Persistent hyperplastic primary vitreous (PHPV) is a rare vitreoretinal disorder which accounts for up to 5% of blindness (1). PHPV's pathophysiology occurs during the embryonic stage, with vessel development occurring in the third week of pregnancy. The hyaloid artery system expands and extends to the anterior part of the eye forming the iridohyaloid or capsulopupillary artery during this period. At this point of development, the posterior tunica vasculosa lentis, which is an anastomosis of vessels at the back of the lens begins to develop and nourishes the lens. Secondary vitreous begins to develop in place of primary vitreous during the second trimester of pregnancy. The pathological persistence of fetal intraocular vessels including the hyaloid artery in embryonic vitreous causes this congenital eye disease (2, 3). Apoptosis or macrophage activation causes hyaloidal artery regression which is accompanied by vasa hyaloidal propria, iridohyaloid, and tunica vasculosa lentis (4). White retrolental tissue, an anteriorly swollen lens, centrally dragging ciliary structures, and varying degrees of lenticular opacification are the most prominent clinical symptoms (5). These vascular remnants can hinder the normal retinal development leading to retinal detachment and optic nerve or macula anomalies, and it can also appear in anterior, lateral, or combined forms in various patients (5, 6). PHPV is usually detected in infants within the first 3 months of life due to leukocoria, microphthalmos, and strabismus (7). Figure 1 represents the typical morphology of a PHPV subject. PHPV is also known as persistent fetal vasculature (PFV) (5). Bilateral PHPV is rare and sporadic compared to unilateral PHPV; however, it is an autosomal dominant or recessive trait that may be inherited (8, 9).

A large number of genes are involved in the development and regression of the hyaloid artery. PHPV traits have been observed in human and animal models. In humans, PHPV incidence was found to be an autosomal dominant inheritance pattern in an Egyptian family (10). Mutations in the NDP gene and the COX15 gene on chromosome 10 have been found in cases of bilateral PHPV (11–14). The ZNF408 gene, which had previously been found in retinitis pigmentosa and autosomal dominant familial exudative vitreoretinopathy (ADFEVR) was also identified in PHPV cases of microcornea, posterior megalolenticonus, and coloboma syndrome (MPPC syndrome) (15). FZD4 (frizzled-type receptor 4) was reported to be associated in certain PHPV cases as well as a gene linked to familial exudative vitreoretinopathy (FEVR) (16). In animal models, various signaling pathways have been implicated in the pathogenesis of PHPV including protooncogene ski, p53, tumor suppressor gene Arf, ephrin-B2, βA3/A1-crystallin, LRP5, ang-2, Bax and Bak, FZD4, and ephrin-A5. FEVR, incontinentia pigmenti, retinoblastoma, and retinopathy of prematurity (RoP) are some of the conditions that mimic PHPV-like symptoms (17–23). However, the regulatory mechanisms responsible and genes involved in the process of fetal vascular regression continue to be unclear, as does the underlying cause of failure of regression.

The current surgical management of PHPV is primarily based on the pathological presence of individual cases. Depending on the ocular pathology of PHPV, the limbal and pars plicata incisions are the two most frequent surgical incision methods (24). The most common criteria for surgical intervention are severe media opacities due to cataract or retrolental membranes, progressive anterior chamber shallowing due to cataract, uncontrolled glaucoma or secondary ocular hypotony related to ciliary process dragging, vitreous hemorrhage, and retinal detachment following vitreoretinal traction (5, 25). In cases with advanced pathology, such as acute optic nerve hypoplasia, severe retinal detachment, or microphthalmia, surgery is not a preferred choice since post-operative vision is often low (24). Non-surgical management is currently used in non-progressive conditions and patients with non-central opacity that does not cause any visual impairment. If a non-surgical alternative is used, diligent follow-up should be carried out to detect any potential risks, such as cataract progression or glaucoma (26). The disease's heterogeneity continues to render PHPV diagnosis and treatment challenging.

Since PHPV is a rare disease, understanding the mechanisms that constitute a group of phenotypes is often restricted by small sampling sizes. Therefore, comprehending the molecular mechanisms underlying the expression of the mutated gene, which leads to improper vascular remodeling and the formation of PHPV is often individual-specific and critical for diagnosis, prevention, and therapeutic management. The assessment and analysis of molecular pathways and genetic variant analysis using conventional variant detection approaches such as Sanger sequencing, next generation sequencing, FISH, aCGH, and GTG banding can be time-consuming, expensive, and results in complicated data analysis for unspecified variants (27–31). Text mining is an effective tool for generating a hypothesis since it can reveal novel correlations between genes and the disease pathologies (32). Integration of text mining with biological knowledge and a bioinformatic approach provides new insights into the potential to reconfigure existing drugs (33). By integrating biological databases and in silico tools, the present paper aims to explore possible molecular mechanisms (if any) and classify the causative genes responsible for the heterogenic disease PHPV, thus discovering new drug targets for the treatment of the disease.

PHPV is a disease that leads to blindness or severe vision loss, although there are currently few therapeutic choices (24, 25). On the other hand, PHPV patients are more likely to develop cataracts and closed-angle glaucoma early on in life. Terminal glaucoma, uveitis, retinal detachment, and intra-ocular hemorrhage can be inevitable for these patients (35). As a consequence, the molecular mechanisms that contribute to PHPV must be established. Our analysis discloses that the molecular mechanism of PHPV overlaps with various other signaling pathways contributing to a broader range of therapeutic targets and prognostic biomarkers. The present paper reports 35 genes that might be involved in the development of the eye's vasculature process in the PHPV condition. The enriched GO and BP terms assigned to these genes were associated mainly with extracellular matrix-cell signaling, multicellular organism development, regulation of transcription by RNA polymerase ii, Norrin signaling pathway, and retina vasculature morphogenesis in camera-type eyes. The PPI network and enrichment analysis identified 14 hub genes, TP53, VEGFA, SMAD2, CDKN2A, FOXC, FZD4, LRP5, KDR, FZD5, PAX6, MYCN, NDP, PITX2, and PAX2 that were involved in camera-type eye morphogenesis, pancreatic cancer, the apoptotic process involved in morphogenesis, and the VEGF receptor signaling pathway (Figure 7). The functional analysis and pathways of the key genes in module 1 and module 2 illustrated using ClueGO are displayed in Figure 7A. Figure 7B displays the distribution of functions and pathways among core genes, while Figure 7C reveals KEGG pathways and enriched GO terms, with colors allocated to each pathway.

Based on the evaluations, four genes such as FZD4, LRP5, FZD5, NDP were involved in the process of eye development, retina vasculature development, retinal blood vessel morphogenesis, and the Wnt signaling pathway [Figure 6B; (43, 44)]. The architecture of the retinal vasculature is dependent on highly organized signaling between various cell types of retina, combining internal metabolic conditions with external influences such as oxygen and nutrient supply. In various organs, including the eye, the Wnt signaling pathway is essential for vascular morphogenesis. During eye development, and in vascular eye disorders, Wnt ligands and receptors are key regulators of ocular angiogenesis and also control the development of structured layers of vasculature in retinas as well as the regression of hyaloid vessels (45). FEVR (an inherited disease in which the peripheral retina is hypovascularized to varying degrees) has been attributed to mutations in Wnt pathway components FZD4, LRP5, and the secreted cysteine-knot protein Norrin (46, 47). Norrin is a non-Wnt ligand with a high affinity FZD4 receptor located in the retina and activates the Wnt/β-catenin pathway. Norrie disease, retinopathy of prematurity, and Coats disease are vascular retinopathies caused by defects in the Norrin gene (48). In humans, mutations in NDP and FZD4 have been identified in a limited number of unilateral and bilateral PHPV patients (14, 49–51). ATOH7 mutation (N46H-homozygous) in a family of autosomal recessive PHPV disease traits linked to 10q21 has been identified (52). These variations include deletions, insertions, and missense and nonsense mutations. However, individuals with X-linked FEVR, autosomal dominant FEVR, retinopathy of prematurity, and Norrie disease have also been reported to have mutations in NDP and FZD4 genes (53). According to the GO analysis, five genes TP53, VEGFA, SMAD2, CDKN2A, and KDR (Figure 6A) are involved in the process of regulation of cell migration by the VEGF signaling pathway, angiogenesis, regulation of muscle cell apoptotic process, and embryonic organ development process. Apoptosis is another crucial process in eye development involving extensive programmed cell death associated with morphogenesis (54).

Previous research on transgenic mice models supports our in silico analysis of PHPV to validate the function of these hub genes in hyaloid vasculature regression such as knockouts of the Arf tumor suppressor gene (23, 55, 56), p53 (21, 57), and Frizzled-5 (57) which were associated with PHPV-like phenotypes in mouse models. During mouse eye development, the arf tumor suppressor gene promoted hyaloid vasculature regression and its deficiency may cause a retrolental membrane with persistent hyaloid vessels (9, 23). In Atoh7 knockout mice, hyaloid vessels persist in the vitreous and proliferate to supply the retina which lacks intrinsic vasculature (58, 59). The ephirin-A5 family of receptor tyrosine has been demonstrated to be significant in the regression of the primary vitreous in mouse models (60). Furthermore, in mice lacking LRP5, a Wnt receptor displayed hyaloid vasculature that lasted throughout their lives (61, 62). Given the correlation between transgenic mouse PHPV phenotypes and the hub genes in human congenital defects affecting the eye morphogenesis or retinal vasculature and molecular signaling pathways in module 1 and module 2, it suggests that the pathogenesis of PHPV is regulated by genes in modules 1 and 2.

Twenty-six drugs identified by the drug-gene interaction analysis were classified as anti-neoplastic agents, ocular vascular disorder agents, kinase inhibitors, immune system functioning agents, or corticosteroids. Among them, four potential drugs such as Ranibizumab, Dinutuximab, Pegaptanib Sodium, and Sonidegib were identified based on their high drug-gene interaction score (Table 4). Ranibizumab is a recombinant humanized monoclonal antibody fragment that binds to human vascular endothelial growth factor A (VEGF-A) and thereby prevents it from binding to its receptor and blocking the development of new blood vessels (63). Pegaptanib Sodium is an anti-angiogenic drug used to treat neovascular diseases. It specifically binds to the 165 isoform of VEGF, a protein that is involved in angiogenesis and increased blood vessel leakage (64). Ranibizumab and Pegaptanib Sodium are typically used to treat wet age-related macular degeneration, a type of eye disease (61, 62, 65, 66). They are also used to treat macular edema after retinal vein occlusion, diabetic macular edema, and diabetic retinopathy. Dinutuximab is a GD2-binding human/mouse chimeric monoclonal antibody. It has been proven that the action of pro and anti-angiogenic factors regulates angiogenesis in the development of new capillaries from a pre-existing capillary network (67). Dinutuximab binds to GD2 on the cell surface and induces GD2 expressing cells to lyse by antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity (68, 69). Sonidegib is an anticancer drug that inhibits the hedgehog (Hh) pathway which is involved in cell differentiation, tissue polarity, and stem cell maintenance during embryonic growth. Hh is essential for the development of the hyaloid loop on the lens's ventral surface by promoting VEGF-mediated angiogenesis. In a zebrafish model, the loss of Hh signaling induced excess sprouting of blood vessels in the dorsal eye and impaired the growth of blood vessels in the ventral eye (70). Regulation of the Hh signaling pathway has been associated with the growth and progression of cancers such as basal cell carcinoma, medulloblastoma (71), and periocular basal cell carcinoma (72).

In PHPV, the ocular fetal vasculature does not go through normal developmental regression. The reasons could be due to presumed loss of apoptosis in PHPV; these natural apoptotic pathways could be pathologically disrupted (22, 73). Apoptosis is a process of cell death that is regulated by a number of gene families. In mice models, the macrophage has been established as a key mediator in studies examining the mechanisms of regression (74, 75). In silico drug-gene analysis using the hub genes of PHPV revealed high interaction with anticancer compounds in the present study. It is understood that vascular quiescence can be regulated by a combination of pro and anti-angiogenic factors. Previous reports have demonstrated that the equilibrium of angiogenic factors such as VEGF and placental growth factor is crucial in vascular regression and mice lacking angiopoietin 2 which regulates angiogenesis by binding to the Tie2 receptor, maintaining fetal vessels in the eyes (18, 76). In addition, the identified drugs can be used for pharmacological screening in mice and zebrafish models to identify compounds affecting vasculature development that could be of therapeutic importance. The results of the study could lead to a better understanding of the potential molecular pathway and possible hyaloid vasculature mechanism, as well as the development of novel therapeutics to prevent or cure this blinding disease, PHPV. Since the current paper focuses on the appropriate path for understanding molecular pathways and therapeutic options for PHPV through in silico analysis, further experimental analysis using animal models is highly recommended to confirm the significance of the candidate genes and pro- and antiangiogenic factors in hyaloid vasculature development and physiology. This continues to be a limitation of the study.

To conclude, no specific candidate genes, molecular pathways, or drug targets have been associated with PHPV until now. TP53, VEGFA, SMAD2, CDKN2A, FOXC, FZD4, LRP5, KDR, FZD5, PAX6, MYCN, NDP, PITX2, and PAX2 were identified for the first time as hub genes using in silico tools that may be involved in the development of retinal vasculature and dysfunction of these genes, leading to PHPV. These genes appear to be predominantly associated with functions related to eye morphogenesis, cancer, apoptosis, and VEGF receptor signaling pathways. Previous reports in knockout TP53, VEGFA, FZD4, and NDP transgenic mouse models confirmed the failure of regression of hyaloid vessels and abnormalities in the retinal vasculature. In the future, these in silico analyses will be validated by mutation screening of the hub genes in PHPV patients in order to identify pathogenic variants and gene product expressivity. Clearly, more research is warranted on animals and in human patients as the phenotypic differences will differ from individual to individual based on the expressivity of the gene product. In addition, we identified four genes that may be potential drug targets. Precision medicine for a fetal ocular condition like PHPV presents new challenges but with a possibility. Since PHPV is a rare and often autosomal recessive condition, the present paper is useful when there is little pathological knowledge about the disease or where there is substantial pathway heterogeneity, underlying the clinical phenotype. As a result, a combination of therapeutic methods such as surgical intervention and candidate gene identification may be used not only to analyze biological pathways unique to specific cases, but also to propose potential drug combinations based on gene products annotated to the disease associated with PHPV. This research sheds light on the potential of personalized intervention in the treatment of PHPV indicating a substantial advancement in management strategy.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Written informed consent was obtained from the individual(s), and minor(s)' legal guardian/next of kin, for the publication of any potentially identifiable images or data included in this article.

DT, CK, and DB conceived, designed the study, and wrote the manuscript. All authors contributed and wrote parts of the manuscript and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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