Contraindications of VEGF-based therapeutic angiogenesis: Effects on macrophage density and histology of normal and ischemic brains

Abstract

Therapeutic angiogenesis by vascular endothelial growth factor (VEGF) is advocated as a promising treatment strategy for brain ischemic stroke. However, data in the literature demonstrating the benefit of therapeutic angiogenesis are contradictory. In this paper, we describe the effects of non-angiogenic and angiogenic doses of VEGF₁₆₅ on macrophage density and histology of normal and ischemic brains of adult rats. VEGF₁₆₅ was administered intra-arterially for 7 days following temporary occlusion of the middle cerebral artery. In contrast to ischemic brains treated with non-angiogenic doses of VEGF₁₆₅ which showed preserved neuropil and reduced numbers of macrophages, ischemic brains treated by an angiogenic dose showed phagocytized neuropil and high macrophage density. Though neither non-angiogenic nor angiogenic doses caused macrophage infiltration in normal brains, damage of the brain matrix occurred with the angiogenic dose. These results suggest an angiogenic dose of VEGF₁₆₅ injures the nervous tissue rather than promote recovery. Angiogenesis by VEGF monotherapy for ischemic stroke should be viewed with caution, or avoided. Since our data show intravascular administration of VEGF₁₆₅ does not cause macrophage inflammation, in contrast to reports in the literature whereby VEGF₁₆₅ was applied directly to the brain, our findings also indicate the relationships between VEGF, angiogenesis, and macrophage inflammation are governed by the route VEGF is administered to the brain.

Introduction

Vascular endothelial growth factor (VEGF) is neuroprotective and angiogenic (Sun et al., 2003, Carmeliet and Storkebaum, 2002, Ferrara and Gerber, 2001). VEGF also promotes neurogenesis, learning, memory and inhibition of apoptosis (Cao et al., 2004, Greenberg and Jin, 2004, Jin et al., 2002). Consequently, VEGF earned wide attention as a promising pharmacological agent for the treatment of ischemic stroke (Bellomo et al., 2003, Krupinski et al., 2003, Zhang and Chopp, 2002). VEGF could simultaneously protect the injured neurons and augment blood flow to the ischemic tissue. On the other hand, VEGF is notorious for causing increased vascular permeability, inflammation, vasodilation and alterations of systemic hemodynamics (Croll et al., 2004a, Bates and Harper, 2002, Forstreuter et al., 2002). VEGF-induced angiogenic vessels, particularly in the formative stages, are leaky (Bates et al., 2002, Schoch et al., 2002, Dobrogowska et al., 1998). These effects of VEGF can lead to compromise of the blood–brain barrier (BBB) and edema and damage of the nervous tissue (Schoch et al., 2002, Pettersson et al., 2000, van Bruggen et al., 1999). Thus, it is unclear whether neuroprotection and angiogenesis by exogenous VEGF can simultaneously be induced for the treatment of ischemic stroke (Carmeliet and Storkebaum, 2002, Ferrara, 2002, Zhang and Chopp, 2002). Though angiogenesis by VEGF may show therapeutic value for limb and heart ischemia (Losordo et al., 1998, Isner, 1998, Isner et al., 1996), it is possible that angiogenesis by VEGF monotherapy (i.e., VEGF treatment without inclusion of other pharmacologic agents to counteract the side-effects and/or enhance the positive effects of the growth factor) may not be beneficial for the treatment of brain ischemia. Rather, angiogenesis by VEGF monotherapy may pose the risk of greater harm to the stroke patient. We have shown that neuroprotection and angiogenesis by exogenous VEGF₁₆₅ monotherapy are not necessarily concurrent events (Manoonkitiwongsa et al., 2004). Instead, the relationship between neuroprotection and angiogenesis appears inversely related. Our findings regarding the inverse relationship between neuroprotection and angiogenesis with VEGF monotherapy are supported by later findings of other investigators (Wang et al., 2005, Valable et al., 2005).

The purpose of this report is to evaluate the tissues of our earlier work (Manoonkitiwongsa et al., 2004) to characterize changes in the histology of the brain matrix and density of macrophages of normal and ischemic brains exposed to non-angiogenic and angiogenic doses of VEGF₁₆₅. This information is vital if angiogenesis by VEGF is to be applied for the treatment of ischemic stroke. The histopathologic endpoints are indispensable for elucidating the therapeutic and adverse effects of VEGF on the nervous tissue. With respect to macrophage inflammation, it is currently believed that administration of exogenous VEGF₁₆₅ to the brain causes migration of macrophages (Croll et al., 2004b, Proescholdt et al., 1999). However, in these reports, VEGF₁₆₅ was applied directly to the brain through invasive techniques. Thus, it is unclear whether VEGF administered intravascularly would produce similar relationships between exogenous VEGF, angiogenesis and macrophage inflammation. In clinical practice, presumably, VEGF would be administered intravascularly rather than directly to the brain through invasive approaches such as cortical penetration, intraventricular or topical. The presence of the blood–brain barrier (BBB), which VEGF would encounter when administered intravascularly, could change the manners in which exogenous VEGF influences the migration of macrophages. The route through which VEGF is administered to the brain can produce different outcomes (Kaya et al., 2005). For our study, we targeted the macrophages as markers of inflammation because they are the characteristic inflammatory cells of ischemic brain (Croll et al., 2004b, Tanaka et al., 2003, Persson et al., 1989). It is known that VEGF attracts macrophages and that macrophages secrete VEGF and express VEGF receptors (Malaguarnera et al., 2004, Sawano et al., 2001, Moore et al., 2001, Heil et al., 2000, Waltenberger et al., 2000).