ABCA1 Deletion Does Not Affect Aqueous Humor Outflow Function in Mice

Background ATP binding cassette transporter A1 (ABCA1) is a candidate gene within a POAG susceptibility locus by GWAS analysis, and it is involved in IOP modulation via the Cav1/eNOS/NO signaling pathway. We aim to examine the phenotype of ABCA1 deletion in the ABCA1 gene knockout (Abca1−/−) mice. Methods The anterior segments of Abca1−/− eyes were imaged by slit-lamp microscopy and anterior segment OCT. IOPs were measured by rebound tonometry. By perfusing enucleated eyes at various pressures, the aqueous humor outflow facility was determined. The mRNA expressions of ABCA1, Cav1, and eNOS were measured by RT-qPCR. The protein expressions were analyzed by western blot and immunofluorescence staining. Results There was no significant difference in the anterior segment morphology of Abca1−/− mice. IOP and aqueous humor outflow facility did not change in Abca1−/− mice compared with wild-type mice. mRNA and protein expressions of ABCA1 were significantly lower in the outflow tissue of Abca1−/− eyes. The expressions of Cav1 and eNOS were both significantly upregulated in the outflow tissue of Abca1−/− eyes. Conclusion ABCA1 deletion does not affect IOP and aqueous humor outflow function but the Cav1/eNOS/NO pathway is changed in Abca1−/− mice. The function of ABCA1 in aqueous humor outflow still requires further research.


Introduction
Glaucoma is an irreversible and progressive degenerative disease of the optic nerve, and it is the second blinding eye disease in the world [1,2].Primarily inherited as a complicated trait, primary open-angle glaucoma (POAG) is one of the most common types of glaucoma in the world.Te main risk factor for POAG is elevated intraocular pressure (IOP) [3], which is characterized by the increased resistance to drainage of aqueous humor [4].
ATP binding cassette transporter A1 (ABCA1) is a member of a large superfamily of ABC transmembrane transporters that facilitates the efux of cholesterol to lipidfree apolipoproteins A-I and E [5].As a transmembrane protein that is widely expressed in many tissues, ABCA1 may have a variety of functions and contribute to the pathophysiology of numerous diseases [6,7].Te reverse cholesterol transport (RCT) process is its most researched function [8].Additionally, ABCA1 regulates the plasma membrane's levels of phospholipids and cholesterol and plays a role in particle production and cell signal transduction [9,10].
We and other groups have found that ABCA1 is a potential gene for the POAG susceptibility locus [11][12][13].
To further understand the function of ABCA1 in controlling IOP and aqueous humor drainage In Vivo, this study examined the phenotype of anterior segment morphology and aqueous humor outfow function of Abca1 −/− mice.

Materials and Methods
2.1.Animals.Our study complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and was approved by the Eye and ENT Hospital Ethics Committee for Animal Experiments (IACUC-DWZX-2021-002).Abca1 −/− mice were purchased from the Model Animal Research Center (Nanjing University, Nanjing, China).Tese genetically modifed mice's preceding generations have been described [16].Wild-type (WT) DBA/2 mice were used as controls.Male mice aged 6-8 weeks were used in this study.Mice were bred and housed professionally in the animal house of the Eye and ENT hospital, Fudan University.

Slit-Lamp Microscopic Examination.
Because of the next noncontact procedures, mice were hypnotized by intraperitoneal injections of 10 mL/kg of 4% chloral hydrate before examination.Te slit-lamp microscopic examination (KSL-H3, Keeler, Windsor, UK) required two researchers.One ensured the position of the anesthetized mouse and the other observed and photographed the cornea, iris, conjunctiva, and anterior chamber of mice.

Anterior Segment Optical Coherence Tomography (AS-OCT).
Mice were examined by AS-OCT (Optovue, Fremont, CA) after slit-lamp microscopic examination under hypnotized status.We measured the central anterior chamber depth from the images recorded.After examination, the mice were placed on the thermal blanket until awakening from hypnosis.

IOP Measurement
. By using rebound tonometry (TonoLab; ICare, Espoo, Finland), we measured the IOPs of live mice.IOPs were measured every day between 9:00 and 11:00 AM without anesthesia.For approximately 2 weeks before the experiments, the mice were acclimatized for IOP measurements.Te researcher used one hand to gently keep the mouse's body still, and IOPs were measured by the other hand.Te average of the three measurements of IOP was used as the IOP measurement for each eye.

Mouse Eye Perfusion.
By perfusing enucleated mouse eyes, the aqueous humor outfow facility was determined.Our lab developed the experimental setup, which was extensively detailed elsewhere [17].Briefy, a 33-gauge bevelled tip needle (stainless steel tip material; Nanofl; World Precision Instruments, Stevenage, UK) was used to cannulate the eye.Te needle tip was placed in the anterior chamber.Te eye was preperfused for 10 minutes under the pressure of 10 mmHg.Ten the eye was repeatedly perfused for 15 minutes at four diferent pressures (6, 9, 11, and 16 mmHg).Te measurement was performed twice for each pressure.At equilibrium, we assume that the total infow rate and total outfow rate are equal.Goldmann's equation was used to compute the conventional outfow facility (C con ): F � (IOP − EVP) C con + F u , where IOP is the perfusion pressure, episcleral venous pressure (EVP) equals zero due to the enucleated eyes, C con is the conventional outfow facility, and F u is the unconventional outfow rate.On a fow rate-pressure response graph, a regression was ftted, and its slope was C con .
2.6.Western Blot.Mice were killed by neck dislocation, and within 5 minutes of death, the eyes were enucleated.Outfow tissue was dissected under a microscope.Trabecular meshwork (TM), Schlemm's canal, and perhaps some iris root were present in the dissected tissue.RIPA lysate (Beyotime, Shanghai, China) was used for preparing the outfow tissue samples.Te Bradford method was used to estimate the protein concentration.By 12-20% gradient sodium dodecylsulfate polyacrylamide gel electrophoresis, equal amounts of protein (20 μg protein/lane for mouse outfow tissue each) were separated.Electrophoresis was used to transfer the isolated proteins to PVDF membranes.Te membranes were blocked using 5% nonfat dry milk in TBST (Sigma-Aldrich) at room temperature for 1 hour.Ten, membranes were incubated using primary antibodies that specifcally recognized ABCA1 (1 : 1000; Abcam, ab66217), eNOS (1 : 1000; Abcam, ab199956), Cav1 (1 : 1000; Cell Signaling Technology, #3238), or β-actin (1 : 3000; Cell Signaling Technology, 3700).β-actin was used as a loading control.Ten blots were incubated with the secondary antibodies for 90 minutes.Te blots were visualized by chemiluminescence.

RT-qPCR.
Te total RNA of outfow tissue was extracted using Tissue RNA Purifcation Kit (EZB, RN5).And mRNA was reverse transcribed using RT Mix Kit with gDNA clean for qPCR (ACCURATE BIOLOGY, AG11728).Next, quantitative real-time PCR was performed using SYBR Green Premix Pro Taq HS Kit (ACCURATE BIOLOGY, AG11701).PCR procedure was as follows: step 1, 30 sec was at 95 °C; step 2, 40 cycles, done for 5 sec at 95 °C, 30 sec at 60 °C and collected fuorescent signals (Bio-Rad).Te reference gene was GAPDH.Te mRNA's relative expression levels were analyzed using the 2 −ΔΔCq method.

H&E Staining.
Ocular sections were stained with hematoxylin solution for 5 min and then rinsed in distilled water.After being dipped in 1% acid ethanol and rinsed in distilled water, the sections were stained with eosin solution for 5 min, then dehydrated with graded alcohol, and cleared in xylene.An inverted confocal microscope (Leica) was used for imaging.

Te IOP of Abca1
−/− Mice.Our previous research showed after anterior chamber injection of the ABCA1 agonist GW3965 in mice, IOP could be lowered [14].In this study, we tried to investigate the IOP of Abca1  2(a)).

Expressions of ABCA1 and Related
Protein.We investigated ABCA1 and related protein expressions in the outfow tissue of Abca1 −/− , Abca1 +/− , and WT eyes.As expected, RT-qPCR and western blot analysis showed mRNA and protein expressions of ABCA1 in Abca1 −/− eyes were signifcantly lower than those in WT eyes (Figures 3(a) and 3(d)).Ten we measured the downstream-related protein expressions as the previous study published [14].
Te mRNA expressions of endothelial NO synthase (eNOS) and caveolin-1 (Cav1) were both signifcantly upregulated in the outfow tissue of Abca1 −/− eyes compared to WT eyes by RT-qPCR analysis (p < 0.05, Figures 3(e) and 3(f )).Furthermore, the protein expressions of eNOS and Cav1 were also signifcantly increased in the outfow tissue of Abca1

Discussion
Our study examined the anterior segment in morphology and aqueous humor outfow function in Abca1 −/− eyes.Te morphology and aqueous humor outfow did not signifcantly change in Abca1 −/− eyes compared to WT eyes.Next, we tried to investigate how ABCA1 was involved in IOP regulation and aqueous humor outfow in the mouse model.Our previous study [14] has found that ABCA1 upregulation in angular aqueous plexus (AAP) cells can decrease Cav1 expression and increase eNOS expression.Conversely, ABCA1 downregulation can increase Cav1 expression and decrease eNOS expression.According to the RT-qPCR, WB, and IF results of our study, Abca1 −/− mice lacked the expression of ABCA1 and the expression of Cav1 in outfow tissue was upregulated compared to WT mice.However, the expression of eNOS was also upregulated in Abca1 −/− mice, which was diferent from the above-mentioned result [14].Our study showed that ABCA1 deletion increased Cav1 expression.ABCA1 and Cav1 proteins colocalize in cellular membranes in mouse peritoneal macrophages and Cav1 participates in the distribution of ABCA1 within the plasma membrane [18].By interacting with ABCA1, Cav1 promotes ABCA1 internalization and degradation [19].In aortic endothelial cells, ABCA1 expression is regulated by Cav1.Suppression of Cav1 decreased ABCA1 expression and reduced cholesterol efux [20].ABCA1 overexpression stabilizes Cav1 in colorectal cancer, leading to increased invasive capacities [21].All the data above indicated ABCA1 and CAV1 can be regulated by each other; however, their underlying mechanism is still unclear.
Te regulation of eNOS is complex.A direct proteinprotein interaction between eNOS and Cav1 causes a decrease in NO release [22,23].Studies using a reconstituted cell culture reveal that eNOS and Cav1 co-transfected cells emit less NO than eNOS alone [24].Cav1 residues 82-101 carry the functional inhibitory impact on eNOS, whereas the F92 residue is in charge of eNOS inhibition [22,23].In endothelial cells, Cav1 siRNA can reduce eNOS protein expression in association with increasing eNOS phosphorylation and nitrate production, which is reversed in cells overexpressing Cav1.So Cav1 maintains eNOS expression and regulates its activity [25].Also, it has been reported that eNOS knockout mice have elevated IOP and their conventional drainage is signifcantly lower [26], which is consistent with human evidence revealing glaucoma-related polymorphisms in the eNOS gene [27][28][29].And Cav1 defciency results in increased eNOS activity, which means the control of IOP requires the interplay of eNOS and Cav1 [30,31].In our study, we found ABCA1 deletion increased Cav1 expression, which should decrease eNOS expression based on the research stated above, but we saw an increase in eNOS in our ABCA1 knockout mouse model.Te reason for this discrepancy might be the increased Cav1 is not fully functional and the dysfunction of Cav1 leads to the increase in eNOS.So the outfow rate of aqueous humor did not decrease and IOP was not elevated in Abca1 −/− mice with the increased eNOS activity.
Compared with previous research [14], we edited the genome for ABCA1 gene knockout instead of RNA interference(RNAi)-mediated knockdown using short hairpin RNA (shRNA).ABCA1 is one of the several proteins involved in cholesterol homeostasis [5].Numerous organs may be impacted by ABCA1 deletion, which also plays a role in the pathophysiology of a wide range of disorders.So eliminating ABCA1 gene function might cause changes in homeostasis.Moreover, In Vivo models seemed to be a more complex system than In Vitro studies and functional protein knockout in the whole body caused diferent results compared to using the ABCA1 agonist or lentiviral ABCA1-shRNA in the cell studies.Te use of In Vivo models enables the investigation of whole-organism complexity because the visual system and other body systems (immune function, nervous system, etc.) are intact [32].Glaucoma is a complex systemic disease so physiological responses should involve many cell types and extracellular efects [2].Now it is still not clear how ABCA1 afects aqueous humor outfow.At present, there are few studies on the function and mechanism of ABCA1 in glaucoma.Understanding the physiological and pathological involvement of ABCA1 in glaucoma and the potential benefts of targeting ABCA1 for the treatment of glaucoma still require more research.
In our study, we employed chloral hydrate as the sole sedative in two procedures: slit-lamp microscopic examination and AS-OCTscanning, because both procedures were noncontact and noninvasive in nature.Intraperitoneal administration of chloral hydrate could result in adverse efects such as adynamic ileus, gastric ulcers, and peritonitis in laboratory animals [33].We observed no systemic adverse efects following intraperitoneal administration of chloral hydrate in our research.Additionally, no abnormal signs were noted upon awakening from the induced state of hypnosis.

Conclusion
ABCA1 deletion does not afect IOP and aqueous humor outfow function but the Cav1/eNOS/NO pathway is changed in Abca1 −/− mice.