Pericyte dysfunction is a key mediator of the risk of cerebral ischemia

Pericytes are critical yet understudied cells that are a central component of the neurovascular unit. They are connected to the cerebrovascular endothelium and help control vascular contractility and maintain the blood–brain barrier. Pericyte dysfunction has the potential to mediate many of the deleterious vascular consequences of ischemic stroke. Current therapeutics are designed to be administered after stroke onset and limit damage, but there are few options to target vascular risk factors pre‐ischemia which likely contribute to stroke outcomes. Here, we focus on the role of pericytes in health and disease, and discuss how pericyte dysfunction can increase the risk of ischemic injury. Additionally, we note that despite the importance of pericytes in cerebrovascular disease, there are relatively few current therapeutic options that target pericyte function.

by responding to EC signaling to promote vasoconstriction or vasodilation (Attwell et al., 2016;Birbrair, 2019;Brown et al., 2019;Mishra et al., 2016;Tilton, 1979).In the brain, pericytes are critical for maintaining resting blood flow and neurovascular coupling, the critical process by which active neuronal regions receive increased blood flow as needed (Hall et al., 2014;Kisler et al., 2017).
The neurovascular unit (NVU) is a group of cell types that function together to connect the cerebral vasculature to the brain and control cerebral blood flow (CBF) (Muoio et al., 2014).The NVU is composed of neurons, astrocytes, pericytes, and EC that line the luminal surface of the cerebrovasculature (Iadecola, 2017).Pericytes and astrocytes combine on the endothelial surface to form the BBB, a system that utilizes tight junctions to limit the movement of molecules into and out of the vessels and brain (Abbott et al., 2006;Armulik et al., 2005).The BBB is critical for prevention of neural toxicity and unchecked neuroinflammation (Jin et al., 2021).
During development, and as part of adult angiogenesis, pericytes play a key role in the creation and expansion of new and growing vessels.Pericytes migrate toward new vessels in response to endothelial-released signals to form and stabilize vascular basement membranes, as well as promote further growth through the release of vascular endothelial growth factor (VEGF) (Hellberg et al., 2010;Kemp et al., 2020;Nehls, 1992).Additionally, pericytes have a neuroimmunological role, and can phagocytose cells that express damage associated molecular patterns, and are capable of acting as antigen presenting cells to other immune cells in the brain, that is, astrocytes, microglia, and leukocytes (Balabanov, 2001;Yang et al., 2016).
Given the diverse roles of pericytes in the brain including regulation of CBF, BBB maintenance, angiogenesis, and inflammatory responses, it stands to reason that a dysfunction of these cells could result in a wide variety of neurological impairments.

| Pericyte responses to neurovascular injury
Much of our understanding of how pericyte function affects the blood flow and BBB permeability come from research in the fields of traumatic brain injury (TBI) and ischemic stroke models (Dore-Duffy et al., 2000;Liu, 2012).TBI induces both acute and chronic changes in how pericytes communicate with EC and surrounding glia, characterized by the release of vasoconstrictive signals like endothelin-1, which can promote vasospasms (Dore-Duffy et al., 2000;Liu, 2012).
Additionally, following mild TBI, release of hypoxia-inducible factor 1 (HIF-1) by EC promotes pericyte detachment from vasculature in conjunction with oxidative stress damage (Choi et al., 2016;Hirota & Semenza, 2006).This detachment acutely promotes vascular survival and growth following an injury, with later reattachment contributing to the stabilization of the new vessels (Conway, 2001).Additionally, TBI generally results in an overall decrease in the total populations of pericytes (Main et al., 2018), as well as dysfunctional communication between the endothelium and pericytes due to loss of paracrine cell-cell signaling and compromised gap junctional proteins (Bhowmick et al., 2019).Cerebral blood vessels that lack pericytes or have impaired pericyte-endothelial communication often display lasting BBB leakage and dilatory impairments, resulting in diminished overall blood flow (Hellstrom, 2001;Rust et al., 2019;Steinman et al., 2019).
Depending on the severity of the TBI, many of these acute responses resolve within a few days, but more severe injuries can impair pericyte function for at least a week post-injury (Zehendner et al., 2015), or even longer in some cases (Clark et al., 2022).
Ischemic strokes produce similar pericyte responses.TBI and ischemic stroke both increase matrix metalloprotease-9 (MMP9) production that results in pericyte damage indicated by membrane damage due to extracellular matrix degradation and subsequent capillary wall breakdown (Underly et al., 2017;Yang et al., 2013).
MMP9 activity increases in parallel with platelet-derived growth factor ß (PDGFß), nitric oxide, and adenosine release to cause pericyte detachment from the vasculature and can result in lasting BBB permeability (Cai et al., 2017;Chen & Swanson, 2003).
Pericyte damage and detachment slows recovery from stroke by disrupting communication between EC and pericytes (Figure 1), as this communication is essential for continued angiogenic development and neurovascular repair after ischemic stroke (Farahani et al., 2019).Thus, the timing and duration of pericyte detachment is a key determinant of stroke outcomes.Detachment typically occurs early during ischemia, often within an hour of onset, mediated by a disruption in paracrine signaling to pericytes.Pericyte detachment can recover but is dependent on how rapidly the ischemic stroke is resolved via mechanical thrombectomy or thrombolysis (Arimura, 2012;Gonul et al., 2002;Ozen et al., 2014).Conversely, when blood flow returns to ischemic tissue, pericytes often clamp onto capillaries within the damaged regions, restricting blood flow and potentially contributing to the lack of reperfusion (noreflow) that occurs after ischemia (Cai et al., 2017;Fernandez-Klett & Priller, 2015;Su et al., 2019).The no-reflow phenomenon

Significance
Stroke is an ongoing health crisis that continues to worsen as the US population ages.Therapies to prevent or protect against stroke have been stagnant for several years, requiring new research into treatments.Pericytes are cells that have potential to play a significant role in stroke research.
Stroke is an ongoing health crisis that continues to worsen as the US population ages.Therapies to prevent or protect against stroke have been stagnant for several years, requiring new research into treatments.Pericytes are cells that have potential to play a significant role in stroke research.As such, we highlight pericytes as central players in neurovascular function with special emphasis on pericyte function in acquired brain injuries.We also discuss how pericyte function is modified by common stroke risk factors such as aging, smoking, hypertension, and small vessel disease.
comes from the combination of decreased blood flow at the time of stroke, as well as the changes in vascular permeability and the presence of neurovascular uncoupling (the loss of communication among neural/glial cells and the blood vessels within the brain) (Hartmann et al., 2021;Kisler et al., 2020;Williamson et al., 2021).
Following resolution of ischemia, pericytes play a role in both repair and recovery.Similar to the signaling cascades that take place after brain injury, there are angiogenic cascades that initiate once the ischemic blockage has been resolved and continue over the course of recovery.After ischemia, pericytes increase VEGF receptor expression, in parallel to increased VEGF release by EC (Gong et al., 2022;LeBlanc et al., 2017).Additionally, pericytes and EC increase the expression of tight junctional proteins like claudin-5 and occludin after ischemia, which stabilizes new vessels and promotes repair of the BBB (Liu, 2012).Taken together, ischemiainduced persistent and lasting damage to pericytes and the surrounding vasculature has been well documented, and literature is beginning to establish a role for pericyte targets as post-stroke therapies (Cheng et al., 2018;Li, Liu, et al., 2017).However, less is known about how a preexisting disruption to the normal function of pericytes may present as a risk factor for ischemic stroke.

| Pericyte roles in ischemic risk factors
Ischemic stroke is a multimodal disease that has a wide array of risk factors, many of which are strongly correlated with pericyte dysfunction; including age, smoking, hypertension, and family history of small vessel disease (Elkind, 1998;Muhammad et al., 2021;Sondergaard et al., 2017).
One of the best predictors for ischemia is age.In the United States, where the population is skewing older, the prevalence and incidence rates of ischemic stroke are predicted to continue to rise across the US and the world over the next several years (CDC.gov/, 2022; Wafa et al., 2020).There has been a limited focus in research on the natural aging of pericytes and how age-related pericyte dysfunction impacts the cerebrovasculature, with conflicting reports regarding changes in pericyte density in aged experimental animals (Montagne et al., 2021;Zhukov et al., 2023).As the body ages, there is a chronic increase in pro-inflammatory markers and reactive oxygen (ROS) and nitrogen species (RNS) (Davalli et al., 2016).
Pericytes respond to sustained exposure to ROS and RNS by increasing calcium release to vasoconstrictive and sometimes cytotoxic levels (Kamouchi et al., 2007;Pieper et al., 2014).Additionally, ROS and RNS production promotes pericyte-mediated vasoconstriction in mouse brains during a hypoxic/ischemic event (Yemisci et al., 2009).Aging also promotes a pro-inflammatory state via free radical release and accumulation, as well as cytokine storms and the slow decline of autophagy of cells (Rea et al., 2018).This state of chronic inflammation can provoke pericytes to undergo a morphological change producing proinflammatory cells with monocyte/ macrophage characteristics (Balabanov, 2001;Hurtado-Alvarado et al., 2014;Pieper et al., 2014).This morphological change and the resulting migration away from the microvasculature generally F I G U R E 1 Physiological pericyte responses are dependent on severity of attachment to vasculature.Injury/Disease states lead to pericyte detachment or clamping, which leads to cerebrovascular impairments that can play a role in initiation of an ischemic stroke.Under normal physiological conditions (center panel), pericytes are adhered to endothelial cells on the vasculature and maintain the blood-brain barrier (BBB), control cerebral blood flow, and function as antigen-presenting cells in inflammatory states.After mild traumatic brain injury (TBI) injury or aging (left panel), pericytes detach away from the vasculature, increasing BBB permeability and decreasing angiogenesis and vasodilatory capacity.After prolonged smoking, severe TBI, or ischemia (right panel), pericytes clamp onto the vasculature which increases vascular rigidity and thrombus formation and decrease angiogenesis.
progresses with age (Duz et al., 2007), can leave the vessels 'undermanned' with fewer pericytes covering the same vascular space, and this resultant pericyte uncoupling may result in a loss of autoregulation (Hughes et al., 2006).
The migration and consequent decrease in pericyte coverage of capillaries is implicated in a number of age-related disease states, including tumor formation and Alzheimer's disease (AD) (Bell et al., 2010).Pericytes attach to amyloid beta fibrils in human brains with AD, as well as induce a high degree of BBB permeability that can have negative impacts on the function of the cerebrovasculature (Wisniewski, 1992).As such, in AD, one of the most prevalent ageassociated diseases, there are several interconnected mechanisms that can induce pericyte dysfunction in the forms of cell signaling changes that result in pericyte migration, inflammatory responses promoting phenotypic changes in pericytes, or pericyte death in brain microvessels.Over time these pericyte dysfunctions compromise vasodilatory ability, increase the risk of thrombosis, and thus increase susceptibility to cerebrovascular disease, such as ischemia.
There is strong evidence linking cigarette smoking to an increased risk of ischemic stroke, with up to a quarter of all strokes being associated with smoking (Hankey, 1999;Hawkins, 2002).
Smoking promotes vascular pathophysiology that correlates with increased risk of ischemia, including reductions in CBF, increased BBB permeability, and cerebral EC dysfunction (Hawkins, 2002;Huang et al., 2013).The inhalation of cigarette smoke is linked to atherosclerotic plaque buildup and arterial stiffening (Chang et al., 2014;Csordas & Bernhard, 2013), often via inflammation and disruption of the endothelium (e.g., matrix metalloprotease activation and upregulation of endothelin receptors) (Vikman, 2009;Xu et al., 2008).The resulting stiffening of arterial walls is caused by prolonged breakdown of endothelial basement membranes and diminished capacity of vascular smooth muscle cells to proliferate and replace older and damaged cells (Xu et al., 2008).The breakdown of basement membranes after the loss of EC-pericyte communication is characterized by the disruption of integrins, collagens and laminins, as well as TIMP-3 which is derived from pericytes, all of which can result in unstable vascular membranes and lead to arterial stiffening (Stratman & Davis, 2012).
Pericytes are central to BBB integrity, inflammatory signaling, and CBF, all of which are dysregulated in individuals who smoke.
Specifically, pericytes respond to endothelin signaling, a prominent vasoconstrictive signal, and a disruption of this pathway also diminishes the proliferation of pericytes (Hibbs et al., 2021;Neuhaus et al., 2017).Additionally, smoke inhalation-initiated disruption of the endothelin-1 pathway, in conjunction with disrupted nitric oxide (NO) signaling, results in loss of bidirectional interactions between pericytes and EC, disrupting the resting balance between vasoconstriction and vasodilation (Dehouck, 1997), increasing the likelihood of forming microthrombi and ultimately increasing the likelihood of a future ischemic event.
In addition to being independent risk factors for ischemia, both aging and smoking promote hypertension, one of the single greatest predictors of ischemia (Endres et al., 2011).Hypertension is typically induced by a decrease in total vascular volume and an increase in vascular resistance due to a number of factors including small thrombotic clots, thickening of the vascular walls, or loss of collateral microvessels (Hajdu, 1994;Sabbatini, 2001).The development of microthrombi due to hypertension typically occurs at sites of endothelial disruption.Exposure of the endothelial basement membrane and glycocalyx promotes von Willebrand Factor (vWF) release, which results in platelet activation and accumulation, with formation of fibrin and thrombin networks that decrease luminal diameters (Albert-Weissenberger et al., 2019;Varga-Szabo et al., 2008).Additionally, vWF release due to shear stress, which can occur within the first few days of injury, can perturb the glycocalyx and promote BBB permeability, leading to damage beyond the structure of the vasculature (Schenck et al., 2021).Thrombotic plaque build-up within the lumens of vessels often originates with shear forces like long-term laminar flow, or from brain injuries that can force pericytes to detach from the endothelium, resulting in exposure of the basement membrane (Albert-Weissenberger et al., 2019).Of note, worsened ischemic outcomes have been seen in mice with pericytes that lack senp1, a small ubiquitination modifying protease that signals other sumoylated proteins to be ubiquitinated (Sun et al., 2020;Zhang et al., 2016).Interestingly, both the rate and scale of thrombus formation are increased in these mice, signifying that dysfunctional pericytes can be directly linked to microthrombus formation.As mentioned previously, atherosclerotic plaque buildup and arterial wall stiffening occur in large part due to basement membrane breakdown and loss of vasoconstrictive integrity (Xu et al., 2008), with many of the dysfunctions seen in smoking also existing in hypertensive patients and each likely resulting from persistent pericyte detachment from vasculature.Therefore, pericyte malfunction is in part responsible for microthrombus formation, arterial stiffening, and persistent increased blood pressure that predisposes to future ischemic events.
One other major risk factor for ischemia is the presence of cerebral small vessel disease, as nearly 25% of all ischemic stroke patients have a history of small vessel disease (Dong et al., 2003;Petty, 1999).Small vessel disease is a broad term for a wide range of hereditary and sporadic diseases that result in arteriosclerosis, small lesion formation, and microthrombus formation in the microvessels within the brain (Pantoni, 2010).There are a number of genetic mutations that result in genetic forms of cerebral small vessel disease, including mutations in Notch3, which results in cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common type of monogenic small vessel disease (Joutel, 1996).Notch3 is expressed by mural cells including vascular smooth muscle cells and pericytes (Boulos et al., 2011;Xu-Dubois et al., 2022).Expression of Notch3 is implicated in pericyte stability, differentiation toward a myofibroblast phenotype, and inflammatory signaling pathways that in turn activate NF-κB signals to regulate immune and inflammatory responses within the brain (Humphreys et al., 2010;Liu et al., 2017).
In addition to genetic mutations, cerebral small vessel disease has been correlated with increased production of ROS.As mentioned above, ROS including NADPH oxidase (Nox4) interact with brain pericytes and increase cell death and BBB leakage via MMP-9 release (Kuroda et al., 2014;Nishimura et al., 2016).Enhanced ROS production is also prevalent in ischemia and can result in pericyte death and subsequent brain damage following stroke (Hall et al., 2014).This wide array of pericyte responses to cerebral small vessel disease is representative of the broad impact that pericyte dysfunction has on capillary mechanics and diseases and displays how pericyte dysfunction may play a role in ischemia outcomes.
Taken together, since pericytes play critical roles in several risk factors that predominantly increase susceptibility and vulnerability to ischemic stroke, pericytes may be a good target for future research into their role in increasing stroke vulnerability and outcomes.

| Pericyte-centered stroke therapeutics and potential future treatments
Pericyte dysfunction is not only a key component of cerebrovascular impairments that increase the risk of ischemic strokes, but is additionally directly linked with post-ischemic vasoconstriction and cell death.Therefore, it is important to identify potential therapies that target the disruption of these cells to prevent future stroke incidence in high-risk individuals.We posit that treatments targeting some of the roles attributed to pericytes, including angiogenic and morphological changes, pericyte adherence, maintaining the BBB, or improving pericyte survival may have important therapeutic potential.
There is evidence that experimentally induced pericyte proliferation helps promote neovascularization and reduce the size of an ischemic infarct (Schuhmann et al., 2015).The resulting increase in angiogenesis allows for collateral flow and lessening of vascular load on each individual blood vessel, which could delay or prevent future ischemia (Freitas-Andrade et al., 2020).Angiogenesis is regulated in part by pericyte and EC interactions, but in several disease states, including ischemia, angiogenic capacity is altered.Unfortunately, in instances of pericyte dysfunction, continued angiogenesis can result in aberrant vessel formation with unstable basement membranes that can exacerbate existing vascular dysfunction (Ribatti et al., 2011).This is especially true following ischemic insults, leading to increased aberrant vessel formation with compromised vascular basement membrane formation, with an increased risk of post-ischemic hemorrhage in these immature vessels (Aronowski & Zhao, 2011).This angiogenic role for pericytes could prove to be a critical therapeutic role in future recovery in hypertensive or ischemic patients, and while growth of new vessels following ischemia is critical, these immature vessels can contribute to an increase in BBB permeability and persistent inflammatory cascades including IL-1, HIF-1α, and NF-ĸB.Several of these inflammatory responses regulate MMP-9 release from pericytes, which in turn increases endothelial permeability and BBB permeability (Takata et al., 2011).
A number of studies have investigated the role of pericyte constriction acutely after initiation of ischemia, and determined that ischemia-induced production of L-amino acid decarboxylase (AADC) via AADC-mediated conversion of tryptophan to tryptamine leading to increased pericyte-specific voltage-gated calcium channel signaling and subsequent prolonged pericyte constriction (Li, Lucas-Osma, et al., 2017).In an experimental spinal cord injury, AADC inhibition in mice increased blood flow and oxygenation within minutes, an effect that persisted for at least 90 min, suggesting that future studies should investigate the role of long-term AADC blockades on pericyte function in the context of ischemia to better understand the processes and provide an avenue for future potential therapeutics.
Another potential treatment approach is cilostazol, an antiplatelet drug and vasodilator which also promotes pericyte proliferation and angiogenesis.Cilostazol treatment in ischemia inhibits pericyte detachment from EC and reduces ischemia-induced pericyte death (Omote et al., 2014;Takagi et al., 2017).Similarly, the lipid lowering drug atorvastatin promotes neurovascular remodeling and angiogenesis after ischemic stroke (Yang, 2020).Both of these drugs are already safely utilized as therapies in humans, and a better understanding of how they can impact pericyte functionality could provide more broad and effective treatments to decrease risk of a future ischemic event.
As mentioned, atorvastatin is neuroprotective and assists in maturing the BBB and protecting against aberrant vessel growth in the regions surrounding the ischemic brain areas in part by decreasing MMP-9 release (Yang, 2020).Additional studies focused on prevention of ischemia-initiated aberrant vessel growth and BBB permeability via increased Gpr124, an endothelial G-protein coupled receptor related to the Wnt signaling pathway.By shutting down the receptor in mice, Gpr124 was shown to be critical for EC-pericyte connection and BBB integrity in the context of ischemic stroke in young adult rodents (Chang et al., 2017).Finally, treatments that reduce HIF-1 led to reduced BBB permeability and pericyte death in ischemia (Tsao et al., 2021), suggesting a larger role for multiple pericyte-linked targets in BBB permeability and angiogenesis in neuroprotection against ischemic damage.As such, there are many potential routes for future expanded studies to focus on pericytespecific responses to treatments and how they impact ischemic stroke outcomes.

| DISCUSS ION/CON CLUDING REMARK S
Pericyte dysfunction is implicated in a number of brain diseases, including brain injury, small vessel disease, and Alzheimer's disease.

ACK N OWLED G M ENTS
We would like to acknowledge the contributions of all of our colleagues in the field whose work has added to the substance of this review article.
Alterations in pericyte morphology, adherence, and cell signaling can promote widespread vascular dysfunction and loss of vascular tone and regulation.Critically, these deficits are promoting the development of ischemic clots, so a focus on pericytes in future research on risk and incidence of stroke is needed to better understand their role as more direct risk factors in ischemia.Mechanisms exist to target pericyte adherence and cell signaling pathways in preclinical settings, but there are no current FDA approved treatments targeting pericytes in human stroke patients.Moreover, more studies are needed to determine the impact of these therapeutic targets on arteriosclerotic plaque and microthrombosis development that leads to ischemia.AUTH O R CO NTR I B UTI O N S Bailey Whitehead: conceptualization (lead); writing -orginal draft (lead); visualization (lead); writing -review and editing (equal).Kate Karelina: writing -review and editing (equal); visualization (supporting).Zachary M. Weil: conceptualization (supporting); writing -review and editing (equal).

FU
This work was funded by the West Virginia Stroke and Alzheimer's Disease Related Dementias T32 (T32 AG052375), the WV Stroke CoBRE grant (5P20GM109098-08), the West Virginia University Experimental Stroke Core, and the West Virginia University Clinical and Translational Science Institute.