Abstract
Purpose :
Sulfur mustard (SM), a chemical warfare agent, and nitrogen mustard (NM), a chemotherapeutic agent, are both potent vesicants. The cornea is particularly sensitive to these agents leading to keratitis, vascularization, ulceration, and perforations. Following acute SM exposure, a subset of patients will experience chronic/delayed-onset ocular complications known as mustard gas keratopathy (MGK). Delayed MGK reportedly can manifest several years after the initial exposure and presents with severe corneal disease with significant visual loss. We hypothesize that NM induces premature senescence in corneal cells which contributes to the progression of chronic MGK and the development of delayed MGK years after the initial SM insult. This study was performed to examine the effect of NM exposure on corneal epithelial and stromal cells, specifically on the development of senescence and senescence-associated secretory phenotype (SASP).
Methods :
Human corneal epithelial (HCLE) cell line was exposed to a range of concentrations of NM for 2h then washed and cultured for another 48 hours. H2O2 was used as positive control to induce senescence. Primary human corneal mesenchymal cells were likewise exposed to a range of concentrations of NM then cultured and passaged up to 3 times. The cells were fixed and subjected to staining for β- galactosidase as a marker of senescence. The number of positive cells were quantified. The expression of senescence-associated p16 and p21 was quantified by qRT-PCR and immunostaining. Proliferation was assessed by Ki67 staining. The expression of SASP factors IL-1, IL-6 and IL-8 was assessed by qRT-PCR.
Results :
Epithelial cells exposed to NM were larger in size, stained more with β-galactosidase staining, and proliferated more slowly as evident by the reduced numbers and ki67 staining. By passage 6, the NM-exposed MSCs show increased signs of senescence morphologically (larger cells), by β-galactosidase staining and Ki67 staining compared to control MSCs. Both p16INK4a and p21 were induced at mRNA level while at the protein level, the expression of p21 by immunostaining was > 2 fold higher. In HCLE cells exposed to a mild NM injury (0.01mM) the expression of SASP factors (IL-6, IL-8, IL-1a and CXCL1) were strongly by qRT-PCR.
Conclusions :
These results support a potential role for senescence in the pathophysiology of chronic and delayed MGK.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.