Progress of the study of pericytes and their potential research value in adenomyosis

Abstract

Pericytes (PCs) are versatile cells integral to the microcirculation wall, exhibiting specific stem cell traits. They are essential in modulating blood flow, ensuring vascular permeability, maintaining homeostasis, and aiding tissue repair process. Given their involvement in numerous disease-related pathological and physiological processes, the regulation of PCs has emerged as a focal point of research. Adenomyosis is characterized by the presence of active endometrial glands and stroma encased by an enlarged and proliferative myometrial layer, further accompanied by fibrosis and new blood vessel formation. This distinct pathological condition might be intricately linked with PCs. This article comprehensively reviews the markers associated with PCs, their contributions to angiogenesis, blood flow modulation, and fibrotic processes. Moreover, it provides a comprehensive overview of the current research on adenomyosis pathophysiology, emphasizing the potential correlation and future implications regarding PCs and the development of adenomyosis.

Background

The pathological features of adenomyosis include the presence of active endometrial glands and stroma surrounded by an enlarged and proliferative myometrial layer, accompanied by fibrosis and neovascularization. However, the exact etiology of this distinct pathological condition remains unclear. Pericytes (PCs), commonly referred to as Rouget cells, were initially characterized in the nineteenth century by Eberth and Rouget as flattened cells with protrusions interspersed among endothelial cells (ECs) and the basement membrane of capillaries.^1,2 As these cellular protrusions are closely associated with or integrate into the surface of the endothelial basement membrane, they are essential for modulating blood flow, ensuring vascular permeability, maintaining homeostasis, and aiding the tissue repair process. Given the involvement of PCs in numerous disease-related pathological and physiological processes, their regulation has emerged as a focal point of research. In this review, a comprehensive overview of the current research on the pathophysiology of adenomyosis was presented, with a particular focus on elucidating the potential correlation of and future implications associated with PCs in the development of adenomyosis.

Origin

The exact process of PC formation remains a topic of discussion. Contemporary literature spanning studies on organs such as the brain, ³ thymus, ⁴ stomach, ⁵ lungs, ⁶ and liver ⁷ and structures such as coronary arteries ⁸ postulate that PC development within organs may encompass epithelial–mesenchymal transition (EMT). This results in their migration to participate as mesenchymal constituents along with other cells such as fibroblasts and vascular smooth muscle cells (VSMCs). ⁹ Furthermore, evolving research has delineated the differentiation of PCs from human-induced pluripotent stem cells.^10,11

Classification

PCs are an eclectic ensemble of cells characterized by varied morphologies and protein profiles. Distinct patterns of protuberances were observed by Zimmermann across capillary beds, with circumferential protuberances located near arterioles, longitudinal protuberances located centrally, and stellate protuberances located near venules. The author classified these variations using the PC nomenclature. Based on their specific location within the capillaries, PCs are categorized into the precapillary, midcapillary, and postcapillary subclasses. ¹² Morphologically, PCs can be classified as sheath PCs, reticular PCs, and thin-stranded PCs.^13,14 The distinct protein expression profiles among these subtypes have been determined via antibody labeling and immunofluorescence techniques.^15,16 The organ-centric categorization scheme comprises brain, retinal, muscle, renal, hepatic, placental, and endometrial PCs. For example, brain PCs play a crucial role in the establishment of the blood–brain barrier, ¹⁷ as well as in the development of the central nervous system, maintenance of immune equilibrium, and regulation of white matter function. ¹⁸ Retinal PCs provide structural support to the vascular system by enveloping EC-aligned vessels, thereby increasing stability and tension to facilitate unobstructed blood flow. ¹⁹ Within kidneys, PCs underpin angiogenesis, regulate medullary and cortical blood dynamics, and function as progenitors during renal fibrosis, metamorphosing into interstitial myofibroblasts. ²⁰ In the liver, PCs are referred to as hepatic stellate cells ²¹ and play a pivotal role in liver fibrosis under pathophysiological conditions.

Markers

With advances in microscopy, multicolor flow cytometry, and lineage mapping, our comprehension of PC attributes and potential has expanded. ²² Common markers used to define PCs in the literature are platelet-derived growth factor receptor β (PDGFRβ),^23–25 CD146, ²⁶ neural glial antigen 2 (NG2),^27–30 desmin, ³¹ nestin, SUSD2, ³² regulator of G-protein signaling 5 (RGS5), ³³ and α-smooth muscle actin (αSMA). ³⁴ Meirelles ³⁵ analyzed the gene expression profile of noncultured, highly purified human adipose tissue PCs, and validated the expression of transcripts known to be PC markers by visually comparing their expression levels with those in other cell types; these markers included PDGFRA, PDGFRB, CXCL12, ANPEP, KCNJ8, and ANGPT1, which may also be expressed in other cell types. When acknowledging the limitations of using protein markers to identify specific subtypes or distinguish cellular states, it is important to note that RNA expression levels do not always correlate with protein expression levels within the cell. An unequivocal molecular beacon to singularly identify PCs and distinguish them from vascular smooth muscle or other mesenchymal cells has not yet been identified. For instance, PCs share PDGFR-β expression with VSMCs, neurons, and neural progenitor cells; ³⁶ NG2 expression with VSMCs, oligodendrocyte progenitors, and neuronal progenitors; ³⁷ and α-SMA expression with myofibroblasts, cardiomyocytes, and VSMCs. ³⁸ In light of this, Crisan et al. devised a targeted flow cytometry protocol for identifying human PCs, ³⁹ distinguishing them via the absence of hematopoietic markers (CD45) and endothelial markers (CD31/CD34) and the presence of CD146.

Notably, PCs exhibit substantial morphological and marker heterogeneity across tissues, with all markers exhibiting dynamic expression ³⁹ that is potentially modulated based on developmental trajectories, pathological stimuli, and ex vivo culture. Therefore, in tissue sections and embedded samples, PC identification is contingent upon a pristine tissue morphology, counterstaining for endothelial markers, and combinations of at least two PC markers. ⁹ Crisan's immunohistochemical exploration of human specimens underscored the heterogeneity in NG2 and αSMA expression across perivascular types. ⁴⁰ For instance, pericapillary PCs are characterized as NG2+/αSMA-, venular PCs as NG2-/αSMA+, and arteriolar PCs as NG2+/αSMA+, whereas CD146 and PDGFRβ expression remains uniform across these types.

Stem cell characteristics

PCs can differentiate into different lineages depending on their location and physiological state within various tissues. ⁴¹ These cells have been demonstrated to differentiate into neurons, ⁴² skeletal muscle fibroblasts, adipocytes, ⁴³ follicular dendritic cells, ⁴⁴ chondrocytes, ⁴⁵ and osteocytes. ⁴⁶ In animal models, PCs have been shown to improve cardiac function postmyocardial infarction, ⁴⁷ participate in tissue regeneration, and serve as niche cells for stem cells, performing specialized functions such as hematopoiesis. ⁴⁸ They also expedite wound healing in injured tissues ⁴⁹ and promote fibrous tissue formation. Tissue-specific PCs play a pivotal role in maintaining the distinct functions of various organs, such as the storage of vitamin A in the liver, ⁵⁰ production of renin in the kidneys, ⁵¹ and maintenance of the blood–brain barrier.

Role in angiogenesis

PCs are integral to microvessels and envelop capillary ECs. They influence the biological properties of capillary ECs through direct interactions and paracrine signaling, acting as pivotal regulators ensuring the stability and quiescence of capillary ECs.^52,53

PCs play a vital role in regulating angiogenesis. Lindahl observed that embryos from platelet-derived growth factor-β (PDGF-β)-knockout mice exhibited lethality, ⁵⁴ with >95% of the PCs lost in the mice, leading to highly aberrant vascular development and hemorrhage. Angiogenesis refers to the process by which existing vascular ECs (ECs) form new vessel sprouts within mature or embryonic tissues. ⁵⁵ The nascent vessel sprouts attract PCs by secreting growth factors such as PDGF-β. ⁵⁶ During early angiogenesis, PCs precede ECs, guiding them to form regular vessels. In the later stages of angiogenesis, PCs exert inhibitory effects on EC growth by activating specific signaling pathways or releasing vascular active substances. This process mediates vessel maturation, improves capillary barriers, and prevents excessive proliferation of ECs in growth factor-rich environments. ⁵⁵ For instance, when recruited PCs overexpress Tie2, ⁵⁷ TGF-β1, ⁵⁸ etc., they significantly negatively impact EC growth However, as the abundance of growth factors decreases, PCs can also promote EC growth, thereby influencing various physiological functions of vessels. Mäe's ⁵⁹ study on PC-deficient PDGFβ^ret/ret mice showed that the loss of 70%–80% of PCs led to excessive angiogenesis and EC transformation toward a venous phenotype, accompanied by overexpression of angiopoietin-2 (Angpt2).

Under pathological conditions, PC signaling pathway dysregulation or excessive detachment of PCs could induce disease development. For instance, Gong ⁶⁰ discovered that after stroke, VEGFR1 was significantly upregulated in PCs. In vitro studies confirmed the critical role of PC-derived VEGFR1 in endothelial vessel formation and PC migration. In proliferative diabetic retinopathy, excessive PC detachment is a hallmark of disease progression. ⁶¹ Clinically, a high percentage of hexokinase (HK2)-positive PCs in vessels is correlated with a lower overall survival rate in patients with non-small cell lung cancer and hepatocellular carcinoma. Treatment with HK2 inhibitors can induce PC-driven tumor vascular remodeling, potentially improving drug delivery and inhibiting tumor growth. ⁶² Precise regulation of ECs by PCs ensures the appropriate differentiation of neovessels under pathological conditions such as wound healing, aiding in vascular adventitial maturation to counteract vascular injury and reduce vascular remodeling. ⁶³

Regulation of blood flow

The VSMCs surrounding cerebral arteries and arterioles play roles in vascular resistance, while PCs located on adjacent capillaries may possess similar regulatory functions. Hall demonstrated that, under pathological conditions, ischemia induces capillary constriction regulated by PCs; when PCs are in a rigid state, their death can permanently constrict capillaries of the blood–brain barrier. Therefore, PCs are the primary cells modulating cerebral blood flow. ⁶⁴ Stefan conducted four-dimensional (x-y-z-t) imaging on microvessels exposed to vasoactive molecules and confirmed the crucial role of precapillary sphincters and PCs as sensors and effectors for ECs or brain-derived vascular signals in primary capillaries. ¹⁴ However, strategies for differentiating between PCs and VSMCs remain controversial. Hartmann et al. ⁶⁵ utilized selective optical ablation or activation of PCs to demonstrate that PCs can modulate capillary diameter and affect blood flow. In contrast to the rapid adjustments typical of arteriolar smooth muscles, the action in capillaries was relatively slow. Similarly, Almaça ⁶⁶ observed in pancreatic islets that PCs are innervated by sympathetic axons, helping regulate local blood flow by modulating the diameter of islet capillaries. Targeted regulation of PCs by drugs, such as the use of drugs to inhibit PC contraction, can improve cerebral microcirculation post-ischemic stroke. ⁶⁷ Research on brain injuries caused by the novel coronavirus (SARS-CoV-2) revealed that the virus spike protein (S protein) binds to the transmembrane receptor angiotensin-converting enzyme 2 (ACE2) to infect host cells. ⁶⁸ The expression of ACE2 in the cerebral vascular PCs of patients increases upon exposure to the S protein, resulting in PC elongation and contraction. This subsequently triggers an immune response that ultimately leads to brain damage. ⁶⁹

Tissue injury repair and fibrosis

Excessive accumulation of extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, leads to fibrosis in injured tissues. Uncontrolled fibrosis inflicts permanent tissue damage and can potentially cause disease. The important role of PCs in organ fibrosis primarily manifests in their provision of a source of myofibroblasts. ⁷⁰ The Glast-CreER transgenic mouse model developed by Göritz et al. ⁷¹ allows recombination of R26R-yellow fluorescent protein specifically in type A spinal cord intrinsic vascular PCs within the R26R subgroup while designating the remaining PCs as type B. Dias ⁷² used in vivo lineage tracing to discover that type A PCs were the origin cells of the matrix myofibroblasts in fibrotic scars post-penetrative spinal injury. Type A PCs were recruited to injured tissues, where they formed fibrotic scar tissue and hindered the regeneration of nerve axons. Birbrair ⁷³ also identified two subtypes of PCs in skeletal muscle interstitial tissue. In vitro experiments revealed that type 1 and type 2 PCs were fibrotic and muscular, respectively. Type 1 PCs differentiated into adipocytes and myofibroblasts, which participated in the fibrosis process, while type 2 PCs promoted the regeneration of blood vessels and muscles. Kramann traced the genetic lineage of vascular periphery Gli1⁺ MSC-like cells (i.e. PCs) and confirmed that they were one of the primary sources of myofibroblasts in various organs. Yamaguchi ⁷⁴ conducted in vitro studies on human lung cells that transformed into PC-like cells in the absence of TGF-β signals and into myofibroblast-like cells (PC–myofibroblast transformation) in the presence of TGF-β signals. In idiopathic pulmonary fibrosis patients, lung lesions were found to be consistently located between the KRT7-expressing lung periepithelium and the CD34-positive capillary endothelium, in which the cells also expressed PDGFRβ and NG2, suggesting that myofibroblasts likely originated from PCs. Using lineage tracing, Humphreys found that in renal fibrosis, PCs detached from vessel walls, migrated, acquired a myofibroblast-like phenotype, differentiated into myofibroblasts, were activated into fibroblasts, and acted as collagen-producing cells. ⁷⁵ Similar studies have been conducted on the impact of hepatic stellate cells on liver fibrosis, ²¹ the role of PCs in systemic sclerosis, ⁷⁶ etc. Blocking signaling pathways involved in PC differentiation to myofibroblasts may offer effective treatment strategy for early fibrosis. By driving PC mobilization to improve tissue regeneration, for example, by utilizing photobiomodulation to facilitate skin regeneration, ⁷⁷ employing PC patches for wound healing promotion, ⁷⁸ and promoting the formation of peri-infarction oligodendrocytes and astrocyte proliferation mediated by PDGFR in postacute ischemic stroke for fibrotic repair, ⁷⁹ functional recovery can be facilitated.

Pathophysiological characteristics of adenomyosis and the potential mechanism of PCs in adenomyosis

Adenomyosis is a prevalent gynecological condition, but a complete understanding of its etiology remains elusive. The current prevailing hypothesis is that invasion of the interstitium into the inner layer of the myometrium, accompanied by infiltration, is promoted by microenvironmental factors that stimulate smooth muscle cell growth. The hypertrophy and proliferation of the myometrial layer are believed to stem from metaplasia of stromal cells. Additional hypotheses that have been proposed to explain adenomyosis pathogenesis include EMT, embryonic remnants, ⁸⁰ vascular PC pluripotency ⁸¹ and adipocyte activation. ⁸² Given the limited research on the role of PCs in adenomyosis, we comprehensively analyzed the existing data to elucidate the relationship between vascular PCs and adenomyosis.

Angiogenesis in adenomyosis

Angiogenesis involves the sprouting of new capillary blood vessels from preexisting vessels, and it occurs in both physiological and pathological contexts. ⁸³ The mean vascular density (MVD) was evaluated, and the results revealed a significantly greater MVD in the ectopic endometrium of adenomyosis patients and significantly greater vascular endothelial growth factor expression than in the control endometrium. ⁸⁴ Tumor-like intravascular proliferation of the adenomyotic stroma (IVSP) was identified in 17.5% of patients in Sieiński's 1993 study. ⁸⁵ IVSP originates from perivascular stromal proliferation and exclusively occurs in deep adenomyosis. In a retrospective analysis of histopathological slides of adenomyosis tissue, Meenakshi discovered that adenomyotic endometrial tissue infiltrates uterine blood vessels and hypothesized that adenomyosis could develop from cells associated with myometrial blood vessels, possibly multipotential perivascular cells. ⁸¹

Tissue injury repair and fibrosis in adenomyosis

Similar to patients with endometriosis, patients with adenomyosis also present with cyclic bleeding from ectopic endometrial tissue, indicating repeated tissue injury and repair (ReTIAR). The etiological factors of adenomyosis can be categorized into exogenous injuries, such as surgical interventions (including curettage, natural childbirth, and cesarean section), and endogenous injuries (excessive uterine muscle motility exerting mechanical force on endometrial cells, leading to microtears, tissue damage, and chronic injury at the endometrial–myometrial junction zone ⁸⁶ ). The basement membrane of the endometrial basal glands may perform a protective function by preserving the structural integrity of these glands. ⁸⁷ During muscular injury in the context of adenomyosis, rupture of the endometrial–myometrial junctional zone occurs, leading to the migration of fibroblasts to the damaged uterus and their differentiation into collagen-producing myofibroblasts. The response of myometrial myofibroblasts to injury and activation from displaced basal endometrial fragments can be aberrant. ⁸⁶ Whether PCs in the endometrium or myometrium play a role in injury repair remains an important topic for further research.

Histologically, endometrial invasion into the myometrium is observed, resulting in a diffusely enlarged uterus. Under microscopic examination, ectopic, nontumorous endometrial glands, and stroma can be observed to be surrounded by hypertrophic and proliferative myometrial layers. ⁸⁸ Fibrosis is a characteristic feature of adenomyosis, as the invasion of the basal endometrial layer into the myometrium can result in extensive fibrotic changes. The presence of ectopic endometrial tissue induces smooth muscle cell hypertrophy and proliferation. ⁸⁹ The distinguishing factor in this fibrotic process is the accumulation of ECM components, such as type I and III collagen and αSMA, which are primarily localized within myofibroblast lesions, ⁹⁰ indicating that activated myofibroblasts may play a crucial role in adenomyosis. ⁹¹ Activated fibroblasts play a crucial role in the pathogenesis of fibrosis and can undergo phenotypic transformation into myofibroblasts expressing αSMA and collagen in various tissues throughout the body. The origin cells of myofibroblasts are diverse and include quiescent fibroblasts (resident fibroblasts or stromal cells), circulating progenitor cells derived from bone marrow stem cells, and cells of different types that have undergone phenotypic transitions, such as EMT in epithelial cells and EMT in ECs.^92–94 However, a recent single-cell analysis performed in 2023 revealed that the predominant cell population in the endometrium consists of densely clustered fibroblast subsets, which appear to originate from PC progenitors. By contrast, the fibroblast population in adenomyosis constitutes a significantly larger proportion (50% compared with 36% in the endometrium) of the total cell population and shows no association with PC progenitors. ⁹⁵

Epithelial–mesenchymal transformation in adenomyosis

Adenomyosis can be classified by MRI as type I (intrinsic), type II (extrinsic), type III (intramural), or type IV (indeterminate). Varying mechanisms underlie these different classifications of adenomyosis. Types I, II, and III are believed to result from the invagination of the endometrial base into the myometrium, serosal infiltration by ectopic endometrial tissue, and neogeneration or differentiation of adult stem cells, respectively.^90,96 In type I adenomyosis, electron microscopy reveals abundant ultrastructural features of myofibroblasts in the endometrial–myometrial junctional zone. ⁸⁶ However, these cells express nonmyogenic markers such as αSMA and type I collagen and lack myogenic differentiation markers. ⁹⁰ They are thus believed to be of nonmyogenic origin and possibly generated through a TGF-β-dependent process. In type II adenomyosis, the upregulation of the TGF-β/Smad signaling pathway leads to the generation of differentiated myofibroblasts with high myogenic potential and extensive ECM deposition. ⁹⁰ Guo's team observed, in studies on mouse uteri and human patients with adenomyosis, that the TGFβ1 signaling pathway is associated with EMT and the transformation of fibroblasts to myofibroblasts, leading to fibrosis and smooth muscle metaplasia.^97,98 The initial step in the development of adenomyotic foci involves the disruption of desmosomal junctions, ⁹⁹ which is facilitated by the elevated levels of TGFβ1 found in uterine lavage fluid from individuals with adenomyosis uteri. ¹⁰⁰

Limitations and prospects

In recent years, increasing research has focused on the role of mesenchymal stem cells in adenomyosis, making it a prominent topic of investigation. Although PCs possess numerous functional characteristics and play crucial roles, considering the role of a single-cell type in the occurrence and development of uterine adenomyosis has limitations. Recently, there has been an increasing focus on heterogeneous populations of stem/progenitor cells (SPCs) found in various blood vessels.^101,102 For instance, adventitial SPCs have been observed to participate in vascular remodeling by differentiating into vascular cells,^103,104 or undergoing differentiation into SMCs for repairing severely damaged vessels.^105–108

Currently, PCs have been determined to play a crucial role via in vitro techniques such as the construction of vascular chips, ¹⁰⁹ microvascular networks, ¹¹⁰ and fibrous networks ¹¹¹ to simulate the progression of disease processes. These techniques allow researchers to assess the interactions among various cell types in vitro. As the understanding of PCs has deepened with the advancement of new technologies, scientists are beginning to recognize that PCs could be able to regulate angiogenesis and promote fibrosis and muscle cell proliferation and that they exhibit characteristics associated with stem cell differentiation, which also plays a pivotal role in the onset and progression of adenomyosis. Given the potential variations in adenomyosis subtypes, it is imperative to investigate angiogenesis, ReTIAR, fibrosis, and EMT in adenomyosis using distinct in vitro and in vivo models tailored to each subtype in future research. Exploring the pathogenesis of adenomyosis from the perspective of PCs may present a groundbreaking opportunity, unveiling potential therapeutic targets for future treatment strategies, such as antiangiogenic interventions or inhibition of fibrosis.

Conclusions

PCs, possessing stem cell potential, are extensively distributed around blood vessels throughout the human body. A substantial body of evidence from both human and animal studies has unequivocally demonstrated the pivotal roles played by PCs in angiogenesis, modulation of blood flow, and facilitation of tissue repair processes. Considering that adenomyosis is characterized by angiogenesis, ReTIAR, fibrosis, and EMT, coupled with limited evidence of mesenchymal changes and the relatively unexplored role of PCs in the initiation and progression of adenomyosis, further comprehensive investigations are warranted to gain deeper insights into this area.