Deadly teamwork: neural cancer stem cells and the tumor microenvironment

Neural cancers display cellular hierarchies with self-renewing tumorigenic cancer stem cells (CSCs) at the apex. Instructive cues to maintain CSCs are generated by both intrinsic networks and the niche microenvironment. The CSC-microenvironment relationship is complex, as CSCs can modify their environment and extrinsic forces induce plasticity in the cellular hierarchy.     

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein–coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase–dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal–regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal–regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction.     

Collagen targeting using multivalent protein-functionalized dendrimers

Collagen is an attractive marker for tissue remodeling in a variety of common disease processes. Here we report the preparation of protein dendrimers as multivalent collagen targeting ligands by native chemical ligation of the collagen binding protein CNA35 to cysteine-functionalized dendritic divalent (AB₂) and tetravalent (AB₄) wedges. The binding of these multivalent protein constructs was studied on collagen-immobilized chip surfaces as well as to native collagen in rat intestinal tissues. To understand the importance of target density we also created collagen-mimicking surfaces by immobilizing synthetic collagen triple helical peptides at various densities on a chip surface. Multivalent display of a weak-binding variant (CNA35-Y175K) resulted in a large increase in collagen affinity, effectively restoring the collagen imaging capacities for the AB₄ system. In addition, dissociation of these multivalent CNA35 dendrimers from collagen surfaces was found to be strongly attenuated.     

Technical approaches for mouse models of human disease

The mouse is the leading organism for disease research. A rich resource of genetic variation occurs naturally in inbred and special strains owing to spontaneous mutations. However, one can also obtain desired gene mutations by using the following processes: targeted mutations that eliminate function in the whole organism or in a specific tissue; forward genetic screens using chemicals or transposons; or the introduction of exogenous transgenes as DNAs, bacterial artificial chromosomes (BACs) or reporter constructs. The mouse is the only mammal that provides such a rich resource of genetic diversity coupled with the potential for extensive genome manipulation, and is therefore a powerful application for modeling human disease. This poster review outlines the major genome manipulations available in the mouse that are used to understand human disease: natural variation, reverse genetics, forward genetics, transgenics and transposons. Each of these applications will be essential for understanding the diversity that is being discovered within the human population.     

Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels

Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.     

UV photoreceptors and UV-yellow wing pigments in Heliconius butterflies allow a color signal to serve both mimicry and intraspecific communication

Mimetic wing coloration evolves in butterflies in the context of predator confusion. Unless butterfly eyes have adaptations for discriminating mimetic color variation, mimicry also carries a risk of confusion for the butterflies themselves. Heliconius butterfly eyes, which express recently duplicated ultraviolet (UV) opsins, have such an adaptation. To examine bird and butterfly color vision as sources of selection on butterfly coloration, we studied yellow wing pigmentation in the tribe Heliconiini. We confirmed, using reflectance and mass spectrometry, that only Heliconius use 3-hydroxy-DL-kynurenine (3-OHK), which looks yellow to humans but reflects both UV- and long-wavelength light, whereas butterflies in related genera have chemically unknown yellow pigments mostly lacking UV reflectance. Modeling of these color signals reveals that the two UV photoreceptors of Heliconius are better suited to separating 3-OHK from non-3-OHK spectra compared with the photoreceptors of related genera or birds. The co-occurrence of potentially enhanced UV vision and a UV-reflecting yellow wing pigment could allow unpalatable Heliconius private intraspecific communication in the presence of mimics. Our results are the best available evidence for the correlated evolution of a color signal and color vision. They also suggest that predator visual systems are error prone in the context of mimicry.     

EGCG, a green tea polyphenol, improves endothelial function and insulin sensitivity, reduces blood pressure, and protects against myocardial I/R injury in SHR

Epigallocatechin gallate (EGCG), a bioactive polyphenol in green tea, may augment metabolic and vascular actions of insulin. Therefore, we investigated effects of EGCG treatment to simultaneously improve cardiovascular and metabolic function in spontaneously hypertensive rats (SHR; model of metabolic syndrome with hypertension, insulin resistance, and overweight). In acute studies, EGCG (1-100 microM) elicited dose-dependent vasodilation in mesenteric vascular beds (MVB) isolated from SHR ex vivo that was inhibitable by N(omega)-nitro-L-arginine methyl ester (L-NAME; nitric oxide synthase antagonist) or wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor]. In chronic studies, 9-wk-old SHR were treated by gavage for 3 wk with EGCG (200 mg.kg(-1).day(-1)), enalapril (30 mg.kg(-1).day(-1)), or vehicle. A separate group of SHR receiving L-NAME (80 mg/l in drinking water) was treated for 3 wk with either EGCG or vehicle. Vasodilator actions of insulin were significantly improved in MVB from EGCG- or enalapril-treated SHR (when compared with vehicle-treated SHR). Both EGCG and enalapril therapy significantly lowered systolic blood pressure (SBP) in SHR. EGCG therapy of SHR significantly reduced infarct size and improved cardiac function in Langendorff-perfused hearts exposed to ischemia-reperfusion (I/R) injury. In SHR given L-NAME, beneficial effects of EGCG on SBP and I/R were not observed. Both enalapril and EGCG treatment of SHR improved insulin sensitivity and raised plasma adiponectin levels. We conclude that acute actions of EGCG to stimulate production of nitric oxide from endothelium using PI 3-kinase-dependent pathways may explain, in part, beneficial effects of EGCG therapy to simultaneously improve metabolic and cardiovascular pathophysiology in SHR. These findings may be relevant to understanding potential benefits of green tea consumption in patients with the metabolic syndrome.     

FK506 binding protein 8 peptidylprolyl isomerase activity manages a late stage of cystic fibrosis transmembrane conductance regulator (CFTR) folding and stability

Cystic fibrosis (CF) is caused by mutations in the apical chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) with 90% of patients carrying at least one deletion of the F508 (ΔF508) allele. This mutant form of CFTR is characterized by a folding and trafficking defect that prevents exit from the endoplasmic reticulum. We previously reported that ΔF508 CFTR can be recovered in a complex with Hsp90 and its co-chaperones as an on-pathway folding intermediate, suggesting that Δ508 CF disease arises due to a failure of the proteostasis network (PN), which manages protein folding and degradation in the cell. We have now examined the role of FK506-binding protein 8 (FKBP8), a component of the CFTR interactome, during the biogenesis of wild-type and ΔF508 CFTR. FKBP8 is a member of the peptidylprolyl isomerase family that mediates the cis/trans interconversion of peptidyl prolyl bonds. Our results suggest that FKBP8 is a key PN factor required at a post-Hsp90 step in CFTR biogenesis. In addition, changes in its expression level or alteration of its activity by a peptidylprolyl isomerase inhibitor alter CFTR stability and transport. We propose that CF is caused by the sequential failure of the prevailing PN pathway to stabilize ΔF508-CFTR for endoplasmic reticulum export, a pathway that can be therapeutically managed.     

Impact Loading and Locomotor-Respiratory Coordination Significantly Influence Breathing Dynamics in Running Humans

Locomotor-respiratory coupling (LRC), phase-locking between breathing and stepping rhythms, occurs in many vertebrates. When quadrupedal mammals gallop, 1∶1 stride per breath coupling is necessitated by pronounced mechanical interactions between locomotion and ventilation. Humans show more flexibility in breathing patterns during locomotion, using LRC ratios of 2∶1, 2.5∶1, 3∶1, or 4∶1 and sometimes no coupling. Previous studies provide conflicting evidence on the mechanical significance of LRC in running humans. Some studies suggest LRC improves breathing efficiency, but others suggest LRC is mechanically insignificant because ‘step-driven flows’ (ventilatory flows attributable to step-induced forces) contribute a negligible fraction of tidal volume. Yet, although step-driven flows are brief, they cause large fluctuations in ventilatory flow. Here we test the hypothesis that running humans use LRC to minimize antagonistic effects of step-driven flows on breathing. We measured locomotor-ventilatory dynamics in 14 subjects running at a self-selected speed (2.6±0.1 ms⁻¹) and compared breathing dynamics in their naturally ‘preferred’ and ‘avoided’ entrainment patterns. Step-driven flows occurred at 1-2X step frequency with peak magnitudes of 0.97±0.45 Ls⁻¹ (mean ±S.D). Step-driven flows varied depending on ventilatory state (high versus low lung volume), suggesting state-dependent changes in compliance and damping of thoraco-abdominal tissues. Subjects naturally preferred LRC patterns that minimized antagonistic interactions and aligned ventilatory transitions with assistive phases of the step. Ventilatory transitions initiated in ‘preferred’ phases within the step cycle occurred 2x faster than those in ‘avoided’ phases. We hypothesize that humans coordinate breathing and locomotion to minimize antagonistic loading of respiratory muscles, reduce work of breathing and minimize rate of fatigue. Future work could address the potential consequences of locomotor-ventilatory interactions for elite endurance athletes and individuals who are overweight or obese, populations in which respiratory muscle fatigue can be limiting.