Histochem Cell Biol (2008) 130:55–70 Antonetti 2005). Levels of VEGF, originally identiWed as an angiongenic factor serving as a mitogen for vascular endothelial cells have been recently correlated with increased vascular permeability (Felinski and Antonetti 2005), compatible with increased VEGF levels detected in the vitreous of diabetic patients for decades. There is enough evidence to show that diabetes and VEGF induce retinal vascular permeability by altering TJ complexes, evi- denced for instance by the decrease in occludin content in the retinas of diabetic rats (Felinski and Antonetti 2005). In cultured primary bovine retinal endothelial cells, this obser- vation was mirrored by the observation of decreased occlu- din contents after VEGF administration (Antonetti et al. 1998). Follow-up investigation underlined this and showed down-regulation of occludin in retinal endothelial cells in culture after VEGF stimulation. VEGF further activates PKC, which leads to increased occludin and ZO-1 phos- phorylation (Harhaj et al. 2006). Phosphorylation of occludin and ZO-1 likely contribute to dysregulated endo- thelial paracellular permeability (Abbott et al. 2006). Regu- lation of PKC activity and TJ protein modiWcations may thus have therapeutic implications for treatment of diabetic retinopathy. Recent investigation has further revealed that altera- tions of the BRB may involve the active proteolytic break- down of the endothelial cell TJs by MMPs as also evidenced in the case of MS (compare above) (Leppert et al. 2001). SpeciWcally, elevated levels of MMP-9 have been seen when retinal endothelial cells were exposed to high glucose conditions as a cell culture model of diabetes, leading to proteloytic degradation of speciWcally the TJ protein occludin followed by disruption of the overall TJ complex (Giebel et al. 2005), rendering the regulation of MMP secretion and activity another therapeutical target in diabetic retinopathy. Multiple sclerosis See above. Tight junction alterations in cancer Decreased expression of claudin-1 correlates with malignant potential in breast cancer Changes in TJ function have been shown to be an early and key aspect in cancer metastasis (Ehler et al. 2001). Recent studies showed a role for claudin-1 in invasion and metastasis in mammary glandular diVerentiation (Förster et al. 2002) and carcinogenesis (Morohashi et al. 2007). Interestingly, there is an absence or signiWcant loss of claudin-1 expression in several established breast cancer cell lines. Claudin-1 expression in primary human mam- 65 mary epithelial cells, in contrast to low or undetectable levels of expression in a number of breast tumors and breast cancer cell lines, points to claudin-1 as a possible tumor-suppressor gene. In sections from surgically resected breast specimens, a signiWcant loss of claudin-1 protein in breast cancer cells could be shown by immuno- histochemistry (Tokes et al. 2005). This Wnding suggests that claudin-1 may play a role in invasion and metastasis. The expression proWle of claudin-1 in non-malignant ver- sus tumor-derived cells has made this gene an interesting candidate for involvement in tumorigenesis, namely by acting as a suppressor of mammary epithelial prolifera- tion. In order to evaluate the CLDN-1 gene in sporadic and hereditary breast cancer, Krämer et al. (2000) characterized its genomic organization and screened the four coding exons for somatic mutations in 96 sporadic breast carci- nomas and for germline mutations in 93 breast cancer patients with a strong family history of breast cancer. In addition, they compared the 5 -upstream sequences of the human and murine CLDN1 genes to identify putative pro- moter sequences involved in down-regulation of the CLDN1 gene under malignant conditions and examined both the promoter and coding regions of the human gene in various breast cancer cell lines showing decreased claudin-1 expression. However, neither in sporadic or hereditary breast cancers, nor in breast cancer cell lines they found evidence for a role of aberrant claudin-1 in breast tumorigenesis and concluded that other regulatory or epigenetic factors could be involved in the down- regulation of the CLDN1 gene during breast cancer development. A correlation between TJ abnormalities and neoplasia can be supported by several observations: alterations in the number, appearance, and permeability of TJs have been demonstrated in various cancer types (Cochand-Priollet et al. 1998; Soler et al. 1999; Swift et al. 1983) and in pre- malignant mammary epithelial cells (Förster et al. 2002). At the molecular level, the Drosophila lethal(1) discs-large- 1 gene (dlg) product, which localises to TJ-related insect septate junctions has been shown to regulate epithelial cell proliferation. Genetic loss of dlg leads to a neoplastic over- growth phenotype (Woods and Bryant 1991). The loss of expression of claudin-1 itself has been demonstrated in several mammary carcinoma cell lines (Swisshelm et al. 1999). The expression of the TJ plaque protein ZO-1 has been shown to be reduced in breast carcinomas or breast cancer cells (Hoover et al. 1998). These Wndings support the hypothesis that claudin-1 might be involved in the development of breast cancer and possibly in other epithe- lial tumors too. The molecular pathways leading toward the loss of clau- din-1 expression however, remain to be explained. 123