Background
The concept of the neurovascular unit is proving to be very useful in efforts to elucidate the pathobiology of stroke [
1]. Brain ischemia is a vascular disorder affecting neuronal function. Indeed, the biological effects of ischemia are not confined to neurons, but impact an ensemble of different cell types in the brain, which, together, form the neurovascular unit. The bimodal response of the neurovascular unit to stroke is characterized by blood-brain-barrier disruption followed later by vascular remodeling and, hence, the switch from brain injury to repair and recovery.
Endothelia form a critical component of the neurovascular unit. Junctional proteins mediating homotypic interendothelial cell-cell contacts or heterotypic interactions with perivascular cells shape endothelial cell behavior [
2]. VE-cadherin is the chief constituent of adherens junctions. Expression of VE-cadherin is strictly confined to endothelia [
3]. By contrast, N-cadherin, the second major endothelial cadherin, is also expressed by other cells including neurons [
4], astrocytes [
5], and pericytes [
6].
So far, surprisingly little experimentation has been done on the role of junctional proteins in cerebrovascular disease. Importantly, the primary focus of this research has been on acute blood-brain barrier protection and maintenance of vascular integrity (e.g. [
7‐
9]). However, a number of studies conducted both in human stroke patients and in experimental rodents have demonstrated that acute lesion size is a poor predictor of overall stroke outcome [
10]. It should also be noted that endothelial junctional proteins represent interesting candidates for further research because they are potentially druggable (e.g. [
11,
12]).
In the current study, we investigated the effects of partial loss of VE-cadherin in VEC+/- mice on both acute and chronic stroke outcome. Acute stroke outcome did not differ between VEC+/- mice and controls. However, chronic lesion sizes were significantly reduced in VEC+/- animals. This was associated with an increase in the overall pattern of mRNA transcription of other junctional proteins and pericyte-related molecules in VEC+/- mice. Taken together, our results suggest that junctional proteins may be harnessed to promote blood flow and recovery after stroke.
Discussion
Stroke is a vascular disorder affecting neuronal function. Notwithstanding, the bulk of experimental stroke research has so far focussed rather one-sidedly on mechanisms to salvage neuronal cells and may thus have contributed to the apparent failure of bench-to-bedside translation plaguing the field in recent years (e.g. [
26]). However, stroke affects different cell types in the brain, which conjointly form the so-called ‘neurovascular unit’. For instance, in the gray matter of the human cerebral cortex, non-neuronal cells outnumber neurons by a factor of approximately 1 to 2 [
27]. In consequence, brain ischemia impacts not only neurons but also astrocytes and other glial cells that support the neurons, the axons of neurons that relay their signals to other cells, and finally, and very importantly, the microvessels that supply oxygen and nutrients to them. These considerations provide a firm basis for shifting the previous focus from neurons alone to the complex of neurons, microvessels and supportive cells (astrocytes, pericytes and resident inflammatory cells) reacting to brain ischemia. Conceptually, the response of the neurovascular unit to brain ischemia may be divided into two distinct phases: (i) damage to the neurovascular unit during the acute phase of stroke is followed by (ii) a phase of regeneration and partial restoration of function. The detailed mechanisms that govern this switch from early injury to delayed recovery are still poorly understood.
Endothelial cells are a crucial component of the neurovascular unit. Early on, endothelial cell injury results in blood-brain barrier dysfunction, which favors tissue swelling and may lead to intracerebral hemorrhage after stroke (e.g. [
28]). At later stages, the formation of new vessels with altered morphology and microvascular structure has been observed in ischemic brain tissue [
29].
Importantly, a number of studies, both by us and others, have established the notion that vascular remodeling after stroke is a critical determinant of chronic stroke outcome (e.g. [
19,
21,
30,
31]).
This study yields a number of new and unexpected insights into the vascular biology of ischemic stroke. In a nutshell, we show that the N-cadherin/VE-cadherin balance can be manipulated such that overall stroke outcome is improved. Contrary to our initial assumption, partial loss of VE-cadherin did not confer an increase in acute lesion size at 72 h. However, none of the junctional proteins investigated including VE-cadherin itself showed a significant difference in mRNA expression between genotypes at baseline. Furthermore, brains from heterozygous mice appeared phenotypically normal, so the genotypic differences were likely not large enough to translate into an early phenotypic difference after MCAo. Furthermore, we also did not detect significant differences in microvessel densities or absolute regional cerebral blood flow in the non-ischemic contralateral hemispheres between VEC+/- mice and wildtype controls.
More interestingly still, our results demonstrate that reduced VE-cadherin expression leads to improved vascular remodeling and a reduction in chronic lesion sizes after stroke. In how far these beneficial effects of reduced VE-cadherin expression also promote functional rehabilitation after stroke (i.e. neuro-behavioral assessment) lies beyond the scope of the present work. The following points in particular, for our purposes, merit further discussion here. Our finding of increased N-cadherin and β-catenin mRNA expression in VEC
+/- mice after stroke recapitulates earlier findings on endothelial cells in development, where N-cadherin mRNA and protein expression was reduced by VE-cadherin via inhibition of β-catenin signaling [
14]. N-cadherin, in turn, recruits pericytes, which also express N-cadherin, to endothelial cells [
6,
14,
32]. Accordingly, we investigated mRNA transcription of a panel of pericyte markers. Although not corroborated at the protein level, we detected a compelling pattern of elevated mRNA levels of pericyte-related molecules after MCAo. On a related note, it is also worth mentioning that the density of perfused microvessels after stroke did not differ between VEC
+/- mice and wildtype controls, but absolute cerebral blood flow did. In line with these seemingly contradictory observations, an in vivo model of brain angiogenesis in the absence of pericytes also yielded normal microvessel density, but abnormalities in microvessel architecture [
33]. It has to be acknowledged that, for this investigation, we did not assess CBF in separate sham controls of either genotype but limited ourselves to the analysis of blood flow in the contralateral striatum, which served as an internal control. Brain ischemia is a strong angiogenic stimulus [
19,
21]. Our study of post-stroke vascular remodeling thus reinforces recent findings indicating that endothelial/pericyte interactions are central processes in the regulation of vessel formation, stabilization, remodeling, and function [
34]. Importantly, pericytes are also beginning to emerge as major regulators of cerebral blood flow [
35,
36]. Targeting endothelial/pericyte interactions through junctional proteins may therefore provide a new avenue for stroke treatment especially during the subacute phase of recovery.
Acknowledgements
We thank Bettina Herrmann, Melanie Kroh, Stefanie Balz and Susann Eigel for excellent technical assistance.