Beebe Lab / Projects

Oxygen and Nuclear Cataracts

The lens exists in a hypoxic environment (Shui et al. 2006). Our studies suggest that increased exposure of the lens to oxygen, especially during later life, contributes to the formation of nuclear cataracts. We are testing this hypothesis in animal models and in human subjects. We are also determining whether maintaining lower-than-normal oxygen levels around the lens protects it from nuclear cataracts.

1. Older individuals develop nuclear cataracts within two years after retinal surgery in which their vitreous body is removed (vitrectomy). Vitrectomy increases the exposure of the lens to oxygen (Holekamp et al. 2005). Diabetics have lower oxygen levels in their vitreous bodies (Holekamp et al. 2006). If our hypothesis is correct, the lower level of oxygen in diabetic eyes will slow the rate of formation of post-vitrectomy nuclear cataracts.


Oxygen consumption by human vitreous humor, as measured with an optical oxygen sensor (optode)

2. Exposing humans and animals to hyperbaric oxygen increases the rate of opacification of the lens nucleus. Glutathione peroxidase (GPX) protects the lens against oxidative damage and GPX knockout mice develop nuclear cataracts at a greater rate than wild type mice. We predict that GPX knockout mice will be protected from oxidative damage and cataracts by keeping them in 11% oxygen, thereby lowering the oxygen level in their eyes.
3. Human vitreous humor degrades oxygen in a catalytic reaction that uses ascorbic acid (vitamin C). This reaction contributes to the low level of oxygen in the human eye and does not occur to an appreciable degree in several other species. We are identifying the catalyst that allows human vitreous to consume oxygen and are determining how this reaction is regulated in the human eye.


A diagram illustrating our model of how the ability of HIF-1 to inhibit lens cell proliferation changes with age. A program (the 'Age Operator'), gradually increases the ability of HIF-1 to suppress proliferation in older lenses. This may be a model for other tissues in which proliferation slows with age.

4. The lens grows throughout life, but the rate of growth slows progressively with age. We found that the hypoxic environment that is normally present in the eye is required to suppress the rate of cell proliferation in the lenses of older animals. Exposing the lens of older rodents to increased oxygen in vivo causes the degradation of the transcription factor HIF-1α and increases the rate of lens cell proliferation to the levels seen at one month of age. HIF-1 slows cell proliferation by increasing the levels of the cyclin-dependent kinase inhibitor, p27^KIP1. We are performing microarray analysis of lens epithelial cells that have been exposed to high or low oxygen in vivo to help identify the mechanism by which HIF-1 regulates the levels of p27^KIP1 to control the growth of the lens.

Growth Factors and Lens Development

For our studies of growth factor signaling in lens development we are using conditional deletion of growth factor receptors to define the contributions of signaling by fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), vascular endothelial growth factor (VEGF) and transforming growth factor-β (TGFβ) to lens induction, lens fiber cell differentiation, lens growth and the fibrosis that often occurs following cataract surgery ('secondary cataract').

1. BMPs are essential for lens induction and the formation of normal lens fiber cells (Belecky-Adams et al. 2002). To determine how BMP signaling affects later lens development, we deleted each of the three type I BMP receptors (Alk2, Alk3 and Alk6), singly or in combination, at the beginning of lens development in the mouse. Lenses form when any one of these receptor genes are deleted, but each knockout has a characteristic phenotype. The primary function of Alk2 signaling is to inhibit the rate of proliferation of lens epithelial cells. Alk3 signaling is most important for the normal differentiation and survival of lens fiber cells (Beebe et al. 2004). By itself, deletion of Alk6 has little effect on lens development. Deletion of both Alk2 and 3, or Alk3 and 6 leads to failure of lens formation. Most of these effects do not require the function of the downstream transducer of BMP signaling, Smad4. We are determining the pathways that are activated by each of these receptors during different stages of lens formation.


Failure of lens fiber cell formation in a lens lacking Fgfr1 and Fgfr2 (LeCre+, right). A normal lens from a littermate in which these genes were not deleted is on the left (LeCre-). The nuclei of proliferating cells are labeled brown after incorporating the DNA base analog, BrdU. Normal fiber cells (left) do not synthesize DNA. Labeling occurs uniformly in the knockout lens (right), showing that fiber cells have not formed.

2. FGFs contribute to lens induction and the formation of lens fiber cells. We are testing the functions of each of the four FGF receptors by deleting them, singly and in combination, at the beginning of lens development in the mouse. Individually deleting Fgfr1, Fgf3 or Fgf4 had no effect on lens development. Fgfr2 contributes to the survival of epithelial and fiber cells and the proper terminal differentiation of fiber cells (Garcia et al. 2005). Removal of Fgfr2, along with Fgfr1 and/or Fgfr3 prevents the formation of lens fiber cells. So far, none of these deletions were found to alter the rate of proliferation of lens epithelial cells. Our preliminary results suggest that signaling through Fgfr4 may cooperate with BMPs during lens induction.
3. As part of our studies of the role of BMPs and FGFs in lens induction, we measured the rate of cell death in the lens placode, the tissue on the surface of the head of the embryo that forms the lens. Although cell density is higher in the placode, we found that cell death was at least twice as high as in the surrounding tissue. Deletion of BMP receptors increased cell death in the lens placode to an even greater extent. These studies reveal an unexpected aspect of placode formation and show that BMP signaling is required to suppress cell death during the first stages of lens formation. We are testing the possibility that increased cell death helps to explain the failure of lens formation when two or more BMP receptors are deleted from the placode.
4. VEGF is produced by the lens throughout its development (Shui et al. 2003). VEGF from the lens is required for the formation of the tunica vasculosa lentis, the network of capillaries that surrounds the posterior of the lens during its development. Although VEGF activates its receptor in lens cells, loss of this signaling has little effect on most aspects of lens development. When Vegf is deleted from the lens, a mild nuclear cataract forms.
5. Deletion of the type II TGFβ receptor had no effect on lens development (Beebe et al. 2004). In spite of evidence that TGFβ signaling is important for secondary cataract formation, the lens fibrosis that characterizes this process still occurred in lenses lacking the TGFβ receptor. We are identifying other pathways that may contribute to the formation of secondary cataracts.

Other Projects

1. Although members of the βϒ-crystallin family were previously thought to be specific to fiber cells, we found that these proteins are abundant in adult lens epithelial cells (Wang et al. 2004). Individual crystallins changed their distribution in the cytoplasm during fiber cell differentiation or when the epithelial cells were stressed. We are testing the hypothesis that the βϒ-crystallins have functions in addition to their importance in maintaining the clarity and high refractive index of lens fiber cells.
2. As part of our studies of the function of HIF-1 in the lens, we deleted the Von Hippel-Lindau gene (Vhlh) from the lens. When oxygen levels are normal, the Von Hippel-Lindau protein targets HIF1α for degradation. When oxygen levels decrease, HIF-1α is no longer recognized by the Von Hippel-Lindau protein, HIF-1α is stabilized and promotes the expression of genes that help cells survive under hypoxic conditions. We were surprised to find that the tissue-specific deletion of Vhlh caused lens degeneration in the embryo. The mechanism responsible for this unexpected result is unknown.
3. Patients with neurofibromatosis type 2 develop acoustic neuromas and gliomas later in life, due to mutations in the tumor suppressor gene Nf2. These patients also have a characteristic type of cataract. To study the mechanism by which inactivation of Nf2 leads to cataract, we deleted Nf2 from the lens. Unexpectedly, this led to early lens degeneration. We are creating lenses in which Nf2 is deleted in only a small number of lens cells to better model the disease that occurs in patients.