Angiogenesis and Blood-Brain Barrier Permeability in Vascular Remodeling after Stroke

Abstract

Angiogenesis, the growth of new blood vessels, is a natural defense mechanism helping to restore oxygen and nutrient supply to the affected brain tissue following an ischemic stroke. By stimulating vessel growth, angiogenesis may stabilize brain perfusion, thereby promoting neuronal survival, brain plasticity, and neurologic recovery. However, therapeutic angiogenesis after stroke faces challenges: new angiogenesis-induced vessels have a higher than normal permeability, and treatment to promote angiogenesis may exacerbate outcomes in stroke patients. The development of therapies requires elucidation of the precise cellular and molecular basis of the disease. Microenvironment homeostasis of the central nervous system is essential for its normal function and is maintained by the blood-brain barrier (BBB). Tight junction proteins (TJP) form the tight junction (TJ) between vascular endothelial cells (ECs) and play a key role in regulating the BBB permeability. We demonstrated that after stroke, new angiogenesis-induced vessels in peri-infarct areas have abnormally high BBB permeability due to a lack of major TJPs in ECs. Therefore, promoting TJ formation and BBB integrity in the new vessels coupled with speedy angiogenesis will provide a promising and safer treatment strategy for improving recovery from stroke. Pericyte is a central neurovascular unite component in vascular barriergenesis and are vital to BBB integrity. We found that pericytes also play a key role in stroke-induced angiogenesis and TJ formation in the newly formed vessels. Based on these findings, in this article, we focus on regulation aspects of the BBB functions and describe cellular and molecular special features of TJ formation with an emphasis on role of pericytes in BBB integrity during angiogenesis after stroke.

Keywords: Cerebral stroke; angiogenesis; barriergenesis; blood-brain barrier permeability; tight junction proteins; vascular remodeling.

Fig. (1)

Differentiation of the blood-brain barrier (BBB). Angiogenesis phase: Vascular sprouts radially invade the embryonic neuroectoderm towards a concentration gradient of VEGF-A, which is produced by neuroectodermal cells located in the ventricular layer. VEGF-A binds to its endothelial receptor, the receptor tyrosine kinase flk-1/KDR/VEGFR2. The EC specific receptor tyrosine kinase Tie-2 and its ligand Ang-1 are involved in angiogenic sprouting early during embryogenesis. The cerebral ECs show Glut-1 evenly distributed and the MECA-32 antigen is highly expressed, contributing to poor barrier characteristics and high paracellular permeability (PP). Differentiation phase: The phenotype of cerebral ECs changes such that they downregulate the expression of the MECA-32 antigen. Glut-1 is now enriched on the abluminal surface of the endothelium. The TJs become complex and thus tight for small polar molecules. Phenotypic changes of ECs are accompanied by their close contact with PCs and astroglial cells. Recruitment of PCs along the differentiating BBB vessels is ensured by several mechanisms. PDGF-BB produced by ECs binds to its receptor PDGFR-β on PCs; N-cadherin enriched at sites of PC-EC contact; Ang-1 expressed by PCs binds to the endothelial receptor tyrosine kinase Tie-2. ECs produce leukemia inhibitory factor (LIF), inducing the maturation of ACs via the LIF-Rb. Furthermore, increased oxygen level and EC-derived PDGF-BB lead to an upregulation of SSeCKS in ACs that in turn upregulates Ang-1. Note: During vascular remodeling after stroke, the newly formed vessels in peri-infarct regions demonstrate cellular features as angiogenesis phase and differential phase as shown above. In the differential phase, the only TJP that formed the TJ strands between ECs is claudin-5, while occludin and ZO-1 are expressed by ACs and PCs. The vascular PCs also express NG2, MMP-3, and other angiogenic factors. The molecular mechanisms involved in crosstalk between ECs, ACs and PCs required for TJ formation and maturation in the newly formed vessels remain under studied. Maturation phase: Despite the fact that the cerebral ECs form the barrier proper, close contact with PCs, ACs and maybe neuronal cells is required for the maintenance of the BBB. The molecular mechanisms involved in this crosstalk required for BBB maintenance in the mature CNS remain unknown to date. Modified from Stefan Liebner et al., Int. J. Dev. Biol. 2011, 55, 467-476. (A higher resolution / colour version of this figure is available in the electronic copy of the article).

Fig. (2)

Blood flow, BBB permeability, and expression of TJPs in the new vessels within peri-infarct regions 3 weeks of reperfusion in spontaneously hypertensive rat subjected to transient middle cerebral artery occlusion. (A) Hyperintensive areas in the anatomical T2 image and ADC map show the lesion extent and tissue ischemia. ven: Ventricle; inf: Core infarct area. Color-coded permeability coefficient maps reconstructed from DCEMRI data demonstrate the regions of high (red) and low (blue) permeability. The parametric image K_i map represents BBB transfer rate. The parametric image V_p map represents plasma volume. Elevated values of K_i and V_p are located in the vicinity of core infarct area (arrows). There are no signals of K_i and V_p in the core infarct area. The color scales used for the permeability and plasma volume signal intensity. ASL map shows higher CBF in peri-infarct areas (arrows). RECA1 immunostaining shows the increased density of new vessels in the peri-infarct area (arrows), corresponding to the elevated BBB transfer rate, plasma volume and blood perfusion (arrows). H&E staining shows red blood cells located inside of the new microvessels (arrows). (B) Left panel: double-immunostaining of occludin (red) with astrocytes (GFAP, green) shows that occludin was co-localized with reactive astrocytes adjacent to or within the peri-infarct region. Middle and right panels: triple-immunostaining of occludin (red) with ACs (blue) and ECs (green). The 3D confocal images demonstrate that ACs expressing occludin (shown in purple when co-localized) with end-feet closely surrounding vessels. (C) Double immunostaining of ZO-1 with astrocytes, endothelial cells, and PCs (PDGFR), respectively. ZO-1 co-localized with reactive astrocytes (GFAP) but not with ECs (RECA1). Z-stack confocal image shows PCs (green) surrounding vessels express ZO-1 (arrows). Arrowheads indicate DAPI-stained endothelium. (D) Double immunostaining shows no co-localization (left panel) between ACs (green) and claudin-5 (red). The 3D confocal image (middle panel) demonstrates that claudin-5 was co-localized with ECs (CD31, green) of microvessels within the peri-infarct region. Confocal image (right panel) of triple-immunostaining shows that ACs (blue) surround a vessel with claudin-5 in endothelial cells (green). Scale bars=20 or 50 μm. Cited from Yi Yang et al., Journal of Cerebral Blood Flow & Metabolism (2013), 1104-1114. (A higher resolution / colour version of this figure is available in the electronic copy of the article).