Characterization of physiological characteristics and pathophysiological role of mechanosensitive channels in corneal epithelium

Namen: Roženični epitel je površinski roženični sloj, ki ščiti roženico pred zunanjim okoljem in je podvržen številnim mehaničnim, kemičnim, biološkim in fizikalnim dejavnikom. Nekateri izmed omenjenih dejavnikov so znani aktivatorji ali modulatorji mehanosenzitivnih kationskih kanalov, med katere prištevamo receptor prehodnega potenciala vaniloida tipa 4 (TRPV4) in Piezo1 kanale. Aktivacija mehanoreceptorjev vodi v aktivacijo znotrajceličnih poti in procesov, ki so povezani z motnjami v delovanju roženice, vnetjem, hiperalgezijo/nocicepcijo in disfunkcijo tkiva. V doktorski disertaciji smo se zato osredotočili na identifikacijo in lokalizacijo mehanosenzitivnih kationskih ionskih kanalov v celicah roženičnega epitela, ovrednotili njihov funkcionalni pomen in opredelili vlogo TRPV4 kanalov v parakrinem signaliziranju ter izločanju vnetnih in bolečinskih mediatorjev. Metode: Roženicam miši linije C57BL/6, transgene miši z izničenim genom za TRPV4 (TRPV4-/-) in transgene miši, ki smo jim gene za TRPV4 zamenjali s fluorescenčno označenimi bakterijskimi geni, ki kodirajo TRPV4 (TRPV4eGFP), smo encimsko odluščili sloj roženičnega epitela in jih uporabili za in situ poskuse. Celice roženičnega epitela, ki smo jih uporabili za in vitro poskuse, smo disociirali, nasadili na silikonske membrane ali objektna stekelca in jih gojili v inkubatorju 4–7 dni pri 37 °C. Celice roženičnega epitela smo izpostavili različnim specifičnim agonistom in antagonistom, cikličnemu vzdolžnemu mehaničnemu raztezku in ultravijoličnemu B (UVB) sevanju. S pomočjo optičnega slikanja smo ovrednotili njihov vpliv na homeostazo kalcijevih ionov (Ca2+) v različnih eksperimentalnih pogojih. Za določitev izražanja genov za izbrane proteine in njihovo lokalizacijo smo uporabili tehniki imunooznačevanja in semikvantitativne verižne reakcije s polimerazo v realnem času (qRT-PCR). Izločanje ATP iz celic roženičnega epitela po izpostavitvi različnim dražljajem smo v supernatantu kvantificirali z luciferin-luciferazno reakcijo, izločanje vnetnih molekul in citokinov pa s pomočjo kemiluminiscence. Rezultati: Celice roženičnega epitela močno izražajo TRPV4 in Piezo1 kanale. Prvi se v roženičnem epitelu pojavljajo v neenakomernem, drugi pa v bolj enakomernem vzorcu glede na sklade celic roženičnega epitela. Skladno s tem sta specifična agonista GSK101 in YODA1 povzročila povečanje koncentracije prostih znotrajceličnih kalcijevih ionov ([Ca2+]i) v celicah roženičnega epitela. Signali Ca2+ so bili zmanjšani po izpostavitvi mehanskemu hipotoničnemu stresu ob uporabi specifičnega TRPV4 antagonista (HC-06) in po izpostavitvi mehanskemu raztezku ob uporabi specifičnega Piezo1 antagonista (GsMTx4). Izpostavitev temperaturi 37 °C je v celicah roženičnega epitela vodila v od TRPV4 kanalov odvisno povečanje [Ca2+]i. Aktivaciji TRPV4 kanalov je sledilo od Ca2+ odvisno izločanje ATP, ki je bilo zavrto ob farmakološki blokadi paneksinskih hemikanalov s probenecidom. Farmakološka aktivacija proteazno aktiviranih receptorjev 2 (PAR-2) je senzibilizirala TPRV4 kanale za aktivacijo z agonistom. UVB sevanje je v celicah roženičnega epitela vodilo do reverzibilnega povečanja [Ca2+]i, učinek je bil delno zmanjšan ob farmakološki blokadi TRPV4 kanalov ali njihovi genetski odstranitvi. Povečanje [Ca2+]i ob stimulaciji z UVB sevanjem je bilo primarno posledica sprostitve Ca2+ iz znotrajceličnih rezerv, predvsem iz endoplazemskega retikuluma. Tako farmakološka aktivacija TRPV4 kanalov kot UVB sevanje sta v celicah roženičnega epitela povzročila povečano izločanje nekaterih provnetnih citokinov in vnetnih mediatorjev, ki je bilo specifično glede na dražljaj in izničeno ob farmakološki blokadi TRPV4 kanalov. Zaključki: Naši rezultati kažejo, da je pri mehanotransdukciji v celicah roženičnega epitela udeleženih več različnih mehanoreceptorjev. Eni izmed najpomembnejših mehanoreceptorjev so TRPV4 kanali, ki so aktivirani z osmotskim stresom. Preko homeostaze znotrajceličnega Ca2+ so povezani z od Ca2+ odvisnim izločanjem ATP iz celic preko paneksinskih hemikanalov in so tako vključeni v purinergično avtosignalizacijo. V celicah roženičnega epitela TRPV4 kanali niso povezani z zaznavanjem mehanskega raztezka, ki ga zaznavajo Piezo1 kanali. TRPV4 kanale aktivirata tudi povečana temperatura in UVB sevanje, senzibilizira pa jih aktivacija PAR-2 receptorjev. Na škodljivo UVB sevanje se celice roženičnega epitela odzovejo z aktivacijo TRPV4 kanalov in sprostitvijo Ca2+ iz znotrajceličnih zalog. Molekule, ki se nahajajo v solzah, lahko prispevajo k od PAR-2 receptorjev odvisni regulaciji TRPV4 kanalov in tako posredno, tudi preko TRPV4 kanalov, vplivajo na delovanje roženičnega epitela v patoloških pogojih. Na roženično okolje najverjetneje vplivajo tudi vnetne molekule, sproščene iz celic roženičnega epitela ob aktivaciji TRPV4 kanalov oziroma ob izpostavitvi UVB sevanju. Vnetne molekule imajo lahko učinek na nocicepcijo, celjenje in prozornost roženice. V splošnem ta študija opredeljuje Piezo1 kanale kot pomembne mehanoreceptorje in TRPV4 kanale kot potencialno novo tarčo pri procesih, povezanih z vnetjem očesne površine in tkivno poškodbo v celicah roženičnega epitela.Purpose: Corneal epithelium – the outermost corneal layer that protects the cornea from the environment – is subjected to various mechanical, biological, chemical, and physical stressors. Many are known activators or modulators of mechanosensitive cation channels. Mechanoreceptors\u27 activation can lead to downstream pathways that are linked to impaired corneal physiology, inflammation, hyperalgesia/nociception, and tissue dysfunction. We localised mechanotransducing cation permeable TRPV4 and Piezo1 ion channels to corneal epithelial cells, characterized their functional properties and assessed their functions in paracrine signalling and the release of inflammatory and nociceptive transmitters. Methods: Corneal epithelia from C57BL/6, TRPV4-/- and TRPV4eGFP mice were enzymatically detached from the stroma and used for in situ experiments. In vitro, epithelial cells were dissociated, plated onto silicon membranes or cover slips, and cultured for 4-7 days. Cells were subjected to either assorted agonists and antagonists, cyclic equiaxial mechanical stretch, or UVB radiation. Optical imaging assessed the properties of Ca2+ homeostasis under different experimental conditions. Immunolabeling and qRT-PCR were performed to determine the changes in gene and protein expression, and localization. For ATP and cytokine release assays, cells were dissociated, subjected to assorted stimuli and supernatants were evaluated by luciferin-luciferase and chemiluminescence reaction, respectively. Results: Corneal epithelial cells strongly express TRPV4 and Piezo1 channels, the former being localized in a non-uniform and the latter in a uniform pattern. Accordingly, the selective agonists GSK101 and YODA1 increased [Ca2+]i in corneal epithelial cells, whereas swelling and pressure induced Ca2+ signals were averted by the selective TRPV4 (HC-06) and Piezo1 inhibitor (GsMTx4), respectively. Heating of corneal epithelium to 37°C evoked TRPV4 dependent [Ca2+]i elevations. The epithelial cells responded to TRPV4 activation with massive release of ATP that was sensitive to the pannexin 1 blocker probenecid and showed Ca2+ dependence. Pharmacological activation of the protease-activated receptor-2 (PAR-2) sensitized TRPV4 for agonist activation. UVB radiation reversibly elevated [Ca2+]i, principally reflected release of Ca2+ from the pool stored intracellularly within the endoplasmic reticulum and was modestly attenuated by TRPV4 ablation or inhibition. UVB radiation and TRPV4 activation evoked stimulus-specific release of proinflammatory cytokines and inflammatory substances, effects that were antagonized by pharmacological blockade of TRPV4. Conclusions: We found that corneal mechanotransduction involves activation of multiple force-activated channels which regulate fundamental aspects of corneal epithelial biology. A principal mechanosensor is TRPV4, which is activated by osmotic stress and contributes to [Ca2+]i homeostasis through Ca2+-dependent release of ATP, purinergic auto-feedback and hemichannel (pannexin) activation. Interestingly, TRPV4 fails to mediate stretch-evoked Ca2+ responses, which are instead mediated by another mechanochannel, Piezo1. In addition to osmotic gradients, TRPV4 channels in the corneal epithelium are activated by moderate heat, UVB radiation and sensitized by the PAR-2 receptor. Our data suggest that corneal epithelial cells respond to harmful UVB radiation with parallel and additive activation of TRPV4 channels and release of Ca2+ from intracellular stores. It is possible that substances residing in tears contribute to PAR-2-mediated TRPV4 regulation in corneal epithelial cells under pathological conditions. In addition, the corneal milieu is likely to be influenced by TRPV4- and/or UVB-dependent release of cytokines and inflammatory substances, with potential effects on transparency, wound healing, and nociception. Overall, this study identifies Piezo1 as an important corneal epithelial mechanosensor that may be triggered by acute impact and TRPV4 as a potential target of ocular surface inflammation, UVB impact and tissue damage

The Comparison of Retinal Microvascular Findings in Acute COVID-19 and 1-Year after Hospital Discharge Assessed with Multimodal Imaging—A Prospective Longitudinal Cohort Study

This study aimed to quantify possible long-term impairment of the retinal microcirculation and microvasculature by reassessing a cohort of patients with acute COVID-19 without other known comorbidities one year after their discharge from the hospital. Thirty patients in the acute phase of COVID-19 without known systemic comorbidities were enrolled in this prospective longitudinal cohort study. Fundus photography, SS-OCT, and SS-OCTA using swept-source OCT (SS-OCT, Topcon DRI OCT Triton; Topcon Corp., Tokyo, Japan) were performed in the COVID-19 unit and 1-year after hospital discharge. The cohort’s median age was 60 years (range 28–65) and 18 (60%) were male. Mean vein diameter (MVD) significantly decreased over time, from 134.8 μm in the acute phase to 112.4 μm at a 1-year follow-up (p p = 0.047) and inferior (mean diff. 1.56 95% CI 0.50–2.61, p p p < 0.001) quadrants of the outer ring. There were no statistically significant differences between the groups regarding vessel density of the superior and deep capillary plexuses. The transient dilatation of the retinal vessels in the acute phase of COVID-19, as well as RNFL thickness changes, could become a biomarker of angiopathy in patients with severe COVID-19

The Role of ACE, ACE2, and AGTR2 Polymorphisms in COVID-19 Severity and the Presence of COVID-19-Related Retinopathy

The proposed SARS-CoV-2-induced dysregulation of the renin-angiotensin-aldosterone (RAAS) system results in endothelial dysfunction and microvascular thrombosis. The retinal plexuses contain terminal vessels without anastomotic connections, making the retina especially susceptible to ischemia. This study aimed to determine the role of selected polymorphisms of genes in the RAAS pathway in COVID-19 severity and their association with the presence of COVID-19 retinopathy. 69 hospitalized patients in the acute phase of COVID-19 without known systemic comorbidities and 96 healthy controls were enrolled in this prospective cross-sectional study. The retina was assessed with fundus photography using a Topcon DRI OCT Triton (Topcon Corp., Tokyo, Japan) in the COVID-19 unit. Genotyping of selected polymorphisms in the genes for ACE (rs4646994), ACE2 (rs2285666), and AGTR2 (rs1403543) was performed. The COVID-19 group was divided into mild (n = 12) and severe (n = 57), and then further divided according to the presence of COVID-19 retinopathy (Yes, n = 50; No, n = 19). The presence of the AGTR2 rs1403543-AA genotype was associated with a 3.8-fold increased risk of COVID-19 retinopathy (p = 0.05). The genotype frequencies of selected gene polymorphisms were not significantly associated with either the presence of COVID-19 or its severity. This is the first study demonstrating a borderline association of the AGTR2 rs1403543-AA genotype with COVID-19 retinopathy in males; hence, the AGTR2 rs 1403543 A allele might represent a genetic risk factor for COVID-19 retinopathy in males

The Relative Preservation of the Central Retinal Layers in Leber Hereditary Optic Neuropathy

(1) Background: The purpose of this study was to evaluate the thickness of retinal layers in Leber hereditary optic neuropathy (LHON) in the atrophic stage compared with presumably inherited bilateral optic neuropathy of unknown cause with the aim of seeing if any LHON-specific patterns exist. (2) Methods: 14 patients (24 eyes) with genetically confirmed LHON (LHON group) were compared with 13 patients (23 eyes) with negative genetic testing results (mtDNA + WES) and without identified etiology of bilateral optic atrophy (nonLHON group). Segmentation analysis of retinal layers in the macula and peripapillary RNFL (pRNFL) measurements was performed using Heidelberg Engineering Spectralis SD-OCT. (3) Results: In the LHON group, the thickness of ganglion cell complex (GCC) (retinal nerve fiber layer (RNFL)&mdash;ganglion cell layer (GCL)&mdash;inner plexiform layer (IPL)) in the central ETDRS (Early Treatment Diabetic Retinopathy Study) circle was significantly higher than in the nonLHON group (p &lt; 0.001). In all other ETDRS fields, GCC was thinner in the LHON group. The peripapillary RNFL (pRNFL) was significantly thinner in the LHON group in the temporal superior region (p = 0.001). Longitudinal analysis of our cohort during the follow-up time showed a tendency of thickening of the RNFL, GCL, and IPL in the LHON group in the central circle, as well as a small recovery of the pRNFL in the temporal region, which corresponds to the observed central macular thickening. (4) Conclusions: In LHON, the retinal ganglion cell complex thickness (RNFL-GCL-IPL) appears to be relatively preserved in the central ETDRS circle compared to nonLHON optic neuropathies in the chronic phase. Our findings may represent novel biomarkers as well as a structural basis for possible recovery in some patients with LHON