The HAT Inhibitor ISOX‐DUAL Diminishes Ischemic Areas in a Mouse Model of Oxygen‐Induced Retinopathy

ABSTRACT

Retinal ischemic disease results in significant visual impairment due to the development of fragile and disorganized, pathologically running blood vessels in the eye. Currently, the mainstay treatment for this disease is the intravitreal administration of anti‐VEGF drugs targeting vascular endothelial growth factor (VEGF), which induces angiogenesis. However, current anti‐VEGF drugs do not diminish the ischemic areas that lead to angiogenesis, making fundamental treatment challenging. Since retinopathy is an acquired disease caused by hypoxic stimulation from ischemia, we paid particular attention to histone acetylases. We conducted a drug screening experiment using a mouse model of oxygen‐induced retinopathy (OIR), which replicates retinal ischemic disease, through the intraperitoneal administration of 17 distinct inhibitors targeting histone acetyltransferases (HAT). The results indicated that, among the 17 inhibitors, only ISOX‐DUAL decreased neovascularization and ischemic regions. Furthermore, microarray analysis was conducted on the drug‐treated samples to refine genes altered by the administration of ISOX‐DUAL. There were 21 genes associated with angiogenesis, including Angpt2, Hmox1, Edn1, and Serpine1, exhibited upregulation in OIR mice and downregulation following treatment with ISOX‐DUAL. Furthermore, STRING analysis confirmed that the aforementioned four genes are downstream factors of hypoxia‐inducible factors and are assumed to be important factors in retinal ischemic diseases.

ISOX DUAL was intraperitoneally administered to OIR mice for 5 days starting from P12, and retinas were excised at P17 to create flat mounts. The percentage of ischemic and neovascular areas relative to the total area was calculated, and a decrease in ischemic and neovascular areas was observed following ISOX DUAL administration (b) compared with controls (a).

1. Introduction

The common, underlying cause of ischemic retinal diseases such as proliferative diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and neovascular glaucoma that lead to acquired blindness is ischemia due to vascular occlusion of the retina (Ding and Wong 2012; Duh, Sun, and Stitt 2017; McAllister 2012; Neo et al. 2020; Senthil et al. 2021; Takamura, Yamada, and Inatani 2023). Vascular endothelial growth factor (VEGF), whose intraocular levels increase as areas of no perfusion expand, plays a central role in the pathogenesis of ischemic retinal disease by enhancing vascular permeability and promoting the growth of neovascular vessels (Ahmad and Nawaz 2022). Intravitreal administration of anti‐VEGF drugs can ameliorate macular edema and regress neovascularization by inhibiting both hyperpermeability and angiogenesis (Higashiyama et al. 2013; Kreutzer et al. 2008; Kriechbaum et al. 2008). Anti‐VEGF treatment results in the disappearance of edema and improvement in vision, but multiple injections must be continued in order to maintain these anatomic and functional improvements. Anti‐VEGF medications are costly and place a substantial economic burden on both patients and society's healthcare systems (Chakravarthy et al. 2013; Mintz‐Hittner et al. 2011). The ischemic area is a source of VEGF (Apte, Chen, and Ferrara 2019; Gozawa et al. 2017), but importantly, existing treatments such as anti‐VEGF drugs do not result in reduction of retinal ischemia itself, as shown by analysis using optical coherence tomography (OCTA) (Chatziralli et al. 2022; Ritter et al. 2006). Laser photocoagulation is employed to impede the progression to proliferative changes in ischemic areas; however, it can lead to damage in retinal choroidal tissue, resulting in nerve atrophy and constriction of the visual field (Cui et al. 2009; Stefansson 2001). Anti‐VEGF therapy is a symptomatic treatment that suppresses VEGF produced from ischemic areas, and its current limitation is that it cannot reduce the ischemic areas themselves. In place of anti‐VEGF drugs, new treatments are required that reduce the underlying cause, ischemia, and promote normal vascular growth.

Seven VEGF ligands have been identified: VEGF‐A, B, C, D, E, placental growth factor (PlGF), and snake venom VEGF (svVEGF). Among these, VEGF‐A selectively acts on vascular endothelial cells and is most deeply involved in angiogenesis. In humans, VEGF‐A exists in four isoforms: 121 (VEGF‐A121), 165 (VEGF‐A165), 189 (VEGF‐A189), and 206 (VEGF‐A206) amino acids in length. VEGF‐A165, in particular, is the most abundant among its isoforms and is considered highly physiologically active, thus strongly implicated in pathological angiogenesis. VEGF‐A binds to the receptors VEGFR‐1 and VEGFR‐2, promoting lumen formation, endothelial cell migration, and affecting pathological neovascularization. While VEGF promotes normal vessel growth during developmental stages, it is also induced by ischemia and contributes to abnormal angiogenesis. In normal angiogenesis, blood vessels extend horizontally across the retina. In contrast, neovascular vessels formed due to pathological vascular occlusion extend from the retina into the vitreous cavity, leading to proliferative membranes and vitreous hemorrhage. In pathological conditions, hypoxic stimulation induces abnormal angiogenesis by triggering the overproduction of endothelial cells through excessive secretion of VEGF. Pathological angiogenesis extends toward the vitreous cavity rather than into the retina, where it should properly grow, leading to vitreous hemorrhage, hyperproliferation, and retinal detachment due to traction. Though both exhibit retinal vascular elongation, the mechanisms underlying this elongation differ significantly. Hence, it is crucial to elucidate the molecular distinctions in physiological and pathological vascular elongation. It has been reported that senescent cells accumulate in the retinas of patients with diabetic retinopathy and are implicated in pathological angiogenesis in animal models of retinopathy. UBX1967, a small molecule compound targeting senescent cells, led to a reduction in retinal ischemic areas in animal models of retinopathy. UBX1967 has been demonstrated to selectively inhibit the apoptotic pathway, suppress pathological angiogenesis, and concurrently promote physiological vascular repair in retinopathy (Crespo‐Garcia et al. 2021). Moreover, CITED2, a transcript of hypoxia‐inducible factor (HIF), is known to negatively regulate the hypoxic response by binding to CBP/p300 and competing allosterically with HIF‐1α (Berlow, Dyson, and Wright 2017; Bhattacharya et al. 1999). The coadministration of CITED2 peptides and anti‐VEGF drugs in animal models of retinopathy resulted in the inhibition of pathological angiogenesis and the reduction of ischemic areas (Usui‐Ouchi et al. 2020). It is possible that the CITED2 peptide may modulate the hypoxic response to CBP/p300 through direct competition with HIF, resulting in the reduction of ischemic areas, but the detailed molecular mechanism is not clear.

In this study, we focused on histone acetylation, a well‐known epigenetic regulatory mechanism (Ling and Ronn 2019; Marmorstein and Zhou 2014). Histone acetyltransferases (HAT) acetylate histones, relaxing chromatin structure, which allows transcription factors to bind readily to DNA and activate gene transcription. The HAT family is classified into three main groups: the GNAT (Gcn5‐related N‐acetyltransferase) family, MYST (Moz, Ybf2/Sas3, Sas2, Tip60) family, and CBP (CREB‐binding protein)/p300 family (Kanada et al. 2019). It has also been reported that bromodomain protein 4 (BRD4) functions to activate HAT (Nagaya, Yamaoka, et al. 2022). We have previously demonstrated that epigenetic regulation of gene expression is implicated in another ocular condition, cataract (Hillyar, Rallis, and Varghese 2020; Kanada et al. 2019; Manzo et al. 2009; Nagaya, Yamaoka, et al. 2022). The ischemia observed in retinopathy under pathological conditions and the anoxic state in physiological conditions share a common hypoxic response. However, the morphology of the blood vessels that form after the hypoxic response differs between physiological and pathological conditions (Moore and Christoforidis 2023). This disparity may be regulated by epigenetic mechanisms. We believe that elucidating the regulatory mechanism mentioned above could contribute to restoring pathological vascular structures to a physiological state. While previous studies have explored the role of histone acetyltransferase (HAT) in retinal development and disease, the specific epigenetic mechanisms in ischemic retinal diseases have not been fully elucidated (Wang et al. 2023).

Mice, rats, and rabbits have been employed as animal models in retinopathy research (Khayat et al. 2017; Olivares et al. 2017). Specifically, the retinal vein occlusion (RVO) model, where retinal veins in mice and rabbits are occluded through laser photocoagulation (Fuma et al. 2017; Neo et al. 2020). In the pathogenesis of diabetic retinopathy (DR), prolonged hyperglycemia induces hyper‐vascular permeability and vascular occlusion. Although the animal models administrated with streptozotocin show hyperglycemia, human‐like typical DR is not easily induced (Olivares et al. 2017; Yin et al. 2023). Akimba mice, generated by crossing a hyperglycemic model of Akita mice and VEGF‐overexpressing Kimba mice exhibits pericyte and vessel loss and retinal neovascularization with diffuse vascular leakage that is observed in late stage of DR (Wisniewska‐Kruk et al. 2014). In this study, we utilized oxygen‐induced retinopathy (OIR) mice. These mice were chosen due to their retinal structure being relatively similar to that of humans, their ease of breeding and reproduction, and their capability to serve as a highly reproducible experimental system that can simulate retinopathy in a short and straightforward manner (Connor et al. 2009; Smith et al. 1994; Stahl et al. 2010).

2. Results

2.1. Drug Screening Experiments Involving Intraperitoneal Administration Demonstrated Therapeutic Efficacy Exclusively With ISOX‐DUAL

We initially investigated whether the regulation of epigenetic mechanisms could alleviate abnormal vessel formation and the development of vascular‐free areas during the pathogenesis of retinal ischemic disease. The OIR mouse was employed as a model animal that can easily reproduce retinal ischemic disease in a short period of time. Mice are exposed to hyperoxia (75% O₂) in a chamber for 5 days starting on postnatal day 7 (P7) to induce ischemic regions. Subsequently, upon returning them to room air, hypoxic stimulation induces neovascularization around the ischemic area. OIR mice were employed in an intraperitoneal drug screening experiment. Intraperitoneal administration was performed once a day for a total of 5 days starting on P12, immediately after the end of hyperoxia (Figure 1a). At 17 days of age, when the neovascularized area (NV) was at its peak, retinas were extracted from the mice, immunostained with isolectin, and retinal flat‐mount specimens were prepared. In this study, our focus was on HAT, one of the factors regulating epigenetic expression that has shown preventive effects in studies of cataracts, an eye disease (Kanada et al. 2019). We explored whether intraperitoneal administration of HAT inhibitors could diminish neovascularization formation and mitigate the development of avascular areas. The 17 HAT inhibitors utilized in this study, along with their target factors and the corresponding concentrations, are listed in Table S1. Intraperitoneal drug screening results confirmed that only ISOX‐DUAL, inhibiting both p300/CBP and BRD4, predominantly reduces ischemic regions and NV areas (Figures 1b–d and S1). Equal variance tests were performed to evaluate differences between the OIR and ISOX DUAL. If there was equal variance, a two‐tailed Student t‐test was used; if there was no equal variance, a two‐tailed Welch t‐test was used. Statistical analysis was performed using Microsoft Office Excel; p < 0.05 was considered statistically significant (Nagaya, Kanada, et al. 2022).

FIGURE 1.

Exploration of the therapeutic efficacy of intraperitoneal administration of ISOX‐DUAL. (a) Schematic of the intraperitoneal administration experiment utilizing OIR mice: P7 mice were subjected to hyperoxia for 5 days and subsequently reintroduced to room air. The drug was administered intraperitoneally once daily from P12 to P16, and extraction was conducted on P17. (b) Retinas were extracted at P17, fixed in 4% paraformaldehyde, and retinas were prepared as flat mounts. After examination with a fluorescence microscope, the regions of neovascularization (NV) were delineated manually in pink using ImageJ, while ischemic regions were outlined in blue. (c) The percentage of NV was assessed by delineating the NV area with ImageJ, calculating the total area, and comparing it to the overall retinal area. (d) The percentage of ischemic areas was determined by delineating the ischemic regions with ImageJ, calculating the total area, and comparing it to the overall retinal area. The data are presented as the mean ± SE. * denotes significance at p < 0.05 compared with the Control. All results are shown in Data S3.

2.2. Therapeutic Efficacy Was Also Demonstrated With the Vitreous Administration of ISOX‐DUAL

Intraperitoneal administration is likely to significantly dilute the concentration of the drug reaching the retina, and the drug may affect the entire body, not just the retina. Therefore, to further investigate the impact of ISOX‐DUAL on the retina, vitreous administration of ISOX‐DUAL were conducted. As in the previous intraperitoneal administration, OIR mice were utilized. For vitreous administration, Since the optimal concentration was unknown, concentrations of 10 μM, 150 μM, and 1 mM were tested, respectively. ISOX‐DUAL (10 μM, 150 μM, 1 mM) was injected into OIR mice only once on P 12, immediately after the end of hyperoxia. Mice were subsequently reared in room air, and retinas were harvested at P17. Following immunostaining with Isolectin, retinal flat‐mount specimens were prepared (Figure 2a). Fluorescence microscopy revealed that vitreous administration of ISOX‐DUAL (10, 150 μM) significantly decreased the number of neovascularizations. In addition, the vitreous administration of ISOX‐DUAL (1 mM) showed a tendency toward a reduction in NV, although the difference was not statistically significant (Figures 2b,c and S2). In the ischemic area, a significant decrease was observed with the administration of ISOX‐DUAL (10 μM, 150 μM, 1 mM) into the vitreous body (Figures 2b,d and S2). Our experiments demonstrated that ISOX‐DUAL, unlike existing anti‐VEGF drugs, not only suppresses areas of NV but also diminishes ischemic regions. The lowest concentration tested, 10 μM, which showed a significant effect, was chosen for further experiments.

FIGURE 2.

Investigation of the therapeutic efficacy of ISOX‐DUAL in vitreous administration. (a) Schematic of the vitreous administration experiment using OIR mice: P7 were exposed to hyperoxia for 5 days and then returned to room air. A single dose of ISOX‐DUAL was administered to the vitreous at P12, and extraction was conducted at P17. (b) Retinas were extracted at P17, fixed in 4% paraformaldehyde, and retinas were prepared as flat mounts. After examination with a fluorescence microscope, the regions of NV were delineated manually in pink using ImageJ, while ischemic regions were outlined in blue. From left to right: Control, ISOX‐DUAL (10 μM), (150 μM), (1 mM). (c) The percentage of NV was assessed by delineating the NV area with ImageJ, calculating the total area, and comparing with to the overall retinal area. (d) The percentage of ischemic areas was determined by delineating the ischemic regions with ImageJ, calculating the total area, and comparing it to the overall retinal area. The data are presented as the mean ± SE. * denotes significance at p < 0.05 compared with the Control. All results are shown in Data S4.

2.3. Analysis of Factors Involved in the Development of Retinopathy Using Microarrays

We conducted a microarray analysis to identify genes whose expression varied during the formation of NV and ischemic regions. Control samples (n = 2), OIR samples (n = 2), and vitreous administration samples of ISOX‐DUAL (10 μM) at P17 (n = 2) were employed to identify genes associated with retinal ischemic disease. Initially, the expression level data for all genes were averaged within each sample. Genes with low expression levels (signal values less than 5) were excluded to enhance detectability in subsequent RT‐qPCR and to reduce the number of genes. We identified 120 genes that exhibited a 1.5‐fold increase in expression in the OIR sample compared to the control sample and a 1.5‐fold decrease in expression in the ISOX‐DUAL‐treated sample compared with the OIR sample (Figure 3a and Data S1). A heat map was created for the selected 120 genes (Figure 3b). The dendrogram to the right of the heatmap indicates that the transcriptional profile of the ISOX‐DUAL‐treated sample is more aligned with the expression state of the control sample than that of the OIR sample. The HAT inhibitor, ISOX‐DUAL, has the potential to transition retinal neovascularization from a pathological state to a physiological state at the gene expression level. To identify the genes to prioritize next, we classified the functions of the 120 genes by conducting a Gene Ontology (GO) analysis in AmiGO (Data S2). As a result, we identified numerous factors (26 genes) associated with angiogenesis, and further research was conducted focusing on these 26 genes.

FIGURE 3.

Narrowing down genes with potential therapeutic targets through microarray analysis. We used a sample size of n = 2 for the microarray analysis. (a) The Venn diagram depicting genes whose expression increased in OIR samples and decreased upon treatment with ISOX‐DUAL. OIR_up comprises 411 genes with a signal value increased 1.5‐fold in OIR mice compared to the control, as indicated by the microarray results. Isox_down consists of 389 genes with signal values decreased 1.5‐fold in OIR mice treated with ISOX‐DUAL. (b) The heatmap illustrating alterations in the expression of 120 genes, which exhibited an increase in the OIR sample and a decrease upon ISOX‐DUAL treatment, as previously confirmed in the Venn diagram. Signal values, in order of increasing intensity, are indicated by white, light blue, and blue.

2.4. Identification of Factors in Retinopathy Angiogenesis

For the 26 identified genes, transcript levels were measured using RT‐qPCR for quantitative analysis. Experiments were performed using at least n = 3 control samples, OIR samples, and ISOX DUAL vitreous administration samples at P17. The results of RT‐qPCR indicated that 21 genes exhibited patterns consistent with the increasing and decreasing trends identified in the microarray analysis (Figure 4a). These 21 genes were suggested to be involved in angiogenesis in retinal ischemic diseases. For the 21 genes, we explored the protein‐level associations using STRING analysis (Figure 4b) (Table 1). The analysis revealed that all genes, except for Anxa3, interacted with each other at the protein level. Among the noteworthy factors, four genes Angpt2, Edn1, Hmox1, and Serpine1 were identified as downstream components of the hypoxia‐inducible factor HIF‐1 signaling. Additionally, eight genes Cdh5, Cd34, Eng, Fn1, Itga1, Mcam, Thbs1, and Tnfrsf12a have been identified as factors contributing to cell adhesion (Figure 4b). These factors were found to be likely causative factors in retinal ischemic disease. Furthermore, as anti‐VEGF drugs are currently employed in therapy, the ISOX‐DUAL sample was assessed through RT‐qPCR to explore its dependence on the VEGF pathway. The measurements revealed that the expression level of Vegfa in the ISOX‐DUAL‐treated samples remained constant (Figure 4c). At the transcriptional level, ISOX‐DUAL may have exerted its therapeutic effect without inhibiting the VEGF pathway.

FIGURE 4.

RT‐qPCR results for genes obtained from the analysis using Amigo. (a) RT‐qPCR was conducted on genes categorized under positive regulation of angiogenesis, angiogenesis, and vascular processes in the circulatory system based on the results obtained from Amigo. The expression levels of the 21 genes (Table 1), whose expression trends were consistent with the microarray, were normalized based on Actβ expression. Data were expressed as mean ± SE. The results for n = 3 or more samples drawn independently for all samples. p values < 0.05 and 0.01 were categorized and marked with an asterisk (*, **) for each. (b) STRING was used to confirm protein–protein interactions for 21 genes (https://string‐db.org/). Due to the presence of numerous orthologs between humans and mice, and with consideration for future applications in humans, humans were chosen as the organism. The four genes encircled by black‐patterned circles are postulated to be downstream targets of hypoxia‐inducible factors, while the eight genes encircled by solid black circles are associated with cell adhesion. (c) RT‐qPCR results for Vegfa. mRNA levels were normalized against Actβ expression. Data are expressed as mean ± SE. Equal variance tests were performed and no statistically significant differences were identified.

TABLE 1.

List of genes assumed to be pathological factors of retinopathy.

Gene symbol	Gene name	GO biological process complete	Function
Acta2	Actin alpha 2	Vascular process in circulatory system (GO:0003018)	Cell motility
Adm	Adrenomedullin	Positive regulation of angiogenesis (GO:0045766), vascular process in circulatory system (GO:0003018)	Vasodilation, promotion of angiogenesis
Angpt2	Angiopoietin 2	Positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525)	Inflammation‐related signaling pathways
Anxa2	Annexin A2	Angiogenesis (GO:0001525)	Regulation of cell proliferation
Anxa3	Annexin A3	Positive regulation of angiogenesis (GO:0045766)	Regulation of cell proliferation
Apln	Apelin	Angiogenesis (GO:0001525), vascular process in circulatory system (GO:0003018)	Fluid homeostasis, cardiovascular function, insulin secretion
Cd34	CD34 antigen	Positive regulation of angiogenesis (GO:0045766)	Cell adhesion
Cdh5	Cadherin 5	Positive regulation of angiogenesis (GO:0045766), vascular process in circulatory system (GO:0003018)	Cell adhesion
Edn1	Endothelin 1	Angiogenesis (GO:0001525), vascular process in circulatory system (GO:0003018)	Vasoconstriction
Ednra	Endothelin receptor type A	Angiogenesis (GO:0001525), vascular process in circulatory system (GO:0003018)	Vasoconstriction
Eng	Endoglin	Positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525)	Regulation of vascular endothelial cell migration
Esm1	Endothelial cell‐specific molecule 1	Angiogenesis (GO:0001525)	Angiogenesis
Fn1	Fibronectin 1	Angiogenesis (GO:0001525)	Cell adhesion
Hmox1	Heme oxygenase 1	Positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525)	Heme oxygenase
Itga1	Integrin alpha 1	Vascular process in circulatory system (GO:0003018)	Cell adhesion
Loxl2	Lysyl oxidase‐like 2	Angiogenesis (GO:0001525)	Lysyl oxidase
Mcam	Melanoma cell adhesion molecule	Angiogenesis (GO:0001525)	Cell adhesion
Nr4a1	Nuclear receptor subfamily 4, group A, member 1	Angiogenesis (GO:0001525)	Inflammation
Serpine1	Serine (or cysteine) peptidase inhibitor, clade E, member 1	Positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525)	Serine protease inhibitor
Thbs1	Thrombospondin 1	Positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525)	Cell adhesion
Tnfrsf12a	Tumor necrosis factor receptor superfamily, member 12a	Angiogenesis (GO:0001525)	Apoptosis

3. Discussion

Therapies targeting pathological angiogenesis are effective for diseases associated with angiogenesis, such as ischemic retinopathy and cancer (Apte, Chen, and Ferrara 2019). Anti‐VEGF drugs, utilized in the treatment of ischemic retinopathy, can inhibit the growth of neovascularization, but they do not reduce ischemic areas. However, administering a VEGF antagonist to a mouse model of retinopathy leads to an enlargement of the ischemic area (Zhang et al. 2011). In the current intraperitoneal drug screening experiments, ISOX‐DUAL, inhibiting p300/CBP and BRD4, suppresses neovascularization and reduces the ischemic area (Figure 1b–d). However, therapeutic effects were not observed with other HAT inhibitors. Some of these inhibitors targeted p300/CBP, but the inhibition of p300/CBP did not suppress neovascularization or reduce ischemic areas. ISOX‐DUAL is a dual‐targeted small molecule inhibitor of both BRD4 and p300/CBP. Among the inhibitors used in this study, only ISOX‐DUAL inhibits BRD4, suggesting that simultaneous inhibition of BRD4 and p300/CBP is crucial for suppressing neovascularization and reducing ischemic areas in ischemic retinal disease (Chekler et al. 2015). BRD4 is a known member of the bromodomain and extra‐terminal (BET) protein family, serving as an epigenetic regulator that plays a crucial role in embryogenesis and cancer development (Donati, Lorenzini, and Ciarrocchi 2018; Usui‐Ouchi et al. 2020). The inhibition of BRD4 using BET inhibitors is currently undergoing clinical trials as a promising treatment for malignant neoplastic diseases. This implies that the inhibition of BRD4 is crucial not only in the investigation of malignant diseases like cancer but also in ischemic retinal diseases.

STRING analysis was conducted to explore the interconnections among the 21 genes identified from the results of RT‐qPCR. Consequently, four genes Angpt2, Edn1, Hmox1, and Serpine1 were recognized as putative downstream factors regulated by the hypoxia‐inducible factor HIF‐1 (Figure 4b). Studies conducted with prostate cancer cell lines have revealed that Hmox1 is a target gene of BRD4, and a super‐enhancer associated with BRD4 is reported to be implicated in Serpine1 (Hussong et al. 2014; Stratton et al. 2016) (Figure 5). Moreover, Thbs1, identified as a factor related to cell adhesion, stimulates TGFB, leading to the induction of Serpine1 expression (Figure 4b) (Farberov and Meidan 2016). Plasminogen activator inhibitor 1 (PAI‐1), a transcript of Serpine1, disrupts the interaction among urokinase‐type plasminogen activator (uPA), uPA receptor, and vitronectin, thereby leading to vascular destabilization (Farberov, Basavaraja, and Meidan 2019). This mechanism of action is anticipated to result in vascular destabilization in ischemic retinal diseases, leading to the formation of fragile pathological vessels lacking proper orientation (Figure 5).

FIGURE 5.

Molecular model of possible disruption of vascular structure. THBS1 induces SERPINE1 expression by activating TGF‐B. Plasminogen activator inhibitor 1 (PAI‐1), a transcript of SERPINE1, disrupts the interaction between urokinase‐type plasminogen activator (uPA) and uPA receptor, as well as vitronectin, resulting in the inhibition of cell adhesion. It is conceivable that a super‐enhancer associated with BRD4 plays a role in the regulation of SERPINE1, potentially influencing the expression of SERPINE1.

Another interesting factor, Angpt2, was extracted from the STRING analysis (Figure 4b). Faricimab, an anti‐VEGF drug that also doubly inhibits ANGPT2 and VEGFA, has demonstrated efficacy in ameliorating edema and vision impairment, as well as inhibiting angiogenesis in ischemic retinal diseases, including diabetic macular edema (Regula et al. 2019). Normally, ANGPT1 binds to Tie‐2 receptors, playing a role in stabilizing the normal vascular structure by impeding vascular pericyte adhesion and endothelial cell proliferation. ANGPT2 also acts as an antagonist to ANGPT1 by binding to Tie‐2 receptors, leading to vascular destabilization, including pericyte shedding and endothelial cell proliferation. In ischemic retinal diseases, intraocular levels of ANGPT2 are elevated, implying its participation in the pathogenesis and underscoring the importance of concurrently suppressing both ANGPT2 and VEGF. Indeed, faricimab treatment robustly inhibits heightened vascular permeability and angiogenesis; however, there is no evidence indicating a reduction in the size of the ischemic zone (Akwii et al. 2019; Khalaf et al. 2017). Therefore, the diminution of the ischemic area by ISOX‐DUAL may not be attributed solely to the influence of ANGPT2 but rather to the synergistic effects of multiple factors. Four genes, Angpt2, Edn1, Hmox1, and Serpine1, identified as downstream factors of the hypoxia‐inducible factor HIF‐1, emerged as potential candidates (Figure 4b). In other words, the distinctions in vascular morphology arising from hypoxic responses under pathological and physiological conditions may be modulated by these four genes, which are factors related to HIF‐1. In the present results, a reduction in Angpt2 expression was observed in the ISOX‐DUAL‐treated samples compared with the OIR samples (Figure 4a), with no significant variation noted in Vegfa (Figure 4c). These results suggest that we believe that the inhibition of four genes, including ANGPT2, is important for retinal vascular regeneration by the administration of ISOX DUAL. In this study, among the HIF‐dependent genes, most of the genes involved in angiogenesis were suppressed. Therefore, it is likely that Vegfa expression is also suppressed by Isox‐Dual treatment, and that Vegfa overexpression may occur through a different pathway. Vegfa expression is known to be induced by inflammatory factors, growth factors, and other factors. These factors may sustain Vegfa expression by activating other transcription factors (Ferrara 2004). We also believe that VEGF functions under conditions close to physiological by inhibiting these four genes, promoting vascular regeneration.

In conclusion, we discovered that the administration of ISOX‐DUAL to mouse models of retinopathy, targeting both p300/CBP and BRD4, mitigates neovascularization and diminishes ischemic regions. Additionally, our comprehensive gene analysis suggests that HIF‐1 downstream factors, such as Angpt2, Serpine1, Hmox1, and Edn1, could serve as potential therapeutic targets for ischemic retinal disease.

Although the results of this study demonstrated that administration of ISOX‐dual elongated the blood vessels surrounding the ischemic area and reduced the ischemic area, histological and functional analyses are required to assess whether this treatment induces damage to the neural retina or other tissues and further analysis is warranted.

4. Experimental Procedures

4.1. Animals

C57BL/6J mice were procured from Sankyo Lab Services, and the OIR mouse model comprised 7‐day‐old pups and their mothers exposed to 75% oxygen for a duration of 5 days within an oxygen chamber, followed by subsequent growth in ambient air (Smith et al. 1994; Stahl et al. 2010). All mice used in this experiment were confirmed to weigh 6 g or more at P17 (Connor et al. 2009). All experiments received approval from the University of Fukui Animal Experiment Committee (Approval No. R04055) and were conducted in strict accordance with the University of Fukui Animal Experiment Regulations, as well as the guidelines outlined in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. This study was reported according to ARRIVE guidelines.

4.2. Intraperitoneal Administration

After termination of hyperoxia in the OIR mouse model, intraperitoneal administration was performed once a day for 5 days until 12 ~ 16 days of age. The mice were anesthetized by isoflurane aspiration, and 30 μL of ISOX‐DUAL (150 μM) was administered using 1 mL of TERUMO SYRINGE and UNIEVER needle 34G. Specimens were taken from the Control group (n = 3) and the Isox group (n = 5).

4.3. Intravitreal Injection

Following the cessation of hyperoxia in the OIR mouse model, a single vitreous administration was conducted on 12‐day‐old mice. The vitreous administration protocol was adopted from a prior study (Huang et al. 2017), utilizing 10 μL of NANOFIL SYRINGE and Lab Nano Needles Plus 36G, along with 1 μL of ISOX‐DUAL. Specimens were taken from the Control (n = 4), 10 μM (n = 4), 150 μM (n = 3), and 1 mM (n = 3) groups. The dosage was estimated from the vitreous volume of mice. 0.5, 1, 1.5, and 2 μL were tested, respectively, and 1 μL was administered in this experiment because 1 μL was the maximum dosage that could be administered. The optimal concentrations of 10 μM, 150 μM, and 1 mM were tested, and the lowest concentration of 10 μM was found to be effective, so a concentration of 10 μM was used in this experiment. This information was added to the experimental procedure.

4.4. Retinal Flat Mount Specimen Preparation and Immunofluorescent Staining

P17 mice were euthanized with CO₂ gas, their eyes were extracted, and the retinas were meticulously isolated. Fixed in 4% paraformaldehyde (PFA), retinal whole mounts were stained with Isolectin GS‐IB4‐Alexa 594 overnight at 4°C (Connor et al. 2009). Images were captured using FLUOVIEW FV10i or Axio Observer Z1 fluorescence microscopy and analyzed with ImageJ.

4.5. Vascular Quantification of Images

All vascular quantification was performed using Image J. Retinal specimen images captured with Axio Observer Z1 fluorescence microscopy were opened in Image J, and brightness was adjusted using the Brightness tool. The entire retina, ischemic areas, and abnormal blood vessels were manually outlined using the Polygon tool in the Area Selection Tools. Subsequently, these areas were quantified using the Measure function. The ratio of avascular area to total retinal area (avascular area %) and the ratio of neovascular tuft area to total retinal area (NV/Retina %) were calculated. The abnormal vascular area was determined by summing the values of all abnormal vessels (Ichiyama et al. 2021).

4.6. Total RNA Extraction and RT‐qPCR

RT‐qPCR analysis was performed on at least n = 3 independently extracted samples for each group. After removal of the eye and isolation of the retina, total RNA extraction and quantitative RT‐qPCR were performed as previously reported (Kanada et al. 2019; Neo et al. 2020). RT‐qPCR used SYBR Green (Applied Biosystems) and gene‐specific primers to amplify target genes and Actβ (Actin Beta). Primer sequences are specified in Table S2. Expression levels were normalized against Actβ expression levels. Statistical analysis of the RT‐qPCR results was conducted using Microsoft Excel Office. Two‐tailed Student t‐tests were employed to assess differences between the OIR group and the drug treatment group at each time point. p < 0.05 was considered statistically significant.

4.7. Microarray Analysis

Microarray analysis was conducted on a total of six samples, with two samples each from control and OIR retinas, as well as drug‐treated retinas at P17 (Kanada et al. 2019; Neo et al. 2020). The RNA integrity number (RIN) and total RNA sample concentrations were assessed before conducting the microarray analysis using an Agilent 2100 Bioanalyzer. The RIN values for the control, OIR, and drug treatment samples all exceeded 7.6. A ClariomTM D Array mouse 10 Array (Thermo Fisher Scientific) was employed, and all experimental procedures were conducted following the manufacturer's guidelines. Before analysis, the data were normalized using the RMA (Robust Multi‐array Average) algorithm in R. In addition, unnamed genes and genes identified as having low signal (maximum signal < 5.00 in all samples) were removed.

Author Contributions

Kengo Nakanishi: conceptualization, methodology, data curation, investigation, validation, formal analysis, visualization, writing – original draft, writing – review and editing. Yoshihiro Takamura: conceptualization, methodology, data curation, investigation, validation, formal analysis, funding acquisition, project administration, resources, writing – original draft, writing – review and editing. Yusei Nakano: conceptualization, methodology, data curation, investigation, validation, visualization, writing – original draft, writing – review and editing. Masaru Inatani: conceptualization, methodology, investigation, supervision, funding acquisition, project administration, resources, writing – original draft, writing – review and editing. Masaya Oki: conceptualization, methodology, data curation, investigation, validation, formal analysis, supervision, funding acquisition, project administration, resources, writing – original draft, writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Data S1. Supporting Information.

Data S2. Supporting Information.

Data S3. Supporting Information.

Data S4. Supporting Information.

Data S5. Supporting Information.

Data Availability Statement

The data that supports the findings of this study are available in the Supporting Information of this article.