Regulation of endothelial nitric oxide synthase activation in endothelial cells by S1P1 and S1P3.

Endothelial nitric oxide synthase (eNOS) plays a crucial role in vascular homeostasis. Lysophospholipid interaction with sphingosine 1-phosphat (S1P) receptors results in eNOS activation in different cells. In endothelial cells, eNOS activation via S1P1 or S1P3 was shown controversially. The aim of this study is to investigate the meaning of both S1P receptors for eNOS activation in human endothelial cells. Therefore, several S1P1 and S1P3 agonists in combination with antagonists and specific RNAi approach were used. eNOS activation was measured in human umbilical vein endothelial cells (HUVEC) via DAF2-DA-based fluorescence microscopy. For investigation of the signaling pathway, agonists/antagonist studies, RNAi approach, Luminex™ multiplex, and Western Blot were used. In HUVEC, both the S1P1 agonist AUY954 as well as the S1P1,3 agonist FTY720P induced eNOS activation in a time- and dose-dependent manner. Other S1P1 agonists activated eNOS to a lesser extent. The AUY954-induced eNOS activation was blocked by the S1P1 antagonist W146, the combination of W146 and the S1P3 antagonist CAY10444 and the S1P1,3 antagonist VPC23019, but not by CAY10444 indicating the meaning of S1P1 for the AUY954-induced eNOS activation. The FTY720P-induced eNOS activation was blocked only by the combination of W146 and CAY10444 and the combined S1P1,3 antagonist VPC23019, but not by W146 or CAY10444 indicating the importance of both S1P1 and S1P3 for FTY720-induced eNOS activation. These results were confirmed using specific siRNA against S1P1 and S1P3. The S1P1,3 activation results in Akt phosphorylation and subsequent activation of eNOS via phosphorylation at serine(1177) and dephosphorylation at threonine(495). Beside former investigations with rather unspecific S1P receptor activation these data show potent selective S1P1 activation by using AUY954 and with selective S1P receptor inhibition evidence was provided that both S1P1 and S1P3 lead to downstream activation of eNOS in HUVEC in the same experimental setting. Inhibition or knockdown of one of these receptor subtypes did not abolish the eNOS activation and subsequent NO production.

Cardiomyocyte progenitor cell-derived exosomes stimulate migration of endothelial cells.

Patients suffering from heart failure as a result of myocardial infarction are in need of heart transplantation. Unfortunately the number of donor hearts is very low and therefore new therapies are subject of investigation. Cell transplantation therapy upon myocardial infarction is a very promising strategy to replace the dead myocardium with viable cardiomyocytes, smooth muscle cells and endothelial cells, thereby reducing scarring and improving cardiac performance. Despite promising results, resulting in reduced infarct size and improved cardiac function on short term, only a few cells survive the ischemic milieu and are retained in the heart, thereby minimizing long-term effects. Although new capillaries and cardiomyocytes are formed around the infarcted area, only a small percentage of the transplanted cells can be detected months after myocardial infarction. This suggests the stimulation of an endogenous regenerative capacity of the heart upon cell transplantation, resulting from release of growth factor, cytokine and other paracrine molecules by the progenitor cells--the so-called paracrine hypothesis. Here, we focus on a relative new component of paracrine signalling, i.e. exosomes. We are interested in the release and function of exosomes derived from cardiac progenitor cells and studied their effects on the migratory capacity of endothelial cells.

Spacer-modified oligosaccharides with basic anchoring groups are inhibitors for endo-glycanases: porcine pancreatic alpha-amylase as model enzyme.

By coupling methyl 2,3,6-tri-O-acetyl-4-O-(5-azido-6-p-tolylsulfonyloxyhexyl)-alpha-D - glucopyranoside with 2-benzoylthioethyl 2,3,4,6-tetra-O-acetyl-alpha-D-glucopyranoside, a spacer-modified disaccharide derivative, methyl 4-O-(5-azido-9-alpha-D-glucopyranosyloxy-7-thianonyl)-alpha- D-glucopyranoside, was obtained and then enzymatically glucosylated to yield the spacer-modified tri- and tetra-saccharide methyl 4-O-(5-azido-9-alpha-maltosyloxy-7-thianonyl)-alpha-D-glucop yranoside and methyl 4-O-(5-azido-9-alpha-maltotriosyloxy-7-thianonyl)-alpha-D-gl ucopyranoside, respectively, the extended spacer spanning the length of two (1-->4)-linked pyranosyl units. The corresponding amines methyl 4-O-(5-amino-9-alpha-D-glucopyranosyloxy-7-thianonyl)-alpha- D-glucopyranoside, methyl 4-O-(5-amino-9-alpha-maltosyloxy-7-thianonyl)-alpha-D-glucop yranoside and methyl 4-O-(5-amino-9-alpha-maltotriosyloxy-7-thianonyl)-alpha-D-gl ucopyranoside, obtained by catalytic reduction, carry the basic functionality in a spacer position to allow ionic interaction with a catalytically active acidic group in porcine pancreatic alpha-amylase (PPA). Optimal inhibition of enzymic activity is by methyl 4-O-(5-amino-9-alpha-maltosyloxy-7-thianonyl)-alpha-D-glucop yranoside where three of the five subsites are occupied by glucosyl units and the spacer spanning the remaining subsites positions the amino group near the catalytic site.

Differential chemical modification of substrate binding areas in porcine-pancreatic alpha-amylase by three regioisomeric photolabile ligands.

Three regioisomeric radiolabelled spacer-modified oligosaccharides: methyl 4'-O-[4-S-(3-azi-4-alpha-D-glucopyranosyloxy-1-[3H]butyl)-6- deoxy- 4-thio-alpha-D-xylo-hex-5-enopyranosyl]-alpha-maltoside (12a, G1-G3*), methyl 4-O-[4-S-(3-azi-4-alpha-maltosyloxy-1-[3H]butyl)-6-deoxy-4-t hio- alpha-D-xylo-hex-5-enopyranosyl]-alpha-D-glucopyranoside (15a, G2-G2*) and methyl 4-S-(3-azi-4-alpha-maltotriosyloxy-1-[3H]butyl)-6-deoxy-4-th io-alpha- D-xylo-hex-5-enopyranoside (16a, G3-G1*) were synthesised and used as photoaffinity probes for the chemical modification of porcine-pancreatic alpha-amylase (PPA). Incorporation of covalently attached radioactivity amounted to 25-38% of the stoichiometric value. Tryptic digestion of the three labelled protein preparations PPA-G1-G3*, PPA-G2-G2*, and PPA-G3-G1* and the purification of the labelled peptides by fractional HPLC yielded altogether six pure components. On the basis of the published three-dimensional structure peptides G1-G3-II, G2-G2-II, and G2-G2-III were part of the catalytic site. G1-G3-I and G2-G2-I were part of the surface binding site. The major component derived from PPA, labelled by G3-G1*, corresponded to an area that is neither close to the active site nor to the surface starch-binding domain, which clearly indicates the presence of a third, hitherto undetected, substrate-binding site.

Selective defucosylation by mercaptolysis. A potential step in analyzing branched oligosaccharides.

A method of stepwise chemical degradation was elaborated on a microgram quantity of 3-O-alpha-L-fucosyllactose. The key step, TiCl4-catalysed dithioacetal formation from the permethylated N-4-nitrophenyl triosylamine (4) was accompanied by quantitative defucosylation. [14C]Acetylation of the dried mercaptalation mixture gave radiolabelled 3,5-di-O-[14C]acetyl-4-O-(2,3,4,6-tetra-O-methyl-beta-D-galactopyranosyl )-2 , 6-di-O-methyl-D-glucose diethyl dithioacetal (7) and 5-O-[14C]acetyl-2,3,4-tri-O-methyl-L-fucose diethyl dithioacetal (8). The former was further degraded via the bis(sulfone), and thereby 2,3,4,6-tetra-O-methyl-D-galactose (13) was expelled. The monosaccharide branches, fucose and galactose, were identified as derivatives 8 and 13, respectively, by comparison with authentic samples. Isolation of microquantities of products was carried out by preparative TLC.

Strong competitive inhibition of porcine pancreatic alpha-amylase by aminodeoxy derivatives of maltose and maltotriose.

The syntheses are described of 6-amino-6-deoxymaltose (2), the 6-amino-6-deoxy (4), 6'-amino-6'-deoxy (6), and 6"-amino-6"-deoxy (8) derivatives of maltotriose, and the methyl alpha- (10) and beta-glycoside (12) and the 1-deoxy derivative (16) of 4. The Ki values (microM) of these competitive inhibitors of porcine pancreatic alpha-amylase were: 2, 88; 4, 1.9; 6, 2.0; 8, 175; 10, 360; 12, 9000; 16, 7600 (cf. 1800 for maltotriose and 3000 for methyl alpha-maltotrioside). The low values for 4 and 6 reflect reinforcement of the normal binding by ionic attraction and, possibly, interaction of the reducing end groups with the protein.

A comprehensive immunotopographic map of human thymus.

We have achieved a comprehensive immunotopographic mapping of human thymus by using a large battery of monoclonal antibodies and the methodological refinement of comparative serial tissue section immunohistochemistry, allowing analysis of multiple phenotypes in the same tissue site. Previous immunohistochemical studies of thymus have concentrated on the majority T-cell and epithelial cell populations. Besides demonstrating the complexity of T-cell antigenic expression (e.g., simultaneous cortical expression of Leu 2, Leu 3, CALLA, Tdt, and Leu 6), we delineate surprisingly complex B-cell zones (e.g., septal B-follicles with DRC+C3d+ dendritic cells and zonal maturation of B-cells). Whereas septal B-follicles were found in 25% of cases, medullary B-cells were universally present as a substantial minority component. This expanded immunotopographic knowledge of the complex T-, B-, epithelial, and reticulum cell neighborhoods suggests that the thymus is an organ capable of a broad repertoire of immunological responses, not limited to T-cell development.

The immunoarchitecture of cutaneous pseudolymphoma.

The immunoarchitecture of five cutaneous pseudolymphomas was studied by staining serial sections for T- and B-cell and dendritic reticulum cell (DRC) antigens with monoclonal antibodies, and compared with that of reactive lymph nodes and cutaneous lymphoma. In four cases compartmentalization of B and T cells was observed, analogous to findings in reactive lymph nodes. In two of these cases the immunoarchitectural features were strikingly similar to those of reactive lymph nodes. Both had distinct follicles with germinal centers, and in one distinct mantle zone formation was seen. B cells in the follicles were polyclonal, with kappa chain predominance. The germinal centers showed the expected intercellular and/or dendritic pattern of immunoglobulin heavy chain, B2, and DRC-antigen expression. T cells admixed in the germinal centers were overwhelmingly of the T-helper type. The B-cell compartments in the other two cases showed some subtle immunologic evidence of aberrance, but the weight of evidence suggested reactive/aberrant rather than malignant processes. The T-cell compartments in all four cases showed a predominance of T-helper and a minority of T-suppressor/cytotoxic cells. All contrasted with the lymphomas, which showed B-cell monoclonality, markedly deranged T-subset proportions, or novel T-cell phenotypes. Although the main focus of this study was cases involving substantial populations of both B and T cells, preliminary observations were made in one case in which a predominance of T cells and prominent epidermotropism simulated mycosis fungoides. Quantitative ultrastructural analysis in this case suggested a reactive T-cell process. Leu-6-positive Langerhans cells were increased in the epidermis and dermis in all five cases, and in the dermis they were found almost exclusively in T-cell compartments. It is proposed that this distribution is the anatomic correlate to the known functional role of Langerhans cells in antigen processing/presentation and T-cell activation. In the cutaneous "lymph node equivalent," Langerhans cells are analogous to interdigitating reticulum cells of reactive lymph nodes in distribution and, probably, in function. The DRC found in the germinal centers in two cases were probably antigenically identical and functionally analogous to those in germinal centers of reactive lymph nodes. Immunologic phenotyping of serial cutaneous sections may aid in distinguishing reactive from neoplastic lymphoid lesions. Immunoarchitectural analysis promises to be a powerful tool for the study of lymphoproliferative disease.

The probable origin of an anemone cell tumor: metastatic transitional cell carcinoma producing HCG.

A neoplasm of unknown origin in cervical and axillary lymph nodes was diagnosed as anemone cell tumor by ultrastructural examination. Three years after the initial diagnosis of anemone cell tumor, a high-grade transitional cell carcinoma of the bladder was discovered. The results of immunoperoxidase staining of the cervical lymph node, axillary lymph node, and bladder tumors for keratin, carcinoembryonic antigen, and human chorionic gonadotropin (HCG) strongly suggest that the anemone cell tumors in this case represent metastases of bladder carcinoma cells capable of producing HCG.