Internalization and degradation of heparin is not required for stimulus of heparan sulfate proteoglycan synthesis

Abstract

In vitro, heparin and antithrombotic drugs specifically stimulate the synthesis of an antithrombotic heparan sulfate proteoglycan (HSPG) produced by endothelial cells. The putative heparin binding site(s) that may be related to this phenomenon were investigated. In the preceding article, using various heparin probes, it was shown that the heparin does not bind to the endothelial cell surface, but only to the extracellular matrix. The present study demonstrated that, when the cells were exposed to heparin at 37 degrees C, the heparin was internalized and with time was localized in lysosomes. However, endocytosis of heparin was not required for the stimulation of HSPG synthesis. The requirement for heparin degradation in the stimulus of HSPG synthesis was also investigated. When the cells were incubated with chloroquine, a lysosomotropic amine that raises the lysosomal pH thus inhibiting enzymatic degradation of internalized compounds, stimulation of HSPG synthesis was still observed. These combined results indicate that neither internalization nor degradation of heparin is required for stimulation of HSPG synthesis, and suggests that its binding to the extracellular matrix could be responsible for this effect.

Heparin Differentially Impacts Gene Expression of Stromal Cells from Various Tissues

“…To enable heparin-free cell culture, fibrinogen was depleted mechanically from pHPL-based medium as described 33 and stromal cells were cultured in the presence and absence of heparin. By flow cytometry and immunocytochemistry a distinct cellular internalization of fluoresceinamine-labeled heparin mainly in the lysosomal compartment could be detected as described previously for other cell types 34–38 . Comparing gene and protein expression profiles of stromal cells from BM, UC and WAT in the presence and absence of heparin we observed distinct significantly influenced sets of genes, signaling cascades and proteins as well as posttranslational phosphorylation of proteins associated with WNT, PDGF, NOTCH and TGFbeta signaling pathways.…”

“…1C). Since data exist indicating that heparin is internalized by lysosomes 38,40 , we next incubated UC-derived stromal cells simultaneously with F-heparin and LysoTracker. Z-stack images obtained by confocal laser microscopy indicated that heparin’s intracellular distribution was associated with lysosomes (Fig.…”

Increased cellular uptake of lauryl gallate loaded in superparamagnetic poly(methyl methacrylate) nanoparticles due to surface modification with folic acid

“…All assays performed corroborate with cytotoxicity assays, in which a higher uptake cellular was observed when the lauryl gallate loaded in superparamagnetic PMMA nanoparticles surface modified with FA was incubated at 37 °C. When the nanoparticles were incubated at 4 °C, assuming that the energy-dependent pathways are inhibited, the nanoparticles were not able to diffuse at 4 °C, suggesting that the nanoparticles were uptake by some energy-dependent pathways [62][63][64]. Lastly, our results suggest that the higher cellular uptake observed by confocal microscopy and flow cytometry of lauryl gallate loaded in superparamagnetic PMMA nanoparticles surface modified with FA were delivered into HeLa cells via folate receptor-mediated endocytosis.…”

Hepatitis B e antigen as a predictor for hepatitis B e antigen-positive chronic hepatitis B patients with peginterferon alfa-2a therapy

“…The synthesis of sulfated glycosaminoglycans was assessed as described previously (Trindade et al, 2008a), in the presence or absence of cell signaling pathway inhibitors: 10 µM PD98059 (Calbiochem, USA); 25 µM FPT II (Calbiochem/Merk, Darmstadt, Germany); 10 µM KN‐62 (Sigma‐Aldrich, St Louis, MO); 10 µM 2‐APB (Calbiochem); 5 µM U73122 (Tocris, Bristol, UK); 10 µM BAPTA/AM (Invitrogen, Carlsbad, CA); 10 µM LNNA (Tocris, Bristol, UK) (Gazit et al, 1989; Grote et al, 2007; Paredes‐Gamero et al, 2010). The concentration of 100 µg/ml was used since it induces the maximal up‐regulation in the expression of HSPG (Nader et al, 1989, 1991).…”