A local proinflammatory signalling loop facilitates adverse age-associated arterial remodeling

Background

The coincidence of vascular smooth muscle cells (VSMC) infiltration and collagen deposition within a diffusely thickened intima is a salient feature of central arterial wall inflammation that accompanies advancing age. However, the molecular mechanisms involved remain undefined.

Methodology/Principal Findings

Immunostaining and immunoblotting of rat aortae demonstrate that a triad of proinflammatory molecules, MCP-1, TGF-β1, and MMP-2 increases within the aortic wall with aging. Exposure of VSMC isolated from 8-mo-old rats (young) to MCP-1 effects, via CCR-2 signaling, both an increase in TGF-β1 activity, up to levels of untreated VSMC from 30-mo-old (old) rats, and a concurrent increase in MMP-2 activation. Furthermore, exposure of young VSMC to TGF-β1 increases levels of MCP-1, and MMP-2 activation, to levels of untreated VSMC from old rats. This autocatalytic signaling loop that enhances collagen production and invasiveness of VSMC is effectively suppressed by si-MCP-1, a CCR2 antagonist, or MMP-2 inhibition.

Conclusions/Significance

Threshold levels of MCP-1, MMP-2, or TGF-β1 activity trigger a feed-forward signaling mechanism that is implicated in the initiation and progression of adverse age-associated arterial wall remodeling. Intervention that suppressed this signaling loop may potentially retard age-associated adverse arterial remodeling.     

Activation of hypersensitive cell death by pathogen-induced receptor-like protein kinases from <Emphasis Type="Italic">Arabidopsis</Emphasis>

In Arabidopsis, there is a family of receptor-like protein kinases (RLKs) containing novel cysteine-rich repeats in their extracellular domains. Genes encoding many of these cysteine-rich RLKs (CRKs) are induced by pathogen infection, suggesting a possible role in plant defense responses. We have previously generated Arabidopsis plants expressing four pathogen-regulated CRK genes (CRK5, 6, 10 and 11) under control of a steroid-inducible promoter and found that induced expression of CRK5, but not the other three CRK genes, triggered hypersensitive response-like cell death in transgenic plants. In the present study, we have analyzed the structural relationship of the CRK family and identified three CRKs (CRK4, 19 and 20) that are structurally closely related to CRK5. Genes encoding these three CRKs are all induced by salicylic acid and pathogen infection. Furthermore, induced expression of CRK4, 19and 20 all activates rapid cell death in transgenic plants. Thus, the activity of inducing rapid cell death is shared by these structurally closely related CRKs. We have also performed yeast two-hybrid screens and identified proteins that interact with the kinase domains of CRKs. One of the identified CRK-interacting proteins is the kinase-associated type 2C protein phospohatase known to interact with a number of other RLKs through its kinase-interacting FHA domain. Other CRK-interacting proteins include a second protein with a FHA domain and another type 2C protein phosphatase. Interactions of CRKs with these three proteins in vivo were demonstrated through co-immunoprecipitation. These CRK-interacting proteins may play roles in the regulation and signaling of CRKs.     

Improving conformational searches by geometric screening

MOTIVATION: Conformational searches in molecular docking are a time-consuming process with wide range of applications. Favorable conformations of the ligands that successfully bind with receptors are sought to form stable ligand-receptor complexes. Usually a large number of conformations are generated and their binding energies are examined. We propose adding a geometric screening phase before an energy minimization procedure so that only conformations that geometrically fit in the binding site will be prompted for energy calculation. RESULTS: Geometric screening can drastically reduce the number of conformations to be examined from millions (or higher) to thousands (or lower). The method can also handle cases when there are more variables than geometric constraints. An early-stage implementation is able to finish the geometric filtering of conformations for molecules with up to nine variables in 1 min. To the best of our knowledge, this is the first time such results are reported deterministically. CONTACT: mzhang@mdanderson.org.     

Restoration of NK T cell development in fyn-mutant mice by a TCR reveals a requirement for Fyn during early NK T cell ontogeny

NK T cells are a unique lymphocyte population that have developmental requirements distinct from conventional T cells. Mice lacking the tyrosine kinase Fyn have 5- to 10-fold fewer mature NK T cells. This study shows that Fyn-deficient mice have decreased numbers of NK1.1(-) NK T cell progenitors as well. 5-Bromo-2'-deoxyuridine-labeling studies indicate that the NK T cells remaining in fyn(-/-) mice exhibit a similar turnover rate as wild-type cells. The fyn(-/-) NK T cells respond to alpha-galactosylceramide, a ligand recognized by NK T cells, and produce cytokines, but have depressed proliferative capacity. Transgenic expression of the NK T cell-specific TCR alpha-chain Valpha14Jalpha18 leads to a complete restoration of NK T cell numbers in fyn(-/-) mice. Together, these results suggest that Fyn may have a role before alpha-chain rearrangement rather than for positive selection or the peripheral upkeep of cell number. NK T cells can activate other lymphoid lineages via cytokine secretion. These secondary responses are impaired in Fyn-deficient mice, but occur normally in fyn mutants expressing the Valpha14Jalpha18 transgene. Because this transgene restores NK T cell numbers, the lack of secondary lymphocyte activation in the fyn-mutant mice is due to the decreased numbers of NK T cells present in the mutant, rather than an intrinsic defect in the ability of the other fyn(-/-) lymphoid populations to respond.     

A protocol for dissecting Drosophila melanogaster brains for live imaging or immunostaining

This protocol describes a basic method for dissection and immunofluorescence staining of the Drosophila brain at various developmental stages. The Drosophila brain has become increasingly useful for studies of neuronal wiring and morphogenesis in combination with techniques such as the 'mosaic analysis with a repressible cell marker' (MARCM) system, where single neurons can be followed in live and fixed tissues for high-resolution analysis of wild-type or genetically manipulated cells. Such high-resolution anatomical study of the brain is also important in characterizing the organization of neural circuits using genetic tools such as GAL4 enhancer trap lines, as Drosophila has been intensively used for studying the neural basis of behavior. Advantages of fluorescence immunostaining include compatibility with multicolor labeling and confocal or multiphoton imaging. This brain dissection and immunofluorescence staining protocol requires approximately 2 to 6 d to complete.     

Development of a single mode electromagnetic resonant cavity for rewarming of cryopreserved biomaterials

An electromagnetic (EM) heating system is developed to achieve the rapid and uniform warming of cryopreserved biomaterials. Using the heating system, a rectangular resonant cavity is excited in TE101 mode at frequencies near 434 MHz. In experiments, a spherical phantom of biomaterial with a diameter of 36 mm is placed at the center of the cavity. The phantom is first cooled down to about -80 degrees C within the cavity and then thawed by EM absorption. Results show that EM warming can produce much higher warming rate than conventional water-bath warming method. The spatial temperature distribution in the phantom during EM warming is also more uniform than that during the water-bath warming.     

Acid-sensitive TWIK and TASK Two-pore Domain Potassium Channels Change Ion Selectivity and Become Permeable to Sodium in Extracellular Acidification

Two-pore domain K⁺ channels (K2P) mediate background K⁺ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K⁺ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH_o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH_o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na⁺-based bath solutions with physiological K⁺ gradients, lowered pH_o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K⁺ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na⁺ to K⁺ relative permeability. Low pH_o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na⁺-based bath solutions than in channel-impermeable N-methyl-d-glucamine-based bath solutions, consistent with increases in the Na⁺ to K⁺ relative permeability and decreases in electrochemical driving forces of outward K⁺ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K⁺ channels change ion selectivity in response to lowered pH_o, provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.     

MFG-E8 activates proliferation of vascular smooth muscle cells via integrin signaling

An accumulation of milk fat globule EGF-8 protein (MFG-E8) occurs within the context of arterial wall inflammatory remodeling during aging, hypertension, diabetes mellitus, or atherosclerosis. MFG-E8 induces VSMC invasion, but whether it affects VSMC proliferation, a salient feature of arterial inflammation, is unknown. Here, we show that in the rat arterial wall in vivo, PCNA and Ki67, markers of cell cycle activation, increase with age between 8 and 30 months. In fresh and early passage VSMC isolated from old aortae, an increase in CDK4 and PCNA, an increase in the acceleration of cell cycle S and G2 phases, decrease in the G1/G0 phase, and an increase in PDGF and its receptors confer elevated proliferative capacity, compared to young VSMC. Increased coexpression and physical interaction of MFG-E8 and integrin αvβ5 occur with aging in both the rat aortic wall in vivo and in VSMC in vitro. In young VSMC in vitro, MFG-E8 added exogenously, or overexpressed endogenously, triggers phosphorylation of ERK1/2, augmented levels of PCNA and CDK4, increased BrdU incorporation, and promotes proliferation, via αvβ5 integrins. MFG-E8 silencing, or its receptor inhibition, or the blockade of ERK1/2 phosphorylation in these cells reduces PCNA and CDK4 levels and decelerates the cell cycle S phase, conferring a reduction in proliferative capacity. Collectively, these results indicate that MFG-E8 in a dose-dependent manner coordinates the expression of cell cycle molecules and facilitates VSMC proliferation via integrin/ERK1/2 signaling. Thus, an increase in MFG-E8 signaling is a mechanism of the age-associated increase in aortic VSMC proliferation.