Omega-3 polyunsaturated fatty acids enhance cerebral angiogenesis and provide long-term protection after stroke

Highlights

• n − 3 PUFAs elicit long-term protection against cerebral ischemic injury.

• n − 3 PUFAs enhance post-stroke angiogenesis.

• n − 3 PUFAs promote astrocyte-mediated endothelial proliferation/barrier formation.

• Angiopoietin 2 potentiates VEGF-induced endothelial proliferation/barrier formation.

• VEGF-Src signaling participates in n − 3 PUFA-mediated revascularization.

Abstract

Stroke is a devastating neurological disorder and one of the leading causes of death and serious disability. After cerebral ischemia, revascularization in the ischemic boundary zone provides nutritive blood flow as well as various growth factors to promote the survival and activity of neurons and neural progenitor cells. Enhancement of angiogenesis and the resulting improvement of cerebral microcirculation are key restorative mechanisms and represent an important therapeutic strategy for ischemic stroke. In the present study, we tested the hypothesis that post-stroke angiogenesis would be enhanced by omega-3 polyunsaturated fatty acids (n − 3 PUFAs), a major component of dietary fish oil. To this end, we found that transgenic fat-1 mice that overproduce n − 3 PUFAs exhibited long-term behavioral and histological protection against transient focal cerebral ischemia (tFCI). Importantly, fat-1 transgenic mice also exhibited robust improvements in revascularization and angiogenesis compared to wild type littermates, suggesting a potential role for n − 3 fatty acids in post-stroke cerebrovascular remodeling. Mechanistically, n − 3 PUFAs induced upregulation of angiopoietin 2 (Ang 2) in astrocytes after tFCI and stimulated extracellular Ang 2 release from cultured astrocytes after oxygen and glucose deprivation. Ang 2 facilitated endothelial proliferation and barrier formation in vitro by potentiating the effects of VEGF on phospholipase Cγ1 and Src signaling. Consistent with these findings, blockade of Src activity in post-stroke fat-1 mice impaired n − 3 PUFA-induced angiogenesis and exacerbated long-term neurological outcomes. Taken together, our findings strongly suggest that n − 3 PUFA supplementation is a potential angiogenic treatment capable of augmenting brain repair and improving long-term functional recovery after cerebral ischemia.

Introduction

Stroke is the fourth leading cause of death and the leading cause of serious long-term disability in adults in the United States (Towfighi and Saver, 2011). Intravenous thrombolysis therapy with tissue plasminogen activator (tPA), the only FDA-approved treatment for ischemic strokes, is severely limited by its short therapeutic time window of less than 4.5 h (Hacke et al., 2008). To date, neuroprotective strategies that only target injured tissue during acute stages of stroke have not been successfully translated to the clinic. In contrast, neurorestorative therapies targeting the tissue spared from direct injury have recently gained attention for their potential to enhance post-stroke brain repair processes and to promote functional recovery with a longer therapeutic time window (Zhang and Chopp, 2009).

Vascular remodeling triggered by reduction of blood flow is a major endogenous defense mechanism in the brain following stroke. Revascularization is induced by shear fluid stress and consists of arteriogenesis, or an improvement in collateral blood flow through pre-existing vasculature in the early phase after ischemic injury. In the late stages after ischemic injury, revascularization also consists of a surge in angiogenesis through endothelial cell proliferation and the subsequent formation of new blood vessels (Liu et al., 2014). Post-stroke angiogenesis greatly improves tissue perfusion and endothelial cells release a plethora of neurotrophic factors, supporting the activity of neurons and neural progenitor cells and promoting long-term functional recovery (Zhang and Chopp, 2009). Thus, post-stroke angiogenesis is an important target for therapeutic interventions. Numerous experiments have attempted to enhance post-stroke angiogenesis, such as the transplantation of endothelial progenitor cells (EPCs), which are of great therapeutic value and already under clinical investigation (Fan et al., 2010, Ishikawa et al., 2013, Liu et al., 2014, Yin et al., 2013).

Omega-3 polyunsaturated fatty acids (n − 3 PUFAs) are known to protect against ischemic brain injury in several stroke models (Belayev et al., 2009, Hu et al., 2013, Zhang et al., 2010). However, the underlying mechanisms are not fully understood. Multiple mechanisms have been proposed for n − 3 PUFA-mediated protection, including reduction of oxidative stress (Bazan, 2005), anti-inflammatory effects (Musiek et al., 2008, Zhang et al., 2010), induction of heme oxygenase 1 by nuclear factor E2-related factor 2 (Zhang et al., 2014), and potentiation of neurogenesis and oligodendrogenesis (Hu et al., 2013). As neurogenesis is thought to only occur within an angiogenic microenvironment (Palmer et al., 2000), it seems likely that n − 3 PUFAs also promote the formation of new blood vessels. However, the effect of n − 3 PUFAs on vascular remodeling after acute ischemic stroke remains to be explored.

In order to fill this gap in the field, the present study examined the impact of stroke in transgenic (Tg) mice expressing the C. elegans fat-1 gene, which encodes an n − 3 fatty acid desaturase. We demonstrated that n − 3 PUFA levels were elevated in these mice through increased conversion from their endogenous n − 6 forms. Overproduction n − 3 PUFAs in fat-1 Tg mice provided remarkable protection against focal cerebral ischemia compared to wild type littermates. Interestingly, endogenous post-stroke angiogenesis was robustly enhanced by n − 3 PUFAs, in a process involving angiopoietin 2 (Ang 2) and vascular endothelial growth factor (VEGF) signaling. Our results strongly support the view that n − 3 PUFA supplementation is a potential prophylactic treatment to improve tissue repair and enhance long-term functional recovery after stroke.