Pericyte loss and deficient vascular platelet-derived growth factor receptor-β (PDGFRβ) signaling are prominent features of the blood-brain barrier breakdown described in Alzheimer's disease (AD) that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid β-protein (Aβ) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aβ deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRβ in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ 40 and Aβ 42 burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRβ and Aβ 40 levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRβ loss accompanying increased vascular amyloidosis in Alzheimer's retina implies compromised blood-retinal barrier integrity and provides new targets for AD diagnosis and therapy.

The authors have previously documented decreased epithelial basement membrane (BM) components and alpha3beta1 epithelial integrin, and increased expression of matrix metalloproteinase (MMP)-10 in corneas of patients with diabetic retinopathy (DR) compared to normal corneas. The purpose of this study was to examine if organ-cultured DR corneas exhibited the same alterations in wound healing and diabetic marker distribution as the autopsy DR corneas. Twenty normal and 17 DR corneas were organ-cultured in serum-free medium over agar-collagen gel at the air-liquid interface for up to 45 days. Circular 5 mm central epithelial wounds were made with n-heptanol, the procedure that will preserve fragile diabetic corneal BM. Wound healing was monitored microscopically every 12 hr. Distribution of diabetic corneal epithelial markers including laminin-10 alpha5 chain, nidogen-1/entactin, integrin alpha3beta1, and MMP-10, was examined by immunofluorescence. Normal corneas healed the central epithelial defect within 3 days (mean=2.3 days), whereas DR corneas on average healed about two times slower (mean=4.5 days). In wounded and completely healed organ-cultured corneas, the patterns of studied markers were the same as in the unwounded organ-cultured corneas. This concerned both normal and DR corneas. As in vivo, normal organ-cultured corneas had continuous staining for laminin-10 and nidogen-1/entactin in the epithelial BM, strong and homogeneous staining for both chains of alpha3beta1 integrin in epithelial cells, and little if any staining for MMP-10. Organ-cultured DR corneas also had marker patterns specific for in vivo DR corneas: interrupted to no staining for laminin-10 and nidogen-1/entactin in the epithelial BM, areas of weak or disorganized alpha3beta1 integrin in epithelial cells, and significant MMP-10 staining in the epithelium and keratocytes. Fibrotic extracellular matrix and myofibroblast markers were largely absent. Thus, epithelial wound healing was much slower in organ-cultured DR corneas than in normal corneas, in complete accordance with clinical data in diabetic patients. DR corneas in organ culture preserved the same marker abnormalities as in vivo. The marker distribution was unchanged in wounded and healed organ-cultured corneas, compared to unwounded corneas. The established corneal organ culture provides an adequate system for elucidating mechanisms of epithelial alterations in human DR corneas.

Purpose Diabetic corneas display altered basement membrane and integrin markers, increased expression of proteinases, decreased hepatocyte growth factor receptor, c-met proto-oncogene, and impaired wound healing. Recombinant adenovirus (rAV) driven c-met overexpression in human organ-cultured corneas was tested for correction of diabetic abnormalities. Methods Forty-six human autopsy corneas from twenty-three long-term diabetic donors (five with diabetic retinopathy) were organ-cultured and transduced with rAV expressing c-met gene (rAV-cmet) under the cytomegalovirus promoter at about 108 plaque-forming units per cornea for 48 hr. Each control fellow cornea received control rAV (rAV expressing β-galactosidase gene or vector alone). After additional 4-5 days incubation, 5-mm epithelial wounds were created with n-heptanol, and healing was monitored. Corneas were analyzed afterwards by immunohistochemistry and Western blotting. Signaling molecule expression and role was examined by immunostaining, phosphokinase antibody arrays, Western blotting, and inhibitor analysis. Results rAV-cmet transduction led to increased epithelial staining for c-met (total, extracellular and phosphorylated) and normalization of the patterns of select diabetic markers compared to rAV-vector transduced control fellow corneas. Epithelial wound healing time in c-met transduced diabetic corneas decreased two-fold compared to rAV-vector transduced corneas and became similar to normal. C-met action apparently involved increased activation of p38 mitogen-activated protein kinase. C-met transduction did not change tight junction protein patterns suggesting unaltered epithelial barrier function. Conclusions rAV-driven c-met transduction into diabetic corneas appears to restore HGF signaling, normalize diabetic marker patterns, and accelerate wound healing. C-met gene therapy could be useful for correcting human diabetic corneal abnormalities.

Elevated cathepsin F and the ability of its inhibitor to produce a more normal phenotype in diabetic corneas suggest increased proteolysis in these corneas. Proteinase changes may result from abnormalities of growth factors, such as HGF and FGF-3, in DR corneas. Specific modulation of proteinases and growth factors could reduce diabetic corneal epitheliopathy.

Ubiquitous protein kinase CK2 participates in a variety of key cellular functions. We have explored CK2 involvement in angiogenesis. As shown previously, CK2 inhibition reduced endothelial cell proliferation, survival and migration, tube formation, and secondary sprouting on Matrigel. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. In this model, CK2 inhibitors had an additive effect with somatostatin analog, octreotide, resulting in marked dose reduction for the drug to achieve the same effect. CK2 inhibitors may thus emerge as potent future drugs aimed at inhibiting pathological angiogenesis. Immunostaining of the retina revealed predominant CK2 expression in astrocytes. In human diabetic retinas, mRNA levels of all CK2 subunits decreased, consistent with increased apoptosis. Importantly, a specific CK2 inhibitor prevented recruitment of bone marrow-derived hematopoietic stem cells to areas of retinal neovascularization. This may provide a novel mechanism of action of CK2 inhibitors on newly forming vessels.