Triple Modification of Alginate Hydrogels by Fibrin Blending, Iron Nanoparticle Embedding, and Serum Protein-Coating Synergistically Promotes Strong Endothelialization

Stent therapy can reduce both morbidity and mortality of chronic coronary stenosis and acute myocardial infarction. However, delayed re-endothelialization, endothelial dysfunction, and chronic inflammation are still unsolved problems. Alginate hydrogels can be used as a coating for stent surfaces; however, complete and fast endothelialization cannot be achieved. In this study, alginate hydrogels are modified by fibrin blending, iron nanoparticle (Fe-NP) embedding, and serum protein coating (SPC) while surface properties and endothelialization capacity are monitored. Only a triple, synergetic modification of the alginate coating by simultaneous I) fibrin blending, II) Fe-NP addition complemented by III) SPC is found to significantly improve endothelial cell viability (live–dead-staining) and proliferation (WST-8 assay). These conditions yield formation of closed endothelial cell monolayers and an up to threefold increase (p < 0.01) in viability, while, interestingly, no effect is found when the modifications (I)–(III) are conducted individually. This synergetic effect is attributed to an accumulation of agglomerated Fe-NP and serum proteins along fibrin fibers, observed via laser scanning microscopy tracking nanoparticle scattering and tetramethylrhodamine (TRITC)-albumin fluorescence. These synergetic effects can pave the way toward a novel strategy for the modification of various hydrogel-based biomaterials and biomaterial coatings

Chemically induced hypoxia by dimethyloxalylglycine (dmog)-loaded nanoporous silica nanoparticles supports endothelial tube formation by sustained vegf release from adipose tissue-derived stem cells

Inadequate vascularization leading to insufficient oxygen and nutrient supply in deeper layers of bioartificial tissues remains a limitation in current tissue engineering approaches to which prevascularization offers a promising solution. Hypoxia triggering pre-vascularization by enhanced vascular endothelial growth factor (VEGF) expression can be induced chemically by dimethyloxalylglycine (DMOG). Nanoporous silica nanoparticles (NPSNPs, or mesoporous silica nanoparticles, MSNs) enable sustained delivery of molecules and potentially release DMOG allowing a durable capillarization of a construct. Here we evaluated the effects of soluble DMOG and DMOG-loaded NPSNPs on VEGF secretion of adipose tissue-derived stem cells (ASC) and on tube formation by human umbilical vein endothelial cells (HUVEC)-ASC co-cultures. Repeated doses of 100 mM and 500 mM soluble DMOG on ASC resulted in 3- to 7-fold increased VEGF levels on day 9 (P<0.0001). Same doses of DMOG-NPSNPs enhanced VEGF secretion 7.7-fold (P<0.0001) which could be maintained until day 12 with 500 mM DMOG-NPSNPs. In fibrin-based tube formation assays, 100 mM DMOG-NPSNPs had inhibitory effects whereas 50 mM significantly increased tube length, area and number of junctions transiently for 4 days. Thus, DMOG-NPSNPs supported endothelial tube formation by upregulated VEGF secretion from ASC and thus display a promising tool for prevascularization of tissue-engineered constructs. Further studies will evaluate their effect in hydrogels under perfusion

Changes of Proteases, Antiproteases, and Pathogens in Cystic Fibrosis Patients&apos; Upper and Lower Airways after IV-Antibiotic Therapy

Background. In cystic fibrosis (CF) the upper (UAW) and lower airways (LAW) are reservoirs for pathogens like Pseudomonas aeruginosa. The consecutive hosts&apos; release of proteolytic enzymes contributes to inflammation and progressive pulmonary destruction. Objectives were to assess dynamics of protease : antiprotease ratios and pathogens in CF-UAW and LAW sampled by nasal lavage (NL) and sputum before and after intravenous-(IV-) antibiotic therapy. Methods. From 19 IV-antibiotic courses of 17 CF patients NL (10 mL/nostril) and sputum were collected before and after treatment. Microbiological colonization and concentrations of NE/SLPI/CTSS (ELISA) and MMP-9/TIMP-1 (multiplex bead array) were determined. Additionally, changes of sinonasal symptoms were assessed (SNOT-20). Results. IV-antibiotic treatment had more pronounced effects on inflammatory markers in LAW, whereas trends to decrease were also found in UAW. Ratios of MMP-9/TIMP-1 were higher in sputum, and ratios of NE/SLPI were higher in NL. Remarkably, NE/SLPI ratio was 10-fold higher in NL compared to healthy controls. SNOT-20 scores decreased significantly during therapy ( = 0.001). Conclusion. For the first time, changes in microbiological patterns in UAW and LAW after IV-antibiotic treatments were assessed, together with changes of protease/antiprotease imbalances. Delayed responses of proteases and antiproteases to IV-antibiotic therapy were found in UAW compared to LAW

Establishment of a Modular Hemodynamic Simulator for Accurate In Vitro Simulation of Physiological and Pathological Pressure Waveforms in Native and Bioartificial Blood Vessels

Purpose!#!In vitro stimulation of native and bioartificial vessels in perfusable systems simulating natural mechanical environments of the human vasculature represents an emerging approach in cardiovascular research. Promising results have been achieved for applications in both regenerative medicine and etiopathogenetic investigations. However, accurate and reliable simulation of the wide variety of physiological and pathological pressure environments observed in different vessels still remains an unmet challenge.!##!Methods!#!We established a modular hemodynamic simulator (MHS) with interchangeable and modifiable components suitable for the perfusion of native porcine-(i.e. the aorta, brachial and radial arteries and the inferior vena cava) and bioartificial fibrin-based vessels with anatomical site specific pressure curves. Additionally, different pathological pressure waveforms associated with cardiovascular diseases including hyper- and hypotension, tachy- and bradycardia, aortic valve stenosis and insufficiency, heart failure, obstructive cardiomyopathy and arterial stiffening were simulated. Pressure curves, cyclic distension and shear stress were measured for each vessel and compared to ideal clinical pressure waveforms.!##!Results!#!The pressure waveforms obtained in the MHS showed high similarity to the ideal anatomical site specific pressure curves of different vessel types. Moreover, the system facilitated accurate emulation of physiological and different pathological pressure conditions in small diameter fibrin-based vessels.!##!Conclusion!#!The MHS serves as a variable in vitro platform for accurate emulation of physiological and pathological pressure environments in biological probes. Potential applications of the system include bioartificial vessel maturation in cardiovascular tissue engineering approaches as well as etiopathogenetic investigations of various cardiovascular pathologies

Analysis of naturally occurring variants (Negishi, 1993a,b; Hasegawa, 1996), of receptor hybrids between different pro-stanoid receptors (Dorn, 1997; Kobayashi, 1997; Neuschä fer-Rube, 1997a,b), and of (Funk, 1993; Irie

ABSTRACT Prostaglandin E 2 receptors (EP-Rs) belong to the family of heterotrimeric G protein-coupled ectoreceptors with seven transmembrane domains. They can be subdivided into four subtypes according to their ligand-binding and G protein-coupling specificity: EP1 couple to G q , EP2 and EP4 to G s , and EP3 to G i . The EP4-R, in contrast to the EP3␤-R, shows rapid agonist-induced desensitization. The agonist-induced desensitization depends on the presence of the EP4-R carboxyl-terminal domain, which also confers desensitization in a G i -coupled rEP3hEP4 carboxyl-terminal domain receptor hybrid (rEP3hEP4-Ct-R). To elucidate the possible mechanism of this desensitization, in vivo phosphorylation stimulated by activators of second messenger kinases, by prostaglandin E 2 , or by the EP3-R agonist M&amp;B28767 was investigated in COS-7 cells expressing FLAGepitope-tagged rat EP3␤-R (rEP3␤-R), hEP4-R, or rEP3hEP4-Ct-R. Stimulation of protein kinase C with phorbol-12-myristate-13-acetate led to a slight phosphorylation of the FLAG-rEP3␤-R but to a strong phosphorylation of the FLAG-hEP4-R and the FLAG-rEP3hEP4-Ct-R, which was suppressed by the protein kinase A and protein kinase C inhibitor staurosporine. Prostaglandin E 2 stimulated phosphorylation of the FLAG-hEP4-R in its carboxyl-terminal receptor domain. The EP3-R agonist M&amp;B28767 induced a time-and dose-dependent phosphorylation of the FLAG-rEP3hEP4-Ct-R but not of the FLAGrEP3␤-R. Agonist-induced phosphorylation of the FLAGhEP4-R and the FLAG-rEP3hEP4-Ct-R were not inhibited by staurosporine, which implies a role of G protein-coupled receptor kinases (GRKs) in agonist-induced receptor phosphorylation. Overexpression of GRKs in FLAG-rEP3hEP4-Ct-R-expressing COS-7 cells augmented the M&amp;B28767-induced receptor phosphorylation and receptor sequestration. These findings indicate that phosphorylation of the carboxyl-terminal hEP4-R domain possibly by GRKs but not by second messenger kinases may be involved in rapid agonist-induced desensitization of the hEP4-R and the rEP3hEP4-Ct-R. Prostaglandin E 2 receptors (EP-Rs), like most prostanoid receptors, belong to the class of G protein-coupled ectoreceptors (GPCR) with seven transmembrane domain

Stimulation by lithium of the interaction between the transcription factor CREB and its coactivator TORC

International audienceLithium salts are clinically important drugs used to treat bipolar mood disorder. The mechanisms accounting for the clinical efficacy are not completely understood. Chronic treatment with lithium is required to establish mood stabilization, suggesting the involvement of neuronal plasticity processes. CREB (cAMP response element binding protein) is a transcription factor known to mediate neuronal adaptation. Recently, the CREB-coactivator TORC (transducer of regulated CREB) has been identified as a novel target of lithium and shown to confer an enhancement by lithium of cAMP-induced CREB-directed gene transcription. TORC is sequestered in the cytoplasm and its nuclear translocation controls CREB activity. In the present study, the effect of lithium on TORC function was investigated. Lithium did affect neither the nuclear translocation of TORC nor TORC1 transcriptional activity, but increased the promoter occupancy by TORC1 as revealed by chromatin immunoprecipitation assay. In a mammalian two-hybrid assay as well as in a cell-free GST pull-down assay, lithium enhanced the CREB-TORC1 interaction. Magnesium ions strongly inhibited the interaction between GST-CREB and TORC1 and this effect was reversed by lithium. Thus, our data suggest that, once TORC has entered the nucleus, lithium as a cation directly stimulates the binding of TORC to CREB, leading to an increase in cAMP-induced CREB target gene transcription. This novel mechanism of lithium action is likely to contribute to the clinical mood stabilizing effect of lithium salts

Identification by site-directed mutagenesis of amino acids contributing to ligand-binding specificity or signal transduction properties of the human FP prostanoid receptor.

Prostanoid receptors belong to the class of heptahelical plasma membrane receptors. For the five prostanoids, eight receptor subtypes have been identified. They display an overall sequence similarity of roughly 30%. Based on sequence comparison, single amino acids in different subtypes of different species have previously been identified by site-directed mutagenesis or in hybrid receptors that appear to be essential for ligand binding or G-protein coupling. Based on this information, a series of mutants of the human FP receptor was generated and characterized in ligand-binding and second-messenger-formation studies. It was found that mutation of His-81 to Ala in transmembrane domain 2 and of Arg-291 to Leu in transmembrane domain 7, which are putative interaction partners for the prostanoid's carboxyl group, abolished ligand binding. Mutants in which Ser-263 in transmembrane domain 6 or Asp-300 in transmembrane domain 7 had been replaced by Ala or Gln, respectively, no longer discriminated between prostaglandins PGF(2alpha) and PGD(2). Thus distortion of the topology of transmembrane domains 6 and 7 appears to interfere with the cyclopentane ring selectivity of the receptor. PGF(2alpha)-induced inositol formation was strongly reduced in the mutant Asp-300Gln, inferring a role for this residue in agonist-induced G-protein activation

Lives of Skin Lesions in Monkeypox: Histomorphological, Immunohistochemical, and Clinical Correlations in a Small Case Series

Monkeypox (mpox), a former rare viral zoonosis, has increasingly made it into the public eye since the major outbreak that started in May 2022. Mpox presents with skin lesions that change over time and go through different stages (macular, papular, pustular, and early and late ulceration). In this study, we evaluated skin biopsies of all stages. Therefore, five biopsies from four patients were analyzed histologically, immunohistochemically with anti-Vaccinia virus antibodies, and electron-microscopically. Notably, the early macular stage only showed subtle viropathic changes; it did not express of Orthopoxvirus proteins in immunohistochemistry and therefore can easily be missed histologically. In later stages, immunohistochemistry with anti-Vaccinia virus antibodies might be useful to distinguish mpox from differential diagnoses such as herpes virus infections. In the ulcerative stages, the identified occlusive vasculopathic changes could be an explanation for the severe pain of the lesions reported by some patients. Despite the small number of samples examined, our analysis suggests that the histological findings of mpox are highly dependent on the stage of the biopsied lesion. Therefore, knowledge of all different stages of histology is necessary to reliably diagnose mpox histologically, especially when molecular testing is not available

CRE/CREB-driven up-regulation of gene expression by chronic social stress in CRE-luciferase transgenic mice: reversal by antidepressant treatment.

BACKGROUND: It has been suggested that stress provokes neuropathological changes and may thus contribute to the precipitation of affective disorders such as depression. Likewise, the pharmacological therapy of depression requires chronic treatment and is thought to induce a positive neuronal adaptation, presumably based on changes in gene transcription. The transcription factor cAMP-responsive element binding protein (CREB) and its binding site (CRE) have been suggested to play a major role in both the development of depression and antidepressive therapy. METHODOLOGY/PRINCIPLE FINDINGS: To investigate the impact of stress and antidepressant treatment on CRE/CREB transcriptional activity, we generated a transgenic mouse line in which expression of the luciferase reporter gene is controlled by four copies of CRE. In this transgene, luciferase enzyme activity and protein were detected throughout the brain, e.g., in the hippocampal formation. Chronic social stress significantly increased (by 45 to 120%) CRE/CREB-driven gene expression measured as luciferase activity in several brain regions. This was also reflected by increased CREB-phosphorylation determined by immunoblotting. Treatment of the stressed mice with the antidepressant imipramine normalized luciferase expression to control levels in all brain regions and likewise reduced CREB-phosphorylation. In non-stressed animals, chronic (21 d) but not acute (24 h) treatment with imipramine (2x10 mg/kg/d) reduced luciferase expression in the hippocampus by 40-50%. CONCLUSIONS/SIGNIFICANCE: Our results emphasize a role of CREB in stress-regulated gene expression and support the view that the therapeutic actions of antidepressants are mediated via CRE/CREB-directed transcription