Anti-angiogenesis Drug Discovery and Development provides an excellent compilation of well-written reviews on various aspects of the anti-angiogenesis process. These reviews have been contributed by leading practitioners in drug discovery science and highlight the major developments in this exciting field in the last two decades.
The feast of these reader-friendly reviews on topics of great scientific importance — many of which are considered significant medical breakthroughs, makes this series excellent reading both for the novice as well as for expert medicinal chemists and clinicians.
This volume brings together 5 reviews on the following topics: - Retinal angiogenesis - Effects of brief daily EMF therapy on tumor growths - Evolution of the role of angiogenesis in cancer treatments over six decades - Anti-angiogenesis drugs - Anti-angiogenesis therapy for multiple sclerosis - Update on the link between angiogenesis and portal hypertension.
Ocular angiogenesis: mechanisms and recent advances in therapy.
Ocular angiogenesis is a major cause of disease and permanent blindness. The introduction of anti-vascular endothelial growth factor anti-VEGF agents has revolutionized the treatment of retinal vasogenic conditions. These drugs are now commonly employed for the treatment of a group of ocular pathologies, including exudative macular degeneration, diabetic macular edema, and retinal vein occlusion.
Multicenter, randomized, controlled clinical trials have been designed to study the efficacy of anti-VEGF therapy in common retinal angiogenic diseases. However, when it comes to extremely rare or uncommon retinal diseases, we must often rely on case studies or small series to elucidate a potential beneficial effect of anti-VEGF treatment.
Louis, MO. Choroidal flat-mounts were washed with 0. Negative control sections were similarly treated, but the primary antibodies were omitted.
Ocular Angiogenesis Diseases Mechanisms and Therapeutics
Fluorescent microphotography was performed on a Zeiss Axioplan2 epifluorescence microscope. Image-Pro Plus software Media Cybernetics, Silver Spring, MD was used to measure the total area of CNV at each rupture site by personnel blinded as to study treatments and groups and the numbers of positive cells were counted and representative images were photographed.
The known cell types that can be labeled with each of the markers are shown in Table 1. Quantification of retinal NV was carried out as described previously  , . Mice were euthanized 12 hours after injection and eyes were fixed in PBS-buffered formalin for 5 hours. Retinas were dissected, washed and incubated with goat anti-rat polyclonal antibody conjugated with Alexa Fluor or were labeled with griffonia simplicifolia Invitrogen, Carlsbad, CA for 45 min. Images of each of the 4 quadrants of whole-mounted retina were taken at 5x magnification on a Zeiss Axioplan 2 microscope and imported into Adobe Photoshop.
Retinal segments were merged to produce an image of the entire retina.
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Neovascular tuft formation was quantified on retinal flat mounts with fluorescence microscopy using Image Pro Plus software. The quantitative BRB assay was performed according to a previously described technique . Mice were sedated as above and given an i.
One hr after injection, the mice were sedated and retinas from experimental and control eyes were rapidly removed. The posterior portion of the globe was firmly grasped with forceps and a razor blade was used to cut across the cornea and extrude the lens, vitreous, and retina. Retinas were dissected free from lens, vitreous, and any RPE that was extruded, and were placed within pre-weighed scintillation vials within 30 seconds of sacrifice. The thoracic cavity was opened and the left superior lobe of the lung was removed, blotted free of excess blood, and placed in another pre-weighed scintillation vial.
A left dorsal incision was made and the retroperitoneal space was entered without entering the peritoneal cavity.
The renal vessels were clamped with a forceps and the left kidney was removed, cleaned of fat, blotted, and placed into a pre-weighed scintillation vial. Superficial liquid was allowed to evaporate over 20 min from the open vials. The vials containing the tissue were weighed and tissue weights were calculated and recorded. P-values for comparison of treatments were adjusted for multiple comparisons using Dunnett's method.
For data sets with two groups, statistical analyses were performed by the unpaired t -test for Excel Microsoft, Redmond, WA. Ten-twelve CNV lesions were created in one eye of each mouse for gene expression analysis and the fellow eye, which was not treated, served as a control. The results were expressed as normalized relative gene expression or the log 2 scale of the mean change fold over control from 5 mice. Laser-induced ruptures of Bruch's membrane were performed as described in Methods. Mice were perfused with FITC-labeled dextran and choroidal flat mounts were prepared and examined by fluorescence microscopy.
The dosages evaluated included MF1: MF1 or DC Fig. Grossly visible side effects were not observed with either antibody, but tissue analysis was not performed. The insert at the lower left of panel j is a higher magnification of the cell in the box, showing the morphological appearance of a microglial cell.
Arrows define the limits of the CNV lesions. The quantification of lectin-positive area is shown in panel n. The merged images are shown in c, f and i.
Because we didn't include detergent when we immuno-stained the choroidal flat mounts, we expected only the cells at the superficial CNV area on the retinal side , close to the sub-retinal space, the RPE, and the outer segments of the photoreceptors of the CNV lesions to be labeled. We first stained them with the endothelial cell marker, CD31, because we speculated they were vascular components, which had failed to assemble into neo vessels due to the lack of VEGFR1 and 2 signaling. Very often, these cells have multiple processes and microglia-like morphology one example is shown in the insert of Fig.
Neovascular tufts were labeled with griffonia simplicifolia lectin The results are expressed as normalized relative gene expression or the log 2 scale of mean change fold over control from 5 mice. The dosages evaluated were the same as those for CNV. MF1: The results demonstrated that blockade of VEGFR1 inhibited the expression of some of these genes to various degrees.
The BRB assay was performed at P The dosing and treatment schedules for MF1 and DC were the same as described above and the vascular leakage was quantified by measuring [ 3 H]-mannitol leakage from blood vessels into the retina as described in Methods. Previous reports have demonstrated that both VEGFR1 and 2 play very important roles in mediating adverse complications of ischemic and inflammatory disorders, particularly in cancers  —  , and our present study gave further insight into their respective roles and mechanisms in pathological angiogenesis and vascular leakage with the two widely-used mouse models of ocular angiogenesis: laser-induced CNV and OIR.
Our observations in this study included i consistent with earlier findings  —  , VEGFR1 is involved in pathological angiogenic processes in ischemic retina and targeting VEGFR1 is a potential therapeutic treatment strategy for ischemic retinopathies such as AMD and ROP, ii both VEGFR1 and 2 are involved in the angiogenic process through differential mechanisms; combined administration of MF1 and DC had additive effects on pathological angiogenesis and vascular leakage, suggesting that combination therapy targeting both receptors would have better efficiency, and iii in addition to its role in promoting inflammation associated with ischemia, VEGFR1 signaling is likely involved in the regulation of ischemia-induced BRB breakdown, which makes it an attractive target for the treatment of macular edema.
For future prospective therapy, additional studies should be conducted to address the following questions. Is the intraocular administration, which is local and requires less antibody, but is more invasive, and is the widely-used approach for drug delivery in eye diseases, more effective, compared to systemic delivery?go to link
What are the safety profiles? Are the two antagonists, MF1 and DC, effective in other pre-clinical models? Answers to these questions could reveal the potential of VEGF receptor antibodies as therapeutic agents for vasoproliferative ocular disorders. Inflammation is one of the important mechanisms that promotes CNV formation and two immune responses are associated with its pathogenesis.
One is the translocation of resident microglia from the inner to the outer retina and the other is the mobilization and homing of bone marrow-derived leukocytes, such as macrophages  , . Griffonia simplicifolia isolectin-B4 staining is not only a marker for vascular endothelial cells, but it has been identified as a marker for microglia  , .
It would be reasonable that these cells were classified as retinal microglia that migrated from the inner retina, rather than macrophages that were mobilized from bone marrow and the lectin positivity supports this contention. This phenomenon suggested that recruitment of retinal microglia from the inner retina to areas of CNV is separated into two distinct steps, which may be dependent on different signaling molecules.
Currently, we don't have evidence to support the possibility and additional studies will be needed to elucidate that. The present results are not designed to dispute the involvement of circulating macrophages in CNV, but to demonstrate the involvement of retinal microglia.
The hypothesized mechanisms regulating recruitment of retinal microglia to CNV is shown in Fig. The inflammatory cells in CNV could be recurited retinal microgila, which settle in the inner retina under normal conditions or macrophages from bone marrow that access CNV lesions via blood flow through the choroid. The exact mechanms controling the recruitment of macrophages is unclear. Line: activation; dished lines: blockade or inhibition.
To quantitatively examine gene expression during CNV pathogenesis, we generated 10—14 CNV average 12 lesions per eye, which is 9 more than the widely-used mouse model of laser-induced CNV typically 3 CNV lesions in each eye , and separated the retina and sclera-choroid-RPE. Interestingly, the up-regulation of the two receptors was more pronounced in retina than in choroid at days 7 and 14 Fig. PlGF showed a consistent increase at all three stages 3, 7 and 14 days after lasering , but VEGF-B only showed a transient up-regulation in choroid at the early stage 3 days after lasering.
Despite these relevant findings obtained from the quantitative analysis of gene expression, we recognized that some limitations and potential artifacts may exist in these approaches. First, the fold change of gene expression in the laser treatment groups was not substantial, but only showed moderate increases from 1. Second, expression of VEGF, in contrast to predications, was not significantly up-regulated, but even down-regulated at some points i.
The total RNA prepared was under-representative for CNV and the assay may not be sensitive enough for detection of the changes. To overcome these limitations, the isolation of targeted or precise CNV areas, such as by Laser Capture Microdissection LCM , would be a more preferable approach to quantify the gene expression in this model, which will lead to more significant results.
Natural product inhibitors of ocular angiogenesis
As stated in the Introduction , the discovery of novel targets for anti-angiogenic therapy, in addition to VEGF, is highly desired and the targeting therapy against VEGFR1 and its ligand PlGF, which can become a potential therapeutic alternative or supplement to anti-VEGF therapy, has drawn increasing attention. Several additional strategies should be explored to overcome the limitations of anti-VEGF therapy [see Introduction , 7—21].
The first strategy would be to target molecules other than VEGF itself. These potential targets include the upstream molecules that regulate VEGF expression i. Second, given that angiogenesis is a complex process involving multiple cell types and signaling pathways, it would be more efficient to target multiple molecules or pathways simultaneously rather than one individual molecule or signaling pathway.
The third strategy would be to develop more efficient agents i. Last, since the ischemic tissues are starving for vessels to supply oxygen and nutrients, which very often leads to the formation of abnormal vasculatures or NV, the inhibition or regression of NV by anti-angiogenic factors has a potential risk of stroke, myocardial infarction, and neuronal toxicity, etc.
Therefore, a safe means of preventing or reversing pathological NV and BRB breakdown or normalizing pathological NV into functional vasculatures with normal perfusion and barrier function would be desirable for anti-angiogenic therapy. The present study has demonstrated that both VEGFR1 and R2 are implicated in pathological angiogenesis and BRB breakdown in ocular disease models and has shown that blocking both receptors is superior to blocking only one.
This approach, alone or in combination with targeting other molecules or signaling pathways, could provide greater benefit than currently implemented therapies. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field.
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Conclusion Our study provides further evidence that both VEGFR1 and 2 mediate pathological angiogenesis and vascular leakage in these models of ocular disease and suggests that antagonist antibodies to these receptor tyrosine kinases RTKs are potential therapeutic agents. Download: PPT. Table 1. Known cell types that can be labeled with each of the markers. Figure 1. Figure 3. Figure 4. Figure 5.