Degraded Carrageenan Causing Colitis in Rats Induces TNF Secretion and ICAM-1 Upregulation in Monocytes through NF-κB Activation

Carrageenan (CGN) is a high molecular weight sulphated polysaccharide derived from red seaweeds. In rodents, its degraded forms (dCGN) can induce intestinal inflammation associated with macrophage recruitment and activation. The aim of this study was: 1) to analyze the size-dependent effects of dCGN on colon inflammation in vivo, and 2) to correlate these effects with monocyte/macrophage proliferation, cytokine production and expression of various cell surface antigens including ICAM-1 adhesion molecule. Peripheral blood monocytes (PBM) and THP-1 monocytic cells were cultured in the presence of either 10 or 40 kDa, dCGN. The 40 kDa, but not the 10 kDa dCGN, induced colitis in in vivo. Degraded CGN inhibited THP-1 cell proliferation in vitro, arresting the cells in G1 phase. In addition, dCGN increased ICAM-1 expression in both PBM and THP-1 cells with a major effect seen after 40 kDa dCGN exposure. Also, dCGN stimulated monocyte aggregation in vitro that was prevented by incubation with anti-ICAM-1 antibody. Finally, dCGN stimulated TNF-α expression and secretion by both PBM and THP-1 cells. All these effects were linked to NF-κB activation. These data strongly suggest that the degraded forms of CGN have a pronounced effect on monocytes, characteristic of an inflammatory phenotype

Degraded CGN activated the NF-kB pathway in monocytes.

<p><b>A</b>: THP-1 cells were transfected with a NF-κB reporter plasmid driving expression of luciferase. Cells were then treated with various concentrations of 10 kDa (triangles), or 40 kDa dCGN (squares). <b>B</b>: THP-1 cells treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) were lysed after various periods of time. Proteins in cell extracts were resolved by SDS-PAGE and then Western blotted for IκBα or α−tubulin as loading control. <b>C</b>: Degraded carrageenans (dCGN) induced activation of NF-κB. THP-1 cells were treated with nothing (control), or with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Nuclei were isolated and lysed. Proteins in nuclear extracts were resolved by SDS-PAGE and then Western blotted for NF-κB p50 subunit (p50) or NF-κB p65 subunit (p65). Lower panels show Western blots of nuclear ERK revealing equivalent amount of protein in each sample. Data are representative of three separate experiments. <b>D</b>: Degraded carrageenan (dCGN) induced activation of NF-κB. Nuclei isolated from THP-1 cells were fluorescence-stained for NF-κB p50 subunit or NF-κB p65 subunit before (filled area) or after cells were treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Dashed line corresponds to nuclei stained only with secondary fluorescence antibody. Fluorescence intensity was analyzed by flow cytometry as described.</p

Degraded CGN induced THP1 cell cycle arrest in G1 phase.

<p>THP-1 cells in exponential growth phase were incubated in the presence or absence of carrageenan for 24 h before being stained with propidium iodide. Cell DNA content was then analyzed by flow cytometry. <b>A:</b> Histograms of cells treated with medium only (control), 10 kDa dCGN (C10), or 40 kDa dCGN (C40). <b>B:</b> Percentage of cells in each phase of the cell cycle when treated with medium only (control), different concentrations of 10 kDa dCGN (C10), of 40 kDa dCGN (C40), or of native CGN (Native).</p

Degraded CGN stimulated ICAM-1 and TNF-α gene expression in monocytes.

<p>Representative samples of RT-PCR analysis showing over expression of ICAM-1 and TNF-α after stimulation of monocytes with 1 g/l of degraded CGN. β-actin expression was used as normalization gene.</p

Degraded CGN induced colon inflammation in rats.

<p>Histograms showing the effect of degraded CGN on: colon length (<b>A</b>); macroscopic (<b>B</b>) and histological (<b>C</b>) inflammation score of colon; Myeloperoxidase (MPO) activity (<b>D</b>). Control rats (white bars); 10 kDa degraded CGN-treated rats (grey bars); 40 kDa degraded CGN-treated rats (black bars). * p<0.05 from control. ** p<0.01 from control. Histological analysis of colon from control rats (<b>E</b>), and from 40 kDa dCGN-treated rats (<b>F</b>).</p

Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease

Bone-marrow-derived EPCs (endothelial progenitor cells) play an integral role in the regulation and protection of the endothelium, as well as new vessel formation. Peripheral circulating EPC number and function are robust biomarkers of vascular risk for a multitude of diseases, particularly CVD (cardiovascular disease). Importantly, using EPCs as a biomarker is independent of both traditional and non-traditional risk factors (e.g. hypertension, hypercholesterolaemia and C-reactive protein), with infused ex vivo-expanded EPCs showing potential for improved endothelial function and either reducing the risk of events or enhancing recovery from ischaemia. However, as the number of existing cardiovascular risk factors is variable between patients, simple EPC counts do not adequately describe vascular disease risk in all clinical conditions and, as such, the risk of CVD remains. It is likely that this limitation is attributable to variation in the definition of EPCs, as well as a difference in the interaction between EPCs and other cells involved in vascular control such as pericytes, smooth muscle cells and macrophages. For EPCs to be used regularly in clinical practice, agreement on definitions of EPC subtypes is needed, and recognition that function of EPCs (rather than number) may be a better marker of vascular risk in certain CVD risk states. The present review focuses on the identification of measures to improve individual risk stratification and, further, to potentially individualize patient care to address specific EPC functional abnormalities. Herein, we describe that future therapeutic use of EPCs will probably rely on a combination of strategies, including optimization of the function of adjunct cell types to prime tissues for the effect of EPCs.Shaundeep Sen, Stephen P. McDonald, P. Toby H. Coates, and Claudine S. Bonde