The Impact of Coronavirus Disease 19 on the Retina and Retinal Microvessels

Znanstvena izhodišča Marca 2020 je bila razglašena pandemija koronavirusne bolezni 19 (COVID-19), ki jo povzroča virus SARS-CoV-2. Bolezen primarno prizadane respiratorni sistem, najpogosteje v obliki hude obojestranske pljučnice, v sklopu okužbe pa so lahko prizadeti številni organski sistemi (Guan et al., 2020). Virus SARS-CoV-2 v celico vstopa z vezavo na angiotenzin konvertazo 2 (ACE2), ki se nahaja na površini gostiteljevih celic (Hoffmann, Kleine-Weber et al. 2020). ACE2 je prisotna v številnih tkivih, vključno s pljuči, ledvicami, srcem, krvnimi žilami, veznico, roženico in mrežnico (de Figueiredo et al., 2020). V mrežnici je izražena v žilnem endotelu v Müllerjevih celicah in v nevronih notranje jedrne plasti (Piippo, Korhonen et al. 2018, Hoffmann, Kleine-Weber et al. 2020). Z vezavo na ACE2 SARS-CoV-2 zmanjša njeno aktivnost, kar vodi v kopičenje angiotenzina II v serumu in s tem vazokonstrikcijo, vnetje, fibrozo, endotelno disfunkcijo, mikrovaskularne tromboze in nastanek reaktivnih kisikovih spojin (ROS) (de Almeida-Pititto, Dualib et al. 2020). Izrazit vnetni odziv s hiperproduckijo vnetnih citokinov in kemokinov (npr. TNF- ?, IL-1ß in IL-6) lahko vodi v citokinsko nevihto in večorgansko prizadetost (Guan, Ni et al. 2020). Predpostavlja se, da kombinacija izrazitega vnetnega odgovora in okvare endotela povzroča hiperkoaguabilno stanje, ki bolnike predisponira za trombembolične zaplete, ki bi se lahko odražali tudi na mrežnici. Disregulacija sistema renin-angiotenzin-aldosteron in s tem spremenjeno razmerje med ACE in ACE2 naj bi vplivalo na potek, resnost ter izid COVID-19. Zato bi lahko s polimorfizmi genov za ACE, ACE2 in AGTR2 pojasnili, zakaj imajo nekateri posamezniki brez drugih pridruženih bolezni hujši potek COVID-19 (Asselta, Paraboschi et al. 2020, Devaux, Rolain et al. 2020, Hou, Zhao et al. 2020, Zheng and Cao 2020, Abobaker, Nagib et al. 2021). Polimorfizem rs4646994 gena za ACE je funkcionalni polimorfizem, ki ga najdemo na intronu 16 v obliki insercije in/ali delecije (I/D) 289 baznih parov (Alu sekvence) (Zmorzynski, Szudy-Szczyrek et al. 2019). Za raziskovanje tega polimorfizma smo se odločili, ker izsledki dosedanjih raziskav kažejo, da naj bi vplival na resnost bolezni, medtem ko naj bi imeli nosilci genotipa D/D večje tveganje prizadetosti pljuč in nastanka pljučne embolije v sklopu okužbe s SARS-CoV-2 v evropski populaciji (Howe, Achuthan et al. 2020). Polimorfizem rs2285666 (8790 G/A) na genu za ACE2 se nahaja na začetku introna 3. Čeprav v dosedanjih raziskavah niso uspeli dokazati povezave med prisotnostjo polimorfizma rs2285666 in težjim potekom COVID-19, smo ga zaradi udeleženosti ACE2 v patogenezi COVID-19 vključili v raziskavo. Polimorfizem rs1403543 v genu za AGTR2 (1332A/G) se nahaja na intronu 1. Polimorfizem rs1403543 smo izbrali, ker so v objavljenih raziskavah predpostavljali, da bi lahko bil povezan z večjim premerom arteriol v mrežnici (Liu, Kuznetsova et al. 2011). Namen V raziskavi smo z uporabo optične koherentne tomografije (OCT) in OCT angiografije (OCTA) želeli ugotoviti, ali virus SARS-CoV-2 prizadene mrežnico pri bolnikih s COVID-19 in brez pridruženih bolezni, ki bi lahko vplivale na mrežnico in mrežnično mikrožilje. Želeli smo raziskati tudi vpliv izbranih polimorfizmov v genih anigotenzin konvertaze (ACE), anigotenzin konvertaze 2 (ACE2) in angiotenzin II receptorja tipa 2 (AGTR2) ter njihovo povezavo s prisotnostjo mrežničnih sprememb. Metode V raziskavo smo vključili bolnike v akutni fazi COVID-19, hospitalizirane v diagnostično-terapevtskem centru UKC Ljubljana, in brez pridruženih bolezni, ki bi se lahko odražale tudi na mrežnici. Primerjali smo jih s kontrolno skupino zdravih, po starosti ujemajočih se preiskovancev. Bolnike s COVID-19 smo glede na potek bolezni v skladu z uveljavljeno klasifikacijo bolezni razdelili v dve skupini (tj. blažji oz. težji potek). V skupino z blažjim potekom smo uvrstili asimptomatske bolnike in bolnike z blago obliko bolezni, ki so imeli simptome in znake COVID-19 brez kratke sape, dispneje ali nenormalne slikovne preiskave pljuč. V skupino s težjim potekom smo uvrstili bolnike, ki so imeli glede na klinično oceno ali slikovne preiskave okužbo spodnjih dihal in so potrebovali zdravljenje s kisikom. Iz dokumentacije bolnikov smo povzeli demografske podatke, laboratorijske izvide in potek zdravljenja. V prvem delu raziskave smo z uporabo multimodalnih slikovnih metod (fotografije očesnega ozadja, OCT, OCTA, Topcon DRI OCT Triton, Topcon, Inc, Tokyo, Japonska) opredelili vpliv COVID-19 na mrežnico in na mrežnično mikrožilje. V analizo smo vključili oko z boljšim indeksom kakovosti slike. Na fotografijah očesnega ozadja smo ocenjevali prisotnost mrežničnih krvavitev, mehkih eksudatov ter razširjenih in zvijuganih mrežničnih žil. Premer mrežničnih žil smo analizirali s prej opisano metodo (Invernizzi et al., 2020), pri čemer smo uporabili programsko opremo ARIA (angl. Automated Retinal Image Analyser, ARIA, V1-09-12-11), dostopno na odprtokodni platformi MATLAB (MathWorks Inc., Natick, MA, ZDA). Za izračun srednjega premera ven (angl. mean vein diameter, MVD) in srednjega premera arterij (angl. mean artery diameter, MAD) smo uporabili premere žil štirih glavnih ven in štirih glavnih arterij. Slike OCT in OCTA so bile samodejno segmentirane z vgrajeno programsko opremo (Topcon Corp., Tokio, Japonska). Na slikah smo ocenjevali pravilnost segmentacije in jo po potrebi ročno prilagodili. Debelino sloja RNFL in sloja GCL smo ocenjevali v štirih kvadrantih: zgornjem kvadrantu (angl. superior, S), spodnjem kvadrantu (angl. inferior, I), nazalnem kvadrantu (angl. nasal, N) in temporalnem kvadrantu (angl. temporal, T) notranjega (3 mm) in zunanjega obroča (6 mm) mreže zgodnjega zdravljenja diabetične retinopatije (angl. early treatment diabetic retinopathy study, ETDRS). Slike povrhnjega kapilarnega pleteža (angl. superficial capillary plexus, SCP) in globokega kapilarnega pleteža (angl. deep capillary plexus, DCP) ter fovealne avaskularne cone (angl. foveal avascular zone, FAZ) smo analizirali s pomočjo odprtokodne platforme MATLAB (MathWorks Inc., Natick, MA, ZDA) z uporabo validirane, predhodno objavljene metode (Díaz et al., 2019). Najprej smo določili središče FAZ in nato na središče FAZ superponirali krog ETDRS ter izračunali gostoto kapilarnega žilja v štirih kvadrantih znotraj 3 mm kroga okoli središča FAZ. Vsak kvadrant smo ločeno binarizirali z metodo Otsu (Nicolo et al., 2017). Gostoto kapilarnega žilja smo izrazili v odstotkih, izpeljanih iz razmerja med celotno površino žilja (bele slikovne pike) in celotno površino analizirane regije (število slikovnih pik v kvadrantu) z metodo, ki so jo opisali Nicoló et al. (Nicolo et al., 2017). Pri kvantitativni analizi smo uporabili povprečne gostote žil SCP in DCP. V drugem delu raziskave smo z multimodalnimi slikovnimi metodami opredelili vpliv dolgotrajnih posledic COVID-19 na mrežnico in na mrežnično mikrožilje, pri čemer smo primerjali mrežnične spremembe v akutni fazi COVID-19 ter eno leto po odpustu iz bolnišnice ter slike analizirali na opisani način. V tretjem delu raziskave smo analizirali vpliv izbranih polimorfizmov v genih za ACE (rs4646994), ACE2 (rs2285666) in AGTR2 (rs1403543) na resnost poteka COVID-19 in njihovo povezavo s prisotnostjo COVID-19 retinopatije. DNK smo izolirali v Raziskovalnem laboratoriju za histologijo in genetiko ateroskleroze in mikrovaskularnih bolezni Inštituta za histologijo in embriologijo Medicinske fakultete Univerze v Ljubljani. Postopek izolacije DNK iz 200 µl krvi smo opravili s kompletom QIAamp DNA Blood Mini Kit (250) (Qiagen GmbH, Hilden, Nemčija). Izbrane polimorfizme smo določili z metodo verižne reakcije s polimerazo (PCR) v realnem času. Statistično analizo smo opravili s programom R statistical software (version 4.1.3, R Development Core Team, Vienna, Austria). Uporabili smo ustrezno korekcijo za multiplo testiranje (Benjamini-Hochbergov postopek) in kot statistično značilno upoštevali vrednost p, manjšo od 0,05 (p < 0,05). Rezultati V prvi del raziskave smo vključili 75 bolnikov v akutni fazi COVID-19 brez pridruženih bolezni ter jih primerjali s 101 preiskovancem iz starostno primerljive kontrolne skupine. Na slikah očesnega ozadja so bile vidne sledeče spremembe: mrežnične krvavitve pri 5 bolnikih (8,5 %), mehki eksudati pri 3 bolnikih (4,0 %) in širše vene pri 50 bolnikih (66,6 %) bolnikih. Mrežnične spremembe so bile bolj pogoste v skupini bolnikov s hudo boleznijo (p = 0,006). Bolniki s težjim potekom bolezni so imeli tudi statistično značilno povečan premer mrežničnih ven (Coef. = 19,2895% CI: 7,34–31,23p = 0,002) in mrežničnih arterij (Coef. = 11,0795% CI: 0,84–21,67p = 0,044). Debelina žilja ni bila povezana z nobeno od spremenljivk (starost, spol, zdravljenje s kisikom, LDH, feritin). Bolniki s težjim potekom bolezni so imeli z OCT izmerjen značilno debelejši sloj živčnih vlaken (angl. retinal nerve fiber layer, RNFL) v zgornjem in spodnjem kvadrantu parafoveolarno (S: p = 0,046I: p = 0,016) in perifoveolarno (S: p = 0,026I: p = 0,014). Ob tem so imeli tudi značilno debelejši sloj gangljiskih celic (angl. ganglion cell layer, GCL) v zunanjem temporalnem kvadrantu (p = 0,038). Med skupinama bolnikov s COVID-19 ter zdravimi preiskovanci ni bilo razlik v gostoti povrhnjenga ali globokega kapilarnega pleteža, merjenih z OCTA. Poleg tega tudi ni bilo razlik v velikosti ali obliki fovealne avaskularne cone. V drugi del raziskave smo vključili 30 bolnikov v akutni fazi COVID-19, v starosti 18–65 let, brez znanih pridruženih sistemskih bolezni. Z uporabo multimodalnih slikovnih metod (fotografije očesnega ozadja, OCT in OCTA, Topcon DRI OCT Triton, Topcon, Inc, Tokyo, Japonska) smo primerjali mrežnične spremembe v akutni fazi COVID-19 ter eno leto po odpustu iz bolnišnice. Povprečni premer ven se je po enoletnem spremljanju s 134,8 µm v akutni fazi zmanjšal na 112,4 µm (p < 0,001). Leto kasneje je prišlo tudi do značilnega stanjšanja RNFL v spodnjem kvadrantu notranjega ETDRS obroča (p = 0,047) ter v spodnjem kvadrantu (p < 0,001), nazalnem kvadrantu (p < 0,001) in zgornjem kvadrantu (p < 0,001) zunanjega ETDRS obroča. Med skupinama ni bilo statistično značilnih razlik v gostoti žil povrhnjega kapilarnega pleteža in globokega kapilarnega pleteža. V tretji del raziskave smo vključili 69 hospitaliziranih bolnikov in jih primerjali s 96 zdravimi kontrolami. Skupino bolnikov s COVID-19 smo glede na potek bolezni razdelili v skupino z blažjim potekom bolezni (N = 12) in v skupino s težjim potekom bolezni (N = 57). Bolnike smo glede na prisotnost COVID-19 retinopatije razdelili v dve skupini (Da: N = 50 in Ne: N = 19). Prisotnost genotipa AGTR2 rs1403543-AA je bila pri moških povezana s 3,8-krat večjim tveganjem COVID-19 retinopatije (p = 0,05). Frekvence genotipov v ostalih izbranih polimorfizmih niso bile povezane z resnostjo poteka COVID-19 ali prisotnostjo COVID-19 retinopatije. Zaključek Z raziskavo smo dokazali, da so mrežnične spremembe pogostejše pri bolnikih s težjim potekom COVID-19. Razširitev mrežničnih žilter zadebelitev mrežničnih slojev RNFL in GCL v akutni fazi COVID-19 bi bili lahko biomarkerji sistemske angiopatije pri bolnikih s težjim potekom COVID-19. Z analizo povezave mrežničnih sprememb z izbranimi polimorfizmi smo dokazali, da je tveganje za prizadetost mrežnice večje pri moških z genotipom AGTR2 rs1403543-AA. Prisotnost genotipa AGTR2 rs1403543-AA pri moških bi bila lahko torej genetski dejavnik tveganja za pojav COVID-19 retinopatije. Na podlagi rezultatov raziskave smo torej prepoznali štiri biomarkerje: i) razširitev mrežničnih žil, ii) debelejši sloj RNFL, iii) debelejši sloj GCL in iv) prisotnost genotipa AGTR2 rs1403543-AA pri moških. Z njimi bi v akutni fazi COVID-19 lahko napovedali težji potek bolezni in potencialno večje tveganje sistemske angiopatije. Za oceno morebitnih dolgoročnih zapletov bodo potrebne nadaljnje longitudinalne raziskave.Background Coronavirus disease 19 (COVID-19) caused by the SARS-CoV-2 virus primarily affects the respiratory system, most often in the form of severe bilateral pneumonia, but many organ systems can be affected (Guan et al., 2020). The SARS-CoV-2 virus enters the cell by binding to angiotensin convertase 2 (ACE2), which is located on the surface of host cells (Hoffmann, Kleine-Weber et al. 2020). ACE2 is present in many tissues, including the lung, kidney, heart, blood vessels, conjunctiva, cornea, and retina (de Figueiredo et al., 2020). In the retina, it is expressed in the vascular endothelium in Müller cells and in neurons of the inner nuclear layer (Piippo, Korhonen et al. 2018, Hoffmann, Kleine-Weber et al. 2020). By binding to ACE2, SARS-CoV-2 reduces its activity, which leads to the accumulation of angiotensin II in the serum and thus vasoconstriction, inflammation, fibrosis, endothelial dysfunction, microvascular thrombosis and the formation of reactive oxygen species (de Almeida-Pititto, Dualib et al. 2020). A pronounced inflammatory response with hyperproduction of inflammatory cytokines and chemokines (e.g. TNF-α, IL-1β and IL-6) can lead to a cytokine storm and multi-organ involvement (Guan, Ni et al. 2020). It is assumed that the combination of a pronounced inflammatory response and endothelial damage causes a hypercoagulable state that predisposes patients to thromboembolic complications, which could also be reflected in the retina. Dysregulation of the renin-angiotensin-aldosterone system and, thus the altered ratio between ACE and ACE2 are thought to influence the course, severity, and outcome of COVID-19. Therefore, ACE, ACE2, and AGTR2 gene polymorphisms could explain why some individuals have a more severe course of COVID-19 (Asselta, Paraboschi et al. 2020, Devaux, Rolain et al. 2020, Hou, Zhao et al. 2020, Zheng and Cao 2020, Abobaker, Nagib et al. 2021). The rs4646994 polymorphism of the ACE gene is a functional polymorphism found on intron 16 in the form of an insertion and/or deletion (I/D) of 289 base pairs (Alu sequences) (Zmorzynski, Szudy-Szczyrek et al. 2019). We decided to investigate this polymorphism because the results of previous research show that it could influence the severity of the disease, while carriers of the D/D genotype are said to have a higher risk of lung involvement and pulmonary embolism as part of SARS-CoV-2 infection in the European population (Howe, Achuthan et al. 2020). The rs2285666 polymorphism (8790 G/A) on the ACE2 gene is located at the beginning of intron 3. Although previous studies have not been able to demonstrate a connection between the presence of the rs2285666 polymorphism and a more severe course of COVID-19, we decided to investigate because of its presumed involvement in the pathogenesis of COVID-19. The rs1403543 polymorphism in the AGTR2 gene (1332A/G) is located in intron 1. We chose the rs1403543 polymorphism because previous studies have shown a possible association with a larger diameter of arterioles in the retina (Liu, Kuznetsova et al. 2011). Purpose The aim of this dissertation was to determine the effect of SARS-CoV-2 on the retina in patients with COVID-19, without associated diseases, using optical coherence tomography (OCT) and OCT angiography (OCTA). In addition, we aimed to investigate the influence of selected polymorphisms in the ACE, ACE2, and AGTR2 genes and their association with the presence of retinal findings. Methods Patients in the acute phase of COVID-19, hospitalized in the COVID-19 unit of the University medical center Ljubljana, and without associated diseases that could also be reflected on the retina were included in the study. We compared them with a control group of healthy, age-matched subjects. Patients with COVID-19 were divided into two groups according to the course of the disease in accordance with the established classification of the disease (i.e. milder and severe course). Asymptomatic patients and patients with a mild form of the disease who had symptoms and signs of COVID-19 without shortness of breath, dyspnea or abnormal lung imaging were included in the mild course group. The group with a severe course included patients who, according to clinical assessment or imaging tests, had a lower respiratory tract infection and needed oxygen treatment. Demographic data, laboratory results, and the course of treatment were obtained from the patient\u27s documentation. In the first part of the study, we used multimodal imaging methods (photographs of the fundus of the eye, OCT, OCTA, Topcon DRI OCT Triton, Topcon, Inc, Tokyo, Japan) to determine the impact of COVID-19 on the retina and retinal microvessels. The eye with a better image quality index was included in the analysis. We evaluated the fundus phtographs for the presence of retinal hemorrhages, soft exudates, and dilated and tortuous retinal vessels on photographs of the fundus of the eye. The diameter of the retinal vessels was analyzed using the previously described method (Invernizzi et al., 2020), using the ARIA software (Automated Retinal Image Analyser, ARIA, V1-09-12-11), available on the open source platform MATLAB ( MathWorks Inc., Natick, MA, USA). To calculate the mean vein diameter (MVD) and the mean artery diameter (MAD), we used the diameters of the veins of the four main veins and the four main arteries. OCT and OCTA images were automatically segmented with built-in software (Topcon Corp., Tokyo, Japan). We evaluated the correctness of the segmentation in the images and adjusted it manually if necessary. The thickness of the RNFL layer and the GCL layer was evaluated in four quadrants: the upper quadrant (S), the lower quadrant (I), the nasal quadrant (N) and the temporal quadrant (T ) of the inner (3 mm) and outer ring (6 mm) of the early treatment diabetic retinopathy study (ETDRS). Images of the superficial capillary plexus (SCP) and deep capillary plexus (DCP) as well as the foveal avascular zone (FAZ) were analyzed using the open-source MATLAB platform (MathWorks Inc. , Natick, MA, USA) using a validated, previously published method (Díaz et al., 2019). We first determined the center of the FAZ and then superimposed the ETDRS circle on the center of the FAZ and calculated the capillary vessel density in the four quadrants within a 3 mm circle around the center of the FAZ. Each quadrant was binarized separately using the Otsu method (Nicolò et al., 2017). The density of the capillary vessel was expressed in percentages derived from the ratio between the total area of the vessel (white pixels) and the total area of the analyzed region (number of pixels in the quadrant) using the method described by Nicoló et al. (Nicolò et al., 2017). In the quantitative analysis, we used the mean densities of SCP and DCP veins. In the second part of the research, we used multimodal imaging methods to determine the impact of the long-term consequences of COVID-19 on the retina and retinal microvessels, comparing the retinal changes in the acute phase of COVID-19 and one year after discharge from the hospital, and analyzing the images as described. In the third part of the research, we analyzed the influence of selected polymorphisms in the genes for ACE (rs4646994), ACE2 (rs2285666) and AGTR2 (rs1403543) on the severity of the course of COVID-19 and their association with the presence of COVID-19 retinopathy. DNA was isolated in the Research Laboratory for Histology and Genetics of Atherosclerosis and Microvascular Diseases of the Institute of Histology and Embryology, Faculty of Medicine, University of Ljubljana. DNA isolation from 200 μl of blood was performed using the QIAamp DNA Blood Mini Kit (250) (Qiagen GmbH, Hilden, Germany). Selected polymorphisms were determined using the polymerase chain reaction (PCR) method in real-time. Statistical analysis was performed with R statistical software (version 4.1.3, R Development Core Team, Vienna, Austria). We applied the appropriate correction for multiple testing (Benjamini-Hochberg procedure) and considered a p-value of less than 0.05 (p < 0.05) as statistically significant. Results In the first part of the study, we included 75 patients in the acute phase of COVID-19 without associated diseases and compared them with 101 subjects from an age-matched control group. The following changes were seen on fundus images: retinal hemorrhages in 5 patients (8.5%), soft exudates in 3 patients (4.0%), and dilated veins in 50 patients (66.6%) patients. Retinal changes were more frequent in the group of patients with severe disease (p = 0.006). Patients with a more severe course of the disease also had a statistically significantly increased diameter of retinal veins (Coef. = 19.2895% CI: 7.34–31.23p = 0.002) and retinal arteries (Coef. = 11.0795 %CI: 0.84–21.67p = 0.044). Vascular thickness was not associated with any of the variables (age, gender, oxygen therapy, LDH, ferritin). Patients with a more severe course of the disease had a significantly thicker retinal nerve fiber layer (RNFL) measured by OCT in the upper and lower quadrants, parafoveolarly (S: p = 0.046I: p = 0.016) and perifoveolarly (S: p = 0.026I: p = 0.014). In addition, they also had a significantly thicker ganglion cell layer in the outer temporal quadrant (p = 0.038). There were no differences in the density of the superficial or deep capillary plexus measured by OCTA between the groups of patients with COVID-19 and healthy subjects. In addition, there were no differences in the size or shape of the foveal avascular zone. In the second part of the research, we included 30 patients in the acute phase of COVID-19, aged 18-65 years, without known associated systemic diseases. Using multimodal imaging methods (photographs of the fundus of the eye, OCT and OCTA, Topcon DRI OCT Trit