Jill Roberts

Perlecan and the blood-brain barrier: beneficial proteolysis?

The cerebral microvasculature is important for maintaining brain homeostasis. This is achieved via the blood-brain barrier (BBB), composed of endothelial cells with specialized tight junctions, astrocytes, and a basement membrane (BM). Prominent components of the BM extracellular matrix (ECM) include fibronectin, laminin, collagen IV, and perlecan, all of which regulate cellular processes via signal transduction through various cell membrane bound ECM receptors. Expression and proteolysis of these ECM components can be rapidly altered during pathological states of the central nervous system. In particular, proteolysis of perlecan, a heparan sulfate proteoglycan, occurs within hours following ischemia induced by experimental stroke. Proteolysis of ECM components following stroke results in the degradation of the BM and further disruption of the BBB. While it is clear that such proteolysis has negative consequences for the BBB, we propose that it also may lead to generation of ECM protein fragments, including the C-terminal domain V (DV) of perlecan, that potentially have a positive influence on other aspects of CNS health. Indeed, perlecan DV has been shown to be persistently generated after stroke and beneficial as a neuroprotective molecule and promoter of post-stroke brain repair. This mini-review will discuss beneficial roles of perlecan protein fragment generation within the brain during stroke.

Perlecan Domain V Induces VEGf Secretion in Brain Endothelial Cells through Integrin α5β1 and ERK-Dependent Signaling Pathways

Perlecan Domain V (DV) promotes brain angiogenesis by inducing VEGF release from brain endothelial cells (BECs) following stroke. In this study, we define the specific mechanism of DV interaction with the α5β1 integrin, identify the downstream signal transduction pathway, and further investigate the functional significance of resultant VEGF release. Interestingly, we found that the LG3 portion of DV, which has been suggested to possess most of DV’s angio-modulatory activity outside of the brain, binds poorly to α5β1 and induces less BEC proliferation compared to full length DV. Additionally, we implicate DV’s DGR sequence as an important element for the interaction of DV with α5β1. Furthermore, we investigated the importance of AKT and ERK signaling in DV-induced VEGF expression and secretion. We show that DV increases the phosphorylation of ERK, which leads to subsequent activation and stabilization of eIF4E and HIF-1α. Inhibition of ERK activity by U0126 suppressed DV-induced expression and secretion of VEGR in BECs. While DV was capable of phosphorylating AKT we show that AKT phosphorylation does not play a role in DV’s induction of VEGF expression or secretion using two separate inhibitors, LY294002 and Akt IV. Lastly, we demonstrate that VEGF activity is critical for DV increases in BEC proliferation, as well as angiogenesis in a BEC-neuronal co-culture system. Collectively, our findings expand our understanding of DV’s mechanism of action on BECs, and further support its potential as a novel stroke therapy.

Stroke neuroprotection revisited: Intra-arterial verapamil is profoundly neuroprotective in experimental acute ischemic stroke.

While clinical trials have now solidified the role of thrombectomy in emergent large vessel occlusive stroke, additional therapies are needed to optimize patient outcome. Using our previously described experimental ischemic stroke model for evaluating adjunctive intra-arterial drug therapy after vessel recanalization, we studied the potential neuroprotective effects of verapamil. A calcium channel blocker, verapamil is often infused intra-arterially by neurointerventionalists to treat cerebral vasospasm. Such a direct route of administration allows for both focused targeting of stroke-impacted brain tissue and minimizes potential systemic side effects. Intra-arterial administration of verapamil at a flow rate of 2.5 µl/min and injection volume of 10 µl immediately after middle cerebral artery recanalization in C57/Bl6 mice was shown to be profoundly neuroprotective as compared to intra-arterial vehicle-treated stroke controls. Specifically, we noted a significant (P ≤ 0.05) decrease in infarct volume, astrogliosis, and cellular apoptosis as well as a significant increase in neuronal survival and functional outcome over seven days. Furthermore, intra-arterial administration of verapamil was well tolerated with no hemorrhage, systemic side effects, or increased mortality. Thus, verapamil administered intra-arterially immediately following recanalization in experimental ischemic stroke is both safe and neuroprotective and merits further study as a potential therapeutic adjunct to thrombectomy.

Juvenile Traumatic Brain Injury Induces Long-Term Perivascular Matrix Changes Alongside Amyloid-Beta Accumulation:

In our juvenile traumatic brain injury (jTBI) model, emergence of cognitive dysfunctions was observed up to 6 months after trauma. Here we hypothesize that early brain injury induces changes in the neurovascular unit (NVU) that would be associated with amyloid-beta (Aβ) accumulation. We investigated NVU changes for up to 6 months in a rat jTBI model, with a focus on the efflux protein P-glycoprotein (P-gp) and on the basement membrane proteins perlecan and fibronectin, all known to be involved in Aβ clearance. Rodent-Aβ staining is present and increased after jTBI around cerebral blood microvessels, and the diameter of those is decreased by 25% and 34% at 2 and 6 months, respectively, without significant angiogenesis. P-glycoprotein staining in endothelium is decreased by 22% and parallels an increase of perlecan and fibronectin staining around cerebral blood vessels. Altogether, these results strongly suggest that the emergence of long-term behavioral dysfunctions observed in rodent jTBI may be related to endothelial remodeling at the blood–brain barrier alongside vascular dysfunction and altered Aβ trafficking. This study shows that it is important to consider jTBI as a vascular disorder with long-term consequences on cognitive functions.

Intra-arterial verapamil post-thrombectomy is feasible, safe, and neuroprotective in stroke:

Large vessel ischemic stroke represents the most disabling subtype. While t-PA and endovascular thrombectomy can recanalize the occluded vessel, good clinical outcomes are not uniformly achieved. We propose that supplementing endovascular thrombectomy with superselective intra-arterial (IA) verapamil immediately following recanalization could be safe and effective. Verapamil, a calcium channel blocker, has been shown to be an effective IA adjunct in a pre-clinical mouse focal ischemia model. To demonstrate translational efficacy, mechanism, feasibility, and safety, we conducted a group of translational experiments. We performed in vivo IA dose–response evaluation in our animal stroke model with C57/Bl6 mice. We evaluated neuroprotective mechanism through in vitro primary cortical neuron (PCN) cultures. Finally, we performed a Phase I trial, SAVER-I, to evaluate feasibility and safety of administration in the human condition. IA verapamil has a likely plateau or inverted-U dose–response with a defined toxicity level in mice (LD50 16–17.5 mg/kg). Verapamil significantly prevented PCN death and deleterious ischemic effects. Finally, the SAVER-I clinical trial showed no evidence that IA verapamil increased the risk of intracranial hemorrhage or other adverse effect/procedural complication in human subjects. We conclude that superselective IA verapamil administration immediately following thrombectomy is safe and feasible, and has direct, dose–response-related benefits in ischemia.

Mice deficient in endothelial α5 integrin are profoundly resistant to experimental ischemic stroke

Stroke is a disease in dire need of better therapies. We have previously shown that a fragment of the extracellular matrix proteoglycan, perlecan, has beneficial effects following cerebral ischemia via the α5β1 integrin receptor. We now report that endothelial cell selective α5 integrin deficient mice (α5 KO) are profoundly resistant to ischemic infarct after transient middle cerebral artery occlusion. Specifically, α5 KOs had little to no infarct 2–3 days post-stroke, whereas controls had an increase in mean infarct volume over the same time period as expected. Functional outcome is also improved in the α5 KOs compared with controls. Importantly, no differences in cerebrovascular anatomy or collateral blood flow were noted that could account for this difference in ischemic injury. Rather, we demonstrate that α5 KOs have increased blood-brain barrier integrity (increased expression of claudin-5, and absent brain parenchymal IgG extravasation) after stroke compared with controls, which could explain their resistance to ischemic injury. Additionally, inhibition of α5 integrin in vitro leads to decreased permeability of brain endothelial cells following oxygen-glucose deprivation. Together, these findings indicate endothelial cell α5 integrin plays an important role in stroke outcome and blood-brain barrier integrity, suggesting that α5 integrin could be a novel therapeutic target for stroke.

Intra-arterial nitroglycerin as directed acute treatment in experimental ischemic stroke.

Background Nitroglycerin (also known as glyceryl trinitrate (GTN)), a vasodilator best known for treatment of ischemic heart disease, has also been investigated for its potential therapeutic benefit in ischemic stroke. The completed Efficacy of Nitric Oxide in Stroke trial suggested that GTN has therapeutic benefit with acute (within 6 hours) transdermal systemic sustained release therapy. Objective To examine an alternative use of GTN as an acute therapy for ischemic stroke following successful recanalization. Methods We administered GTN IA following transient middle cerebral artery occlusion in mice. Because no standard dose of GTN is available following emergent large vessel occlusion, we performed a dose–response (3.12, 6.25, 12.5, and 25 µg/µL) analysis. Next, we looked at blood perfusion (flow) through the middle cerebral artery using laser Doppler flowmetry. Functional outcomes, including forced motor movement rotor rod, were assessed in the 3.12, 6.25, and 12.5 µg/µL groups. Histological analysis was performed using cresyl violet for infarct volume, and glial fibrillary activating protein (GFAP) and NeuN immunohistochemistry for astrocyte activation and mature neuron survival, respectively. Results Overall, we found that acute post-stroke IA GTN had little effect on vessel dilatation after 15 min. Functional analysis showed a significant difference between GTN (3.12 and 6.25 µg/µL) and control at post-stroke day 1. Histological measures showed a significant reduction in infarct volume and GFAP immunoreactivity and a significant increase in NeuN. Conclusions These results demonstrate that acute IA GTN is neuroprotective in experimental ischemic stroke and warrants further study as a potentially new stroke therapy.

Internal carotid artery stenosis: A novel surgical model for moyamoya syndrome

Moyamoya is a cerebrovascular disorder characterized by progressive stenosis of the intracranial internal carotid arteries. There are two forms: Disease and Syndrome, with each characterized by the sub-population it affects. Moyamoya syndrome (MMS) is more prominent in adults in their 20’s-40’s, and is often associated with autoimmune diseases. Currently, there are no surgical models for inducing moyamoya syndrome, so our aim was to develop a new animal model to study this relatively unknown cerebrovascular disease. Here, we demonstrate a new surgical technique termed internal carotid artery stenosis (ICAS), to mimic MMS using micro-coils on the proximal ICA. We tested for Moyamoya-like vasculopathies by fluorescently labelling the mouse cerebrovasculature with Di I for visualization and analysis of vessel diameter at the distal ICA and anastomoses on the cortical surface. Results show a significant narrowing of the distal ICA and anterior cerebral artery (ACA) in the Circle of Willis, as observed in humans. There is also a significant decrease in the number of anastomoses between the middle cerebral artery (MCA) and the ACA in the watershed region of the cortex. While further characterization is needed, this ICAS model can be applied to transgenic mice displaying co-morbidities as observed within the Moyamoya syndrome population, allowing a better understanding of the disease and development of novel treatments.

Bilateral carotid artery stenosis causes unexpected early changes in brain extracellular matrix and blood-brain barrier integrity in mice

Bilateral carotid artery stenosis (BCAS) is one experimental model of vascular dementia thought to preferentially impact brain white matter. Indeed, few studies report hippocampal and cortical pathology prior to 30 days post-stenosis; though it is unclear whether those studies examined regions outside the white matter. Since changes in the blood-brain barrier (BBB) permeability precede more overt brain pathology in various diseases, we hypothesized that changes within the BBB and/or BBB-associated extracellular matrix (ECM) could occur earlier after BCAS in the hippocampus, cortex and striatum and be a precursor of longer term pathology. Here, C57Bl/6 mice underwent BCAS or sham surgeries and changes in the BBB and ECM were analyzed by collagen IV (vascular basement membrane component), α5 integrin (marker of endothelial activation), claudin-5 and occludin (tight junction proteins), Evans blue (permeability marker), Ki-67 (cell proliferation marker), and GFAP and CD11b (glial cell markers) immunohistochemistry after 14 days. Significant changes in markers of cerebrovascular integrity and glial activation were detected, not only in the striatum, but also in the hippocampus and cortex. In conclusion, this study demonstrates for the first time that changes in the BBB/ECM occur shortly after BCAS and within multiple brain regions and suggests such changes might underlie the gradual development of BCAS non-white matter pathology.

The Blood And Clot Thrombectomy Registry And Collaboration (BACTRAC) protocol: novel method for evaluating human stroke

Background Ischemic stroke research faces difficulties in translating pathology between animal models and human patients to develop treatments. Mechanical thrombectomy, for the first time, offers a momentary window into the changes occurring in ischemia. We developed a tissue banking protocol to capture intracranial thrombi and the blood immediately proximal and distal to it. Objective To develop and share a reproducible protocol to bank these specimens for future analysis. Methods We established a protocol approved by the institutional review board for tissue processing during thrombectomy (www.clinicaltrials.gov NCT03153683). The protocol was a joint clinical/basic science effort among multiple laboratories and the NeuroInterventional Radiology service line. We constructed a workspace in the angiography suite, and developed a step-by-step process for specimen retrieval and processing. Results Our protocol successfully yielded samples for analysis in all but one case. In our preliminary dataset, the process produced adequate amounts of tissue from distal blood, proximal blood, and thrombi for gene expression and proteomics analyses. We describe the tissue banking protocol, and highlight training protocols and mechanics of on-call research staffing. In addition, preliminary integrity analyses demonstrated high-quality yields for RNA and protein. Conclusions We have developed a novel tissue banking protocol using mechanical thrombectomy to capture thrombus along with arterial blood proximal and distal to it. The protocol provides high-quality specimens, facilitating analysis of the initial molecular response to ischemic stroke in the human condition for the first time. This approach will permit reverse translation to animal models for treatment development.