In this study we have developed a novel angiogenesis model by implanting 7KCh-containing wafers in the anterior chamber of the rat eye. This model has several advantages over other angiogenesis models which make it particularly useful for drug testing. It is relatively inexpensive, technically easy to generate and doesn't distort the cornea. This facilitates imaging and quantification of the neovessels (, , ). The main difference between the corneal pocket and the anterior chamber models is reliability. We could obtain 100% angiogenesis in all anterior chamber implanted eyes using 7KCh concentrations at or above 5% while implants placed in corneal pockets only generated angiogenesis in 50% of the animals regardless of 7KCh concentration. We believe this is due to the ability of the rats to rub the implant off their corneas. In addition, corneal distortion in the corneal pocket model makes it difficult to generate the high quality images needed for accurate neovessel area quantification. This study also demonstrates that 7KCh is pro-inflammatory and pro-angiogenic in vivo
. The cytotoxic and inflammatory effects of 7KCh have been extensively studied and published in vitro
but to our knowledge this is the first report directly implicating 7KCh in angiogenesis in vivo
7-KCh was able to induce angiogenesis as early as 4 days post implantation. As mentioned above, at concentrations of 5% or greater 7KCh generated neovessels in 100% of all of the implanted rats (). The neovessel area peaked at 7 to 10 days and was significantly reduced by 21 days (). Histological sections through the implant area indicate that the blood vessels originate at the limbus and grow through the cornea ( and ). No vessels were seen in the Ch control (). The 7KCh implants attract large numbers of cells and by day 14 are several times larger than the Ch implants (). Both implants contain CD68 positive cells (presumed macrophages) but in 7KCh implants these cells concentrate around the periphery (). The 7KCh implants were firmly attached to the cornea and iris and have numerous blood vessels growing through and around them ( and ) while roughly doubling in diameter. By contrast, the Ch implants have much fewer cells and remain unattached to the cornea or iris ( and ). Unlike the 7KCh implant, the Ch implants only seem to have CD68 positive cells (macrophages) and are reduced to roughly half of their original size (). The appearance of CD68 positive cells in the Ch implant seems to be the result of trauma to the iris epithelial cells during implantation. Some pigmented cells can be seen attached to the Ch implant (). The introduction of the wafer implant into the anterior chamber and its subsequent temporal displacement seems to scrape the iris and detach some cells that subsequently attract macrophages. Nevertheless, the immune response caused by this trauma is clearly minor when compared to the response generated by the 7KCh implants.
The role of VEGF in angiogenesis has been well established 
, as well the use of anti-VEGF therapies for the treatment of ocular angiogenesis 
. Histological sections of the implants demonstrated anti- VEGF immunoreactivity in the 7KCh () but not in Ch-implants (). This is consistent with the VEGF induction previously demonstrated in vitro
. However, since VEGF is a secreted protein we examined the AH of the implanted rats. The first thing we noticed was the markedly elevated protein concentration in the AH of the implanted eyes (, ). In order to get a better understanding of the angiogenic response, the chemokines MIP-1α and GRO/KC and the cytokines VEGF, IL-1β, IL-6 and TNF- α were measured at 4 and 7 days post implantation. These molecules are known to be involved in inflammatory responses and angiogenesis. The results clearly show that the implantation process itself can raise the levels of these molecules as seen with the Ch implants (). However, the 7KCh-containing implants cause a significantly more robust and prolonged response, which is consistent with the neovessels formation and the histological data. MIP-1α, IL-6 and TNF- α were not detectable in AH (data not shown), but VEGF, IL-1β and GRO/KC were markedly induced in the 7KCh-implant (). VEGF essentially doubled by day 4 and this level was sustained through day 7 (). IL-1β levels tripled over controls by day 4 and further increased to 21-fold by day 7 (). The GRO/KC increased approximately 32-fold over controls by day 4 but the levels significantly dropped by day 7 to only 2-fold (). VEGF has a relatively minor induction (2-fold) when compared to IL-1β and GRO/KC which warrants further investigation. Both of these cytokines are known to be synthesized by macrophages and are involved in angiogenesis 
. However, the IL-1β was considerably greater at day 7 than at day 4. Since neovessels are already present by day 4 this suggests IL-1β may not be playing a primary role in the 7KCh-mediated angiogenesis. The fact that VEGF is constitutively expressed in the AH also suggests that it may be playing a “housekeeping” function and its role in inflammatory angiogenesis is dependent on the induction of other factors, like GRO/KC. GRO/KC responds vigorously early-on (day 4) but is nearly back to basal levels by day 7 when the neovessel growth is reaching its peak. This suggests that GRO/KC may be playing a more direct role in the 7KCh-mediated angiogenesis. On a side note, the retinas of the 7KCh implanted rats do not seem to be affected or show any signs of neovessel formation.
The amount of 7KCh in these implants is very high but surprisingly within the physiological range found in atheromatous plaques. Most of the studies that have reported the 7KCh levels in atheromatous plaques have unfortunately used saponification 
. Saponification (methanolic KOH hydrolysis) or even mild alkaline treatments significantly destroys 7KCh 
forming cholesta-3,5-diene-7-one which also further breaks down into other artifacts. This has resulted in significant underestimation of 7KCh content in many of the published studies. Saponification under argon in the presence of EDTA and BHT does not prevent the breakdown of 7KCh (unpublished results). A recent study analyzed the atheromatous plaques without saponification and has reported considerably higher levels of 7KCh 
. In this study, human atheromatous lesions were reported to contain 25 mg of 7KCh per gram of wet weight or 2.5% 
. In dry weigh equivalent, the levels in these plaques would likely be in double digit percent. Moreover, this study 
did not quantify the 7KCh-fatty acid esters, which previous studies 
have shown to be abundant in oxidized LDL. Therefore, while our implants are obviously designed to induce an acute inflammatory response, our results demonstrate the dangers that oxidized lipid deposits with high 7KCh content could pose to cells.
Recently, Lutty and colleagues 
developed a choroidal neovascularization model in rats and rabbits by subretinal injections of 13(s)-hydroperoxy-9z-11e-octadienoic acid. This compound is an oxidized form of linoleic acid which has previously been reported to be present in abnormally high levels in aged Bruch's membrane and choriocapillaris 
. This is an elegant model that further supports our overall hypothesis that oxidized lipids present in oxidized lipoprotein deposits may be age-related risk factors in the pathogenesis of age-related macular degeneration.
In summary our in vivo data supports the previously published in vitro work regarding the cytotoxicity and inflammatory responses to 7KCh. This model also establishes an in vivo platform for further studies into the mechanisms of7KCh- and oxidized LDL-related angiogenesis and inflammation. In addition this model can also be used to study anti-angiogenic drugs. In preliminary studies we have successfully used this model to test various anti-angiogenic drugs by either mixing the drug with the 7KCh in the implant or by topical delivery via eye drops.