Angiogenesis & Eye Technology

Ocular Vascular Demarcations

One of the key basic science goals of our laboratory is to elucidate molecular foundations of barriers to vascular ingrowth from vascularized to non-vascularized regions of the eye eye. In 2006, we answered the foremost outstanding question in vascular biology: “What is responsible for avascularity of the cornea?” This work (Ambati et al., Nature 2006) demonstrated that, contrary to prevailing dogma that a multitude of anti-angiogenic molecules was required for corneal avascularity, a single protein – soluble VEGF receptor-1 (also known as sFlt) – was uniquely responsible. This work was hailed by Science as a “Signaling Breakthrough of the Year” (Adler et al., Science STKE 2007), and is far-reaching because the cornea is the default platform for testing therapies for cancer, atherosclerosis, and other diseases driven by angiogenesis.

We are now focusing on which nuclear factors & events control the alternative splicing of VEGF receptor-1 into its soluble vs. membrane isoforms, and how this process can be therapeutically manipulated at a pre-mRNA level to promote either an anti- or pro-angiogenic tissue microenvironment. Manipulating the splicing event towards anti-angiogenesis would be of great value in cancer, while the converse (promoting pro-angiogenic events) could benefit stroke, heart disease, and wound healing. Furthermore, we are analyzing what controls the subretinal vascular barrier between the choroid and the outer retina, which is breached in macular degeneration. This line of inquiry is also helping us develop a novel murine model of macular degeneration which structurally correlates with the human model much more than laser injury.

Intraceptors and Novel Anti-Angiogenics

Current anti-angiogenic therapies include Avastin and Lucentis, an antibody and Fab fragment targeting VEGF (vascular endothelial growth factor). Both of these drugs bind VEGF outside of cells. However, many cell types (including cancer cells and blood vessel cells) produce their own VEGF and own VEGF receptors which could enable intracellular autocrine loops conferring resistance to extracellular anti-VEGF therapy. Indeed, clinically, many patients do not respond or stop responding after a period of time to anti-angiogenic drugs, very likely due to intracellular loops.

To sabotage the target of intracellular VEGF autocrine loops, we have developed plasmids which express siRNA (short interfering RNA) against VEGF (Singh et al. 2007), and plasmids expressing a recombinant molecule (Flt23K – which consists of VEGF binding domains 2 and 3 coupled with KDEL, an endoplasmic reticulum retention signal) which functions as an intracellular receptor, or “intraceptor”, serving to “handcuff” VEGF before it can exit the endoplasmic reticulum, where it is eventually degraded. We have shown that Flt intraceptors can inhibit and regress corneal neovascularization (Singh et al. 2005; Singh et al. 2006). We have also used the intraceptor strategy with the PDGF (platelet-derived growth factor) pathway to inhibit corneal fibrosis after laser corneal injury (Kaur et al. 2009). We are currently testing this gene therapy approach in macular degeneration and developing agents targeting other growth factor pathways in diabetic retinopathy.

Drug Delivery Strategies

Drug delivery to the back of the eye is a challenge. Patients receiving Avastin or Lucentis for macular degeneration or other retinal disorders must receive the drug via intravitreal injection (a needle placed into the middle of the eyeball), which has significant risks of bleeding, infection, and retinal detachment, as well as pain. Clearly, we must do better.

In collaboration with Dr. Uday Kompella (a world expert in nanoparticles and drug delivery), we have developed a long-term systemic therapeutic strategy consisting of biodegradable nanoparticles loaded with plasmids expressing intraceptors which can be injected to target tissues for extended anti-angiogenic effect (Jani et al. 2007). We have conjugated nanoparticles with RGD peptide (which targets alpha-v-beta-3 integrin, a cell surface marker selective for neovessels) which enables targeted systemic delivery via tail vein injection to areas of laser-induced choroidal neovascularization. This “guided missile” approach shows great promise in inhibiting rodent models of macular degeneration (Singh et al. Gene Therapy. 2009).

For long-term intraocular drug delivery, we are developing a completely new intraocular drug delivery relying on cataract extraction (the most common eye surgery and very common in the elderly) to permit the space for a drug reservoir coupled with a semipermeable membrane loaded with a polymer matrix containing drugs of interest. This device has the potential to lift the burden of repeated intravitreal injections and complex topical eyedrop regimens (Molokhia et al. Vision Research. In press).

Bioimaging

The current therapeutic paradigm is one of reacting to tissue damage and symptoms of disease. With the growth of genetic knowledge, epidemiologic understanding of risk stratification, and quantitative imaging techniques, a future of predictive imaging enabling preventing intervention can be envisioned. We are developing non-toxic fluorescent labels for disease-related biomarkers to identify “hotspots” of pathogenic phenomena before angiogenesis or apoptosis occur. Further, we are using the state-of-the-art Heidelberg Spectralis imaging modalities, including 3-dimensional optical coherence tomography to perform in vivo volumetric analysis of lesions and enable targeted contrast imaging of pathology within retinal layers.

Other Projects

We are also involved in projects investigating:

• Room temperature storage of corneal transplant tissue
• Nonviral genomic integration of recombinant cDNA
• Minimally invasive ocular bioimaging of molecular markers of disease to enable diagnosis of pre-symptomatic conditions and early intervention