The Ocular Vasculogenesis and Angiogenesis Laboratory at the Wilmer Eye Institute is the laboratory of Gerard "Jerry" Lutty, Ph.D., the G. Edward and G. Britton Durrell Professor of Ophthalmology. Dr. Lutty's lab, with its highly skilled personnel, studies the development of blood vessels in the eye and how they change in diseases like retinopathy of prematurity (ROP; formerly called retolental fibroplasia or RLF), sickle cell and diabetic retinopathies, and age-related macular degeneration (AMD). Dr. Lutty believes that a better understanding of the normal development and function of blood vessels will result in a greater understanding of what happens to them in disease processes like diabetes and AMD.
Ischemic retinopathies, one group of diseases which includes diabetic and sickle cell retinopathies, occur because blood vessels in the retina become blocked or occluded by blood cells sticking to the endothelial cells which line the blood vessel wall. This change in blood flow causes the neural tissue in the retina to have insufficient nutrients and oxygen, i.e. become ischemic. Ischemic retina releases substances that stimulate the growth of abnormal new blood vessels (neovascularization), which can leak and break and eventually can cause blindness.
Our lab examines the factors that stimulate new blood vessels, angiogenic factors, and naturally occurring inhibitors of angiogenesis, antiangiogenic factors. The ultimate goal of the lab is to develop therapies to stop the blood vessels from being blocked in ischemic retinopathies and inhibit the growth of neovascularization. This is being accomplished by merging nanotechnology with biological therapies. These efforts are designed to build a new generation of therapeutics.
The therapeutics that are being developed utilize nanofabrication techniques to generate therapeutic nanoparticles that, when delivered to the eye, allow the tissues of the eye to essentially treat themselves only when needed. The goal of these studies is to have the tissues generate their own therapeutics when needed and stop production when the condition is resolved. These therapies will help reduce the need for repeated treatment and provide focused therapy, rather than treating the body with chemicals.
There is still controversy over the mechanisms by which normal blood vessels develop. The Lutty lab has demonstrated that retinal blood vessels develop initially from vascular precursor cells (angioblasts) that exist in retina early in development.
Our lab has recently identified a factor that may attract these angioblasts to inner retina and may stimulate their assembly into blood vessels. This assembly of and differentiation or maturation of the precursors is called vasculogenesis.
In Picture 1, the blood vessels and precursors are stained red with a marker that we found for angioblasts called CD39. The individual cells outside of the blood vessels are the angioblasts and the endothelial cells in the formed blood vessels continue to express CD39 through life. Our lab has grown these angioblasts in culture and found that they have the potential to differentiate or change into two different cell types (i.e. angioblasts are multipotent). The two cell types are the components of normal blood vessels: endothelial cells that line the lumen of the blood vessels and pericytes that are the contractile cell on the outside of the blood vessel. We have also discovered that the choroidal vasculature develops by hemovasculogenesis: the formation of blood cells (white blood cells and red blood cells) and blood vessel cells from a common precursor, the hemangioblast.
The normal appearance of a newborn retina (top). The retina of an animal exposed to to hyperoxia after the new blood vessels have grown (middle). There is a y-shaped veil of neovascularization growing above the retina (arrow). A section through the ROP retina shown in the middle picture that shows the new blood vessels over the retina (arrow) that are tugging on the retina, causing the retina to detach in two areas from the back of the eye (bottom).
When children are born prematurely, they are placed into an incubator in which they breath oxygen levels that are higher than normal (hyperoxic). This is because their lungs are not fully developed and require supplemental oxygen. This hyperoxic environment stops the process of retinal vasculogenesis and causes newly formed retinal blood vessels to die. This poorly vascularized, ischemic retina makes angiogenic factors that cause abnormal growth of retinal blood vessels (angiogenesis). The new blood vessels grow up and out of the retina where they can bleed and cause blindness; this is called retinopathy of prematurity (ROP).
The Lutty lab uses an animal model of ROP and retinal cells in culture to study the death of retinal endothelial cells in hyperoxia and their abnormal regrowth afterwards. Our lab has identified inhibitors of neovascularization that can limit the growth of the new blood vessels.
Dr. Lutty's lab has also studied why blood vessels get blocked in diabetic retina as well. They found that a type of white blood cell called a neutrophil appears to get stuck in diabetic retina. An example of a normal retinal vasculature stained with the ADPase technique that was developed in the Lutty labs as shown (image 1). The retinal blood vessels will be removed and the blood vessels of choroid will come into focus (image 2).
We have also developed a technique to study the choroidal blood vessels in two dimensions. The choroid provides nutrition and oxygen to keep the photoreceptors healthy; photoreceptors are cells that see light and record images which are sent to the brain. We have demonstrated that not only are retinal blood vessels occluded in diabetes (image 3) but that diabetic choroidal vessels become blocked as well (image 4).
The same cells, neutrophils, appear to block the choroidal blood vessels (image 5), as well as retinal blood vessels (image 6) in diabetic subjects. Prevention of neutrophil binding to the blood vessel wall in diabetes could be a therapeutic target in the future.
Sickle cell disease is caused by a small or point mutation in the hemoglobin molecule that is found in red blood cells (RBCs). Hemoglobin and the RBCs are important for carrying oxygen throughout the body in blood vessels. Because of the mutation, the hemoglobin makes polymers in the RBCs, which causes them to become rigid and abnormally shaped. Often RBCs appear sickle-shaped, giving the disease its name. These sickle RBCs (sRBCs) get stuck in blood vessels in retina and in many other organ systems, causing the tissue to become ischemic.
Dr. Lutty's lab has developed a rat animal model for the occlusions caused by sRBCs in which human donor's sRBCs are labeled with a fluorescent dye and injected into the blood of a rat. The number of cells that get stuck in retina can be counted at the end of the experiment (above), allowing therapies to prevent them from getting stuck to be evaluated.
The Lutty lab, with a generous donation from the Reginald Lewis Foundation, has acquired an instrument called a scanning laser ophthalmoscope (SLO) that allows movies to be taken of individual sRBCs moving through the rat retina.
When the retinal blood vessels get occluded and the retina becomes ischemic, angiogenic factors are made in retina. In sickle cell disease, this occurs mostly in the peripheral retina, which does not obscure vision at first. Eventually, the entire peripheral retina of the sickle cell patient becomes occluded and many neovascular formations occur. Using a technique developed by Scott McLeod to look at the retinal vasculature from cadaver eyes in two dimensions, the Lutty lab has studied these occlusion sites and neovascular formations (NV) in great detail. The work has helped clinicians better understand where and how the NV forms and determine their structure, which is very complex and unique (example of NV in sickle cell patient to the right). This information is useful in treating these patients.
Age-related macular degeneration (AMD) is a disease that affects human retina and choroids and eventually causes loss of central vision in the elderly. Using our technique to study the human choroid, the Lutty lab has evaluated and quantified the loss of small choroidal blood vessels or capillaries (called choriocapillaris) and the cells that lie just above the choriocapillaris, the retinal pigment epithelium (RPE), to determine which cells die first and what conditions are associated with their degeneration.
When either of these cell types dies, the photoreceptors also die because they lack proper nutrition and their cellular waste cannot be removed. Using this approach, our lab has found that RPE cells appear to die first in one form of AMD called Geographic Atrophy (dry AMD), and then the blood vessels of choroid die later. In wet or exudative AMD, choroidal neovascularization (CNV) forms in the macular area, the center of vision. In wet AMD, it appears that the choriocapillaris dies first, causing the RPE to become unhealthy, and then choroidal neovascularization formsin an attempt to compensate for the loss of choriocapillaris.
The picture at the top shows the appearance of the retina and choroid in a wet AMD subject. With our technique (bottom picture), the choroidal blood vessels are stained blue and the RPE cells are brown; a fan-like CNV formation can be clearly seen, and it is apparent that the RPE are still present over the growing tips of the CNV (edge of the fan-like formation). We have recently found that three inhibitors of neovascularization that are normally found in RPE cells and choriocapillaris are greatly reduced in AMD, perhaps creating an environment where the choroidal neovascularization can form and invaded retina.
Dr. Lutty's Lab is using nanotechnology to develop unique approaches to treating the retinopathies mentioned in this section. Many labs are using viruses to deliver genes that can be used to treat diseases. Viruses, however, can themselves cause problems in that they may initiate inflammation and the genes may be expressed at too high a level or for too long. Our goal is delivery of therapeutic genes without a virus, using nanoparticles to deliver the genes. The particles can be made with multiple layers so the outer layer will have a peptide that can target the particles to cells of interest. A schematic of an iron nanoparticle with multiple layers is seen in the pitcure above. These nanoparticles can be delivered to cells in the retina.
Once the particles get into the target cell (see above), the genes are expressed for a limited time period. Using the genes for naturally occurring antiangiogenic factors, we hope to treat neovascularization without using a virus or a drug. The nanoparticles can also carry a biosensor DNA that will allow the gene to be expressed only when it is needed.
Imran Bhutto, M.D., Ph.D.
Experimental Pathologist, Expert on Choroid
D. Scott McLeod
Experimental Pathologist, Histologist, Histochemist and Microscopist
Debasish Sinha, Ph.D.
Assistant Professor: Cellular and Molecular Biologist
Expert on Lens, Astrocytes and Nanoparticles