CD44, a cell surface adhesion receptor and stem cell biomarker, is recently implicated in chronic metabolic diseases. Ablation of CD44 ameliorates adipose tissue inflammation and insulin resistance in obesity. Here, we investigated cell type-specific CD44 expression in human and mouse adipose tissue and further studied how CD44 in preadipocytes regulates adipocyte function. Using Crispr Cas9-mdediated gene deletion and lentivirus-mediated gene re-expression, we discovered that deletion of CD44 promotes adipocyte differentiation and adipogenesis, whereas re-expression of CD44 abolishes this effect and decreases insulin responsiveness and adiponectin secretion in 3T3-L1 cells. Mechanistically, CD44 does so via suppressing Pparg expression. Using quantitative proteomics analysis, we further discovered that cell cycle-regulated pathways were mostly decreased by deletion of CD44. Indeed, re-expression of CD44 moderately restored expression of proteins involved in all phases of the cell cycle. These data were further supported by increased preadipocyte proliferation rates in CD44-deficient cells and re-expression of CD44 diminished this effect. Our data suggest that CD44 plays a crucial role in regulating adipogenesis and adipocyte function possibly through regulating PPARγ and cell cycle-related pathways. This study provides evidence for the first time that CD44 expressed in preadipocytes plays key roles in regulating adipocyte function outside immune cells where CD44 is primarily expressed. Therefore, targeting CD44 in (pre)adipocytes may provide therapeutic potential to treat obesity-associated metabolic complications.

Agonists at μ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve β-arrestin2. Agonists biased against β-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed μ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted μ receptor availability in PathHunter CHO cells using the irreversible antagonist, β-funaltrexamine (β-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in β-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating β-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to β-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between β-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the β-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished β-arrestin2 recruitment stimulated by DAMGO (1 μM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against β-arrestin2 recruitment through interactions with μ receptors outside the orthosteric agonist site. This article is part of the Special Issue on "Ligand Bias".

Radiotherapy (RT) plays a fundamental role in the treatment of glioblastoma (GBM). GBM are notoriously invasive and harbor a subpopulation of cells with stem-like features which exhibit upregulation of the DNA damage response (DDR) and are radioresistant. High radiation doses are therefore delivered to large brain volumes and are known to extend survival but also cause delayed toxicity with 50%-90% of patients developing neurocognitive dysfunction. Emerging evidence identifies neuroinflammation as a critical mediator of the adverse effects of RT on cognitive function. In addition to its well-established role in promoting repair of radiation-induced DNA damage, activation of poly(ADP-ribose) polymerase (PARP) can exacerbate neuroinflammation by promoting secretion of inflammatory mediators. Therefore, PARP represents an intriguing mechanistic link between radiation-induced activation of the DDR and subsequent neuroinflammation. PARP inhibitors (PARPi) have emerged as promising new agents for GBM when given in combination with RT, with multiple preclinical studies demonstrating radiosensitizing effects and at least 3 compounds being evaluated in clinical trials. We propose that concomitant use of PARPi could reduce radiation-induced neuroinflammation and reduce the severity of radiation-induced cognitive dysfunction while at the same time improving tumor control by enhancing radiosensitivity.

Abstract PARP inhibitors (PARPi) enhance radiation sensitivity in multiple cancer models, both in vitro and in vivo. Our observation that the radiosensitizing properties of PARPi are most pronounced in rapidly proliferating cells is reflected in early phase clinical trial data showing exacerbation of acute radiation toxicity in rapidly proliferating tissues such as oropharyngeal and esophageal mucosa. Lack of radiosensitization in late responding, slowly proliferating normal tissues indicates that PARPi may be more effectively combined with radiation therapy (RT) in patients with brain tumors. We are therefore evaluating the oral PARPi olaparib in combination with RT and/or temozolomide (TMZ) in the treatment of glioblastoma (GBM), the most prevalent and most aggressive primary brain tumor. Patients with GBM experience very poor outcomes in terms of median survival (c.1 year) and neurocognitive decline caused primarily by RT. Olaparib was initially evaluated in combination with daily low-dose TMZ in patients with recurrent GBM in the OPARATIC trial. Pharmacokinetic studies revealed that olaparib penetrates both core and margin regions of GBM, indicating that the BBB is significantly disrupted throughout these tumors. Olaparib could be safely combined with daily TMZ (75 mg/m2), but intermittent olaparib dosing (150 mg three days per week) was required to avoid dose-limiting hematological toxicity. Early phase testing of the olaparib-radiotherapy combination is now underway in three populations of patients with newly diagnosed GBM. Patients aged >65 with MGMT unmethylated GBM are being recruited to a randomized, placebo-controlled phase II study (PARADIGM) after a phase I dose escalation study showed that olaparib (200 mg twice daily) was extremely well tolerated when combined with brain irradiation (40 Gray in 15#). Good performance status patients aged <70 are being recruited to two parallel phase I dose escalation studies: patients with MGMT unmethylated tumors are receiving daily olaparib with RT (60 Gy in 30#) without TMZ, while patients with MGMT methylated tumors are receiving intermittent olaparib with standard chemoradiation (60 Gy). The impact of PARPi on RT induced neurotoxicity is being investigated in preclinical studies. In vitro data show that PARPi reduce proliferation of neural stem cells and protect them against RT induced apoptosis, while in vivo studies support the emerging concept that RT induced neuroinflammation is important in the pathogenesis of neurotoxicity. Importantly, preliminary PET and immunohistochemical studies have shown robust anti-neuroinflammatory effects of PARPi in this context. Ongoing experiments are defining the roles of microglia, astrocytes and neurogenesis in this phenomenon. These diverse data sets provide support for our hypothesis that combining PARPi with RT has potential to improve outcomes for GBM patients by enhancing tumor control while simultaneously suppressing neuroinflammation and alleviating RT related neurocognitive decline. Citation Format: Anthony J. Chalmers, Rodrigo Gutierrez-Quintana, David J. Walker, Karin Williams, Duncan Forster, Mark R. Jackson, Sarah Derby, Jon Stobo, Lorna Sweeting, Caroline Kelly, Stephen Durant, Kaye J. Williams. Enhancing the therapeutic ratio for glioblastoma by combining radiation therapy with PARP inhibitors [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-006.

Abstract While radiotherapy (RT) is fundamental for the treatment of brain tumors, irradiation of the brain frequently causes devastating effects on cognitive function and quality of life. DNA damage within neural stem cells (NSC) is a key factor in the pathogenesis of radiation-induced cognitive dysfunction. The ataxia telangiectasia mutated (ATM) kinase is a central protein in the DNA damage response and a critical determinant of tumor cell survival after radiation. ATM inhibition potently radiosensitizes preclinical models of GBM in vitro and in vivo. A novel, brain penetrant ATM inhibitor AZD1390, which is predicted to achieve brain tumor concentrations in the range of 1-5nM, is currently in early phase clinical evaluation in combination with RT. In marked contrast to observations in tumor models, genetic knockdown of ATM has radioprotective effects on NSC in vitro; the proposed mechanism is via suppression of p53 mediated apoptosis. The purpose of this study was to investigate the impact of AZD1390 on survival responses and mode of death in NSCs exposed to RT in vitro and in vivo. NSCs were derived from the telencephalon of E13 mouse embryos. Cells were treated with AZD1390 (0.1-10nM) 1 hour prior to ionizing radiation (IR; 0-5 Gy). Mode and timing of cell death was interrogated using IncuCyte live cell analysis to measure proliferation, cytotoxicity and apoptosis up to 72 hours post-IR. Cell viability and neurosphere formation assays were also used to measure radiation sensitivity in vitro. C57BL/6 mice received 20Gy hemibrain irradiation +/- 7-day treatment with AZD1390 (10mg/kg). Immunohistochemistry for Ki67 and Sox2 was used to assess effects on NSC in the subventricular zone (SVZ) 50 days post-irradiation. In vitro AZD1390 (1-10nM) inhibited ATM kinase function within 1 hour, evidenced by abrogation of KAP1 and p53 phosphorylation. NSCs primarily undergo apoptosis in response to IR. AZD1390 at 1 and 3nM significantly reduced apoptosis in irradiated NSCs (ratios of annexin V area under the curve 1.95 and 2 respectively); 10 nM had no effect on this parameter. Proliferation rates and cell viability after radiation were preserved at all drug concentrations. AZD1390 at 1nM did not modulate radiation effects on neurosphere formation whereas at 10nM a radiosensitizing effect was observed (ratio of SF[3Gy]=0.25). In vivo, IR decreased the number of Ki67 positive proliferating cells (92% reduction) and Sox2-positive cells (24% reduction) in the SVZ after 50 days; these effects were not exacerbated by addition of AZD1390. Acute effects (24 hours post-IR) are under investigation. We demonstrate in vitro that AZD1390 has radioprotective effects on NSCs at clinically achievable concentrations. In vivo, treatment with AZD1390 did not enhance the effects of radiation on NSCs in the SVZ. In the context of its profound radiosensitizing effects on GBM models, the absence of radiosensitization of NSCs both in vitro and in vivo strengthens the rationale for evaluating AZD1390 in combination with RT in GBM patients. Citation Format: Rodrigo Guttierez-Quintana, David J. Walker, Mark R. Jackson, Natividad Gomez-Roman, Sandeep Chahal, Stephen T. Durant, Anthony J. Chalmers. Radiotherapy in combination with the brain penetrant ATM inhibitor AZD1390 does not exacerbate radiation toxicity of neural stem cells in vitro or in vivo [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-017.

Stress exposure during prenatal and postnatal development can have persistent and often dysfunctional effects on several physiological systems, including immune function, affecting the ability to combat infection. The neuroimmune response is inextricably linked to the action of the hypothalamic-pituitary-adrenal (HPA) axis. Cytokines released from neuroimmune cells, including microglia, activate the HPA axis, while glucocorticoids in turn regulate cytokine release from microglia. Because of the close links between these two physiological systems, coupled with potential for persistent changes to HPA axis activity following developmental stress, components of the neuroimmune system could be targets for developmental programming. However, little is known of any programming effects of developmental stress on neuroimmune function. We investigated whether developmental stress exposure via elevated prenatal corticosterone (CORT) or postnatal unpredictable food availability had long-term effects on pro- (IL-1β) and anti-inflammatory (IL-10) cytokine and microglia-dependent gene (CSF1R) expression within HPA axis tissues in a precocial bird, the Japanese quail (Coturnix japonica). Following postnatal stress, we observed increased IL-1β expression in the pituitary gland, reduced IL-10 expression in the amygdala and hypothalamus, and reduced CSF1R expression within the hypothalamus and pituitary gland. Postnatal stress disrupted the ratio of IL-1β:IL-10 expression within the hippocampus and hypothalamus. Prenatal stress only increased IL-1β expression in the pituitary gland. We found no evidence for interactive or cumulative effects across life stages on basal cytokine and glia expression in adulthood. We show that postnatal stress may have a larger impact than elevated prenatal CORT on basal immunity in HPA-axis-specific brain regions, with changes in cytokine homeostasis and microglia abundance. These results provide evidence for postnatal programming of a pro-inflammatory neuroimmune phenotype at the expense of reduced microglia, which could have implications for central nervous system health and subsequent neuroimmune responses.

Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear sub population of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3' sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S1OK14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-la activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14o- by shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.

Sprouty2 (Spry2) acts as a central regulator of tubular growth and branch patterning in the developing mammalian lung by controlling both magnitude and duration of growth factor signalling. To determine if this protein coordinates airway and vascular growth factor signalling, we tested the hypothesis that Spry2 links the primary cue for airway outgrowth, fibroblast growth factor-10 (FGF-10), to genomic events underpinning the expression and release of vascular endothelial growth factor-A (VEGF-A). Using primary fetal distal lung epithelial cells (FDLE) from rat, and immortalised human bronchial epithelial cells (16HBE14o-), we identified a nuclear subpopulation of Spry2 which interacted with regions of the rat and human VEGF-A promoter spanning the hypoxia response element (HRE) and adjacent 3′ sites. In FDLE cultured at the PO2 of the fetal lung, FGF-10 relieved the Spry2 interaction at the HRE region by promoting clearance of a 39 kDa form and this was accompanied by histone-3 S10K14 phosphoacetylation, promoter de-methylation, hypoxia inducible factor-1α activation and VEGF-A expression. This repressive characteristic of nuclear Spry2 was relieved in 16HBE14oby shRNA knockdown, and stable expression of mutants (C218A; C221A) that do not interact with the VEGF-A promoter HRE region. We conclude that nuclear Spry2 acts as a molecular link which co-ordinates airway and vascular growth of the cardiopulmonary system. This identifies Spry2 as a contributing determinant of design optimality in the mammalian lung.

Throughout life physiological systems strive to maintain homeostasis and these systems are susceptible to exposure to maternal or environmental perturbations, particularly during embryonic development. In some cases, these perturbations may influence genetic and physiological processes that permanently alter the functioning of these physiological systems; a process known as developmental programming. In recent years, the neuroimmune system has garnered attention for its fundamental interactions with key hormonal systems, such as the hypothalamic pituitary adrenal (HPA) axis. The ultimate product of this axis, the glucocorticoid hormones, play a key role in modulating immune responses within the periphery and the CNS as part of the physiological stress response. It is well-established that elevated glucocorticoids induced by developmental stress exert profound short and long-term physiological effects, yet there is relatively little information of how these effects are manifested within the neuroimmune system. Pre and post-natal periods are prime candidates for manipulation in order to uncover the physiological mechanisms that underlie glucocorticoid programming of neuroimmune responses. Understanding the potential programming role of glucocorticoids may be key in uncovering vulnerable windows of CNS susceptibility to stressful experiences during embryonic development and improve our use of glucocorticoids as therapeutics in the treatment of neurodegenerative diseases.

Serum and glucocorticoid‐inducible kinase 1 (SGK1) is a protein kinase that contributes to the hormonal control of renal Na+ retention by regulating the abundance of epithelial Na+ channels (ENaC) at the apical surface of the principal cells of the cortical collecting duct (CCD). Although glucocorticoids and insulin stimulate Na+ transport by activating SGK1, the responses follow different time courses suggesting that these hormones act by different mechanisms. We therefore explored the signaling pathways that allow dexamethasone and insulin to stimulate Na+ transport in mouse CCD cells (mpkCCDcl4). Dexamethasone evoked a progressive augmentation of electrogenic Na+ transport that became apparent after ~45 min latency and was associated with increases in SGK1 activity and abundance and with increased expression of SGK1 mRNA. Although the catalytic activity of SGK1 is maintained by phosphatidylinositol‐OH‐3‐kinase (PI3K), dexamethasone had no effect upon PI3K activity. Insulin also stimulated Na+ transport but this response occurred with no discernible latency. Moreover, although insulin also activated SGK1, it had no effect upon SGK1 protein or mRNA abundance. Insulin did, however, evoke a clear increase in cellular PI3K activity. Our data are consistent with earlier work, which shows that glucocorticoids regulate Na+ retention by inducing sgk1 gene expression, and also establish that this occurs independently of increased PI3K activity. Insulin, on the other hand, stimulates Na+ transport via a mechanism independent of sgk1 gene expression that involves PI3K activation. Although both hormones act via SGK1, our data show that they activate this kinase by distinct physiological mechanisms.

The existence of a nutrient sensitive “autocatakinetic” regulator of embryonic tissue growth has been hypothesised since the early 20th century, beginning with pioneering work on the determinants of foetal size by the Australian physiologist, Thorburn Brailsford-Robertson. We now know that the mammalian target of rapamycin complexes (mTORC1 and 2) perform this essential function in all eukaryotic tissues by balancing nutrient and energy supply during the first stages of embryonic cleavage, the formation of embryonic stem cell layers and niches, the highly specified programmes of tissue growth during organogenesis and, at birth, paving the way for the first few breaths of life. This review provides a synopsis of the role of the mTOR complexes in each of these events, culminating in an analysis of lung branching morphogenesis as a way of demonstrating the central role mTOR in defining organ structural complexity. We conclude that the mTOR complexes satisfy the key requirements of a nutrient sensitive growth controller and can therefore be considered as Brailsford-Robertson's autocatakinetic centre that drives tissue growth programmes during foetal development.

Development of stem cell therapies to treat cardiovascular disease is limited by the ability to regulate stem cell differentiation and by poor cell survival once grafted to sites of damage in the heart [[1]Zhang J. Wilson G.F. Soerens A.G. et al.Functional cardiomyocytes derived from human induced pluripotent stem cells.Circ Res. 2009; 104: e30-e41Crossref PubMed Scopus (1107) Google Scholar]. SUR2A belongs to a group of “atypical” ABC proteins as, although possessing a structure of an ABC protein, it does not seem to mediate transport. Instead, SUR2A binds to inward rectifier Kir6.2 to form cardiac sarcolemmal ATP-sensitive K+ channels. The binding of SUR2A to Kir6.2 serves a dual purpose: 1) it allows translocation of the channel to the sarcolemma and 2) contributes to the channel regulation (reviewed in ref. [[2]Burke M.A. Mutharasan R.K. Ardehali H. The sulfonylurea receptor, an atypical ATP-binding cassette protein, and its regulation of the KATP channel.Circ Res. 2008; 102: 164-176Crossref PubMed Scopus (107) Google Scholar]). In vivo, KATP channels exist as a multiprotein complex that, besides pore-forming Kir6.1/Kir6.2 and regulatory SUR2A subunits, also contain a string of glycolytic and ATP-producing enzymes including creatine kinase, GAPDH and M-LDH. It has been shown that the changes in levels of SUR2A alone have a profound effect on myocardial susceptibility to different types of metabolic stresses including hypoxia, ischemia, ischemia-reperfusion and stimulation with β-adrenergic agonists (reviewed in ref. [[3]Jovanović A. Jovanović S. SUR2A targeting for cardioprotection?.Curr Opin Pharmacol. 2009; 9: 189-193Crossref PubMed Scopus (16) Google Scholar]). Increase in intracellular SUR2A raises the number of fully-assembled cardioprotective KATP channels resulting in 1) earlier opening of KATP channels in response to stress and 2) increased subsarcolemmal ATP due to increased recruitment of creatine kinase and glycolytic enzymes to the KATP channel protein complex. Improved timing of KATP channel opening as well as increased subsarcolemmal production of ATP seems to mediate cardioprotection afforded by SUR2A [[3]Jovanović A. Jovanović S. SUR2A targeting for cardioprotection?.Curr Opin Pharmacol. 2009; 9: 189-193Crossref PubMed Scopus (16) Google Scholar]. The efficacy and safety of SUR2A led to suggestion that manipulation of its expression in cardiac tissue could be a promising therapeutic strategy against ischemic heart disease [[3]Jovanović A. Jovanović S. SUR2A targeting for cardioprotection?.Curr Opin Pharmacol. 2009; 9: 189-193Crossref PubMed Scopus (16) Google Scholar]. On the other hand, it has been also shown that cardiac stem cells regenerate infarcted myocardium and improve cardiac function [[1]Zhang J. Wilson G.F. Soerens A.G. et al.Functional cardiomyocytes derived from human induced pluripotent stem cells.Circ Res. 2009; 104: e30-e41Crossref PubMed Scopus (1107) Google Scholar]. Whether SUR2A-based therapeutic strategy of heart ischemia is compatible/complementary with stem cell therapy is at the present unknown. To assess possible relationship between SUR2A and cardiac stem cells, we have collected mouse fetal hearts at E12.5 stage (for details of heart harvesting at this stage see ref. [[4]Scott C.L. Walker D.J. Cwiklinski E. Tait C. Tee A.R. Land S.C. Control of HIF-1alpha and vascular signaling in fetal lung involves cross talk between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching periodicity clock.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L455-L471Crossref PubMed Scopus (27) Google Scholar]) and infected them with adenovirus containing SUR2A (AV-SUR2A) or luciferase (control; method of infection is described in ref. [[5]Du Q. Jovanović S. Sukhodub A. Jovanović A. Infection with AV-SUR2A protects H9C2 cells against metabolic stress: a mechanism of SUR2A-mediated cytoprotection independent from the KATP channel activity.Biochim Biophys Acta-Mol Cell Res. 1803; 2010: 405-415Google Scholar]). Hearts were used 24 h later for biochemical assessments. Infection of the heart with AV-SUR2A increased SUR2A mRNA levels for ~10 times showing that infection was sufficient to produce significant increase in SUR2A (Fig. 1). At the same time, expression of genes indicating differentiation of cardiomyocytes, as measured by quantitative real time RT-PCR (for detailed methodology see ref. [[5]Du Q. Jovanović S. Sukhodub A. Jovanović A. Infection with AV-SUR2A protects H9C2 cells against metabolic stress: a mechanism of SUR2A-mediated cytoprotection independent from the KATP channel activity.Biochim Biophys Acta-Mol Cell Res. 1803; 2010: 405-415Google Scholar]), was dramatically decreased; troponin C for ~30 times and GATA 4 for ~4 times. BMZ, a housekeeping gene, was not affected at all (Fig. 1). These findings suggest that SUR2A shifted embryonic cardiomyocytes towards less differentiated state. It is well established that ERK1/2 pathway is responsible for heart embryonic development (reviewed in ref. [[6]Wang Y. Mitogen-activated protein kinases in heart development and diseases.Circulation. 2007; 116: 1413-1423Crossref PubMed Scopus (243) Google Scholar]). In order to determine whether SUR2A affects this signaling pathway we have measured phosphorylation of ERK following infection with AV-SUR2A. Using Western blotting (for details of methodology see ref. [[4]Scott C.L. Walker D.J. Cwiklinski E. Tait C. Tee A.R. Land S.C. Control of HIF-1alpha and vascular signaling in fetal lung involves cross talk between mTORC1 and the FGF-10/FGFR2b/Spry2 airway branching periodicity clock.Am J Physiol Lung Cell Mol Physiol. 2010; 299: L455-L471Crossref PubMed Scopus (27) Google Scholar]), we have found significant decrease in ERK1/2 phosphorylation in hearts infected by AV-SUR2A (Fig. 2), which was associated with dramatic increase in expression of stem cell pluripotency marker mRNAs (Oct-4, Sox2 and NANOG; Fig. 2) whose abundance is known to promote non-differentiating, proliferative growth in cardiac and other tissues [[7]Kleger A. Seufferlein T. Malan D. et al.Modulation of calcium-activated potassium channels induces cardiogenesis of pluripotent stem cells and enrichment of pacemaker-like cells.Circulation. 2010; 122: 1823-1836Crossref PubMed Scopus (100) Google Scholar]. It is well established that pluripotent stem cells can be generated from somatic cells by expression of reprograming factors, such as Oct-4 and Sox2. It has been proposed that it's the ratio of these genes to one another that really matters in reprogramming [[8]Papapetrou E.P. Tomishima M.J. Chambers S.M. et al.Stoichiometric and temporal requirements of Oct4, Sox2, Klf4, and c-Myc expression for efficient human iPSC induction and differentiation.Proc Natl Acad Sci USA. 2009; 106: 12759-12764Crossref PubMed Scopus (227) Google Scholar]. Here we have found that although SUR2A increased the expression of all 3 genes, the effect was greatest for Sox2 and Oct-4 strongly suggesting that cardiomyocytes were indeed reprogrammed into stem cells. Ratio of Sox2/Oct-4/NANOG seems to be a hallmark of cardiomyocyte non-differentiation and recent demonstration that NANOG over-expression raises Oct-4 and Sox2 and hinders cardiomyocyte differentiation strongly supports our data [[9]Otaka S. Nagura S. Koike C. et al.Selective isolation of nanog-positive human amniotic mesenchymal cells and differentiation into cardiomyocytes.Cell Reprogram. 2013; 15: 80-91PubMed Google Scholar]. These results suggest that SUR2A “tip” cardiomyocytes towards a primed state capable of non-differentiating growth which is characteristic to cardiomyocyte precursor cells. When considering that, in addition to findings from this study, increased SUR2A levels are also efficient in protecting both embryonic and adult heart cells against severe metabolic stress (including ischemia; refs. [3Jovanović A. Jovanović S. SUR2A targeting for cardioprotection?.Curr Opin Pharmacol. 2009; 9: 189-193Crossref PubMed Scopus (16) Google Scholar, 5Du Q. Jovanović S. Sukhodub A. Jovanović A. Infection with AV-SUR2A protects H9C2 cells against metabolic stress: a mechanism of SUR2A-mediated cytoprotection independent from the KATP channel activity.Biochim Biophys Acta-Mol Cell Res. 1803; 2010: 405-415Google Scholar]), this protein seems perfect to be used as a tool to 1) keep stem cells into non-differentiated state, while 2) increasing their resistance to metabolic stress. Stem cells overexpressing SUR2A would be easier to maintain in non-differentiated state, while such cells would survive better when they are grafted to treat ischemia/myocardial infarction. As methodologies securing long-lasting expression of a gene are now well developed [[10]Huang S. Kamihira M. Development of hybrid viral vectors for gene therapy.Biotechnol Adv. 2013; 31: 208-223Crossref PubMed Scopus (65) Google Scholar], there are no any technical obstacles in obtaining cardiac stem cells overexpressing SUR2A for therapeutic purposes. The properties of SUR2A to keep stem cells non-differentiated and to increase their resistance to metabolic stress suggest that SR2A overexpressing stem cells deserve to be seriously tested as a potential therapy against heart ischemia, including the myocardial infarction.Fig. 2Infection of fetal heart by SUR2A results in suppressed ERK activity and heart shifted towards less differentiated state. Fetal hearts were collected at E12.5 stage. A. Original Western blotting and corresponding graphs of fetal hearts infected with luciferase (control; luciferase was used as a control as we have determined that infection with luciferase does not affect phospho- or total ERK1/2) and SUR2A. While total ERK1/2 was not affected by SUR2A, phosphorylation of ERK1/2 was. Each bar represents mean ± S.E.M. (n = 3; n is defined as the number of infected fetal hearts). *P < 0.05. B. Original real time RT-PCR progress curves for Oct4, Sox 2 and nanog and corresponding graphs depicting cycling threshold. Each bar represents mean ± S.E.M. (n = 3). *P < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Lung development requires co‐ordinated growth of airway and vascular structures to form the branched network of tubes which supply the blood/gas barrier. We have shown that the inducer of airway outgrowth, Fibroblast Growth Factor‐10 (FGF‐10), promotes vascular signalling from fetal airway epithelium through Sprouty2 cleavage and consequent mTORC1‐dependent activation of HIF‐1α. However, fetal airway epithelium displays a nuclear sub‐population of Sprouty2 which associates with chromatin. We therefore hypothesised that uncleaved Sprouty2 represses HIFα interaction with the VEGF‐A promoter by competitive interaction with the hypoxia response element (HRE). Chromatin immunoprecipitation (ChIP) revealed that Sprouty2 interacts with the VEGF promoter in fetal airway epithelium by: 1) weak, FGF‐10‐dependent, binding to the HRE promoter region, and, 2) constitutive, FGF‐10‐independent binding to GC‐rich regions near the transcriptional start site. This interaction was inhibitory as modulation of Sprouty2 activity by siRNA knockdown or null (Y55F) mutation augmented HIF‐dependent VEGF‐A production. Moreover, immunoprecipitation revealed an FGF‐10‐dependent interaction between Sprouty2 and the HIF‐competitive inhibitor, HIF‐3α. We conclude that Sprouty2 represses HIF‐mediated VEGF‐A production in fetal lung by direct association with critical sequences within the VEGF‐A promoter.

According to inference to the best explanation (IBE), scientists infer the loveliest of competing hypotheses, 'loveliness' being explanatory virtue. This generates two key objections: that loveliness is too subjective to guide inference, and that it is no guide to truth. I defend IBE using Thomas Kuhn's notion of exemplars: the scientific theories, or applications thereof, that define Kuhnian normal science and facilitate puzzle-solving. I claim that scientists infer the explanatory puzzle-solution that best meets the standard set by the relevant exemplar of loveliness. Exemplars are the subject of consensus, eliminating subjectivity; divorced from Kuhnian relativism, they give loveliness the context-sensitivity required to be truth-tropic. The resulting account, 'Kuhnian IBE', is independently plausible and offers a partial rapprochement between IBE and Kuhn's account of science. (C) 2011 Elsevier Ltd. All rights reserved.

Lung development requires coordinated signaling between airway and vascular growth, but the link between these processes remains unclear. Mammalian target of rapamycin complex-1 (mTORC1) can amplify hypoxia-inducible factor-1α (HIF-1α) vasculogenic activity through an NH(2)-terminal mTOR binding (TOS) motif. We hypothesized that this mechanism coordinates vasculogenesis with the fibroblast growth factor (FGF)-10/FGF-receptor2b/Spry2 regulator of airway branching. First, we tested if the HIF-1α TOS motif participated in epithelial-mesenchymal vascular signaling. mTORC1 activation by insulin significantly amplified HIF-1α activity at fetal Po(2) (23 mmHg) in human bronchial epithelium (16HBE14o-) and induced vascular traits (Flk1, sprouting) in cocultured human embryonic lung mesenchyme (HEL-12469). This enhanced activation of HIF-1α by mTORC1 was abolished on expression of a HIF-1α (F99A) TOS-mutant and also suppressed vascular differentiation of HEL-12469 cocultures. Next, we determined if vasculogenesis in fetal lung involved regulation of mTORC1 by the FGF-10/FGFR2b/Spry2 pathway. Fetal airway epithelium displayed distinct mTORC1 activity in situ, and its hyperactivation by TSC1(-/-) knockout induced widespread VEGF expression and disaggregation of Tie2-positive vascular bundles. FGF-10-coated beads grafted into fetal lung explants from Tie2-LacZ transgenic mice induced localized vascular differentiation in the peripheral mesenchyme. In rat fetal distal lung epithelial (FDLE) cells cultured at fetal Po(2), FGF-10 induced mTORC1 and amplified HIF-1α activity and VEGF secretion without induction of ERK1/2. This was accompanied by the formation of a complex between Spry2, the cCBL ubiquitin ligase, and the mTOR repressor, TSC2, which abolished GTPase activity directed against Rheb, the G protein inducer of mTORC1. Thus, mTORC1 links HIF-1α-driven vasculogenesis with the FGF-10/FGFR2b/Spry2 airway branching periodicity regulator.