Antisense down regulation of connexin31.1 reduces apoptosis and increases thickness of human and animal corneal epithelia

Abstract

The roles of the gap junction protein connexin31.1 (Cx31.1) are poorly understood, especially as the protein appears to form non-functional channels. Cx31.1 specific antisense oligodeoxynucleotides (ODNs) were designed to evaluate its roles in a corneal epithelium model. Expression of Cx31.1 in corneal epithelium extends from the suprabasal layers of polyhedral wing cells through to the flat squamous cells of superficial layers which are shed into the tear film. Deoxyribozymes (Dzs) were tested for cleavage efficacy using in vitro transcribed Cx31.1 mRNA. Cleavage results showed a putative tertiary structure for Cx31.1 mRNA with one region appearing to have a higher potential for antisense targeting. Application of antisense ODNs designed to this region caused Cx31.1 knockdown in rat and human corneal organotypic culture models, leading to a reduction in apoptosis and a thickening of the corneal epithelium (p=0.0045). Cx31.1 appears to play a role in triggering cell death; knocking it down may provide a novel approach for tissue repair and engineering.

Connexin expression patterns in diseased human corneas

“…In the current experiment, it was identified that Cx31.1 expression levels were significantly higher at the mRNA and protein levels in the chemically burned and infected corneas. These findings support the results of Chang et al (6) , as following chemical burns and infection, apoptosis is triggered both in corneal epithelial cells and corneal keratocytes (40). The present results, together with those of Chang et al further indicate that antisense Cx31.1 is likely to be a promising candidate for the treatment of corneal wounds.…”

“…Following antisense Cx31.1 treatment in rat and human corneal organotypic culture models, not only was there evidence for Cx31.1 knockdown, but also the cornea epithelium appeared significantly thicker within just 24 h and a marked reduction in epithelial apoptotic cell numbers was observed. The results indicated that Cx31.1 may play a role in triggering apoptosis, leading to cell sloughing into the tear film (6). In the current experiment, it was identified that Cx31.1 expression levels were significantly higher at the mRNA and protein levels in the chemically burned and infected corneas.…”

“…Antisense Cx43 and antisense Cx31.1 have been developed to modulate Cx43 expression at the cellular and tissue levels and have been identified to accelerate skin wound healing in various animal models and clinical trials (4). There are also studies that indicate antisense Cx43 and Cx31.1 are novel therapeutic candidates for ocular wounds, particularly corneal wounds (5,6). …”

Cardiac to cancer: Connecting connexins to clinical opportunity

“…Application of Cx31.1 antisense oligonucleotides in ex vivo rodent and human corneal models results in decreased epithelial cell apoptosis and increases in corneal thickness. The mechanism of action is likely linked to roles for Cx31.1 in the reduction in superficial cell death and shedding [101]. Additional research investigating the mechanism is required to fully elucidate the clinical potential of targeting Cx31.1 in the cornea.…”

In vitro release characteristics and cellular uptake of poly(D,L-lactic-co-glycolic acid) nanoparticles for topical delivery of antisense oligodeoxynucleotides

“…Connexin 31.1 (Cx31.1) protein is predominantly expressed in the corneal epithelium and the skin epidermis (Chang et al, 2009;Goliger and Paul, 1994;Hennemann et al, 1992). It leads to formation of nonfunctional gap junction channels between cells and is therefore associated with cell apoptosis.…”