Žilna prizadetost in dolžina telomer v levkocitih pri bolnikih s Fabryjevo boleznijo

[doktorska disertacija]

Andreja Cokan (Author), Mišo Šabovič (Mentor), Aleš Blinc (Thesis defence commission member), Katarina Trebušak Podkrajšek (Thesis defence commission member), Borut Peterlin (Thesis defence commission member), Srdjan Novaković (Co-mentor)

Abstract

Izhodišča Fabryjeva bolezen (FB) je redka, na X-kromosom vezana dedna bolezen metabolizma maščob. Posledica genetske spremembe v GLA-genu je odsotna ali zmanjšana aktivnost lizosomskega encima ?-galaktozidaze A (?-Gal A). Posledično se v številnih celicah v telesu kopičijo glikosfingolipidi, predvsem globotriaosilceramid (Gb3) in njegov deacilirani toksični metabolit lyso-Gb3, kar privede do okvare številnih organov. Življenjska doba bolnikov je pomembno skrajšana, pri moških za približno 15 let in pri ženskah za približno 5 let. Vzrok umrljivosti so predvsem srčno-žilni zapleti. V literaturi obstajajo nasprotujoči si in pomanjkljivi podatki o prizadetosti srčno-žilnega sistema pri FB, še posebej glede prisotnosti ateroskleroze. Telomere so specializirane DNA-strukture, ki so locirane na koncih kromosomov in jih ščitijo pred poškodbami ter preprečujejo zlepljanje s sosednjimi kromosomi. Telomeraza je encim, ki ohranja dolžino telomere. Dolžina telomer v levkocitih (DTL) in telomerazna aktivnost (TA) se uporabljata kot označevalca »celičnega« staranja. Obstaja več dokazov o povezavi DTL in s starostjo povezanih boleznimi, kot so rak, ateroskleroza, sladkorna bolezen in srčno popuščanje. Glavni vzrok za krajšanje DTL pri kroničnih boleznih naj bi bila vnetje in oksidativni stres. Kljub temu da je za FB značilna prezgodnja smrt, možno prezgodnje in/ali pospešeno staranje pri FB še ni bilo preučeno. Pospešeno staranje so opisovali pri kroničnih boleznih s kroničnim vnetjem, oksidativnim stresom, disfunkcijo imunskega sistema, krajšanjem telomer in razvojem progresivne ledvične in srčne bolezni. Vsi ti dejavniki, razen krajšanje telomer, dokazano igrajo pomembno vlogo pri patofiziologiji in pri progresu FB. Namen in hipoteze naloge Dolžina telomer je dokazano krajša pri različnih stanjih, ki so povezana s celičnim stresom, vključno z oksidativnim stresom in vnetjem. Pri nezdravljenih bolnikih s FB kronična ledvična odpoved, srčno-žilni in možgansko-žilni zapleti povzročajo prezgodnjo umrljivost. Osrednji namen doktorskega dela je opredeliti srčno-žilno ogroženost bolnikov s FB in njeno povezavo z biološkimi označevalci vnetja, oksidativnega stresa in dolžinami telomer. Pri bolnikih s FB bi želeli opredeliti povezave teh parametrov glede na spol, zdravljenje in stopnjo bolezni, ki se najpogosteje izraža s stopnjo ledvične prizadetosti. S tem želimo preveriti naslednje hipoteze: 1. Dolžina telomer je pri bolnikih s FB krajša kot pri enako starih zdravih preiskovancih. 2. Bolniki s FB se razlikujejo od zdrave populacije po prizadetosti srca in ožilja, ocenjeni z neinvazivnimi preiskavami. 3. Prizadetost srca in ožilja in DTL se razlikujejo tudi med bolniki s FB glede na spol, zdravljenje z encimskim nadomestnim zdravilom (ENZ) in stopnjo bolezni, ki je izražena s stopnjo ledvične prizadetosti. Pričakujemo, da bomo z meritvami DTL dokazali starejšo biološko starost, kot pa je njihova kronološka. DTL bi lahko bila bioindikator srčno-žilnega tveganja in biološkega staranja v žilah pri FB. Z merjenjem DTL in TA, ki pri FB še nista bili proučeni, bi lahko skupaj s kronološkimi leti razložili interindividualne variacije v dovzetnosti za srčno-žilne bolezni pri FB. Z meritvami DTL bi lahko sklepali tudi na prognozo obolenja pri posameznem bolniku. Zasnova preiskave, opis metode in preiskovancev V raziskavo smo vključili 33 bolnikov s FB (13 moških in 20 žensk, povprečne starosti 44,6 let in 45,3 let) in 66 zdravih preiskovancev, ki so se ujemali glede na starost, spol, indeks telesne mase, kadilski status in prisotnost arterijske hipertenzije. Vsem smo opravili antropološke meritve, odvzeli kri za določitev osnovnih laboratorijskih preiskav krvi, seroloških markerjev vnetja in za določitev DTL in TA. Opravili smo ultrazvočne preiskave vratnih arterij, arterij okončin in srca, obremenitveno testiranje na kolesu. Funkcijsko stanje žil smo ocenili z merjenjem endotelne funkcije (FMD). Opisane parametre pri bolnikih s FB smo primerjali z zdravimi kontrolami. Poleg tega smo bolnike s FB primerjali tudi med seboj glede na spol, zdravljenje z ENZ in stopnjo bolezni oziroma ledvične prizadetosti. Rezultati Dolžina telomer in telomerazna aktivnost Bolniki s FB so imeli statistično krajšo (p = 0,015) povprečno DTL (0,68; IQR 0,58–0,81) kot zdrave kontrole (0,72; IQR 0,52–0,98). Ta razlika je bila prisotna le pri moških bolnikih (p = 0,02), ne pa pri ženskih bolnicah, kar bi lahko bila posledica dejstva, da so moški s FB v splošnem bolj prizadeti. TA je bila statistično pomembno višja pri bolnikih (1,55 ± 1,002) v primerjavi s kontrolno skupino (1,19 ± 0,104) (p = 0,047). A razlike so bile statistično pomembno (p = 0,005) višje vrednosti le pri bolnicah, medtem ko pri bolnikih razlike niso bile statistično pomembne. Med bolniki in bolnicami ni bilo statistično pomembnih razlik v DTL in TA. Ledvična prizadetost ni vplivala na DTL, TA pa je bila statistično pomembno (p = 0,003) znižana pri skupini bolnikov z napredovalo ledvično prizadetostjo. Zdravljenje z ENZ ni vplivalo na DTL in TA. DTL bolnikov s FB se je statistično pomembno zniževala s starostjo, a le pri moških bolnikih. DTL in TA nista bili povezani z nobenim izmed opazovanih srčno-žilnih ali vnetnih parametrov. TA je bila pri bolnikih s FB obratno povezana s starostjo. Morfološke in funkcijske preiskave srčno-žilnega sistema Bolniki so imeli statistično večje premere skupne karotidne arterije (ACC), notranje karotidne arterije (ACI) (p < 0,001) in premer bulbusa aorte (p = 0,001). IMT (debelina intima – medija) je bila zadebeljena pri večini merjenih arterij, pomembno pa le pri ACC (p = 0,021) v primerjavi s kontrolami. Aterosklerotične naplastitve v karotidnih arterijah smo našli le pri treh starejših ženskih bolnicah s FB, kar je bilo pomembno manj kot pri kontrolah (p < 0,001). FMD (merjenje od endotelija odvisne dilatacije) je bilo statistično nižje kot pri kontrolah (p = 0,001). Pri bolnikih je bila prisotna statistično pomembna hipertrofija srčne mišice in diastolična disfunkcija levega prekata. Sistolna funkcija je bila ohranjena. Parametri funkcijskih sposobnosti (maksimalna fizična obremenitev, delež maksimalne obremenitve, delež maksimalne srčne frekvence) so bili pri bolnikih pomembno znižani. Pri moških s FB so bile spremembe izrazitejše kot pri ženskah. Pri bolnikih z napredovalo ledvično prizadetostjo smo beležili pomembne razlike s povečanimi premeri v vseh pregledovanih žilah in s prisotnimi biokemičnimi, morfološkimi in funkcionalnimi znaki napredovale srčne bolezni. Bolniki so imeli statistično višjo plazemsko vsebnost naslednjih cirkulirajočih markerjev vnetja: TNF-?, IL-6, hsCRP, VCAM-1 in nižjo vsebnost E-selektina kot kontrole. Vrednosti teh parametrov so bile pri bolnikih višje kot pri bolnicah. Pri bolnikih z napredovalo ledvično prizadetostjo v primerjavi s tistimi z blago so bili vsi markerji vnetja povišani. Zaključki Naša raziskava je prva na področju redkih metabolnih bolezni, kjer smo dokazali, da imajo bolniki s FB pomembno krajšo DTL in višjo TA od zdrave populacije. Ob skrajšani DTL je bila povečana TA, vendar je bilo to povečanje statistično pomembno le pri bolnicah, ne pa tudi pri bolnikih, ki so bili bolezensko tudi bolj prizadeti. Na verjeten vpliv stopnje bolezni kaže tudi to, da so bile povišane vrednosti TA prisotne le pri bolnikih z blažjo obliko bolezni, medtem ko so bile pri bolnikih z napredovalo stopnjo bolezni vrednosti TA pomembno znižane. Ena od možnih razlag bi lahko bila, da je povečana TA zaščitni mehanizem pred skrajšanjem DTL, ki se ob napredovanju bolezni z leti izčrpa. Rezultati raziskave so pokazali tudi na razlike v izmerjenih vrednostih TA glede na stopnjo ledvične okvare. Ta raziskava je tudi prvi prikaz morfoloških in funkcionalnih značilnosti srčno-žilnega sistema pri bolnikih s FB v Sloveniji. Prizadetost srčno-žilnega sistema pri naših bolnikih s FB se kaže z razširjenimi premeri arterij, zadebeljeno IMT, okrnjeno endotelijsko funkcijo ob odsotnosti aterosklerotičnih plakov, s hipertrofijo levega prekata in diastolično disfunkcijo ter s povišano vsebnostjo TNF-?, IL-6, hsCRP, VCAM-1 in znižano vsebnostjo E-selektina. Večina teh sprememb je bila izrazitejša pri moških bolnikih in pri bolnikih z bolj napredovalo stopnjo ledvične prizadetosti. FB praviloma bolj prizadene moške kot ženske in tudi pri naših preiskovancih smo pričakovano ugotavljali pri moških bolj izraženo obliko bolezni. Glede na dokazane obsežne morfološke in funkcijske spremembe arterij (zlasti oslabljeno delovanje endotelija) bi pri bolnikih s FB pričakovali visoko stopnjo aterosklerotičnih sprememb, pri naših bolnikih pa aterosklerotični plaki niso bili prisotni. Možno je, da gre pri FB za drugačen fenotip prizadetosti ožilja in tudi ateroskleroze. Ker je znano, da gre pri napredovali obliki bolezni za obsežne ireverzibilne fibrotične spremembe tudi na ožilju, smo si razlagali, da te spremembe v intimi in medii onemogočajo nalaganje holesterola in tvorbo plakov. Vendar pa bodo za potrditev te hipoteze potrebne še nadaljnje raziskave, saj so ta opažanja tudi klinično zelo pomembna. Ugotovili smo, da so pri bolnikih s FB prisotne nekatere spremembe, ki kažejo znake prezgodnjega staranja: znaki celičnega staranja (znižan DTL), fenotip arterijskega (razširjene arterije z zadebeljeno steno in endotelno disfunkcijo) in srčnega staranja (hipertrofija levega prekata in diastolična disfunkcija) in znaki aktivacije patofizioloških mehanizmov staranja (aktivacija vnetja in oksidativnega stresa). V raziskavi smo ugotovili nekatere nove značilnosti FB, ki bi lahko bile koristne pri patofiziologiji in oceni napredovanja FB.

Keywords

interna medicina;Dedne bolezni;Disertacije;Fabryjeva bolezen;Motnje metabolizma maščob;Posledice;Patofiziologija;Dolžina telomer;

Data

Language:	Slovenian
Year of publishing:	2020
Typology:	2.08 - Doctoral Dissertation
Organization:	UL MF - Faculty of Medicine
Publisher:	[A. Cokan Vujkovac]
UDC:	616-056.7:577.125(043.3)
COBISS:	61155587
Views:	521
Downloads:	86
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	Vasculopathy and leukocyte telomere length in patients with Fabry disease
Secondary abstract:	Background Fabry disease (FD) is a rare, X-linked hereditary disease of lipid metabolism. The genetic change in the GLA gene results in the absence or reduced activity of the lysosomal enzyme α-galactosidase A (α-Gal A). As a result, glycosphingolipids, mainly globotriaosylceramide (Gb3) and its deacylated toxic metabolite lyso-Gb3, accumulate in many cells in the body, leading to damage of many organs. The lifespan of patients is significantly shortened, in men by about 15 years and in women by about 5 years. The cause of mortality is mainly cardiovascular complications. There are conflicting and deficient data in the literature on the involvement of the cardiovascular system in FD, especially regarding the presence of atherosclerosis. Telomeres are specialized DNA structures that are located at the ends of chromosomes and protect them from damage and prevent them from sticking to adjacent chromosomes. Telomerase is an enzyme that maintains the length of the telomere. Leukocyte telomere length (LTL) and telomerase activity (TA) are used as markers of “cellular” aging. There is more evidence of an association between LTL and age-related diseases, such as: cancer, atherosclerosis, diabetes, and heart failure. The main cause of LTL shortening in chronic diseases is thought to be inflammation and oxidative stress. Despite the fact that FD is characterized by premature death, the possible premature and / or accelerated aging in FD has not yet been studied. Accelerated aging has been described in chronic diseases with chronic inflammation, oxidative stress, immune system dysfunction, telomere shortening, and the development of progressive kidney and heart disease. All of these factors, except telomere shortening, have been shown to play an important role in pathophysiology and in FD progression. Aim and hypotheses of study Telomere length has been shown to be shorter in a variety of conditions associated with cellular stress, including oxidative stress and inflammation. In untreated patients with FD, chronic renal failure, cardiovascular and cerebrovascular complications cause premature mortality. The central purpose of the doctoral thesis is to define the cardiovascular risk of patients with FD and its association with biological markers of inflammation, oxidative stress, and telomere lengths. In patients with FD, we would like to define the links between these parameters according to gender, treatment, and the degree of disease most commonly expressed by the degree of renal impairment. With this we want to test the following hypotheses: 1. Telomere length is shorter in patients with FD than in healthy subjects of the same age. 2. Patients with FD differ from the healthy population in cardiovascular impairment assessed by noninvasive examinations. 3. Cardiovascular impairment and LTL also differ between patients with FD according to gender, enzyme replacement therapy (ERT) and the degree of disease expressed by the degree of renal impairment. We expect to use LTL measurements to prove an older biological age than their chronological one. LTL could be a biomarker of cardiovascular risk and biological vascular aging in FD. By measuring LTL and TA, which have not yet been studied in FD, we could, together with chronological years, explain interindividual variations in susceptibility to cardiovascular disease in FD. LTL measurements could also be used to infer the prognosis of the disease in an individual patient. Methods and subjects The study included 33 patients with FD (13 men and 20 women, mean ages 44.6 years and 45.3 years) and 66 healthy subjects matched by age, sex, body mass index, smoking status, and presence. arterial hypertension. We performed anthropological measurements on all of them, took blood to determine basic laboratory blood tests, serological markers of inflammation, and blood to determine LTL and TA. We performed ultrasound examinations of the carotid arteries, arteries of the extremities and heart in addition to stress testing on a bicycle. The functional status of the vessels was assessed by measuring endothelial function (FMD). The described parameters in Fabry patients were compared with healthy controls. In addition, patients with FD were also compared with each other according to gender, enzyme replacement therapy (ERT) and disease stage defined as the degree of renal impairment. Results Telomere length and telomerase activity Patients with FD had a statistically shorter (p = 0.015) mean LTL (0.68; IQR 0.58–0.81) than healthy controls (0.72; IQR 0.52–0.98). This difference was present only in male patients (p = 0.02) and not in female patients, which could be due to the fact that men with FD are generally more affected. TA was statistically significantly higher in patients (1.55 ± 1.002) compared to the control group (1.19 ± 0.104) (p = 0.047). However, the differences were statistically significant (p = 0.005) higher values only in patients, while the differences were not statistically significant in the patients. There were no statistically significant differences in LTL and TA between patients. Renal impairment did not affect LTL, and TA was statistically significantly (p = 0.003) reduced in the group of patients with advanced renal impairment. LTL and TA were not affected by ERT treatment. The LTL of patients with FD decreased statistically significantly with age, but only in male patients. LTL and TA were not associated with any of the observed cardiovascular or inflammatory parameters. TA was inversely related to age in patients with FD. Morphological and functional examinations of the cardiovascular system Patients had statistically larger common carotid artery (ACC), internal carotid artery (ACI) diameters (p <0.001), and aortic bulb diameter (p = 0.001). IMT (intima - media thickness) was thickened in most of the measured arteries, but important only in ACC (p = 0.021) compared to controls. Atherosclerotic deposits in the carotid arteries were found in only three elderly female patients with FD, which was significantly less than in controls (p <0.001). FMD (measurement of endothelium-dependent dilatation) was statistically lower than in controls (p = 0.001). Patients had statistically significant cardiac muscle hypertrophy and left ventricular diastolic dysfunction. Systolic function was preserved. Functional parameters (maximum physical activity, maximum load, maximum heart rate) were significantly reduced in patients. In men with FD, the changes were more pronounced than in women. In patients with advanced renal impairment, significant differences were noted with increased diameters in all examined vessels and biochemical, morphological, and functional signs of advanced heart disease present. Patients had statistically higher plasma levels of the following circulating markers of inflammation: TNF-α, IL-6, hsCRP, VCAM-1, and lower levels of E-selectin than controls. The values of these parameters were higher in patients than in female patients. All markers of inflammation were elevated in patients with advanced compared with those with mild renal impairment. Conclusions Our study is the first in the field of rare metabolic diseases, where we demonstrated that patients with FD have significantly shorter LTL and higher TA than the healthy population. With shortened LTL, TA was increased, but this increase was statistically significant only in female patients and not in male patients who were also more affected by the disease. The probable influence of the disease stage is also indicated by the fact that elevated TA values were present only in patients with a milder form of the disease, while in patients with more advanced disease the TA values were significantly reduced. One possible explanation could be that the increased TA is a protective mechanism against shortening of the LTL, which is depleted as the disease progresses over the years. The results of the study also showed differences in the measured TA values according to the degree of renal impairment. This study is also the first presentation of the morphological and functional characteristics of the cardiovascular system in patients with FD in Slovenia. Cardiovascular involvement in our patients with FD is manifested by dilated arterial diameters, thickened IMT, impaired endothelial function in the absence of atherosclerotic plaques, left ventricular hypertrophy and diastolic dysfunction, and elevated TNF-h, ILP 6 -1 and reduced E-selectin content. Most of these changes were more pronounced in male patients and in patients with a more advanced stage of renal impairment. As a rule, FD affects men more than women, and we also found a more pronounced form of the disease in men as expected in our subjects. Given the proven extensive morphological and functional changes of the arteries (especially impaired endothelial function), a high rate of atherosclerotic changes would be expected in patients with FD, while atherosclerotic plaques were not present in our patients. It is possible that FD has a different phenotype of vascular involvement as well as atherosclerosis. Since it is known that the advanced form of the disease involves extensive irreversible fibrotic changes also in the blood vessels, we explained that these changes in the intima and media prevent the accumulation of cholesterol and the formation of plaques. However, further research will be needed to confirm this hypothesis, as these observations are also clinically very important. We found that in patients with FD there are some changes that show signs of premature aging: signs of cellular aging (decreased LTL), arterial phenotype (dilated arteries with thickened wall and endothelial dysfunction) and cardiac (left ventricular hypertrophy and diastolic dysfunction) and signs of activation of pathophysiological mechanisms of aging (activation of inflammation and oxidative stress). The study identified some new features of FD that could be useful in the pathophysiology and assessment of FD progression.
Secondary keywords:	Dedne bolezni;Presnovne bolezni;Fabry disease;Physiopathology;Diagnosis;Therapy;Telomere;Telomerase;Biomarkers;Laboratory techniques and procedures;Ultrasonics;Exercise test;Chronic kidney disease-mineral and bone disorder;Atherosclerosis;Inflammation;Aging;Oxidative stress;Fabryjeva bolezen;Patofiziologija;Diagnostika;Terapija;Telomera;Telomeraza;Biološki označevalci;Laboratorijske tehnike in postopki;Ultrazvok;Obremenitveni test;Kronična ledvična bolezen-mineralna in kostna bolezen;Ateroskleroza;Vnetje;Staranje;Oksidativni stres;
Type (COBISS):	Dissertation
Study programme:	0
Embargo end date (OpenAIRE):	1970-01-01
Thesis comment:	Univ. v Ljubljani, Medicinska fak.
Pages:	XXI, 23-127 str.
ID:	12717727