ssc ) is   a chronic , multifactorial disease which was first 
 described by goetz .
the disease can run either a 
 chronic or violent course which depends on the degree of internal 
 organs dysfunction .
literature 
 data point out that small and large 
 blood vessel disturbances and fibrosis development play a key 
 role in connective tissue changes of immunological character 
 . in healthy tissues
angiogenesis does not take place because of 
 equilibrium levels of proangiogenic and antiangiogenic factors . 

the following factors   are known to   trigger angiogenesis , that is , vascular endothelial 
 growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , and 
 interleukin-18 ( il-18 ) [ 5 , 6 , 
 7 ] , whereas endostatin , 
 tgf- , tnf- , interleukin-10 ( il-10 ) , as well as tissue 
 inhibitor of metalloproteinases 1 , 2 , 3 ( timp1 , 2 , 3 ) have 
 antiangiogenic activity . literature data reporting 
 inhibitory effects on angiogenesis exerted by monocytes and 
 lymphocytes obtained from systemic sclerosis patients could 
 further support the importance of angiogenesis disturbances in 
 the development of systemic sclerosis [ 8 , 
 9 ] .
impairment of collagen i , collagen iii , collagen v , collagen vi , 
 collagen vii synthesis in ssc leads to skin and internal organ 
 fibrosis .
recent research data underline the important role of 
 proteases in extracellular matrix degradation , including 
 collagen , in the course of this disease .
it is worth 
 pointing out that   matrix metalloproteinases ( mmps ) belong to the 
 group of proteases .
mmps are synthesized and released 
 by fibroblasts , monocytes , granulocytes , t lymphocytes , dendritic 
 cells , macrophages , endothelial cells , microglia , epithelial 
 cells , myocytes , and astrocytes .
tissue inhibitors of 
 proteinases ( timps ) are suggested to play a more important role 
 in fibrosis development than proteolytic enzymes .
what is more , 
 timps are synthesized by the same cells as their proteolytic 
 enzymes .
they exert an inhibitory effect on 
 metalloproteinases activation and further also on active forms of 
 those enzymes . up till now
, four timps have been 
 described , out of which the role of three has been fairly well 
 elucidated , that is , timp1 , 2 , and 3 .
homeostasis 
 disturbances between mmps and their tissue inhibitors could be 
 responsible for changes in extracellular matrix composition .
heart involvement could be one of the most important prognostic 
 factors in the course of ssc .
heine , already in 1926 , 
 was the first scientist to describe heart disturbances in ssc 
 .
it should be noted that cardiovascular changes could 
 develop at any stage of   the disease .
however , clinical 
 symptoms have not been characteristic and manifest 
 fairly late thus leading to late diagnosis and subsequent delay 
 in proper treatment .
the most common complaints reported 
 by the patients with heart involvement are palpitations , 
 dyspnoea , fatigability , and vague pain in the chest . 

myocardial ischaemia , resulting from coronary vessels spasms , 
 observed in ssc patients may resemble peripheral raynaud 's 
 phenomenon . in ssc patients , more often than in the 
 general population , morphological changes of heart structures , 
 specially of the mitral valve , are observed .
employment 
 of echocardiography , spiroergometry , perfusion scintigraphy of the 
 myocardium , 24-hour heart rhythm monitoring allow to detect 
 clinically  silent  changes [ 21 , 
 22 ] .
the aim of our study was to evaluate cardiovascular changes in 
 ssc patients and assess serum levels of vegf , endostatin , and timp2 
 in those patients .
the study involved 34 ssc patients , 26 women and 8 men , of age 
 range 1669 years . in our group 15 patients presented diffuse 
 form ( dssc ) ( mean 45.4  12.5 years ) and 19 limited ssc ( lssc ) 
 ( mean age 50.7  13.3 years ) .
all the   patients were 
 diagnosed according to american college of reumatology 
 ( acr ) criteria . because of the illness course , all dssc subjects were on 
 disease - modifying drugs , that is , cyclophosphamide ( 50  mg 
 daily ) , prednisone ( 1520  mg daily ) or combination of both 
 agents in the same doses as in monotherapies ( treatment duration : 
 3168 months , mean : 27  48 months ) .
patients from both subgroups 
 were treated with nifedipine ( 10  mg daily ) and vitamin e 
 ( 400  mg daily ) for at least 5 months .
the degree of skin sclerosis was assessed using the total skin 
 score ( tss ) according to kahaleh et al ( 066 points ) .
functional evaluation of four internal organs and systems , that 
 is , oesophagus , cardiovascular system , joint   and bone 
 system , and lungs was   performed by oesophagal 
 scintigraphy , 24-hour ecg monitoring ( 3-channel 
 oxford medilog excel-2 ) , doppler echocardiography ( h - p 2500 
 apparatus ) , x - ray examination   of feet and hand bone , x - ray chest 
 examination , and dlco ( diffusing capacity of the lung for carbon 
 monoxide ) ( expressed in percentages of the 
 predicted value ; pulmonary changes were diagnosed when dlco was 
 below 80% of the predicted value ) .
blood was collected in the morning into the pyrogen - free tubes and 
 stored in 20c until further evaluated .
vegf levels were 
 measured in 33 patients ( 19 with lssc and 14 with dssc ) and 
 endostatin in 30 patients ( 16 with lssc and 14 with dssc ) .
the 
 control group consisted of 20 healthy persons ( 19 female , 1 male ; 
 aged 2564 years , mean age : 46.3  13.2 years ) .
timp2 serum 
 levels were measured in 21 patients ( 9 with lssc and 12 with dssc ) 
 and 10 healthy persons ( 8 women and 2 men ; aged 2064 years , mean 
 age : 47.2  12 years ) .
vegf , endostatin , and timp2 serum levels were evaluated by enzyme 
 immunoassay technique ( elisa ) ( quantikine r & d systems inc , 
 minneapolis minn ; oncogene research products , boston , mass ; 
 amersham , pharmacia biotech , england , uk ) .
detection sensitivity 
 of the examined cytokines was 9.0  pg / ml for vegf , 
 1.953  ng / ml for endostatin , and 0.011  ng / ml for 
 timp2 .
this timp-2 assay detects approximately 50% of 
 recombinant human timp-2 when complexed with recombinant human 
 active mmp-9 in a 1:1 molar ratio .
greater than 2.5  ng / ml . antinuclear antibody titres were examined by indirect immunofluorescence 
 test on hep-2 cells .
all patients and individuals from the control group gave their informed 
 consent to participate in our study according to the bioethic committee of 
 medical university of lodz requirements .
the   obtained results were expressed as mean , maximum , 
 minimum , and median values together with standard deviation ( sd ) . 

mann - whitney , cochran - cox , and two independent sample tests 
 were employed for comparison of mean or median values . 

correlations between   serum levels of vegf , 
 endostatin and heart involvement were evaluated by fisher 
 precisely test with correlation coefficient q. a p value less 
 than .05 was considered to be statistically significant .
clinical characteristics of the patients are presented in 
 table 1 we revealed disturbances in different internal organs , that is , 
 oesophagus in 26/34 ssc patients ( 15 with lssc and 11 with dssc ) ; 
 lungs in 20/34 ssc patients ( 10 with lssc and 10 with dssc ) ; heart 
 in 15/34 ssc patients ( 8 with lssc and 7 with dssc ) ; 
 osteoarticular changes in 27/34 ssc patients ( 15 with lssc and 12 
 with dssc ) .
cardiovascular 
 changes were observed in 15 ssc patients ( 8 with lssc and 7 with dssc ) . 

they were of   different character and developed as single events or 
 coexisted   with each other .
mean heart rate in ssc patients was 81  11 
 in comparison to 71  9 observed in the control group . 

conduction disturbances were found in 3 patients , 6 presented 
 heart rhythm disturbances , and other 6 presented episodes of 
 ischaemia .
statistically significantly lower values of 24-hour 
 heart rhythm monitoring were observed in ssc patients in 
 comparison to the control group .
cardiographic examination did not reveal any impairment of 
 systolic function of the left ventricle in any of the examined 
 patients ; however 6 patients presented impairment of diastolic 
 function of this ventricle .
eight patients presented organic 
 changes such as fibrosis of the mitral valve leaflets and/or 
 aortic cusps ; however only in two patients the changes were of 
 hemodynamic importance .
it was demonstrated that mean levels of vegf were lower than 
 those in the control group ( 194.0  196.8 
pg / ml and 
 271.2  201.0  pg / ml , respectively at p > .05 ) ; endostatin 
 were higher than those in the control group ( 113.6  60.7  ng / ml and 73.6  25.9  ng / ml , respectively at 
 p < .05 ) ; and timp2 were not different from the control group 
 ( 80.0  31.5  ng / ml and 91.6  20.0  ng / ml , 
 respectively at p > .05 ) ( table 1 ) .
using fisher 
 test , we did not find any statistically significant correlation 
 between serum levels of the examined parameters and 
 cardiovascular changes ( detailed data not presented ) .
however , 
 comparison of mean serum levels of vegf , endostatin , and timp2 in 
 patients with heart involvement showed higher levels of 
 endostatin than in the control group ( p < .05 ) ; also such a 
 tendency , however still statistically insignificant , was observed 
 in the   selected 14 
 ssc patients ( sssc ) , in whom all 3 substances were assessed 
 ( table 2 ) .
it was also demonstrated that mean serum 
 levels of endostatin and timp2 were statistically significantly 
 higher in patients with heart changes than in the group of 
 patients without such changes ( p < .05 ) ( table 3 ) .
antinuclear antibodies ( ana ) were found in 34 patients . in 19 
 patients with
lssc the following ana were observed : 
 anti - topoisomerase i ( 10 patients ) , u3rnp antibodies ( 1 patient ) , 
 u1rnp antibodies ( 2 patients ) , anti - rna polimerase i antibodies ( 1 
 patient ) , and antinuclear antibodies of unidentifiable specificity 
 ( 5 patients ) . in dssc patients
the following antinuclear 
 antibodies were identified : anti - scl 70 antibodies ( 10 
 patients , in two patients coexisted with anticentromere 
 antibodies , aca , and in one patient coexisted with ku antibodies ) 
 and u3rnp ( 5 patients ) .
coronary vessel changes and fibrosis of the myocardium are 
 features of heart involvement in the course of systemic sclerosis . 

myocardial fibrosis usually develops in 
 subendocardial layer and does not depend on blood supply , as in 
 atherosclerosis .
kucharz also revealed that ecg changes were 
 observed in about 50% of systemic sclerosis patients . 

thickening of the left ventricular wall , asymmetrical hypertrophy 
 of interventricular septum , cardiomegaly , and exudative changes in 
 the pericardial sac have been described .
rodnan et al 
 was the first group who demonstrated that apart from vessel 
 spasms , inflammatory changes and fibrosis of coronary vessel walls 
 were important factors in myocardial ischaemia 
 development .
endostatin is found in blood , urine , and basement membranes of many 
 organs but its activity has not been fully elucidated . 
 however , it is known that endostatin specifically inhibits 
 endothelial cell proliferation and migration stimulated by vegf 
 .
endostatin also vastly reduces inflow of endothelial 
 cells to newly developed basal membrane of the blood vessels 
 . in vitro ,
incubation of endostatin with endothelial 
 cells leads to cell apoptosis . increased levels of endostatin were found in systemic sclerosis 
 patients in comparison to the control group .
our results 
 demonstrated significantly increased serum endostatin levels in 
 scleroderma patients when compared with the control group , too . 

collagen type xviii can be found in the basement membrane of skin 
 blood vessels and internal organs involved in systemic 
 sclerosis .
other authors demonstrated increased endostatin 
 serum levels only in a small group of patients ; however they did 
 not find any correlation with clinical parameters . in ssc 
 patients , especially with cardiovascular disturbances , timp2 and 
 endostatin action could be dependent on tissue microenvironment . 

timp2 and endostatin may act synergistically exerting their 
 antiangiogenic action via not yet fully elucidated pathways in 
 ssc course .
this action could be also triggered by the treatment 
 methods employed and could further result from systemic response 
 of the organism . however , despite not fully elucidated endostatin 
 action , it could be assumed that this factor inhibits activation 
 of proenzymatic metalloproteinase 2 form and catalytic actions of 
 membrane metalloproteinases type 1 and type 2 .
thus , 
 endostatin exerts a considerable effect on accumulation of 
 extracellular matrix components and fibrosis development in the 
 internal organs .
our results point out statistically 
 significantly increased endostatin levels in scleroderma patients 
 presenting cardiovascular disturbances in comparison to the 
 patients without such abnormalities .
probably increased levels of 
 this factor may also lead to inhibitory effect of angiogenesis in 
 anoxied heart tissue .
tissue inhibitor of metalloproteinase 2 is a protein of 21-  kd 
 molecular weight , released in a soluble form .
this protein exerts 
 an inhibitory effect on all active metalloproteinases forms and 
 competes with them as regards regulation of the extracellular 
 matrix amount .
yazawa et al demonstrated statistically 
 significantly increased serum levels of both timp1 and timp2 in 
 ssc patients than in the control group .
the current study results did not reveal statistically significant 
 differences as for timp2 serum levels between ssc patients and the 
 control group .
however , our results demonstrated statistically 
 significant differences of this protein level in the serum of lssc 
 patients with / or without heart involvement .
so , this observation 
 could further support the importance of timp2 in the development 
 of cardiovascular changes .
excessive accumulation of collagen 
 type i could lead to heart muscle fibrosis and restrictions in the 
 systo - diastolic function of the heart ventricles .
it can 
 be further assumed that increased levels of timp2 may lead to 
 angiogenesis inhibition , thus intensifying already triggered 
 dysfunction of microcirculation .
imbalance of heart 
 extracellular matrix components and decreased number of 
 capillaries could also lead to oxygen diffusion restrictions and 
 myocyte hypoxia .
structural changes of heart walls probably result 
 from imbalance between production and destruction of extracellular 
 matrix components , proliferation and migration of cells and their 
 apoptosis and necrosis .
heart fibroblasts take part in the above 
 processes via production of growth factors and cytokines , 
 which exert auto and paracrine action
thus , it seems reasonable to conclude that the currently observed 
 correlation between timp2 levels and heart dysfunction may be much 
 more important in   disturbances of extracellular matrix degradation than 
 ecm excessive accumulation .
the obtained results seem to confirm an important role of 
 angiogenic and fibrotic factors in the course of systemic 
 sclerosis , including development of cardiovascular disturbances . 

they also suggest that evaluation of endostatin and timp2 serum 
 levels in ssc patients could be helpful in the assessment of 
 the cardiovascular system .
 fibrosis of oesophagus , lungs , heart , and kidney in the course of 
 systemic sclerosis ( ssc ) may lead to dysfunction of the above 
 organs or even patients death . 
 recent studies point out 
 the role of angiogenesis and fibrosis disturbances in the 
 pathogenesis of ssc . 
 heart fibrosis is one of the most important 
 prognostic factors in ssc patients . 
 so , the aim of our study was 
 to examine cardiovascular dysfunction in ssc patients and its 
 correlation with serum levels of vascular endothelial growth 
 factor ( vegf ) , endostatin , and tissue inhibitor of 
 metalloproteinase 2 ( timp2 ) . 
 the study group comprised 34 
 patients ( 19 with limited scleroderma ( lssc ) and 15 with diffuse 
 scleroderma ( dssc ) ) . 
 the control group consisted of 20 healthy 
 persons , age and sex matched . 
 internal organ involvement was 
 assessed on the basis of specialist procedures . 
 serum vegf , 
 endostatin , and timp2 levels were evaluated by elisa . 
 we found 
 cardiovascular changes in 15 patients with ssc ( 8 with lssc and 
 7 with dssc ) . 
 the observed symptoms were of different characters 
 and also coexisted with each other . 
 higher endostatin serum 
 levels in all systemic sclerosis patients in comparison to the 
 control group were demonstrated ( p < .05 ) . also higher serum 
 levels of endostatin and timp2 
 were observed in patients with 
 cardiovascular changes in comparison to the patients without 
 such changes ( p < .05 ) . 
 the obtained results support the notion 
 that angiogenesis and fibrosis disturbances may play an important 
 role in ssc . 
 evaluation of endostatin and timp2 serum levels 
 seems to be one of the noninvasive , helpful examinations of heart 
 involvement in the course of systemic sclerosis . 
INTRODUCTION
MATERIALS AND METHODS
STATISTICAL ANALYSIS
RESULTS
DISCUSSION