in the pathogenesis of multiple sclerosis ( ms ) , which is an autoimmune disease , pathologic remodelling of the blood - brain barrier precedes the formation of focal demyelination in the brain white matter [ 13 ] .
the remodelling of the blood - brain barrier in the course of ms is controlled by the activated t cd4 + lymphocytes and cytokines they release . in the initial phases , endotheliopathy caused by the adhesion of activated t lymphocytes
is observed within the blood - brain barrier , and these lesions can lead to vascular dysregulation .
vascular dysregulation can be congenital , or primary ( pvd ) or secondary ( svd ) .
the latter is often a result of autoimmune disease , including ms [ 46 ] .
the vascular dysregulation syndrome , earlier referred to as the vasospastic syndrome , is characterized by increased spasticity of blood vessels manifested as excessive vasoconstriction or insufficient vasodilation .
endothelial dysfunction is thought to play the main role in its pathogenesis as it results in the impairment of vasodilation and in increased blood levels of endothelin-1 ( et-1 ) .
et-1 is one of the most potent and longest acting vasospastic substances [ 810 ] produced by the vascular endothelium .
apart from endothelial dysfunction , impaired humoral or neurogenic mechanisms , which additionally may be interrelated , can play a role in the pathogenesis of the vasospastic syndrome .
in the course of severe clinical attacks of ms increased concentrations of et-1 were measured in the cerebrospinal fluid . the few studies published to date found increased levels of et-1 also in the blood of ms patients .
a transient increase of et-1 plasma level has been reported in a patient with optic neuritis which very often is the first sign of ms .
ms - dependent optic neuropathy is characterized by a mild increase in the cup to disc ratio [ 1517 ] and pallor of the optic disk suggesting ischaemia of the initial segment of the optic nerve which can result from a spasm of the supplying blood vessels .
et-1 has been shown to have a potent vasoconstrictive effect in the eye and hence to decrease the ocular blood flow [ 7,1820 ] , including blood supply to the optic disc .
studies using color doppler imaging have shown in ms patients a decreased blood flow velocity and increased vascular resistance index ( ri ) in the extraocular vessels [ 13,2325 ] , which indicates a reduction of the ocular blood flow in the course of ms .
the aim of this study was to determine the et-1 levels in the plasma of ms patients in view of a potential role of et-1 in the vascular dysregulation observed in the course of ms .
the study protocol was approved by the bioethics committee at the medical centre for postgraduate education in warsaw .
the patients were requested to sign written informed consent to participate in the research study .
the exclusion criteria were as follows : a history of cardiovascular disease , tobacco smoking , dyslipidemia , and current treatment with vasoconstrictors or vasodilators .
lipid fractions ( total cholesterol , triglycerides , ldl cholesterol , hdl cholesterol ) in the blood were determined in the study group and controls to exclude subjects with dyslipidemia , i.e. , those whose measurements were outside any of the following ranges : total cholesterol : 130200 mg / ml ; triglycerides : 35131 mg / ml ; ldl cholesterol : < 135 mg / ml ; hdl cholesterol : 4280 mg / ml .
following that , 20.41% and 12.9% of subjects were excluded from the initial ms group of 49 and from among 31 prospective controls , respectively .
additionally , subjects with a history of diseases known to be associated with increased blood levels of et-1 were also excluded .
ultimately , the study group consisted of 39 ms patients ( 9 males , 30 females ) aged 2262 years , mean age : 38.810.02 .
the diagnosis of ms was established using the mcdonald criteria   and the patients were recruited at the department of neurology and epileptology , medical centre for postgraduate education in warsaw .
the control group consisted of 27 healthy volunteers , free of any systemic disease , recruited mainly from among the hospital staff : 3 males and 24 females aged 2062 years , mean age 37.410.88 . of the study group ,
12.8% of the patients were diagnosed with primary progressive multiple sclerosis ( ppms ) , 15.4% with secondary progressive multiple sclerosis ( spms ) , 20.5% with progressive relapsing multiple sclerosis ( prms ) , and 51.3% with relapsing remitting multiple sclerosis ( rrms ) . at the time of the study ,
a history and/or medical records revealed a relapse in the previous 2 years in 12 patients , in the previous year in 9 patients , and in the previous 6 months in 2 patients .
however , in none of the patients a clinical relapse occurred in the 3 months preceding et-1 measurements within the study .
the mean duration of ms ( the medically documented diagnosis ) was 4.4 years ( range : 1 year to 22 years ) .
of the 46.2% of patients in the study group who received treatment , 13 were treated with interferon -1b , 4 patients with glatiramer acetate and 1 patient with azathioprine .
the remaining patients did not take any medication either because of low activity of their disease or because their disease was newly diagnosed and no treatment had yet been instituted .
the levels of clinically observed disabilities in the study patients were measured with the kurtzke expanded disability status scale ( edss ) ( 30 ) , the mean score being 2.55 ( range : 1.06.5 ) .
blood was drawn from the basilic vein after at least a 30-minute rest in a relaxed position ( supine ) , at room temperature , in the morning hours ( 9 a.m.10 a.m. ) . in patients on interferon therapy , blood
et-1 plasma level determinations were conducted at the department of neuroendocrinology , medical centre for postgraduate education .
blood samples from 27 controls and 39 study patients were placed in test - tubes containing edta and aprotinin ( protease inhibitor ) .
after centrifugation of the blood samples at 4c , plasma samples were frozen and stored at 70c until endothelin extraction .
et-1 was extracted from 1.5 ml of plasma . prior to plasma sample injections , sep - pak c18 extraction columns ( phoenix pharmaceuticals , inc . ;
usa ) were rinsed with 1 ml 60% acetonitrile with 1% trifluoroacetic acid , followed by 9 ml 1% trifluoroacetic acid .
the sample extracts were stored at 70c until endothelin-1 determination . immediately prior to the quantitative determination of et-1 ,
the extracts were dissolved in 0.15 ml of pbs buffer containing 1% bsa and 0.05% tweed 20 .
the et-1 concentration was determined using the endothelin-1 elisa kit ( immuno - biological laboratories co. , japan ; code no .
the efficiency of the extraction procedure was tested by adding a specified amount of exogenous endothelin-1 standard ( 20 pg / ml and 10 pg / ml ) to 1.5 ml of plasma with the endogenous endothelin content below the analytical sensitivity of the method .
the final measurements of et-1 concentrations in the study samples were adjusted by the extraction efficiency .
the et-1 concentrations were below the limit of detection in 29.0% of the control samples and in 36.7% of the samples from ms patients .
it was assumed that in such samples , endothelin-1 concentrations were below 8 fmol / ml .
the differences between the measurements were analyzed with the nonparametric mann - whitney u test for 2 independent groups of observations .
statistical significance was established at p<0.05 . additionally , to assess the homogeneity of the study group , the et-1 plasma levels were statistically analyzed in patients receiving immunomodulatory treatment vs. untreated patients , and in patients with rrms vs. progressive courses of multiple sclerosis ( spms , ppms , prms ) .
statistical analysis of potential differences in the et-1 levels between the patients with progressive courses of multiple sclerosis ( spms , ppms , prms ) could not be performed due to small numbers of patients in these subgroups .
the differences between the measurements were analyzed with the nonparametric mann - whitney u test for 2 independent groups of observations .
statistical significance was established at p<0.05 . additionally , to assess the homogeneity of the study group , the et-1 plasma levels were statistically analyzed in patients receiving immunomodulatory treatment vs. untreated patients , and in patients with rrms vs. progressive courses of multiple sclerosis ( spms , ppms , prms ) .
statistical analysis of potential differences in the et-1 levels between the patients with progressive courses of multiple sclerosis ( spms , ppms , prms ) could not be performed due to small numbers of patients in these subgroups .
the mean et-1 ( sd ) plasma level in ms patients was significantly lower than in healthy subjects : 0.550.44 pg / ml ( 146.05118.27 fmol / ml ) vs. 0.950.48 pg / ml ( 252.83127.16 fmol / ml ) ( p=0.012 ) ( figure 1 ) .
patients receiving immunomodulatory treatment tended to have lower et-1 plasma levels than untreated patients , but the difference was not statistically significant : 0.510.51 pg / ml ( 135.98136.66 fmol / ml ) vs. 0.580.39 pg / ml ( 155.20104.75 fmol / ml ) ( p=0.44 ) .
also , there were no statistically significant differences in the et-1 plasma levels between the patients with rrms and those with the other progressive courses of ms ( ppms , spms , prms ) : 0.640.45 pg / ml ( 170.85118.67 fmol / ml ) vs. 0.450.44 pg / ml ( 118.77117.72 fmol / ml)(p=0.46 ) .
no correlation was established between the et-1 plasma levels and age ( control group p=0.69 , ms group p=0.77 , all p=0.50 ) or the edss scores ( p=0.66 ) .
weak correlation between the et-1 plasma levels and the disease duration was found ( p=0.044 ) .
the pathogenetic changes in ms result from the remodelling of the blood - brain barrier associated with endotheliopathy due to the effect of activated cd4 + t - cells .
et-1 not only regulates the vascular wall tension , but may also act as a proinflammatory mediator , including its effect on the proliferation of astrocytes with an associated increase in the production of metalloproteases , which are involved in the remodeling of the extracellular matrix and the blood - brain barrier . in the present study we observed significantly lower plasma levels of et-1 in ms patients compared to healthy subjects , unlike earlier studies   that found significantly higher et-1 blood levels in ms patients than in controls .
interestingly , the groups of ms patients in those studies and in our study are comparable in terms of age and size ( haufschild et al . :
20 patients , 10 females and 10 males , mean age 43.8 years 14.7 , pache et al . :
30 patients , 22 females and 8 males , mean age 38 years 7.5 years , the present study : 39 patients , 30 females and 9 males , mean age 38.8 years 10.02 .
the ms patients in our study , as compared to the study by pache et al . had a slightly lower level of disease activity and almost twice as short disease duration ( 4.4 years vs. 8.4 years ) , which might account for the lower blood levels of et-1 . moreover ,
46.2% of the patients in our study group were treated with immunosuppressants , which decrease the activity of t lymphocytes   and the production of t cell - dependent mediators involved in the pathophysiologic process .
the earlier studies also found that untreated ms patients   had higher blood levels of et-1 compared to patients who received treatment , a tendency which we confirmed .
, the percentage of patients receiving treatment was similar ( pache et al . 56.7% . ) , which makes explanation of the differences between the studies difficult .
since et-1 blood levels are increased in cardiovascular disease , we excluded from our study not only patients with clinically diagnosed cardiovascular disease , as it had been done in earlier studies , but also subjects with dyslipidemia confirmed by laboratory investigations . with these criteria ,
20.41% of the ms patients and 12.9% of the prospective controls were excluded at baseline , which could account for lower et-1 levels in the ms patients .
additionally , the et-1 plasma levels might be related to a particular clinical course of ms .
however , we did not find any statistically significant differences in the et-1 plasma levels between rrms patients and those with the other forms of ms .
it could be speculated that , compared to other studies , lower disease activity and shorter duration of ms , and exclusion of patients with cardiovascular disease associated with endothelial dysfunction , including clinically 
silent dyslipidemia , may have accounted for the plasma levels of et-1 found in this study . however , these factors seem unlikely to cause the decrease of et-1 plasma levels in the ms patients to values below the normal range
. this finding may suggest another type of vascular dysregulation different from secondary vascular dysregulation , dependent on the autoimmune process in the course of ms , in which increased levels of et-1 in blood were observed .
the finding of lower than normal , et-1 plasma levels in the study group is in agreement with the results of studies in patients with optic neuropathy not associated with ms ( glaucomatous optic neuropathy ) , which found lower blood levels of et-1 in glaucoma patients compared to healthy subjects [ 3235 ] .
increased spasticity of blood vessels in the course of primary vascular dysregulation ( pvd ) has been considered a vasogenic risk factor for glaucomatous optic neuropathy for over 20 years [ 4,26,3646 ] .
endothelial dysfunction with increased et-1 blood levels can play a role in the pathogenesis of pvd syndrome and normal tension glaucoma ( ntg ) .
a recent meta - analysis of studies concerning et-1 levels in the blood of primary open - angle glaucoma ( poag ) patients found that only ntg patients had significantly higher levels of et-1 compared to healthy individuals .
however , the results of studies measuring the et-1 levels in the serum of ntg patients are inconsistent as various authors have shown in ntg patients compared to healthy individuals either increased blood levels of et-1 [ 4750 ] or no difference at all   or decreased levels .
these discrepancies in measurements of et-1 blood concentration in ntg patients may be due to many factors .
each study was carried out in a different age group , while et-1 levels are known to increase with age   and the associated progression of endothelial dysfunction .
lower et-1 levels in patients with glaucomatous optic neuropathy were observed mainly in the japanese population .
these observations suggest that the decreased blood levels of et-1 in ntg patients can be associated with the pvd syndrome .
the lower blood levels of et-1 in blood could be a result of interactions between neurogenic and endothelium - dependent vascular tone control mechanisms , associated with the already present dysregulation of the autonomous nervous system .
yoshida et al . suggest that the low levels of et-1 in the serum of glaucoma patients can be related to the up - regulation of endothelin receptors ( et - a , et - b1 , etb2 ) .
other researchers observed in poag patients a significantly higher increase in the serum levels of et-1 following exposure to cold , particularly in patients with ntg . in this situation ,
a transient increase of et-1 level in the blood serum due to cold can intensify the et 1-induced vasospastic reaction .
such a response may indicate an imbalance between the release of et-1 which is a vasoconstrictor and no which is a vasodilator , and can be related to the impairment of the vasodilating function of endothelium in ntg patients .
the above observations may suggest that as a result of impaired neural - endothelial control of vascular tone in pvd syndrome blood levels of et-1 can be low under normal conditions , while an increase in the et-1 concentration occurs only on a cold challenge test , which would provoke a vasospastic reaction .
it has been also noted that ms patients , similar to ntg patients , tend to develop pvd syndrome .
patients with ms experience problems typical of pvd , i.e. , a tendency to peripheral vasospastic reactions due to exposure to cold or emotional stress which often precedes ms symptoms .
this is confirmed by our observations as 76.92% of the ms patients had a history of peripheral vasospastic reactions before the actual diagnosis of ms .
it is possible that the lower levels of et-1 in ms patients found in the present study are accounted for by the et-1 measurements being performed at the time when the levels of et-1 had not yet increased as such increases are a consequence of svd , which results from the progression of the underlying autoimmune process .
the results showing significantly lower levels of et-1 in the plasma of patients with multiple sclerosis compared to healthy subjects suggest that some form of endothelium - dependent vascular dysregulation could occur in these patients .
this finding is in agreement with the reports of low blood levels of et-1 from other studies in patients with primary open - angle glaucoma , especially normal tension glaucoma .
this may suggest that similar pathophysiologic mechanisms may be involved in both , multiple sclerosis and primary open - angle glaucoma .
the role of et-1 in the pathogenesis of these 2 diseases , characterized by optic neuropathy , remains to be explained .
 backgroundmultiple sclerosis ( ms ) is a demyelinating autoimmune disease of the central nervous system with possible involvement of vascular dysregulation secondary to endothelial dysfunction caused by destruction of the vessel wall . 
 vascular dysregulation leads to excessive vasoconstriction or insufficient vasodilatation , resulting in vasospasm mediated by endothelin-1 ( et-1 ) , the most potent and long - lasting mediator . 
 vascular dysregulation can play an important role in the pathogenesis of some eye disorders and it has been hypothesized that it is a vascular risk factor for glaucomatous optic neuropathy . 
 the aim of this study was to estimate endothelin-1 ( et-1 ) plasma levels in patients with ms.material/methodsthe ms group consisted of 39 patients ( 9 males , 30 females ) , mean age : 38.810.02 years , range : 2262 . 
 the control group consisted of 27 healthy volunteers ( 3 males and 24 females ) , mean age : 37.410.88 years , range : 2062 ; clinically , in a non - active stage of the disease . et-1 
 plasma levels were measured using the endothelin-1 elisa kit ( immuno - biological laboratories co. , japan ) . 
 statistical analysis was performed with the nonparametric mann - whitney u test for independent groups.resultsendothelin-1 ( et-1 ) plasma levels were significantly lower in ms patients compared to healthy controls : mean value 0.550.44 pg / ml ( 146.05118.27 fmol / ml ) vs. 0.950.48 pg / ml ( 252.83127.16 fmol / ml ) ; p=0.012.conclusionssignificantly decreased et-1 plasma levels in the ms patients could reflect the non - active disease at the time of et-1 measurements or the effects of immunomodulatory treatment , but it can not be excluded that decreased et-1 plasma levels in these patients might result from vascular dysregulation . 
Background
Material and Methods
Statistical analysis
Results
Discussion
Conclusions