Regulation of vascular smooth muscle cells and mesenchymal stem cells by mechanical strain

Vascular smooth muscle cells (SMCs) populate in the media of the blood vessel, and play an important role in the control of vasoactivity and the remodeling of the vessel wall. Blood vessels are constantly subjected to hemodynamic stresses, and the pulsatile nature of the blood flow results in a cyclic mechanical strain in the vessel walls. Accumulating evidence in the past two decades indicates that mechanical strain regulates vascular SMC phenotype, function and matrix remodeling. Bone marrow mesenchymal stem cell (MSC) is a potential cell source for vascular regeneration therapy, and may be used to generate SMCs to construct tissue-engineered vascular grafts for blood vessel replacements. In this review, we will focus on the effects of mechanical strain on SMCs and MSCs, e.g., cell phenotype, cell morphology, cytoskeleton organization, gene expression, signal transduction and receptor activation. We will compare the responses of SMCs and MSCs to equiaxial strain, uniaxial strain and mechanical strain in three-dimensional culture. Understanding the hemodynamic regulation of SMC and MSC functions will provide a basis for the development of new vascular therapies and for the construction of tissue-engineered vascular grafts.     

Nectarless flowers: ecological correlates and evolutionary stability

In animal-pollinated flowers, the pollinators cannot detect the presence of nectar before entering flowers, and therefore flowers may cheat by not producing nectar. An earlier model suggested that a mixed strategy of producing nectarful and nectarless flowers would be evolutionarily stable. Here we compare nectarless flowers as a cheating strategy with three competing hypotheses namely "visit-more-flowers", "cross-pollination enhancement" and "better contact". We collected field data on 28 species of plants to test some of the differential predictions of the hypotheses. Nectarless flowers were detected in 24 out of 28 plant species. Correlations of percent nectarless flowers with floral and ecological variables support the cheater flower hypothesis. We further model the cost-benefits of cheating and show that an evolutionary stable ratio of nectarless to nectarful flowers can be reached. The equilibrium ratio is mainly decided by factors associated with pollinator density and pollinator learning.     

Effects of shear stress on endothelial cell haptotaxis on micropatterned surfaces

Endothelial cell (EC) migration plays a critical role in vascular remodeling. Here we investigated the interactions between haptotaxis (induced by extracellular matrix gradient) and mechanotaxis (induced by mechanical forces) during EC migration. A micropatterning technique was used to generate step changes of collagen surface density. Due to haptotaxis, ECs developed focal adhesions and migrated into the area with higher surface density of collagen. Different levels of fluid shear stress were applied on ECs in the direction perpendicular to collagen strips. Shear stress at 2 dyn/cm2 did not affect haptotaxis, while shear stress at 3 dyn/cm2 or higher was sufficient to drive the migration of most ECs in the flow direction and against haptotaxis. Immunostaining revealed the increase of focal adhesions and lamellipodial protrusion in the direction of flow. These results suggest that shear stress beyond a certain threshold can be a predominant factor to determine the direction of EC migration.     

Optimization of single-step purification of alkaline protease using different hydrophobic ligands

The adsorption and desorption pattern of alkaline protease was studied using different aliphatic and aromatic hydrophobic ligands. Overall, higher adsorption was obtained on ligands coupled to 6% cross-linked gel than the 4% gel. The highest adsorption was obtained on butyl (94%) and phenyl (98.4%) of 6% cross-linked gel. The adsorption was dependent on concentration and nature of the ligand. In a single-step operation, almost 20-fold purification with 40% yield of the enzyme was obtained using all the optimized experimental parameters. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cytokines and growth factors cross-link heparan sulfate

The glycosaminoglycan heparan sulfate (HS), present at the surface of most cells and ubiquitous in extracellular matrix, binds many soluble extracellular signalling molecules such as chemokines and growth factors, and regulates their transport and effector functions. It is, however, unknown whether upon binding HS these proteins can affect the long-range structure of HS. To test this idea, we interrogated a supramolecular model system, in which HS chains grafted to streptavidin-functionalized oligoethylene glycol monolayers or supported lipid bilayers mimic the HS-rich pericellular or extracellular matrix, with the biophysical techniques quartz crystal microbalance (QCM-D) and fluorescence recovery after photobleaching (FRAP). We were able to control and characterize the supramolecular presentation of HS chains—their local density, orientation, conformation and lateral mobility—and their interaction with proteins. The chemokine CXCL12α (or SDF-1α) rigidified the HS film, and this effect was due to protein-mediated cross-linking of HS chains. Complementary measurements with CXCL12α mutants and the CXCL12γ isoform provided insight into the molecular mechanism underlying cross-linking. Fibroblast growth factor 2 (FGF-2), which has three HS binding sites, was also found to cross-link HS, but FGF-9, which has just one binding site, did not. Based on these data, we propose that the ability to cross-link HS is a generic feature of many cytokines and growth factors, which depends on the architecture of their HS binding sites. The ability to change matrix organization and physico-chemical properties (e.g. permeability and rigidification) implies that the functions of cytokines and growth factors may not simply be confined to the activation of cognate cellular receptors.     

The microRNA miR-29a is associated with human immunodeficiency virus latency

Background

Latent reservoirs of HIV-1 provide a major challenge to its cure. There are increasing reports of interplay between HIV-1 replication and host miRNAs. Several host miRNAs, which potentially target the nef-3′LTR region of HIV-1 RNA, including miR-29a, are proposed to promote latency.

Findings

We used two established cellular models of HIV-1 latency – the U1 monocytic and J1.1 CD4+ T cell lines to show an inverse relationship between HIV-1 replication and miR-29a levels, which was mediated by the HIV-1 Nef protein. Using a miR-29a responsive luciferase reporter plasmid, an expression plasmid and an anti-miR29a LNA, we further demonstrate increased miR-29a levels during latency and reduced levels following active HIV replication. Finally, we show that miR-29a levels in the PBMCs and plasma of HIV infected persons also correlate inversely with latency and active viral replication.

Conclusions

The levels of miR-29a correlate inversely with active HIV-1 replication in cell culture models and in HIV infected persons. This links miR-29a to viral latency and suggests another approach to activate and destroy latent HIV-1 reservoirs.

     

Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry

Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions.The transport of macromolecules across the nuclear envelope occurs through large proteinaceous structures called nuclear pore complexes (NPCs).¹ NPCs are composed of ∼30 nucleoporins that occur in copy numbers of eight or multiples of eight, leading to a complex with a total size of ∼125 MDa in vertebrate cells (1, 2). Active nucleocytoplasmic transport of proteins is a signal- and energy-dependent process that is mostly mediated by transport receptors of the importin β-superfamily called karyopherins or importins/exportins (3, 4). These proteins interact not only with their cargos, but also with certain nucleoporins, facilitating the translocation of the transport complex across the NPC. For nuclear export, at least seven nuclear export receptors/exportins have been identified (3, 4). Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the most important export receptor for proteins in yeast and vertebrates, and it is also involved in the export of several RNA species (5). Very little is known about the interaction of CRM1 with nucleoporins. Binding to cargo molecules, in contrast, is very well described. Exported proteins typically carry a nuclear export signal (NES) that is enriched with leucines or other hydrophobic amino acids. Such leucine-rich NESs were first discovered in the HIV type 1 Rev protein (6) and the cAMP-dependent protein kinase inhibitor (7). The consensus sequence consists of four key hydrophobic amino acids (leucine, isoleucine, valine, and phenylalanine or methionine; denoted by Φ¹–Φ⁴) following the sequence Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ₄, with x preferentially being a charged polar or small amino acid (for a review, see Ref. 8). A structural analysis of different NES peptides revealed a fifth hydrophobic amino acid in some substrates involved in CRM1 recognition, leading to a revised consensus sequence of Φ⁰-(x)-Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ⁴ (9). In a very recent study, Chook and coworkers established a novel database, NESdb, for NES-containing proteins and analyzed the sequence requirements for proteins in that database in detail (10, 11). “Supraphysiological ” substrates with NESs that fulfill all criteria bind CRM1 with very high affinity and can outcompete other substrates (9, 12). Apart from linear sequences, CRM1 might recognize more complex export signals, such as in fatty acid binding protein 4, in which a functional NES is established only in the tertiary structure of the protein (13), or in snurportin 1 (SPN1), in which sequences outside of the NES proper contribute to CRM1 binding (14–16). This high level of complexity in the recognition sequence for the export receptor makes it very difficult to predict potential CRM1-dependent export cargos using bioinformatics tools. Nevertheless, >200 potential CRM substrates have been described so far (11, 17–19; see also NESbase 1.0 and NESdb).The small GTP-binding protein Ran also plays an essential role in CRM1-mediated nuclear export, as it binds cooperatively to the export receptor, together with the NES cargo. As the affinity of many NES substrates for CRM1 is rather low, the formation of this trimeric transport complex seems to be a rate-limiting step in nuclear export (20). On the nuclear side of the NPC, a number of accessory factors such as RanBP3 (21, 22), Nup98 (23), and NLP1 (24) can further promote the formation of export complexes. Following export, RanBP1 and RanGAP initiate the disassembly of the export complex (for a review, see Ref. 3).A powerful tool for the analysis of CRM1-mediated export is the fungal metabolite leptomycin B (LMB). LMB originally was discovered as an antifungal antibiotic in Streptomyces (25) and later turned out to be a specific and selective inhibitor of the CRM1-mediated nuclear export pathway (26, 27). It binds covalently to cysteine 528 in the NES-binding region of human CRM1 (28), preventing the formation of trimeric export complexes (for a review, see Ref. 5).We used a quantitative MS-based approach (stable isotope labeling with amino acids in cell culture (SILAC)) to evaluate the nuclear export characteristics of proteins by measuring changes in their relative abundance in subcellular fractions after blocking the CRM1-mediated nuclear export with LMB. Using this approach, we identified known and novel CRM1-targets and characterized the NES of one cargo, cytosolic carboxypeptidase 1 (CCP1), in detail.     

Overexpression of prothymosin alpha predicts poor disease outcome in head and neck cancer

Background

In our recent study, tissue proteomic analysis of oral pre-malignant lesions (OPLs) and normal oral mucosa led to the identification of a panel of biomarkers, including prothymosin alpha (PTMA), to distinguish OPLs from histologically normal oral tissues. This study aimed to determine the clinical significance of PTMA overexpression in oral squamous cell hyperplasia, dysplasia and head and neck squamous cell carcinoma (HNSCC).

Methodology

Immunohistochemistry of PTMA protein was performed in HNSCCs (n = 100), squamous cell hyperplasia (n = 116), dysplasia (n = 50) and histologically normal oral tissues (n = 100). Statistical analysis was carried out to determine the association of PTMA overexpression with clinicopathological parameters and disease prognosis over 7 years for HNSCC patients.

Results

Our immunohistochemical analysis demonstrated significant overexpression of nuclear PTMA in squamous cell hyperplasia (63.8%), dysplasia (50%) and HNSCC (61%) in comparison with oral normal mucosa (p_trend<0.001). Chi-square analysis showed significant association of nuclear PTMA with advanced tumor stages (III+IV). Kaplan Meier survival analysis indicated reduced disease free survival (DFS) in HNSCC patients (p<0.001; median survival 11 months). Notably, Cox-multivariate analysis revealed nuclear PTMA as an independent predictor of poor prognosis of HNSCC patients (p<0.001, Hazard''s ratio, HR = 5.2, 95% CI = 2.3–11.8) in comparison with the histological grade, T-stage, nodal status and tumor stage.

Conclusions

Nuclear PTMA may serve as prognostic marker in HNSCC to determine the subset of patients that are likely to show recurrence of the disease.     

Quantitative set analysis for gene expression: a method to quantify gene set differential expression including gene-gene correlations

Enrichment analysis of gene sets is a popular approach that provides a functional interpretation of genome-wide expression data. Existing tests are affected by inter-gene correlations, resulting in a high Type I error. The most widely used test, Gene Set Enrichment Analysis, relies on computationally intensive permutations of sample labels to generate a null distribution that preserves gene–gene correlations. A more recent approach, CAMERA, attempts to correct for these correlations by estimating a variance inflation factor directly from the data. Although these methods generate P-values for detecting gene set activity, they are unable to produce confidence intervals or allow for post hoc comparisons. We have developed a new computational framework for Quantitative Set Analysis of Gene Expression (QuSAGE). QuSAGE accounts for inter-gene correlations, improves the estimation of the variance inflation factor and, rather than evaluating the deviation from a null hypothesis with a P-value, it quantifies gene-set activity with a complete probability density function. From this probability density function, P-values and confidence intervals can be extracted and post hoc analysis can be carried out while maintaining statistical traceability. Compared with Gene Set Enrichment Analysis and CAMERA, QuSAGE exhibits better sensitivity and specificity on real data profiling the response to interferon therapy (in chronic Hepatitis C virus patients) and Influenza A virus infection. QuSAGE is available as an R package, which includes the core functions for the method as well as functions to plot and visualize the results.