Modified Anthrax Toxin Activated By MMPs: Therapeutic Approach for Treatment of Corneal Neovascular Disease

Kashiwabuchi, Fabiana

Modified Anthrax Toxin Activated By MMPs: Therapeutic Approach for Treatment of Corneal Neovascular Disease

Friday, April 25, 2014

KIOSKS (Boston Convention and Exhibition Center)

Fabiana K. Kashiwabuchi, MD, Himo, Sao Jose do Rio Preto- SP, Brazil

Murilo W. Rodrigues Jr., MD, Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA

Thomas Bugge, PhD, NIH, Bethesda, Maryland, USA

Xiaoban Xin, PhD, Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA

Stephen Leppla, PhD, NIH, Bethesda, MD, USA

Akrit Sodhi, MD, PhD, Wilmer Eye Institute, Johns Hopkins Medical Institutions, Baltimore, MD, USA

Narrative Responses:

Purpose
Corneal neovascularization (NV) is often insensitive to VEGF inhibition, prompting the exploration of other genes that contribute to this process. Our goal is to identify matrix metalloproteinase (MMPs) upregulated by activated endothelial cells (ECs) to develop targeted therapies as an approach for the treatment of corneal NV.

Methods
Human microvascular ECs (HMVECs) were exposed to recombinant human (rh)VEGF protein in culture, and expression of MMPs was assessed by quantitative PCR, and confirmed by zymography. These results were then corroborated in vivo using the rat corneal pocket (rCP) assay by immunohistochemistry (IHC). Expression of MMPs was confirmed in human tissue from patients with vascularized corneal grafts (IHC). A modified bacterial anthrax toxin (mBAT) engineered to be active only in the presence of specific MMPs was then assessed in vitro (Cell Titer Glo and TUNEL) and in vivo (TUNEL) to evaluate its potential as a therapeutic approach for corneal NV.

Results
We observed that treatment of HMVECs with rhVEGF results in an increase in the gelatinase, MMP-2 RNA levels (approximately 4 fold; p < 0.05) and enzymatic activity (approximately 6 fold; p < 0.05) in treated cells in a dose and time-dependent manner. We further observed an increase in the expression of MMP-2 in ECs in the rCP assay in response to VEGF and in human failed (vascularized) corneal grafts. Use of a mBAT specifically targeted and killed activated (dividing) ECs in vitro (p < 0.05) and effectively prevented and treated corneal neovascular blood vessels in vivo (p < 0.05).

Conclusion
Corneal NV is associated with the leading causes of corneal blindness in the developing (e.g., trachoma) and developed (e.g., herpetic keratitis) world. Our studies suggest that a mBAT that specifically targets MMP-2-expressing ECs may be an effective therapeutic approach for the treatment of patients with corneal NV.