Hye Shin Lee

Brain angiogenesis in developmental and pathological processes: regulation, molecular and cellular communication at the neurovascular interface

The vascular network of the brain is formed by the invasion of vascular sprouts from the pia mater toward the ventricles. Following angiogenesis of the primary vascular network, brain vessels experience a maturation process known as barriergenesis, in which the blood–brain barrier is formed. In this minireview, we discuss the processes of brain angiogenesis and barriergenesis, as well as the molecular and cellular mechanisms underlying brain vessel formation. At the molecular level, angiogenesis and barriergenesis occur via the coordinated action of oxygen‐responsive molecules (e.g. hypoxia‐inducible factor and Src‐suppressed C kinase substrate/AKAP12) and soluble factors (e.g. vascular endothelial growth factor and angiopoietin‐1), as well as axon guidance molecules and neurotrophic factors. At the cellular level, we focus on neurovascular cells, such as pericytes, astrocytes, vascular smooth muscle cells, neurons and brain macrophages. Each cell type plays a unique role, and works with other types to maintain environmental homeostasis and to respond to certain stimuli. Taken together, this minireview emphasizes the importance of the coordinated action of molecules and cells at the neurovascular interface, with regards to the regulation of angiogenesis and barriergenesis.

The astrocyte-expressed integrin αvβ8 governs blood vessel sprouting in the developing retina.

The mouse retina is vascularized after birth when angiogenic blood vessels grow and sprout along a pre-formed latticework of astrocytes. How astrocyte-derived cues control patterns of blood vessel growth and sprouting, however, remains enigmatic. Here, we have used molecular genetic strategies in mice to demonstrate that αvβ8 integrin expressed in astrocytes is essential for neovascularization of the developing retina. Selective ablation of αv or β8 integrin gene expression in astrocytes leads to impaired blood vessel sprouting and intraretinal hemorrhage, particularly during formation of the secondary vascular plexus. These pathologies correlate, in part, with diminished αvβ8 integrin-mediated activation of extracellular matrix-bound latent transforming growth factor βs (TGFβs) and defective TGFβ signaling in vascular endothelial cells, but not astrocytes. Collectively, our data demonstrate that αvβ8 integrin is a component of a paracrine signaling axis that links astrocytes to blood vessels and is essential for proper regulation of retinal angiogenesis.

αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion

Experiments with human cancer glioblastoma multiforme cell lines, primary patient samples, and preclinical mouse models are performed to show that αvβ8 integrin and RhoGDI1 are components of a signaling axis that drives brain tumor cell invasion via regulation of Rho GTPase activation.

Endothelial Expression of TGFβ Type II Receptor Is Required to Maintain Vascular Integrity during Postnatal Development of the Central Nervous System

TGFβ signalling in endothelial cells is important for angiogenesis in early embryonic development, but little is known about its role in early postnatal life. To address this we used a tamoxifen inducible Cre-LoxP strategy in neonatal mice to deplete the TypeII TGFβ receptor (Tgfbr2) specifically in endothelial cells. This resulted in multiple micro-haemorrhages, and glomeruloid-like vascular tufts throughout the cerebral cortices and hypothalamus of the brain as well as in retinal tissues. A detailed examination of the retinal defects in these mutants revealed that endothelial adherens and tight junctions were in place, pericytes were recruited and there was no failure of vascular smooth muscle differentiation. However, the deeper retinal plexus failed to form in these mutants and the angiogenic sprouts stalled in their progress towards the inner nuclear layer. Instead the leading endothelial cells formed glomerular tufts with associated smooth muscle cells. This evidence suggests that TGFβ signalling is not required for vessel maturation, but is essential for the organised migration of endothelial cells as they begin to enter the deeper layers of the retina. Thus, TGFβ signalling is essential in vascular endothelial cells for maintaining vascular integrity at the angiogenic front as it migrates into developing neural tissues in early postnatal life.

Meteorin promotes the formation of GFAP-positive glia via activation of the Jak-STAT3 pathway

Meteorin is an orphan ligand which has been previously reported to control neuritogenesis and angiogenesis, as well as gliogenesis. However, the precise function of this factor in CNS development and the underlying molecular mechanisms are poorly understood. Here, we demonstrate that meteorin is involved in GFAP-positive glial differentiation through activation of the Jak-STAT3 pathway, by using neurosphere and retinal explant culture systems. During embryonic brain development, meteorin is highly expressed in neural stem and radial glia cells of the ventricular zone and immature neurons outside the ventricular zone but its expression disappears spontaneously as development proceeds except in GFAP-positive astrocytes. In cultured neurospheres, meteorin activates STAT3, and in turn increases the transcriptional activity of GFAP by enhancing the binding of STAT3 to the promoter. By meteorin stimulation, differentiating neurospheres show increased numbers of GFAP-positive cells, but the effect is abrogated by a blockade of the Jak-STAT3 pathway using either a Jak inhibitor or STAT3 siRNA. Furthermore, we expand our findings to the retina, and show that meteorin increases GFAP expression in Müller glia. Together, our results suggest that meteorin promotes GFAP-positive glia formation by mediating the Jak-STAT3 signaling pathway during both cortical stem cell differentiation and retinal glia development.

Meteorin regulates angiogenesis at the gliovascular interface

Brain microvasculature requires a coordinated interaction between endothelial cells and astrocytes at the gliovascular interface. However, the role of the factors involved in that interaction and expressed by these cells is poorly understood. In this study, we demonstrate that Meteorin is highly expressed in astrocytes of the brain and retina during the late embryonic and postnatal stages of mouse development. Most notably, Meteorin is localized to the astrocyte endfeet that surround the blood vessels. To investigate the role of Meteorin in perivascular astrocytes, we depleted endogenous levels of Meteorin in cultured astrocytes using siRNA, and found that Meteorin attenuates angiogenic activity indirectly via astrocyte‐derived thrombospondin‐1/‐2 (TSP‐1/‐2). Exogenous treatment of astrocytes with Meteorin protein also promotes astrocyte expression and secretion of TSP‐1/‐2. The conditioned media from the Meteorin‐treated astrocytes attenuated angiogenic activity of microvascular endothelial cells. This activity was reversed by inhibiting the binding of TSP‐1/‐2 to its receptor. Furthermore, we found that TSP‐1/‐2 was co‐localized with Meteorin in the developing brain. Therefore, our data strongly suggests that Meteorin is expressed and secreted by perivascular astrocytes and the secreted protein upregulates TSP‐1/‐2 to attenuate angiogenesis in the surrounding endothelial cells and to promote vascular maturation.

Clusterin protects H9c2 cardiomyocytes from oxidative stress-induced apoptosis via Akt/GSK-3β signaling pathway

Clusterin is a secretory glycoprotein, which is highly up-regulated in a variety of normal and injury tissues undergoing apoptosis including infarct region of the myocardium. Here, we report that clusterin protects H9c2 cardiomyocytes from H2O2-induced apoptosis by triggering the activation of Akt and GSK-3β. Treatment with H2O2 induces apoptosis of H9c2 cells by promoting caspase cleavage and cytochrome c release from mitochondria. However, co-treatment with clusterin reverses the induction of apoptotic signaling by H2O2, thereby recovers cell viability. The protective effect of clusterin on H2O2-induced apoptosis is impaired by PI3K inhibitor LY294002, which effectively suppresses clusterin-induced activation of Akt and GSK-3β. In addition, the protective effect of clusterin is independednt on its receptor megalin, because inhibition of megalin has no effect on clusturin-mediated Akt/GSK-3β phosphoylation and H9c2 cell viability. Collectively, these results suggest that clusterin has a role protecting cardiomyocytes from oxidative stress and the Akt/GSK-3β signaling mediates anti-apoptotic effect of clusterin.

Neuropilin 1 balances β8 integrin-activated TGFβ signaling to control sprouting angiogenesis in the brain.

Angiogenesis in the developing central nervous system (CNS) is regulated by neuroepithelial cells, although the genes and pathways that couple these cells to blood vessels remain largely uncharacterized. Here, we have used biochemical, cell biological and molecular genetic approaches to demonstrate that β8 integrin (Itgb8) and neuropilin 1 (Nrp1) cooperatively promote CNS angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells. β8 integrin in the neuroepithelium promotes the activation of extracellular matrix (ECM)-bound latent transforming growth factor β (TGFβ) ligands and stimulates TGFβ receptor signaling in endothelial cells. Nrp1 in endothelial cells suppresses TGFβ activation and signaling by forming intercellular protein complexes with β8 integrin. Cell type-specific ablation of β8 integrin, Nrp1, or canonical TGFβ receptors results in pathological angiogenesis caused by defective neuroepithelial cell-endothelial cell adhesion and imbalances in canonical TGFβ signaling. Collectively, these data identify a paracrine signaling pathway that links the neuroepithelium to blood vessels and precisely balances TGFβ signaling during cerebral angiogenesis. Summary: Neuropilin 1 and β8 integrin cooperatively promote cerebral angiogenesis by mediating adhesion and signaling events between neuroepithelial cells and vascular endothelial cells in the mouse brain.

Protein tyrosine phosphatase-PEST and β8 integrin regulate spatiotemporal patterns of RhoGDI1 activation in migrating cells.

ABSTRACT Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cells leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells.

ARD1-mediated Hsp70 acetylation balances stress-induced protein refolding and degradation

Heat shock protein (Hsp)70 is a molecular chaperone that maintains protein homoeostasis during cellular stress through two opposing mechanisms: protein refolding and degradation. However, the mechanisms by which Hsp70 balances these opposing functions under stress conditions remain unknown. Here, we demonstrate that Hsp70 preferentially facilitates protein refolding after stress, gradually switching to protein degradation via a mechanism dependent on ARD1-mediated Hsp70 acetylation. During the early stress response, Hsp70 is immediately acetylated by ARD1 at K77, and the acetylated Hsp70 binds to the co-chaperone Hop to allow protein refolding. Thereafter, Hsp70 is deacetylated and binds to the ubiquitin ligase protein CHIP to complete protein degradation during later stages. This switch is required for the maintenance of protein homoeostasis and ultimately rescues cells from stress-induced cell death in vitro and in vivo. Therefore, ARD1-mediated Hsp70 acetylation is a regulatory mechanism that temporally balances protein refolding/degradation in response to stress.