Involvement of Epigenetic Mechanisms and Non-coding RNAs in Blood-Brain Barrier and Neurovascular Unit Injury and Recovery After Stroke

Way beyond my ability to decode. We need our fucking failures of stroke associations to translate all stroke research into usable stroke rehab protocols. But that is way beyond their abilities, they are that fucking incompetent.   I would fire everyone in every stroke association for not actually serving survivors.  The boards of directors are the most complicit in such incompetency.

Involvement of Epigenetic Mechanisms and Non-coding RNAs in Blood-Brain Barrier and Neurovascular Unit Injury and Recovery After Stroke

Svetlana M. Stamatovic¹, Chelsea M. Phillips², Gabriela Martinez-Revollar¹, Richard F. Keep^3,4 and Anuska V. Andjelkovic^1,3*

• ¹Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, United States
• ²Neuroscience Graduate Program, University of Michigan Medical School, Ann Arbor, MI, United States
• ³Department of Neurosurgery, University of Michigan Medical School, Ann Arbor, MI, United States
• ⁴Department of Molecular Integrative Physiology, University of Michigan Medical School, Ann Arbor, MI, United States

Cessation of blood flow leads to a complex cascade of pathophysiological events at the blood-vascular-parenchymal interface which evolves over time and space, and results in damage to neural cells and edema formation. Cerebral ischemic injury evokes a profound and deleterious upregulation in inflammation and triggers multiple cell death pathways, but it also induces a series of the events associated with regenerative responses, including vascular remodeling, angiogenesis, and neurogenesis. Emerging evidence suggests that epigenetic reprograming could play a pivotal role in ongoing post-stroke neurovascular unit (NVU) changes and recovery. This review summarizes current knowledge about post-stroke recovery processes at the NVU, as well as epigenetic mechanisms and modifiers (e.g., DNA methylation, histone modifying enzymes and microRNAs) associated with stroke injury, and NVU repair. It also discusses novel drug targets and therapeutic strategies for enhancing post-stroke recovery.

Introduction

Stroke is defined as an abrupt onset of focal or global neurological symptoms caused by a blockage of cerebral vessels (ischemic stroke), rupture of blood vessels (hemorrhage), or transient occlusion of small blood vessels (transient ischemic attack). It is particularly prevalent in the aging population, with people over 65 years old accounting for ∼75% of all registered cases (Hollander et al., 2003). Ischemic stroke is further subdivided based on the caliber of occluded vessels into macro- and microvascular (i.e., lacuna stroke), and based on the origin of clot-causing blockage into (a) thrombotic stroke where clot form inside brain blood vessels, and (b) thromboembolic/embolic stroke where clots form elsewhere in the body and travel toward and lodge in brain blood vessels (Reed et al., 2014; Topcuoglu et al., 2018; Tsai et al., 2018).

Pathophysiologically, cessation of blood flow leads to a complex cascade of events at the blood-vascular-parenchymal interface which evolves over time and space, and results in damage to neural cells and edema formation (Dirnagl et al., 1999; Allen et al., 2012). The central events in the hyperacute phase (within minutes and up to 6 h) include compromised mitochondrial function, anaerobic glycolysis, decreased pH (acid condition), impaired ATP production and reduced ion pump activity. As a consequence, cells swell and die, predominantly due to accumulation of lactic acid, ions (Ca²⁺ and Na⁺) and increased water influx (Dirnagl et al., 1999; Ginsberg, 2008; Nagy and Nardai, 2017).

(The following sounds an awful lot like the neuronal cascade of death. Why doesn't anyone solve this death cascade?)

There follows a cascade of events in the acute and subacute phase (hours to 7 days), including blood-brain barrier (BBB), and neurovascular unit (NVU) damage characterized by mitochondrial failure, robust production of reactive oxygen species (ROS) (superoxide O₂, hydrogen peroxide, and peroxynitrite), excitotoxicity (release of glutamate from dying neurons), activation of matrix metalloproteinases (MMP2, astrocytes; MMP3, MMP9 endothelial cells and neutrophils), BBB damage triggering inflammation and blood cell infiltration (neutrophils, monocytes) that can lead to further cell death, and cellular and vasogenic edema (Enzmann et al., 2013; Posada-Duque et al., 2014; Sifat et al., 2017). The secondary damage mostly takes place in the penumbra surrounding the core infarct and its progression can extend into the chronic phase after stroke.

Paralleling these injury processes, there is activation of endogenous protective and repair mechanisms that include vascular remodeling, angiogenesis, and neurogenesis (Chopp et al., 2007; Venna et al., 2014). The degree of these ongoing repair processes on one side and persistent inflammation/damaging processes on the other determines stroke recovery and the potential risk of another stroke.

There is mounting evidence on the importance of epigenetic factors in stroke. The purpose of this review is to examine how such factors impact the cerebrovasculature in stroke injury and recovery, focusing on effects at the BBB, and NVU. There is debate over whether non-coding RNAs should be included as an epigenetic mechanism and this review will cover their effects as well as other epigenetic mechanisms.

The Blood-Brain Barrier and Neurovascular Unit in Stroke

The blood-brain barrier is a highly complex and dynamic barrier, formed by an interdependent network of brain capillary endothelial cells, endowed with barrier properties. The BBB strictly regulates paracellular permeability due to the presence of tight junctions (TJs) between endothelial cells. Those TJs are built by intricate interactions between transmembrane proteins (claudins -5, -3, -1, -12, occludin, and JAM-A), important for paracellular space occlusion, scaffolding proteins (ZO-1, -2), and the actin cytoskeleton vital for physical support and TJ function (Daneman et al., 2010; Stamatovic et al., 2016). The transcellular interactions of claudin-5 play the major role in occluding the paracellular space (Nitta et al., 2003; Ohtsuki et al., 2007). Any loosening of its adhesive interactions directly affects BBB integrity and increases paracellular permeability. The BBB role of other claudins with lower expression is still uncertain and under investigation (Tietz and Engelhardt, 2015; Sladojevic et al., 2019). BBB function is also dependent on the perivascular microenvironment, which contains cells (e.g., pericytes, astrocytes, and perivascular macrophages), neuronal endings and tissue unique extracellular matrix (Mae et al., 2011; Muoio et al., 2014). Because of this functional integration, nearly two decades ago, the concept of a BBB was broadened to a new structure, the NVU.

The neurovascular unit is composed of BBB-endowed endothelial cells and a perivascular milieu composed from cells including pericytes, smooth muscle cells, astrocytes, perivascular macrophages/microglia, neurons/neuronal endings, and extracellular matrix. The NVU mediates neurovascular coupling, modulating vessel tone (Mae et al., 2011; Muoio et al., 2014). These intimately and reciprocally linked cells and matrix generate a complex structure that regulates exchange between blood and brain, oxygen and nutrient delivery, and regional cerebral blood flow. It is essential for maintaining circulatory and brain homeostasis.

In stroke, blood-brain barrier, and neurovascular unit dysfunction actively contributes to injury pathogenesis, being a “solid substrate” for ongoing injuring processes (oxidative stress, inflammation, and cytotoxicity), contributing to ischemic core (infarct) formation in the acute phase of stroke, and facilitating the progression of injury in the subacute and chronic phases. For example, in the early (acute) phase of stroke, NVU dysfunction is characterized by disruption of BBB integrity/BBB breakdown (disassembly of TJ complex, decreases in the TJ proteins claudin-5, occludin, and ZO-1) that leads to vasogenic brain edema, a life-threatening acute stroke complication (Bauer et al., 2010; Zehendner et al., 2011; Sladojevic et al., 2014). The cell components of the NVU undergo a series of transformations during injury. Brain endothelial cells, for example, are affected very early by cytotoxic effects with dysfunction of ion channels and transporters (e.g., Na⁺-K⁺-Cl^– cotransporter, and Na⁺/H⁺ exchanger), release of extracellular vesicles and conversion of brain endothelial cells toward a proinflammatory and prothrombotic phenotype due to upregulation of protease-activated receptor 1 (PAR-1), tissue factor, and matrix metalloproteinases (MMPs) (Zhu et al., 2008; Bauer et al., 2010; Yamashita and Abe, 2011; Chen et al., 2015). The proinflammatory phenotype of brain endothelial cells involves an upregulation of endothelial adhesive molecules (ICAM-1, VCAM-1, P-, and E- selectins) that guide leukocyte infiltration in the acute inflammatory phase response and T and B cells infiltration in the late phase (Kleinschnitz et al., 2013; Zhou et al., 2013; Sladojevic et al., 2014). Overall, inflammation is thought to worsen acute ischemic injury, contributing to chronic focal inflammation and restricting functional recovery. However, inflammation is also involved in tissue repair.

In the hyperacute phase after stroke, pericytes may be involved in vasoconstriction, causing capillary occlusion (no-reflow phenomenon), while later, by switching to pro-inflammatory phenotype, they may enhance BBB permeability, and brain edema formation (Hall et al., 2014; Underly et al., 2017). Ischemia triggers a series of damaging reactions in astrocytes including mitochondrial dysfunction, energy depletion, ion disequilibrium, increased glutamate and Ca²⁺, aquaporin 4 (AQP4) channel activation, increased water permeability, and cell swelling (Friedman et al., 2009; Ito et al., 2009; Hertz et al., 2014; Mogoanta et al., 2014). It results in the release of oxidative stress products and inflammatory cytokines/chemokines (IL1, IL6, IL15, CCL2, CXCL1, CXCL10, CXCL12, and IP-10), proinflammatory associated small molecules [S100 Ca²⁺-binding protein B (S100B) and nitric oxide (NO)] that enhance the inflammatory post-stroke response (Yamashita et al., 2000; Hill et al., 2004; Mori et al., 2008; Shin et al., 2014; Liu H. et al., 2015; Chen et al., 2018).

Perivascular macrophages and microglia play an important role in the stroke-induced inflammatory response via production of proinflammatory cytokines (IL1|upbeta TNFα IL6, IL12) and ROS (Drake et al., 2011; Liu H. et al., 2015; Wu et al., 2016; He et al., 2019). They trigger the first line of inflammation at the NVU in the acute phase of stroke. Notable changes also occur in the extracellular matrix. At early time points (within hours), there is MMP-related basement membrane degradation with reductions in agrin, SPARC, perlecan, laminin, and fibronectin (Sole et al., 2004; Castellanos et al., 2007; Lee et al., 2011; Ji and Tsirka, 2012; Lloyd-Burton et al., 2013). This ultimately leads to increased BBB disruption, accumulation of new extracellular matrix proteins (i.e., chondroitin sulfate proteoglycan neurocan and osteopontin) and leakage of plasma proteins, such as fibrinogen, into the CNS. This mediates inflammation, edema, and potentially hemorrhagic transformation (Figure 1).

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