Microglial activation state exerts a biphasic influence on brain endothelial cell proliferation by regulating the balance of TNF and TGF-β1

Abstract

BackgroundStudies of cerebral ischemia and other neuroinflammatory states have demonstrated a strong association between new vessel formation and microglial recruitment and activation, raising the possibility that microglia may be involved in promoting angiogenesis. As endothelial cell proliferation is a fundamental early step in angiogenesis, the aim of this study was to test this hypothesis by examining the influence of microglial secreted factors on brain endothelial cell (BEC) proliferation using BrdU incorporation.MethodsPrimary cultures of mouse BEC, microglia and astrocytes were used in this study. Proliferation of BEC was examined by BrdU incorporation. ELISA was used to quantify TNF and TGF-β1 levels within cell culture supernatants.ResultsMicroglia regulated BEC proliferation in a biphasic manner; microglia conditioned medium (MG-CM) from resting microglia inhibited, while that from activated microglia promoted BEC proliferation. A screen of microglial cytokines revealed that BEC proliferation was inhibited by TGF-β1, but promoted by TNF. ELISA showed that TNF and TGF-β1 were both present in MG-CM, and that while TGF-β1 dominated in resting MG-CM, TNF levels were massively increased in activated MG-CM, shifting the balance in favor of TNF. Antibody-blocking studies revealed that the influence of MG-CM to inhibit or promote BEC proliferation was largely attributable to the cytokines TGF-β1 and TNF, respectively.ConclusionThis data suggests that microglial activation state might be an important determinant of cerebral angiogenesis; inhibiting BEC proliferation and neovascularization in the normal central nervous system (CNS), but stimulating the growth of new capillaries under neuroinflammatory conditions.

Extensive vascular remodeling in the spinal cord of pre-symptomatic experimental autoimmune encephalomyelitis mice; increased vessel expression of fibronectin and the α5β1 integrin

“…In light of our current findings, showing similar upregulation of endothelial α5 integrin on remodeling cerebral blood vessels during the early stages of EAE, we next examined in vitro whether α5 integrin mediates the BEC proliferative response to a pro-inflammatory stimulus. Previously, we found that TNF-α strongly stimulates BEC proliferation (Welser et al 2010), and our current experiments, using bromodeoxyuridine (BrdU) incorporation, confirmed this finding, with TNF-α increasing proliferation (from 20.7 ± 1.8% under control conditions to 43.3 ± 2.9%, p < 0.001; see Figure 7D). Furthermore, incubation with an α5 integrin function-blocking antibody significantly reduced brain endothelial cell proliferation, both under non-stimulated conditions (from 20.7 ± 1.8% to 16.3 ± 2.1%, p < 0.05) and in response to TNF-α (from 43.3 ± 2.9% to 22.5 ± 4.6%, p < 0.005).…”

Cerebral Vascular Disease and Neurovascular Injury in Ischemic Stroke

“…296 The M2 phenotype microglia, in contrast, enhance BBB integrity by increasing TJ expression after stroke. 297 …”

The Yin and Yang of Microglia

“…During this period, microglia not only have key functions such as the clearance of dying or dead cells. Several studies are emerging that demonstrate a role in developmental processes such as apoptosis, elimination of excess axons, promotion of neuroaxonal growth, axonal guidance, neuronal differentiation, regulation of embryonic cortical precursor cell development, astrocyte proliferation and angiogenesis [20,30,34,35,36,37,38,39,40,41,42,43,44,45]. Especially the role of microglia in neurogenesis is intriguing as, within the framework of CNS inflammation, the impact of this feature on the developing brain can be enormous.…”