Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis - PubMed (original) (raw)

Combination angiostatic therapy completely inhibits ocular and tumor angiogenesis

Michael I Dorrell et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Angiostatic therapies designed to inhibit neovascularization associated with multiple pathological conditions have only been partially successful; complete inhibition has not been achieved. We demonstrate synergistic effects of combining angiostatic molecules that target distinct aspects of the angiogenic process, resulting in the complete inhibition of neovascular growth associated with development, ischemic retinopathy, and tumor growth, with little or no effect on normal, mature tissue vasculature. Tumor vascular obliteration using combination angiostatic therapy was associated with reduced tumor mass and increased survival in a rat 9L gliosarcoma model, whereas individual monotherapies were ineffective. Significant compensatory up-regulation of several proangiogenic factors was observed after treatment with a single angiostatic agent. In contrast, treatment with combination angiostatic therapy significantly reduced compensatory up-regulation. Therapies that combine angiostatic molecules targeting multiple, distinct aspects of the angiogenic process may represent a previously uncharacterized paradigm for the treatment of many devastating diseases with associated pathological neovascularization.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Combination therapy enhances angiostasis in a neonatal eye model. (A) During the first three postnatal weeks, the mouse retinal vasculature forms three distinct planar plexuses. At postnatal day (P)8, vessels in the superficial plexus branch and form a deep plexus. To assess angiostasis, intravitreal injections were performed at P7, and inhibition of the deep vessels was scored 5 days later. Neural retina and previously formed superficial plexus were evaluated for toxicity. (B) Combination angiostatic therapy dramatically increases the percentage of treated retinas with high levels of neovascular inhibition. (C) Images from a representative experiment directly comparing angiostatic monotherapy and combination therapy at optimal doses (1×). sup., superficial; I.A., integrin antagonist; V.A., VEGF aptamer.

Fig. 2.

Fig. 2.

Combination angiostatic therapy is synergistic. (A) Triple combination therapy is potent at highly diluted concentrations. (B) Doses that exhibit minimal angiostatic activity as monotherapies (0.1× optimal dose) still exhibit potent angiostatic activity when used in combination. I.A., integrin antagonist; V.A. VEGF aptamer; ant., antagonist; apt., aptamer.

Fig. 3.

Fig. 3.

Combination angiostatic therapy inhibits ischemia-induced, pathological neovascularization. (A) In the mouse OIR model, hyperoxia (75% oxygen from P7 to P12) results in vascular obliteration. When mice are returned to normoxia (P12), retinas becomes hypoxic because of a lack of vessels, resulting in pathological neovascularization (P17). (B) Combination therapies significantly inhibit pathological neovascularization compared with vehicle control and angiostatic monotherapies (asterisks indicate P values <0.01 vs. PBS and each monotherapy, error bars represent SEM). (C) Many retinas treated with T2/VEGF aptamer combination (Center), or triple combination (Right) demonstrated nearly complete inhibition of neovascular tuft formation, seen as brightly stained areas in the PBS control-treated retinas (Left). int., integrin; ant., antagonist; apt., aptamer; I.A., integrin antagonist; V.A., VEGF aptamer.

Fig. 4.

Fig. 4.

Combination angiostatic therapy obliterates tumor vasculature, decreases tumor size, and increases survival. (A) Vessels are absent in tumors of animals treated for 3 days with triple combination therapy (Lower). Tumor vasculature is normal in control PBS treated tumors (Upper). (B) PBS treated tumors are highly proliferative (Upper) as indicated by ki-67 staining, whereas no proliferation is seen in the avascular triple combination-treated tumors (Lower). (C) Massive infiltrates of mononuclear cells are observed in avascular tumor regions after treatment with triple combination (Lower). In PBS controls (Upper) small areas of mononuclear cell infiltration are observed within large areas of normal tumor growth. (D) After 6 days of triple combination treatment, empty cavities (star), areas of mononuclear infiltrate (arrowhead), and smaller areas of reduced vasculature (diamond) are all observed within the tumor implantation areas (T). Normal brain vasculature in adjacent regions is not affected (N). (E) Rats with PBS-treated tumors have extensive, highly vascular tumors. (F) Triple combination significantly increases survival. Treatments were initiated 6 days after tumor implantation by using constant, local, convection-enhanced delivery to the central tumor. (Scale bars: 0.5 mm.)

Fig. 5.

Fig. 5.

Angiostatic monotherapy results in compensatory up-regulation of proangiogenic factors; combination therapy reduces such up-regulation. (A) Solutions consisting of PBS, monotherapies, or triple combination angiostatic therapy were injected into developing mouse eyes (P7) and protein expression levels of angiogenic factors were analyzed by an ELISA-based assay (P11). Stars and crosses indicate individual P values <0.05 compared with PBS-injected controls, or triple combination-treated retinas, respectively. (B) Statistical analyses of overall protein expression changes demonstrate that T2 and VEGF aptamer monotherapy treatments resulted in significant global increases in proangiogenic factor expression compared with PBS control-treated and triple combination-treated retinas.

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