Hypertension secondary to nitric oxide depletion produces oxidative imbalance and inflammatory/fibrotic outcomes in the cornea of C57BL/6 mice.

Vol.:(0123456789) 1 3 https://doi.org/10.1007/s13105-022-00916-2 ORIGINAL ARTICLE Hypertension secondary tonitric oxide depletion produces oxidative imbalance andinflammatory/fibrotic outcomes inthecornea ofC57BL/6 mice ÁlvaroSantana‑Garrido1,2,3· ClaudiaReyes‑Goya1· AnaArroyo‑Barrios1· HelderAndré3· CarmenM.Vázquez1,2,3· AlfonsoMate1,2 Received: 7 March 2022 / Accepted: 28 July 2022 © The Author(s) 2022 Abstract Arterial hypertension (AH) leads to oxidative and inflammatory imbalance that contribute to fibrosis development in many target organs. Here, we aimed to highlight the harmful effects of severe AH in the cornea. Our experimental model was established by administration of NG-nitro-L-arginine-methyl-ester (L-NAME) to C57BL/6 mice, which were monitored weekly for arterial blood pressure and intraocular pressure (IOP). Morphological studies of ocular tissues were accompanied by analyses of reactive oxygen species generation, and localization/expression of NAPDH oxidase isoforms (NOX1, NOX2, NOX4) and inflammatory biomarkers (PPARα, PPARγ, IL-1β, IL-6, IL-10, TNF-α, and COX-2). Masson’s trichrome and Sirius Red staining were used to explore the fibrotic status of the cornea. The expression of collagen isoforms (COL1α1, COL1α2, COL3α1, COL4α1, COL4α2) and relevant metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) were also quantified to evaluate the participation of collagen metabolism in AH-related corneal damage. Hypertensive animals showed an increase in IOP values, and a thinner cornea compared with normotensive controls. Moreover, AH increased NADPH oxidase activity and reactive oxygen species generation in the cornea, which was accompanied by transcriptional upregulation of NOX isoforms and inflammatory biomarkers, while reducing PPAR expression. L-NAME-treated animals also developed corneal fibrosis with overexpression of collagen isoforms and reduction of factors responsible for collagen degradation. This is the first study reporting structural changes in the cornea and elevated IOP in L-NAME-treated mice. Overexpression of the NADPH oxidase system and collagen deposition might play a substantial role in the pathogenic mechanisms contributing to ocular disturbances in a context of severe hypertension. Keywords L-NAME hypertension· Intraocular pressure· Cornea· Fibrosis· Inflammation· Oxidative stress Introduction Arterial hypertension (AH) is a systemic disease considered as one of the major risk factors for the development and progression of cardiovascular, renal, and neurological diseases, among others [61]. With a global prevalence of around 30–45%, AH is defined as a sustained rise of systolic blood pressure (SBP) and/or diastolic blood pressure Key points • L-NAME administration in mice increases intraocular pressure. • Nitric oxide deprivation induces oxidative stress, inflammation, and corneal fibrosis. • L-NAME damage in the eye suggests a link between arterial and ocular hypertension. • The importance of monitoring corneal structure in the setting of high blood pressure. * Carmen M. Vázquez v[email protected] * Alfonso Mate [email protected] 1 Departamento de Fisiología, Facultad de Farmacia, Universidad de Sevilla, 41012Seville, Spain 2 Epidemiología Clínica y Riesgo Cardiovascular, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen del Rocío/Consejo Superior de Investigaciones Científicas/Universidad de Sevilla, 41013Seville, Spain 3 Department ofClinical Neuroscience, St. Erik Eye Hospital, Karolinska Institutet, 11282Stockholm, Sweden / Published online: 9 August 2022 Journal of Physiology and Biochemistry (2022) 78:915–932 1 3 (DBP) values over 140/90mmHg, respectively, with different hypertension grades depending on the precise values [59]. Despite the potential and well-documented harmful effects of hypertension on the eye, specifically in the retina and choroid [16], additional outcomes in the cornea in this context remain unexplored. Oxidative stress, defined as an excessive production of reactive oxygen species (ROS), is known to produce tissue damage, cell death, and acceleration of the aging process. In turn, inflammation is a complex process aiming to protect the tissues from damage and infectious agents [52]. Both processes contribute to the pathogenesis of various ocular diseases, including not only the development of retinopathies but also corneal and conjunctival disorders, such as dry eye syndrome, pterygium, or keratoconus [40]. Regarding oxidative stress, many reports support that, in a context of AH, the enzyme nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) is a major enzymatic source for ROS production (mainly superoxide anion (O2.−) and/or peroxide hydrogen (H2O2)), with subsequent organ damage [31]. The expression of different NOX isoforms (NOX1-5 and Duox1-2) has been found in various tissues with differential functions [70]. Although NAPDH oxidase seems to be highly implicated in the oxidative imbalance induced by AH in the retina [48, 49], no link between AH and oxidative stress has been established so far in the cornea. Along with the rest of the ocular surface, and due to continuous exposure to environmental oxidizing agents, the cornea has an effective antioxidant defense mechanism. However, in some circumstances, an oxidative imbalance develops that favors the pathogenesis of corneal diseases and can even lead to blindness [38]. Therefore, it is important to investigate potential mechanisms capable of altering the oxidative status of the cornea. The oxidative imbalance generated by overproduction of ROS can lead to inflammatory processes, thus playing a key function in the pathophysiology of many diseases. The low-grade inflammation involved in AH modulates the expression of the main inflammatory biomarkers, including tumor necrosis factor alpha (TNF-α), cyclooxygenase 2 (COX-2), and some interleukin isoforms [57]. In relation to eye diseases, corneal inflammation has been well associated with the development and progression of dry eye disease and keratoconus [34, 64]. Along with fibrosis, the importance of inflammation has been studied in corneal pathological events, such as corneal wounds [66]. However, in spite of the importance of AH in inflammatory and fibrotic ocular events, e.g., in retinopathies, its possible pathological role in the cornea is still unknown. Fibrogenic processes may be aggravated by inflammatory and oxidative events in AH. Arterial fibrosis and extracellular matrix (ECM) deposition related to AH contribute to hypertensive target organ damage, possibly because AH leads to a modification of the turnover of ECM components [56]. ECM components, among which collagen is especially relevant, provide structural integrity to the cornea for proper functionality. A balanced composition of collagen fibers at the corneal stroma (which constitutes nearly 90% of the corneal thickness) is very important to maintain its transparency [35]. Type I collagen is the main collagen isoform in the cornea, accompanied by type III and type IV collagen fibers in smaller amounts. Fibrotic processes in the cornea, including wound healing and inflammation, have been associated with an upregulation of collagens that can alter the transparency of cornea and produce visual loss [33]. Maintenance and repair of the corneal ECM are coordinated by a brittle balance between matrix metalloproteinases (MMPs) and its tissue inhibitors (TIMPs), which are altered in some corneal disorders [46]. Maintaining this balance is so important that therapies based on corneal collagen administration have been approved for treatment of corneal ecstatic disorders [14]. Surprisingly, despite the importance of collagen metabolism for an adequate corneal function, the implication of AH at this level has not been studied yet. To investigate the possible role of AH in the development and progression of corneal pathologies, here, we measured different biomarkers of oxidative stress, inflammation, and fibrosis in a mice model of AH induced by administration of NG-nitro-L-arginine-methyl-ester (L-NAME, an inhibitor of nitric oxide (NO) synthesis). Blood pressure and intraocular pressure (IOP) monitoring, morphometric analyses of the cornea, measurement of ROS and NO levels, and the activity and location/expression of NOX components, inflammatory biomarkers (PPARα, PPARγ, TNF-α, COX-2, IL-1β, IL-6, and IL-10), and fibrosis-related parameters (including collagen profile and MMP/TIMP enzymes) were assayed in the cornea of L-NAME-treated mice and compared with a parallel normotensive control group. Materials andmethods Animal characteristics andblood pressure The experimental design was conducted in accordance with the European Union (EU) Directive 2010/63/EU and the National (RD 53/2013) guidelines for the care and use of Laboratory animals and was approved by the competent Institutional Animal Care and Use Committee (approval reference #13/03/2019/031, issued by Junta de Andalucía, Dirección General de la Producción Agrícola y Ganadera). Male C57BL/6 mice aged 10–12weeks were obtained from 916 Á. Santana-Garrido et al. 1 3 the Center for Animal Production and Experimentation at the University of Seville (Spain). Mice were housed with free access to food and drink and exposed to 12:12-h light/ dark, and randomly assigned into two groups: (1) normotensive mice fed a standard pellet diet (control group), and (2) hypertensive mice (via administration of 45mg L-NAME/ kg body weight/day in the drinking water) fed the same commercial diet (L-NAME group). The concentration of L-NAME in the water bottles was calculated weekly considering the evolution of body weight and water intake, and treatment was maintained for 6weeks. SBP and DBP were recorded weekly by the noninvasive tail-cuff technique with a pressure recorder (NIPREM 645, Cibertec, Spain). Values were estimated as the average of 3–4 consecutive records. Intraocular pressure measurements Intraocular pressure (IOP) was measured using a rebound tonometer (Tonolab, Icare), following protocols described previously [58]. Briefly, tonometry was performed in awaked and unrestrained mice between 8:00 and 11:00 a.m. to minimize the effects of the circadian rhythm on IOP. IOP readings were obtained at the beginning (week 1), middle (week 3), and at the end (week 6) of the treatment with L-NAME. Mice were habituated to the tonometer from the week before Table 1 Primers used for real-time PCR Gene Forward primer (5′ → 3′) Reverse primer (5′ → 3′) Accession number Nox1 GGT TGG GGC TGA ACA TTT TTC TCG ACA CAC AGG AAT CAG GAT NM_172203.2 Nox2 CCC TTT GGT ACA GCC AGT GAA GAT CAA TCC CAC GTC CCA CTA ACA TCA FJ168469.1 Nox4 ATC ACA GAA GGT CCC TAG CA TAA CCA TGA GGA ACA ATA CCAC AF276957.1 TNF-α CCA CGC TCT TCT GTC TAC TG ACT TGG TGG TTT GCT ACG AC D84199.2 COX-2 ACC CCC TGC TGC CCG ACA CCT CCA GCA ACC CGG CCA GCA ATC NM_011198.4 IL1β CCG TGG ACC TTC CAG GAT GA GGG AAG GTC ACA CAC CAG CA M15131.1 IL6 CTC TGC AAG AGA CTT CCA TCC TTC TGC AAG TGC ATC ATC GT DQ788722.1 IL10 CTG GAC AAC ATA CTG CTA ACCG GGG CAT CAC TTC TAC CAG GTAA BC120612.1 COL1α1 GCT CCT CTT AGG GGC CAC T CCA CGT CTC ACC ATT GGG G NM_007742.4 COL1α2 GGT GAG CCT GGT CAA ACG G ACT GTG TCC TTT CAC GCC TTT NM_007743.3 COL3α1 CCT GGC TCA AAT GGC TCA C GAC CTC GTG TTC CGG GTA T NM_009930.2 COL4α1 CTA ACG GTT GGT CCT CAC TG CGT GGG CTT CTT GAA CAT CTC NM_009931.2 COL4α2 GGC TTC ATC AAA GGA GTC AAGG CCC AAT GTC ACC AAA GTC CC NM_009932.4 MMP2 CAA GTT CCC CGG CGA TGT C TTC TGG TCA AGG TCA CCT GTC NM_008610.3 MMP3 GTC TTT GAA GCA TTT GGG TTT CTC GGT GTC ATC CAT AGC TCC TG NM_010809.2 MMP9 CTG GAC AGC CAG ACA CTA AAG CTC GCG GCA AGT CTT CAG AG NM_013599.5 TIMP1 CCA GAG CAG ATA CCA TGA TGG CCA CAG AGG CTT TCC ATG AC NM_001044384.1 TIMP2 GAA TCC TCT TGA TGG GGT TG CGT TTT GCA ATG CAG ACG TA NM_011594.3 TIMP3 TAG ACC AGA GTG CCA AAG GG CCA GGA TGC CTT CTG CAA C NM_011595.2 GAPDH GCC AAA AGG GTC ATC ATC TCCGC GGA TGA CCT TGC CCA CAG CCTTG XM_017321385.2 Table 2 Antibodies used for Western blotting analyses 1st antibody Origin Dilution 2nd antibody Dilution Reference Anti-iNOS Mouse monoclonal 1:1000 Goat Anti-Mouse 1:2000 sc-7271, Santa Cruz Biotechnology, Santa Cruz, CA Anti-Nitrotyrosine Mouse monoclonal 1:2000 Goat Anti-Mouse 1:3000 sc-32757, Santa Cruz Biotechnology, Anti-NOX1 Mouse monoclonal 1:2000 Goat Anti-Mouse 1:3000 sc-518023, Santa Cruz Biotechnology, Anti-NOX2 Rabbit monoclonal 1:7000 Goat Anti-Rabbit 1:8000 ab129068, Epitomics-Abcam, Burlingame, CA Anti-NOX4 Rabbit monoclonal 1:7000 Goat Anti-Rabbit 1:8000 ab133303, Epitomics-Abcam Anti-PPARα Mouse monoclonal 1:1000 Goat Anti-Mouse 1:2000 sc-398394, Santa Cruz Biotechnology, Anti-PPARγ Mouse monoclonal 1:1000 Goat Anti-Mouse 1:2000 sc-271392, Santa Cruz Biotechnology, Anti-β-actin Mouse monoclonal 1:10,000 Goat Anti-Mouse 1:20,000 sc-47778, Santa Cruz Biotechnology, 917Hypertension secondary to nitric oxide depletion produces oxidative imbalance and… 1 3 (A)(B) (D)(E) iNOS (140 kDa) -actin (42 kDa) Nitrotyrosine -actin 0 20 40 60 80 100 120 ControlL-NAME NO noitartnecnoc(%) * 0.0 0.5 1.0 1.5 2.0 ControlL-NAME iNOS protein expresión (RU) * 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ControlL-NAME Nitrotyrosine expresión (RU) * (F)(G)(H) (C) * ** ** * ** ** * * * * 918 Á. Santana-Garrido et al. 1 3 the start of the measurements, and the readings were taken on the central cornea of both eyes. Cornea isolation andhomogenization Mice were euthanized by cervical dislocation. Corneas were rapidly dissected under a binocular stereoscopic microscope, snap-frozen in liquid nitrogen, and stored at − 80°C until measurement of NAPDH oxidase activity. Fifty millimolar PBS (pH 7.4) containing protease inhibitors (Sigma Aldrich- Roche, Madrid, Spain) was used to obtain cornea homogenates (which included the two pooled corneas from each mouse) using a Potter–Elvehjem tissue grinder. Homogenates were centrifuged for 10min at 10,000 × g and the supernatants were recovered to determine the protein concentration by the Bradford method. NADPH oxidase activity measurements NADPH oxidase activity was measured in cornea homogenates by lucigenin-enhanced chemiluminescence, following routine protocols in our laboratory [48]. Potential sources of superoxide anion (O2.−) production in corneal samples were discriminated at 37°C after a 5-min preincubation with 0.1mmol/L DPI, oxypurinol, or rotenone (respective inhibitors of flavoproteins, xanthine oxidase, and mitochondrial electron transport chain; Sigma-Aldrich, Madrid, Spain). Similar protocols were followed when using an inhibitor of NOX1/4 (0.1µmol/L GKT136901; Sigma- Aldrich, 492,000), specific NOX1 inhibitor (0.5µmol/L ML171; Sigma-Aldrich, 175,226), and the pan-NADPH oxidase inhibitor (10µmol/L VAS2870; Sigma-Aldrich, 5,340,320,001) to determine the relative contribution of each NOX isoform in total O2.− production. All measurements were referred to the samples’ protein content, and results were always expressed as relative to those in the control group. Nitric oxide concentration The concentration of NO in corneal homogenates was estimated following the Griess method [32]. The readings were normalized to the corresponding protein content of each sample and the results were plotted relative to the control group. Real‑time PCR RNA isolation from corneal tissue was performed using TRIzol® RNA isolation method (Thermo Fisher Scientific, Madrid, Spain) with the two pooled corneas from each animal. Reverse transcription and polymerase chain reactions were run as previously described [50] and using specific primers listed in Table1, which were designed using the application PerlPrimer v. 1.1.20 (Marshall OJ). The integrity of isolated RNA and PCR products was confirmed by observing the appearance and quality of the relevant bands on agarose gels and determining A260/280 and A260/230 ratios in a NanoDrop (Thermo Fisher Scientific). Western blotting Aliquots of corneal homogenates containing equal amounts of proteins (20μg) were used to perform Western blot experiments following protocols previously described [48]. The primary antibodies used are listed in Table2. Quantitative optical densitometry (Cytive Europe GmbH, Barcelona, Spain) was performed using β-actin as a loading control. Histological sections andstaining Corneal tissue sections (5μm) were obtained following a protocol previously reported by Santana-Garrido etal. [48]. Paraffin-embedded sections were used for morphometric analysis, Masson’s trichrome, Sirius Red staining, DHE staining, and immunofluorescence, as detailed below. Deparaffinized hematoxylin/eosin-stained sections (H/E) were photographed using an Olympus BX41 microscope coupled to an Olympus DP73 camera. The thickness of the corneal layers was always measured at the central height of the cornea using ImageJ-NIH freeware (v. 2.0.0). Furthermore, collagen fibers in the corneas were visualized using Masson’s trichrome (MTS; Merk, Germany, 1.00485) and picro Sirius Red (SRS; Sigma-Aldrich Inc, St Louis, USA) staining. MTS and SRS pictures were quantified using Image J-NIH freeware (v. 2.0.0) and following protocols detailed elsewhere [17]. Corneal ROS measurements Paraffin-embedded eye sections were used to estimate corneal ROS production insitu by dihydroethidium staining (DHE; MedChemExpress, Madrid, Spain, Cat. No. HY-D0079), following a protocol previously used in our laboratory [48]. The specificity of DHE staining in Fig. 1 Animal general characteristics. Evolution of (A) systolic blood pressure (SBP), (B) diastolic blood pressure (DBP), and (C) intraocular pressure (IOP) values measured by rebound tonometry, in normotensive (control, black squares) and hypertensive (L-NAME, gray squares) animals throughout the 6-week experimental period. D Weight gain and E water/food intake at the end of the treatment. F Nitric oxide concentration, G iNOS protein expression, and H protein nitrosylation in corneal homogenates. Values in F–H are presented as relative to those of the control group, and all values are expressed as mean ± SEM of seven animals per group. *p < 0.05 vs control. RU relative units ◂ 919 Hypertension secondary to nitric oxide depletion produces oxidative imbalance and… 1 3 0 0.5 1 1.5 2 2.5 CEP DHE staining(RFU/area) Control CT+PEG-SOD CT+VAS2870 CT+GKT136901 CT+ML171 L-NAME L-NAME+PEG-SOD L-NAME+VAS2870 L-NAME+GKT136901 L-NAME+ML171 *,# *,# *,# *,# * # *,#*,# *,# 0 2 4 6 8 10 12 ST DHE staining(RFU/area) Control CT+PEG-SOD CT+VAS2870 CT+GKT136901 CT+ML171 L-NAME L-NAME+PEG-SOD L-NAME+VAS2870 L-NAME+GKT136901 L-NAME+ML171 *,# *,# # *,# * # # # # 0 20 40 60 80 100 120 CEN DHE relative staining (RFU/area) Control CT+PEG-SOD CT+VAS2870 CT+GKT136901 CT+ML171 L-NAME L-NAME+PEG-SOD L-NAME+VAS2870 L-NAME+GKT136901 L-NAME+ML171 2 0 20 40 60 80 100 120 140 CEPSTCEN TOTAL Corneal relative thickness (%) Control L-NAME * * * * L-NAME CEP ST CEN Control (A) (B) (C) )E()D( (F) *,# *,# *,# *,# * # *,#*,# *,# 920 Á. Santana-Garrido et al. 1 3 deparaffinized sections was evaluated by preincubation with 100 U/mL polyethylene glycol-conjugated superoxide dismutase (PEG-SOD; Sigma Aldrich, S9549) for 30min at 37°C. Parallel deparaffinized corneal sections were preincubated with specific NOX inhibitors VAS2870, GKT136901, and ML171 for 30min at 37°C. The slides were then mounted with DAPI Fluoromount- G® (SouternBiotech Associates, Inc, Birmingham, AL; Cat. No. 0100–20) after 20min incubation with DHE at 37°C. An Olympus DP73 fluorescence microscope (Tokyo, Japan) and Image J-NIH freeware (v. 2.0.0) were used to measure the intensities of the staining. Central corneal sections from normotensive and hypertensive animals were processed in parallel under similar conditions. Images were taken using the same exposure time and a × 10 objective. Results were expressed as relative to the control group. Immunohistofluorescence The corneal expression of NOX and PPAR isoforms was evaluated by immunohistofluorescence in deparaffinized eye sections. Diva Decloaker (Biocare Medical, LLC, Pacheco, CA), an antigen retrieval reagent, and the following specific primary antibodies were used for immunostaining: mouse monoclonal anti-NOX1 (Santa Cruz Biotechnology, Santa Cruz, CA; Cat. No. sc-518023; 1:200 dilution); rabbit monoclonal anti-NOX2 (Epitomics-Abcam, Burlingame, CA; Cat. No. ab129068; 1:100 dilution); rabbit monoclonal anti-NOX4 (Epitomics-Abcam; Cat. No. ab133303; 1:500 dilution); mouse monoclonal anti- PPARα (Santa Cruz Biotechnology; Cat. No. sc-398394; 1:200 dilution); mouse monoclonal anti-PPARγ (Santa Cruz Biotechnology; Cat. No. sc-271392; 1:200 dilution). Goat anti-rabbit Alexa Fluor® 555 (Cohesion Biosciences Ltd., London, UK; Cat. No. CSA3411) and Goat anti-mouse Alexa Fluor® 647 (Cat. No. CSA3808) were used as fluorescent secondary antibodies, where appropriate, and sections were mounted with DAPI Fluoromount-G®. Image J-NIH freeware (v. 2.0.0) was used to measure the intensity of the staining on parallel images of both animal groups, which were acquired from central corneal sections and processed with the same settings. Results were expressed as relative to the control group. Statistical analyses Data are presented as means ± SEM. Unless more than two groups were compared (where one-way ANOVA followed by Tukey multiple comparison test was performed), results were subjected to unpaired Student’s t-test using the Graph- Pad InStat software (San Diego, CA) and were considered significant at p < 0.05. Results Hypertensive general data andNOconcentration As expected, elevated SBP and DBP values were found in the L-NAME group (196/104mmHg, respectively) at the end of the 6-week treatment (Fig.1A–B). Remarkable differences with the control group were found from the first week of L-NAME administration. As shown in Fig.1C, IOP values (which showed no interocular variations) also increased during treatment with L-NAME; significant differences were observed from mid-treatment and became substantial at the end of the experimental period (13 vs 17mmHg in control and L-NAME groups, respectively). On the other hand, the intake of L-NAME did not affect the animals’ weight gain or their drinking and feeding behaviors throughout the treatment (Fig.1D–E). The estimated concentration of NO in corneal homogenates was reduced by 40% in L-NAME-treated animals compared to the control group (Fig.1F). This alteration was accompanied by an upregulation of the inducible isoform of nitric oxide synthase, iNOS (Fig.1G). As an additional marker of oxidative stress, the degree of nitrosylated proteins was also quantified in corneal homogenates, showing a significant overexpression of 3-nitrotyrosine residues in NO-depleted mice (Fig.1H). Arterial hypertension induces histomorphometric changes andROS production inthecornea Figure2Ashows representative corneal images stained with H/E. Morphometric analysis revealed a noticeable reduction in the thickness of the corneal layers (about 42%) in L-NAME-treated animals (Fig.2B). Specifically, this group presented significant reductions of 21%, 51%, and 28% in Fig. 2 Histomorphometric analyses and ROS measurements in the cornea. A Representative images of hematoxylin/eosin staining and B corneal layer thickness in normotensive (control) and hypertensive (L-NAME) mice. C Reactive oxygen species (ROS) production in the cornea visualized by DHE labeling (red color) with or without preincubation with specific NOX inhibitors: NOX pan-inhibitor (VAS2870), dual NOX1/NOX4 inhibitor (GKT136901), and NOX1 inhibitor (ML171). The specificity of DHE staining was confirmed by preincubation with 100 U/mL polyethylene glycol-conjugated superoxide dismutase (PEG-SOD). Corneal layers can be distinguished with 4′,6-diamidino-2-phenylindole (DAPI, blue color) nuclei staining. D–F Relative fluorescence intensity in C relative to that of the control group and quantified using the Image J software. Magnification: × 10. ANOVA followed by Tukey multiple comparison test was performed to analyze the results, and values are expressed as mean ± SEM of seven animals per group. *p < 0.05 vs control; #p < 0.05 vs L-NAME. CEN, corneal endothelium; CEP, corneal epithelium; RFU, relative fluorescence units; ST, stroma ◂ 921 Hypertension secondary to nitric oxide depletion produces oxidative imbalance and… 1 3 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX1 mRNA expression(RU) * 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX2 mRNA expression(RU) * 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX4 mRNA expression (RU) * 0 50 100 150 200 250 300 350 400 450 ControlL-NAME esadixoHPDA Na ctivity(%) * (A)(B) (C) (D) (E) (F) 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX1 protein expression(RU) * NOX1 (59 kDa) -actin (42 kDa) 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX2 protein expression(RU) * NOX2 (65 kDa) -actin (42 kDa) 0 0.5 1 1.5 2 2.5 3 3.5 ControlL-NAME NOX4 protein expression(RU) * NOX4 (67 kDa) -actin (42 kDa) 0 50 100 150 200 250 300 350 400 450 O2·- generation (%) ## *** *, # *, # # *, # *, # ##*,# CEN 50m ST CEP CEN IPAD1XON IPAD4XON 2XON IPAD CEP ST CEN CEP ST CEN ControlL-NAME 922 Á. Santana-Garrido et al. 1 3 the corneal epithelium (CEP), stroma (ST), and corneal endothelium (CEN), respectively, compared with the equivalent layers of the control group. Representative images of DHE-stained corneas are shown in Fig.2C. DHE signal was intensified in L-NAME-treated mice in CEP (approximate twofold increase; Fig.2D) and especially in ST and CEN layers (10- and 100-fold increases, respectively; Fig.2E–F), because the staining was hardly visible in the latter in normotensive animals. To clarify the relative contribution of NOX isoforms to corneal ROS production, additional sections from control and L-NAME- administered mice were preincubated with different NOX inhibitors. The pan-inhibitor VAS2870 induced a major reduction of DHE-dependent O2.− signal in the cornea of hypertensive mice (83%, 90%, and 99% in CEP, ST, and CEN, respectively, compared to the L-NAME group). Smaller reductions in DHE staining were observed in corneas of L-NAME-treated mice after incubation with the dual NOX1/NOX4 inhibitor, GKT136901 (36%, 70%, and 51% in CEP, ST, and CEN, respectively), or with the inhibitor of NOX1, ML171 (19%, 70%, and 35%, respectively). In turn, the inhibitory action on DHE-related baseline signals detected in normotensive mice was much lower. Taken together, these results suggest a higher release of ROS in corneas with a preferential role for NADPH oxidase isoforms NOX2 and NOX4, and also regional differences with the CEN being more affected than the CEP and the ST. NADPH activity andNOX isoforms are upregulated inhypertensive corneas To confirm the pivotal role of the NADPH oxidase system in local O2.− production, corneal homogenates were obtained and used to measure NADPH oxidase activity and to check the source(s) of O2.− generation locally in this tissue. A significant (fourfold) increase in the activity of NADPH oxidase was found in the L-NAME group relative to the control group (Fig.3A). Corneal O2.− production in hypertensive mice remained unaltered when homogenates from these animals were preincubated with either oxypurinol or rotenone, while DPI abolished superoxide overproduction completely (Fig.3B). Also shown in Fig.3B, preincubation with NOX inhibitors reduced superoxide production in L-NAME animals by 21%, 56%, and 77% when ML171 (NOX1-inhibitor), GKT136901 (dual NOX1/NOX4 inhibitor), and VAS2870 (NADPH oxidase pan-inhibitor) were used, respectively. On the other hand, preincubation with these inhibitors had essentially no effect on corneal homogenates obtained from normotensive animals, except for DPI and VAS2870 (40% and 22% reductions, respectively). Isoforms NOX1, NOX2, and NOX4 were detected in corneal sections with a higher immunofluorescence signal in CEP, ST, and CEN from L-NAME-administered animals (Fig.3C). The most remarkable differences between normotensive and hypertensive animals were found at the CEN level (especially for NOX4 signal). At the ST, NOX- related signals were weak in L-NAME animals and absent in the control group. In addition, the results of gene and protein expression confirmed the upregulation of these three NOXes in the L-NAME group; thus, our experimental model of hypertension by nitric oxide deprivation presented with significant increases in the mRNA and protein expression of NOX1, NOX2, and NOX4 (~ 2–2.5-fold; Fig.3D–F). Collectively, our results indicate that NOX4 might be the main isoform of NADPH oxidase involved in excess O2.− release at the corneal level in hypertensive mice. Profile ofinflammatory biomarkers inL‑NAME‑hypertensive corneas Peroxisome proliferator-activated receptors (PPARs), isoforms PPARα and PPARγ, could be detected by immunofluorescence in CEP, ST, and CEN (Fig.4A–B). A decrease in PPAR expression was clearly visible in all corneal layers in hypertensive animals. Signal immunofluorescence quantification confirmed downregulation of PPARα (60%, 96%, and 30% reductions compared to the control group in CEP, ST, and CEN, respectively; Fig.4C), and PPARγ (63%, 98%, and 78% reductions in CEP, ST, and CEN, respectively; Fig.4D). These results paralleled the relative quantification of PPAR protein expression estimated by Western blotting (Fig.4E–F). Concerning cytokines and modulators of inflammation, L-NAME induced the corneal expression of proinflammatory biomarkers TNF-α, COX-2, IL-1β, and IL-6 (~ 2–threefold increase compared to the control group), whereas the anti-inflammatory IL-10 was downregulated (56% reduction) in hypertensive mice (Fig.4G). Fig. 3 NADPH oxidase activity and NOX expression in the cornea. A Relative NADPH oxidase activity in cornea homogenates and B characterization of the primary source of superoxide anions after preincubation with different inhibitors: DPI (diphenyleneiodonium chloride, inhibitor of flavoproteins); OXI (oxypurinol, xanthine oxidase inhibitor); ROT (rotenone, inhibitor of mitochondrial electron transport chain); VAS2870 (NOX pan-inhibitor); GKT136901 (dual NOX1/NOX4 inhibitor); and ML171 (NOX1 inhibitor). C NOX expression (red color) and nuclei staining with DAPI (blue color) of NOX1 (top), NOX2 (middle), and NOX4 (bottom) in corneal layers from each experimental group. Magnification: × 10. mRNA and protein expression of (D) NOX1, (E) NOX2, and (F) NOX4 in cornea homogenates from normotensive (control) and hypertensive (L-NAME) mice. Quantitative fold changes in gene expression were determined relative to the corresponding value for the housekeeping gene GAPDH. ANOVA followed by Tukey multiple comparison test was performed to analyze the results depicted in B. Values are expressed as mean ± SEM of seven animals per group. *p < 0.05 vs control; #p < 0.05 vs L-NAME. CEN, corneal endothelium; CEP, corneal epithelium; RU, relative units; ST, stroma ◂ 923 Hypertension secondary to nitric oxide depletion produces oxidative imbalance and… 1 3 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. References 1. Amadi PU, Agomuo EN, Adumekwe CW (2020) Modulatory properties of cardiac and quercetin glycosides from Dacryodes edulis seeds during L-NAME-induced vascular perturbation. J Basic Clin Physiol Pharmacol 31. https:// doi. org/ 10. 1515/ jbcpp- 2019- 0116 2. Atilano S, Lee D, Fukuhara P etal (2019) Corneal oxidative damage in keratoconus cells due to decreased oxidant elimination from modified expression levels of SOD enzymes, PRDX6, SCARA3, CPSF3, and FOXM1. J Ophthalmic Vis Res 14:62–70. https:// doi. org/ 10. 4103/ jovr. jovr_ 80_ 18 3. Baltanás A, Miguel-Carrasco JL, San José G etal (2013) A synthetic peptide from transforming growth factor-β1 type III receptor inhibits NADPH oxidase and prevents oxidative stress in the kidney of spontaneously hypertensive rats. Antioxidants Redox Signal 19:1607–1618. https:// doi. org/ 10. 1089/ ars. 2012. 4653 4. Barbier O, Torra IP, Duguay Y etal (2002) Pleiotropic actions of peroxisome proliferator–activated receptors in lipid metabolism and atherosclerosis. Arterioscler Thromb Vasc Biol 22:717–726. https:// doi. org/ 10. 1161/ 01. ATV. 00000 15598. 86369. 04 5. Belovay GW, Goldberg I (2018) The thick and thin of the central corneal thickness in glaucoma. Eye 32:915–923. https:// doi. org/ 10. 1038/ s41433- 018- 0033-3 6. Bisogni V, Cerasari A, Pucci G, Vaudo G (2020) Matrix metalloproteinases and hypertension-mediated organ damage: current insights. Integr Blood Press Control 13:157–169. https:// doi. org/ 10. 2147/ IBPC. S2233 41 7. Boegehold MA (2013) The effect of high salt intake on endothelial function: reduced vascular nitric oxide in the absence of hypertension. J Vasc Res 50:458–467. https:// doi. org/ 10. 1159/ 00035 5270 8. Chan EC, van Wijngaarden P, Chan E etal (2016) NADPH oxidase 2 plays a role in experimental corneal neovascularization. Clin Sci 130:683–696. https:// doi. org/ 10. 1042/ CS201 50103 9. Chelladurai P, Seeger W, Pullamsetti SS (2012) Matrix metalloproteinases and their inhibitors in pulmonary hypertension. Eur Respir J 40:766–782. https:// doi. org/ 10. 1183/ 09031 936. 00209 911 10. Creemers EEJM, Cleutjens JPM, Smits JFM, Daemen MJAP (2001) Matrix metalloproteinase inhibition after myocardial infarction. Circ Res 89:201–210. https:// doi. org/ 10. 1161/ hh1501. 094396 11. Cui N, Hu M, Khalil RA (2017) Biochemical and biological attributes of matrix metalloproteinases. Prog Mol Biol Transl Sci 147:1–73. https:// doi. org/ 10. 1016/ bs. pmbts. 2017. 02. 005 12. Dornas WC, Silva ME (2011) Animal models for the study of arterial hypertension. J Biosci 36:731–737. https:// doi. org/ 10. 1007/ s12038- 011- 9097-y 13. Dziedziak J, Zaleska-Zmijewska A, Szaflik JP, Cudnoch- Jȩdrzejewska A (2022) Impact of arterial hypertension on the eye: a review of the pathogenesis, diagnostic methods, and treatment of hypertensive retinopathy. Med Sci Monit 28:1–12. https:// doi. org/ 10. 12659/ MSM. 935135 14. Evangelista CB, Hatch KM (2018) Corneal collagen cross-link- ing complications. Semin Ophthalmol 33:29–35. https:// doi. org/ 10. 1080/ 08820 538. 2017. 13538 09 15. Fan D, Kassiri Z (2020) Biology of tissue inhibitor of metalloproteinase 3 (TIMP3), and its therapeutic implications in cardiovascular pathology. Front Physiol 11:1–16. https:// doi. org/ 10. 3389/ fphys. 2020. 00661 16. Fraser-Bell S, Symes R, Vaze A (2017) Hypertensive eye disease: a review. Clin Experiment Ophthalmol 45:45–53. https:// doi. org/ 10. 1111/ ceo. 12905 17. Gewehr DM, Giovanini AF, Munhoz SI etal (2021) Softwareassisted image analysis for identification and quantification of hepatic sinusoidal dilatation and centrilobular fibrosis. Arq Bras Cir Dig 34:e1608. https:// doi. org/ 10. 1590/ 0102- 67202 02100 02e16 08 18. Hakami NY, Dusting GJ, Chan EC etal (2020) Wound healing after alkali burn injury of the cornea involves Nox4-type NADPH oxidase. Investig Ophthalmol Vis Sci 61:1–3. https:// doi. org/ 10. 1167/ IOVS. 61. 12. 20 19. Han S, Makareeva E, Kuznetsova NV etal (2010) Molecular mechanism of type i collagen homotrimer resistance to mammalian collagenases. J Biol Chem 285:22276–22281. https:// doi. org/ 10. 1074/ jbc. M110. 102079 20. Han B, Song M, Li L etal (2021) The application of nitric oxide for ocular hypertension treatment. Molecules 26:7306. https:// doi. org/ 10. 3390/ molec ules2 62373 06 21. Herndon LW (2004) Central corneal thickness as a risk factor for advanced glaucoma damage. Arch Ophthalmol 122:17. https:// doi. org/ 10. 1001/ archo pht. 122.1. 17 22. Hong JW, Liu JJ, Lee JS etal (2001) Proinflammatory chemokine induction in keratocytes and inflammatory cell infiltration into the cornea. Invest Ophthalmol Vis Sci 42:2795–2803 23. Kang KB, Tu EY, Aref AA etal (2019) Corneal ectasia associated with postoperative hypotony. Can J Ophthalmol 54:e40–e43. https:// doi. org/ 10. 1016/j. jcjo. 2018. 03. 014 24. Khatol P, Saraf S, Jain A (2018) Peroxisome proliferated activated receptors (PPARs): opportunities and challenges for ocular therapy. Crit Rev Ther Drug Carr Syst 35:65–97. https:// doi. org/ 10. 1615/ CritR evThe rDrug Carri erSyst. 20170 20231 25. Kvanta A, Sarman S, Fagerholm P, Seregard SSB (2000) Expression of matrix metalloproteinase-2 ( MMP-2) and vascular endothelial growth factor ( VEGF ) in inflammation-associated. Exp eye 4:419–428. https:// doi. org/ 10. 1006/ exer. 1999. 0790 26. Lerman LO, Kurtz TW, Touyz RM etal (2019) Animal models of hypertension: a scientific statement from the American Heart Association. Hypertension 73:e87–e120. https:// doi. org/ 10. 1161/ HYP. 00000 00000 000090 27. Lesk MR, Hafez AS, Descovich D (2006) Relationship between central corneal thickness and changes of optic nerve head topography and blood flow after intraocular pressure reduction in open-angle glaucoma and ocular hypertension. Arch Ophthalmol 124:1568–1572. https:// doi. org/ 10. 1001/ archo pht. 124. 11. 1568 28. Levy A, Georgeon C, Knoeri J, etal (2022) Corneal epithelial thickness mapping in the diagnosis of ocular surface disorders involving the corneal epithelium: a comparative study. Cornea 1–9. https:// doi. org/ 10. 1097/ ico. 00000 00000 003012 29. Li B, He X, Lei SS etal (2020) Hypertensive rats treated chronically with Nω-Nitro-L-Arginine Methyl Ester (L-NAME) induced disorder of hepatic fatty acid metabolism and intestinal pathophysiology. Front Pharmacol 10:1–17. https:// doi. org/ 10. 3389/ fphar. 2019. 01677 930 Á. Santana-Garrido et al. 1 3 30. Liu R, Yan X (2021) Oxidative stress in corneal stromal cells contributes to the development of keratoconus in a rabbit model. Eur J Ophthalmol 31:3518–3524. https:// doi. org/ 10. 1177/ 11206 72121 10287 45 31. Loperena R, Harrison DG (2017) Oxidative stress and hypertensive diseases. Med Clin North Am 101:169–193. https:// doi. org/ 10. 1016/j. mcna. 2016. 08. 004 32. Marzinzig M, Nussler AK, Stadler J etal (1997) Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric oxide Biol Chem 1:177– 189. https:// doi. org/ 10. 1006/ niox. 1997. 0116 33. Massoudi D, Malecaze F, Galiacy SD (2016) Collagens and proteoglycans of the cornea: importance in transparency and visual disorders. Cell Tissue Res 363:337–349. https:// doi. org/ 10. 1007/ s00441- 015- 2233-5 34. McMonnies CW (2015) Inflammation and keratoconus. Optom Vis Sci 92:e35–e41. https:// doi. org/ 10. 1097/ OPX. 00000 00000 000455 35. Meek KM, Knupp C (2015) Corneal structure and transparency. Prog Retin Eye Res 49:1–16. https:// doi. org/ 10. 1016/j. prete yeres. 2015. 07. 001 36. Merjava S, Liskova P, Sado Y etal (2009) Changes in the localization of collagens IV and VIII in corneas obtained from patients with posterior polymorphous corneal dystrophy. Exp Eye Res 88:945–952. https:// doi. org/ 10. 1016/j. exer. 2008. 12. 017 37. Neidhart B, Kowalska M, Valentin JDP, Gall FM, Ren Q, Riedl R, Pot SRM (2021) Tissue inhibitor of metalloproteinase (TIMP) peptidomimetic as an adjunctive therapy for infectious keratitis. Biomacromol 22:629–639. https:// doi. org/ 10. 1021/ acs. biomac. 0c014 73 38. Nita M, Grzybowski A (2016) The role of the reactive oxygen species and oxidative stress in the pathomechanism of the agerelated ocular diseases and other pathologies of the anterior and posterior eye segments in adults. Oxid Med Cell Longev 2016:1–23. https:// doi. org/ 10. 1155/ 2016/ 31647 34 39. O’Brien WJ, Krema C, Heimann T, Zhao H (2006) Expression of NADPH oxidase in rabbit corneal epithelial and stromal cells in culture. Investig Ophthalmol Vis Sci 47:853–863. https:// doi. org/ 10. 1167/ iovs. 05- 1063 40. Oduntan OA, Masige KP (2011) A review of the role of oxidative stress in the pathogenesis of eye diseases. African Vis Eye Heal 70:191–199. https:// doi. org/ 10. 4102/ aveh. v70i4. 116 41. Overby DR, Bertrand J, Tektas OY etal (2014) Ultrastructural changes associated with dexamethasone-induced ocular hypertension in mice. Investig Ophthalmol Vis Sci 55:4922–4933. https:// doi. org/ 10. 1167/ iovs. 14- 14429 42. Pereira BP, do Vale GT, Ceron CS (2022) The role of nitric oxide in renovascular hypertension: from the pathophysiology to the treatment. Naunyn Schmiedebergs Arch Pharmacol 395:121–131. https:// doi. org/ 10. 1007/ s00210- 021- 02186-z 43. Pont C, Ascaso FJ, Grzybowski A, Huerva V (2020) Corneal endothelial cell density during diabetes mellitus and ocular diabetes complications treatment. J Fr Ophtalmol 43:794–798. https:// doi. org/ 10. 1016/j. jfo. 2019. 12. 003 44. Pouw AE, Greiner MA, Coussa RG etal (2021) Cell–matrix interactions in the eye: from cornea to choroid. Cells 10:1–28. https:// doi. org/ 10. 3390/ cells 10030 687 45. Rokicki W, Żaba M, Wyględowska-Promieńska D etal (2015) Inducible and endothelial nitric synthetase expression and nitrotyrosine accumulation in iris vasculature of patients with primary open-angle glaucoma: a pilot study. Med Sci Monit 21:76–81. https:// doi. org/ 10. 12659/ MSM. 891177 46. Sakimoto T, Sawa M (2012) Metalloproteinases in corneal diseases: degradation and processing. Cornea 31(Suppl 1):S50– S56. https:// doi. org/ 10. 1097/ ICO. 0b013 e3182 69ccd0 47. Sakimoto T, Shoji J, Sawa M (2003) Active form of gelatinases in tear fluid in patients with corneal ulcer or ocular burn. Jpn J Ophthalmol 47:423–6. https:// doi. org/ 10. 1016/ s0021- 5155(03) 00138-2 48. Santana-Garrido Á, Reyes-Goya C, Pérez-Camino MC etal (2020) Retinoprotective effect of wild olive (Acebuche) oilenriched diet against ocular oxidative stress induced by arterial hypertension. Antioxidants 9:885. https:// doi. org/ 10. 3390/ antio x9090 885 49. Santana-Garrido Á, Reyes-Goya C, Fernández-Bobadilla C, etal (2021) NADPH oxidase-induced oxidative stress in the eyes of hypertensive rats. Mol Vis 27:161–178. molvis.org/molvis/ v27/161/ 50. Santana-Garrido Á, Reyes-Goya C, Milla-Navarro S etal (2021) Anti-Inflammatory action of dietary wild olive (Acebuche) oil in the retina of hypertensive mice. Foods 10:1993. https:// doi. org/ 10. 3390/ foods 10091 993 51. Sharif R, Bak-Nielsen S, Hjortdal J, Karamichos D (2018) Pathogenesis of keratoconus: the intriguing therapeutic potential of Prolactin-inducible protein. Prog Retin Eye Res 67:150–167. https:// doi. org/ 10. 1016/j. prete yeres. 2018. 05. 002 52. Sies H, Jones DP (2020) Reactive oxygen species (ROS) as pleiotropic physiological signalling agents. Nat Rev Mol Cell Biol 21:363–383. https:// doi. org/ 10. 1038/ s41580- 020- 0230-3 53. Singh RP, Goldberg I, Graham SL etal (2001) Central corneal thickness, tonometry, and ocular dimensions in glaucoma and ocular hypertension. J Glaucoma 10:206–210. https:// doi. org/ 10. 1097/ 00061 198- 20010 6000- 00011 54. Skrzypecki J, Grabska-Liberek I, Przybek J, Ufnal M (2018) A common humoral background of intraocular and arterial blood pressure dysregulation. Curr Med Res Opin 34:521–529. https:// doi. org/ 10. 1080/ 03007 995. 2017. 14152 03 55. Smith VA, Matthews FJ, Majid MA, Cook SD (2006) Keratoconus: matrix metalloproteinase-2 activation and TIMP modulation. Biochim Biophys Acta - Mol Basis Dis 1762:431–439. https:// doi. org/ 10. 1016/j. bbadis. 2006. 01. 010 56. Stanchev S, Stamenov N, Kirkov V etal (2020) Differential collagen expression in kidney and heart during hypertension. Bratislava Med J 121:73–78. https:// doi. org/ 10. 4149/ BLL_ 2020_ 011 57. Tanase DM, Gosav EM, Radu S etal (2019) Arterial hypertension and interleukins: potential therapeutic target or future diagnostic marker? Int J Hypertens 2019:1–17. https:// doi. org/ 10. 1155/ 2019/ 31592 83 58. Tribble JR, Otmani A, Sun S etal (2021) Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction. Redox Biol 43:101988. https:// doi. org/ 10. 1016/j. redox. 2021. 101988 59. Unger T, Borghi C, Charchar F etal (2020) 2020 International Society of Hypertension global hypertension practice guidelines. Hypertension 75:1334–1357. https:// doi. org/ 10. 1161/ HYPER TENSI ONAHA. 120. 15026 60. Vallabh NA, Romano V, Willoughby CE (2017) Mitochondrial dysfunction and oxidative stress in corneal disease. Mitochondrion 36:103–113. https:// doi. org/ 10. 1016/j. mito. 2017. 05. 009 61. van Oort S, Beulens JWJ, van Ballegooijen AJ etal (2020) Association of cardiovascular risk factors and lifestyle behaviors with hypertension. Hypertension 76:1971–1979. https:// doi. org/ 10. 1161/ HYPER TENSI ONAHA. 120. 15761 62. Wang X, Khalil RA (2018) Matrix metalloproteinases, vascular remodeling, and vascular disease. Adv Pharmacol 81:241–330. https:// doi. org/ 10. 1016/ bs. apha. 2017. 08. 002 63. Wang X, Tang L, Zhang Z etal (2020) Keratocytes promote corneal neovascularization through VEGFr3 induced by PPAR α 931 Hypertension secondary to nitric oxide depletion produces oxidative imbalance and… 1 3 -inhibition. Exp Eye Res 193:107982. https:// doi. or g/ 10. 1016/j. exer. 2020. 107982 64. Yamaguchi T (2018) Inflammatory response in dry eye. Invest Ophthalmol Vis Sci 59:DES192–DES199. https:// doi. org/ 10. 1167/ iovs. 17- 23651 65. Yang Y-L, Liu DD, Hsieh N-K, Chen HI (2008) Endothelin and gelatinases in renal changes following blockade of nitric oxide synthase in hypertensive rats. Chin J Physiol 51(186–95):0304–4920 66. Yin H, Lu Q, Wang X etal (2019) Tissue-derived microparticles reduce inflammation and fibrosis in cornea wounds. Acta Biomater 85:192–202. https:// doi. org/ 10. 1016/j. actbio. 2018. 12. 027 67. Zambrano S, Blanca AJ, Ruiz-Armenta MV etal (2013) The renoprotective effect of L-carnitine in hypertensive rats is mediated by modulation of oxidative stress-related gene expression. Eur J Nutr 52:1649–1659. https:// doi. org/ 10. 1007/ s00394- 012- 0470-x 68. Zambrano S, Blanca AJ, Ruiz-Armenta MV etal (2013) L-Carni- tine protects against arterial hypertension-related cardiac fibrosis through modulation of PPAR-γ expression. Biochem Pharmacol 85:937–944. https:// doi. org/ 10. 1016/j. bcp. 2012. 12. 021 69. Zhang S, Gu H, Hu N (2015) Role of peroxisome proliferatoractivated receptor γ in ocular diseases. J Ophthalmol 2015:1–10. https:// doi. org/ 10. 1155/ 2015/ 275435 70. Zhang Y, Murugesan P, Huang K, Cai H (2020) NADPH oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets. Nat Rev Cardiol 17:170–194. https:// doi. org/ 10. 1038/ s41569- 019- 0260-8 71. Zhang J, Wang S, He Y etal (2020) Regulation of matrix metalloproteinases 2 and 9 in corneal neovascularization. Chem Biol Drug Des 95:485–492. https:// doi. org/ 10. 1111/ cbdd. 13529 Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. 932 Á. Santana-Garrido et al.