formerly used as an ultimate ratio in end - stage pulmonary diseases , lung transplantation has become a commonly accepted therapy with an increasing number of procedures performed annually . despite the considerable advantages of perioperative medical care , two main problems remain unresolved : rejection of the graft and primary graft failure ( pgf ) of the allograft defined by a noncardiogenic pulmonary oedema appearing shortly after reperfusion of the transplanted organ . large clinical trials report an incidence of between 22% and 57% in patients receiving a lung transplant [ 1 , 2 ] . a central factor involved in the development of pgf seems to be the expression of adhesion molecules on the surface of pulmonary endothelial cells . they represent a group of different glycoproteins and carbohydrates expressed on the surface of a wide variety of cell types , including endothelial cells . by interfering with receptors on the leukocytes , firm adhesion and transendothelial migration follow , resulting in a sequestration of leukocytes at the endothelium and the later infiltration of the interstitial space . the release of proteolytic enzymes and oxygen - free radicals contributes to damage of the alveolar membrane , resulting in noncardiogenic pulmonary oedema . furthermore , some adhesion molecules seem to be involved in allograft rejection and the development of obliterative bronchiolitis . different investigators have demonstrated a positive correlation between the intensity of adhesion molecule expression on the endothelium of the allograft vasculature and clinical and histological signs of organ rejection [ 3 , 4 ] . therefore , suppression of adhesion molecule expression on the vascular endothelium ( thereby interrupting the leukocyte - endothelial interaction ) seems to be a promising tool to improve the quality of life of allograft recipients . e - selectin from the selectins group modulates the initial establishment of contact and causes rolling of the leukocytes along the vascular endothelium . intercellular adhesion molecule 1 and vascular adhesion molecule belong to the immunoglobulins group and mediate the fixed connection between leukocytes and endothelium . other groups of investigators have already demonstrated that adhesion molecules play a key role when it comes to recruitment of leukocytes in lungs , thus modulating the subsequent inflammatory reaction . using small interfering rna ( sirna ) to develop new therapies seems to show promise . originally identified as an intermediate of the rna interference pathway in the late 1980s , sirna has become a serious alternative in medical research and , therefore , therapeutic application . for practical use , analogous to the gene sequence , double - stranded sirna is manufactured by commercial providers and brought in the cytoplasm by transfection . one strand of sirna becomes part of a ribonucleoprotein complex called rna - induced silencing complex ( risc ) . risc can cleave rna sequences complementary to the sirna strand , thereby causing the rapid degradation of the target mrna . importantly , and in contrast to longer double - stranded rnas , sirnas in most cases do not induce an interferon response leading to a general cytotoxicity . the vision of our working group consists of using sirnas for effecting temporary suppression of endothelial adhesion molecules in pulmonary allografts in order to reduce the early and longer - lasting losses in functionality described above as well as the increase in patient morbidity and mortality resulting therefrom . the aim of the present study was to evaluate the potency of specific sirna to block the expression of intercellular adhesion molecule 1 ( icam1 , cd 54 ) , the vascular cell adhesion molecule 1 ( vcam1 , cd 106 ) , and e - selectin ( cd 62e ) in cultured human pulmonary microvasculature cells . stimulation of hlmecs with tnf resulted in a significant increase in receptor expression of all three adhesion proteins measured by facs . maximal expression was found 4  h and 12  h post - tnf- stimulation for esele and vcam-1 , respectively . icam-1 expression stayed high between 8 and 48  h ( see figure 1 ) . concerning all three types of adhesion molecules , the time span of twelve hours after stimulation ensured a high expression level of all adhesion molecules investigated . facs analysis showed a significant increase of the expression of all three adhesion proteins after hlmecs stimulation with tnf- ( figure 2 ) . for esele expression ( figure 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% ( p < .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% ( p < .001 ) , and treatment of hlmecs with vcam-1 sirna ( figure 2(c ) ) was 35.65  6.11% compared to tnf- stimulated cells ( p < .001 ) ( see figure 2 ) . hlmecs were transfected with the 3 sirnas in a similar manner as that described in the experiments shown above . however , this was not performed individually but by combining all 3 sirnas into a cocktail ( figure 3 ) . 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)4(c ) ) . normalized gene expression was calculated by the ct method ; gapdh was used as a reference , and group 1 ( nontreated and nonstimulated ) was set to the value of 1.00 . 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 ( p < .05 ) . in contrast , the control groups treated with siscr showed no reduction in respective mrna levels ( figure 4 ) . additionally , hlmecs were transfected with the 3 different sirnas in a similar manner as that described in the experiments shown above . however , this was not performed individually , but by combining all 3 sirnas into a cocktail ( figure 5 ) . the application of this sirna cocktail targeting all three different adhesion receptors resulted in a high , significant reduction of all adhesion receptor mrnas ( figure 5 ) . two million pmn - neutrophils were added to each well containing confluent layers of hlmecs , and after 1 hour , the adherent neutrophils were quantified using a casy cell counter system ( figure 6 ) . on the nonactivated cell layer , the stimulation of the hlmecs with tnf- led to a more than 4-fold increase ( up to 7,0009,000 ) in the number of adherent granulocytes ( figures 6(a)6(d ) ) . groups transfected with specific sirna showed significantly reduced adhesion of neutrophils ( p < .05 ) . transfection with icam-1 sirna ( figure 6(b ) ) resulted in a diminished adhesion of 4,824  57 , while transfection with vcam-1 sirna ( figure 6(c ) ) resulted in an adhesion of 6,375  492 . finally , the cocktail transfection ( 3 different sirnas ) resulted in 4,325  113 adherent neutrophils ( figure 6(d ) ) . lung transplantation carries a higher perioperative morbidity than other transplant procedures   including more frequent occurrence of primary graft failure . 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 . in particular , the vascular endothelium seems to be the graft component that is most vulnerable to the stimulus exerted by ischemia . endothelial activation leads to expression of adhesion molecules , which , in turn , exert a considerable influence on the interaction between leukocytes and endothelium [ 1013 ] . have demonstrated a correlation between intercellular adhesion molecule 1 ( icam 1 ) and recruitment of leukocytes within pulmonary parenchyma . have shown that blocking of adhesion molecules exerts a positive influence on reperfusion - associated hyperpermeability of transplanted lungs . these findings indicate that adhesion molecules play an essential role in the modulation of acute as well as chronic graft rejection reactions [ 15 , 16 ] , thus exerting a significant influence on the further development of perioperative morbidity and mortality of transplanted patients . therefore , suppressing expression of adhesion molecules in pulmonary allografts in order to maintain a high level of graft function seems to be a very promising therapeutic approach .   to gain a systematic insight into the expression patterns of adhesion molecules on pulmonary microvascular endothelial cells , tumour necrosis factor alpha , a well - known and highly potent cytokine , we have been able to show that various adhesion molecules from the selectin and immunoglobulin families can be stimulated to a significant degree but within different time windows ( see figure 1 ) , which partly differs from the findings published by other investigators . in analogy to the fact that different adhesion molecules exert different functions , we have been able to demonstrate , by means of our experiments , that expression of different adhesion molecules on human lung microvascular endothelial cells reaches a maximum after different latency periods following application of the stimulus . the fact that according to several authors , up to 40% of the pulmonary parenchymal mass might be composed of endothelial cells underlines the particular importance of these findings when it comes to protecting and preserving pulmonary allografts . furthermore , we were able to show that human lung microvascular endothelial cells can in fact be effectively transfected using a cationic liposomal transfection medium . we decided not to use a viral vector so as to work as closely as possible to actual clinical applicability while at the same time preventing the restriction of our method to a safe laboratory environment . in the course of practical application , however , it will not be sufficient to choose a single target or a single adhesion molecule . instead , it will be necessary to suppress a whole series of adhesion molecules in order to achieve a sustainable effect , and therefore , we have also performed what we call  cocktail transfection . this means that we have performed transfection of cells with a protective medium containing a combination of the three specific sirna sequences against esele , icam , and vcam and subsequently examined the effect achieved with respect to the individual adhesion molecules . this resulted in a highly significant reduction of expression of the individual adhesion molecules , as shown by facs analysis , qrt - pcr , as well as by functional assays quantifying neutrophil adhesion . it remains to be shown , of course , whether the results achieved in a laboratory setting under static conditions will be confirmed under dynamic conditions in animal experiments . the present study has demonstrated that transfection with specific sirna in each case achieved a significant degree of suppression of the adhesion molecules examined . to the best of our knowledge , so far , this has not been described in the literature on lung microvascular cells . the silencing is sustained for a period of approximately 2 weeks only ( and internal datas ) , after this period , a normal vascular endothelium showing regular physiological reactions is once again available .   on the basis of both the results described above and the knowledge of the consequences of pathological interaction between leukocytes and endothelium in transplanted organs , we believe that protecting pulmonary allografts by means of sirna transfection may represent a promising therapy for improving both short and long - term function of pulmonary grafts . human lung microvascular endothelial cells ( hlmecs ) and the mvecgm medium ( microvascular endothelial cell growth medium ) were purchased from sciencell ( carlsbad , calif , usa ) . these primary hlmecs are isolated from human lung tissue and are characterized by immunofluorescent methods with antibodies to vwf / factor viii and cd31 ( p - cam ) and by uptake of dii - ac - ldl . one day before sirna transfection , 150,000  hlmecs per well were cultured in gelatine - coated 12-well plates without antibiotics . after reaching 80% confluence , the cells were transfected with 100  nm specific sirna targeting e - selectin ( esele ) intercellular adhesion molecule-1 ( icam-1 ) or vascular adhesion molecule-1 ( vcam-1 ) or a nonspecific sirna ( negative control = scrambled sirna , siscr ) for 2 hours in a serum - free medium , followed by the replacement of the supernatants with ecm without antibiotics . transfections of sirna were carried out with cellfectin , 1  ng / ml ( invitrogen , karlsruhe , germany ) . twelve hours later , hlmecs were stimulated with tumour necrosis factor- ( tnf- ) ( immunotools , friesoythe , germany ) . keeping in mind the time dependent expression curves of the three adhesion molecules the time span of 12 hours after tnf stimulation ensured a high expression level of all three adhesion molecules . for esele and icam-1 , 2.5  ng / ml tnf- , and for vcam-1 , 5  ng / ml tnf- were used as described previously [ 2123 ] . using these concentrations experimental group setup 
 negative controls : hlmecs without sirna transfection and without tnf- stimulation.positive controls : hlmecs without sirna transfection , but with tnf- stimulation.sirna groups : hlmecs transfected with a receptor specific sirna ( or a cocktail out of three sirna ) and with tnf- stimulation.scrambled-sirna groups : hlmecs transfected with a nonspecific sirna and with tnf- stimulation . negative controls : hlmecs without sirna transfection and without tnf- stimulation.positive controls : hlmecs without sirna transfection , but with tnf- stimulation.sirna groups : hlmecs transfected with a receptor specific sirna ( or a cocktail out of three sirna ) and with tnf- stimulation.scrambled-sirna groups : hlmecs transfected with a nonspecific sirna and with tnf- stimulation . sirna groups : hlmecs transfected with a receptor specific sirna ( or a cocktail out of three sirna ) and with tnf- stimulation . scrambled - sirna groups : hlmecs transfected with a nonspecific sirna and with tnf- stimulation . three to five different sequences ( each targeting icam-1 , vcam-1 , or esele ) were designed by using various internet - based software tools . the sequences ( all synthesized by qiagen gmbh , hilden , germany ) showing the highest knockdown were chosen for all further experiments . the following sirna sequences and concentrations were used for transfection : sense : 5-gguugaaugcaccacucaadtdt-3 antisense : 5-uugaguggugcauucaaccdtdt-3 sense : 5-gccucagcacguaccucuadtdt-3 antisense : 5-uagagguacgugcugaggcdtdt-3 sense : 5-aaugcaacucucaccuuaadtdt-3 antisense : 5-uuaaggugagaguugcauudtdt-3 siscr ( scrambled sirna ) : sense : 5-uucuccgaacgugucacgudtdt-3 antisense : 5-acgugacacguucggagaadtdt-3 eight hours after stimulation with tnf- , the cells were stained for 1 hour at 4c with the respective antibodies in 1,000  l pbs containing 0.5% fcs in a 1  :  300 dilution for esele and icam-1 and 1  :  200 for vcam-1 . antibodies ( esele : pe - cy5 antihuman cd62e ( igg1 mouse , clone 68 - 5h11 ) , icam-1 : pe antihuman cd54 ( igg1 mouse , clone ha 58 ) , and vcam-1 : fitc antihuman cd106 ( igg1 mouse , clone 51 - 10c9 ) ) were all purchased from bd biosciences pharmingen ( heidelberg , germany ) . after the staining step , the cells were washed first with 1,000  l 0.5% fcs in pbs and then with 800  l hepes - bbs and detached . the cells were centrifuged at 220  g and fixed with 2.5% paraformaldehyde in pbs . flow cytometric analyses ( 5,000 cells / measurement ) were performed with a facscan ( becton dickinson gmbh ) and evaluated with cellquestpro software ( becton dickinson gmbh ) . eight hours after stimulation with tnf- , the cells were prepared for qrt - pcr . total rna from cultured hlmecs was purified by aurum total rna mini kit ( bio - rad laboratories , inc . 200  ng of each rna sample was reverse transcribed by iscript cdna synthesis kit ( bio - rad laboratories , inc . all qrt - pcrs contained iq sybr green supermix ( bio - rad laboratories , inc . , hercules , calif , usa ) , 400  nm forward and reverse primer and 2  ng of cdna in a total volume of 15  l . primer sequences 
							 esele : sense : 5-gcccagagccttcagtgtacc-3antisense : 5-ggaatggctgcacctcacag-3icam-1:sense : 5-cttgagggcacctacctctgtc-3antisense : 5-cggctgctaccacagtgatg-3vcam-1:sense : 5-acactttatgtcaatgttgcccc-3antisense : 5-gaggctgtagctccccgt-3 
 
							 esele : sense : 5-gcccagagccttcagtgtacc-3antisense : 5-ggaatggctgcacctcacag-3 sense : 5-gcccagagccttcagtgtacc-3 antisense : 5-ggaatggctgcacctcacag-3 icam-1:sense : 5-cttgagggcacctacctctgtc-3antisense : 5-cggctgctaccacagtgatg-3 sense : 5-cttgagggcacctacctctgtc-3 antisense : 5-cggctgctaccacagtgatg-3 vcam-1:sense : 5-acactttatgtcaatgttgcccc-3antisense : 5-gaggctgtagctccccgt-3 sense : 5-acactttatgtcaatgttgcccc-3 antisense : 5-gaggctgtagctccccgt-3 all qrt - pcrs were performed in triplicates . preliminary , not in this paper , demonstrated data showed a dose - dependent fashion of the different adhesion molecules , analogous to already published cell lines . they show a concentration of 2.5  g tnf for esele und icam and 5  g tnf for vcam to get a expression profile which was not increasable any more . for the sirna cocktail , we used a tnf concentration of 2.5  g . one hundred and fifty thousand hlmecs per well were cultured and stimulated with tnf-. every 2 hours for the next 24 ( or 48 ) hours , the expression of icam-1 , vcam-1 , or esele was measured by facs . healthcare bio - science ab , uppsala , sweden ) was formed by adding 0.18  g nacl to 20  ml percoll . the percoll solution was then diluted with 0.15  m nacl to final concentrations of 55% and 74% ; 3  ml of each solution was used to form the gradient . human blood was diluted 1  :  1 with pbs , and 3  ml of this solution was placed on the top of each gradient . the gradients were centrifuged at 350  g for 20 minutes ; cells were collected from the interface and washed with 6  ml of egm-2 . erythrocytes were lysed by incubating the cells with 5  ml of lysing buffer ( 8.29 g / l ammonium chloride , 1  g / l potassium hydrogen carbonate , 0.037  g / l na2-edta-2h2o ) . after stimulating the endothelial cells with tnf- , the medium was removed and 2 million granulocytes in 1  ml egm-2 were added to each well . after 1 hour , the granulocyte suspension was gently removed , and the wells were washed with medium twice . the adherent granulocytes were detached ( detachment kit , promocell gmbh , heidelberg , germany ) , resuspended in 100  l medium and quantified using a casy cell counter system ( schrfe system , reutlingen , germany ) . the results represent the average of three independent experiments ( each performed in triplicate ) . adhesion assay : the number of attached neutrophils on tnf- stimulated hlmecs was adjusted to 100% ( positive control ) , and the number of attached cells from the experiments with specific or scrambled sirna was calculated in relation to the 100% of the control . statistical analyses were performed using the statistics software package spss ( spss software inc . , differences between the groups were calculated by f - test followed by a post hoc test ( tukey hsd ) .
background . 
 adhesion molecules are known to influence postoperative organ function , they are hardly involved in the inflammatory response following the ischemia - reperfusion injury . 
 we sought to investigate the potency of small interfering rnas to suppress adhesion molecule expression in human pulmonary microvascular endothelial cells . 
 methods . 
 human lung microvascular endothelial cells were transfected with specific sirna followed by a stimulation of the cells with an inflammatory cytokine . 
 adhesion molecule expression was determined by facs - analysis , and reduction of intracellular mrna was determined by qrt - pcr . 
 furthermore , the attachment of isolated neutrophils on the endothelial layer was determined after sirna transfection . 
 results . in summary , 
 sirna transfection significantly decreased the percentage positive cells in a single cocktail transfection of each adhesion molecule investigated . 
 adhering neutrophils were diminished as well . 
 conclusion . 
 sirna might be a promising tool for the effective suppression of adhesion molecule expression on pulmonary microvascular cells , potentially minimizing leukocyte - endothelial depending interactions of a pulmonary allograft .