Background Multiple sclerosis (MS) is an autoimmune disease which results from the invasion of the brain by activated immune cells across the endothelial cells (ECs) of the blood-brain barrier (BBB), due to loss of immune self-tolerance. Many reports define the metabolic profile of immune cells in MS, however little is known about the metabolism of the BBB ECs during the disease. We aim to determine whether circulating factors in MS induce metabolic alterations of the BBB ECs compared to a healthy state, which can be linked with disruption of BBB integrity and subsequent immune cell extravasation. Methods and results In this report, we used an in vitro model to study the effect of sera from naïve-to-treatment, relapsing-remitting MS (RRMS) patients on the human brain microvascular endothelium, comparing effects to age/sex-matched healthy donor (HD) sera. Our data show that RRMS serum components affect brain endothelial cells by impairing intercellular tightness through the down-modulation of occludin and VE-cadherin, and facilitating immune cell extravasation through upregulation of intercellular adhesion molecules (ICAM-1) and P-glycoprotein (P-gp). At a metabolic level, the treatment of the endothelial cells with RRMS sera reduced their glycolytic activity (measured through the extracellular acidification rate-ECAR) and oxygen consumption rate (oxidative phosphorylation rate-OCR). Such changes were associated with the down-modulation of endothelial glucose transporter 1 (GLUT-1) expression and by altered mitochondrial membrane potential. Higher level of reactive oxygen species released from the endothelial cells treated with RRMS sera indicate a pro-inflammatory status of the cells together with the higher expression of ICAM-1, endothelial cell cytoskeleton perturbation (stress fibres) as well as disruption of the cytoskeleton signal transduction MSK1/2 and β-catenin phosphorylation. Conclusions Our data suggest that circulating factors present in RRMS patient serum induce physiological and biochemical alterations to the BBB, namely reducing expression of essential tightness regulators, as well as reduced engagement of glycolysis and alteration of mitochondrial potential. As these last changes have been linked with alterations in nutrient usage and metabolic function in immune cells; we propose that the BBB endothelium of MS patients may similarly undergo metabolic dysregulation, leading to enhanced permeability and increased disease susceptibility.
To explore the mechanism of increased collagen synthesis by scleroderma skin fibroblasts in vitro, control and scleroderma fibroblasts were compared in confluent monolayer cultures growth-arrested by serum deprivation; responses to optimal mitogenic doses of platelet-derived growth factor, fibroblast growth factor, epidermal growth factor and nerve growth factor were compared. Plateletderived growth factor had a selective mitogenic effect on control skdn fibroblasts not observed with scleroderma skin fibroblasts. None of the factors studied had a selective effect on collagen synthesis independent of cell replication; scleroderma and control fibroblasts responded similarly. Therefore, the growth factors studied may not be involved in generating the activated scleroderma fibroblast directly; platelet-derived growth factor may play an indirect role in fibroblast replication in human fibrotic disorders.More than a decade ago, it was observed that fibroblasts from involved skin of patients with scleroderma synthesized increased levels of collagen in vitro (1, 2); today the mechanism remains unknown (3-6). During this period, observations ofthe pathogenesis of scleroderma have focused on vascular and microvascular lesions; on evidence of endothelial damage; on platelet adhesion, aggregation, and release; and on the subsequent activation both of smooth muscle cells to produce the intimal proliferation and of interstitial fibroblasts to produce fibrosis (7-10). A unifying hypothesis would propose the presence offactors released from plasma or platelets which act as stimuli to fibrosis; among such factors, the potent mitogen, plateletderived growth factor (PDGF), is released during endothelial injury and platelet adhesion to the subendothelium (11). Therefore, the influence of PDGF on skin fibroblast collagen synthesis was studied.Endothelial damage and platelet interaction with the exposed subendothelium, resulting in release of platelet-derived mitogenic factors (including PDGF), have been proposed, at least indirectly, to play a role in the evolution of the scleroderma vascular lesion. Events preceding endothelial injury are poorly understood; altered immunity leading to humoral or lymphocyte/monocyte mediators ofinjury has been proposed with supporting evidence (12). If PDGF is the direct mediator of fibroblast activation in scleroderma, the enhanced collagen synthesis of scleroderma fibroblasts should be accompanied by increased cell replication at some stage in the process. Thus, a PDGF mechanism in scleroderma would imply replication offibroblast populations instead of, or in addition to, the selective stimulation ofcollagen synthesis per cell; in earlier studies, increased collagen synthesis per cell was observed in vitro without evidence of increased cell replication (1, 2). If PDGF is the mediator of fibroblast activation in scleroderma, its action should account for the initial observations of increased collagen synthesis per cell.A second characteristic of scleroderma fibroblasts in culture has been ...
Fibroblast mitogenic activity (MA) has been identified in scleroderma (SD) sera. Control and scleroderma skin fibroblasts in early passage were observed for replication (cell counts) after 72 h of serum exposure. SD sera at 15% concentration induced a significant increase in control fibroblast numbers when compared with control sera; this effect was not seen with SD cells while at higher serum concentrations (30%); SD cells were slightly responsive to the MA. MA was completely abrogated by the proteinase inhibitors STI and TLCK which did not affect mitogens in healthy sera. Circulating mitogenic proteinases selective for fibroblasts could play a role in the fibrosis of SD by modulating fibroblast replication.
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