Effects of ranibizumab (Lucentis®) and bevacizumab (Avastin®) on human corneal endothelial cells.

BACKGROUND: Ingrowth of newly formed blood and lymph vessels (angiogenesis) from the limbus region into the cornea can be treated successfully by subconjunctival application of antiangiogenic agents. Currently, there are several angiogenesis inhibitors from various manufacturers available, such as vascular endothelial growth factor (VEGF) antibodies. The aim of the study was to investigate potential cytotoxic effects of two anti-VEGF agents, ranibizumab (Lucentis®) and bevacizumab (Avastin®) on the human corneal endothelium. METHODS: Human donor corneas, not suitable for corneal transplantation, were organ-cultured in the presence of either ranibizumab (Lucentis®) or bevacizumab (Avastin®) at different concentrations (group 1: 250 µg / ml, group 2: 25 µg / ml, group 3: 2.5 µg / ml) for a period of up to 4 weeks. Microscopic imaging for endothelial cell counting, detection of morphologic alterations of the endothelium, and molecular biology testing (Enzyme-linked Immunosorbent Assay [ELISA]) for metabolic changes was performed. RESULTS: Background-corrected results showed neither a significant lactate dehydrogenase (LDH) change with increasing culturing time nor a significant difference between ranibizumab (Lucentis®) and bevacizumab (Avastin®) treatment. The endothelial cell density revealed also no statistically significant difference between the two treatment groups with ranibizumab (Lucentis®) and bevacizumab (Avastin®) at all concentrations tested in this study. CONCLUSIONS: In this study, the anti-angiogenic agents ranibizumab (Lucentis®) and bevacizumab (Avastin®) demonstrated no cytotoxic effects on the corneal endothelium of human organ-cultured donor corneas over the limited study time period of 4 weeks. However, based on the study design (in-vitro) and the limited follow-up period, no conclusions on potential long-term effects can be drawn.

Elaborate spatial patterning of cell-wall PME and PMEI at the pollen tube tip involves PMEI endocytosis, and reflects the distribution of esterified and de-esterified pectins.

In dicots, pectins are the major structural determinant of the cell wall at the pollen tube tip. Recently, immunological studies revealed that esterified pectins are prevalent at the apex of growing pollen tubes, where the cell wall needs to be expandable. In contrast, lateral regions of the cell wall contain mostly de-esterified pectins, which can be cross-linked to rigid gels by Ca(2+) ions. In pollen tubes, several pectin methylesterases (PMEs), enzymes that de-esterify pectins, are co-expressed with different PME inhibitors (PMEIs). This raises the possibility that interactions between PMEs and PMEIs play a key role in the regulation of cell-wall stability at the pollen tube tip. Our data establish that the PME isoform AtPPME1 (At1g69940) and the PMEI isoform AtPMEI2 (At3g17220), which are both specifically expressed in Arabidopsis pollen, physically interact, and that AtPMEI2 inactivates AtPPME1 in vitro. Furthermore, transient expression in tobacco pollen tubes revealed a growth-promoting activity of AtPMEI2, and a growth-inhibiting effect of AtPPME1. Interestingly, AtPPME1:YFP accumulated to similar levels throughout the cell wall of tobacco pollen tubes, including the tip region, whereas AtPMEI2:YFP was exclusively detected at the apex. In contrast to AtPPME1, AtPMEI2 localized to Brefeldin A-induced compartments, and was found in FYVE-induced endosomal aggregates. Our data strongly suggest that the polarized accumulation of PMEI isoforms at the pollen tube apex, which depends at least in part on local PMEI endocytosis at the flanks of the tip, regulates cell-wall stability by locally inhibiting PME activity.