Introduction Diabetic retinopathy is fundamentally a vascular disease. Compelling evidence suggests that defects in mural cells--smooth muscle cells (SMC) and pericytes, may play an important role in this disease process. The loss of pericytes is the earliest morphological lesion observed in diabetic retinopathy. Furthermore, mice null for genes required for mural cell recruitment develop microaneurysms and leaky vessels, the hallmarks of early phase diabetic retinopathy. My long-term goal is to develop therapeutic strategies to treat this disease, based on a more complete understanding of mural cell biology. Specifically, I am interested in the following questions: how are mural cells recruited to the newly formed vessels? How can the recruitment of mural cells be manipulated? What is the nature of mural cell/endothelial cell interactions? What are the effects of mural cell degeneration on adult vasculature? Can this degeneration process be slowed or even reversed? Mouse retinal vasculature is an ideal system to study these questions because of its well-defined structure, accessibility to experimental intervention, potential for transgenic manipulation and availability of many mutations. Thus, our immediate goal has been to develop animal models and tools to facilitate our studies of questions listed above. To this end, we have been doing the following: (1) Creating transgenic mice whose mural cells express GFP in vivo. (2) Creating transgenic mice that allow conditional ablation of mural cells in vivo. (3) Identifying specific molecular markers for mouse retinal pericytes. (4) Identifying novel pericyte surface molecules through large-scale sequencing of a human pericyte library. Finished work (1) Creating SMAA-GFP mice whose retinas contain mural cells expressing GFP in vivo The ability to visualize mural cells in vivo via GFP fluorescence should greatly facilitate the study of angiogenesis and the role of pericyte recruitment in this process. Since no promoter is available for driving gene expression specifically in all mural cell lineages, we reason that SMAA promoter is the best for this purpose for two reasons. First, SMAA has been the marker of choice for cultured SMCs and pericytes. Second, the SMAA promoter has been extensively characterized in tissue culture cells and in mice. We have created transgenic mice expressing EGFP under the control of the SMAA promoter. The vascular SMCs in this mouse are brightly labeled. The pericyte labeling appears heterogeneous. Some pericytes are intensely labeled while others show little or no GFP signal. In capillaries of the deep plexus, we also observed two unknown types of GFP-positive cells located adjacent to vessels. One has a round shape and does not make contact with vessels. The other appears to extend processes to nearby vessels. The identities of these cells are currently under investigation. (2) Creating transgenic mice for conditional ablation of mural cells To ablate the mural cells conditionally, two reversible/inducible toxin systems are used. First is the aldose reductase (AR)/ galactose system. In vivo studies show that lens cells and Schwann cells degenerate in galactose-fed mice over-expressing AR in those cells. Since the endogenous AR activity of wild type mice is very low, the galactose-induced toxicity will be limited to cells in which AR is expressed transgenically. A construct (SMAA-hAR) containing human AR (hAR) cDNA under the control of SMAA promoter was used to produce three lines of SMAA-HAR mice. Using antibodies against hAR, hAR is found in mural cells of retina vessels. In one line, the hAR is also expressed strongly in a subset of neurons in the inner nuclear layer. The distribution and synaptic arborization patterns of these cells suggest that they are cone bipolar cells. We are currently testing this hypothesis by immuno-labeling with anti-recoverin antibodies. Another approach is to apply a tetracycline inducible diphtheria toxin A (DTA) strategy. This system requires generation of two separate transgenic lines carrying different genetic elements. The first element uses SMAA promoter to direct expression of the tetracycine repressible transactivator (tTA) into mural cells. The second element is a toxin-containing construct (TetP-DTA) in which DTA sequence is under the control of a tetracycline responsive promoter (TetP). In transgenic mice carrying both elements, the TetP-DTA is turned on by tTA, but this activation can be completely repressed by tetracycline. We have made 8 independent lines of SMAA- tTA mice, and obtained the TetP-DTA mice from Dr. Ronald Depinho (Harvard). The expression pattern of tTA in SMAA-tTA mice is currently being examined by immunohistochemstry. (3) Identification of NG2 as a marker for mouse retinal pericytes One major problem hampering the progress of mural cells research, particularly pericytes, is the lack of definitive in vivo markers. Conflicting reports abound in literature. For example, although smooth muscle alpha actin (SMAA) is the marker of choice for cultured pericytes, its presence in pericytes in vivo has remained unclear. NG2 is a transmembrane proteoglycan and was originally reported to localize to vascular endothelial cells in CNS tissues and mural cells in blood vessels of non-CNS tissues. However, using immunohistochemistry on flat mount and elastase digest retina preparation, we have founded that anti-NG2 antibodies exhibit a unique intense peri-nuclear staining pattern in pericytes, which makes it the best marker to identify pericytes in retina. (4) Human pericytes EST projects As a part of NEI EYEBank project, we have recently sequenced 3000 random clones from a human pericyte cDNA library. Totally 1600 different genes are identified in this collection. The preliminary analysis indicates that the expression pattern of pericytes is similar to those of SMCs. Interestingly, many genes controlling endothelial cells proliferation and migration, such as matrix metalloproteinases (MMPs), pigment epithelium derived factor (PEDF), insulin growth factor (IGF) and IGF binding proteins, are well represented in this library, which is consistent with the regulatory roles of pericyte on endothelial cells.
| Sakurai, Shinichi; Alam, Shahabuddin; Pagan-Mercado, Glorivee et al. (2002) Retinal capillary pericyte proliferation and c-Fos mRNA induction by prostaglandin D2 through the cAMP response element. Invest Ophthalmol Vis Sci 43:2774-81 |
