Vascular regrowth following photodynamic therapy in the chicken embryo chorioallantoic membrane

Abstract

Photodynamic therapy (PDT) induces damage to the endothelium, which can lead to increased vascular permeability and, under intensive PDT conditions, even to platelet aggregation, vasoconstriction, and blood flow stasis. Eventually, ischemia, hypoxia, and inflammation can occur, resulting in angiogenesis. We studied the sequence of the vascular events after Visudyne®-PDT in the chicken chorioallantoic membrane (CAM) at day 11 of development. Using epi-fluorescence microscopy, we monitored the regrowth of capillaries in the PDT treated area. Immediately after irradiation, the treatment resulted in blood flow arrest. And 24 h post PDT, sprouting of new blood vessels was observed at the edge of the PDT zone. Neovessels looping out from the edge of the PDT zone gave rise to specialized endothelial tip structures guiding the vessels towards the center of the treated area. At 48 h almost all of the treated area was repopulated with functional but morphologically altered vasculature. These observations also showed reperfusion of some of the vessels that had been closed by the PDT treatment. CAM samples were immunohistochemically stained for Ki-67 showing proliferation of endothelial cells in the PDT area. Also, several markers of immature and angiogenic blood vessels, such as αVβ3-integrin, vimentin and galectin-1, were found to be enhanced in the PDT area, while the endothelial maturation marker intercellular adhesion molecule (ICAM)-1 was found to be suppressed. These results demonstrate that the new vascular bed is formed by both neo-angiogenesis and reperfusion of existing vessels. Both the quantitative real-time RT–PCR profile and the response to pharmacological treatment with Avastin®, an inhibitor of angiogenesis, suggest that angiogenesis occurs after PDT. The observed molecular profiling results and the kinetics of gene regulation may enable optimizing combination therapies involving PDT for treatment of cancer and other diseases.

Angiostatic kinase inhibitors to sustain photodynamic angio-occlusion

“…Illumination was provided by a 100 W high pressure Hg-arc lamp, as described before [23]. Light doses were adjusted with neutral density filters and measured with a calibrated Field-Master GS power meter (Coherent, Santa Clara, CA, USA).…”

“…The in ovo CAM model for Visudyne ® -PDT Fertilized chicken eggs were labelled and transferred into a hatching incubator with a relative air humidity of 65% and a temperature of 37ЊC, as described previously [23,25]. On EDD3, a hole of approximately 3 mm in diameter was opened in the eggshell and covered with a laboratory wrapping film, Parafilm ® (Pechiney, Menasha, WI, USA) to prevent dehydration and possible infections.…”

“…The PDT conditions we used led to angio-occlusion efficiency for the CAM vessels similar to those observed clinically after Visudyne ® -PDT in the human eye, that is similar diameters of vessels were closed in the CAM as in the human CNV. Photodynamic therapy induced closure of the CAM-vasculature is defined as optimal when vessels with a diameter Յ70 m are closed, while leaving the larger vessels open [23,29]. Fluorescein isothiocyanate based fluorescence angiographies were taken 24 and 48 hrs after PDT after i.v.…”

“…Prior to the fluorescence angiography, an India ink injection in the extra-embryonic cavity was performed to block optical interference from the autofluorescence originating in the embryo below the CAM. For these angiographies, we used light from a filtered Hg-arc lamp for excitation ( ex ϭ 470 Ϯ 20 nm), and a longpass emission filter ( em Ͼ 520 nm), as described later [23].…”

“…Study of photodynamic angio-occlusion and reperfusion, as well as neoformation of vessels can be performed in situ and in real time [23][24][25][26]. This in vivo pre-clinical model has the advantage of having a thin, planar vascular network, which is well-accessible to PDT and PDT in combination with adjuvants, added either topically or intravenously (i.v.)…”