Background/Aims: Dysfunction of the arterial endothelial cells promotes the progression of atherosclerosis. We studied how exposure of human arterial endothelial cells to atherosclerotic serum from patients with peripheral artery disease changes the secretory activity of these cells, and whether that reaction is modified by sulodexide. Methods: Endothelial cells in in vitro culture were exposed to standard culture medium ± 100pg/mL Interleukin-1(IL-1) or to medium supplemented with 20% atherosclerotic serum. Afterwards, the expression of genes responsible for the synthesis of Interleukin-6 (IL-6), Vascular Cell Adhesion Protein-1 (VCAM-1) and Von Willebrand Factor (VWF) was evaluated, together with the secretion of these compounds. Additionally, the effect of sulodexide on these processes was studied. Results: Atherosclerotic serum stimulated the expression of IL6, VCAM-1 and VWF genes in endothelial cells, which was followed by increased secretion of these compounds by 179%, 121% and 116%, respectively. Sulodexide (0.5 LRU/mL) reduced atherosclerotic serum-induced increased expression of genes for IL-6 (-32%), VCAM-1 (-20%) and VWF (-42%), and lowered secretion of these molecules: IL-6 (-27%), VCAM-1(-27%), VWF (-25%). Sulodexide also reduced, in a dose- dependent manner, secretion of IL6 from unstimulated and stimulated with IL-1 endothelial cells. Conclusions: Atherosclerotic serum induces proinflammatory and prothrombotic phenotype in arterial endothelium, which is partially reduced by sulodexide, via inhibition of genes expression, and in consequence lower secretory activity.

Background/Aims: Aging of the arterial endothelial cells results in the appearance of their inflammatory phenotype, which may predispose patients to the acceleration of arteriosclerosis. We studied the effect of serum from patients with peripheral artery disease (PAD) on the senescence of human aortic endothelial cells (HAEC) and how that process is modulated by sulodexide. Methods: HAEC replicative aging in vitro was studied in the presence of 10% PAD-serum (PAD Group) or10%PAD serum and Sulodexide 0.5 LRU/mL (PAD-SUL group). In control group cells were cultured in medium supplemented with 10% fetal bovine serum. All studied parameters were evaluated at the beginning and at the end of the study, in all experimental groups. Population doubling time (PDT) was studied from the cells growth rate after repeated passages, and senescence-associated beta- galactosidase activity (SA-β gal activity) was measured with the fluorescence flow cytometry. Expression of IL6, vWF, p21 and p53 genes was measured with the real-time polymerase chain reaction (Real-Time PCR). Concentrations of IL6 and vWF were measured with the standard ELISA kits. Results: PAD serum accelerated the senescence of HAEC as reflected by increased, compared to control, expression of the IL6 gene (+43%, p<0.05) vWF gene (+443%, p<0.01), p21 gene (+ 124%, p<0.01) and p53 gene (+ 85%, p<0.01). Secretion of IL6 and vWF was higher in that group: + 101%, p<0.01 and + 78%, p<0.01, respectively, as compared to control. Also, SA-β gal activity was higher in the PAD group (+33%, p<0.05) than in the control group. In the PAD group PDT was longer (+108%, p<0.01) as compared to control. Simultaneous use of Sulodexide with PAD serum significantly reduced all the above described senescent changes in HAEC. Conclusions: PAD serum accelerates the aging of HAEC which may result in the faster progression of arteriosclerosis. Sulodexide reduces PAD induced senescence of HAEC, which results in lower inflammatory and thrombogenic activity of these cells.

Background/Aims: Thromboembolic episodes are a frequent problem in end stage renal failure patients. The pathomechanism of the disorder is complex, including bioincompatibility of renal replacement therapy, endothelial dysfunction, increased blood level of procoagulant factors and uremic toxins. We studied changes in the functional properties of venous endothelial cells (VEC) in the presence of uremic serum and evaluated their possible modulation by N-acetylcysteine (NAC) or sulodexide (SUL). Methods: Serum samples from 12 uremic patients treated with hemodialysis were studied ex vivo on in vitro cultured VEC. In separate experiments, NAC 1 mmol/L or SUL 0.5 LRU/mL were added to uremic serum samples. Both changes in the gene expression and secretory activity of VEC were studied. Results: Uremic serum increased the expression of the following genes: IL6 +97%, p < 0.002; VEGF +28%, p < 0.002; vWF +47%, p < 0.002; PECAM +76%, p < 0.002; ICAM-1 +275%, p < 0.002; t-PA +96%, p < 0.002. Changes in gene expression were reflected by the increased secretory activity of VEC treated with the uremic serum. Exposure of VEC to uremic serum supplemented with NAC or SUL resulted in weaker stimulation of the studied genes’ expression. Also, secretion of the studied solutes, with the exception of ICAM-1, was reduced in the presence of NAC: IL6 –34%, p < 0.01; VEGF –40%, p < 0.005; vWF –25%, p < 0.001; t-PA –47%, p < 0.01, and MMP9 –37%, p < 0.001. SUL reduced the uremic serum-induced secretion of all solutes: IL6 –24%, p < 0.05; ICAM-1 –43%, p < 0.01; VEGF –38%, p < 0.01; vWF –23%, p < 0.01; t-PA –49%, p < 0.01, and MMP9 –25%, p < 0.05. Conclusions: Uremic serum induces prothrombotic changes in VEC, which may cause a predisposition to thrombotic disorders in patients with renal failure. NAC and SUL reduce the effects of the uremic serum in VEC, which suggests their potential therapeutic application in uremic patients.

Background/aims: Hemodialysis causes the systemic inflammatory response, which may affect the function of endothelial cells. Methods: We studied the effect of the serum obtained after a hemodialysis session, compared to serum collected before the start of the treatment, on the gene expression and secretory activity of arterial endothelial cells (AECs) and venous endothelial cells (VECs) in in vitro culture. Results: Serum collected at the end of the hemodialysis session increased expression of the studied genes in VECs, and at the same time decreased their expression in AECs. Secretory activity was increased in VEC: (interleukin-6 [IL-6] +29%, p < 0.05, von Willebrand factor +23%, p < 0.02; tissue plasminogen activator [t-PA] +35%, p < 0.002, t-PA/plasminogen activator inhibitor-1 [PAI-1] ratio + 57%, p < 0.005). In AEC, synthesis of IL-6 and vascular endothelial growth factor were reduced (–36%, p < 0.02, –34%, p < 0.05, respectively) and the tPA/PAI-1 ratio was increased (+22%, p < 0.01). Conclusions: Hemodialysis induces the inflammatory, procoagulant, and profibrinolytic activity of VEC, whereas suppression of AEC is observed at the same time. Video Journal Club ‘Cappuccino with Claudio Ronco’ at https://www.karger.com/Journal/ArticleNews/223997?sponsor=52

Background/Aims: Hemodialysis induces an intravascular inflammatory reaction which may further deteriorate renal function. We studied changes of serum interleukin 6 (IL6) and hepatocyte growth factor (HGF) concentrations during dialysis sessions, and at 12 month intervals. The synthesis of these cytokines in arterial endothelial cells in the presence of serum obtained from dialyzed patients was studied. Changes of the inflammatory reaction during 12 months of treatment were correlated with GFR. Methods: The study was performed on a group of 30 uremic patients treated with hemodialysis. Serum samples were collected before the start of dialysis, 15 minutes, and 4 hours later, when the session was finished. Serum levels of IL6 and HGF were measured with ELISA, as was the effect of serum samples on the synthesis of these cytokines in arterial endothelial cells. Results: At baseline hemodialysis induced an increase of serum IL6 (+10%) and HGF (+164%) levels at the end of the session. After 12 months of treatment predialysis serum IL 6 level was increased as compared to the beginning of the study (+22%), but no change in serum HGF level was observed. At that time the dialysis-induced rise of serum IL6 level was stronger than at the start (+18%), but the observed effect for HGF was weaker (+116%). An inverse correlation was observed between the dialysis-induced increase of HGF level and decrease of GFR after 12 months of study. The same relation was seen for HGF synthesis in the endothelium, but opposite for IL6 synthesis in the endothelium. Conclusions: We found that a higher HGF serum level during hemodialysis treatment is associated with a slower loss of residual renal function.