Among a variety of other findings, histological examinations have shown increased infiltration of leukocytes into the parenchyma, causing damage to the graft and thus reducing graft function [8]. all three adhesion proteins after HLMECs activation with TNF-(Physique 2). For ESELE expression (Physique 2(a)), the baseline value (no TNF-and no siRNA application) was 7.02 4.36% compared to the TNF-group (100%). Treatment of HLMECs with ESELE specific siRNA resulted in a significant diminished receptor expression of 68.09 3.89% ( .001), whereas the siSCR control group remained at 97.68 0.89%. Application of ICAM-1-specific siRNA led to a significant reduction of the ICAM-1 expression to only 43.80 8.98% ( .001), and treatment of HLMECs with VCAM-1 siRNA (Figure 2(c)) was 35.65 6.11% compared to TNF-stimulated cells ( .001) (see Physique 2). Open in a separate window Physique 2 Effects of siRNA and TNF treatment around the portion of cells positive for ESELE, ICAM-1, and VCAM-1. The (group 2) were set to 100%. activation; group 2: HLMVECs without siRNA transfection, but with TNF-stimulation; group 3: HLMVECs transfected with a receptor-specific siRNA and with TNF-stimulation; group 4: HLMVECs transfected with Longdaysin a nonspecific siRNA and with TNF-stimulation. Each bar represents the imply SD of = 3 impartial experiments. 2.1.2. Suppression of Receptor Expression by Application of an siRNA Cocktail Targeting All Three Different Adhesion Receptors HLMECs were transfected with the 3 siRNAs in a similar manner as that explained in the experiments shown above. However, this was not performed individually but by combining all 3 siRNAs into a cocktail (Physique 3). Open in a separate window Physique 3 Application of the mixture of 3 different siRNAs targeting ESELE, ICAM-1, and VCAM-1 exhibited the practicability and efficacy of transfection of multiple siRNAs simultaneously. The (group 2) were set to 100%. activation; group 2: HLMECs without siRNA transfection, but with TNF-stimulation; group 3: HLMECs transfected with 3 different receptor specific siRNAs and with TNF-stimulation; group 4: HLMECs transfected with a nonspecific siRNA and with TNF-stimulation. Each bar represents the imply SD of = 3 impartial experiments. 2.1.3. qRT-PCR In addition to the determinations of the protein level of the adhesion receptor expressions, the effects of siRNA transfection on ESELE, ICAM-1 and VCAM-1 mRNA transcript levels were analysed by qRT-PCR (Figures 4(a)C4(c)). Normalized gene expression Longdaysin was calculated by the Ct method; GAPDH was used as a reference, and group 1 (nontreated and nonstimulated) was set to the value of 1 1.00. Open in a separate windows Physique 4 Effects of siRNA and TNF-treatment on ESELE, ICAM-1, and VCAM-1 mRNA levels. Transcript levels were examined by real-time RT-PCR. Each bar represents the imply SD of three impartial experiments. Gene expression was normalized in relation to group 1, which was set to the value of 1 1.00. Group 1: HLMECs without siRNA transfection and without TNF-stimulation; group 2: HLMECs without siRNA transfection, but with TNF-stimulation; group 3: HLMECs transfected with a receptor specific siRNA and with TNF-stimulation; group 4: HLMECs transfected with a nonspecific siRNA and with TNF-stimulation. The effects of siESELE, siICAM-1, and siVCAM-1 on target protein expression were strongly reflected by changes in the corresponding transcript levels. All three siRNAs (siESELE, siICAM-1 and siVCAM-1) significantly reduced the levels of their corresponding target mRNA compared to the groups treated with TNF-only ( .05). In contrast, the control groups treated with siSCR Longdaysin showed no reduction in respective RXRG mRNA levels (Physique 4). Additionally, HLMECs were transfected with the 3 different siRNAs in a similar manner as that explained in the experiments shown above. However, this was not performed individually, but by combining all Longdaysin 3 siRNAs into a cocktail (Physique 5). The application of this siRNA cocktail targeting all three different adhesion receptors resulted in a high, significant reduction of all adhesion receptor mRNAs (Physique 5). Open in a separate windows Physique 5 Effects of siRNA and TNF-treatment on ESELE, ICAM-1, and VCAM-1 mRNA levels. Experiments and groups were as explained in Physique 4. 2.1.4. Neutrophil Adhesion Assay Two million PMN-neutrophils were added to each well made up of confluent layers of HLMECs, and after 1 hour, the adherent neutrophils were quantified using a CASY cell counter system (Physique 6). Around the nonactivated cell layer, only approximately 2,000 neutrophils attached. The activation.