Endothelial Cells and the Blood–Brain Barrier: Critical Determinants of Ineffective Reperfusion in Stroke

ABSTRACT

Ineffective reperfusion remains a critical challenge in neurointerventional treatment following ischemic stroke, with the integrity of the blood–brain barrier (BBB) being a key determinant of patient outcomes. This review explores the distinctive characteristics and roles of brain endothelial cells (ECs) in the context of stroke and ineffective reperfusion. We examine the unique properties of brain ECs compared to their counterparts in other tissues, focusing on their pathophysiological changes, functional impairments and the inflammatory cascades that follow stroke. Differences in gene expression between brain ECs and those in other organs offer deeper insights into their role in neuroprotective therapies. Additionally, drawing parallels between brain ECs and ECs from organs with similar ischemia–reperfusion injury profiles may inspire novel therapeutic approaches. This review highlights the critical importance of understanding the nuanced roles of ECs in BBB regulation, which ultimately impacts reperfusion outcomes.

The blood–brain barrier (BBB) is fundamental for maintaining cerebral homeostasis and vascular function. Cerebral endothelial cells are an important part of the BBB. Ineffective reperfusion may occur during ischemia–reperfusion in stroke. Ineffective reperfusion can damage endothelial cells through a variety of mechanisms (including disturbance of energy metabolism, destructive effects of ROS, inflammatory response, synthesis and secretion of nitric oxide and neutrophil aggregation), leading to degeneration or death of endothelial cells, breakdown of tight junctions between endothelial cells, disturbance of microcirculation, apoptosis and eventually causes brain injury.

List of Abbreviations

AIF

apoptosis‐inducing factor

AIS

acute ischemic stroke

AP

alternative complement pathway

BBB

blood–brain barrier

BMVECs

brain microvascular endothelial cells

CDK

cyclin‐dependent kinase 5

circHIPK

circular RNA homeodomain‐interacting protein kinase

CNS

central nervous system

DAMP

danger‐associated molecular pattern

EC

endothelial cell

ECM

extracellular matrix

EPC

endothelial progenitor cell

EVT

endovascular treatment

GMVEC

glomerular microvascular endothelial cell

HB‐EGF

heparin‐binding epidermal growth factor

hCMEC

human cerebral microvascular endothelial cell

HERMES

Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials

HIF‐1α

hypoxia‐inducible factor‐1 alpha

ICAM‐1

intracellular adhesion molecule‐1

JAM

junctional adhesion molecules

LAM

leukocyte adhesion molecule

MCAO

middle cerebral artery occlusion

MMP

matrix metalloproteinas

MS

multiple sclerosis

NO

nitric oxide

NOX

NADPH oxidase

NVU

neurovascular unit

OGD/R

oxygen–glucose deprivation/reoxygenation

PC

perivascular cells

ROS

reactive oxygen species

TJ

tight junction

TNF

tumour necrosis factor

TYMP

thymidine phosphorylase

tPA

tissue plasminogen activator

VCAM‐1

vascular cell adhesion molecule‐1

VEGFA

vascular endothelial growth factor A

vWF

von Willebrand factor

ZO

zonula occludens

1. Introduction

Acute ischemic stroke (AIS) remains a significant global health challenge, consistently ranking as a leading cause of mortality and long‐term disability. Endovascular treatment (EVT), with its precise approach to recanalizing occluded large vessels, has emerged as a vital intervention for AIS, significantly reducing stroke‐related deaths and improving recovery prospects for patients (Su et al. 2022; Mirbolouk et al. 2023). However, stroke management in clinical settings reveals a more intricate narrative. Achieving timely thrombus clearance, though essential, does not always correlate with favourable patient outcomes. A perplexing phenomenon has been observed in stroke patients who, despite successful EVT and recanalization, experience neurological deterioration. This conundrum is exemplified in the Highly Effective Reperfusion Evaluated in Multiple Endovascular Stroke Trials (HERMES), where 71% of the cohort achieved successful vessel reopening, yet only 46% of these cases resulted in favourable clinical outcomes (Goyal et al. 2016).

While the blood–brain barrier (BBB) and endothelial cells (ECs) are fundamental to maintaining cerebral homeostasis and vascular function, their potential roles in the context of ineffective reperfusion following stroke are not fully understood and warrant further exploration. Ineffective reperfusion occurs when, despite successful restoration of blood flow poststroke, expected clinical improvements are not observed. This discrepancy can sometimes result in severe clinical outcomes such as substantial cerebral edema and hemorrhagic transformation, which can exacerbate neurological damage and even lead to death (Nie et al. 2023). These adverse outcomes are intimately linked to the impairment of the BBB’s integrity and the dysfunction of ECs. After recanalization, some patients may experience no improvement in neurological function due to extensive neuronal ischemia and hypoxia that preclude recovery. However, for others, reperfusion can cause significant disruption to the BBB, mediated by the ECs. This disruption facilitates cerebral edema and hemorrhagic transformation, leading to further deterioration.

By focusing on the BBB and ECs, this review is aimed at examining their roles during these complex poststroke events. Understanding how the BBB and ECs contribute to ineffective reperfusion could unveil critical insights into post‐stroke recovery dynamics, thereby guiding future research and therapeutic strategies aimed at enhancing patient outcomes after EVT.

2. BBB, Neurovascular Units (NVUs) and ECs

The BBB represents the specialized microvascular system of the central nervous system (CNS). Unlike most blood vessels, CNS vessels are continuous and nonfenestrated and exhibit unique properties that strictly regulate the movement of molecules, ions and cells between the blood and the brain (Zlokovic 2008; Alvarez, Cayrol, and Prat 2011; Sandoval and Witt 2008). This stringent control is essential for maintaining CNS homeostasis, which is critical for proper function. The BBB also protects the CNS from harmful agents such as toxins, pathogens and inflammation. In various neurological conditions, including stroke, multiple sclerosis (MS), brain trauma and neurodegenerative diseases, a compromised BBB plays a crucial role in disease progression (Zlokovic 2008; Alvarez, Cayrol, and Prat 2011; Sandoval and Witt 2008; Zhang et al. 2024; Li et al. 2024). When the BBB is impaired, it can lead to ion imbalances, disrupted signalling and the infiltration of immune cells and inflammatory agents into the CNS, resulting in neuronal dysfunction and degeneration.

ECs, along with perivascular cells (PCs), the basement membrane, astrocytes (ACs), microglia (MGs) and nerve endings, form the NVU (Figure 1). As the foundation of the BBB, the NVU plays vital roles in maintaining local homeostasis, supporting neurons, regulating neurotransmitter levels, selectively transporting substances, preventing the entry of plasma macromolecules and shielding the brain from neurotoxic impacts. Morphologically, the BBB is characterized by the presence of tight interendothelial junctions and the absence of pinocytotic vesicles and fenestrations (Brightman and Reese 1969; Reese and Karnovsky 1967). Cerebrovascular ECs form a highly restrictive barrier, with low ion permeability due to their high membrane resistance (Crone and Olesen 1982).

FIGURE 1.

Composition of neurovascular units. A diagrammatic representatiosn of the neurovascular unit (NVU) in cross‐section, highlighting the intricate cellular and structural components essential for maintaining the functional integrity of the blood–brain barrier (BBB). Key elements include neurons, astrocytes, microglia, pericytes, endothelial cells (ECs), tight junctions (TJs), and the basement membrane. Neurons are depicted interacting with blood vessels and other NVU cells. ECs form the blood vessel walls, encased by a basement membrane and connected by TJ proteins that maintain the BBB. Astrocytes, with their end feet, make contact with both ECs and pericytes, Pericytes are located between the astrocytic end feet and ECs, supporting vascular stability and BBB function. Microglia, dispersed throughout the brain, occasionally extend processes directly to blood vessels, contributing to immune responses and inflammation regulation within the NVU.

Tight junctions (TJs) interconnect CNS ECs, significantly limiting the extracellular movement of solutes. In addition, CNS ECs exhibit a much lower rate of transcellular transport compared to peripheral ECs, which curbs vesicle‐mediated solute transport (Coomber and Stewart 1985). This strict regulation of paracellular and transcellular pathways results in polarized cell structures, with distinct luminal and abluminal domains that enable precise regulation of transport between the blood and brain (Betz and Goldstein 1978).

CNS ECs contain a higher number of mitochondria than ECs in other tissues, providing the energy required to maintain vital ion gradients essential for transport functions. Additionally, CNS ECs express low levels of leukocyte adhesion molecules (LAMs), reducing the likelihood of immune cell infiltration into the CNS (Shenoda 2015; Luo et al. 2007; Cui et al. 2018). This combination of physical properties (such as TJs and reduced transcellular transport) and molecular features (including efflux transporters, specialized metabolism and reduced LAM expression) equips ECs to effectively regulate CNS homeostasis.

Two predominant subsets of ECs in the brain, namely EC (Ptn) and EC (Col6a1), identified by their preferentially expressed genes, were juxtaposed with renal ECs. According to the differential gene expression analysis of kidney and brain ECs, it was found that two genes, namely Ptn and Col6a1, were specifically expressed in brain ECs. Pleiotrophin is a kind of secreted growth factor encoded by the Ptn gene on chromosome 7, arm q33, is at least 42 kb and contains seven exons. Ptn induces ECs migration and differentiation into tubes in various substrates in vitro, through its receptor PTPRZ1, leading to the translocation of nucleolin. Ptn has also been shown to enhance vascular endothelial growth factor A (VEGFA) expression, and it may be helpful to angiogenic activities. Ptn has a moderate stimulatory effect on ECs via VEGFA but also limits the angiogenic effects of VEGFA through VEGFR2. These studies unveiled the positive role in these subsets in the context of stroke (Huang et al. 2022; Bates et al. 2023; Lui et al. 2023). Furthermore, two genes—Ube2g1 and Pdcd4—emerged as pivotal players in stroke development (Huang et al. 2022). Ube series genes are involved in the regulation of the HIF‐1 signalling pathway, which is important for the maintenance of oxygen homeostasis through sensing hypoxia. They also play a role in inducing genes and are involved in angiogenesis, glycolysis and other processes. Pdcd4 is a gene associated with programmed cell death that mediates neuronal death after ischemia and reperfusion injury. These two genes are also expressed in brain ECs after stroke (Perez‐Pinera, Berenson, and Deuel 2008; Zhang et al. 2023).

Further research (Sartain, Turner, and Moake 2018) explored the differences in immune complement activation between the brain and kidney ECs. Brain microvascular endothelial cells (BMVECs) displayed superior control over alternative complement pathway (AP) activation compared to glomerular microvascular endothelial cells (GMVECs), especially under tumour necrosis factor (TNF) stimulation. This resilience of BMVECs was linked to higher levels of activated protein C and reduced thrombosis risk. Additionally, von Willebrand factor (vWF) expression was higher in brain ECs compared to liver and kidney ECs, aligning with the propensity of brain, heart and lung ECs to maintain haemostasis more efficiently (Przysinda, Feng, and Li 2020).

ECs are crucial within the NVU due to their direct involvement in regulating vascular tone, permeability and BBB integrity. However, other components of the NVU, such as PCs, ACs and neurons, also play significant roles during reperfusion. For instance, ACs, which maintain close associations with ECs, contribute to BBB function and regulate water and ion balance, which is crucial during the reperfusion phase to prevent edema and maintain ionic homeostasis. PCs, including pericytes, are essential for capillary stability and blood flow regulation. They respond to ischemic conditions by altering their coverage of capillaries, which can affect tissue survival during reperfusion by modulating blood flow and barrier integrity.

In addition to detailing the specific roles of these cells, parallels between cardiac and cerebral reperfusion highlight the sensitivity of ECs to reperfusion injury. Studies in cardiac models have shown that coronary ECs can trigger proapoptotic pathways that adversely affect surrounding tissues (Scarabelli et al. 2001). Similarly, in the brain, damage to ECs during reperfusion can lead to BBB breakdown, resulting in cerebral edema and hemorrhagic transformation (Bernardo‐Castro et al. 2020; Sadeghian et al. 2019). Furthermore, the release of soluble mediators from damaged ECs can exacerbate neuronal damage in the ischemic penumbra, impeding functional recovery (Scarabelli et al. 2001).

Taken together, while ECs are critical, the collaborative interactions within the entire NVU significantly influence the outcomes of ischemia–reperfusion events. Understanding these interactions provides crucial insights into developing targeted therapies that could alleviate reperfusion injury and improve recovery following stroke. This full view of the NVU during reperfusion emphasizes the complex interplay of various cell types that are essential for a comprehensive understanding of stroke pathology and therapy.

3. Mechanisms of EC Injury After Ischemia and Reperfusion in Stroke

This section explores the complex mechanisms of EC damage during ischemia–reperfusion events in stroke. Endothelial injury results from multiple interconnected factors, including energy metabolism disturbances, oxidative damage from reactive oxygen species (ROS) and inflammatory responses. These mechanisms collectively drive the pathology of endothelial dysfunction in stroke, underscoring its multifaceted nature (Figure 2).

FIGURE 2.

Mechanisms of endothelial cell injury. A conceptual diagram illustrating the key factors contributing to endothelial cell (EC) injury. It highlights disruptions in energy metabolism, the damaging effects of reactive oxygen species (ROS), and the inflammatory response. Additionally, the diagram emphasizes the role of nitric oxide (NO) synthesis and secretion, along with the mechanisms leading to EC damage, such as neutrophil aggregation and the promotion of apoptosis in vascular ECs. These interconnected pathways collectively underscore the complexity of endothelial injury in vascular pathologies.

3.1. Energy Metabolism Disturbances

During ischemia and hypoxia, the mitochondrial respiratory chain’s ability to synthesize ATP is impaired (Yu et al. 2021). ATP depletion disrupts sodium–potassium pump function in ECs, causing intracellular sodium accumulation and cellular edema. Ischemia‐induced acidosis further lowers intracellular pH, inhibiting sodium–hydrogen exchange and activating sodium–calcium exchange, leading to calcium overload (Shenoda 2015). Excessive calcium activates phospholipase C and A2, degrading the lipid membranes and generating free radicals. These processes, along with fatty acids, prostaglandins, leukotrienes, thrombin and platelet activators, trigger local inflammation and irreversible cellular damage (Luo et al. 2007). Studies show that melatonin mitigates endothelial injury through the AMPK/SERCA2a pathway (Cui et al. 2018), while lithium chloride reduces calcium overload by inhibiting inositol 3‐phosphate‐sensitive Ca2+ release from the endoplasmic reticulum (Bosche et al. 2013).

3.2. Destructive Effects of ROS

Under normal conditions, the body maintains a balance between oxidants and antioxidants. ROS, while essential for cellular signalling and immune protection (Babior et al. 2003), become harmful when overproduced in pathological states. Enzyme‐induced or spontaneously formed ROS, including superoxide and hydrogen peroxide (Forrester et al. 2018), can directly damage cellular structures and trigger apoptosis through mitochondrial pathways and transcription factors like NF‐κB (Fu et al. 2016). Furthermore, ROS reacts with nitric oxide (NO) to form peroxynitrite, which induces EC apoptosis (Liu et al. 2020).

3.3. Inflammatory Response

ECs are central to neuroinflammation following ischemic stroke. Under hypoxic conditions induced by ischemia, neurons and glial cells release molecules known as danger‐associated molecular patterns (DAMPs), which signal cellular distress (Gadani et al. 2015a; Gadani et al. 2015b). These DAMPs activate ECs to recruit inflammatory cells, initiating a cascade that disrupts the integrity and permeability of the BBB and the glycocalyx (Sieve, Münster‐Kühnel, and Hilfiker‐Kleiner 2018). The inflammatory response at the BBB involves several coordinated steps: Leukocytes roll along the endothelium, adhere firmly and then transmigrate. This diapedesis involves passing through the endothelial layer and its basement membrane into the perivascular space, followed by migration through the glial basement membrane. This process is facilitated by the endothelial expression of selectins, chemokines such as CCL2 and integrin ligands like intracellular adhesion molecule‐1 (ICAM‐1), which mediate rolling and adhesion respectively. Furthermore, proinflammatory cytokines such as tumour necrosis factor‐α, IFN‐γ and interleukin‐17 enhance MMP‐2 and MMP‐9 activities, promoting leukocyte migration through basement membranes, particularly at the glial limitans (Galea 2021). Another critical component is the endothelial sphingosine‐1‐phosphate receptor 2, which has been shown to increase BBB permeability and promote neutrophil infiltration via upregulation of endothelial E‐selectin, underscoring the complex interplay of signals that regulate inflammation at the neurovascular interface (Galea 2021).

3.4. NO Synthesis and Secretion

NO is a key regulator of vascular tone and blood flow (Lundberg, Weitzberg, and Gladwin 2008; An et al. 2021). Under ischemic conditions, NO production helps dilate blood vessels, improving blood flow to deprived areas and reducing inflammation (Kan et al. 2023), However, excess NO can increase ROS production in mitochondria, exacerbating oxidative stress and cellular damage (Gladwin and Tejero 2011). Thus, while NO has protective effects, its overaccumulation can be detrimental.

3.5. Endothelial Injury and Neutrophil Aggregation

EC damage during ischemia–reperfusion disrupts normal vascular function and promotes neutrophil aggregation. Neutrophils, drawn to the site of injury by chemokine gradients (Zhao et al. 2023), activated by the damaged ECs, neutrophils release certain chemicals known as vasoactive agents, such as (1) vasoactive amines (e.g., histamine and serotonin), (2) peptide (e.g., bradykinin) and (3) eicosanoids (e.g., thromboxanes, leukotrienes and prostaglandins). These mediators can cause vasoconstriction or narrowing of blood vessels, which can worsen local blood flow disturbances. This aggravates the initial ischemic injury, creating a vicious cycle of worsening tissue damage (Hidalgo et al. 2007; Oliveira et al. 2018; Duilio et al. 2001; Jang et al. 2009).

3.6. Apoptosis of ECs

Cells can undergo death through several pathways. Ischemia–reperfusion injury, for example, can trigger both necrosis, which is an uncontrolled form of cell death, and apoptosis, which is a regulated and programmed cell death process (Zhang et al. 2005). Specifically, the activation of poly (ADP‐ribose) polymerase‐1 (PARP‐1) and a PARP‐1‐dependent, caspase‐independent pathway lead to the nuclear translocation of apoptosis‐inducing factor (AIF), which contributes to the apoptotic death of cerebral ECs following ischemia–reperfusion (Marqués et al. 2022). Moreover, overexpression of NADPH Oxidase 5 (NOX5) has been found to inhibit proliferation and promote apoptosis in human cerebral microvascular endothelial cells (hCMEC/D3), contributing to endothelial dysfunction. Additionally, oxygen–glucose deprivation/reoxygenation (OGD/R) induces activation of the hypoxia‐inducible factor‐1 alpha (HIF‐1α)/VEGFA pathway, leading to decreased cell viability and increased permeability and apoptosis in HBMECs. Furthermore, the circular RNA homeodomain‐interacting protein kinase 3 (circHIPK3) acts as an endogenous sponge of miR‐148b‐3p to modulate its activity, resulting in the upregulation of cyclin‐dependent kinase 5 regulatory subunit 1 (CDK5R1) and CDK5 expression, and downregulation of sirtuin 1 (SIRT1). This regulation contributes to apoptosis and mitochondrial dysfunction in BMEVCs (Ni et al. 2022; Chen et al. 2022). These findings underscore the potential of circHIPK3 as a therapeutic target for ischemic stroke, providing translational evidence of its significance (Yang et al. 2014).

In summary, ischemia–reperfusion injury in stroke leads to a cascade of events starting with ATP depletion that disrupts cellular energy metabolism, causing ionic imbalances and cellular edema. This metabolic disruption triggers an excessive influx of calcium into cells, activating destructive enzymes and increasing the production of inflammatory mediators. Concurrently, oxidative stress from ROS damages cellular structures and further activates mitochondrial pathways leading to apoptosis. The inflammatory response exacerbates endothelial damage by increasing vascular permeability and attracting immune cells, which further disrupts the BBB and enhances local inflammation. Additionally, NO, which normally acts as a vasodilator, contributes to oxidative stress under pathological conditions. These processes collectively exacerbate endothelial dysfunction, contributing significantly to the progression and severity of stroke‐related injuries. This integrated understanding can help in developing targeted interventions to mitigate these effects and improve stroke outcomes.

4. EC Damage and Dysfunction in Ineffective Reperfusion

The consequences of stroke extend beyond the cessation and restoration of blood flow; they initiate a complex cascade of physiological disruptions at cellular and molecular levels. A key aspect of this process is the profound impact on ECs, which play a critical role in maintaining cerebral health (Xiao et al. 2023). Following a stroke, the degeneration and death of cerebral ECs are well‐documented (Gong et al. 2021). As ischemia progresses, these cells gradually deteriorate, starting with edema and potentially advancing to complete structural collapse, which can result in hemorrhagic transformation. This deterioration affects not only the local tissue but also disrupts overall blood recirculation in the brain.

Additionally, stroke‐induced increases in transcytosis compromise the BBB, leaving the brain more vulnerable to external threats (Yan et al. 2023; Uzuner and Uzuner 2023). A particularly concerning aspect is the disruption of TJs within the BBB (Winkler et al. 2021). Poststroke, alterations in the structure and expression of TJ proteins significantly weaken this barrier, and proteolytic enzymes further degrade it, undermining its protective functions.

Beyond the direct cellular damage, stroke also impairs the brain’s microcirculation. ECs, which are vital for maintaining vascular health, become overwhelmed by ischemic events, leading to vasomotor dysfunction and reduced perfusion. This disruption impacts both major and microvessels, which are crucial for delivering oxygen and nutrients to brain tissue (Eaton, Duru, and Powers 2023; Yang, Lee, and Wu 2023).

Perhaps the most critical consequence is the heightened inflammatory response that follows a stroke (Ma et al. 2021). ECs, in coordination with glial cells, participate in a bidirectional inflammatory exchange that amplifies the stroke’s damaging effects. The release of inflammatory mediators from ECs weakens vascular integrity, attracting white blood cells into the brain, which further exacerbates the inflammatory response and contributes to ineffective reperfusion (Yang, Lee, and Wu 2023; Kang et al. 2020).

4.1. Degeneration and Death of Cerebral ECs

4.1.1. Degeneration Progression

Using electron microscopy, researchers have identified a four‐stage progression of EC degeneration in ischemic brain tissue (Krueger et al. 2015). In the initial stage, endothelial edema occurs with a reduction in cytoplasmic content, though the ECs maintain their barrier function, preventing tracer extravasation. In the second stage, the ECs begin to lose their protective barrier functions, allowing tracers to enter the cells. By the third stage, there is a complete loss of endothelial integrity, with tracers leaking into the surrounding parenchymal tissue. Finally, in the fourth stage, structural breakdown extends to the basement membrane, leading to hemorrhagic transformation as red blood cells escape from the vessels. These findings are significant in both ischemia–reperfusion and permanent ischemia models, highlighting the critical role of EC damage in stroke pathology.

4.1.2. Endothelial Transport Dysfunction

Following cerebral ischemia, there is a marked increase in EC pits and vesicles, signifying compromised endothelial integrity (Ma et al. 2021). This rise in transcytosis contributes to increased permeability of the BBB (Ma et al. 2021). Recent animal studies have shown that the poststroke surge in BBB permeability is linked to both transcellular and paracellular transport mechanisms (Knowland et al. 2014). In experiments with genetically modified mice lacking Caveolin1, there was a notable reduction in transcellular permeability in cortical vessels after transient middle cerebral artery occlusion (t‐MCAO), highlighting the distinct regulatory pathways for different transport modes. Stroke also affects ion channels in ECs, disrupting processes like the Na+/K+‐ATPase, especially in response to elevated extracellular potassium levels (Schielke, Moises, and Betz 1991).

4.2. Breakdown of EC TJs Leading to BBB Dysfunction

4.2.1. Molecular Composition of TJs

TJs are essential for maintaining cell–cell adhesion and controlling permeability in endothelial tissues (Figure 3). They are primarily composed of proteins from the claudin (CLDN) family, the MARVEL protein (TAMP) family, the junctional adhesion molecules (JAM) family and Zonula occludens (ZO) proteins (Winkler et al. 2021). Among these, CLDN‐5 is critical, as it shows the highest mRNA expression compared to other CLDN proteins (Castro Dias et al. 2019; Daneman et al. 2010). The TAMP family also plays a significant role in reinforcing TJ complexity and strengthening the BBB (Tsukita and Furuse 1999; Cording et al. 2013). JAM‐A interactions rely on cooperation with other TJ scaffold proteins and auxiliary molecules for full functionality (Aurrand‐Lions et al. 2001; Severson et al. 2008; Monteiro et al. 2014). ZO‐1 is indispensable, stabilizing CLDN‐5 expression and facilitating proper TJ assembly in microvascular ECs (Van Itallie, Tietgens, and Anderson 2017).

FIGURE 3.

The tight and adherens junctions. This schematic illustrates the structure of cell–cell junctional complexes, focusing on the key components of tight junctions (TJs) and adherens junctions. Transmembrane proteins like claudin, occludin, and junction adhesion molecules (JAMs) are highlighted, along with cytoplasmic proteins such as ZO‐1, ZO‐2, and cadherin. CLDN‐5 plays a critical role in maintaining the integrity of TJs, with ZO‐1 stabilizing its expression to ensure proper TJ assembly in microvascular ECs. JAM‐A requires interactions with scaffold proteins and other auxiliary molecules for full functionality. Adherens junctions, structurally similar to TJs, rely on cadherins and alpha‐actinins, which regulate adhesions and out‐in signalling processes, including interactions with platelet endothelial cell adhesion molecule‐1 (PECAM‐1).

4.2.2. Impact of Cerebral Ischemia on TJs

Cerebral ischemia profoundly alters the molecular structure of TJs. Ischemia‐induced inflammation leads to phosphorylation and posttranslational modifications in TJ proteins (Jiang et al. 2018). This structural turmoil is not the only casualty, and ischemia also triggers regulatory disruptions in protein distribution and expression within TJs, which have been confirmed in vitro cell studies, including occludin, CLDN, ZO‐1 and ZO‐2. Besides, actin, which plays a crucial role in BBB function, was found to be upregulated under deprivation/reoxygenation conditions (Mark and Davis 2002). As another example, poststroke abnormalities in the TJ chain become clearly apparent around 48–58 h after a transit MCAO, coinciding with a second peak of escalated biphasic BBB permeability (Knowland et al. 2014). This pattern suggests delayed TJ remodelling in response to ischemic events. Additionally, proteolytic enzymes, including tissue plasminogen activator (tPA), matrix metalloproteinases (MMPs), cathepsin and heparinase, degrade the BBB’s extracellular matrix (ECM), further destabilizing TJs through integrin‐driven mechanisms (Sandoval and Witt 2008).

4.3. Microcirculation Disorders With EC Injury

4.3.1. Dynamics of Microcirculation Dysfunction

1. Microcirculation becomes severely disrupted following ischemia, with ECs bearing the brunt of these effects. Several manifestations of this dysfunction include the following: Arterioles display impaired vasomotor function, with both endothelium‐dependent and independent dilation compromised, increasing the risk of thrombosis.

2. Capillaries exhibit constriction, obstruction and reduced perfusion, limiting oxygen and nutrient delivery to ischemic tissue.

3. Venules experience endothelial barrier dysfunction, progressing to severe vascular injury, allowing blood cell extravasation. Postischemia/reperfusion (I/R) conditions further exacerbate this by upregulating adhesion molecules in venules, increasing leukocyte adhesion and migration, which intensifies tissue damage (Arbaizar‐Rovirosa et al. 2023).

4.3.2. Factors Influencing Microcirculation

The disturbance in poststroke microcirculation is coordinated by various factors. Cerebral edema exerts pressure on capillaries, impairing their function (Jerome, Akimitsu, and Korthuis 1994; Xu, Wang, and Ji 2023). Pericytes, which regulate microvessel diameter and blood flow, are also implicated (Guo et al. 2022). However, there is a debate regarding whether ischemic damage stems from pericyte contraction in the early stages of ischemia or from increased ROS production through the Nox4 enzyme pathway, which leads to pericyte destruction (Nishimura et al. 2016). As these factors converge, the BBB integrity is compromised, and capillary function is further degraded, complicating the recanalization process. While larger vessels may partially recover, capillaries remain vulnerable to ongoing pathological changes.

4.4. Inflammatory Processes Associated With ECs

4.4.1. Significance of Endothelial–Glial Interactions

Stroke is characterized by a significant inflammatory response, which activates both endothelial and glial cells. These chemokines/cytokines released by MG and ACs include proinflammatory factors interleukin‐1b (IL‐1b), IL‐6, IL‐17, heparin‐binding epidermal growth factor (HB‐EGF), VEGF and thymidine phosphorylase (TYMP). In addition, the reactive AC‐derived IL‐1b limits astrocytic sonic hedgehog production. This is a signalling pathway that can have an impact on the integrity of the BBB, while triggering the astrocytic release of proinflammatory chemokines in vitro (Wang et al. 2014). The damaging effects of AC‐derived IL‐6 on the BBB have also been well established that IL‐6 specifically reduces barrier function through stimulating the proteasomal degradation of the junctional protein ZO‐1 (Chang et al. 2015). These interactions create a reciprocal inflammatory communication between ECs and glial cells, with far‐reaching consequences for the poststroke inflammatory landscape (Didier et al. 2003).

4.4.2. Repercussions of Inflammation on the Endothelial Surface

At the forefront of the inflammatory response is the endothelial glycocalyx layer, which releases inflammatory mediators under stress. Degradation of the glycocalyx exposes ICAM‐1 and vascular cell adhesion molecule‐1 (VCAM‐1), facilitating the adhesion of inflammatory cells (Mulivor and Lipowsky 2004; Vestweber 2015; Bui, Wiesolek, and Sumagin 2020; Liew et al. 2021). Additionally, ECs express selectins that assist in the rolling of white blood cells, which then undergo transendothelial migration into the tissue. Once inside, these cells release proteases, ROS and other inflammatory compounds, exacerbating reperfusion injury and complicating the recovery process (Nishimura et al. 2016).

Taken together, the degeneration of ECs—from initial edema to complete structural collapse—impedes the restoration of effective blood flow during reperfusion. Poststroke alterations in TJ proteins compromise the BBB, making the brain vulnerable even when reperfusion is achieved. Furthermore, disrupted microcirculation, marked by vasomotor impairments and reduced capillary perfusion, hinders the efficient delivery of oxygen and nutrients. Coupled with the intense inflammatory response involving endothelial–glial interactions and the infiltration of white blood cells, this chain of events exacerbates ineffective reperfusion. Although therapies such as thrombolysis and thrombectomy can reopen occluded vessels, the underlying cellular and molecular disruptions often result in suboptimal reperfusion outcomes. Therefore, addressing EC damage and dysfunction is crucial for improving poststroke recovery.

5. Clinical Application of EC Protection

There have been promising studies indicating potential treatments, such as Dl‐3‐n‐butylphthalide (NBP) enhancing the expression of longevity genes like SIRT3 to reverse endothelial damage in models of OGD/R; these findings have not yet translated into clear clinical strategies (Liu et al. 2022). Additionally, while endothelial progenitor cell (EPC)‐based therapies have shown benefits in animal models for AIS, clinical applications remain limited due to challenges such as immune rejection, infection risks and a lack of significant evidence from clinical trials to support their effectiveness (Custodia et al. 2022; Fang et al. 2019). Moreover, agomelatine, primarily used for treating depression, shows potential in preventing macrophage infiltration and protecting brain ECs in stroke mouse models by enhancing TJs between ECs. However, its direct impact on stroke recovery is still under investigation, and more specific clinical trials are needed (Cao et al. 2021).

Other proposed strategies involve the use of drugs like GM‐CSF, SDF‐1α and statins to promote EPC levels (Wang et al. 2020). Yet, these treatments also affect other progenitor cells and pathways, complicating their direct attribution to improved outcomes in AIS patients.

In conclusion, while the potential for targeting EC injury in stroke with various treatments is promising, current clinical applications are in the early stages. More research is needed to develop specific, effective therapies that can be validated in clinical settings.

6. Conclusion and Future Directions

Ineffective reperfusion remains a major challenge in neurointerventional medicine, with endothelial injury and BBB disruption being central concerns. This review underscores the critical role of brain ECs, particularly in stroke cases with inefficient reperfusion. It highlights the unique characteristics of brain ECs compared to those in other tissues and explores their pathological changes and inflammatory responses poststroke.

Given the pivotal role brain ECs play in neuroprotection, ongoing research into these cells offers significant promise for developing new therapeutic strategies. Looking forward, drawing analogies between brain ECs and those from other organs exposed to ischemia–reperfusion injuries may provide valuable insights. Despite interventions such as thrombolysis or thrombectomy, the cellular and molecular disruptions discussed in this review contribute to continued challenges in achieving optimal reperfusion outcomes.

Author Contributions

Yuchuan Ding, Xiang Li, and Qianhui Zhao contributed to writing the manuscript. Leticia Simo and Enoch Gene Kim edited the language. Yuchuan Ding and Xiaokun Geng contributed to study design and manuscript revision.

Conflicts of Interest

The authors declare no conflicts of interest.

Peer Review

The peer review history for this article is available at https://www.webofscience.com/api/gateway/wos/peer‐review/10.1111/ejn.16663.

Data Availability Statement

This review paper does not involve new data generation. All data discussed in this paper are derived from previously published articles, which are available on PubMed (pubmed.gov).