Expression of vascular endothelial growth factor(VEGF) in the developing Avian lung: and the effect of Efavirenz on embryonic development and Angiogenesis in chick chorioallantoic membrane

Abstract

The first part of this work characterized the pattern of endogenous VEGF expression in the developing avian lung while the second experiment evaluated the effect of Efavirenz on chick CAM (the domestic fowl, Gallus gallus variant domesticus). Introduction: Developmental angiogenesis is a complex process that requires a sequential interplay between embryonic cells, angiogenic factors such as VEGF, and extracellular matrix (ECM) components. VEGF has also been implicated in pathological angiogenesis such as neovascularization and carcinogenesis (e.g. Ferrara, 2000; Hagedorn et al. 2004; Losordo et al., 2004; Maharaj, 2006; Makanya et al., 2007). Anti-angiogenic drugs are therefore potential anti-cancer agents. Spontaneous resolution of HIV-associated lung cancers has been observed in patients on “highly active antiretroviral therapy” (HAART).and Efavirenz, a Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI), is a first line drug used in the management of HIV-1 infection in both adults and children. Efavirenz has not been adequately studied e.g. in patients with advanced HIV disease (CD4 (T- cell) counts < 50 cells/mm3), and on it use after failure of other regimens. Thus the aim of this work is to evaluate he anti-angiogenic property of Efavirenz, marketed as Sustiva using Chick Chorioallantoic Membrane (CAM) of the domestic fowl (Gallus gallus variant Domesticus) as an in vivo vascular test environment. Material and Methods: Animal ethics clearance was obtained (Ethics clearance certificate number 2008/7/1). Freshly laid, fertile eggs of the New Hampshire breed of chicken (Gallus gallus variant domesticus) and of the same clutches, were used for each aspect of the experiment. All the eggs were incubated in thermostatically regulated incubators and with humidity maintained using sponges soaked in water baths. The eggs were turned twice each day. In the first part 42 eggs were routinely processed in an automatic tissue processor (Shandon citadel 1000) and paraffin sections of 5 μm thickness were immunolocalized for VEGF using standard immunofluorescence technique (Polyclonal goat anti-rabbit antibodies 1:100 dilutions were detected with secondary Alexa Fluor® 568 red Goat Anti-Rabbit IgG at 1:300 dilution, Santa Cruz). Nuclear staining was done with 1:1000 dilutions of DAPI. The sections were viewed on an Olympus IX71 inverted fluorescent microscope and images captured using Olympus analysis Software. In the second aspect of the work, 30 Fertile eggs at the stage of onset for VEGF expression (day 3 of incubation; estimated Hamburger and Hamilton’s stage 21) were used as in vivo vascular test environment. They were randomly allocated (n=6) to Human recombinant VEGF (R&D Systems), Thalidomide (Sigma-Aldrich) and Efavirenz, (Sequoia Research Products). Control 1 group had the diluent (70% ethanol) but Control 2 were not manipulated. The eggs were treated by a single injection into the air cells under sterile conditions. CAMs were captured on day 8 and 18 of incubation, using a Nikon microscope camera and compared. Results were analysed using one-way ANOVA and Fisher exact test. Results: VEGF immune-reactivity was found in both airway epithelium and the cytoplasm of specific mesenchyme cells from as early as stage 22 (Day 3.5) of development. This expression gradually increased with lung maturation, peaking at stage 40 (Day 14). In the second experiment, it was observed that exogenous VEGF caused vascular haemorrhage. Thalidomide-treated embryos had poorly patterned CAM blood vessel while Efavirenz had absolute anti-angiogenic effect on chick CAM, and also suppressed haematopoiesis. Embryonic viability was 80% for VEGF group, 20% in the thalidomide and 0% in the efavirenz group. Discussion: VEGF is useful in the early processes of avian lung development and is particularly useful in pulmonary vasculogenesis. Its expression increased with lung maturation, structural complexity and function efficiency, in preparation for gas exchange and flight. Efavirenz showed more anti-angiogenic properties than thalidomide. Conclusion: Since cancer cell growth and viability depends on neo-vascularization, the anti-angiogenic property of Efavirenz, a member of the HAART regimen, may cause spontaneous resolution of HIV associated cancers.