The effect of rhAPC on contractile tension : an in-vitro sepsis model of cardiomyocytes and endothelial cells

Sepsis is the most common cause of death among patients in intensive care, due to multiple organ dysfunction. It is difficult to treat, and its incidence has steadily increased over the years. Since it is characterized by hypotension due to endothelial barrier disruption, multiple organ dysfunction and impaired cardiac contractility, hundreds of clinical trials have focused on endothelial cell dysfunction and cardiac depression. Both endothelial dysfunction and cardiac depression are regulated by mechanical properties of the cells such as contractile tension, and also involve numerous signaling pathways. Sepsis is the systemic inflammatory response to infection caused by excessive stimulation of endotoxin. The immune system is rapidly activated, resulting in the release of cytokines such as TNF-alpha, IL-6 and IL-1Beta. These cytokines help to control the infection by promoting a number of pathways, including coagulation, oxidation, nitric oxide, reactive oxygen species production, and activation of tissue factors. These responses disrupt endothelial cells, resulting in dysfunction, activation of coagulation and inflammation. Endothelial dysfunction causes microvascular thrombosis, leading to cardiac dysfunction. Myocardial depression is a clear and widely recognized sign of organ dysfunction in sepsis. Once endothelial cells are activated, their surface becomes prothrombotic. Thrombomodulin expression and EPCR expression are down-regulated, resulting in a decrease in the anticoagulant and anti-inflammatory effects of the protein C pathway. rhAPC has emerged as a novel therapeutic agent, indicated to improve survival in patients with severe sepsis. In-vitro studies have shown that rhAPC has antiapoptotic, anticoagulant and anti-inflammatory effects which cause cellular changes such as the enhancement of barrier function and cytoprotection. Moreover, recent experimental studies have shown that APC induces systemic and tissue inflammation and preserves cardiovascular function during experimental endotoxemia. In addition, one group indicated that APC induces a positive inotropic effect on cardiomyocytes which is dependent upon EPCR and PAR-1 pathways. Although the mechanisms underlying these pathways are not fully understood, sudden depletion of protein C 8 during sepsis might help us to understand better the importance of rhAPC. The administration of rhAPC reduced mortality among treated versus placebo patients in the PROWESS trial. As previously indicated, many studies have investigated the effect of lipopolysaccharide (LPS) and rhAPC through signaling pathways. Our study benefits from this earlier research, which proves both our in-vitro sepsis model and the effect of rhAPC on this model. However, we have chosen specifically to investigate the effect of LPS and the beneficial effect of rhAPC on endothelial cells and the cardiomyocytes establishing an in-vitro sepsis model. The aim of this project was to use the contractile tension measurements as a marker of mechanical properties of cells. The mechanical properties of cells are strongly associated with diseases or syndromes like sepsis. The multiple molecular mechanisms underlying disease offer many pathways which may be used for diagnostic and therapeutic purposes, but which may also be very complex. This can make it difficult to focus on one particular effect, whereas mechanical properties can give direct information about diseases and the body’s response to treatments. It is also necessary to measure contractile tension directly in order to understand how mechanical tension modulates a number of cell-dependent processes during sepsis. For this reason, we have used the CellDrum® system to measure the contractile tension of endothelial cells and cardiomyocytes after LPS application, and after rhAPC treatment following LPS application. The reliability of the CellDrum® system was validated by our preview studies. Our results support those of the previous studies, which showed that LPS caused endothelial cell contraction that causes endothelial permeability increase and cardiac depression that causes cardiogenic shock. Moreover, novel data was found to demonstrate that APC inhibits the effect of LPS through the mechanical tension of cardiomyocytes in vitro. We believe that the mechanical tension at the cellular level is an important parameter to understand the full process and the problem and might help us in diagnosis, treatment and prognosis of the diseases. These new results with endothelial cells and cardiomyocytes are believed to shed new light on the mechanical properties of cells during septicemia, which is crucial for improving the success of therapy