Aging microvasculature: Effects on immune cell trafficking and inflammatory diseases

Aging is defined as time-dependent deterioration of organs and biological systems at structural, functional, and molecular level. As such, aging disrupts all components of the immune system, compromising the host’s defense system. Additionally, this promotes a low-grade state of systemic chronic inflammation that ultimately forms the basis of many age-related diseases (Furman et al., 2019; Nikolich-Žugich, 2018). While the impact of aging on immunity is multifactorial, a crucial element is progressive dysregulation of leukocyte recruitment from the blood circulation to peripheral tissues. In line with this, single-cell RNA sequencing analysis of aged mouse tissues has illustrated immune cell infiltration at steady state in multiple organs, and numerous studies have reported impaired innate and adaptive immune cell recruitment in inflammatory settings in mice and humans (Tabula Muris Consortium, 2020; Hopkin et al., 2021; Zhang et al., 2025). Collectively, an increase in human longevity coupled to a surge in the incidence of chronic inflammatory disorders highlights the need for better understanding of the molecular mechanisms that underscore age-related changes in leukocyte trafficking and the implications of this to disease progression.

Effective shuttling of leukocytes between the bloodstream and interstitial tissues is commonly triggered by locally generated damage-associated molecular patterns and pathogen-associated molecular patterns as induced by sterile and infectious insults, respectively (Zindel and Kubes, 2020). Damage-associated molecular pattern and pathogen-associated molecular pattern stimulation of tissue sentinel cells (e.g., mast cells and macrophages) lead to generation of pro-inflammatory mediators, such as certain cytokines and chemokines, that drive immune cell trafficking. Soluble or immobilized, these molecular signals act on cognate receptors expressed on cellular components of the microvasculature or are presented to blood leukocytes via cellular/matrix scaffolding structures, respectively. Physiological leukocyte extravasation primarily occurs via postcapillary venules and, as such, involves breaching of endothelial cells (ECs), pericytes, and the venular basement membrane (BM) that is generated by both of these cell types (Nourshargh and Alon, 2014). The molecular events that mediate leukocyte passage through the endothelium have been extensively studied and are described by the classical leukocyte adhesion cascade (Ley et al., 2007). Briefly, this involves several leukocyte–EC interactions within the vascular lumen, initiating with leukocyte rolling along the endothelium, followed by leukocyte arrest and crawling. This series of sequential but overlapping steps is mediated by different adhesion receptors, including selectins, leukocyte integrins (β2 and β1), and their counter receptors on ECs (e.g., ICAM-1 and VCAM-1). During crawling, leukocytes seek and engage with EC junctions that are the primary sites of traversing the endothelial barrier and begin the process of breaching venular walls (Nourshargh and Alon, 2014; Reglero-Real et al., 2012).

Migration through the EC barrier in a luminal-to-abluminal direction (transendothelial cell migration, TEM) is exquisitely regulated by the expression and reorganization of junctional adhesion molecules and is predominantly driven by locally generated and presented chemokines (Muller, 2016; Nourshargh and Alon, 2014; Vestweber, 2015). Leukocyte TEM can occur via both transcellular and paracellular modes, and while the former is the most prevalent in vivo (Woodfin et al., 2011), significant use of the transcellular route has been reported across the blood–brain barrier (BBB) during inflammatory pathologies (Mapunda et al., 2022). Additionally, there is now ample evidence for the ability of neutrophils to exhibit reverse TEM, a phenomenon linked to disrupted molecular composition and/or functionality of EC junctions and chemokine localization (Colom et al., 2015; Girbl et al., 2018; Owen-Woods et al., 2020; Woodfin et al., 2011). Beyond the endothelium, leukocytes are required to traverse the pericyte layer as guided by pericyte-expressed adhesion molecules and chemokines (Girbl et al., 2018; Proebstl et al., 2012) and finally through the venular BM using permissive sites in the dense matrix structure (Voisin et al., 2010; Wang et al., 2006). As well as initiating the whole process of immune cell extravasation, tissue sentinel cells such as mast cells and macrophages can facilitate continued migration of immune cells across venular walls and within inflamed tissues (De Filippo et al., 2013). Mechanistically, a notable example of this is the ability of mast cells to regulate expression of pro-adhesive and/or pro-migratory molecules on the pericyte layer (Joulia et al., 2022).

As briefly summarized above, we now have a good understanding of the mode, dynamics, and molecular basis of immune cell trafficking, but whether and how this is dysregulated by age requires further exploration. In this context, age-related alterations in leukocyte production and behavior have been studied in-depth and comprehensively discussed in recent articles (Lu et al., 2021; Nikolich-Žugich, 2018; Van Avondt et al., 2023). However, a growing body of evidence now indicates that age-linked aberrations in vascular and stroma cells can dramatically influence the trafficking of immune cells, particularly neutrophils (Barkaway et al., 2021) (Fig. 1). Below, we provide an overview of such concepts and present their associated implications to aging-linked pathologies.

Vascular aging plays a central role in the development of cardiovascular disorders and hence is closely linked to enhanced morbidity and mortality of the elderly population. The aged microvasculature is characterized by molecular, cellular, and structural changes, including an increased pro-inflammatory state, altered circadian rhythms, disabled autophagy pathways, and changes in BM components. Many of these perturbations may be linked to cellular senescence, a response aligned with dysfunctional vascular responses in aged organs. The impact of these aging-linked vascular changes on the trafficking of immune cells, particularly of neutrophils, is discussed (Fig. 2).

Cellular senescence, a key hallmark of aging (Baker et al., 2016; Camell et al., 2021; López-Otín et al., 2013; López-Otín et al., 2023; Suda et al., 2021; Xu et al., 2018), initially observed by Hayflick in fibroblasts (Hayflick and Moorhead, 1961), is a state of stable cell cycle arrest resulting from the accumulation of DNA damage, oncogene activation, and organelle dysfunction (d’Adda di Fagagna, 2008; Schumacher et al., 2021). This response manifests at every stage of life, playing pivotal roles in regulating embryogenesis, wound healing, and tumor suppression (Burton and Krizhanovsky, 2014; Huang et al., 2022). However, excessive accumulation of senescent cells contributes to numerous inflammatory conditions, such as metabolic, neurodegenerative, and cardiovascular conditions, and the pathogenesis of various age-related syndromes (Muñoz-Espín and Serrano, 2014). Indeed, transplantation of senescent cells into young mice results in persistent physical dysfunction, reminiscent of aging-linked frailty (Xu et al., 2018). Conversely, pharmacological removal of senescent cells through use of senolytics or senostatics (see below for further details) alleviates age-related physical weakness (Camell et al., 2021; Suda et al., 2021; Xu et al., 2018). Although cellular senescence is considered a heterogenous phenomenon that is cell and context dependent, common features include activation of a DNA damage response and induction of cell cycle inhibitors, such as p16^Inka and p21^Waf1 (d’Adda di Fagagna, 2008; Ogrodnik et al., 2024; Young and Narita, 2009). Critically, a prominent feature of senescent cells is their increased capacity to secrete a wide range of soluble pro-inflammatory mediators, such as cytokines, chemokines, growth factors, proteases, and extracellular vesicles. This response is termed the senescence-associated secretory phenotype (SASP) and likely plays a prominent role in mediating key pathological effects of senescent cells (Coppé et al., 2010; Estévez-Souto et al., 2023).

Vascular senescence and its consequences are supported by a combination of in vitro and in vivo investigations. In vitro, models of cultured senescent macrovascular ECs (e.g., human umbilical vein endothelial cells [HUVEC]), as induced by insults, such as H₂O₂ and shear stress, have provided valuable phenotypic and mechanistic insights. These include identifying stress-induced premature senescence signaling pathways involving, among other factors, cyclin-dependent kinase inhibitors (p16^Inka and p21^Waf1) and DNA damage response pathways (Di Micco et al., 2021). Crucially, senescent HUVECs in culture show a general shift toward a pro-inflammatory state, exhibiting increased expression of cell adhesion molecules, NF-kB activation (Coleman et al., 2013), and a pronounced SASP. The latter involves enhanced secretion of a wide range of soluble bioactive molecules, such as TNF, IL-6, CXCL8, MCP-1/2, PGE2, vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), colony stimulating factors (CSFs), urokinase/tissue-type plasminogen activator (u-/tPA) and matrix metalloproteinases (MMPs), and extracellular vesicles. These factors can collectively modulate multiple inflammatory and vascular functions, including recruitment and activation of immune cells (Carracedo et al., 2019; Chen et al., 2020). Intriguingly, the Gamble group additionally observed that H₂O₂-, disturbed flow-, and hypoxia-induced HUVEC senescence, unlike replicative senescence, can induce a subpopulation of anti-inflammatory senescent ECs (Coleman et al., 2010; Coleman et al., 2013). While this concept needs explorations in vivo, it highlights the complexity and heterogenous nature of EC senescence induction as triggered by different stimuli.

The lack of specific markers has rendered the study of cellular senescence in vivo challenging (Ogrodnik et al., 2024). While physiologically aged mice are commonly employed, this strategy does not enable functional investigations of defined senescent cells, such as ECs. Nonetheless, numerous single-cell RNA sequencing studies offer comprehensive transcriptomic atlas of aged mouse tissues, as compared with young, enabling researchers the opportunity to analyze multiple cell types in different organs, including senescent vascular cells (Tabula Muris Consortium, 2020; Saul et al., 2022; Zhang et al., 2025). The Tabula Muris Senis or “Mouse Aging Cell Atlas,” and other similar data sets (Zhang et al., 2025), have provided insights into the common and varied transcriptomic profiles of senescent ECs in diverse vascular beds and as triggered by different stimuli (Saul et al., 2022; Uyar et al., 2020).

The use of senescent-ablator transgene mouse models, senescent reporter mice, and senolytic reagents (further details are provided in later section) have shed light on the localization and consequences of senescent cells in vivo (Ogrodnik et al., 2024). In particular, mouse models of senescent ablation have proven to be highly valuable in initial assessment of cellular senescence as a potential therapeutic target. This is most notable in the context of p16^high cells, where ablation extends the lifespan of aged mice (Baker et al., 2016). Most of these models rely on targeting cells that express high levels of p16 either through targeted activation of caspase (INK-ATTAC [Baker et al., 2011]), ganciclovir-induced apoptosis (p16-3MR [Haston et al., 2023]), or diphtheria toxin–mediated ablation (p16-FDR [Haston et al., 2023]). In the latter, by intercrossing a p16-Cre knockin mouse line with a Cre-reporter line (Rosa26-mTmG) and an ablator line (Rosa26-DTA), researchers identified the majority of aging-induced p16^high cells as being ECs, especially in the liver (Grosse et al., 2020). Elevated EC p16 was also observed in lungs in a murine model of lung cancer (Haston et al., 2023) and in mouse livers subjected to nonalcoholic steatohepatitis (Omori et al., 2020). Interestingly, the ablation of p16^high elicits a reduction in vessel density in multiple organs (Grosse et al., 2020). More recently, the p21-ATTAC model has been developed to eliminate cells expressing high levels of p21 (Chandra et al., 2022). Curiously, distinct phenotypes have emerged from the ablation of p16^high or p21^high senescent cells that highlights the complexity of such a strategy. Furthermore, of importance, although removing certain types of senescent cells leads to their efficient replacement that restores effective organ function and promotes increased lifespan, the situation is likely more complicated for poorly proliferative ECs. Specifically, genetic models of senescent EC ablation may be prone to disruptive vascular properties and hence may not be informative for analyzing the consequence of senescent ECs on immune cell migration. Furthermore, an essential consideration is that these genetically modified mice must undergo aging to accumulate sufficient senescent ECs for ablation. These factors contribute to the limited use of these models in investigating the functional role of vascular senescence in leukocyte trafficking in vivo. As such, models of cellular senescent induction (as opposed to ablation) could be more valuable for in vivo studies of EC senescence. This would include taking advantage of models overexpressing cyclin-dependent kinase inhibitors (e.g., p21) (Sturmlechner et al., 2021) when intercrossed with EC-specific Cre-expressing mice.

Additionally, conditional progeroid mouse models, in particular mouse models based on the genetics of the fatal premature aging disorder Hutchinson–Gilford progeria syndrome, have proven useful (Yousefzadeh et al., 2019). Hutchinson–Gilford progeria syndrome is caused by a point mutation in the LMNA gene that results in the production of progerin, a truncated form of the nuclear protein lamin A. At the molecular level, progerin causes numerous senescence-associated responses, such as genomic instability, DNA damage, and nuclear deformation, and as such affects multiple aging-related processes, such as mTOR signaling, inflammation, microRNA activation, and stress response mechanisms (Cenni et al., 2020). Using a conditional mouse model, we found progerin-expressing microvascular ECs to exhibit profound features of senescence and show pro-inflammatory characteristics (Rolas et al., 2024). Functionally, inflamed postcapillary venules that expressed progerin^+ve ECs promoted excessive neutrophil attachment to the luminal aspect of ECs and mediated neutrophil-dependent vascular permeability in models of inflammation. Mechanistically, senescent progerin-expressing ECs were overtly pro-adhesive for neutrophils in a cell autonomous manner that was CXCL1 dependent (Rolas et al., 2024). Further evidence for the ability of senescent microvascular ECs to activate immune cells was obtained using a mouse model of ischemic retinopathy, where senescent vascular units were found to stimulate neutrophil NETosis, a process that led to clearance of senescent ECs and reparative vascular regeneration (Binet et al., 2020). In contrast, though not directly investigating the involvement of vascular cells, activated ROS-generating neutrophils have been aligned with induction of cellular senescence in a murine model of acute liver injury and in livers of aged mice (Lagnado et al., 2021). Overall, while there is evidence for the ability of senescent ECs to promote neutrophil-mediated inflammation, the interplay between senescent ECs and immune cell migration and activation is complex and requires further exploration. Collectively, senescent ECs exhibit an elevated level of key EC adhesion molecules and a range of pro-inflammatory mediators (e.g., both neutrophilic and non-neutrophilic chemokines) at transcriptome and/or protein level in different settings. As such, senescent ECs could support rapid activation and recruitment of immune cells, driving an acute inflammatory response toward a chronic state.

To date, the field of vascular senescence has predominantly focused on vascular smooth muscle cells and ECs, with few studies investigating this phenomenon in pericytes. In this context, pericytes become senescent in vivo under conditions such as fibrosis, oxygen-induced retinopathy, and brain injury, contributing to the pathology of these diseases through their SASP (Cherry et al., 2023; Crespo-Garcia et al., 2021; Gil, 2023; Luo et al., 2024; Ting et al., 2023). The extent to which pericytes undergo cellular senescence during aging and the potential influence of senescent pericytes on regulation of immune cell trafficking and function remain largely unexplored. Additionally, there is emerging interest in the role of senescent perivascular cells in regulation of inflammation. For example, perivascular mast cells exhibit features of cellular senescence in aged mouse tissues, such as SASP, that directly impacts leukocyte trafficking (Barkaway et al., 2021).

Among the many molecular changes associated with old age, compromised macroautophagy, hereafter referred to as autophagy, has recently emerged as a hallmark of aging across diverse species (Aman et al., 2021; Kaushik et al., 2021). The nature of the autophagic process is to contribute to intracellular homeostasis by delivering cytoplasmic content to the lysosome for degradation. This includes the elimination or recycling of protein aggregates, membranes, whole organelles, or microorganisms, and the generation of metabolic precursors in response to starvation (Herzig and Shaw, 2018; Lin and Hardie, 2018; Morishita and Mizushima, 2019; Liu and Sabatini, 2020). Autophagy is highly regulated by autophagy-related genes (Atgs) that encode proteins implicated in the formation of double-membrane autophagosomes. Among these, the complex composed by the proteins Atg5, Atg12, and Atg16L is crucial for the initiation of autophagy (Yu et al., 2018; Morishita and Mizushima, 2019).

Progressive impaired autophagy has been detected in the aged cardiovascular system of mice and humans (LaRocca et al., 2012; Sasaki et al., 2017; Donato et al., 2018). Pharmacological approaches that restore autophagy, such as rapamycin and spermidine, can reverse aspects of arterial aging and vascular dysfunction (LaRocca et al., 2013; Lesniewski et al., 2017). Likewise, autophagy inducing drugs, such as rapamycin and resveratrol, limit neutrophil recruitment in murine models of LPS-induced acute lung injury (Fielhaber et al., 2012) and attenuate both macro and microvascular EC inflammation (Patella et al., 2016; Li et al., 2021; Mameli et al., 2022). These drugs are known to exert multifaceted effects on inflammation, including antioxidant activity, inhibition of immune cell proliferation, and modulation of cytokine production (Malaguarnera, 2019). As such, their influence on immune cell trafficking is likely driven by a combination of direct anti-inflammatory properties and their ability to enhance autophagy. In general, activation of autophagy remains a key mechanism through which ECs respond to local perturbations and modulate their survival, angiogenic, thrombotic, metabolic, and overall inflammatory functions (Sprott et al., 2019; Torisu et al., 2013; Verhoeven et al., 2021; Vion et al., 2017). Regarding the latter, we have provided in vivo evidence for microvascular EC autophagy acting as a negative regulator of neutrophil recruitment during acute physiological inflammation (Reglero-Real et al., 2021). Analysis of neutrophil–EC interactions by 4-D intravital microscopy revealed enhanced and faster neutrophil TEM across autophagy-compromised EC junctions, both in a paracellular and transcellular manner. Collectively, mice with selective deletion of Atg5 in ECs exhibit increased neutrophil extravasation, a finding that was reproduced in a diverse range of tissues and in response to several inflammatory stimuli (including sterile and non-sterile inflammation). Of note, this observation extended to other polymorphonuclear immune cells, such as eosinophils, suggesting a broader functionality. Overall, since excessive neutrophil extravasation in EC Atg5-deficient tissues was associated with enhanced tissue damage, and neutrophil trafficking was suppressed by local pharmacological induction of EC autophagy, these findings present activation of EC autophagy as a potential anti-inflammatory strategy. Mechanistically, ablation of EC autophagy led to increased cell surface expression of adhesion molecules and membrane projections at EC contact sites. These findings suggest that EC autophagy can curb neutrophil TEM by restricting the availability of cell surface adhesion receptor–enriched membranous pools that support leukocyte TEM (Mamdouh et al., 2003; Reglero-Real et al., 2012). In line with these results, autophagy increases barrier function of inflamed microvascular pulmonary ECs. This response has been linked to degradation of activated Src kinase and hence reduced VE-cadherin phosphorylation and internalization that collectively promote increased integrity of AJs (Dong et al., 2018). Autophagy modulation of EC junctional composition therefore arises as a key regulator of vascular permeability and neutrophil trafficking during acute inflammation. In addition to junctional remodeling, EC autophagy may regulate neutrophil TEM through additional mechanisms. An intriguing possibility relates to the potential role of EC autophagy in regulation of neutrophil/platelet interactions (Torisu et al., 2013; Yau et al., 2017; Verhoeven et al., 2021), a phenomenon linked to efficient neutrophil TEM (Sreeramkumar et al., 2014). Furthermore, impaired EC autophagy is linked to the induction of cellular senescence, both in vitro and in vivo, as observed in Caenorhabditiselegans, where it is associated with premature aging and reduced lifespan (Zhang et al., 2023b). Moreover, mice with selective deletion of Atg5 in ECs exhibit a senescent and pro-inflammatory phenotype in models of atherosclerosis (Vion et al., 2017), providing an additional link between autophagy and senescence. Finally, the functional role of diminished microvascular EC autophagy in promoting immune cell trafficking appears to extend to cells of the adaptive immune system, as observed in the context of T cells in a model of melanoma (Verhoeven et al., 2023).

Although autophagy in pericytes remains less explored than in ECs, emerging evidence points to this pathway as a regulator of microvascular functions in response to various stresses and pathological conditions. Autophagy supports the migration of retinal pericytes during diabetic retinopathy, preserves BBB integrity, and modulates pericyte inflammatory responses as well as their transition into a senescent state (Lin et al., 2022; Luo et al., 2024; Milani et al., 2024). Additionally, recent findings highlight an essential role for pericyte autophagy in establishing immune tolerance during cancer progression (Valdor et al., 2019). Overall, while an underdeveloped topic, impairment of autophagy pathways in pericytes may compromise their functions with implications for immune cell recruitment during both healthy aging and disease states.

The microvascular milieu features cells capable of generating high levels chemokines during acute inflammation, most notably ECs, pericytes, perivascular sentinel cells, macrophages, and mast cells (De Filippo et al., 2013; Girbl et al., 2018; Proebstl et al., 2012). Classically, the transcription factor NF-κB plays a pivotal role in driving a pro-inflammatory phenotype in aged vascular cells, orchestrating pathological upregulation of numerous genes associated with vascular inflammation, including adhesion molecules, cytokines, and chemokines. Interestingly, the application of deep-learning methods to analysis of blood immune biomarkers of a large cohort of individuals established a direct link between excessive chemokine production and health decline with age. Among these biomarkers, CXCL9 was identified as the strongest contributor (Sayed et al., 2021). More directly, and at single-cell level, excessive microvascular EC-expressed CXCL1 enhances luminal neutrophil–vessel wall interactions in inflamed aged murine venules (Barkaway et al., 2021). This phenomenon was also observed in a mouse model of EC senescence (Rolas et al., 2024). Furthermore, enhanced production of mast cell–derived CXCL1 in aged tissues promoted elevated levels of neutrophil reverse TEM (rTEM) that were subsequently detected in remote organs (most notably lungs) aligned with sites of vascular leakage (Barkaway et al., 2021). Mechanistically, this was mediated through extreme localization of CXCL1 at microvascular EC junctions, as supported by increased EC expression of the atypical chemokine receptor 1. Functionally, this sequence of events leads to desensitization of the chemokine receptor CXCR2 on neutrophils undergoing diapedesis, and consequentially, their loss of directional motility within EC junctions. Hence, while atypical chemokine receptor 1 has a pivotal role in supporting physiological luminal-to-abluminal neutrophil TEM (Girbl et al., 2018), its ability to bind a plethora of chemokines and its upregulation on microvascular ECs in aged and chronically inflamed tissues suggest a potential pathological role for this receptor in aging-linked disorders.

A key feature of aged microvessels is compromised barrier function to macromolecules both in health and disease, a phenomenon observed in mice and humans (Oakley and Tharakan, 2014; Stamatovic et al., 2019; Verheggen et al., 2020). As well as promoting pathogenic edema, increased vascular permeability can induce aberrant immune cell trafficking. Specifically, enhanced microvascular permeability can disrupt localized chemokine gradients at the vessel wall, and consequently, promote neutrophil rTEM (Owen-Woods et al., 2020). As such, it is plausible to speculate that aging-associated excessive microvascular leakage may be a causative trigger of aberrant immune cell diapedesis. Although our knowledge of the molecular basis of age-related vascular hyperpermeability remains limited, several factors, including increased oxidative stress, disrupted expression of EC adhesion molecules, and EC apoptosis, are potential contributors to this response (Oakley and Tharakan, 2014). Furthermore, we have recently shown that increased neutrophil attachment to senescent microvascular ECs can promote neutrophil activation and neutrophil-dependent microvascular leakage (Rolas et al., 2024). These findings identify EC senescence as an additional driver of microvascular hyperpermeability during aging.

Circadian rhythms refer to biological processes exhibiting oscillations over a 24-h period, typically synchronized with the Earth’s rotation through environmental cues such as light exposure (Scheiermann et al., 2012). These rhythms govern a multitude of physiological functions, including sleep cycles, metabolism, thermoregulation, vascular tone, and immune responses (Adrover et al., 2019; Han et al., 2021; Mohawk et al., 2012). However, with advancing age, the circadian system undergoes marked disruption and fragmentation, leading to adverse consequences for health (Hood and Amir, 2017; Nakamura et al., 2011). Critically, aging is associated with the loss of diurnal regulation of innate immune responses (Blacher et al., 2022; Casanova-Acebes et al., 2013). Aged mice, for instance, exhibit diminished diurnal monocyte egress from the bone marrow and reduced macrophage phagocytic capacity, rendering them more vulnerable to sepsis-induced inflammation during daylight hours (Blacher et al., 2022). Mechanistically, these phenomena have been linked to reduced circadian gene transcription in aged macrophages, driven by downregulation of Kruppel-like factor 4. Beyond innate immunity, age-related alterations in leukocyte trafficking may also stem from circadian disruptions within the vasculature itself. Indeed, a molecular clock within ECs orchestrates leukocyte recruitment during the dark phase (He et al., 2018). Deletion of the circadian regulator Bmal1 in ECs abolishes rhythmic leukocyte trafficking and enhances the nocturnal expression of adhesion molecules, such as ICAM-1 and VCAM-1. Altered expression of these molecules in aged ECs could contribute to impaired rhythmic leukocyte trafficking. Furthermore, the circadian clock machinery and age-related vascular changes appear to be reciprocally regulated. In support of this, Bmal1-deficient mice exhibit a pronounced premature aging phenotype (Acosta-Rodríguez et al., 2021; Khapre et al., 2014; Kondratov et al., 2006; Yu and Weaver, 2011) and circadian clock alterations have been detected in the aorta of aged mice (Gao et al., 2020). Moreover, mutations in circadian genes exacerbate hallmarks of vascular aging, including cellular senescence (Wang et al., 2008). While the identity of such senescent cells remains unclear, in addition to ECs, a potential role for senescent pericytes in this process cannot be excluded. Taken together, these findings suggest that targeting the molecular clock within the vasculature could represent a promising therapeutic strategy for modulating leukocyte trafficking and ameliorating inflammatory diseases in aged individuals.

Besides cellular components, extracellular matrix (ECM) is an integral element of all blood vessel types that is essential for their structural and functional properties (Hallmann et al., 2020). The venular BM, predominantly composed of laminin-411, laminin-511, and type IV collagen, linked through a network of other ECMs, is located at the basal surface of ECs and encases the pericytes. Functionally, the venular BM provides a formidable barrier to migrating immune cells, limits macromolecular passage, provides a depot for presentation of bioactive molecules, and preserves vascular integrity. Additionally, it is important for cell polarity, cell migration, and cell signaling and is highly dynamic in terms of its spatial and temporal remodeling during development and inflammation (Töpfer, 2023). Of relevance, with age, the venular BM experiences significant structural and molecular modifications, including increased thickness, accumulation of advanced glycation end products, and heightened levels of collagen and laminin deposition across various tissues (Koester et al., 2021; Leclech et al., 2020; Uspenskaia et al., 2004). These changes can lead to enhanced stiffness and establish a pro-oxidative stress environment within the BM, potentially impeding efficient leukocyte transmigration (Scioli et al., 2014).

While early in vitro studies suggested breaching of the venular BM by immune cells requires enzymatic degradation of its constituents (Heck et al., 1990), subsequent in vivo investigations revealed the essential role of integrins in this process (Dangerfield et al., 2002). Importantly, we have previously reported on the existence of permissive regions within the venular BM structure characterized by reduced matrix protein content. Termed “low expression regions” (LERs), these sites act as preferential exit points for extravasating immune cells (Voisin et al., 2009; Voisin et al., 2010; Voisin et al., 2019; Wang et al., 2006). As LERs are located to gaps between pericytes, it is plausible to speculate that the heterogeneous expression profile of the venular BM components is governed by the sheath-like pericyte coverage of venular walls (Voisin et al., 2010; Wang et al., 2006). Hence, since the pericyte coverage of the microvasculature diminishes with age (Berthiaume et al., 2022; Chen et al., 2021), aged venular BMs could potentially exhibit increased levels of LERs. The latter would facilitate greater and faster extravasation of immune cells into inflamed aged tissues, an interesting notion that warrants further exploration. Collectively, it can be speculated that age-associated alterations in BM composition and structure can differentially regulate immune cell breaching of venular walls, impairing or facilitating this process depending on the local microenvironment.

In addition to microvascular changes, tissue sentinel cells, including fibroblasts, mast cells, and macrophages, undergo significant molecular and functional alterations with age that can profoundly influence immune cell recruitment within tissues. For example, cellular senescence is prevalent across mesenchymal stromal cells, fibroblasts, and perivascular macrophages within various aged tissues (Feng et al., 2019; Reyes et al., 2022; Saito et al., 2020; Saito et al., 2024; Sturmlechner et al., 2021). Here, the associated SASP is a notable driver of inflammation and a potential cause of disrupted immune cell trafficking. In support of this, human senescent fibroblasts in the skin of older adults recruit inflammatory monocytes through the secretion of CCL2 (Chambers et al., 2021). Similarly, adipose-derived mesenchymal stromal cells and synovial fibroblasts in aged mice adopt a pro-inflammatory phenotype and contribute to leukocyte recruitment in conditions such as obesity and arthritis (Acar et al., 2020; Croft et al., 2019). Furthermore, we have obtained evidence of mast cell senescence in aged tissues, a phenomenon linked to excessive CXCL1 release and induction of neutrophil rTEM (Barkaway et al., 2021). Additionally, we and other groups have reported on increased number of mast cells in aged stroma (Barkaway et al., 2021; Kundu et al., 2020; Pilkington et al., 2019), a response that likely contributes to aging-linked dysregulated pro-inflammatory responses. In contrast, there remains no consensus regarding alterations in macrophage numbers with age, with reports of varied outcomes depending on the tissue type and the context (Duong et al., 2021; Moss et al., 2023). Reports of functional changes in macrophages with age are similarly inconsistent, indicating both pro-inflammatory and anti-inflammatory phenotypes in aged tissues in an organ-specific manner. For example, in the aged brain, microglia promote T cell infiltration by releasing CCL3 and upregulating adhesion molecules on venular ECs (Zhang et al., 2022b). In contrast, aging in skeletal muscle is associated with an increased presence of anti-inflammatory M2a macrophages, a subset linked to enhanced fibrotic remodeling (Wang et al., 2015). Nevertheless, certain age-related functional impairment of macrophages, such as a decline in phagocytic capacity and compromised autophagic activity, appear to be universal across tissues. These profiles potentially contribute to increased susceptibility to infections and defective inflammation and wound healing with age (Wang et al., 2015; Arnardottir et al., 2014; Franceschi et al., 2000).

Overall, there is ample evidence to suggest that age-related changes in stromal cells are context and tissue dependent. Furthermore, cellular senescence and enhanced pro-inflammatory profile of stromal cells appear to constitute key elements of their dysregulated behaviors in inflamed aged tissues.

Aging of the vasculature plays a central role in increased morbidity and mortality of older individuals and significantly contributes to age-related health deterioration (Abdellatif et al., 2023; Abdellatif et al., 2024; Ungvari et al., 2018). Vascular aging is commonly associated with arterial stiffening and heightened oxidative stress, both of which drive endothelial dysfunction and are major contributors to cardiovascular disorders in the elderly (Donato et al., 2018). Emerging evidence suggests that beyond these mechanical and oxidative changes, aberrant leukocyte infiltration into tissues and dysregulated immunosurveillance can contribute to the pathogenesis of numerous age-related chronic inflammatory diseases. Here, by focusing on selective tissues (brain, heart, muscle, adipose tissue, liver, lung, and skin), we explore the mechanisms through which organ-specific vasculature and perivascular stroma cells impact immunosurveillance and immune cell tissue infiltration in aged-linked disorders.

Immunosurveillance, the process by which the immune system recognizes and destroys foreign pathogens and other harmful cells, has been most extensively studied in aging in the context of cancer (Wang and Nakanishi, 2024; Zitvogel et al., 2024). Such studies have largely focused on interaction of immune cells with precancerous and cancerous cells, and to date relatively little is known about how the aged vasculature influences immune surveillance. In contrast, emerging evidence highlights a role for senescent perivascular stroma cells, most notably fibroblasts, as regulators of immunosurveillance. Of particular interest, senescent fibroblasts were found to exhibit several cellular features that render them efficient in activating dendritic cells and antigen-specific CD8⁺ T cells. These include the release of alarmins, activation of IFN signaling, enhanced MHC class I machinery, and presentation of senescence-associated self-peptides that can activate CD8 T cells (Marin et al., 2023). In skin, resident CD4⁺ cytotoxic T cells also eliminate senescent fibroblasts that express human cytomegalovirus glycoprotein B in an HLA-II–dependent manner (Hasegawa et al., 2023). Additionally, senescent fibroblasts activate a p21-retinoblastoma protein-dependent transcriptional program, generating a bioactive known as the p21-activated secretory phenotype. This secretome includes the chemokine CXCL14 that recruits macrophages to stressed cells, ultimately triggering a cytotoxic T cell response that promotes clearance of senescent cells (Sturmlechner et al., 2021). Harnessing these principles, chimeric antigen receptor T cells targeting the urokinase plasminogen activator receptor have been developed to ablate senescent cells, extend survival in murine lung adenocarcinoma, and restore tissue homeostasis in liver fibrosis mouse models (Amor et al., 2020). Despite these immunogenic properties, senescent cells also develop mechanisms to evade immune surveillance. For example, in the lung, p16⁺ senescent alveolar macrophages stabilize the immune checkpoint protein programmed death-ligand 1, preventing its ubiquitin-dependent degradation and promoting an immunosuppressive microenvironment that supports senescent cell accumulation in chronic inflammation (Majewska et al., 2024). Additionally, senescent cells in the liver, lung, kidney, and bone upregulate ganglioside GD3 that suppresses natural killer cell-mediated immunosurveillance. Targeting GD3⁺ senescent cells with anti-GD3 immunotherapy in mice reduces lung and liver fibrosis and pathological bone remodeling. These findings highlight a potential therapeutic avenue (Iltis et al., 2024). Together, the results of these studies underscore the dual role of senescent cells in immune surveillance, acting as both targets of immune clearance and drivers of immune evasion. The latter, in conjunction with age-related compromised immunity (immunosenescence), can lead to accumulation of senescent cells in aged tissues, contributing to development of chronic disorders.

As discussed in previous sections, age-related microvascular dysfunction profoundly alters immune cell trafficking, contributing to onset of acute inflammation and its progression toward a chronic state across multiple organ systems.

Significant work has investigated the impact of age in the central nervous system (CNS) and neurocognitive diseases. Microvascular aging is closely linked to BBB dysfunction and cognitive decline, facilitating pathological immune cell infiltration and compromising the brain’s immune-privileged status (Bennett et al., 2023, Preprint; Montagne et al., 2020; Nation et al., 2019). Cellular senescence within the CNS has been identified as a major driver of these changes, as clearance of p16⁺ senescent cells in aged mice restores immune homeostasis and preserves cognitive function (Zhang et al., 2022a). Age-related shifts in immune cell dynamics are particularly evident in stroke models, where alterations in granulopoiesis promote excessive neutrophil trafficking into the ischemic brain, leading to increased vascular occlusion, oxidative stress, and tissue damage (Gullotta et al., 2023). Similarly, aged T cells have been linked to larger infarct volumes and poorer neurological recovery following stroke (Lu et al., 2023). In contrast, CNS-resident macrophages in aged mice limit excessive neutrophil and T cell infiltration by suppressing endothelial adhesion molecule expression, highlighting a protective role for these cells during stroke pathology (Levard et al., 2024). In Alzheimer’s disease, amyloid β accumulation disrupts endothelial junctions by downregulating VE-cadherin and claudin-5, leading to increased vascular permeability and immune cell infiltration (Li et al., 2024; Ting et al., 2023). Aged brain ECs further exacerbate neuroinflammation by adopting a pro-inflammatory transcriptional profile, characterized by increased VCAM-1 expression that promotes immune cell recruitment and impairs neurogenesis (Chen et al., 2020; Yousef et al., 2019). Neutrophil infiltration into the CNS accelerates neurodegeneration, and inhibition of LFA-1–mediated neutrophil adhesion improves cognitive function in Alzheimer’s disease models (Zenaro et al., 2015). Additionally, increased CD8⁺ T cell infiltration, driven by endothelial C3a receptor signaling, promotes vascular inflammation and microglial activation, further contributing to neurodegenerative pathology (Propson et al., 2021).

Beyond the CNS, age-related changes in the vasculature and perivascular stroma impact immune cell trafficking in multiple organs, though these mechanisms remain less explored. In the heart, vascular aging drives leukocyte infiltration into aged myocardium, with CD4⁺ T cell accumulation contributing to chronic myocardial inflammation and functional decline (Ramos et al., 2017). Additionally, an increased population of cardiac-resident macrophages in aged hearts has been implicated in dysregulation of electrical conduction in models of endotoxemia (Esfahani et al., 2021). In the aged liver, CXCL2⁺ intrahepatic macrophages acquire a SASP phenotype, promoting excessive neutrophil recruitment and neutrophil extracellular trap formation, thereby exacerbating tissue damage following ischemia-reperfusion injury (Liu et al., 2024). Similarly, age-related macrophage dysfunction is evident in fractured bones, where these cells exhibit an M1/pro-inflammatory phenotype, impairing fracture healing (Clark et al., 2020). In skeletal muscle, macrophage-derived TNF-α promotes age-related sarcopenia, while reduced Treg cell recruitment following muscle injury impairs tissue repair (Kuswanto et al., 2016; Wang et al., 2018). Conversely, in adipose tissue, regulatory T cells accumulate with age, promoting insulin resistance, while aged adipose mast cells drive monocyte infiltration and exacerbate metabolic inflammation (Bapat et al., 2015; Liu et al., 2009; Yabut et al., 2020). Aged lungs also exhibit elevated pro-inflammatory cytokine levels and an expanded population of resident macrophages with enhanced Mycobacteriumtuberculosis uptake (Canan et al., 2014). Additionally, aging skews the lung immune microenvironment toward a premetastatic state, characterized by an accumulation of IL-17–producing γδ T cells and tumor-promoting neutrophils that suppress CD8⁺ T cell function and facilitate melanoma lung metastasis (Duan et al., 2024).

Age-related immune alterations also shape tumor microenvironments, influencing cancer progression and therapeutic response. In melanoma, aged mice exhibit reduced levels of intratumoral Tregs in the skin, correlating with enhanced responses to anti-PD1 therapy (Kugel et al., 2018). In contrast, prostate cancer progression in aged mice is associated with an increase in tumor-associated macrophages that rapidly adopt a pro-tumorigenic phenotype upon tumor cell engraftment (Bianchi-Frias et al., 2019).

Collectively, these findings highlight the tissue-specific consequences of age-related vascular and stromal changes on immune cell trafficking. While leukocyte infiltration is essential for immune surveillance, its dysregulation by the aged microvasculature and perivascular cells contributes to chronic inflammation, impaired tissue repair, cancer development, and the progression of neurodegenerative and cardiovascular diseases. Understanding the mechanisms governing these interactions within the aged vasculature and its microenvironment will be crucial for developing targeted interventions to restore immune homeostasis and counteract age-related disease.

Targeting the hallmarks of aging to attenuate cardiovascular disease and overall improve healthy lifespan is a topic of much interest (Abdellatif et al., 2023). In particular, there is much focus on epigenetic rejuvenation, metabolic reprogramming, pharmacological removal of senescent cells, and activation of autophagy pathways. These strategies are all aimed at therapeutically regulating pathological immune cell trafficking during aging. Here, we focus on the specific impact of drugs targeting cellular senescence and autophagy on the vasculature and their role in modulating leukocyte trafficking. While such interventions exert systemic effects, their influence on the aged microvasculature is a key component of their overall therapeutic benefit, normalizing vascular–immune cell interactions and broader health outcomes.

Senolytics are small molecules that selectively remove senescent cells by disabling pro-survival pathways and principally targeting BCL-2, p53, or p38 MAPK to induce apoptosis (Chaib et al., 2022). Dasatinib and quercetin, two of the most widely used senolytic drugs, were initially used for different purposes: dasatinib, a tyrosine kinase inhibitor, is primarily used to suppress the proliferation, adhesion, and migration of cancer cells, while quercetin, a natural organic compound, is valued for its anti-inflammatory and antioxidant properties, though its full therapeutic potential remains to be fully explored. Their senolytic effects were identified when Kirkland and Tchkonia (Kirkland and Tchkonia, 2020) conducted high-throughput screening of existing drugs, revealing that the combination of dastinib and quercetin (D+Q) could promote the clearance of senescent cells. Subsequent studies demonstrated that the senolytic actions of D+Q involves the transient disruption of the pro-survival network in senescent cells, which, in the context of cardiovascular diseases, was shown to improve cardiac function (Fan et al., 2022; Zhu et al., 2015). Since then, additional senolytic drugs have been identified, with diverse modes of action, and their development has been extensively reviewed (Kirkland and Tchkonia, 2020). Although the impact of removing vascular senescent cells on leukocyte recruitment was not investigated in these studies, senolytics have been described to limit inflammation and improve frailty in aged rodents. Such findings have formed the basis of extending this combination drug therapy to clinical trials, speculating an efficacy in modulating vascular–immune cell interactions in aging-related phenotypes (Baker et al., 2016; Camell et al., 2021; Xu et al., 2018). Consistent with this, genetic ablation of senescent macrovascular ECs or pharmacological targeting of senescent tissue macrophages attenuates plaque burden in mouse models of atherosclerosis (Childs et al., 2016; Suda et al., 2021). However, as discussed earlier, the ablation of senescent cells has been shown to reduce vessel density across multiple organs (Grosse et al., 2020), suggesting that this approach may not be optimal for modulating immune cell recruitment to tissues. Of note, since in addition to senescence, aged cells exhibit numerous other hallmarks, such as decreased autophagy (see below), the benefits of senolytics likely extend beyond senescence-specific mechanisms.

An alternative strategy to alleviate the deleterious consequences of vascular senescent cells in aging relies on the use of senomorphic drugs. These reagents target pathways implicated in the molecular expression of specific SASP components or their related transcription factors and include inhibitors of a variety of signaling hubs, such as mTOR, NF-κB, JAK-STAT, p38 MAPK, HSP90, NADH metabolism, and mitochondrial complexes (Zhang et al., 2023a). Despite the interest in this class of drugs, to date, there is limited mechanistic information on potential effects of senomorphics on aging-linked pathological leukocyte trafficking. A promising innovative approach in this area relates to the use of monoclonal antibodies or pharmacological inhibitors aimed at selective targeting of distinctive pro-inflammatory components of SASP. Consistent with this strategy, and since dysregulated chemokine expression in aged microvessels may be linked to SASP, pharmacological inhibition of CXCL1 led to reduction of neutrophil rTEM and abrogation of remote organ damage in aged mice subjected to muscle ischemia-reperfusion injury (Barkaway et al., 2021). These findings are clinically relevant, as soft-tissue trauma victims often require hospitalization due to lung complications, especially in the elderly (Lefaivre et al., 2010; Vollrath et al., 2022). As such, repurposing CXCL8 (IL-8; human functional homologue of mouse CXCL1) inhibitors that have been tested in clinical trials of metastatic tumors present an interesting therapeutic prospect for treatment of remote organ damage in aged trauma patients.

Manipulating autophagic pathways within the microvasculature presents another promising avenue for modulating aberrant aging-linked leukocyte trafficking. Thus, given that impaired autophagy is a hallmark of cardiovascular aging (Abdellatif et al., 2023), restoring the autophagic activity within aged vascular cells offers a therapeutic approach to mitigating chronic, age-associated inflammation. However, while pharmacological inducers of autophagy, such as resveratrol and rapamycin, are well-recognized anti-inflammatory agents, the value of normalizing autophagy within aged vascular cells remains largely unexplored. In support of such a targeted approach, activation of autophagy in microvascular ECs can significantly restrict neutrophil recruitment, as evidenced using a locally administered Beclin-1–activating peptide in a murine model of ischemia-reperfusion injury (Reglero-Real et al., 2021). Conversely, pharmacological inhibition of autophagy using the PI3K inhibitor 3-MA stimulates EC inflammation and promotes macrophage recruitment in hypercholesterolemic mice (Zhang et al., 2020), highlighting the essential role of autophagy in maintaining vascular homeostasis. Mechanistically, many autophagy-inducing agents work through inhibition of mTOR, directly targeted by drugs, such as rapamycin and torin1, or indirectly by AMP-activated protein kinase (AMPK) activators, such as metformin and trehalose. These compounds, long recognized for their anti-aging potential, have shown promising outcomes in both human and murine models of aging-linked diseases. The latter includes exerting improved immune function and diminished inflammatory responses (Mannick and Lamming, 2023). Furthermore, genetic, and pharmacological induction of autophagy in ECs and pericytes reverses key features of vascular aging, including cellular senescence (Luo et al., 2024; Zhang et al., 2023b). These findings underscore the intertwined nature of autophagy and cellular senescence and crucially suggest that therapeutic modulation of autophagy could provide a multifaceted approach to addressing the broader spectrum of vascular aging.

In summary, aged microvasculature and its associated milieu strongly impact the trafficking of immune cells, with significant implications to local and remote organ damage. Mechanistically, to date, there is much evidence for altered distribution and signaling of vascular chemokines, disabled autophagy, and enhanced senescence with age. Despite our growing understanding of the associated molecular and cellular players, many pivotal questions remain unanswered. Here, exemplar avenues include elucidating whether microvascular aging progresses uniformly across different tissues, vessel subtypes (i.e., venules versus capillaries), or at single-cell level. Given that age-related functional decline across organs does not occur in synchrony (Schaum et al., 2020), it is plausible to speculate a similar differential aging profile across different vascular beds. Such a mechanism would potentially establish localized pro-inflammatory vascular regions that may propagate inflammation to the local microenvironment and ultimately systemically. Moreover, the extent and dynamics of microvascular aging appears to be highly context dependent, driven to varied levels by different triggers, and may differ between male and females. The application of spatial transcriptomics and development of better reporter models of aging-linked molecular aberrations will be powerful means of investigating these avenues.

Beyond ECs, exploring the impact of age on molecular and functional profile of stromal cells will be crucial areas to explore. This includes investigating the impact of increased mast cell numbers in regulating inflammatory responses in aged tissues, in particular neutrophil TEM dynamics. Similarly, impaired pericyte remodeling, which compromises capillary flow in aging models (Berthiaume et al., 2022), stimulates an interest in further research into aged pericytes. This includes analysis of pericytes in different aged organs in terms of their numbers, localization, senescence, and autophagic machinery. An exciting possibility relates to how pericyte SASP may potentially impact the composition of the vascular BM and EC functionality, factors that could influence immune cell trafficking to aged organs. Interestingly, alterations in the ECM, a feature of aging, reportedly reduce inflammation and extend lifespan in rodent models (Zhang et al., 2023c). These findings emphasize the need for a more comprehensive understanding of how aging affects BM composition in different organs and how such changes impact leukocyte migration and activation.

Therapeutic interventions targeting the aging vasculature, including senolytics, autophagy enhancers, VEGF overexpression, and other drugs targeting different hallmarks of aging, are anticipated to alleviate endothelial dysfunction, enhance angiogenesis, and mitigate inflammation in elderly individuals. While such pharmacological strategies are anticipated to extend healthspan (Grunewald et al., 2021), there is an urgent need for greater understanding of how lifestyle modifications, such as exercise and dietary interventions, may influence microvascular senescence and delay vascular aging. Ultimately, greater understanding of the extrinsic and intrinsic factors, and associated processes that drive microvascular aging will enhance our ability to develop innovative strategies to combat age-related multi-morbidities.

Figures were created with https://BioRender.com.

This work was principally funded by the Wellcome Trust (098291/Z/12/Z and 221699/Z/20/Z to S. Nourshargh). N. Reglero-Real is a Ramón y Cajal awardee (RYC2021-031221-I) funded by the Spanish Ministry of Innovation and Science (MCIN) and Spanish State Research Agency (AEI), cofunded by ‘‘NextGenerationEU,’’ and additionally supported by ‘‘Generación de Conocimiento’’ projects program of MCIN/AEI (PID2022-137552OA-100). L. Rolas is supported by funding from the British Heart Foundation (FS/IBSRF/22/25121).

Author contributions: N. Reglero-Real: conceptualization, funding acquisition, project administration, supervision, and writing—original draft, review, and editing. L. Rolas: conceptualization, funding acquisition, visualization, and writing—original draft, review, and editing. S. Nourshargh: conceptualization, funding acquisition, and writing—review and editing.