Long-term persistent exposure to cigarette smoke induces AhR driven corneal endothelial dysfunction in mice

Abstract

Epidemiological studies show cigarette smoking enhances corneal endothelial dysfunction, but mechanisms remain unclear. Our study reveals that prolonged smoke exposure activates the aryl hydrocarbon receptor (AhR), increasing CYP1B1 expression and accelerating senescence and fibrosis in corneal endothelium, potentially reflecting adaptive responses to maintain corneal resilience. Although these molecular modifications indicate early endothelial dysfunction, no pathological changes were observed. The findings indicate that while chronic cigarette smoke exposure triggers initial molecular alterations and endothelial dysfunction, the progression to Fuchs endothelial corneal dystrophy likely requires additional environmental or genetic factors beyond smoke exposure alone.

The cornea is the outermost tissue of the eye which facilitates the refraction of incoming light onto the retina. Its transparency is therefore paramount for ensuring unimpeded vision. The corneal endothelium is the posterior layer of the cornea comprising of a monolayer of hexagonal corneal endothelial cells (CEnCs). CEnCs are important in regulating ion and water exchange between the stromal layer and the aqueous humor thereby maintaining the hydration and transparency of the tissue. Dysfunction or loss of these cells can disrupt the hydro-balance, leading to corneal edema and subsequent vision impairment. A spectrum of abnormalities, including stress-induced aging, DNA damage, and augmented production of reactive oxygen species (ROS) can induce endothelial dysfunction, which can also be exacerbated by genetic and environmental factors (Ong Tone et al., 2021; Zhang et al., 2013).

Although epidemiological cross-sectional analyses have demonstrated an association between cigarette smoking and elevated risk of advanced corneal endothelial dysfunction, the underlying mechanisms remain unexplored (Zhang et al., 2013). Herein, we investigated whether chronic cigarette smoke exposure induces aryl hydrocarbon receptor (AhR)-driven corneal endothelial dysfunction. AhR has shown to be activated upon binding with polycyclic aromatic hydrocarbons present in the tobacco smoke, and are implicated in modulating molecular responses (Vogel et al., 2020). Although it has been shown that persistent cigarette smoke causes CEnC loss and extracellular matrix modulation (Ali et al., 2021), whether AhR signaling modulates CEnC dysfunction has not yet been elucidated.

We exposed 10 weeks old female C57BL/6J mice to cigarette smoke (CS) for 1.5 hours/day over 6 days/week for 6 months (Fig. 1A). The CS consisting of 150 mg/m³ of total particulate matter was generated using the JB2069 cigarette smoking machine with a mixture of main and side stream. Control mice were exposed to normal air. After 6 months of CS exposure, mice were euthanized, and the corneas were harvested for staining or RT-PCR analysis.

Figure 1: Chronic cigarette smoke exposure induces AhR driven corneal endothelial dysfunction.

A) Experimental timeline. 10 weeks old female C57BL/6J mice exposed to CS or normal air for 6 months. RT-PCR analysis showing B) significant upregulation of AhR, and C) its downstream target CYP1B1. D) AhR activation correlated with upregulation of p53. CS exposure resulted in significant upregulation of E) CDKN1A and F) CDKN2A, and G) increase in percentage of H3K9me3 positive nuclei (H3K9me3=green, DAPI=blue) indicating cellular senescence. H) CS exposure resulted in significant upregulation of ACTA2 along with I) an increase in fibronectin-1 deposition (FN-1=green, DAPI=blue). J) Collagen deposition was noted by COL4A1 mRNA expression and K) Masson’s Trichrome stain showed higher collagen intensity (dark blue: collagen on DM in insert) in CS exposed mice compared to control. L) Cell densities counted based on ZO-1 (green) staining did not differ between CS exposed and control mice. M) Periodic-acid Schiff staining showed no difference in DM thickness between CS exposed and control groups as shown in insert.

RT-PCR: real-time polymerase chain reaction; CS: cigarette smoke; DM: Descemet’s membrane; ECD: endothelial cell density. Data expressed as Mean±SEM. *p<0.05; **p<0.01; ns: not significant. Two-tailed unpaired t-test was performed using GraphPad Prism version 10.0.0 (Boston, MA, USA). n=8 mice (16 eyes) per group were analyzed. Three eyes from control and 3 from smoke exposure were used for the following - n=6 eyes for H3K9me3 staining, n=6 eyes for FN1 staining, n=6 eyes for ZO-1 staining; n=6 eyes for Masson’s Trichrome and PAS staining; and n=8 eyes were used for RT-PCR analysis. At least 4 images (histochemical/immunofluorescence) or 3 technical replicates (RT-PCR) per eye were performed. Each dot represents one biological value.

RT-PCR analysis showed a significant increase in mRNA expression levels of AhR (20,620-fold, p<0.01; Fig. 1B) in the corneal endothelium of CS exposed mice compared to air exposed mice. We further investigated the effect of CS exposure on the gene expression of cytochrome P450 1B1 (CYP1B1), an AhR target gene, which has previously shown to be upregulated by environmental pollutants or CS(Jacob et al., 2011). We observed a significant increase in mRNA expression levels of CYP1B1 (155-fold, p<0.05; Fig. 1C). CYP1B1 enzyme is known to catalyze the conversion of estrones and estradiols into catechol estrogens that favor estrogen quinone formation, which in turn reacts with DNA and forms depurinating DNA adducts and apurinic sites leading to DNA damage. It is also known that CYP1A1 metabolizes aromatic hydrocarbons while CYP1A2 metabolizes aromatic amines and heterocyclic compounds (Zanger and Schwab, 2013). However, CS exposure did not upregulate these enzymes (Supp. Fig. 1). Consistent with the increase in CYP1B1 activity, we observed a significant upregulation of p53 (2.8-fold; p<0.05; Fig. 1D). Although p53 is activated by phosphorylation, the activation of downstream pathway leading to senescence reflect the possible involvement of p53 in DNA damage recovery mechanism in the CS exposed mice. p53 has been shown to activate several genes that regulate cell-cycle arrest and senescence(Lakin and Jackson, 1999). In addition, CDKN1A gene that encodes p21, the downstream target of p53 and a marker of cellular senescence induction, was significantly upregulated in CS exposed mice (3.6-fold; p<0.05; Fig. 1E). Concurrently, we observed an uptrend in expression of CDKN2A gene that encodes p16, a known permanent senescence maintenance marker, with CS exposure (1.8-fold; p>0.05; Fig. 1F) suggesting that although cellular senescence may have been induced by CS exposure, it was not enough to retain permanent senescence in CEnCs and could have been reversed due to the repair mechanism. Consistent with the RT-PCR results, immunostaining against H3K9me3 (senescence marker) showed a significant increase in percentage of senescent cells (H3K9me3 +ve) (Schleich et al., 2020) in CS exposed group (20.5±7.6%) compared to the controls (6.9±2.2%; p<0.05; Fig. 1G).

Additionally, we observed a significant upregulation in mRNA expression levels of ACTA2, a known fibrosis marker (Rockey et al., 2013) in CS-exposed mice compared to the controls (2.6-fold; p<0.01; Fig. 1H). Histochemical analysis against Fibronectin-1 (FN-1; fibrosis marker) showed significantly higher FN-1 deposition in the corneal endothelium of CS exposed mice (99±14) compared to control group (62±13%; p<0.05; Fig. 1I). Fibronectin deposition in CS exposed mice correlated with an upregulation of COL4A1 (collagen deposition marker) (374-fold, p<0.01; Fig. 1J) in the CS-exposed mice tissues compared to the controls. Collagen deposition was further confirmed by Masson’s Trichrome staining to quantify the intensity of collagen deposition in the Descemet’s membrane (DM). The staining showed a higher collagen intensity in DM of CS exposed mice (194±4 a.u.) compared to the control mice (171±5, p<0.01; Fig. 1K) indicating that persistent smoke exposure could lead to corneal tissue fibrosis and collagen deposition.

However, endothelial cell counts did not differ between the CS-exposed mice (60±6 cells/microscopic field) and the controls (61±12 cells/microscopic field; Fig. 1L) with intact intercellular tight junctions observed by zonula-occludens 1 (ZO-1) staining. DM thickness from the CS-exposed mice tissues (1.8±0.2 μm) did not differ compared to the controls (1.6±0.2 μm; p>0.05), as observed after Periodic-acid Schiff (PAS) staining (Fig. 1M).

In conclusion, prolonged exposure to cigarette smoke led to significant molecular changes, but expected phenotypic outcomes, such as endothelial cell loss, were not observed. The increased expression of AhR, CYP1B1, senescence and fibrosis markers may also represent adaptive responses that contribute to the cornea’s resilience against pathological changes. Our findings suggest that chronic CS-exposure in mice triggers early molecular alterations, culminating in dysfunctional CEnCs, potentially influencing the onset of Fuchs endothelial corneal dystrophy. However, while this response may contribute to endothelial dysfunction, it is likely that additional environmental or genetic factors are required to drive the progression of the disease.

Supplementary Material

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Supplementary Figure 1 RT-PCR analysis showing downregulation of A) CYP1A1 and B) CYP1A2 genes. Data expressed as Mean±SEM. **p<0.01; ****p<0.0001. Two-tailed unpaired t-test was performed using GraphPad Prism version 10.0.0 (Boston, MA, USA).