose of this study was to test the effects of a novel small molecule inhibitor of phosphatidylinositol-3 kinase (PI3K) for the treatment of glioblastoma multiforme (GBM). The key hurdles to effective treatment for GBM, the “most deadly” form of brain cancer, are resistance to chemotherapy, to radiation, and to immune check point inhibitor therapy. The overarching goal of our experiments has been to determine if a well-characterized small molecule inhibitor of PI3K, GCT.Glio.1, would improve treatment outcomes for GBM patients. The PI3K pathway has been shown to confer both chemotherapy and radiation resistance in GBM. GCT.Glio.1 (NPT520-337), a small molecule compound known to cross the blood brain barrier (BBB), was developed to target PI3K to treat GBM. GBMs, like other “difficult to treat” tumors, often over-express and actively utilize the PI3K pathway for survival, cell growth, and cell division. We used this small molecule inhibitor to determine how targeting the PI3K pathway impacts treatment resistance in GBM. We hypothesized that targeting PI3K would result in growth arrest, increase cell surface expression of a key immune check-point inhibitor target PD-L1, and would sensitize the GBM to treatment with radiation. Treatment of GL261 mouse GBM and U251 human GBM cell lines with GCT.Glio.1 resulted in profound growth arrest at the G2/M checkpoint, increased the cell surface expression of PD-L1, and synergized with radiation to cause growth arrest and subsequent cell death. In conclusion, these results indicate that GCT.Glio.1 may be a strong candidate for treatment of GBM, and suggest that GCT.Glio.1 is a candidate for rationally designed combinations with currently available radiation and immune checkpoint inhibitor therapies. Currently, the animal studies using GCT.Glio.1 in the GL261 mouse model of GBM have been initiated. Based on our promising pre-clinical data and early encouraging results from pre-clinical pharmacology and safety studies, we anticipate filing a pre-Investigational New Drug application (pre-IND) as the next step toward our goal of a clinical trial using GCT.Glio.1 for GBM patients. Citation Format: Ekokobe Fonkem, M. Karen Newell-Rogers, Richard Tobin, Debbie Healey, Mita Das, Sara Bowen, Chad Quarles, John Clark. Characterization of a novel PI3Kinase inhibitor for treatment of glioblastoma multiforme [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB136.

Pulmonary arterial (PA) wall modifications are key pathological features of pulmonary hypertension (PH). Although such abnormalities correlate with heightened phosphorylation of c-Jun N-terminal kinases 1/2 (JNK1/2) in a rat model of PH, the contribution of specific JNK isoforms to the pathophysiology of PH is unknown. Hence, we hypothesized that activation of either one, or both JNK isoforms regulates PA remodeling in PH. We detected increased JNK1/2 phosphorylation in the thickened vessels of PH patients’ lungs compared to that in lungs of healthy individuals. JNK1/2 phosphorylation paralleled a marked reduction in MAP kinase phosphatase 1 (JNK dephosphorylator) expression in patients’ lungs. Association of JNK1/2 activation with vascular modification was confirmed in the calf model of severe hypoxia-induced PH. To ascertain the role of each JNK isoform in pathophysiology of PH, wild-type (WT), JNK1 null (JNK1-/-), and JNK2 null (JNK2-/-) mice were exposed to chronic hypoxia (10% O2 for six weeks) to develop PH. In hypoxic WT lungs, an increase in JNK1/2 phosphorylation was associated with PH-like pathology. Hallmarks of PH pathophysiology, i.e. excessive accumulation of extracellular matrix and vessel muscularization with medial wall thickening, was also detected in hypoxic JNK1-/- lungs, but not in hypoxia-exposed JNK2-/- lungs. However, hypoxia-induced increases in right ventricular systolic pressure (RVSP) and in right ventricular hypertrophy (RVH) were similar in all three genotypes. Our findings suggest that JNK2 participates in PA remodeling (but likely not in vasoconstriction) in murine hypoxic PH and that modulating JNK2 actions might quell vascular abnormalities and limit the course of PH.

Current trends in preclinical medical education are moving away from traditional discipline based courses and towards integrative learning sessions centered around organ or system based courses. Elaborate mapping approaches are often developed to demonstrate linkages between and within courses. Yet curriculum design in preclinical medical education often fails to model interdisciplinary reasoning or provide sufficient time for students to practice integrative reasoning skills. The purpose of this report is to describe an approach to developing an integrated preclinical physiology/pathology curriculum by a faculty at a new college of osteopathic medicine. The Burrell College of Osteopathic Medicine will welcome its inaugural class in Fall 2016 into an educational environment based upon a spiral curriculum. The preclinical years will consist of two passes through the all organ systems of the human body. Faculty in our department are working to develop integrated sessions that form clear connections between normal physiology and the pathologic mechanisms of disease. In doing so, we are identifying core principles that can be revisited throughout in the organ system courses(Selinfreund, et al, 2016). Fabricated longitudinal clinical patient profiles guide the integration of course content (Osborne, et al, 2016). To assist students with learning within the integrated physiology and pathology framework, we are incorporating core principles of physiology, pathology and osteopathic medicine into spirally integrated learning objectives that function to guide student learning and ensure congruent faculty participation throughout preclinical curriculum. In this model, core principles are derived from discipline specific learning objectives (e.g., American Physiological Society [APS]; Group for Research in Pathology Education [GRIPE]]; National Board of Osteopathic Medical Examiners [NBOME]) and merged into session objectives that focus learning outcomes on application of knowledge. The objectives are designed to promote scaffolding within the spiral curriculum and are linked to the principles of osteopathic medicine. Session objectives in first year system courses are built around weekly themes that integrate core concepts centered on physiology and general pathology. Session objectives in the second year are built around systemic pathology and relevant physiological material in clinical medicine textbooks. The multi‐organ nature of systemic pathology will be underscored using longitudinal virtual patients. The result is a fully integrated physiology/pathology curriculum within a program of osteopathic medical education.

Rationale Lack of an experimental model of portopulmonary hypertension (POPH) has been a major obstacle in understanding of pathophysiological mechanisms underlying the disease. Objective We investigated the effects of CCl4-mediated cirrhosis on the pulmonary vasculature, as an initial step towards an improved understanding of POPH. Methods And Results Male C57BL/6 mice received intraperitoneal injection of either sterile olive oil or CCl4 3 times/week for 12 weeks. Cirrhosis and portal hypertension were confirmed by evidence of bridging fibrosis and nodule formation in CCl4-treated liver determined by trichrome/picrosirius red staining and an increase in spleen weight/body weight ratio, respectively. Staining for the oxidative stress marker, 4-hydroxynonenal (4-HNE), was strong in the liver but was absent in the lung, suggesting that CCl4 did not directly induce oxidative injury in the lung. Pulmonary acceleration time (PAT) and the ratio of PAT/pulmonary ejection time (PET) measured by echocardiography were significantly decreased in cirrhotic mice. Increase in right ventricle (RV) weight/body weight as well as in the weight ratio of RV/(left ventricle + septum) further demonstrated the presence of pathological changes in the pulmonary circulation in these mice. Histological examination revealed that lungs of cirrhotic mice have excessive accumulation of perivascular collagen and thickening of the media of the pulmonary artery. Conclusion Collectively, our data demonstrate that chronic CCl4 treatment induces pathological changes in pulmonary circulation in cirrhotic mice. We propose that this murine cirrhotic model provides an exceptional tool for future studies of the molecular mechanisms mediating pulmonary vascular diseases associated with cirrhosis and for evaluation of novel therapeutic interventions.

Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O 2 ) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

Hanying Zhang, Miyako Okamoto, Evgeniy Panzhinskiy, W. Michael Zawada, and Mita Das Department of Animal Sciences, University of Wyoming, Laramie, Wyoming; Kawahara Clinic, Tokyo, Japan; Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia, Canada; Department of Geriatrics, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and Department of Internal Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Reactive oxygen species (ROS) have been reported to affect neural stem cell self-renewal and therefore may be important for normal development and may influence neurodegenerative processes when ROS activity is elevated. To determine if increasing production of superoxide, via activation of NADPH oxidase (Nox), increases neural stem cell proliferation, 100 nM angiotensin II (Ang II) - a strong stimulator of Nox - was applied to cultures of a murine neural stem cell line, C17.2. Twelve hours following a single treatment with Ang II, there was a doubling of the number of neural stem cells. This increase in neural stem cell numbers was preceded by a gradual elevation of superoxide levels (detected by dihydroethidium fluorescence) from the steady state at 0, 5, and 30 min and gradually increasing from 1 h to the maximum at 12 h, and returning to baseline at 24 h. Ang II-dependent proliferation was blocked by the antioxidant N-acetyl-L-cysteine. Confocal microscopy revealed the presence of two sources of intracellular ROS in C17.2 cells: (i) mitochondrial and (ii) extramitochondrial; the latter indicative of the involvement of one or more specific isoforms of Nox. Of the Nox family, mRNA expression for one member, Nox4, is abundant in neural stem cell cultures, and Ang II treatment resulted in elevation of the relative levels of Nox4 protein. SiRNA targeting of Nox4 mRNA reduced both the constitutive and Ang II-induced Nox4 protein levels and attenuated Ang II-driven increases in superoxide levels and stem cell proliferation. Our findings are consistent with our hypothesis that Ang II-induced proliferation of neural stem cells occurs via Nox4-generated superoxide, suggesting that an Ang II/Nox4 axis is an important regulator of neural stem cell self-renewal and as such may fine-tune normal, stress- or disease-modifying neurogenesis.

Genetically modified mouse models have unparalleled power to determine the mechanisms behind different processes involved in the molecular and physiologic etiology of various classes of human pulmonary hypertension (PH). Processes known to be involved in PH for which there are extensive mouse models available include the following: (1) Regulation of vascular tone through secreted vasoactive factors; (2) regulation of vascular tone through potassium and calcium channels; (3) regulation of vascular remodeling through alteration in metabolic processes, either through alteration in substrate usage or through circulating factors; (4) spontaneous vascular remodeling either before or after development of elevated pulmonary pressures; and (5) models in which changes in tone and remodeling are primarily driven by inflammation. PH development in mice is of necessity faster and with different physiologic ramifications than found in human disease, and so mice make poor models of natural history of PH. However, transgenic mouse models are a perfect tool for studying the processes involved in pulmonary vascular function and disease, and can effectively be used to test interventions designed against particular molecular pathways and processes involved in disease.

AIMSPulmonary hypertension (PH) is a devastating condition for which no disease-modifying therapies exist. PH is recognized as proliferative disease of the pulmonary artery (PA). In the experimental newborn calf model of hypoxia-induced PH, adventitial fibroblasts in the PA wall exhibit a heightened replication index. Because elevated platelet-derived growth factor β receptor (PDGFβ-R) signalling is associated with PH, we tested the hypothesis that the activation of PDGFβ-R contributes to fibroblast proliferation and adventitial remodelling in PH.METHODS AND RESULTSNewborn calves were exposed to either ambient air (P(B) = 640 mmHg) (Neo-C) or high altitude (P(B) = 445 mm Hg) (Neo-PH) for 2 weeks. PDGFβ-R phosphorylation was markedly elevated in PA adventitia of Neo-PH calves as well as in cultured PA fibroblasts isolated from Neo-PH animals. PDGFβ-R activation with PDGF-BB stimulated higher replication in Neo-PH cells compared with that of control fibroblasts. PDGF-BB-induced proliferation was dependent on reactive oxygen species generation and extracellular signal-regulated kinase1/2 activation in both cell populations; however, only Neo-PH cell division via PDGFβ-R activation displayed a unique dependence on c-Jun N-terminal kinase1 (JNK1) stimulation as the blockade of JNK1 with SP600125, a pharmacological antagonist of the JNK pathway, and JNK1-targeted siRNA selectively blunted Neo-PH cell proliferation.CONCLUSIONSOur data strongly suggest that hypoxia-induced modified cells engage the PDGFβ-R-JNK1 axis to confer distinctively heightened proliferation and adventitial remodelling in PH.

BACKGROUND:Reactive oxygen species (ROS), superoxide and hydrogen peroxide (H2O2), are necessary for appropriate responses to immune challenges. In the brain, excess superoxide production predicts neuronal cell loss, suggesting that Parkinson's disease (PD) with its wholesale death of dopaminergic neurons in substantia nigra pars compacta (nigra) may be a case in point. Although microglial NADPH oxidase-produced superoxide contributes to dopaminergic neuron death in an MPTP mouse model of PD, this is secondary to an initial die off of such neurons, suggesting that the initial MPTP-induced death of neurons may be via activation of NADPH oxidase in neurons themselves, thus providing an early therapeutic target.METHODS:NADPH oxidase subunits were visualized in adult mouse nigra neurons and in N27 rat dopaminergic cells by immunofluorescence. NADPH oxidase subunits in N27 cell cultures were detected by immunoblots and RT-PCR. Superoxide was measured by flow cytometric detection of H2O2-induced carboxy-H2-DCFDA fluorescence. Cells were treated with MPP+ (MPTP metabolite) following siRNA silencing of the Nox2-stabilizing subunit p22phox, or simultaneously with NADPH oxidase pharmacological inhibitors or with losartan to antagonize angiotensin II type 1 receptor-induced NADPH oxidase activation.RESULTS:Nigral dopaminergic neurons in situ expressed three subunits necessary for NADPH oxidase activation, and these as well as several other NADPH oxidase subunits and their encoding mRNAs were detected in unstimulated N27 cells. Overnight MPP+ treatment of N27 cells induced Nox2 protein and superoxide generation, which was counteracted by NADPH oxidase inhibitors, by siRNA silencing of p22phox, or losartan. A two-wave ROS cascade was identified: 1) as a first wave, mitochondrial H2O2 production was first noted at three hours of MPP+ treatment; and 2) as a second wave, H2O2 levels were further increased by 24 hours. This second wave was eliminated by pharmacological inhibitors and a blocker of protein synthesis.CONCLUSIONS:A two-wave cascade of ROS production is active in nigral dopaminergic neurons in response to neurotoxicity-induced superoxide. Our findings allow us to conclude that superoxide generated by NADPH oxidase present in nigral neurons contributes to the loss of such neurons in PD. Losartan suppression of nigral-cell superoxide production suggests that angiotensin receptor blockers have potential as PD preventatives.

How reactive oxygen species (ROS) affect neural stem cell (NSC) self‐renewal is poorly understood, but might be important for normal development and during neurodegenerative conditions. We investigated whether production of superoxide via activation of NADPH oxidase (Nox) affects NSC proliferation. In the vascular smooth muscle cells, angiotensin II (Ang II) activates Nox‐driven superoxide generation to induce proliferation. We hypothesized that Ang II activates one of the Nox isoforms to regulate NSC self‐renewal. In a murine neural stem cell line, C17.2, Ang II (100nM) stimulated superoxide production detected by dihydroethidium (DHE) fluorescence. Ang II increased proliferation and an antioxidant N‐acetyl‐cysteine blocked Ang II‐induced increase in cell numbers. Confocal microscopy using DHE and a mitochondrial marker (MitoTracker) revealed that C17.2 cells contain two sources of ROS: mitochondrial and extramitochondrial, indicating that Nox might be involved. Nox4 was found to be constitutively expressed and Ang II increased protein levels of Nox4. SiRNA targeting Nox4 reduced Nox4 expression and attenuated Ang II‐induced ROS production and proliferation. Our findings suggest that Ang II‐induced proliferation of NSCs requires Nox4‐generated superoxide production. This mechanism might be important for normal and stress‐ or disease‐modified neurogenesis. Support: AA016654 and HL64917.

Objective Platelet derived growth (PDGF)-BB plays a pivotal role in the pathophysiology of pulmonary hypertension (PH). Pulmonary arterial adventitial fibroblasts (PAAF) are the earliest cells activated during the disease, playing important role in vessel wall remodeling. Therefore, we tested the hypothesis that PDGF-BB-induced c-Jun N-terminal kinase 1 (JNK1) activation is a key regulator of this process. Results Newborn calves were exposed to either control (C) or high altitude (4750 m, HA) for 2 weeks to induce PH. PAAF were purified from the pulmonary arteries for the cell culture studies. Phosphorylated PDGFβ receptor (PDGFβ-R) levels were elevated in the PAAF from the HA lungs compared to C lungs. PDGF-BB stimulated greater proliferation rate and reactive oxygen species (ROS) production in HA cells compared to C cells. ROS scavengers, N-acetyl cysteine (NAC), and superoxide dismutase mimetic, TEMPOL, selectively inhibited PDGF-BB-induced proliferation and ROS production in HA cells. PDGF-BB stimulated JNK1 activation only in the HA cells, a response that was attenuated by both NAC and TEMPOL. SP600125, an inhibitor of JNK1/2, and siRNA targeting JNK1 expression attenuated the PDGF-BB-induced proliferation of HA cells. Conclusions Our data strongly suggest that PDGF-BB-induced proliferation of PAAF during the vessel wall remodeling is mediated through ROS production and JNK1 activation. Funded by HL64917; P20RR016474

Epithelial‐to‐mesenchymal transition (EMT) plays an important role during progression of lung cancer. Recently, hypoxia and midkine (MK) have been shown to be key regulators of EMT in various types of cancer cells. Signal transducer and activator of transcription 3 (STAT3) also plays a critical role in this process. Therefore, we hypothesize that MK will enhance EMT in lung cancer cells grown in a hypoxic microenvironment through STAT3 activation. Human lung cancer cells (A549) were exposed to 1% oxygen in the presence of MK to test our hypothesis. EMT was evaluated by assessment of cell morphology as well as expression of marker proteins. Cell morphology was altered from cuboidal to elongated shape after 48 hours of treatments. Levels of E‐cadherin, marker of epithelial cells, were decreased. However, expression of alpha smooth muscle actin (αSMA) and vimentin, markers of mesenchymal cells, were augmented. Hypoxia/MK treatment stimulated STAT3 phosphorylation at Ser727 as well as Tyr705 residues. Interestingly, the two phosphospecies had unique subcellular localization patterns. PhophoSTAT3(Tyr705) was restricted to focal points within the cytoplasm, whereas Ser727 phosphoSTAT3 was present predominantly in the nuclear compartment. Therefore, our data strongly suggest that hypoxia/MK potentiates EMT process and this process might be mediated through STAT3 activation in human lung cancer cells.Funded by HL64917 (MD); P20RR016474 (NCRR & Wyoming INBRE)

PDGF‐BB contributes to vascular remodeling during the development of hypoxia‐induced pulmonary hypertension (HPH). Adventitial fibroblasts (AdFibs) are the first cells activated in HPH. We tested the hypothesis that PDGF‐BB‐induced activation of AdFibs is a key regulator of HPH development. Newborn calves were exposed to high altitude (4750 m) (HA) for 2 weeks to induce HPH. Phosphorylated PDGFβ receptor (PDGFβ‐R) levels were markedly elevated in the thickened adventitia of the vessels in HA lungs compared to that of control. PDGFβ‐R expression was similarly activated in AdFib isolated from the main pulmonary artery of HA animals as evaluated by immunoblot analysis. PDGF‐BB stimulated greater proliferation rate and reactive oxygen species (ROS) production in HA cells compared to the AdFibs of control animals. ROS scavengers, N‐Acetyl Cysteine (NAC), and superoxide dismutase mimetic, TEMPOL, selectively inhibited PDGF‐BB‐induced proliferation and ROS production in HA cells. PDGF‐BB induced activation of ERK1/2 and JNK1/2, but not p38 MAP kinase in both cell types. However, both NAC and TEMPOL attenuated PDGF‐BB‐stimulated heightened phosphorylation levels of ERK1/2 and JNK1/2 in HA cells only. Our results suggest that dramatic adventitial remodeling during the progression of HPH depends on PDGF‐BB‐induced AdFib proliferation mediated through ROS production and ERK/JNK MAP kinase activation.Funded by HL64917; P20RR016474

Although mitogen-activated protein kinase phosphatase-1 (MKP-1) is a key deactivator of MAP kinases, known effectors of lung vessel formation, whether it plays a role in the expression of proangiogenic vascular endothelial growth factor (VEGF) in hypoxic lung is unknown. We therefore hypothesized that MKP-1 is a crucial modulator of hypoxia-stimulated vessel development by regulating lung VEGF levels. Wild-type MKP-1(+/+), heterozygous MKP-1(+/-), and deficient MKP-1(-/-) mice were exposed to sea level (SL), Denver altitude (DA) (1609 m [5280 feet]), and severe high altitude (HYP) (∼5182 m [∼17,000 feet]) for 6 weeks. Hypoxia enhanced phosphorylation of p38 MAP kinase, a substrate of MKP-1, as well as α smooth muscle actin (αSMA) expression in vessels, respiratory epithelium, and interstitium of phosphatase-deficient lung. αSMA-positive vessel (<50 μm outside diameter) densities were markedly reduced, whereas vessel wall thickness was increased in hypoxic MKP-1(-/-) lung. Mouse embryonic fibroblasts (MEFs) of all three genotypes were isolated to pinpoint the mechanism involved in hypoxia-induced vascular abnormalities of MKP-1(-/-) lung. Sustained phosphorylation of p38 MAP kinase was observed in MKP-1-null MEFs in response to hypoxia exposure. Although hypoxia up-regulated VEGF levels in MKP-1(+/+) MEFs eightfold, only a 70% increase in VEGF expression was observed in MKP-1-deficient cells. Therefore, our data strongly suggest that MKP-1 might be the key regulator of vascular densities through the regulation of VEGF levels in hypoxic lung.

Introduction Regulated neurotransmitter actions in the mammalian central nervous system determine brain function and control peripheral organs and behavior. Although drug-seeking behaviors, including alcohol consumption, depend on central neurotransmission, modification of neurotransmitter actions in specific brain nuclei remains challenging. Herein, we report a novel approach for neurotransmission modification in vivo by transplantation of stem cells engineered to take up the neurotransmitter dopamine (DA) efficiently through the action of the human dopamine transporter (hDAT). As a functional test in mice, we used voluntary alcohol consumption, which is known to release DA in nucleus accumbens (NAC), an event hypothesized to help maintain drug-seeking behavior. We reasoned that reducing extracellular DA levels, by engrafting into NAC DA-sequestering stem cells expressing hDAT, would alter alcohol intake. Methods We have generated a neural stem cell line stably expressing the hDAT. Uptake kinetics of DA were determined to select a clone for transplantation. These genetically modified stem cells (or cells transfected with a construct lacking the hDAT sequence) were transplanted bilaterally into the NAC of wild-type mice trained to consume 10% alcohol in a two-bottle free-choice test for alcohol consumption. Alcohol intake was then ascertained for 1 week after transplantation, and brain sections through the NAC were examined for surviving grafted cells. Results Modified stem cells expressed hDAT and uptaken DA selectively via hDAT. Mice accustomed to drinking 10% ethanol by free choice reduced their alcohol consumption after being transplanted with hDAT-expressing stem cells. By contrast, control stem cells lacked that effect. Histologic examination revealed surviving stem cells in the NAC of all engrafted brains. Conclusions Our findings represent proof of principle suggesting that genetically engineered stem cells can be useful for exploring the role of neurotransmitters (or other signaling molecules) in alcohol consumption and potentially in other aspects of brain function.