Clinical And Experimental Research On Alteration Of Vascular Permeability In Severe Sepsis

Objective Septic shock remains the leading cause of death in intensive care units. Cardiovascular symptoms of septic shock are characterized as intrinsic myocardial dysfunction as well as a marked vascular dysfunction including increased vasopermeability and decreased vasoconstrictor tone. We conducted a retrospective clinical trial to determine the correlation of fluid balance with changes of hemodynamics, pulmonary vascular permeability and tissue perfusion in septic shock patients achieving a central venous pressure (CVP) of 8-12 mmHg after early resuscitation, and evaluate its potential influence on clinical outcome. Then we will study the mechanisms involved in sepsis-induced vascular hyperpermeability, and examine the beneficial effects of glucocorticoid in improving vascular hyperpermeability using isolated rat aorta treated with LPS in vivo.Methods A total of 105 septic shock patients with continuous hemodynamic monitoring achieving CVP of 8-12 mmHg (To) after early resuscitation in 6-12 hours followed shock happening were included in this study. Data were collected from three time points:To and 24 hours (T24),48 hours (T48) later. All patients were divided into two groups based on 28-day mortality, or four groups based on cumulative fluid balance quartile of T48, to determine the correlation of fluid balance with hemodynamics [global end diastolic volume index (GEDVI), cardiac index (CI), and stroke volume index (SVI)], tissue perfusion [lactate level and central venous blood oxygen saturation (ScvO2)], and pulmonary vascular permeability [extravascular lung water index (EVLWI), pulmonary leak index (PLI), pulmonary vascular permeability index (PVPI), and oxygen index (OI)], evaluate the potential effects on patients’ prognosis.Male Sprague-Dawley rats were given either a bolus intraperitoneal injection of a nonlethal dose of LPS (escherichia coli 055:B5,10 mg/kg, Sigma) or vehicle (pyrogen-free water).30 minutes before or after the injection, groups of animals were also given either dexamethasone (4 mg/kg) or the MMPs inhibitor doxycycline (4 mg/kg). All animals were then killed humanely by pentobarbital sodium overdose (100 mg/kg ip) at 6 hours. Blood samples and aortas were obtained and used for functional and biochemical analysis. Activities and protein levels of MMP-2 and -9 were measured by gelatin zymography, Western blot and immunohistochemistry. Expression of tight junction (TJ)-associated protein (ZO-1) was measured by Western blot, and Glycosaminoglycans (GAGs)-containing proteoglycan, Syndecan-1 was measured with enzyme-linked immunosorbent assay (ELISA).Results (1) 57.1% (60/105) studied patients died within the 28 days followed shock happening. Based on 28-day mortality, APACHE II score (p=0.013) and lactate level (p=0.03l) were significantly different between survivors and non-survivors, no difference was found in hemodynamics and pulmonary vascular permeability indicators except that at To. The differences between survivors and non-survivors in net fluid balance (p T24=0.011,pT48<0.000), tissue perfusion (lactate level,pT0=0.031, pT24=0.002,pT48<0.001; ScvO2 pT0=0.079,pT24=0.003,pT48<0.001) and pulmonary permeability (EVLWI,pT0=0.233,pT24=0.003,pT48=0.001; PLIpT0=0.672,pT24=0.001, pT48<0.001; and PVPI pT0=0.161,pT24=0.001,pT48<0.001) were increased persistently from To to T48. Compared to survival patients, the growing trend of pulmonary vascular permeability (PVPI) of non-survivors was similar with tissue perfusion (lactate level) (RT48 = 0.227,p＜0.001), opposite to the decreasing trend of patients’ survival time (R=0.102, p<0.001). (2) The growing trends of net fluid balance at T24 and T48 were associated with pulmonary vascular permeability (PVPI, RT24=0.301, p<0.001; RT48=0.346,p<0.001) but not with cardiac preload (GEDVI,pT24=0.089,pT48=0.194) or cardiac output (CI,pT24=0.796;pT48=0.532; SVI, pT24=0.294, pT48=0.131). The result also showed that the net fluid balance and pulmonary vascular permeability tend to the same tendency in patients with and without pulmonary infection (pulmonary infection: R=0.291, p＜0.001; non-pulmonary infection:R=0.518,p＜0.001). (3) Multivariate regression analysis revealed that net fluid balance at T48 [odds ratio (OR) 3.15; 95% confidence interval (CI) 3.01-3.32], tissue perfusion (lactate level and ScvO2) (OR 7.47, 95% CI 2.51-9.14; OR 0.31,95% CI 0.13-0.94) and pulmonary vascular permeability (EVLWI, PLI and PVPI)(OR 2.49,95% CI 2.23-2.87; OR 1.92,95% CI 1.83-1.95; OR 1.88,95% CI 1.78-1.99) at T48 were all independent predictors for 28-day mortality in study patients of septic shock. (4) After correcting for pulmonary permeability, Cox survival analysis provided evidence that a more positive net fluid balance after early resuscitation was associated with higher risk and increased mortality in septic shock patients.The experimental study showed that both activities and protein levels of MMP-2 (p<0.001) and MMP-9 (p<0.001) were up-regulated significantly in aortic homogenates from LPS-treated rats, correlated with decreased ZO-1 (p＜0.001) and Syndecan-1 (p=0.011) expressions. Both Treatments of doxycycline and dexamethasone could inhibit MMPs activity and reserve the proteins of ZO-1 and Syndecan-1 significantly. Compared to doxycycline, the MMPs inhibition of dexamethasone was significantly lower while the improvement of Syndecan-1 expression was higher in aortas from LPS-induced endotoxemic rats (p=0.03).Conclusions For septic shock patients achieving CVP of 8-12 mmHg after early resuscitation, the deteriorated tissue perfusion always correlated significantly with increased pulmonary permeability, accompanied with significantly increased net fluid balance. Both deteriorated tissue perfusion and increased pulmonary permeability and net fluid balance were independent predictors for 28-day mortality in septic shock patients. Cox survival analysis provided evidence that a more positive 48-hour net fluid balance after early resuscitation was associated with higher risk and increased mortality in septic shock patients.MMPs activation plays important roles in regulating ZO-1 and Syndecan-1 protein levels in LPS mediated endothelial perturbation. Both doxycycline and dexamethasone exhibited MMPs inhibition that correlated with preservation in expressions of ZO-1 and Syndecan-1. The protective effects of dexamethasone on reserving LPS-induced endothelial dysfunction by preventing tight junction alteration and glycocalyx shedding are partly mediated by MMPs inhibition.