Platelets, thrombospondin-1 and human dermal fibroblasts cooperate for stimulation of endothelial cell tubulogenesis through VEGF and PAI-1 regulation

During cutaneous wound repair, platelets, dermal fibroblasts (DF) and endothelial cells all cooperate. We have presently investigated the regulation of endothelial cell tubulogenesis by human platelet thrombospondin-1 (TSP-1), in comparison to transforming growth factor-beta1 (TGF-beta1) and total platelet lysates (PL), in a fibrin matrix cell culture system incorporating DF. TSP-1, TGF-beta1 and PL all stimulated VEGF expression in DF dose dependently at mRNA and protein level. TSP-1- and PL-treated DF supernatants significantly stimulated capillary-like structure formation (tubulogenesis) by dermal microvascular endothelial cells (HMEC-1 and HDMEC), in part via VEGF, as confirmed with neutralizing anti-VEGF antibodies. In contrast, TGF-beta1-treated DF supernatants did not induce tubulogenesis. This apparent discrepancy could be explained by the differential expression regulation in HMEC-1 of fibrinolysis and metalloproteinase mediators by TSP-1 and TGF-beta1. TSP-1 potently reduced the expression of plasminogen activator inhibitor-1 (PAI-1) (mRNA and protein), whereas TGF-beta1 enhanced it. The crucial role of PAI-1 in tubulogenesis was confirmed via the addition of active recombinant PAI-1, which abrogated tubulogenesis. In contrast, neutralizing PAI-1 antibodies enhanced tubulogenesis. Our results suggest that platelet TSP-1 released in a wound stimulates endothelial cell tubulogenesis through an upregulation of DF VEGF expression and a downregulation of endothelial cell PAI-1 expression.     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Colour constancy in insects

Colour constancy is the perceptual phenomenon that the colour of an object appears largely unchanged, even if the spectral composition of the illuminating light changes. Colour constancy has been found in all insect species so far tested. Especially the pollinating insects offer a remarkable opportunity to study the ecological significance of colour constancy since they spend much of their adult lives identifying and choosing between colour targets (flowers) under continuously changing ambient lighting conditions. In bees, whose colour vision is best studied among the insects, the compensation provided by colour constancy is only partial and its efficiency depends on the area of colour space. There is no evidence for complete ‘discounting’ of the illuminant in bees, and the spectral composition of the light can itself be used as adaptive information. In patchy illumination, bees adjust their spatial foraging to minimise transitions between variously illuminated zones. Modelling allows the quantification of the adaptive benefits of various colour constancy mechanisms in the economy of nature. We also discuss the neural mechanisms and cognitive operations that might underpin colour constancy in insects.     

Relationship between Watershed Infarcts and Recent Intra Plaque Haemorrhage in Carotid Atherosclerotic Plaque

Objective

Watershed infarcts (WSI) are thought to result from hemodynamic mechanism, but studies have suggested that microemboli from unstable carotid plaques may distribute preferentially in watershed areas, i.e., between two cerebral arterial territories. Intraplaque haemorrhage (IPH) is an emerging marker of plaque instability and microembolic activity. We assessed the association between WSI and IPH in patients with recently symptomatic moderate carotid stenosis.

Methods and Results

We selected 65 patients with symptomatic moderate (median NASCET degree of stenosis = 31%) carotid stenosis and brain infarct on Diffusion-Weighted Imaging (DWI) on Magnetic Resonance Imaging (MRI) from a multicentre prospective study. Fourteen (22%) had WSI (cortical, n = 8; internal, n = 4; cortical and internal, n = 2). Patients with WSI were more likely to have IPH than those without WSI although the difference was not significant (50% vs. 31%, OR = 2.19; 95% CI, 0.66–7.29; P = 0.20). After adjustment for degree of stenosis, age and gender, the results remained unchanged.

Conclusion

About one in fifth of brain infarcts occurring in patients with moderate carotid stenosis were distributed in watershed areas. Albeit not significant, an association between IPH - more generally plaque component - and WSI, still remains possible.     

Transmembrane Recognition of the Semaphorin Co-Receptors Neuropilin 1 and Plexin A1: Coarse-Grained Simulations

The cancer associated class 3 semaphorins require direct binding to neuropilins and association to plexins to trigger cell signaling. Here, we address the role of the transmembrane domains of neuropilin 1 and plexin A1 for the dimerization of the two receptors by characterizing the assembly in lipid bilayers using coarse-grained molecular dynamics simulations. From experimental evidence using a two-hybrid system showing the biochemical association of the two receptors transmembrane domains, we performed molecular simulations in DOPC and POPC demonstrating spontaneously assembly to form homodimers and heterodimers with a very high propensity for right-handed packing of the helices. Inversely, left-handed packing was observed with a very low propensity. This mode of packing was observed uniquely when the plexin A1 transmembrane domain was involved in association. Potential of mean force calculations were used to predict a hierarchy of self-association for the monomers: the two neuropilin 1 transmembrane domains strongly associated, neuropilin 1 and plexin A1 transmembrane domains associated less and the two plexin A1 transmembrane domains weakly but significantly associated. We demonstrated that homodimerization and heterodimerization are driven by GxxxG motifs, and that the sequence context modulates the packing mode of the plexin A1 transmembrane domains. This work presents major advances towards our understanding of membrane signaling platforms assembly through membrane domains and provides exquisite information for the design of antagonist drugs defining a novel class of therapeutic agents.     

Differential proteomic analysis of noncardia gastric cancer from individuals of northern Brazil

Gastric cancer is the second leading cause of cancer-related death worldwide. The identification of new cancer biomarkers is necessary to reduce the mortality rates through the development of new screening assays and early diagnosis, as well as new target therapies. In this study, we performed a proteomic analysis of noncardia gastric neoplasias of individuals from Northern Brazil. The proteins were analyzed by two-dimensional electrophoresis and mass spectrometry. For the identification of differentially expressed proteins, we used statistical tests with bootstrapping resampling to control the type I error in the multiple comparison analyses. We identified 111 proteins involved in gastric carcinogenesis. The computational analysis revealed several proteins involved in the energy production processes and reinforced the Warburg effect in gastric cancer. ENO1 and HSPB1 expression were further evaluated. ENO1 was selected due to its role in aerobic glycolysis that may contribute to the Warburg effect. Although we observed two up-regulated spots of ENO1 in the proteomic analysis, the mean expression of ENO1 was reduced in gastric tumors by western blot. However, mean ENO1 expression seems to increase in more invasive tumors. This lack of correlation between proteomic and western blot analyses may be due to the presence of other ENO1 spots that present a slightly reduced expression, but with a high impact in the mean protein expression. In neoplasias, HSPB1 is induced by cellular stress to protect cells against apoptosis. In the present study, HSPB1 presented an elevated protein and mRNA expression in a subset of gastric cancer samples. However, no association was observed between HSPB1 expression and clinicopathological characteristics. Here, we identified several possible biomarkers of gastric cancer in individuals from Northern Brazil. These biomarkers may be useful for the assessment of prognosis and stratification for therapy if validated in larger clinical study sets.     

Platelet type III collagen binding protein (TIIICBP) presents high biochemical and functional similarities with kindlin-3

Type III collagen binding protein (TIIICBP) was previously described as a platelet membrane protein that recognizes the KOGEOGPK peptide sequence within type III collagen. In order to better characterize this protein, we performed different approaches including mass spectrometry sequencing and functional experiments. This study leads to identify high biochemical and functional similarities between TIIICBP and kindlin-3, a member of a family of focal adhesion proteins. Indeed, mass spectrometry surveys indicated that TIIICBP contains several peptides identical to kindlin-3, covering 41% of the amino acid sequence. Polyclonal antibodies raised against a kindlin-3 specific N-terminal sequence, recognized and immunoprecipitated TIIICBP from platelet lysates. Electron microscopy and flow cytometry experiments showed that kindlin-3, as well as TIIICBP, were present associated to platelet membrane and a translocation of cytosolic kindlin-3 to the platelet membrane was observed after platelet activation. Similarly to anti-TIIICBP antibodies and the KOGEOGPK peptide, anti-kindlin-3 antibodies inhibited platelet interactions with type III collagen under flow conditions and slowed down platelet aggregation induced by glycoprotein VI agonists; e.g. collagen-related peptides and convulxin. In addition, the anti-kindlin-3 antibody inhibited platelet aggregation induced by low - but not high - doses of ADP or thrombin which depends on α(IIb)β(3) integrin function. In conclusion, our results show that the peptides identified by mass spectrometry from purified TIIICBP correspond to the kindlin-3 protein and demonstrate biochemical and functional similarities between TIIICBP and kindlin-3, strengthening a key role for TIIICBP/kindlin-3 in platelet interactions with collagen by cooperating with glycoprotein VI activation and integrin clustering in focal adhesion complexes.     

Feline Histoplasmosis in Brazil: Clinical and Laboratory Aspects and a Comparative Approach of Published Reports

The present study described clinical and epidemiological aspects of three cases of feline histoplasmosis and compared them to previously described cases. A detailed mycological identification and antifungal susceptibility profile of each isolate are presented. Secondarily, a serological survey for anti-Histoplasma antibodies was performed with domestic and wild cats. Diseased animals presented nodular to ulcerated skin lesions and respiratory disorders as main clinical signs. H. capsulatum var. capsulatum was isolated and the strains showed to be susceptible to antifungal drugs. Considering that feline histoplasmosis is uncommonly observed in veterinary clinics, diagnosis, and clinical management in endemic areas should be improved.     

β-Cyclodextrin polymer nanoparticles as carriers for doxorubicin and artemisinin: a spectroscopic and photophysical study

The association of doxorubicin (DOX) and artemisinin (ART) to a β-CyD-epichlorohydrin crosslinked polymer (pβ-CyD), organized in nanoparticles of ca. 15 nm size, was investigated in neutral aqueous medium by circular dichroism (CD), UV-vis absorption and fluorescence. The stability constants and the absolute CD spectra of the drug complexes were determined by global analysis of multiwavelength data from spectroscopic titrations. The polymer pβ-CyD proved able to disrupt the DOX dimer when the latter is the predominant form of DOX in solution. The spectroscopic and photophysical properties of the complexes evidenced an alcohol-like environment for ART and an improved inherent emission ability for DOX in the nanoparticle frame.