dmd is an inherited x - linked disorder with an incidence of 1 in 3,500 male births , and is due to the absence of dystrophin , a large protein linking the intracellular cytoskeleton to the extracellular matrix . it follows a predictable clinical course marked by progressive skeletal muscle weakness , myocyte hypertrophy , atrophy , and fibrosis . in general , the involvement of the heart leads to dilative cardiomyopathy ( dcm ) in 90% of patients . death usually occurs in the second or third decade of life and is due to respiratory or circulatory failure . over the last 20 years , respiratory care for these patients has improved because of the development of supportive equipment and techniques . consequently , dcm is increasing as the major cause of death in people with dmd , so that currently 1040% of dmd patients ( 2030% on average ) die due to dcm . the classical animal model for human dmd , the mdx mouse , is marked by a mutation genetically similar to the human deletion in the xp21.1 locus . although the underlying gene defect is the same in humans and the mdx mouse , the clinical picture is quite different . in the mdx mouse , skeletal muscle undergoes an early acute phase of degeneration at about 34 weeks of age , followed by a successful regeneration phase . the defect in the gene encoding dystrophin  one of the cytoskeletal proteins  may influence the susceptibility of the cardiac tissue to hypoxia . myocardial injury secondary to hypoxia has been thoroughly investigated and described in the setting of myocardial infarction , and reflects local acute ischemic hypoxia and can lead to dcm under certain circumstances . collateral blood vessels supplement normal coronary blood flow and coronary blood flow compromised by coronary artery disease , thereby protecting the myocardium from ischemia . collateral vessel formation is the result of angiogenesis , which depends upon the appropriate expression of growth factors [ 1012 ] . hypoxia is the main stimulating factor for the expression of vascular endothelial growth factor ( vegf ) [ 1316 ] . vegf - a , also known as vascular permeability factor , is a potent angiogenic factor and endothelial cell - specific mitogen that is regulated by hypoxia in vitro and in vivo . it also enhances embryonic and postnatal angiogenesis [ 12 , 19 , 20 ] and modulates vascular remodeling , tubulogenesis , and arteriogenesis . available data suggest a relationship between impaired vegf expression , a disturbance in angiogenesis , and the progression of dcm [ 2224 ] . the present study investigated alterations of the secretion of vegf - a in myocardium and heart vessels between healthy mice and dystrophic mice ( c57bl/10scsn mdx mice ) , which are the animal model of dmd . all mice were handled according to the guidelines of the institutional animal care and use committee . c57bl/10scsn and c57bl/10scsn mdx male mice , weighing 2030 grams , were purchased from jackson laboratory ( bar harbor , ma ) . all mice were housed in an individually vented cage system with a 12-hour light / dark cycle and received standard mouse chow and water ad libitum . adult mice c57bi/10scsn ( homozygotes + /+ in the locus encoding dystrophin , referred to herein as  normal  ) and adult mice c57bi/10scsn mdx ( homozygotes / in the locus encoding dystrophin , referred to herein as  dystrophic  or  mdx  ) were used in the experiment . initially , each group was placed in a low - pressure chamber for 1 h. the change from normal air pressure to low pressure corresponded to a rise from the altitude of 113 meters above sea level ( p=1000 hpa , po2=210 hpa ) to 7000 m above sea level ( p=410.25 hpa ; po2= 86.15 hpa ) . the target pressure was achieved within 10 minutes , and the initial pressure was restored within the same timeframe . in a previous study   we showed that 7000 meters corresponds to the sublethal hypobaric hypoxia conditions , and that the dystrophin - deficient mdx mice were more susceptible to acute hypobaric hypoxia than were normal mice of the same strain . hearts were harvested from the control group ( normobaric mice ) and mice exposed to low pressure directly after and 1 , 3 , 7 , and 21 days following the hypobaric exposure and fixed in a 4% solution of paraformaldehyde ( pfa ) in 0.01 m phosphate buffer ( pbs , ph=7.4 ) . the heart specimens were dipped in paraffin and cut into 6 m thick sections along the plane perpendicular to the long axis of the heart ( transverse sections through the ventricles ) . some of the material was frozen in liquid nitrogen and stored at 70c for later analysis . specimens were subjected to western blot analysis , in situ hybridization , and immunohistochemical analysis . vegf - a protein expression was determined using 100 mg of homogenized cardiac tissue suspended in 1 ml of mm tris - hcl buffer ( ph 7.4 ) and centrifuged at 10 000 rpm for 20 min . in order to select proper parameters for electrophoresis , the quantity of protein in the supernatant was assessed by the biuret reaction ( according to mejbaum - katzenellenbogen ) . electrophoresis was carried out on a 12% polyacrylamide gel , chosen according to the molecular weight of vegf - a ( 46 kda ) . the proteins were transferred ( semidry transfer ) to a nitrocellulose membrane ( 0.45 mm pore ; protran , schleicher & schnell bioscience ) using the mini semi - dry - blot device ( roth ) at 12 ma / cm over 12 h and at room temperature . immunodetection of vegf - a was performed using the abc kit ( santa cruz biotechnology ) . the membranes were blocked in 0.05%tween 20 for 1 h , then incubated with primary anti - vegf - a ( santa cruz biotechnology ) at 3 l / ml in 0.05% tween 20 in a humid chamber with orbital shaking for 1.5 h , followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. proteins were visualized using the horseradish peroxidase - diaminobenzidine system ( dab substrate kit for peroxidase ; sk-4100 , vector laboratories ) . specimens were blocked using 1% solution of h2o2 and proteinase k ( 200 g / ml ) was added for cell permeabilization . in order to deactivate any remaining rnase and proteinase k , the sections were fixed a second time in 0.4% pfa at 4c for 20 min . the samples were hybridized with the vegf - a mrna probe ( 5 biotin - tgg gtg cag cct ggg acc act tgg cat ggt gga ggt a - biotin-3 ; dna - gdansk , poland ) overnight under similar conditions . after hybridization , the slides were washed 25 min in 4 ssc/30% formamide , 25 min in 2scc/30% formamide , 115 min in 0.1% triton x-100 in tbs with albumin . the abc staining system kit ( santa cruz biotechnology , sc-2017 ) was used to detect the hybrids . then , the preparations were dehydrated using a set of alcohol solutions and xylene , and mounted in mountex medium . endogenous peroxidase was blocked by incubating samples in 0.1% h2o2 for 30 min , and then incubated with monoclonal mouse anti - vegf antibody ( santa cruz biochemistry , sc-7269 ) at 3 l / ml in 1.5% normal goat serum / pbs at room temperature for 30 min . primary antibody was detected using the abc staining system kit ( santa cruz biochemistry , sc-2017 ) . subsequently , the sections were incubated with biotinylated secondary antibody for 30 min and visualized using the dab - horseradish peroxidase system . 2 ( sigma ) , and then sections were dehydrated using a standard set of alcohol solutions and mounted in mountex medium . adjacent sections used as negative controls were processed in the same manner except that primary antibodies were omitted . the slides were evaluated by optical microscopy using a nikon eclypse 80i equipped with a digital camera ds-5mc ( 5 megapixel ) and software for digital analysis ( nis - elements , nikon ; japan ) . mean optical density was determined under constant light conditions according to a gray scale ranging from 0 to 255 , where 0 = black and 255 = white . nis - elements uses brightness / gray calibration curve evaluation of this parameter , and shows brightness / density ranges that correspond to the range of gray values recalculated via brightness / density calibration curves . five samples from each group were chosen for analysis , and 6 preparations from each mouse were examined . five fields of myocardium measuring 7050 m and not containing blood vessels were evaluated in each preparation ( figure 1 ) . the result recorded for each mouse was the mean of the values obtained for each sample . endothelial specimens on similar field sizes that contained cardiac vessels only were evaluated , and the mean value of all results was calculated for every group of 5 mice . for the control group , the mean optical density was defined as 100% of the change in optical density saturation for preparations containing both myocardium and vessels ( both arterioles / venules and capillaries ) , without distinguishing between these 2 compartments . this allowed us to calculate the mean value for the whole organ ( figure 1 ) . the results obtained in the experimental groups are expressed as percentages of the results for the control group . the values obtained from in situ hybridization analysis were analyzed using the unpaired student s t test . the values obtained from western analysis of vegf were analyzed using the wilcoxon signed rank test . all mice were handled according to the guidelines of the institutional animal care and use committee . c57bl/10scsn and c57bl/10scsn mdx male mice , weighing 2030 grams , were purchased from jackson laboratory ( bar harbor , ma ) . all mice were housed in an individually vented cage system with a 12-hour light / dark cycle and received standard mouse chow and water ad libitum . adult mice c57bi/10scsn ( homozygotes + /+ in the locus encoding dystrophin , referred to herein as  normal  ) and adult mice c57bi/10scsn mdx ( homozygotes / in the locus encoding dystrophin , referred to herein as  dystrophic  or  mdx  ) were used in the experiment . initially , each group was placed in a low - pressure chamber for 1 h. the change from normal air pressure to low pressure corresponded to a rise from the altitude of 113 meters above sea level ( p=1000 hpa , po2=210 hpa ) to 7000 m above sea level ( p=410.25 hpa ; po2= 86.15 hpa ) . the target pressure was achieved within 10 minutes , and the initial pressure was restored within the same timeframe . in a previous study   we showed that 7000 meters corresponds to the sublethal hypobaric hypoxia conditions , and that the dystrophin - deficient mdx mice were more susceptible to acute hypobaric hypoxia than were normal mice of the same strain . hearts were harvested from the control group ( normobaric mice ) and mice exposed to low pressure directly after and 1 , 3 , 7 , and 21 days following the hypobaric exposure and fixed in a 4% solution of paraformaldehyde ( pfa ) in 0.01 m phosphate buffer ( pbs , ph=7.4 ) . the heart specimens were dipped in paraffin and cut into 6 m thick sections along the plane perpendicular to the long axis of the heart ( transverse sections through the ventricles ) . some of the material was frozen in liquid nitrogen and stored at 70c for later analysis . specimens were subjected to western blot analysis , in situ hybridization , and immunohistochemical analysis . vegf - a protein expression was determined using 100 mg of homogenized cardiac tissue suspended in 1 ml of mm tris - hcl buffer ( ph 7.4 ) and centrifuged at 10 000 rpm for 20 min . in order to select proper parameters for electrophoresis , the quantity of protein in the supernatant was assessed by the biuret reaction ( according to mejbaum - katzenellenbogen ) . electrophoresis was carried out on a 12% polyacrylamide gel , chosen according to the molecular weight of vegf - a ( 46 kda ) . the proteins were transferred ( semidry transfer ) to a nitrocellulose membrane ( 0.45 mm pore ; protran , schleicher & schnell bioscience ) using the mini semi - dry - blot device ( roth ) at 12 ma / cm over 12 h and at room temperature . immunodetection of vegf - a was performed using the abc kit ( santa cruz biotechnology ) . the membranes were blocked in 0.05%tween 20 for 1 h , then incubated with primary anti - vegf - a ( santa cruz biotechnology ) at 3 l / ml in 0.05% tween 20 in a humid chamber with orbital shaking for 1.5 h , followed by incubation with the secondary biotinylated antibody under similar conditions for 1 h. proteins were visualized using the horseradish peroxidase - diaminobenzidine system ( dab substrate kit for peroxidase ; sk-4100 , vector laboratories ) . specimens were blocked using 1% solution of h2o2 and proteinase k ( 200 g / ml ) was added for cell permeabilization . in order to deactivate any remaining rnase and proteinase k , the sections were fixed a second time in 0.4% pfa at 4c for 20 min . the samples were hybridized with the vegf - a mrna probe ( 5 biotin - tgg gtg cag cct ggg acc act tgg cat ggt gga ggt a - biotin-3 ; dna - gdansk , poland ) overnight under similar conditions . after hybridization , the slides were washed 25 min in 4 ssc/30% formamide , 25 min in 2scc/30% formamide , 115 min in 0.1% triton x-100 in tbs with albumin . the abc staining system kit ( santa cruz biotechnology , sc-2017 ) was used to detect the hybrids . then , the preparations were dehydrated using a set of alcohol solutions and xylene , and mounted in mountex medium . endogenous peroxidase was blocked by incubating samples in 0.1% h2o2 for 30 min , and then incubated with monoclonal mouse anti - vegf antibody ( santa cruz biochemistry , sc-7269 ) at 3 l / ml in 1.5% normal goat serum / pbs at room temperature for 30 min . primary antibody was detected using the abc staining system kit ( santa cruz biochemistry , sc-2017 ) . subsequently , the sections were incubated with biotinylated secondary antibody for 30 min and visualized using the dab - horseradish peroxidase system . nuclei were stained with gill s hematoxylin no . 2 ( sigma ) , and then sections were dehydrated using a standard set of alcohol solutions and mounted in mountex medium . adjacent sections used as negative controls were processed in the same manner except that primary antibodies were omitted . the slides were evaluated by optical microscopy using a nikon eclypse 80i equipped with a digital camera ds-5mc ( 5 megapixel ) and software for digital analysis ( nis - elements , nikon ; japan ) . mean optical density was determined under constant light conditions according to a gray scale ranging from 0 to 255 , where 0 = black and 255 = white . mean brightness / gray value is the statistical mean of brightness values of pixels . nis - elements uses brightness / gray calibration curve evaluation of this parameter , and shows brightness / density ranges that correspond to the range of gray values recalculated via brightness / density calibration curves . five samples from each group were chosen for analysis , and 6 preparations from each mouse were examined . five fields of myocardium measuring 7050 m and not containing blood vessels were evaluated in each preparation ( figure 1 ) . the result recorded for each mouse was the mean of the values obtained for each sample . endothelial specimens on similar field sizes that contained cardiac vessels only were evaluated , and the mean value of all results was calculated for every group of 5 mice . for the control group , the mean optical density was defined as 100% of the change in optical density saturation for preparations containing both myocardium and vessels ( both arterioles / venules and capillaries ) , without distinguishing between these 2 compartments . this allowed us to calculate the mean value for the whole organ ( figure 1 ) . the results obtained in the experimental groups are expressed as percentages of the results for the control group . the values obtained from in situ hybridization analysis were analyzed using the unpaired student s t test . the values obtained from western analysis of vegf were analyzed using the wilcoxon signed rank test . there were significant differences in cardiac vegf expression in response to hypoxia between mdx and normal mice ( figure 2 ) . during the first days following hypoxia , expression of vegf in the hearts of the control group did not differ from that observed in the hearts of the normal and mdx mice ( 1023% vs. 994% , respectively ) ( p>0.05 ) ( figure 2c ) . immediately after exposure to hypoxia , vegf expression was 985% and 966% in normal and mdx mice , respectively ( p>0.05 ) . one day later , the changes in vegf concentrations were similar in normal ( 1688% ) and mdx mice ( 1729% ) ( p>0.05 ) . the decrease in vegf expression was markedly greater among the mdx mice ( 1137% ) than the normal mice ( 1335% ) ( p<0.05 ) . this trend reversed on day 7  vegf expression in normal mice continued to decrease ( 1075% ) , whereas it increased in mdx mice , reaching 1476% ( p<0.05 ) . after day 21 , vegf expression began to decline in both groups , reaching its initial concentration in normal mice ( 1046% ) and falling below the initial concentration in mdx mice ( 704% ) ( p<0.05 ) . effects on vegf mrna expression are depicted in figure 3a for the cardiac vessel endothelium cells and in figure 3b for the myocardium . in the case of cardiac endothelium cells , there was no difference in vegf - a mrna expression between the control groups for normal and mdx mice ( 1018% vs. 997% , respectively ) ( p>0.05 ) or between the normal and dystrophic mice immediately following exposure to hypoxia ( 1039% vs. 988% , respectively ) ( p>0.05 ) , on day 1 ( 22813% vs. 23314% ) ( p>0.05 ) , and on day 3 ( 14410% vs. 1338% ) ( p>0.05 ) . however , on day 1 there was a marked increase in vegf - a mrna expression in normal and mdx mice exposed to hypoxia as compared to normobaric control mice . by day 7 , vegf - a mrna expression levels had returned to their initial levels ( 10610% vs. 9313% , respectively ) ( p>0.05 ) . the difference in vegf - a mrna expression in myocardium between normal and dystrophic animals was significant ( p>0.05 ) ( figure 3b ) . after day 1 , normal mice expressed relatively more vegf mrna than did mdx mice ( 23415 vs. 1239% , respectively ) ( p<0.05 ) . on day 3 , vegf - a mrna expression in normal mice was similar to that in mdx mice ( 1329% vs. 1228% ) ( p>0.05 ) . by day 7 , vegf - a mrna expression in normal mice decreased to the initial level ( 836% ) , whereas in mdx mice , the level of mrna remained stable and twice as high as that in the control group ( 119%8% ) ( p<0.05 ) . twenty - one days following hypoxia , the expression of vegf - a mrna was similar in both groups and similar to the initial levels ( 575% vs. 437% ) ( p>0.05 ) . the expression of vegf - a mrna in control myocardium was lower than that in endothelial cells ( 60% vs. 100% , respectively ) . there were also differences in the expression of vegf - a protein expression between the normal and mdx mice . in the case of endothelium , there were no difference in vegf - a concentrations in normal and mdx mice ( figure 4a ) . in the case of myocardial cells , the initial control levels ( 997% vs. 926% , respectively ) and those measured immediately following hypoxia ( 1047% vs. 964% , respectively ) did not differ between normal and mdx mice ( p>0.05 ) ( figure 4b ) . differences in vegf - a protein concentration became apparent 1 day after hypoxia in the normal group . in these animals , there was an increase in optical density up to 168% ( 9% ) of the control level ( figure 5a ) . in dystrophic mdx mice , the protein concentration remained constant at 957% ( p<0.05 ) ( figure 5b ) . after 3 days , the concentration of vegf - a in normal mice fell to 120% ( 7% ) , but remained elevated relative to the initial level . in the mdx mice , the concentration of vegf remained stable at 868% ( p<0.05 ) . after 7 days , the vegf - a concentration in normal mice returned to the initial value ( 994% ) ( figure 6a ) , whereas in the mdx mice , the level of vegf - a protein began to increase , reaching 1359% ( p<0.05 ) ( figure 6b ) . after 21 days , there was a clear difference between the 2 groups in terms of vegf concentration . in control mice , the vegf concentration remained at its initial level ( 1037% ) but had decreased in mdx mice ( 768% ) ( p<0.05 ) . hypoxia is the primary cause of ischemic cardiomyopathy , dcm . during ischemia , certain factors , such as fgf-2   or vegf and no , are released and cause the collateral vessels to open . in dystrophic mdx mice , the low concentration of no leads to ischemic cardiomyopathy . in normal muscular tissue , hypoxia induces an increase in vegf expression through increased transcription of vegf mrna and improved protein stability . some reports have shown that dystrophinopathy is caused by lesions in the myocardium ( tissue ) but not in endothelial cells , in vessels . the increased expression of vegf enhances both the quantity and density of vessels , and not only in heart the vegf expression disorders in dystrophic mdx mice described in this paper , with the initial fall in the third day and a rise in the seventh day , are no exception . in their experiment with prolonged hypoxia of the brain , kuo et al   described similar changes in vegf expression . after a fall on day 4 , since vegf expression disorders occur only in mdx mice and are not seen in normal mice , they do not have to be related to prolonged ischemia . in our experiment , vegf expression disorders in normal and dystrophic mdx mice were limited to inappropriate expression and transcription of vegf in myocardial cells . it follows that expression of endothelial vegf would have an impact on total vegf expression in the heart only on day 1 after hypoxia ( figures 2 , 3a , 4a ) ; after that , the level of vegf expression in ischemic ( hypoxic ) cardiac tissue is the consequence of changes in the myocardium . in fact , on the third day , the expression of vegf protein and vegf mrna are decreased ( figures 3b , 4b ) , leading to dissimilarities in vegf expression between normal and dystrophic hearts ( figure 2 ) . increased expression of vegf mrna ( figure 4 ) and protein ( figure 4b ) in the myocardium influence this situation . these increases could be due to increased vegf mrna stability , as well as increased transcription . the situation changes between days 1 and 7 , when the relative expression of vegf mrna is higher . by day 21 vegf protein expression levels vary as well . however , the pattern of changes in vegf protein expression does not mirror that of vegf mrna expression . up to the 3rd day after hypoxia , , protein expression increases remarkably but falls again by day 21 . in the heart , the total level of vegf depends on vegf expression in myocardium , not in vessel endothelium , and our research demonstrates that the expression of vegf is dystrophin - dependent . the relationships between vascularization , other growth factors such as bfgf , pdgf , and dystrophin disorders have been documented . the reason for the discrepancies between the levels of vegf protein and mrna expression in myocardium seems to be associated with decreased stimulation of vegf mrna expression in myocytes . in myocardium , higher levels of vegf expression are not associated with increased protein stability , as has been reported . instead , the secretion of vegf from dystrophic myocardium is compromised and vegf is retained in cells . even if the expression of vegf protein is increased only marginally , a negative feedback mechanism blocks secretion of vegf from the cell , resulting in an elevated level of vegf within the cell . thus , the dramatic reduction in vegf mrna transcription on day 21 that we observed in our experiments was the result of the cells receiving incorrect ( inflated ) information about of the level of available vegf . our results suggest that high - altitude hypoxia causes abnormalities in cardiac muscle functioning in dystrophic mdx mice . the mutation in the dystrophin gene results in aberrant vegf secretion , which ultimately leads to ischemia . the disorders localized to non - vascular cardiac tissues related to dystrophic cardiomyopathy have been also described by other authors . impaired myocardial expression of vegf causes a significant loss of vegf to the whole organ . these disturbances are not only quantitative  the timing and speed of expression are affected as well . therefore , in such a setting , the heart is not properly protected against hypoxia , which could promote the gradual progression of cardiomyopathy , eventually manifesting as heart failure [ 23,3739 ] . our study investigated whether increased cardiac muscle susceptibility to ischemia was related to the efficacy of hypoxia - induced vegf expression . we have provided new support for the role of dystrophin in angiogenesis through its interactions with other growth factors . these data may aid in the development of new therapeutic options for dystrophin - related disorders .