s - nitroso - n - acetyl - penicillamine ( snap ) , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt),n - nitro - l - arginine methyl ester ( l - name ) , griess reagent , and interleukin-1(il-1 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) . a microchemoattraction chamber ( no . 3415 ; transwell , sigma - aldrich ) was purchased from corning ( acton , ma , usa ) . fetal bovine serum ( fbs ) , dulbecco 's modified eagle 's medium ( dmem ) , penicillin / streptomycin , and trypsin were purchased from gibco ( invitrogen , carlsbad , ca , usa ) . trizol was purchased from invitrogen and taq green master mix was purchased from promega ( fitchburg , wi , usa ) . tm cell cultures were established from enucleated human eye obtained from the eye bank and transported on ice within two hours after exsanguinations as previously described . briefly , tm tissues were excised by dissecting a continuous strand of tissue between the line of schwalbe and the scleral spur by teasing away the tm tissue using a curette . the excised tm tissues were placed in a sterile culture dish with dmem medium containing 15% fbs , 2 mm glutamine , 50 g / ml gentamicin , and 2.5 mg / ml fungizone and left undisturbed for three to five days in a 37 incubator with a 5% co2 atmosphere . after identifying initial cell growth , the explants were removed and the cultures were maintained with a medium containing 10% fbs . cultures approaching confluency were trypsinized and inoculated into 12-well culture plates ( 110 cells / well ) . after allowing attachment , the cells were washed three times with serum - free medium and cultured for 24 hours in medium lacking fbs but supplemented with 0.5% bovine serum albumin . the no donor snap was added to the medium at concentrations of 0 , 1 , or 10 m with or without co - treatment with the no inhibitor 0.5 mm l - name . to induce certain mmps , 25 ng / ml the mtt assay is based on the tetrazolium salt mtt that detects living but not dead cells . as signals are generated , the optical density is directly proportional to the number of cells . after adding 100 l of a mtt stock solution ( 5 mg mtt / ml phosphated buffered saline ) to each well , all the media was removed from the well after a four - hour incubation at 37. then , 0.5 ml of dimethyl sulfoxide ( dmso ) was added to each well , and 100 l of the solution was transferred from each well to a 96-well plate and read on a multi - well scanning spectrophotometer (  = 570 nm , fluostar optima ; bmg labtech , offenburg , germany ) . a minimum 10-minute exposure to dmso was required to dissolve the mtt formazan crystals after which the absorbance remained constant for 20 minutes . therefore , in all experiments , spectrophotometric readings were taken 15 minutes after the addition of dmso . briefly , media samples were collected from each well following appropriate treatment and reacted with modified griess reagent by mixing equal volumes with each other at room temperature for 15 minutes . optical density was then measured and read on a multi - well scanning spectrophotometer at 540 nm . the nitrite concentration was then determined from a comparison of absorbance with that of a standard solution of sodium nitrite in medium . the background absorbance , measured using the medium alone , was subtracted from all values . the cells were seeded in the presence or absence of snap or l - name in two 96-well plates . after a 60-minute incubation , unattached cells were removed by washing three times with phosphated buffered saline . after removing the unattached cells , cells were quantified by the mtt assay as described above . cell adhesion ( % ) was expressed as the number of adhered cells ( after washing ) divided by the number of total cells ( before washing ) . the migration of tm cells was measured with a transwell migration apparatus as described previously . briefly , cells were trypsinized and resuspended at a density of 210 cells / ml . then , the tm cells were added into the upper wells of a transwell chamber ( insert pore size , 3.0 m ) . the cultures containing media with or without snap were incubated for five hours at 37 in an atmosphere of 95% air and 5% co2 . at the end of the incubation period , cells were stained with trypan blue , and migrated cells attached to the bottom of the filter were counted under a light microscope . cell migration ( % ) was expressed as the number of migrated cells divided by the number of total cells . an rna denaturation mix consisting of isolated rna , oligo dt primers , and nuclease - free water was denatured . rt - pcr was performed using oligonucleotide primers specific to mmp-2 , -3 , -9 , -14 , and tissue inhibitor of metalloproteinase ( timp)-1 and-2 mrna ( table 1 ) . two tubes were run in parallel with the second tube containing only platinum taq polymerase to assure that the source of the rt - pcr product was mrna . taq green master mix and 10 pm each of forward and reverse primers was added to the synthesized cdna . the amplification reaction was carried out for 30 cycles on a dna engine cycler ( bio - rad , hercules , ca , usa ) . the amplified pcr products were analyzed using multi - gauge software ( fujifilm , tokyo , japan ) after electrophoresis . the expression of mrna was scanned , and the relative intensity of bands was determined by densitometry . every experiment was performed in triplicates , and the data are expressed as mean  standard error of the mean corresponding to the number of wells analyzed . experimental differences between the results of control cultures and a single treatment group were evaluated using the student 's t - test . s - nitroso - n - acetyl - penicillamine ( snap ) , 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide ( mtt),n - nitro - l - arginine methyl ester ( l - name ) , griess reagent , and interleukin-1(il-1 ) were purchased from sigma - aldrich ( st . louis , mo , usa ) . a microchemoattraction chamber ( no . 3415 ; transwell , sigma - aldrich ) was purchased from corning ( acton , ma , usa ) . fetal bovine serum ( fbs ) , dulbecco 's modified eagle 's medium ( dmem ) , penicillin / streptomycin , and trypsin were purchased from gibco ( invitrogen , carlsbad , ca , usa ) . trizol was purchased from invitrogen and taq green master mix was purchased from promega ( fitchburg , wi , usa ) . tm cell cultures were established from enucleated human eye obtained from the eye bank and transported on ice within two hours after exsanguinations as previously described . briefly , tm tissues were excised by dissecting a continuous strand of tissue between the line of schwalbe and the scleral spur by teasing away the tm tissue using a curette . the excised tm tissues were placed in a sterile culture dish with dmem medium containing 15% fbs , 2 mm glutamine , 50 g / ml gentamicin , and 2.5 mg / ml fungizone and left undisturbed for three to five days in a 37 incubator with a 5% co2 atmosphere . after identifying initial cell growth , the explants were removed and the cultures were maintained with a medium containing 10% fbs . cultures approaching confluency were trypsinized and inoculated into 12-well culture plates ( 110 cells / well ) . after allowing attachment , the cells were washed three times with serum - free medium and cultured for 24 hours in medium lacking fbs but supplemented with 0.5% bovine serum albumin . the no donor snap was added to the medium at concentrations of 0 , 1 , or 10 m with or without co - treatment with the no inhibitor 0.5 mm l - name . to induce certain mmps , 25 ng / ml the mtt assay is based on the tetrazolium salt mtt that detects living but not dead cells . as signals are generated , the optical density is directly proportional to the number of cells . after adding 100 l of a mtt stock solution ( 5 mg mtt / ml phosphated buffered saline ) to each well , all the media was removed from the well after a four - hour incubation at 37. then , 0.5 ml of dimethyl sulfoxide ( dmso ) was added to each well , and 100 l of the solution was transferred from each well to a 96-well plate and read on a multi - well scanning spectrophotometer (  = 570 nm , fluostar optima ; bmg labtech , offenburg , germany ) . a minimum 10-minute exposure to dmso was required to dissolve the mtt formazan crystals after which the absorbance remained constant for 20 minutes . therefore , in all experiments , spectrophotometric readings were taken 15 minutes after the addition of dmso . briefly , media samples were collected from each well following appropriate treatment and reacted with modified griess reagent by mixing equal volumes with each other at room temperature for 15 minutes . optical density was then measured and read on a multi - well scanning spectrophotometer at 540 nm . the nitrite concentration was then determined from a comparison of absorbance with that of a standard solution of sodium nitrite in medium . the background absorbance , measured using the medium alone , was subtracted from all values . the cells were seeded in the presence or absence of snap or l - name in two 96-well plates . after a 60-minute incubation , unattached cells were removed by washing three times with phosphated buffered saline . after removing the unattached cells , cells were quantified by the mtt assay as described above . cell adhesion ( % ) was expressed as the number of adhered cells ( after washing ) divided by the number of total cells ( before washing ) . the migration of tm cells was measured with a transwell migration apparatus as described previously . briefly , cells were trypsinized and resuspended at a density of 210 cells / ml . then , the tm cells were added into the upper wells of a transwell chamber ( insert pore size , 3.0 m ) . the cultures containing media with or without snap were incubated for five hours at 37 in an atmosphere of 95% air and 5% co2 . at the end of the incubation period , cells were stained with trypan blue , and migrated cells attached to the bottom of the filter were counted under a light microscope . cell migration ( % ) was expressed as the number of migrated cells divided by the number of total cells . total rna was extracted with trizol ( invitrogen ) . an rna denaturation mix consisting of isolated rna , oligo dt primers , and rt - pcr was performed using oligonucleotide primers specific to mmp-2 , -3 , -9 , -14 , and tissue inhibitor of metalloproteinase ( timp)-1 and-2 mrna ( table 1 ) . two tubes were run in parallel with the second tube containing only platinum taq polymerase to assure that the source of the rt - pcr product was mrna . taq green master mix and 10 pm each of forward and reverse primers was added to the synthesized cdna . the amplification reaction was carried out for 30 cycles on a dna engine cycler ( bio - rad , hercules , ca , usa ) . the amplified pcr products were analyzed using multi - gauge software ( fujifilm , tokyo , japan ) after electrophoresis . the expression of mrna was scanned , and the relative intensity of bands was determined by densitometry . every experiment was performed in triplicates , and the data are expressed as mean  standard error of the mean corresponding to the number of wells analyzed . experimental differences between the results of control cultures and a single treatment group were evaluated using the student 's t - test . the no donor snap significantly increased no in the media in a dose - dependent manner . nitrite concentration increased significantly from 0.68 m in the non - exposed control to 6.62 and 13.21 m with exposure to 1 or 10 m snap , respectively ( p = 0.003 , 0.001 ) ( fig . 1 ) . however , 25 ng / ml il-1 had no effect on the production of no compared to the non - exposed control ( p = 0.263 ) . addition of l - name to 10 m snap decreased no production to 7.39 m . snap did not significantly inhibit the survival of tm cells at a concentration of 1 or 10 m ( p > 0.05 ) ( fig . 2 ) . when 0.5 mm l - name or 25 ng / ml il-1 was administered simultaneously with snap , cell survival was also not significantly affected ( p > 0.05 ) . thus , the results of these experiments show that cell survival and proliferation were not affected by a concentration of 10 m snap . to test the possibility that no interferes with cell matrix adhesion , the attachment of tm to the cell matrix was examined in the presence or absence of snap . incubation of tm cells with either snap or the inhibitor of no synthase l - name were plated and allowed to attach for five hours . the percent of adherent cells at 1 or 10 m was 70.1%  1.0% and 71.7%  0.7% , respectively . adhered cells in the controls were 68.4%  0.5% of the total added cells ( fig . increasing the concentration of snap up to 10 m did not increase cell matrix adhesion over control levels compared to the non - exposed control ( p = 0.559 ) . the adhesion of l - name - treated cells was not different from the control cell population , and the percent of adherent cells was 62.1%  0.8 % ( p = 0.452 ) . assessment of transmigration five hours later showed that snap - induced changes in tm cell migration were not significantly different from the non - treated control cells ( fig . the percent of migrated cells at 1 or 10 m was 16.3% and 15.3% , respectively the migration of l - name - treated cells was not different from the control cell population , and the percent of migrated cells was 18.0% . in cultured tm cells , the relative level of mrna expression for four mmps and two timps in response to snap was determined by rt - pcr . , the expression of mmp-2 decreased slightly to an average of 7.5% . at 10 m , a further dose - dependent decrease in mmp-2 expression was observed ( p = 0.041 ) . at 1 m , the expression of mt1-mmp was unchanged ( fig . overall , the expression of mmp-2 decreased and mt1-mmp increased in response to 10 m snap . the expression of mmp-3 decreased at 10 m , but the decrease was not statistically significant ( p = 0.109 ) ( fig . 6 ) . the expression of timp-1 decreased slightly at 10 m , but the decrease was not statistically significant . at 1 m 7b ) to an average of 10.5% at 10 m ( p = 0.005 ) . overall , the expression of timp-1 did not change , whereas timp-2 expression increased in response to snap . the no donor snap significantly increased no in the media in a dose - dependent manner . nitrite concentration increased significantly from 0.68 m in the non - exposed control to 6.62 and 13.21 m with exposure to 1 or 10 m snap , respectively ( p = 0.003 , 0.001 ) ( fig . 1 ) . however , 25 ng / ml il-1 had no effect on the production of no compared to the non - exposed control ( p = 0.263 ) . addition of l - name to 10 m snap decreased no production to 7.39 m . snap did not significantly inhibit the survival of tm cells at a concentration of 1 or 10 m ( p > 0.05 ) ( fig . 2 ) . when 0.5 mm l - name or 25 ng / ml il-1 was administered simultaneously with snap , cell survival was also not significantly affected ( p > 0.05 ) . thus , the results of these experiments show that cell survival and proliferation were not affected by a concentration of 10 m snap . to test the possibility that no interferes with cell matrix adhesion , the attachment of tm to the cell matrix was examined in the presence or absence of snap . incubation of tm cells with either snap or the inhibitor of no synthase l - name were plated and allowed to attach for five hours . the percent of adherent cells at 1 or 10 m was 70.1%  1.0% and 71.7%  0.7% , respectively . adhered cells in the controls were 68.4%  0.5% of the total added cells ( fig . increasing the concentration of snap up to 10 m did not increase cell matrix adhesion over control levels compared to the non - exposed control ( p = 0.559 ) . the adhesion of l - name - treated cells was not different from the control cell population , and the percent of adherent cells was 62.1%  0.8 % ( p = 0.452 ) . assessment of transmigration five hours later showed that snap - induced changes in tm cell migration were not significantly different from the non - treated control cells ( fig . the percent of migrated cells at 1 or 10 m was 16.3% and 15.3% , respectively the migration of l - name - treated cells was not different from the control cell population , and the percent of migrated cells was 18.0% . in cultured tm cells , the relative level of mrna expression for four mmps and two timps in response to snap was determined by rt - pcr . , the expression of mmp-2 decreased slightly to an average of 7.5% . at 10 m , a further dose - dependent decrease in mmp-2 expression was observed ( p = 0.041 ) . at 1 m , the expression of mt1-mmp was unchanged ( fig . overall , the expression of mmp-2 decreased and mt1-mmp increased in response to 10 m snap . the expression of mmp-3 decreased at 10 m , but the decrease was not statistically significant ( p = 0.109 ) ( fig . 6 ) . the expression of timp-1 decreased slightly at 10 m , but the decrease was not statistically significant . at 1 m 7b ) to an average of 10.5% at 10 m ( p = 0.005 ) . overall , the expression of timp-1 did not change , whereas timp-2 expression increased in response to snap . the regulation of gene expression and activity of various mmps is complex and no seems to play a role . mmp-2 ( gelatinase a ) activation is suppressed by no donors in human breast cancer cells . however , no has been shown to inhibit the expression of mmp-9 ( gelatinase b ) but not mmp-2 in rat smooth muscle cells . mmps degrade the basement membrane and ecm , facilitating cell migration . among the mmps , mmp-2 and mmp-9 play a critical role in cell migration . in both ecs and vsmcs no inhibits mmp-2 expression , attenuates endothelial migration , and reversibly inhibits migration independent of proliferation or cytotoxicity in cultured vsmcs . however , there are controversial reports on the effects of no on cell migration . in contrast , other studies demonstrated that no donors increase no production , which leads to inhibition of endothelial migration . in addition , mmp mrna and protein levels are up - regulated in response to mechanical stretching , elevated pressure , and various cytokines or growth factors . it is known that mmp-2 , -14 , -15 , and -16 are constitutively expressed at relatively high levels in the tm . mmp-1 , -3 , and -9 are normally expressed at low levels but are massively up - regulated and activated in response to various stimuli . it is known that no is involved in the regulation of trabecular outflow , and external administration of no donors results in decreased iop . mmps play a significant role in the regulation of aqueous humor outflow facility by controlling ecm turnover in the tm . no modulates the expression of mmps , which may also be important for tissue remodeling characterized by cell proliferation and migration . in tm cells , numerous mmps have been detected , and mmp mrna and protein levels are up - regulated in response to mechanical stretching and elevated pressure . however , the detailed molecular mechanisms of how no affects tm cell migration is still unclear . our results demonstrate that no had no significant effect on the survival of cultured tm cells , in agreement with a previous report . for example , no serves as a switch from a stationary to locomoting phenotype in bs - c-1 cells , and exogenous no has been shown to elicit chemotaxis of neutrophils in vitro . previous studies have demonstrated that no plays a permissive role in endothelin-1-elicited locomotion of ecs . we performed adhesion and migration assays to determine whether no induces stimulation of podokinesis in tm cells . our results suggest that no did not mediate adherence or migration of tm cells in response to snap . lack of an effect by l - name on adherence indicates that tm cells in culture express minimal endothelial nitric oxide synthease activity , and it is possible to elicit an effect by l - name only when cells are stimulated . overall , incubation of tm cells with an no donor did not affect cell adherence . these findings further support that interference by no with respect to tm cell migration was negligible . thus , it appears that no does not cause significant changes on tm cell adherence or migration , unlike ecs or vsmcs . since mmp-2 and mmp-9 are particularly relevant for migration , and mt1-mmp ( mmp-14 ) acts as an mmp-2 activator , we focused on these mmps to evaluate the effect on migration in response to no . mmp-2 and mt1-mmp mrna are known to be highly expressed in tm cells . to gain more insight into how mmp activation is regulated in response to no donors during human tm cell migration , we evaluated the effects of snap on the expression of mmps and timps . we observed that treatment with snap resulted in a decrease of mmp-2 expression compared with control cells . since mt1-mmp has been reported to be a key molecular effector of no during ec migration , we also explored mt1-mmp expression in human tm cells treated with snap . mmp-1 , -3 , and -9 are normally expressed at low levels but are massively up - regulated and activated in response to various types of stimuli including il-1 . although il-1 stimulation was selective for mmp-3 , we observed no significant difference in mmp-3 expression in response to snap . furthermore , we did not observe expression of mmp-9 mrna in control or snap - treated tm cells , which is in agreement with a previous study . the absence of mmp-9 and concurrent up - regulation of timp-2 along with down - regulation of mmp-2 may explain the limited effect of no donors on tm cell migration . the results from our experiments show that timp-2 activation in tm cells in response to an no donor occurs despite increased expression of mt1-mmp mrna . depletion of tm cells is considered to play a pivotal role within the factors conferring the development of primary open angle glaucoma ( poag ) . in fact , age - related tm cell loss is even more pronounced in patients with poag than in age - matched controls . in this context , detachment from the tm and migration from the outflow system stimulated by factors present in the aqueous humor has been suggested as one mechanism conferring meshwork cell depletion in poag . immunoreactions for mmps are stronger in poag samples compared to controls , with the exception of mmp-2 levels . moreover , the staining intensity of mmp-1 or mmp-3 is significantly higher in poag samples . it has been reported that the mmp-2/timp-2 ratio is increased in the aqueous humor of poag eyes , possibly as a result of detachment or loss of tm cells . these results suggest that mmp-1 and mmp-3 are the most likely candidates to be involved in the remodeling of trabecular tissue in poag eyes . it is also likely that cell loss can be explained by local cell death or tm cell migration . if a cell enters the cell cycle to replenish the diminishing tm cell population , it will loosen its contact with the ecm . the present study demonstrates that no elicited a decrease in mmp-2 activity , an absence of mmp-9 activity , and an increase in timp-2 activity without a permissive role of no in tm cell adhesion or migration , which , if occurred , would result in tm cell detachment and subsequent cell loss . an imbalance in the equilibrium between mmp and timp levels may be an important factor in the maintenance and regulation of the ecm in the tm . a decrease in mmp-2 and an increase in timp-2 secretion may play an important role in inhibiting cell migration by altering the ratio of mmps to timps . this shifts the balance of mmp activity , which may favor the inhibition of cell migration . our data suggest that the mmp-2/timp-2 ratio decreased in response to exogenous no . in conclusion , we have demonstrated that no , an iop - lowering agent , does not significantly affect migration of tm cells . thus , no may increase trabecular outflow facility without tm cell loss . in vitro analyses of tm cell migration had the limitation of being remote from physiological and pathological events in vivo . further in vivo analyses
purposeto determine the effect of exogenous nitric oxide ( no ) on the migration of trabecular meshwork ( tm ) cells and its association with expression of matrix metalloproteinases ( mmps).methodsprimary human tm cells treated with 1 or 10 m s - nitroso - n - acetyl - penicillamine ( snap ) and examined for changes in adherence . 
 tm cells were seeded onto transwell culture inserts , and changes in their migratory activity were quantified . 
 reverse transcription polymerase chain reaction was performed to determine the relative changes in mrna expression of mmps and tissue inhibitor of metalloproteinases ( timps).resultstreatment with snap did not significantly suppress tm cell adhesion or migration ( p > 0.05 ) . 
 treatment of tm cells with 10 m snap decreased expression of mmp-2 and increased expression of membrane type mmp-1 and timp-2 . 
 treatment with interleukin-1 triggered mmp-3 expression but did not exert significant effects on mmp-3 activation in response to snap.conclusionsthese data suggest that no revealed no significant effect on the migration of tm cells because no decreased mmp-2 and increased timp-2 expression . 
 although expression of certain mmps and timps change in response to no donors , no may modulate trabecular outflow by changing the cellular production of extracellular matrix without having a significant effect on the migration of tm cells .