1 Introduction

In keratoconus (KC), corneal thinning and keratectasia are the most common symptoms, resulting in severe vision impairment and corneal swelling due to structural disintegration. Keratoconus usually affects one or both eyes bilaterally and generally asymmetrically (Amit et al., 2020). Globally, the incidence of KC ranges from approximately 1:400 to 1:2000 people suffered per annum. In a general population, KC has been reported to affect up to 2.34 % of individuals, 6–10 % of people with KC have a pattern of autosomal dominant inheritance with incomplete penetrance (Atilano et al., 2019; Chaerkady et al., 2013). The KC affects both genders from puberty to early mid-life, therefore it poses a significant socioeconomic burden to the society for both young and working individuals (Yam et al., 2019). Eventually, this causes irregular astigmatism and corneal fibrosis with significant vision loss (Hashmani et al., 2017). There is a very variable and unpredictable progression rate between patients following diagnosis, which can last up to two decades after presentation (Vazirani & Basu, 2013). Studies have concerned, that this prevalence is much higher due to improvements in corneal topography, allowing for earlier diagnosis of KC subjects (Masiwa and Moodley, 2020; Atalay et al., 2021; Li et al., 2021).

The histological examination of KC corneas reveals that changes are primarily noted in the epithelium, Bowman's layer, and the stroma (Kenney et al., 1997). But, the initial changes may occur either in the epithelium or stroma remains unclear. The KC corneas have been studied histopathological for fragmentation of the epithelial basement membrane (EBM), superficial linear ruptures in Bowman's layer filled with fibrotic connective tissue, and folds on Descemet’s membrane (Marino et al., 2017). During early histopathological investigations, abnormalities in the corneal epithelium were noted, and it was postulated that insults to the epithelium caused proteolytic enzyme release, which caused stromal tissue degradation and an ensuing chain of events (Khaled et al., 2017).

Epithelium on the cornea is the outermost layer that acts as a physical barrier to protect pathogens and is in constant contact with tear film. There are distinguished three cellular layers in the epithelium: basal cells, wing cells and superficial cells (Kammergruber et al., 2019). As new epithelial cells are continually being generated at the basal level from the limbus, the corneal epithelium constantly renews itself. The wing cells migrate interiorly towards the cornea to form the superficial squamous cells, which eventually lose their adhesions (desmosomes) and slough off into the tear film (Mobaraki et al., 2019). Continuous loss of epithelium causes keratocyte apoptosis, and an abnormal epithelium might therefore impair collagen synthesis and keratocyte functions (You et al., 2018). Numerous pathological conditions can disrupt the many components that promote corneal epithelium damage. There are several abnormal morphologies of this highly dynamic tissue in KC including epithelial thinning, elongated basal epithelial cells, and breaks in the basement membrane (Grieve et al., 2016).

Moreover, KC has caused extensive oxidative damage to cornea, cultured cells, and tears in numerous molecular and biochemical findings (Atilano et al., 2019). The current therapeutic strategies are focused on lessening oxidative damage in corneal epithelial cells and preventing cell death. Previous studies have shown that oxidative stress plays a pivotal role in corneal epithelial cell apoptosis and KC occurrence (Liu and Yan, 2018; Chwa et al., 2006; Wojcik et al., 2013). Ex vivo studies and in vitro cultured cells have suggested the pathogenic mechanisms for KC, however the molecular pathology is not completely understood (Shetty et al., 2017; Kaluzhny et al., 2020). A therapeutic target for KC does not yet exist because the disease progression is still not fully studied. Among these phenomenon’s, the epithelial cells have been increasingly targeted with key factors that enhance KC-associated damage in corneal integrity. Hence, the present study examined the effects of basement membrane and basal epithelia in KC subjects, whether they delivering proapoptotic factor transductions on peripheral and center corneal epithelium.

2 Materials and methods

3 Results

3.1

3.1 Histological analyses of human normal and KC corneas

Figs. 1 & 2 showed the histology and ultra-structural illustrations from three normal and KC corneas, respectively. Both light and electron microscopic images were revealed broken bowmen's layers, hemidesmosomes, and basement membrane defects on the KC corneas but not occurs in the normal corneas. Comparatively to normal corneal epithelium in H&E stain, the basal cells from KC exhibits single squamous cells in lacking nuclei (Fig. 1B). Also, the KC corneal epithelium illustrated a hallow shape with widely elongated cytosolic bodies, when compared to normal corneal epithelial cells. The high magnification images of KC corneas (Fig. 2 C, D and E, F) from electron microscopy illustrated that the epithelial basement membrane (EBM) had debridement, fragmented hemidesmosomes that were strewn throughout bowmen's layer. On both light and electron microscope images (Figs. 1 & 2), the arrow marks are indicating that the structural and ultrastructural changes in KC and normal corneal tissue sections.

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Fig. 1

Histological changes in human normal and KC corneas. As shown in (A & B), basal epithelial cells and basement membrane damage were assessed in normal and keratoconus corneas. The images were all captured at a 40x magnification. EP-epithelium, BC-basal cells, BM-basement membrane, BL-bowmen’s layer, ST-stroma.

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Fig. 2

Ultra-structural changes in human normal and KC corneas. The electron microscopic ultrastructural images of hemidesmosomes and basement membrane fragmentation were assessed. The (A & B) images shows in low magnification of keratoconus cornea (KC) and normal cornea (5000x). Images (C & D) show the normal and KC cornea sections at high magnification (30000x). Images (E & F) show the KC cornea sections at low (10000x) and high magnification (30000x) in basal epithelial cells basement membrane debridement’s. BC-basal cells, BM-basement membrane, BL-bowmen’s layer, ST-stroma, HD-hemidesmosomes, EBM-epithelial basement membrane.

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3.2

3.2 Pro-apoptotic proteins expression in human normal and KC corneas

Western blot study was examined whether the expression levels of proapoptotic proteins could differ between normal and KC cornea. Immunoblotting studies were performed to determine the expression of apoptosis-associated proteins (such as Bax, Bid, Bad, p-p53 and caspases). In Fig. 3(A & B) histograms are representing the total protein expression of Bax, Bid, Bad, p-p53, cleaved caspase-3 and β-actin levels from three normal and KC subjects in pooled tissue samples. Fig. 3C shows the quantified results of band density using the Image-J software. In KC corneas, Bax, p-p53 and cleaved caspase-3 protein expression levels were increased significantly by 1.5, 5.6 & 2 folds (*p = 0.05) compared with normal corneas (Fig. 3C). According to the results (Fig. 3C), the relative levels of protein expression of Bid and Bad were significantly decreased in KC cornea (more than 40 %, **p = 0.01) as compared to normal cornea. Based on these results, corneal tissues from the KC patients showed increased caspase-3 activation, which may be contribute to enhance apoptosis.

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Fig. 3

Analysis of pro-apoptotic proteins expression in normal and KC corneas. Western blot images (A & B) illustrating the expression of Bax, Bid, Bad, p-p53, cleaved caspase-3, and β-actin proteins. Using Image-J software, these expressions were quantified using densitometry analysis. The graph in (C) plots shows the levels of Bax, Bid, Bad, p53, and cleaved caspase-3 protein expressions. The relative density bands of the target gene were normalized to the expression of the housekeeping gene β-actin. Three replicates were conducted for each experimental point (n = 3). The data represent Mean ± Standard Error (S.E.). Using Mann-Whitney unpaired T-test multiple comparisons, P values of 0.05, 0.01, and 0.001 representing *, **, and *** compared to normal were calculated.

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3.3

3.3 Immuno-histochemical changes of apoptotic proteins in human normal and KC cornea

To further confirm that Bax, Bid, Bad, p-p53, and caspase-3 are associated with pro-apoptotic signaling in the normal and KC corneal epithelium, immunohistochemistry was performed (Figs. 4 and 5). The expression of pro-apoptotic proteins Bax, phospho-p53, and cleaved caspase-3 immuno-staining results indicated that the basal cells and scarred bowmen's layer of KC cornea were absolutely stained more than normal corneal epithelium (Fig. 4A-F). There was no protein expression in either the anterior or posterior stroma of the KC cornea. Comparatively, Bid and Bad proteins expressed moderately have been found in the entire scarred center epithelium of the cornea in KC (Fig. 5A-D). However, no staining was observed on the bowmen's layer, anterior and posterior stromal areas of the KC cornea. Accordingly, the immuno-histochemistry staining of these three KC corneal tissue sections (Bax, phosphor-p53, and cleaved caspase-3) revealed identical results compared to normal corneal sections.

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Fig. 4

Detection of pro-apoptotic proteins in human normal and KC corneas. Using immunohistochemistry, pro-apoptotic proteins Bax, phospho-p53, and cleaved caspase-3 were analyzed. Fig. 4(A-F) shows the light microscopic images of Bax, phospho-p53, and cleaved caspase-3 immunostainings in normal and KC corneal tissue sections. The images were all captured at a 20x magnification.

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Fig. 5

Detection of pro-apoptotic proteins in human normal and KC corneas. Using immunohistochemistry, pro-apoptotic proteins Bid and Bad were analyzed. Fig. 5(A-D), immunostaining of Bad and Bid is shown in sections of normal and KC corneas. The images were all captured at a 20x magnification.

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3.4

3.4 Colocalization of apoptotic proteins in human normal and KC corneas

Based on the above results, apoptosis signaling could be required to find co-localization within the KC corneal epithelium and stroma. For further confirmation, we examined whether pro-apoptotic signaling influences the expression of the apoptosis-related proteins (such as Bax, p-p53, and cleaved caspase-3). For using triple antibody staining, each antibody stain color illustration shows that mouse anti-Bax antibody interacts with mouse AP polymer to formed a permanent red chromogen, A rabbit anti-cleaved caspase-3 antibody interacts with rabbit HRP polymer to formed DAB brown chromogen and mouse p-p53 antibody interacts with mouse HRP polymer to appeared emerald green chromogen. Afterwards, a co-localization color dark blue/purple was revealed, which confirms the co-expression on the same site. The results of Fig. 6, which compares the epithelium of normal corneas (Fig. 6A) and KC corneas (Fig. 6B), indicate that the triple antibody staining illustrated strong protein colocalization (dark blue/purple) between basal cells and basement membrane on the KC corneas. On light microscope images (Fig. 6B), arrow marks indicate that the protein colocalization (dark blue/purple) from basal cells and basement membrane in KC corneal sections. Blue/purple staining was not observed in the bowmen's layer, anterior or posterior stromal areas of the KC cornea. An immuno-staining clearly shows that antibodies are expressed in the cytosolic cellular matrix and basement membrane of basal cells, but not in their nuclei. Thus, triple antibody staining of three KC corneal tissue sections (Bax, phospho-p53, and cleaved caspase-3) revealed identical results compared to normal corneal tissue sections.

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Fig. 6

An analysis of the colocalization of pro-apoptotic proteins in human normal and KC corneas. The light micrographs (A & B) show Bax, phospho-p53, and cleaved caspase-3 immunostaining in normal and KC corneal sections. The dark blue/purple color was indicating the co-localization of the triple protein’s expressions (Bax, phospho-p53, and cleaved caspase-3) in normal and KC corneal tissue sections. The images were all captured at a 40x magnification.

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3.5

3.5 Detection of progenitor cells membrane lipid content in human normal and KC cornea

The Nile red staining in cell membranes allowed us to observe membrane lipids and progenitor cell translocation in cells by using fluorescence microscopy. In Fig. 7A & B, Nile red staining revealed that in normal peripheral corneas, KC corneas had less membrane lipid content and a lower number of progenitor cells occurred. The epithelial and stromal parts of the KC cornea (Fig. 7B) differed from those of the normal cornea (Fig. 7A). The arrow marks (red colors) on fluorescence microscope images (Fig. 7A & B) indicate the presence of lipid contents in both normal and KC corneal sections. From Fig. 7C & D, the DAPI staining (blue color) supports the identification of cellular nuclei from normal and KC corneal tissue sections. In both Nile red/DAPI merged images, the deviation in membrane lipid contents and progenitor cell distribution in normal and KC corneal tissue sections are clearly showing in Fig. 7E & F. This study may help us to identify differences between the pathological changes in normal and KC corneas.

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Fig. 7

Analysis of progenitor cells lipid contents in normal and KC corneal tissue sections. The (A & B) images show the fluorescent dye Nile red staining in normal and KC corneal tissue sections. The image of (C & D) shows the fluorescent dye DAPI nuclear staining in normal and KC corneal tissues. Merged fluorescent images of Nile red/DAPI staining in normal and KC corneal tissue sections is shown in (E & F). The images were all captured at a 20x magnification.

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4 Discussion

Currently, there is no therapeutic target associated with the disease progression of KC, as the pathology needs to be fully elucidated. It has been reported that certain factors contribute to the pathogenesis of KC, including genetic, metabolic, hormonal, environmental influences, oxidative stress, and cytokine signaling (Ates et al., 2021; Wojakowska et al., 2020). Also, a number of systemic diseases have been associated with an increased risk of developing KC, including, but not limited to, diabetes mellitus, connective tissue diseases, hypertension, macular edema, and autoimmune diseases (Anitha et al., 2021). The early research, it has been discovered that KC is a partially systemic disease due to the role that endocrine function plays in regulating metabolism and inflammation, some biomarkers of hormonal regulation have been projected in KC, including metabolites and hormones (McKay et al., 2016). Therefore, the identification of the key molecular players in KC-associated corneal pathogenesis may lead to new therapeutic targets to improve the therapeutic efficacy. These studies have been limited by the fact that either the whole cornea or the whole epithelium of the cornea was used.

Histopathologic changes are apparent in both the epithelium and stroma of the cornea during the progression of KC. One of the earliest detectable changes in KC cornea can be seen in its morphological changes of the corneal epithelium, as well as in other epithelial structures (Khaled et al., 2017). A few studies have also raised concerns about the extracellular matrix (ECM) that fills in the gaps of Bowmen's layer in the early stages of KC (Kenney et al., 1997; Pahuja et al., 2016). There may be a localized fibrotic repair process occurring at the sites of BM breaks based on the accumulation of proteiolytic enzymes via endocrine transductions. The combination of stromal ECM and BM may allow for both keratocytes and epithelia to perform their repair functions (Torricelli et al., 2016). In the present study, the H&E staining and electron microscopy findings illustrated that the BM is irregular shaped, accompanied by breaks in keratoconic corneas as compared to normal corneal sections (Figs. 1 and 2). These findings indicated in dysfunctional interactions between corneal epithelium and stroma, which affects gene expression as well.

In addition to the altered turnover of corneal epithelial cells, corneal thinning may also result from abnormal cell degeneration. The keratocone has a higher number of normal epithelial cells in the periphery whereas the epithelial cells at the conical apex (center) are elongated and arranged in whorls-like fashion (Sherwin & Brookes, 2004). It is generally believed that the KC cornea is primarily affected by apoptosis in the stroma and possibly in the epithelium, which leads to layers of cornea thinning. Using wound healing assays, studies have shown that apoptotic corneal epithelium can increase keratocyte death (Song et al., 2015; Coulson-Thomas et al., 2015). Also, a higher number of apoptotic cells were detected in the deeper levels of KC corneal stroma when compared to those found on the surface of normal corneas (supplementary Fig. 1). The pro-apoptotic cellular signaling mechanisms in KC are mostly unknown, except for the biomechanical, biochemical, and structural changes.

Apoptosis can also be induced by cellular stresses which act on nuclei, mitochondria, the endoplasmic reticulum or lysosomes through paracrine regulations. Despite the heterogeneity of potential cell death inducers, apoptosis exhibits common morphological and biochemical changes (Forrester et al., 2018). In addition, the IL-1 receptor, lysate products from apoptotic cells, and FAS and its ligands are all potential inducers of apoptosis (England et al., 2014; Spadea et al., 2016). In later, this family of proteins contains both pro-apoptotic Bax, Bad, and Bid and anti-apoptotic proteins Bcl2 respectively that also regulate apoptosis signals. In normal cells, Bax resides in the cytosol, but when stimulated it is translocated to the mitochondrial outer membrane. These proapoptotic proteins trigger apoptosis, which activates caspase cascade. There is no consensus on whether apoptotic cell death involves a final death pathway. In this regard, Western blots and immuno-staining were undertaken to determine the levels of apoptotic protein expressions. Based on these findings, KC corneas (Figs. 3 & 4) was overexpressed in the Bax protein, which may decline in Bid and Bad proteins expression, and it shifts to enhance p-p53 and caspase-3 proteins expression. Overall, these results indicate that increasing the pro-apoptosis protein Bax, which regulates cellular apoptosis signals, may be a key risk factor for causing cell death.

P53, another major gene, is responsible for maintaining genomic stability in biological systems (Eischen, 2016). The 53-kd phosphorprotein produced by this gene plays a role in numerous types of abnormal cell proliferation; apoptosis in response to DNA injury, as well as the prevention of replication of genomes damaged by DNA strand break (Williams & Schumacher, 2016). Only a few studies have focused on p53 subcellular expression and function in normal, mature corneal tissues. A high level of p53 protein was previously detected in the cytoplasm of normal C57BL/6 mice's corneal epithelium (Tendler & Panshin, 2020). As a consequence, pathologic p53 proteins bind to mitochondrial outer membranes, induce permeabilization of the membrane, and form complexes with proapoptotic proteins. Upon binding of these factors, cytochrome C is released, and caspase activation triggers apoptotic cell death (Schuler et al., 2000). Accordingly, the triple antibody immuno-staining results showed these proteins Bax-pp53-casp-3 upregulated in the cytoplasm and basal cell membrane of the KC corneal tissues sections (Fig. 6). A study published in 2013 by Tendler et al. found that p53 proteins were colocalized in various subcellular organs and induced apoptosis in cultured corneal epithelial cells after UV radiation exposure (Tendler et al., 2013). According to our knowledge, this is the first report that demonstrated the apoptotic proteins Bax-pp53-casp-3 triple antibody immuno-staining and the co-expression of apoptotic proteins in KC corneal tissues. Additionally, the Nile red fluorescent staining of the KC corneas revealed a less dense plasma membrane and smaller number of progenitor cells than those in peripheral normal corneas (Fig. 7). There were fewer progenitor cells in the epithelium and the stroma of KC corneas, and the lipid content in membrane differed from those in the normal cornea. Observations of these new findings may be helpful in understanding the pathological changes between normal and KC corneal structural integrity. By using new techniques, it will be potential to establish the clinical significance of these pro-apoptotic morphological patterns in the future. Additionally, its potential use for predicting risk factors associated with the progression of KC is to be explored.

5 Conclusions

A combination of protein expressions and histological analysis revealed structural and biological changes in KC corneal epithelia, enhancing our understanding of KC pathophysiology. As a consequence, the collective results indicate that pro-apoptotic proteins occur colocalization through basal epithelial cell damage and fragmented basement membrane, both of which enhance apoptotic induction in the KC epithelia. It has been proposed that phosphorylation of cytoplasmic p53 has a profound effect on caspase-3 activation in KC corneal epithelium and basement membrane. Further studies are needed to fully characterize the role of p53 and caspase-3 proteins and their regulation as part of the corneal epithelium of the KC. These novel findings might help to enhance our understanding of KC's apoptosis signaling pathways, thereby improving its clinical treatment and management.

Author contributions

Substantial contributions to conception and design of this study: RS, OK; Acquisition of data: RS, AAK; Analysis and interpretation of data: RS, AAK; Drafting the article: RS, AIS, SAA; Revising it critically for important intellectual content: AMM, ESA, SAA; Final approval of the version to be published: RS, AMM, ESA, SAA.

Financial support

The authors are grateful to the Deanship of Scientific Research, King Saud University for funding through Vice Deanship of Scientific Research Chairs.