Fractalkine Is Expressed in Early and Advanced Atherosclerotic Lesions and Supports Monocyte Recruitment via CX3CR1

Fractalkine (CX3CL1, FKN) is expressed in the inflamed vascular wall and absence of FKN reduces atherogenesis. Whether FKN is expressed throughout all stages of atherosclerotic disease and whether it directly contributes to monocyte recruitment to atherosclerotic lesions is not known. We collected human atherosclerotic plaque material and blood samples from patients with carotid artery disease undergoing endarterectomy. Plaques were analyzed by immunohistochemistry and qPCR. We found that FKN is expressed at all stages of atherosclerotic lesion formation, and that the number of FKN-expressing cells positively correlates with the number of CX3CR1-positive cells in human carotid artery plaques. In the circulation, soluble FKN levels are significantly elevated in the presence of high-grade (sub-occlusive) stenosis. To determine the role of the FKN-CX3CR1 axis for monocyte adhesion in vivo we then performed intravital videofluorescence microscopy of the carotid artery in ApoE(-/-) mice. Notably, FKN-CX3CR1 interactions are critical for recruitment of circulating monocytes to the injured atherosclerotic vascular wall. Thus, this chemokine dyad could represent an attractive target for anti-atherosclerotic strategies.     

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.     

Mammalian Hippo signalling: a kinase network regulated by protein-protein interactions

The Hippo signal transduction cascade controls cell growth, proliferation and death, all of which are frequently deregulated in tumour cells. Since initial studies in Drosophila melanogaster were instrumental in defining Hippo signalling, the machinery was named after the central Ste20-like kinase Hippo. Moreover, given that loss of Hippo signalling components Hippo, Warts, and Mats resulted in uncontrolled tissue overgrowth, Hippo signalling was defined as a tumour-suppressor cascade. Significantly, all of the core factors of Hippo signalling have mammalian orthologues that functionally compensate for loss of their counterparts in Drosophila. Furthermore, studies in Drosophila and mammalian cell systems showed that Hippo signalling represents a kinase cascade that is tightly regulated by PPIs (protein-protein interactions). Several Hippo signalling molecules contain SARAH (Salvador/RASSF1A/Hippo) domains that mediate specific PPIs, thereby influencing the activities of MST1/2 (mammalian Ste20-like serine/threonine kinase 1/2) kinases, the human Hippo orthologues. Moreover, WW domains are present in several Hippo factors, and these domains also serve as interaction surfaces for regulatory PPIs in Hippo signalling. Finally, the kinase activities of LATS1/2 (large tumour-suppressor kinase 1/2), the human counterparts of Warts, are controlled by binding to hMOB1 (human Mps one binder protein 1), the human Mats. Therefore Hippo signalling is regulated by PPIs on several levels. In the present paper, I review the current understanding of how these regulatory PPIs are regulated and contribute to the functionality of Hippo signalling.     

Cks1 is required for tumor cell proliferation but not sufficient to induce hematopoietic malignancies

The Cks1 component of the SCF(Skp2) complex is necessary for p27(Kip1) ubiquitylation and degradation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27(Kip1) levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27(Kip1) levels but exhibited no impact on tumor onset. This raises the possibility that Cks1 could have other oncogenic activities than suppressing p27(Kip1). To challenge this notion we have targeted overexpression of Cks1 to B cells using a conditional retroviral bone marrow transduction-transplantation system. Despite potent ectopic overexpression, Cks1 was unable to promote B cell hyperproliferation or B cell malignancies, indicating that Cks1 is not oncogenic when overexpressed in B cells. Since Skp2 overexpression can drive T-cell tumorigenesis or other cancers we also widened the quest for oncogenic activity of Cks1 by ubiquitously expressing Cks1 in hematopoetic progenitors. At variance with c-Myc overexpression, which caused acute myeloid leukemia, Cks1 overexpression did not induce myeloproliferation or leukemia. Therefore, despite being associated with a poor prognosis in various malignancies, sole Cks1 expression is insufficient to induce lymphoma or a myeloproliferative disease in vivo.     

Analysis of Metabolic Subnetworks by Flux Cone Projection

Background

Analysis of elementary modes (EMs) is proven to be a powerful constraint-based method in the study of metabolic networks. However, enumeration of EMs is a hard computational task. Additionally, due to their large number, EMs cannot be simply used as an input for subsequent analysis. One possibility is to limit the analysis to a subset of interesting reactions. However, analysing an isolated subnetwork can result in finding incorrect EMs which are not part of any steady-state flux distribution of the original network. The ideal set to describe the reaction activity in a subnetwork would be the set of all EMs projected to the reactions of interest. Recently, the concept of "elementary flux patterns" (EFPs) has been proposed. Each EFP is a subset of the support (i.e., non-zero elements) of at least one EM.

Results

We introduce the concept of ProCEMs (Projected Cone Elementary Modes). The ProCEM set can be computed by projecting the flux cone onto a lower-dimensional subspace and enumerating the extreme rays of the projected cone. In contrast to EFPs, ProCEMs are not merely a set of reactions, but projected EMs. We additionally prove that the set of EFPs is included in the set of ProCEM supports. Finally, ProCEMs and EFPs are compared for studying substructures of biological networks.

Conclusions

We introduce the concept of ProCEMs and recommend its use for the analysis of substructures of metabolic networks for which the set of EMs cannot be computed.     

Autophagy in idiopathic pulmonary fibrosis

Background

Autophagy is a basic cellular homeostatic process important to cell fate decisions under conditions of stress. Dysregulation of autophagy impacts numerous human diseases including cancer and chronic obstructive lung disease. This study investigates the role of autophagy in idiopathic pulmonary fibrosis.

Methods

Human lung tissues from patients with IPF were analyzed for autophagy markers and modulating proteins using western blotting, confocal microscopy and transmission electron microscopy. To study the effects of TGF-β₁ on autophagy, human lung fibroblasts were monitored by fluorescence microscopy and western blotting. In vivo experiments were done using the bleomycin-induced fibrosis mouse model.

Results

Lung tissues from IPF patients demonstrate evidence of decreased autophagic activity as assessed by LC3, p62 protein expression and immunofluorescence, and numbers of autophagosomes. TGF-β₁ inhibits autophagy in fibroblasts in vitro at least in part via activation of mTORC1; expression of TIGAR is also increased in response to TGF-β₁. In the bleomycin model of pulmonary fibrosis, rapamycin treatment is antifibrotic, and rapamycin also decreases expression of á-smooth muscle actin and fibronectin by fibroblasts in vitro. Inhibition of key regulators of autophagy, LC3 and beclin-1, leads to the opposite effect on fibroblast expression of á-smooth muscle actin and fibronectin.

Conclusion

Autophagy is not induced in pulmonary fibrosis despite activation of pathways known to promote autophagy. Impairment of autophagy by TGF-β₁ may represent a mechanism for the promotion of fibrogenesis in IPF.     

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.     

Class B scavenger receptor types I and II and CD36 targeting improves sepsis survival and acute outcomes in mice

Class B scavenger receptors (SR-Bs), such as SR-BI/II or CD36, bind lipoproteins but also mediate bacterial recognition and phagocytosis. In evaluating whether blocking receptors can prevent intracellular bacterial proliferation, phagocyte cytotoxicity, and proinflammatory signaling in bacterial infection/sepsis, we found that SR-BI/II- or CD36-deficient phagocytes are characterized by a reduced intracellular bacterial survival and a lower cytokine response and were protected from bacterial cytotoxicity in the presence of antibiotics. Mice deficient in either SR-BI/II or CD36 are protected from antibiotic-treated cecal ligation and puncture (CLP)-induced sepsis, with greatly increased peritoneal granulocytic phagocyte survival (8-fold), a drastic diminution in peritoneal bacteria counts, and a 50-70% reduction in systemic inflammation (serum levels of IL-6, TNF-α, and IL-10) and organ damage relative to CLP in wild-type mice. The survival rate of CD36-deficient mice after CLP was 58% compared with 17% in control mice. When compensated for mineralocorticoid and glucocorticoid deficiency, SR-BI/II-deficient mice had nearly a 50% survival rate versus 5% in mineralo-/glucocorticoid-treated controls. Targeting SR-B receptors with L-37pA, a peptide that functions as an antagonist of SR-BI/II and CD36 receptors, also increased peritoneal granulocyte counts, as well as reduced peritoneal bacteria and bacterium-induced cytokine secretion. In the CLP mouse sepsis model, L-37pA improved survival from 6 to 27%, reduced multiple organ damage, and improved kidney function. These results demonstrate that the reduction of both SR-BI/II- and CD36-dependent bacterial invasion and inflammatory response in the presence of antibiotic treatment results in granulocyte survival and local bacterial containment, as well as reduces systemic inflammation and organ damage and improves animal survival during severe infections.     

Depth of α-synuclein in a bilayer determined by fluorescence, neutron reflectometry, and computation

α-Synuclein (α-syn) membrane interactions are implicated in the pathogenesis of Parkinson's disease. Fluorescence and neutron reflectometry (NR) measurements reveal that α-syn penetrates ～9–14 Å into the outer leaflet of the bilayer, with a substantial portion of the membrane-bound polypeptide extending into the aqueous solvent. For the first time, to our knowledge, we used NR to obtain direct quantitative evidence of α-syn-induced membrane thinning. To examine the effect of specific residues on membrane penetration depths, we used a series of W4-containing N-terminal peptides. We identified that the first 15 residues (P15) nearly recapitulate the features of the full-length protein (i.e., partition constants, molecular mobility, and insertion of the W4 side chain into the bilayer), and found that as few as the first four N-terminal residues are sufficient for vesicle binding. Although at least one imperfect amphipathic repeat sequence (KAKEGV) is required for α-helical formation, secondary structural formation has little effect on membrane affinity. To develop an N-terminal α-syn model for bilayer interactions, we performed molecular-dynamics simulations of the P15 peptide submerged in a bilayer. The simulation results are highly consistent with experimental data indicating a broad low-energy region (8.5–14.5 Å) for W4 insertion.     

In vivo quantification of cell coupling in plants with different phloem-loading strategies

Uptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.