Kameran Lashkari

Application of Retinal Progenitor Cells Derived From Eyes with Persistent Fetal Vasculature to Replace Lost Retinal Ganglion Cells in Clinical Glaucoma and Retinal Degeneration

Persistent fetal vasculature (PFV) is described as a phenomenon of failed regression of the vascular bed in the eye. In general, the hyaloid vascular system (HVS) nourishes the developing lens and regresses late in human fetal development. In PFV, the remnants of the HVS, together with fibroblasts, and occasional pigmented cells form a fibrovascular membrane that grows behind the lens and can incorporate retinal elements. These membranes are dissected and removed from the eye without disturbing the underlying retinal tissue. Novel findings from our laboratory indicate that the fibrovascular membranes from PFV patients are replete with retinal progenitor cells that form clusters of nestin positive cells within a local niche. Our laboratory has successfully isolated and characterized these progenitor cells (herein, called PFV cells). We have found that PVF cells are programmed to differentiate solely into a neuronal/retinal lineage, with predilection to develop into retinal ganglion cells (that form the optic nerve). In support of this contention, PFV cells transplanted into the vitreous of C57BL/6 mice differentiate into retinal neurons. Additionally, these cells have been maintained in culture for an extended period of time and have continued their progenitor phenotype with no evidence of malignant transformation. This novel finding may have an important impact on regenerative approaches to future management of advanced glaucoma, in which there is selective loss of retinal ganglion cells resulting in optic atrophy and in retinal degeneration, in which there is loss of outer retinal layers. We are characterizing both in vitro and in vivo properties of PFV cells before and after xenotransplantation to replace the lost retinal and ganglion cells, including their survival, connectivity and gain of visual function.

Pathogenesis of Advanced Retinopathy of Prematurity

Advanced retinopathy of prematurity (ROP) is the leading cause of childhood blindness and is characterized by intense retinal neovascularization, resulting in traction retinal detachment and formation of fibrovascular membranes that lead to total retinal detachment. Clinical evidence shows that the process of retinal neovascularization occurs on the border between vascularized and peripheral, avascular retina. The latter is the putative source for the release of VEGF, which is closely involved with this process. It is not clear why in some patients advanced ROP develops, as this intense process of angiogenesis leading to advanced ROP cannot be replicated in any animal model. Our goal is to identify factors that participate in advanced ROP, using tissue collected from patients with this condition. Much of the work is focused on cellular and biochemical processes that contribute to the formation of retinal neovascular membranes (RNM). We have shown that RNM is comprised of two major compartments -- a retinal neural/glial compartment and a vascular compartment. The neural compartment is comprised of adult and progenitor cells that would normally participate in development of the adult retina. RNMs contain nestin-positive cells that form neurospheres in vitro, and that can be induced to differentiate into a mature neuronal/retinal lineage in preference to a glial lineage, as shown by photoreceptor and bipolar cell markers. Fluorescent-tagged or GFP-expressing progenitor cells, introduced by viral vectors injected into the subretinal space of SCID mice, survive and differentiate into morphologically mature neuronal and retina-like elements. We are currently characterizing the expression of several tyrosine kinase receptors in the RNMs, including VEFGR-2 and hMet/ HGFR, as we have found that these markers are expressed very early in the lineage. We have also studied the vascular compartment of RNMs by doublefluorescence imaging. We have shown that there are distinct endothelial cell compartments within the RNM, suggesting that there is a significant contribution from endothelial progenitor cells. These include (1) CD31+/VE-Cadherin+/CD34-/ CD133- endothelial cells associated with mature blood vessels, (2) CD34+/Tal-1+/CD31- endothelial cells representing early, immature blood vessels, and (3) individual cells expressing CD34+/CD133+/GFAP, probably representing endothelial precursors within the stroma of these membranes. Our findings suggest that the RNM is a very complex tissue that contains many different cellular elements. Furthermore, we are analyzing the subretinal fluid (SRF) compartment in eyes with advanced ROP. We have postulated that, as the retina is the target of intense retinal angiogenesis, the SRF may be a good source for analysis of putative pro-angiogenic agents. We have previously shown that VEGF levels were  highly elevated, in excess of HGF on a weight basis, in SRF from stage 4 ROP and equally elevated in stage 5 ROP. We have further sizefractionated SRF and subjected these fractions to an in vitro angiogenesis assay using capillary endothelial cells placed in three-dimensional fibrin clots. This procedure allows us to determine which fraction exhibits the most robust pro-angiogenic activity. We are currently performing proteomic techniques including 2-D gel analysis with mass spectroscopy and luminex analysis on selected albumin and globulin-depleted SRF fractions.

Role of Hepatocyte Growth Factor in RPE Cell Biology, in Response to Wound Healing and Laser Injury

The human eye is extremely vulnerable to direct laser injury and maintaining good vision is an important determinant in success of military operations, and even survival of military personnel in theatre. Laser injury resulting from laser use as a weapon or through inadvertent retinal exposure (range finder, etc.) can immediately and potentially severely impact vision. There is increasing use of Nd- YAG laser in military operations resulting in increased risk of potentially blinding exposure by military personnel. As a result there has been congressional appeal and military interest to develop a treatment modality that could be applied to immediately counteract acute retinal laser injury in the battlefield before debilitating injury results. Injury from laser irradiation comprises a partial disruption of the retinal pigment epithelium (RPE), remodeling of the monolayer into more motile cell types, formation of scar tissue, and eventual loss of vision. The hepatocyte growth factor (HGF) and its receptor (HGFR) have been implicated in wound healing responses. As part of our current department of defense project, we are studying the role of HGF and its receptor in retinal / RPE injury and wound healing in a mouse model of laser injury. Our current results indicate that HGF itself becomes upregulated following laser injury and activates its receptor, HGFR. Anatomically, this process is closely correlated with enhanced RPE motility. In the current study, we hypothesize that HGFR activation is closely associated with RPE responses to laser injury and abrogation of HGFR activity can taper RPE motility and the detrimental wound healing response that ensues. We will employ a transgenic mouse approach to directly manipulate the activity of HGFR following Nd-YAG laser-induced retinal injury. We expect that abrogation of HGFR activity will result in decreased wound healing responses and reduced disruption of retinal anatomy following laser injury. This project directly addresses the mechanisms of retinal injury and wound healing caused by Nd-YAG lasers in combat and non-combat military conditions. We plan to design selective inhibitors for HGFR in order to taper wound healing responses.