Pregnancy and social stress in female rats: influences on blood leukocytes and corticosterone concentrations.

The consequences of pregnancy and social stress on blood immune cells and on plasma corticosterone concentrations were assessed in Long Evans rats. Normal pregnancy in control females was characterized by a progressive increase in corticosterone concentration and increasing numbers of granulocytes. In contrast, CD4 T, CD8 T, and B cell numbers as well as the proliferative response of lymphocytes decreased as pregnancy progressed. Stress was induced in pregnant females by social confrontation for 2 h daily with a female resident opponent over a period of 2 months. Corticosterone concentrations were substantially higher in pregnant stressed than in pregnant control rats. Furthermore, the numbers of monocytes, NK and B cells were lower in stressed females, and there was a strong trend towards suppressed lymphocyte proliferation. Interestingly, pregnant females did not show granulocytosis in response to the stressor. In sum, the social stress paradigm in females appears to be a good model for the investigation of the interactions between stress, pregnancy and the immune system. It also provides an excellent platform for studies on prenatal stress under relatively naturalistic conditions.

Effective silencing of adhesion molecules on venous endothelial cells for protection of venous bypass grafts.

OBJECTIVE: The patency of venous conduits after aortocoronary bypass grafting is still not satisfactory and needs to be improved. Atherosclerotic alterations mediated by adhesion molecules triggering the transmigration of leukocytes are regarded as one of the major causes for venous graft failure. This study deals with short interfering RNA (siRNA)-mediated silencing of adhesion molecule expression on venous endothelial cells, which could lead to a new therapeutic strategy, resulting in improved patency rates by inhibiting early graft alterations. METHODS: Primary human venous endothelial cells (HVECs) were cultured in a newly developed perfusion model and subsequently transfected with specific siRNAs targeting three different adhesion molecules (the E-selectin (ESELE), the intercellular adhesion molecule 1 (ICAM-1), and the vascular adhesion molecule (VCAM-1)), followed by stimulation with tumor necrosis factor-alpha (TNF-α). Isolated leukocytes were perfused under physiological shear stress conditions, and their attachment to HVEC after single and triple transfection was quantified. RESULTS: siRNA transfection effectively knocks down adhesion molecule expression on venous endothelial cells, which subsequently reduces leukocyte attachment. Leukocyte adhesion to activated HVEC was significantly reduced after transfection by specific siRNAs in each case compared to the controls (p<0.05). Transfection with a mixture of all three siRNA sequences improved this effect even more (p<0.05). CONCLUSION: For the first time, a functional protection of HUEC in a model simulating physiologic vascular conditions by using nonviral transfection of the cells in a setup with high relevance for clinical applicability was demonstrated. Therefore, siRNA transfection of bypass material may develop into a new therapeutic option to improve the quality of venous graft material in the future.

Optimized basic conditions are essential for successful siRNA transfection into primary endothelial cells.

RNA interference (RNAi) is a powerful technique in basic research and has a high potential for therapeutic applications. To realize its clinical applicability, introduction of short double-stranded RNA (dsRNA) has to be carried out under physiological conditions. This study evaluates two cationic liposomal transfection reagents on the efficiency of successful silencing of primary human endothelial cells. Transfection efficiency was investigated under different conditions, for example different media during transfection, duration of transfection, siRNA concentration, and the use of serum and antibiotics. Viability after transfection was examined by CASY and MTT assay. Interferon response was examined by real-time PCR. First we revealed that transfection carried out in the presence of serum and antibiotics caused good knockdown results only by the use of the novel lipid cationic transfection reagent. Both lipid cations had slightly the same transfection efficiency over the range of 10-150 nM siRNA concentration. Examination of interferon response showed increasing OAS1 and STAT1 expression, but not as high as if the transfections were carried out with synthetic polyinosinic-polycytidylic acid double-stranded RNA (poly[IC]). The optimized combination of basic conditions for transfection significantly enhanced the efficiency of the siRNA-mediated knockdown, without causing toxicity or stimulation of the interferon pathway.

Graft protection in bypass surgery: siRNA-mediated silencing of adhesion molecules.

The outcome of patients after coronary bypass grafting is greatly influenced by the type of graft material employed, especially regarding the rate of graft restenosis. Besides direct thrombotic events, the leukocyte-endothelial interaction modulated by adhesion molecules is identified to be the central cause leading to graft alterations. This study deals with a new therapeutic concept in order to achieve superior protection of a new bypass graft by blocking the adhesion molecule expression pathway with RNA interference to inhibit the initial leukocyte adhesion and transmigration. Leukocyte binding to adhesion molecules on activated human venous endothelial cells (HVECs) was determined by video-assisted microscopy in a flow chamber mimicking physiological conditions. The cells under study were sequentially transfected in a nonviral manner with specific short interfering RNA-sequences (siRNA) targeting E-selectin, intercellular adhesion molecule, and vascular adhesion molecule. After stimulation of adhesion molecule expression by tumor necrosis factor, a leukocyte-rich suspension was run through the chamber and the attaching leukocytes were counted. Transfection with specific siRNA targeting three different adhesion molecules resulted in a highly significant reduction of leukocyte attachment to activated HVECs in each case compared to the controls (p < 0.05). Transfection with a mixture out of all three siRNA-sequences showed the lowest leukocyte adhesion (p < 0.05) compared to the controls. siRNA-sequences inhibit the adhesion molecule expression on HVECs in an extremely effective way; not only in a single transfection of specific molecules but also in a parallel transfection of multiple sequences in one transfection. Accordingly, siRNA treatment significantly reduced adhesion of leukocyte cells to HVECs compared to controls. This study showed for the first time an effective knockdown of the leukocyte-endothelium interactions by transfection of HVECs with a cocktail consisting of three highly specific siRNAs against three different endothelial adhesion molecules.

Inhibition of adhesion molecule expression on human venous endothelial cells by non-viral siRNA transfection.

OBJECTIVE: Expression of adhesion molecule receptors on venous endothelial cells crucially influences the fate of venous grafts by mediating leukocyte-endothelium interactions. These interactions include adhesion of leukocytes to the endothelium, followed by transendothelial migration, leading to neointimal hyperplasia (NIH) and finally graft occlusion. Therefore, inhibition of adhesion molecule expression may be a promising strategy to improve the quality of venous grafts. We tested the efficiency of non-viral transfection of human venous endothelial cells (HVEC) with short interfering RNA (siRNA) to specifically down-regulate adhesion molecule expression. METHODS: Primary cultures of HVEC were examined for expression of the adhesion molecules ICAM1, VCAM1 and E-selectin (SELE) after non viral siRNA transfection. Adhesion molecule expression was measured by flow cytometry, real-time polymerase chain reaction and immunoblotting after stimulation with TNF-alpha, an inflammatory cytokine. RESULTS: Non-transfected cells showed a strong increase of adhesion molecule expression following cytokine stimulation (P < 0.01). Upon transfection with specific siRNAs a sixfold decrease in ICAM1 (P < 0.001) and SELE expression and cell positivity (P < 0.05) and a twofold decrease in VCAM1 expression and cell positivity (P < 0.01) P could be observed. SiRNA-mediated gene suppression of adhesion molecules was also reflected by corresponding decreases in adhesion protein and transcript levels. CONCLUSIONS: The expression of adhesion molecules on HVECs can be effectively inhibited by specific siRNAs using a safe, non-viral transfection approach. This is a promising tool to pre-condition venous bypass grafts in order to interfere with endothelium-leukocyte interactions and to prohibit neointima thickening or atherosclerosis, which are regarded to be the most important causes of venous graft failure.