The neuronal oxygen-sensing pathway controls postnatal vascularization of the murine brain

Abstract

The contribution of neurons to growth and refinement of the microvasculature during postnatal brain development is only partially understood. Tissue hypoxia is the physiologic stimulus for angiogenesis by enhancing angiogenic mediators partly through activation of hypoxia-inducible factors (HIFs). Hence, we investigated the HIF oxygen-sensing pathway in postmitotic neurons for physiologic angiogenesis in the murine forebrain during postnatal development by using mice lacking the HIF suppressing enzyme prolyl-4-hydroxylase domain (PHD)2 and/or HIF-1/2α in postmitotic neurons. Perinatal activation or inactivation of the HIF pathway in neurons inversely modulated brain vascularization, including endothelial cell number and proliferation, density of total and perfused microvessels, and vascular branching. Accordingly, several angiogenesis-related genes were up-regulated in vivo and in primary neurons derived from PHD2-deficient mice. Among them, only VEGF and adrenomedullin (Adm) promoted angiogenic sprouting of brain endothelial cells. VEGF and Adm additively enhanced endothelial sprouting through activation of multiple pathways. PHD2 deficiency in neurons caused HIF-α stabilization and increased VEGF mRNA levels not only in neurons but unexpectedly also in astrocytes, suggesting a new mechanism of neuron-to-astrocyte signaling. Collectively, our results identify the PHD-HIF pathway in neurons as an important determinant for vascularization of the brain during postnatal development.—Nasyrov, E., Nolan, K. A., Wenger, R. H., Marti, H. H., Kunze, R. The neuronal oxygen-sensing pathway controls postnatal vascularization of the murine brain.

Vascularization of the vertebrate CNS begins early during embryogenesis with the formation of the perineural vascular plexus around the neural tube (1). Subsequently, the developing brain and spinal cord are predominantly vascularized by secondary angiogenesis from the perineural vascular plexus, where new vessel sprouts invade the CNS and extend toward the ventricle (1). Upon reaching the ventricle, vessels form new branches that surround the ventricle and reverse their direction toward the pia. Finally, branches anastomose with other branches, giving rise to a rich capillary plexus (1). Although general and CNS-specific mechanisms of developmental angiogenesis throughout embryogenesis have been extensively studied over the past years (1–3), the growth and refinement of the cerebral microvasculature during postnatal CNS development is only poorly understood. Studies on vascularization of the postnatal rodent retina suggest that VEGF, among other angiogenic factors, is of crucial importance for retinal angiogenesis. Although astrocytes and Müller glia are the major source of VEGF in the retina (4–6), conditional gene deletion and cell ablation approaches have clearly demonstrated that VEGF derived from neurons (including retinal ganglion cells, amacrine, and horizontal cells) rather than glial cells is most important for retinal developmental angiogenesis (7, 8). The pivotal importance of postmitotic neurons for precise spatiotemporal vascularization of the embryonic CNS has also been demonstrated in the developing spinal cord, where motor neurons control blood vessel patterning by an autocrine mechanism that titrates motor neuron-derived VEGF via their own expression of Fms-related tyrosine kinase 1 (Flt1), a soluble isoform of VEGF receptor-1 acting as a VEGF trap (9).

Hypoxia is the principal physiologic stimulus that induces angiogenesis and provides a stimulus-response pathway by which all cells are ensured adequate oxygenation (10). Expression of virtually all of the critical angiogenic growth factors is induced by hypoxia either directly or indirectly through hypoxia-inducible factors (HIFs) (10). HIFs are heterodimeric transcription factors that consist of oxygen-regulated HIF-1α or HIF-2α and constitutively expressed HIF-1β subunits that mediate adaptive responses to hypoxia/ischemia in all nucleated cells of metazoan organisms (10). HIF-α subunits are targeted for proteasomal degradation under nonhypoxic conditions through hydroxylation of conserved proline residues by prolyl-4-hydroxylase domain (PHD) enzymes utilizing molecular oxygen and α-ketoglutarate as cosubstrates (10, 11). Prolyl hydroxylation of HIF-α causes binding to the von Hippel-Lindau protein, an E3 ubiquitin ligase, resulting in immediate HIF-1α and HIF-2α ubiquitination and proteasomal degradation (10, 11). Hydroxylation of a conserved asparagine residue of HIF-1α and HIF-2α catalyzed by factor-inhibiting HIF prevents binding to the transcriptional coactivators CREB-binding protein and p300. Consequently, hypoxia-induced inhibition of prolyl and asparaginyl hydroxylase activity results in a rapid increase in HIF-α levels and transcriptional activity (10, 11). Post-translationally stabilized HIF-α translocates to the nucleus, dimerizes with HIF-1β, and binds to hypoxia response elements, which function as cis-acting elements that determine the target genes for activation by HIF (10, 11).

PHD2 is the most abundant PHD isoform in neurons and the adult murine CNS (12, 13). PHD2 has further been reported to be the critical oxygen sensor setting the low steady-state levels of HIF-1α in normoxia (14). However, although previous experimental studies indicate a pivotal role of postmitotic neurons (7–9) and the molecular PHD-HIF axis in developmental brain angiogenesis (15–17), their importance for postnatal cerebrovascular remodeling is still largely unknown. Thus, the present study elucidated whether and how the oxygen-sensing machinery in mature neurons controls physiologic angiogenesis in the murine forebrain during early postnatal development.