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Director: Victor L. Perez,
M.D.
Department of Ophthalmic Research
Cole Eye Institute
9500 Euclid Avenue, i32
Office telephone: 216/ 444-9830
Fax: 216/ 445-3670
Email: perezv@ccf.org
The staff of the Perez lab (from left) is Fitz Collings, Research Fellow; Xiaping (Annie) Yang, Lead Technician; Victor L. Perez, M.D., Principal Investigator; Guillermo Amescua, M.D., Research Scholar; and Constance Cox, M.D., Fellow, Cornea/External Diseases and Refractive Surgery.
Goals and Projects of the Perez
Laboratory
 |  | Caption: A) Normal cornea | B) Vascularized cornea after an inflammatory response |
The cornea is the part of the eye responsible for allowing
the entrance of external images. Corneal clarity and transparency are the
unique qualities of this tissue that is critical for visual function. Moreover,
the cornea also serves as a mechanical barrier against infections and trauma,
and is considered part of the bio-defense system of the eye. For these reasons,
the immune-regulatory mechanisms used by this immune privilege tissue are key
in maintaining a balance between these two important functions of the cornea.
Failure of these mechanisms to control inflammation will result in corneal
inflammation, vascularization, fibrosis and scarring, which can lead to severe
functional compromise and even blindness. Common and clinically relevant
examples in which the dysregulation of immuno-inflammatory responses of the
cornea are critical for a good visual outcome include microbial keratitis, herpetic
keratitis, contact lens-related ulcers, autoimmune ulcerative keratitis and
corneal transplant allograft rejection. The main goal of the Perez laboratory
is to develop in vivo models of corneal inflammation to study the
mechanisms of immuno-regulation of the ocular surface and develop novel
immunotherapeutic treatment of these disorders.
Innate and Adaptive Immunological Responses in High-Risk
Corneal Transplantation
Corneal transplantation in individuals who have
a vascularized corneal bed represents a high risk of graft rejection,
opacification and blindness. Despite the use of topical steroids and systemic
immunosuppression, the success rate of these transplants is extremely poor.
One of the research projects in our laboratory is dedicated to investigate in
vivo immune responses after orthotopic corneal allograft transplantation in
a murine model of high-risk corneal transplantation. More specifically, we are
interested in understanding how early innate immune responses mediated by
neutrophils, chemokines and macrophages regulate adaptive T-cell responses to
alloantigen. We have developed an in vivo animal model in which
immunological responses during corneal transplantation can be monitored in real
time with time-lapsed image analysis. This is being used to analyze
inflammatory signals involved in the early recruitment of inflammatory cells.
 |
Caption: In vivo
real time imaging of fluorescently labelled inflammatory cells infiltrating
a rejecting murine corneal allograft. Perez Laboratory of Ocular
Immunology and Transplantation |
In Vivo Visualization of Immune Responses in the
Eye
My laboratory has committed an effort in the development of in
vivo animal models, where immune responses could be studied as thoroughly
and controlled as in vitro. The intimate cellular interactions that
occur among inflammatory cells, the endothelium and interstitial space within
the microenvironment of different tissues are difficult to mimic in tissue
culture systems. Furthermore, new development in the generation of transgenic
and knock-out mice, in addition to other forms of gene manipulation in vivo
using gene transfer technology, have given scientists the opportunity to
exploit effectively the use of in vivo systems to study ocular immune
responses.
Our group has utilized some of these techniques to study
antigen-specific systemic immune responses to antigens placed in the eye and to
describe a new route of antigen processing used by the eye. However, the next
level of in vivo biology is the development of models where immune
responses can be traced in real time at tissue-specific sites. The eye
provides a unique window into the body. Its translucent nature permits the
visualization of events, such as cell growth, cell death, migration, and
transformation as they occur in vivo, in a non-invasive manner. In
order to image in vivo the recruitment pattern and track the fate of
inflammatory cells during an immune response in the eye, we have utilized mice
that express green fluorescent protein in the majority of their hematopoietic
cells (GFP mice). To detect the recruitment of inflammatory bone marrow-derived
cells into the eye, we used digital in vivo fluoresecence microscopy in
combination with ex-vivo 3-dimensional reconstruction of corneal whole
mount full thickness confocal images. Ongoing experiments utilize this animal
model to understand recruitment, fate and cellular interaction in the corneal
stroma in models of keratitis, transplantation, limbal stem cell deficiency and
wound healing after refractive surgery.
 |  | Caption: Confocal images of full thickness corneas
from EGFP-chimeric mice injected with red-labeled LPS. Green inflammatory bone
marrow-derived cells can be visualized as they migrate through the corneal
stroma in between the epithelium and endothelium (blue cells) and interact with
the LPS (red). |
Gene Delivery to the Cornea
Our initial interest in developing an in vivo model
of intra-ocular inflammation led us to pursue ways of expressing exogenous
genes of interest in the eye to influence the immunoregulatory mechanisms of
this organ and study inflammation. We have adopted a method of transfecting
and expressing exogenous genes in the stromal cells of the cornea in mice by
performing intrastromal injections of adenoviral vectors containing the DNA
sequence of a gene of interest. These experiments have led us to develop a
research program in gene delivery to the cornea. In our recent work, we report
that gene delivery can be accomplished in a tissue-specific fashion in the
cornea by using the keratocan promoter to drive the expression of any gene of
interest.
We believe this work will lay the groundwork for the development
of gene therapy as a novel therapy for corneal disorders.
 | Caption: Front cover of the IOVS (Carlson EC, Liu CY, Yang X, Gregory
M, Ksander B, Drazba J, Perez VL. In vivo Gene Delivery and
Visualization of Corneal Stromal Cells Using an Adenoviral Vector and
Keratocyte Specific Promoter. Invest Ophthalmol Vis Sci 2004;45:2194-2200)
Demonstrating tissue-specific transfection of corneal keratocytes by
intrastromal injections of Keratoca-EGFP construct. |
The Perez Laboratory staff
Eric C. Carlson, Ph.D.: Post-doctoral Research Fellow
Juan P. Rodriguez, MD: Post-doctoral Research Fellow
Xiaoping (“Annie”) Yang: Senior Research Technician
Anat Galor, M.D.: Ophthalmology Resident
Fitz Collings: Summer Research Fellow, Cornell University
Collaborators
Robert Fairchild, Ph.D.1
Peter Heeger, M.D.1
Steven Wilson, M.D.2
Eric Pearlman, Ph.D.3
Bruce Ksander, Ph.D.4
Luk VanParijs, Ph.D.5
Chia-Yang Liu6
James Funderburgh, Ph.D.7
1 Department of Immunology, CCF
2 Ophthalmic Research, Cole Eye Institute
3 Department of World Health Medicine, Case Western
Reserve University
4 Schepens Eye Research Institute, Harvard Medical
School
5 Center for Cancer Research, MIT
6 University of Miami, Bascom Palmer
7 University of Pittsburgh, Ophthalmology
Publications
1.
Carlson EC, Liu CY, Yang X, Gregory M, Ksander B, Drazba J, Perez VL. In vivo
Gene Delivery and Visualization of Corneal Stromal Cells Using an Adenoviral
Vector and Keratocyte Specific Promoter. Invest
Ophthalmol Vis Sci 2004;45:2194-2200.
2.
Wilson SE, Mohan RR, Netto M, Perez VL, Rodriguez JP, Possin D, Huang J,
Kwon R, Alekseev A. RANK, RANKL, OPG, and M-CSF Expression in Stromal
Cells during Corneal Wound Healing. Invest Ophthalmol Vis Sci 2004;45:2201-2211.
3.
Roche M, Carlson EC, Drazba J, Yang X, Van Parijs L, Perez VL. In
Vivo Visualization and Characterization of Inflammatory Cells
Recruitment and Migration through the Corneal Stroma in Endotoxin-Induced
Keratitis. Invest Ophthalmol Vis Sci (Submitted)
4. Perez VL,
Biuckians AJ, Streilein WJ. In-vivo impaired T helper 1 cell development in
submandibular lymph nodes due to IL-12 deficiency following antigen injection
into the anterior chamber of the eye. Ocul Immunol Inflamm 2000;
8(1):9-24.
5. Lu M,
Perez VL, Ma N, Miyamoto K, Peng HB, Liao JK, Adamis AP. VEGF increases retinal
vascular ICAM-1 expression in vivo. Invest Ophthalmol Vis Sci. 1999;40(8):1808-12.
6. Perez VL,
Heanault L, Lichtman AH. Endothelium antigen presentation: stimulation of
previously activated but not naive TCR transgenic mouse T cells. Cell
Immunol.1998;1889(1):31.
7. Perez VL,
Van Parijs L, Biuckians A, Zheng XX, Strom TB, Abbas AK. Induction of
Peripheral T Cell Tolerance In Vivo Requires CTLA-4 Engagement. Immunity.1997;6:411-417.
8. Van Parijs
L, Perez VL, Biuckians A, Abbas AK. Roles of Interleukin 12 and Costimulators
in T Cell Anergy In Vivo. Journal of Experimental Medicine 1997;186(7):1119.
ARVO Abstracts 2003-2004
1. Perez
VL, Gregory MA, Rothstein AM, Ksander B. Expression of Soluble Fas Ligand in
the Cornea Blocks LPS Induced Keratitis. 2003 ARVO.
2. M.Roche2,
A.Hsia1, L.Van Parijs2 and V.L.Perez1. Cole
Eye Institute, Cleveland Clinic Foundation1 and Center for Cancer
Research, Massachusetts Institute of Technology2. Real
Time Imaging of Bone Marrow-Derived Inflammatory Cell Migration into the Cornea
during Lipopolysaccharide Induced Keratitis. 2003 ARVO.
3. Galor A, Carlson EC, Rodriguez JP, Yang X, Perez VL. Differential
Migration of Inflammatory Cells in the Vascularized High Risk Cornea. 2004
ARVO.
4. Perez VL1, Rodriguez JP1, Carlson EC1,
Cohen D2, Burnett SH2. 1Ophthalmic
Research, Cole Eye Institute, Cleveland Clinic Foundation, Cleveland, OH; 2Microbiology,
Immunology and Molecular Genetics, University of Kentucky, Lexington, KY. In
Vivo Visualization of Early Inflammatory Cell Recruitment in Corneal
Transplantation using MAFIA and EGFP-Chimeric Mice. 2004 ARVO.
5. Rodriguez-Perez JP, Carlson EC, Yang X, Perez VL. Development
and Characterization of a Murine Model of Selective Limbal Stem Cell
Deficiency. 2004 ARVO.
6. Carlson
EC, Burnett SH, Cohen D, Perez VL. Seek and Destroy: The Use of the MAFIA Mouse
to Track and Eliminate In Vivo Endogenous Macrophages and Dendritic
Cells during Immunological Responses in the Eye. 2004 ARVO.
7. David
M. Meisler, M.D.1 Victor L. Perez, M.D.1 James Proudfit2.
1Cole Eye Insitute, and 2Department of Engineering, Cleveland
Clinic Foundation, OH. Device to Facilitate Limbal Stem Cell Harvesting
8. Ouyang
J, Carlson EC, Perez VL, Whey-Yang Kao W, Liu CY. In Vivo
Characterization of the Murine Keratocan (mKera) Promoter. 2004 ARVO.
Funding
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NIH/NEI K08EY014912-01
•
Knights Templar Foundation
•
Cleveland Clinic Foundation
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