The role of endothelial cell–pericyte interactions in vascularization and diseases

Graphical abstract

Highlights

• •Physiologic significance of intact normal microvascular structures.
• •The role of endothelial cells and pericytes in angiogenesis and vascular remodeling.
• •Discuss the complex signaling pathways between endothelial cells and pericytes.
• •The disease manifestations of disordered signaling pathways.
• •Potential therapeutic strategies to correct disordered signaling pathways.

Abstract

Background

Endothelial cells (ECs) and pericytes (PCs) are crucial components of the vascular system, with ECs lining the inner layer of blood vessels and PCs surrounding capillaries to regulate blood flow and angiogenesis. Intercellular communication between ECs and PCs is vital for the formation, stability, and function of blood vessels. Various signaling pathways, such as the vascular endothelial growth factor/vascular endothelial growth factor receptor pathway and the platelet-derived growth factor-B/platelet-derived growth factor receptor-β pathway, play roles in communication between ECs and PCs. Dysfunctional communication between these cells is associated with various diseases, including vascular diseases, central nervous system disorders, and certain types of cancers.

Aim of review

This review aimed to explore the diverse roles of ECs and PCs in the formation and reshaping of blood vessels. This review focused on the essential signaling pathways that facilitate communication between these cells and investigated how disruptions in these pathways may contribute to disease. Additionally, the review explored potential therapeutic targets, future research directions, and innovative approaches, such as investigating the impact of EC-PCs in novel systemic diseases, addressing resistance to antiangiogenic drugs, and developing novel antiangiogenic medications to enhance therapeutic efficacy.

Key scientific concepts of review

Disordered EC-PC intercellular signaling plays a role in abnormal blood vessel formation, thus contributing to the progression of various diseases and the development of resistance to antiangiogenic drugs. Therefore, studies on EC-PC intercellular interactions have high clinical relevance.

Introduction

The establishment of a mature and well-organized vascular system is important for organisms to maintain organismal and cellular homeostasis. The formation of new blood vessels is important for providing cells and tissues with the oxygen and nutrients that are needed for normal growth and function. Angiogenesis plays a critical role during all stages of mammalian development, including during the earliest stages of embryonic development as well as during normal growth and tissue repair. In particular, angiogenesis involves the proliferation and differentiation of blood vessels as well as the formation and remodeling of new blood vessels. Endothelial cells (ECs) and pericytes (PCs) play synergistic roles in angiogenesis. ECs promote vascular development by releasing signaling molecules, such as growth factors and extracellular matrix proteins. Moreover, PCs function as support cells, providing physical support and facilitating extracellular matrix synthesis during angiogenesis. The interactions and crosstalk between ECs and PCs are essential for ensuring proper vascular system development and function. Angiogenesis is a tightly regulated process that is characterized mainly by the proliferation, migration, and vascular remodeling of ECs and PCs via complex crosstalk and interactions [1], [2]. Understanding the roles of ECs and PCs in angiogenesis is critical to our understanding of organismal growth and development and could provide novel insights for vascular-related disease treatment and tissue engineering.

Many human diseases are related to vascular dysfunctions, such as microvascular dilation and vascular wall hyperpermeability phenotypes, and vascular dysfunctions are often characteristics of inflammatory diseases [3]. In recent years, increasing attention has been given to pathological vaso-related diseases that are caused by abnormal angiogenesis in addition to vascular dysfunction. Abnormal activity of PCs and ECs in the vasculature can be observed in many important and common diseases, such as retinal vascular diseases and tumors [2].

Many years ago, antiangiogenesis strategies, which aim to inhibit abnormal or excessive blood vessel formation, emerged as therapeutic approaches. Antiangiogenesis strategies are currently being used in the treatment of many diseases. For example, in cancer, antiangiogenic treatments aim to block the blood supply to the tumor and hinder its growth [4]. In retinal vascular diseases and arthritis [5], these treatments are used to alleviate the symptoms and inflammation that are caused by abnormal blood vessel formation. Researchers have made continuous efforts to develop novel antiangiogenic drugs and treatment methods to provide more effective treatment options.

This review focuses on the role of ECs and PCs in blood vessel formation, with an emphasis on the intercellular communication between ECs and PCs. This study highlights signaling pathways, such as the vascular endothelial growth factor/vascular endothelial growth factor receptor (VEGF/VEGFR), platelet-derived growth factor-B/platelet-derived growth factor receptor-β (PDGF-B/PDGFR-β), angiopoietin/tyrosine kinase with immunoglobulin-like and EGF-like domains 2 (Ang/Tie2), Notch signaling, transforming growth factor-β (TGF-β), and sphingosine-1-phosphate/endothelial differentiation gene receptor (S1P/EDG) pathways, and highlights how disruption of EC–PC communication plays a crucial role in diabetic microvascular disease, central nervous system disorders, cancers, and other diseases. This review also discusses the progress of current research on potential drugs that target these pathways. By revealing the mechanisms that disrupt the crosstalk between PCs and ECs during disease development and progression, this review aims to provide potential new directions for the development of future treatments.

The role of EC–PC interactions in vascular development

Endothelial cells

ECs are flat epithelial cells that line the inner walls of blood vessels. ECs make up the innermost, single-layered, flat epithelial cell structure in the heart and blood vessels (arteries, veins, and capillaries). ECs are referred to as the vascular endothelium, and they line the entire circulatory system from the heart to microvessels [6] (Fig. 1). The vascular endothelium was long considered an inert barrier that merely functions as a barrier between the blood and surrounding tissues; it was not until the late twentieth century that the vascular endothelium gradually became recognized as a multifunctional organ that is involved in the establishment of homeostasis in the body. ECs are important components of the vascular system because of their selective barrier function, their role in regulating vasomotion, their participation in modulating the inflammatory response, and their secretion of various constituent active substances [7]. Moreover, ECs also play a critical role in mediating immune responses at sites of injury and infection [6]. In addition, vascular ECs ensure the normal flow of blood through blood vessels by playing roles in the coagulation, anticoagulation and fibrinolytic systems [8].

Fig. 1.

Spatial structure of ECs and PCs on small terminal arteries. Taking terminal small arteries as an example, PCs have a stellate cellular structure, with a relatively prominent nucleus and a cytosol protruding in all directions (categorized as ring-shaped and longitudinal processes). PCs are located on the surface of the tunica intima of vessels wrapping around the ECs, which are connected by the basement membrane that is shared by the two cell types.

Pericytes

PCs are cells that are embedded in the basement membrane (BM) of blood vessels, and they are wrapped around the ECs of microvessels (capillaries, small postcapillary venules and terminal arterioles) [9]. However, there are several limitations to the definition of PCs that is currently generally accepted. First, the definition does not apply to PCs that are actively exhibiting angiogenic activity; although at this stage, PCs have characteristic cellular features and functions, the recruitment of PCs during angiogenesis is not yet complete, and the basement membrane between ECs and PCs has not yet formed. Thus, although PCs are already functional at this stage, they are limited by their spatial location in the vessel wall, which is outside the scope of this classical definition. Second, in recent years, PC-like cells have been observed in the subendothelium of large blood vessels, which also challenges the traditional definition of the site of PCs [10].

PCs that are present in different tissues and organs are heterogeneous, as reflected by their embryonic origin, morphology and molecular signatures [11]. PCs in different tissues have heterogeneous origins, with most PCs originating in the mesoderm and some originating in the ectoderm. In addition, heterogeneity in the origin of PCs from the same tissue has been described [12]. Furthermore, PCs are morphologically diverse; unlike ECs, PCs consist of a protruding nucleus and a small amount of swollen cytoplasm [13]. The morphology of PCs also correlates with their location in the microvasculature [14], [15], [16]. Moreover, the molecular markers of PCs are diverse, and since no single molecular marker can be used to identify PCs, a multimarker approach must be used to identify PCs. Some common molecular markers include α-smooth muscle actin (α-SMA), PDGFR-β, NG2, alkaline phosphatase (ALP), desmin, aminopeptidases A and N, RGS5, and CD146 (Fig. 2), but the expression of these markers is not limited to PCs [17], [18]. Notably, these key markers exhibit dynamic expression patterns in PCs from different organs, in PCs at different developmental stages, and in PCs under different disease conditions [19], [20], [21]. Therefore, in practical application, despite the use of a combination of multiple markers to distinguish PCs, it is possible that other cells may be labeled. That is, isolated PC populations might include other cells that surround capillaries, and this complication is a significant limitation in PC research. Therefore, a combination of methods, including methods that consider location, morphology, and multiple marker expression, with comprehensive criteria is needed to define or characterize PCs (Table 1).

Fig. 2.

Examples of PC identification using different markers. (A). The basal membrane (BM) of a brain capillary, which is indicated by collagen IV (Col-IV) staining in green, and the PCs surrounding the endothelium, which are indicated by PDGFRβ staining in red, were observed. Notably, the PC is located within the BM of the vessel, as evidenced by the yellow color in the merged image. The white arrow indicates a typical PC cell body, while the arrowheads indicate the PC processes that extend horizontally along the capillary. (B). This image shows brain ECs (CD105, red) and PCs (desmin, green) in 3D. Importantly, the PCs in this image appear different from those in (A) because a different marker was used for identification. In (B), desmin, which is an intracellular intermediate filament protein, was used to visualize clustered intermediate filaments, whereas PDGFRβ, which is a transmembrane cell surface protein, was used in (A) to visualize the contour of the PC membrane. (C). The endothelium (CD31, blue) and mural cells (NG2-green; αSMA-red) in the retina were analyzed using triple immunostaining. vSMCs found on the surface of the vein (v) expressed αSMA and, occasionally, NG2, while PCs surrounding the capillaries did not express αSMA but did express NG2. (D). The retinal vessels of a promoter trap transgenic mouse (XlacZ4) were examined using epifluorescence imaging. PC nuclei were identified through X-gal staining (appearing as dark blue), and PC bodies were visualized through NG2 staining (appearing as green). ECs were visualized by CD31 staining (red). Notably, all the X-gal-positive cells also exhibited positive staining for NG2. vSMCs covering the vein (v) are indicated by red arrowheads, while white arrowheads indicate PCs covering capillaries. (Scale bars indicate distances of 10 μm (A), 20 μm (B), and 50 μm (C and D)) [Reproduced with permission [31]. Copyright © 2011 Elsevier, Inc.]. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 1.

Tissue specific pericytes.

Subpopulations	Molecular markers
Brain pericytes	PDGFRβ+, αSMA+, NG2+, CD146+, CD13+, CD73+, CD140b+, CD105+, CD45-, CD90+/-, Desmin-
Liver pericytes	PDGFRβ+, αSMA+/-, NG2+, CD146+, Vimentin-, CD90-, CD140b+
Tubulointerstitial pericytes	PDGFRβ+, αSMA+/-, NG2+, Coll1a1+, CD73+, PDGFRα+, CD45-
Mesangial cells	PDGFRβ+, αSMA+/-, NG2+, Coll1a1+, CD73+, PDGFRα+, CD45-
Podocytes	Nephrin+, Podocin+, αactinin+, CD2AP+, Podocalyxin+, Synaptopodin+, WT1+, CD10+, GLEPP-1+
Lung pericytes	PDGFRβ+, αSMA+/-, NG2+, CD146+, Desmin+, Calponin+, CD90+, CD73+, CD31-, CD45-
Cardiac pericytes	PDGFβ+, αSMA+, NG2+, CD146+, CD34+, Vimentin+, PDGFα+

Pericytes are specific for different tissues and their surface markers vary. Classification of pericyte subpopulations: brain pericytes [191], [192], liver pericytes [193], tubulointerstitial pericytes [194], [195], mesangial cells [194], [195], podocytes [196], lung pericytes [197], [198], [199], cardiac pericytes [200], [201], [202].

ECs and PCs in specific blood vessels

In addition to their previously described characteristics and functions, ECs and PCs in certain blood vessels often perform other specialized functions. For example, the hepatic sinusoid is a cavity between adjacent liver plates and is a specialized type of capillary. The sinusoidal wall of the hepatic sinusoids consists of liver sinusoidal endothelial cells (LSECs), which form a large, permeable barrier that separates blood cells on one side from hepatocytes on the other side while allowing the exchange of certain substances. There is no single marker that specifically distinguishes LSECs from other ECs, but these cells regulate microvascular hepatic blood flow through interactions with hepatic stellate cells (HSCs) [22]. HSCs, which are located in the Disse lumen between LSECs and hepatocytes, have long been recognized as specialized PCs of the liver. LSEC dysregulation reportedly allows hepatic stellate cells to be regulated by aberrant TGF-β and is involved in the process of liver fibrosis [23].

Specialized subpopulations of PCs (mesangial cells) are located around glomerular arterioles where they stabilize the microvascular system and regulate blood flow; inhibition of mesangial cell recruitment can lead to excessive dilation of glomerular capillaries. In addition, mesangial cells produce renin to regulate blood vessels throughout the body [24]. Similar to stellate cells, mesangial cells are involved in the development of renal fibrosis. When activated by renal injury, mesangial cells can escape from peritubular capillaries into the interstitium and differentiate into matrix-secreting myofibroblasts [23].

High endothelial venules (HEVs) are specialized postcapillary veins that are found in lymph nodes and various lymphoid organs (excluding the spleen). Gowans confirmed that HEVs serve as sites of lymphocyte migration from the bloodstream to the lymph nodes [25]. The characteristic feature of HEV ECs (HECs) that distinguishes them from other vascular ECs is their expression of the 6-sulfo-sialyl Lewis X epitope, which is recognized by many HEV-specific antibodies [25]. More specifically, migrating lymphocytes have been observed in the abluminal compartment of the HEV wall where they are arranged in rows between HECs and the first PC layer [26].

A specialized capillary that couples angiogenic and osteogenic processes, namely, type-H vessels, was recently discovered and has received substantial attention because of its close association with bone development and regeneration. Although the proportion of type-H ECs in the overall skeletal cell population is low, cells such as Osterix osteoblasts and PDGFRβ PCs are selectively present around type-H vessels. Parathyroid hormone has been reported to remodel type-H vessels and the leptin receptor-positive PC network in mice [27]. However, although PCs perform stem cell-like functions and directly contact type H vascular ECs, whether they are directly involved in skeletal development, osteonecrosis of the femoral head and other metabolic skeletal disorders needs to be further studied.

The role of ECs and PCs in vasculogenesis and angiogenesis

Although the concept of angiogenesis was proposed more than 200 years ago [28], PCs were not discovered until nearly a century later [29]. Because of this fact and the limitations of various research tools and techniques, studies related to EC–PC interactions have progressed slowly. The study of ECs and PCs was further developed only in the early 1960s with the widespread use of in vitro culture models and corresponding techniques [28], [29], [30] (Fig. 3). The two major cellular components, namely, ECs and PCs, share the same basement membrane, but these cell types have structures that directly contact areas that are not covered by the basement membrane. Structures that form direct contacts between ECs and PCs include peg-and-socket structures, adhesion plaques and gap junctions. In addition, microparticles (MPs) and exosomes are transferred between ECs and PCs to transport materials and exchange information [31], [32].

Fig. 3.

Important events related to ECs and PCs in the field of angiogenesis research. This timeline shows the development of the concept of angiogenesis encompassing ECs and PCs, the rise of research on EC–PC signaling pathways, and the progress of the approval of related targeted drugs.

We focused on the paracrine signaling pathway between ECs and PCs, which plays a crucial role in vascular development under both physiological and pathological conditions. Various signaling pathways have been extensively studied but only within the last 40 years. The TGF-β signaling pathway was one of the first pathways to be discovered in studies on the EC–PC relationship, but this pathway did not receive much attention at the time because TGF-β signaling performs various functions in a variety of physiological environments. It was not until the 1970 s, when Folkman proposed treating tumors with antiangiogenic agents [4], that the search for other angiogenic factors and pathways began. The discovery of VEGF by Ferrara in 1989 provided a major impetus for the burgeoning of related research [4], [33]. Over time, we have come to understand the roles of several signaling pathways in mammalian embryonic development, especially in terms of angiogenesis and vasculogenesis. Through these factors, both ECs and PCs play crucial roles in regulating vascular development.

Vasculogenesis

Vasculogenesis is defined as the de novo formation of blood vessels from endothelial progenitor cells (i.e., angioblasts) [34]. Angioblasts that originate from the mesoderm differentiate into ECs during the early stages of vascular development, during which they aggregate to form the embryonic vascular labyrinth. This primordial structure serves as the foundation for the formation of the primary network of blood vessels that is located within the yolk sac [19], [35]. Vasculogenesis plays a crucial role in the development of the heart and the initial formation of the primitive vascular plexus in the embryo. This process is essential for the establishment of the embryonic vascular system, which facilitates the proper delivery of nutrients and oxygen to support the developing organism. The differentiated formation of ECs provides the material basis that is necessary for vasculogenesis to proceed, thus enabling the development and regeneration of the vascular system.

In many studies, PCs have been shown to be important for stabilizing and promoting vascular maturation, and during the vascular maturation stage, PCs regulate the development of the vascular wall by binding to ECs. However, the exact time at which PCs and their differentiation-initiating precursor cells become involved in early embryonic vascular development is still relatively understudied, and little is known about the involvement of PCs during the vasculogenesis stage in particular. A recent study indicated the emergence of a separate PC lineage during the period when vasculogenesis occurs. This lineage establishes a direct connection with the developing endothelium through Cx43, thereby playing a role in regulating vasculogenesis [36]. However, related topics still need further investigation.

Based on this foundation, subsequent modulation by various signals leads to arteriovenous differentiation. The initial stage of this process involves the rapid proliferation of ECs to form primitive vascular ducts, which subsequently differentiate into distinct arteries and veins. Studies have demonstrated that the transition from endothelial ducts to arteries depends on increased VEGF and Notch signaling. On the other hand, venous specification requires inhibition of VEGF and COUP transcription factor 2, which is also known as COUP-TFII or NR2F2 [37]. This process is also regulated by VEGF and Notch signaling [38], [39].

In conclusion, PCs play a crucial role throughout the entire process of vasculogenesis. Currently, the role of PCs in the vascular maturation steps of vasculogenesis has been well studied; however, there is a gap in the existing research on how PCs and their precursor cells participate in the initiation of embryonic vasculogenesis and development, and there is a need to explore how PCs and their precursor cells influence vascularization of embryos. How PCs and their precursor cells affect ECs to participate in embryonic vasculogenesis is a promising area for future exploration.

Angiogenesis

Once the vascular plexus is established, both embryonic and adult vascular growth and vascular remodeling depend on angiogenesis, which is the process by which new vessels sprout from existing vessels and extend into a previously avascular area. Angiogenesis involves a complex sequence of vasodilatation, basement membrane degradation, EC migration, perivascular cell recruitment, EC proliferation, and blood vessel formation.

In the preexisting vascular structure, ECs are connected to each other by molecules such as VE-calmodulin and claudins, forming the innermost layer of the original duct. These ECs are then encapsulated by PCs, and quiescent ECs and PCs produce a common basement membrane with multiple forms of direct physical contact between cells, increasing the tightness of the connection between the two cell types and simultaneously permitting the exchange of chemical and mechanical information. When the quiescent state of existing blood vessels is disrupted by hypoxia and inflammation, the cells that are in need of blood supply release relevant angiogenic signals, such as VEGF and Ang2, which contribute to the initiation of angiogenesis. This process can be roughly divided into three steps (Fig. 4), described as follows.

Fig. 4.

Dynamic processes by which ECs and PCs form new blood vessels during angiogenesis. (A). In response to angiogenesis-related factors, the stable vascular structure begins to disintegrate, PCs detach from the vessel wall, and the basement membrane degrades. Subsequently, a specific EC is selected for differentiation into a tip cell. (B). The tip cell is oriented to the EC by repulsive factors, and ECs near the tip cell proliferate in response to the corresponding factors. (C). EC tubes are formed while free PCs are recruited and basement membrane deposition is promoted.

PC separation and EC sprouting

Cells that require blood supply release relevant angiogenic signals that contribute to the initiation of angiogenesis. This process requires PCs to detach from the vessel wall via matrix metalloproteinase (MMP)-mediated protein hydrolysis in response to Ang2 and allows the release of inactive VEGF from the extracellular matrix (ECM) for activation. Immediately thereafter, ECs respond to VEGF by loosening their junctions to promote vasodilatation, thereby increasing the permeability of the EC layer; then, plasma proteins extravasate to form a temporary ECM that serves as a temporary scaffold for the subsequent proliferation and migration of ECs. To ensure orderly EC migration, selected ECs form tip cells, and this process is regulated by multiple signaling molecules, such as VEGFR-2, DLL4, JAGGED1, NRP1, integrins, HIF-1α, MT1-MMP, and PGC-1α. Tip cells promote the migration of newborn ECs [40]. The initiation of angiogenesis depends mainly on VEGF, which induces vascular germination at the sprouting site by binding to VEGFR-2, while VEGF/VEGFR-2-activated ECs express the Notch ligand DLL4 to prevent excessive angiogenesis in order to achieve orderly neovascularization development [40], [41].

Stalk elongation and tip guidance

In newborn vascular sprouts, tip cells play a role in leading the migration of newborn ECs; after sensing the guiding effect of ephrins and semaphorins, tip cells grow along temporary extracellular matrix scaffolds in the direction of the cells that need blood supply. The mode of growth is mainly related to the neighboring ECs around apical cells, which receive NOTCH, VEGFR-1, WMT, PIGF, WNT, and NRARP signals that promote division and proliferation. These newly proliferated cells form immature vascular lumens, also known as stems, in response to VE-cadherin, CD34, VEGF, etc., in a process known as stalk elongation. During this process, PCs detach from the vascular wall and influence the growth of stem ECs, and PCs affect stem ECs via the secretion of S1P and TGFβ [40].

PC recruitment and formation of stable blood vessels

To guide blood flow, neovascularization needs to progress through a process that involves the recruitment of PCs and vascular smooth muscle cells [42]. During angiogenesis, the recruitment of PCs plays a crucial role in maintaining normal blood vessel morphology. Previous studies have reported that the PDGFR-β-mediated paracrine pathway induces ECs to produce PDGF-B and that the production of PDGF-B promotes the migration of PDGFR-β-positive PCs to the EC population; this process is called PC recruitment [17]. Then, vascular remodeling occurs, and Ang1 and Ang2 regulate vascular maturation and stability by binding to the Tie-2 receptor on ECs. TGF-β and NOTCH signaling also participate in PC coverage of the vessel wall. Subsequently, connections between ECs and PCs are formed, and basement membrane deposition occurs to ensure the transportation of blood flow.

During this complex process, an extensive network of early primitive vessels expands as the vessels branch out and establish connections [43]. ECs and PCs play important roles in the complex molecular mechanisms that regulate angiogenesis. Thus, when any aspect of the communication between ECs and PCs is disrupted, abnormalities in vascular structure and function can occur, and it is reasonable to believe that these abnormalities play a role in the pathogenesis of abnormal vascular diseases, both hereditary and acquired. Since circulation occurs throughout the body in all systems, the effects of abnormal angiogenesis are not limited to the vascular system but are very likely to potentially contribute to potential pathological mechanisms underlying other nonvascular diseases.

Signaling pathways that regulate the interactions between ECs and PCs

As mentioned above, ECs and PCs play important roles in the process of blood vessel formation, and they depend on synergistic cooperation to participate in vascularization and remodeling; thus, their interactions are essential for maintaining normal vascular function, for regulating vascular tone, and for maintaining and repairing the vascular wall. Aberrant EC–PC signaling also plays an important role in diseases, such as retinal vascular diseases, neurodegenerative diseases, and tumors. Thus, summarizing EC–PC interactions will increase the in-depth understanding of these disease mechanisms and identify potential therapeutic approaches in this field.

Vascular endothelial growth factor/vascular endothelial growth factor receptor

VEGF is a key factor in the initiation of angiogenesis. VEGF establishes a favorable environment for the detachment of PCs from the existing vasculature and the proliferative migration of ECs via interactions with matrix metalloproteinases (MMPs), thereby promoting the degradation of the basement membrane between ECs and PCs. The VEGF family includes several proangiogenic factors, including VEGF-A (often referred to as VEGF), VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PIGF) [44]. VEGF-A plays a crucial role in governing both normal physiological and pathological processes. VEGFRs are transmembrane tyrosine kinase receptors that can be divided into three types: VEGFR1-3 [45]. VEGFR1 and VEGFR2 are expressed mainly in vascular ECs, and despite the high affinity of VEGFR1 for VEGF-A, intracellular tyrosine kinase activity is significantly decreased after VEGFR1 activation [46]. In contrast, VEGFR2 exhibits high intracellular tyrosine kinase activity upon activation, despite its relatively low affinity for VEGF-A; therefore, VEGFR2 is the most important signaling receptor for vasculogenesis and angiogenesis [47]. This suggests that VEGFR1 likely acts as a ligand trap to reduce VEGFR2 signaling and limit angiogenesis. VEGFR3 is expressed mainly in endothelial lymphocytes and is associated with lymphangiogenesis [47], [48]. The expression of VEGFR on primitive ECs enables these cells to respond to VEGF and regulates the mitosis and repulsion of primitive ECs, thus promoting vasculogenesis [49], [50]. It has been reported that VEGF-knockout mice do not have an organized vascular system during embryonic development, suggesting that VEGF plays a crucial role in vascular development [51], [52]. In addition, VEGF acts as a vascular permeability factor that promotes the extravasation of plasma proteins and provides a temporary functional environment for EC migration during vascular sprouting [40]. VEGF, which is expressed by perivascular cells, including PCs, also plays a key role in stable blood vessel formation [53], [54]. However, unlike soluble VEGF, which promotes EC migration by establishing a concentration gradient, VEGF that is expressed by PCs binds to the cell surface and acts only to promote EC survival and stabilize blood vessels without causing EC migration [54], [55]. This suggests that VEGF functions as a survival factor between ECs and PCs in resting vessels (Fig. 5).

Fig. 5.

Major signaling pathways between ECs and PCs. The signaling pathways involved in the interaction between ECs and PCs involve multiple mediating ligand-receptor complexes, including VEGF/VEGFR, PDGF-B/PDGFRβ, Ang1/Tie-2, TGFβ/TGFβR (which involves differences in the levels of Alk1 and Alk5), Jagged1/Notch, and S1P/S1P1. In contrast, peg–socket, adhesion plaque, and VE–cadherin interactions involve direct contact between PCs and ECs and between ECs and ECs.

Platelet-derived growth factor-B/platelet-derived growth factor receptor-β

Neovessels are extremely unstable, and to perform the function of transporting blood, a stable vascular system needs to be formed, which involves PC recruitment. PDGF-B, which is secreted by ECs, is the key factor in this PC recruitment step. The PDGF-B and PDGFR-β signaling pathways play crucial roles in facilitating PC recruitment throughout the process of neovascularization [56], [57].

PDGFR-β is expressed on PCs [56], and PDGF is mainly released by ECs. Although PDGF-B has been shown to be released by neurons and hematopoietic cells, knocking down PDGF-B in these cell types does not have a substantial effect on the vasculature [58], [59], [60]. However, knockdown of PDGF in ECs or knockdown of PDGF-β in PCs leads to early embryonic death in mice. This result occurs because these mice are broadly deficient in PCs and vascular smooth muscle cells that support the vascular system, indicating the importance of PCs in angiogenesis. This study also highlights the practical significance of the PDGF-B/PDGFR-β signaling pathway in EC–PC communication. PDGF-B, which is secreted by ECs, recruits PCs to participate in angiogenesis by binding to PDGF-β on PCs. Activation of this pathway allows perivascular cells, including PCs, to proliferate and migrate toward the new EC population [61], [62], [63], [64], [65]. PDGF-B production by ECs affects PC development, and mice with null alleles of PDGF-B show reduced numbers of PCs [66]. PDGF-B expression is also spatially specific and occurs only at sites of active angiogenesis [62], [67], and the spatially specific localization of PDGF-B may contribute to the migration of PCs along the microvasculature. Loss of PDGF-B/PDGFR-β signaling pathway activity, in addition to affecting PC recruitment, is accompanied by other secondary consequences, such as EC proliferation, abnormal junctions and compensatory upregulation of VEGF-A [67] (Table 2).

Table 2.

Major roles of VEGF, PDGF, Ang, TGF-β,Notch, S1P signaling pathways in angiogenesis.

Molecule	Effect on PCs recruitment	Functions
VEGF	↓	VEGF interacts with MMPs to promote the degradation of the basement membrane between EC and PC, while also directly stimulating the proliferation and migration of PC in hypoxic environments [203]. VEGF derived from PC supports EC survival and functions as a stabilizing factor for EC. In VEGF knockout mice, embryonic development occurs without the formation of an organized vascular system.
PDGF	↑	Activation of the PDGF receptor triggers activation of the Ras/Rho/Rac, PKC, FAK and ERK pathways. This activation leads to cell proliferation, migration, survival, and expression of vascular endothelial growth factor. Activation of the PDGF receptor assumes an important role in the recruitment of PCs. PDGF knockdown results in reduced PCs recruitment, microaneurysms, and embryonic death.
Ang1	↑	Ang1 plays a role in vascular stabilization, actin remodeling, and cell migration. Loss of Ang1 function results in defective angiogenesis and decreased PC coverage, leading to embryonic death from cardiovascular failure.
Ang2	↓	On the other hand, Ang2 is associated with neovascular sprouting in tumors, and its expression is usually inversely proportional to PC coverage. In contrast to the role of Ang1, in the absence of Ang2 function, blood vessels continue to develop normally but with lymphoid defects.
TGFβ	↓	TGFβ is ubiquitous and promotes ECM protein expression and flow. Lack of it results in disturbed pericyte-neovascular interactions, inadequate coverage, and disruption of the vascular network.
Alk1	↑	Alk1 induces PC proliferation and recruitment. Lack of Alk1 results in embryonic death and defective vascularization.
Alk5	↓	Alk5 induces the expression of PC contractile proteins, promotes ECM generation, and stabilizes nascent capillaries, shifting from inducing the proliferation of ECs and PCs to differentiation in contrast to Alk1. Its defects lead to defective vascular development and defective angiogenesis, resulting in embryonic death.
Notch	↑	Notch receptors on ECs inhibit EC proliferation and promote EC maturation by binding to JAG1 on PCs and downregulating VEGFR2 and upregulating VEGFR1. Activation of Notch receptors on PCs upregulates PDGFR-β expression and increases the recruitment process of PCs to the ECs, leading to the formation of stable and mature vascular structures.
S1P/EDG-1	↑	This pathway regulates the cellular response to PGF and enhances ECM production. Lack of effect of this pathway results in vascular defects and discontinuous vascular coverage.

Angiopoietin/tyrosine kinase with immunoglobulin-like and EGF-like domains 2

The Ang/Tie2 pathway is one pathway that plays an essential role in EC and PC communication. The Ang1/Tie2 pathway and the Ang2/Tie2 pathway are two key pathways within the Ang/Tie2 system. The Ang1/Ang2 balance regulates vascular stabilization and remodeling. Ang1 is expressed by PCs and exerts regulatory effects on ECs by interacting with Tie2 receptors, which are normally considered to be specifically expressed by ECs; thus, Ang1 is clearly a paracrine signal of PCs that originally targets ECs. In contrast, Ang2 is predominantly expressed by ECs, and the Ang1/Tie2 and Ang2/Tie2 pathways perform mutually exclusive functions in vascular regulation; i.e., Ang1 is antagonized by Ang2 [68], [69]. Ang1 mediates capillary PC coverage and regulates adhesion junctions with ECs, whereas Ang2 mediates PC loss and vascular destabilization and is a key factor in PC detachment from the vasculature during the vascular sprouting phase [31], [40].

Studies of Tie2-knockout mice showed that the early steps of vasculogenesis and development could still proceed without Tie2. However, these mice failed to develop to the subsequent stages of the primary capillary plexus, they formed significantly poorer tissues that lacked PCs and vascular smooth muscle cell support, and they presented an early embryonic lethal phenotype. This suggests that Tie2 deletion is incompatible with embryonic vascular development in mice [70], [71], [72]. These findings also suggest that Tie2 receptors play important roles in the formation and maintenance of blood vessel stability. For example, Ang1-deficient mice exhibit defective vascular structures that are similar to those of Tie2-deficient mice, demonstrating the critical importance of Ang1 in the vascular development of early embryos [73]. On the other hand, Ang2 acts as an antagonist of Ang1 by binding to Tie2 but not activating its signaling pathway, thereby reducing the level of Tie2 activation and leading to vascular EC depolymerization and vascular instability [69]. Notably, vascular development was not dramatically affected in Ang2-deficient mice, and early embryos developed into newborn mouse pups; however, persistent vascular defects were still observed, indicating that Ang2 is essential for normal embryonic development [74], [75] (Table 2).

Notch signaling

Unlike those of other pathways, Notch receptors and ligands are membrane proteins that mediate signaling between neighboring cells that are in direct contact, thereby regulating cell differentiation, proliferation and apoptosis [76]. In mammals, including humans, Notch receptors are divided into four types, Notch1-4 [77], [78], and five classes of Notch signaling ligands, namely, Jagged1, Jagged2, Delta1, Delta3 and Delta4, have been identified in humans and mice [77], [79], [80]. Binding of a Notch ligand to its receptor initiates Notch signaling activation. Subsequently, the Notch receptor undergoes two consecutive protein hydrolysis events and then translocates to the nucleus. This translocation leads to the activation of target gene transcription, ultimately promoting various biological functions [81]. In addition, the expression of Notch receptors and ligands differs significantly between ECs and PCs. ECs express all four types of Notch receptors as well as the ligands DLL-4, JAG-1, and JAG-2, whereas PCs express only JAG-1 and Notch1-3 [82].

All of these ligands have unique regulatory properties [80]. Notch receptors on ECs reduce EC proliferation and promote EC maturation by binding to JAG1 on PCs, thus downregulating VEGFR2 and upregulating VEGFR1 [83], [84], [85], [86], [87]. In contrast, activation of Notch receptors on PCs upregulates PDGFR-β expression and increases the recruitment of EC populations, leading to the formation of stable and mature vascular structures [86], [88], [89]. Furthermore, Notch signaling is critical for establishing arteriovenous identity in the early vascular system. Activation of Notch signaling in ECs induces the production of arterial markers, while inhibition of Notch signaling in ECs has been shown to promote the production of venous markers [90], [91], [92]. (Fig. 5).

Transforming growth factor-β

The TGF-β family is a class of signaling molecules that are highly conserved in the animal kingdom, and this family includes more than 30 different molecules, including TGF-β (β1/β2/β3), BMP, GDF, activin, nodal, inhibin, and AMH/MIS [93], [94]. Of these molecules, TGF-β1 has anti-inflammatory and profibrotic activities [95]. ECs and PCs synthesize latent TGF-β, which is maintained in an inactive state in the ECM by specific TGF-β-binding proteins; then, TGF-β is activated in response to direct contact between ECs and PCs [96], [97], [98]. Errors in the conserved TGF-β signaling pathway can occur, including inactivation of TGF-β and genes encoding its receptor, genes encoding activin receptor-like kinases 1 and 5 (Alk1 and Alk5), and the downstream effector Smad2/3/5. These errors can lead to similar cardiovascular defects during embryonic angiogenesis [99], [100], [101]. The function of TGF-β is directly influenced by its local concentration and the expression of its receptors on target cells [102], [103]. Alk-1 and Alk-5 are the two most important receptors for TGF-β that are involved in angiogenesis, and these two receptors exert opposite effects [76]. Alk-1 is mainly expressed on ECs, is preferentially activated in the presence of lower concentrations of TGF-β and promotes EC proliferation through the action of Alk1/Smad1/5 to promote angiogenesis [96], [100], [104]. Alk-5 is expressed on both ECs and perivascular cells, and there is evidence that active TGF-β affects PC and EC differentiation via the Alk-5/Smad2-3 pathway [96]. Alk-5 activation promotes the differentiation of perivascular cells, including PCs [105]. Alk-5 promotes the shift from EC proliferation toward EC differentiation by downregulating VEGFR2 and upregulating VEGFR1 [104], [106]. (Fig. 5).

Sphingosine-1-phosphate/endothelial differentiation gene receptors

S1P is a polar lipid mediator that is generated by the metabolism of sphingolipids [107]. S1P signaling reportedly plays a crucial role in angiogenesis development [108]. S1P is categorized into five types called S1P1-5; these proteins are secreted by PCs and act by binding to the S1P receptor, which functions as a G protein-coupled receptor (GPCR) [109]. Activation of the endothelial differentiation gene-1 (EDG-1) receptor (also known as S1P1) on vascular ECs regulates angiogenesis by modulating cell–cell contacts and cell-matrix adhesion. S1P/EDG signaling activates N-calmodulin-dependent junctions, which are important for the establishment of stable and strong “peg-and-socket” structures that contain N-calmodulin and form direct contacts between ECs and PCs [110], [111]. A lack of S1P leads to a lack of PC-vascular interaction, which subsequently causes vasodilation and hemorrhage [112]. Activation of S1P1 leads to the formation of VE-calmodulin-containing junctions, enhancing tight cellular junctions and vascular stability [113], [114]. Endothelial-specific knockdown of N-calmodulin significantly reduces the expression of VE-calmodulin, resulting in defective endothelial–endothelial junctions that cause vascular system disorders [115]. Notably, the tissue and organ locations where S1P1 is expressed differ during different stages of development. S1P1 is particularly important for normal development of the vascular system and is highly expressed during embryonic development; additionally, in adult tissues, S1P1 is expressed in a variety of tissues and organs, including the brain, lung, liver, heart, and spleen, and it is an important receptor on the surface of lymphocytes [116]. (Fig. 5).

Understanding the interactions between ECs and PCs is very important in the present, and it will continue to be important in the future. First, a deeper understanding of these interactions will provide insights into the mechanisms underlying disease development. Second, understanding the signaling pathways that link ECs and PCs will guide drug development and treatment approaches. These known signaling pathways are involved in only some of the interactions between ECs and PCs, and future research will further explore these pathways to discover new signaling molecules and mechanisms as well as reveal additional details about the cellular interactions that are involved. Additionally, new detection and intervention methods can be developed using techniques such as single-cell sequencing and high-resolution imaging to further enhance our understanding of the interactions between ECs and PCs. These methods can lead to the development of more targeted approaches for disease prevention and treatment as well as personalized medicine. Overall, in-depth research on these interactions will greatly benefit human health and guide further advancements in medical research and practice.

EC–PC crosstalk in inherited and acquired diseases

Retinal vascular diseases

The loss of PCs and ECs due to the dysregulation of EC–PC communication is observed in many retinal vascular diseases, such as retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO) [117]. Among these diseases, DR is one of the most widely studied and clinically important diseases (Fig. 6). DR can be subdivided into nonproliferative DR (NPDR) and proliferative DR (PDR) depending on the development and severity of the disease, and the main difference between the two types of disease is the presence or absence of neovascularization [118].

Fig. 6.

Progression of diabetic nonproliferative retinopathy. Progression of early-stage nonproliferative retinopathy to proliferative retinopathy. Elevated glucose levels cause apoptotic cell death of pericytes. This leads to more permeable blood vessels and subsequent endothelial cells, which further increase vessel leakage. Loss of pericytes and endothelial cells increases fluid leakage as well as immune cell infiltration in the retina. These intraocular vascular changes subsequently contribute to the development of proliferative retinopathy. [Reproduced under terms of the CC-BY license [209]. Copyright © 2016 The Authors. Published by Elsevier Inc.].

DR is the leading cause of vision loss in patients with diabetes [119]. The earliest cellular defect that has been identified in DR is the loss of PCs in the retinal microvasculature; indeed, this is one of the hallmarks of DR [17]. The loss of PCs is associated with disruptions in the crosstalk between retinal vascular ECs and PCs. Chronic hyperglycemia causes decreases in the expression of growth factors by ECs, including the decreased expression of PDGF-B [120]. Research on the inhibition of pathways that include PDGF and its receptor PDGFR-β has revealed that defects in PCs can independently initiate retinopathic pathology, even in the absence of hyperglycemia [121]. Recent reports have indicated that the concurrent inhibition of VEGF and PDGF, particularly PDGF-B, can significantly enhance the inhibition of neovascularization and effectively decelerate the progression of diseases [122]. The Ang/Tie2 signaling pathway is another key signaling pathway that leads to the loss of PCs in DR [123]. Ang-2 is an important regulator of angiogenesis, and its overexpression may contribute to the development of DR. It has been reported that endogenous Ang-2 is strongly upregulated in the retinas of diabetic rats [124]. The VEGF and Notch signaling pathways are also involved in the disease progression of DR. VEGF is thought to be associated with pathological retinal neovascularization and to increase vascular permeability [122]. Studies have shown that VEGF levels in the vitreous may serve as a potential indicator of the severity of DR and that there is a significant correlation between an increase in VEGF levels and the severity of DR [125], [126]. Furthermore, exposure to high glucose levels activates the Notch signaling pathway. This activation occurs through the binding of the Notch1 ligands DLL1 and DLL4 to Notch1 receptors [127]. Consequently, EC dissociation occurs in patients with diabetes. Additionally, abnormal Notch signaling engages in crosstalk with VEGF signaling in the retina, further contributing to EC dysfunction [128]. Both NPDR and PDR may be associated with diabetic macular edema (DME) [118]. The macular thickening that occurs in this disease mainly occurs due to increased extracellular fluid that is produced by the hyperpermeability of the retinal gross vessels, and it is also associated with the loss of PCs and the breakdown of EC–PC junctions. In addition, capillary ECs along with PCs undergo apoptosis, leading to the formation of cell-free capillaries, which is a common lesion in advanced DR. Similar to the pathogenic effects of abnormal EC–PC crosstalk in DR, the formation of cell-free capillaries is one of the pathogenic mechanisms underlying DME, as confirmed by the fact that anti-VEGF therapy has significant therapeutic effects in clinical treatment protocols [129].

Tumors

The tumor microenvironment (TME) refers to the internal and external environment within tissues where tumors are located during growth and metastasis. The TME includes vascular ECs, PCs, immune cells, and matrix components, which play important roles in regulating tumor cell growth, metastasis, tumor angiogenesis, and the development of drug resistance [130]. Researchers have shown that it is possible to regulate tumor progression and outcomes by affecting the tumor microenvironment. Additionally, elucidating the role of crosstalk between ECs and PCs in tumor progression and developing therapeutic modalities that target these cell types will be clinically valuable (Fig. 7).

Fig. 7.

Altered interactions between pericytes and endothelial cells affect tumor angiogenesis. Multiple signaling pathways play important roles in the local angiogenic signaling pathway in tumors. Endothelial cells secrete TGFβ1/2 to initiate pericyte recruitment, and PDGF-BB/DD and ET-1 bind to the corresponding receptors on free pericytes. Angiopoietin-Tie2 regulates tumor angiogenesis and tumor vascular maturation, and ANGPT1/2 competitively bind to the TIE2 receptor; the former inhibits endothelial cell proliferation to stabilize neovascularization, and the latter produces competitive inhibition. NG2 on the surface of pericytes establishes and maintains contact between recruited pericytes and endothelial cells in a β1 integrin-dependent manner and by promoting VI collagen anchoring and IV collagen deposition; thus, it participates in the recruitment of pericytes and vessel maturation during angiogenesis. [Reproduced under terms of the CC-BY license [210]. Copyright © 2023 The Authors. Published by Elsevier B.V.].

It has been reported that in the TME, PCs deregulate ECs and vascular basement membranes, detach from vessel walls, and promote EC sprouting and angiogenesis, and the PCs themselves undergo phenotypic changes [131]. However, unlike neovessels that are generated by normal angiogenesis, these vessels tend to exhibit features of immaturity and high permeability, which are closely associated with abnormal crosstalk between ECs and PCs. The PDGF-B/PDGFR-β signaling pathway plays a critical role in mediating communication between ECs and PCs and thus regulates the recruitment of PCs during angiogenesis. However, within the TME, PDGF-B, which is produced by tumors, promotes the migration of PCs within disorganized tumor vessels by stimulating ECs to secrete SDF-1α [132]. In addition, elevated concentrations of prostaglandin E2 (PGE2) in the TME downregulate N-calmodulin signaling between ECs and PCs. The downregulation of N-calmodulin signaling between ECs and PCs by the EP-4 and EP-1 pathways results in the destabilization of the EC–PC “peg-and-socket” structure, which leads to disruption of the EC–PC cellular connection [133]. In the TME, the function of the Ang/Tie2 pathway is also altered. Under normal conditions, PCs release Ang1, which binds to Tie2 receptors on ECs. This interaction triggers signaling that hinders the EC proliferation and promotes stable angiogenesis. However, in the TME, the inhibitory effect of Ang1 is significantly limited due to increased competitive inhibition by Ang2. Consequently, increased Ang2 leads to tumor blood vessel immaturity and disruption of microcirculation regulation [134], [135], [136].

Taken together, these findings suggest that a set of immature vascular systems with obvious PC deficiency and high permeability forms under conditions of abnormally regulated EC–PC crosstalk. In conclusion, defects in EC–PC interactions during tumor angiogenesis are major factors that lead to dysfunction of the tumor vascular system and hypoxic conditions in the TME; thus, these defects provide a suitable environment for the invasion and metastasis of cancer cells.

Nervous system diseases

The neurovascular unit is composed of brain microvascular ECs (BMECs), PCs, vascular smooth muscle cells, astrocytes, microglia, and neurons [137], [138]. This structure is a structural barrier that is used by the vascular system to control the entry and exit of ions, macromolecules, and cells, and it is called the blood–brain barrier (BBB) [139]. PCs are localized in brain structures, where they influence the regulation of capillary blood flow and BBB permeability via crosstalk with ECs, glial cells, and neurons. This helps to maintain the homeostatic function of the nervous system. Abnormal signaling between ECs and PCs that results in abnormal PC degeneration is also involved in the development of many neurological disorders.

Alzheimer's disease (AD) is a complex neurodegenerative disorder that affects cognitive functions, such as memory, thinking and behavior. There is a widespread consensus among scholars that AD is associated with dysfunctions of the cerebral microvasculature (Fig. 8). Disruption of the BBB and neurovascular unit (NVU) is believed to play a significant role in this context. The pathological hallmarks of AD are amyloid-β (Aβ) deposition and neurofibrillary tangle (NFT) formation (caused by tau hyperphosphorylation) [140]. The loss of PCs in the NVU during AD has also been hypothesized to be related to the pathogenesis of AD and the accumulation of pathological substances that are associated with AD. Previous research on the brains of AD patients and animal models has revealed accelerated degeneration of intracranial microvascular cells, which are PCs [141]. These cells are responsible for regulating capillary blood flow and affecting BBB permeability, which in turn affects the accumulation of Aβ in the brain [142], [143]. A study of human Aβ and PDGFRβ^+/- overexpression in a transgenic mouse line by Sagare et al. showed that PDGFRβ^+/- mice exhibit accelerated age-dependent loss of PCs due to defects in PDGFRβ in PCs. This disrupts the normal function of the PDGF-B/PDGFR-β pathway. Additionally, the mice exhibited robust Aβ levels and tau pathology, suggesting that disruption of the EC–PC PDGF-B/PDGFR-β pathway leads to PC deficiency. This in turn results in reduced clearance of Aβ and the development of severe tau pathology [144]. In recent correlative studies, it was discovered that the retina, which is considered the most effective area for the noninvasive detection of AD outside the brain, is also associated with vascular deposition of Aβ40 and Aβ42. This association was also observed in patients with early and progressive retinal vascular PDGFRβ deficiency and PC loss [145]. However, the underlying mechanisms are still unclear.

Fig. 8.

Components of the neurovascular unit (NVU) and changes that occur in AD. The components of the NVU play an important role in maintaining the blood–brain barrier (BBB) under healthy conditions. In dementia, changes, particularly in endothelial cells and pericytes, lead to loss of function and BBB breakdown. Subsequently, the surrounding astrocytes, neurons, and oligodendrocytes are damaged, contributing to pathological findings. [Reproduced with permission [211]. Copyright © 2011 Elsevier Inc.].

Stroke is a neurological deficit caused by acute focal damage to the central nervous system that occurs due to an interruption or insufficiency of the blood supply to the nerve center as a result of vascular disease; stroke is divided into two types: ischemic stroke and hemorrhagic stroke [146]. There is evidence that PCs are extensively involved in cerebral hemorrhagic disease and are involved in the pathogenesis of hemorrhagic stroke [147]. According to one study, sustained high levels of estrogen (E2) can contribute to maintaining normal PDGF crosstalk between ECs and PCs, which controls microvascular stability and reduces the incidence of hemorrhagic strokes and cerebral aneurysms [148]. PCs are also involved in ischemic stroke, which is the development of ischemic tissue in the brain followed by infarction due to insufficient blood supply. In the case of ischemic stroke, the ischemic tissue sends signals that induce the rapid expression of VEGF in the brain to initiate angiogenesis [149]. In contrast, ECs coordinately express PDGF-B to promote PDGFR-β activation in PCs around infarct foci, enhancing PC survival [150].

Other diseases

In addition to the diseases discussed above, there are other rarer diseases that are associated with disrupted crosstalk between ECs and PCs. Hereditary hemorrhagic telangiectasia (HHT) is caused mainly by mutations in the ENG receptor and the Alk-1 receptor on ECs. These mutations block TGF-β signaling and decrease the expression of N-calmodulin in ECs. As a result, junctions between ECs and perivascular cells are impaired. The disease is characterized by dilated capillaries in multiple mucous membranes and skin, as well as abnormal blood vessel formation [151], [152], [153]. Idiopathic basal ganglia calcification (IBGC), on the other hand, is associated with a mutation that impacts the function of PDGF-B signaling between ECs and PCs. This mutation leads to a reduction in the efficacy of the PDGF-B/PDGFR-β pathway. Consequently, the recruitment of perivascular cells in the brain and the appearance of abnormal calcified deposits are decreased. These deposits occur in the region of the basal ganglia in the brain [154], [155]. Genetic mutations in the TIE2 receptor gene TEK can cause hereditary cutaneomucosal venous malformation. This familial disease causes increased tyrosine kinase receptor activity in vascular ECs, leading to a phenotype of venous vascular walls with variable thickness [156].

In addition to these diseases for which research supports an association with disruptions in EC-PC communication, additional diseases that are related to disruptions in signaling pathways between ECs and PCs may be identified in the future, since the interactions between these cells play key roles in many physiological and pathological processes. For example, disruptions in signaling pathways between ECs and PCs may affect rejection after organ transplantation. In skeletal diseases, dysregulation of signaling between ECs and PCs in H-vessels, which has been shown to be closely related to bone development, may be associated with the development of femoral head necrosis and osteoporosis. However, this needs further confirmation by future studies.

Current therapeutic strategies and potentially relevant drugs that target ECs and PCs

There are two main therapeutic strategies for targeting ECs and PCs: therapeutic approaches that target the cells themselves and therapeutic approaches that modulate signaling pathways between the cells. Therapeutic approaches that target ECs are diverse but fall into two major groups: approaches that modulate EC function and approaches that promote EC repair. In pharmacotherapy, many antiplatelet drugs, antihypertensive drugs, and hypolipidemic drugs functionally regulate ECs and blood vessels by modulating certain mediators that are secreted by ECs (e.g., NO) [157]. Among physical therapies, EC phototherapy can regulate EC metabolism and stimulate EC growth and repair through the administration of specific wavelengths of light [158]. Other treatment modalities include gene therapy [159] and stem cell transplantation therapy [160]. On the other hand, therapies that target PCs are being developed more slowly and involve mainly targeting markers on the membrane of PC cells to overexpress these factors and enhance the efficacy of anticancer drugs [161]. However, these cell-based therapies are still limited in their ability to regulate the functional status of blood vessels, and they have little effect on the regulation of abnormal angiogenesis to inhibit pathological angiogenesis. Current and future antiangiogenic therapies should focus on the signaling pathway between ECs and PCs.

EC–PC signaling pathways are very important for angiogenesis; however, its role in disease development also provides a potential direction for disease treatment. Therefore, targeting these signaling pathways to treat diseases has definite value for the overall development of disease treatment strategies. To date, progress has been made in the study of drugs that regulate ECs and PCs by targeting these pathways; however, relatively well-studied drugs with better efficacy are mainly found in the fields of tumor therapy and eye disease treatment, and no safe and effective drugs that target these pathways with clinical significance have emerged in studies of drugs for other diseases. Research on drugs that target different pathways has also been uneven, with research on VEGF- and PDGF-targeting drugs being relatively mature, while research on drugs that target other pathways is still in its infancy or even nonexistent. Angiogenesis is essential for tumor growth and the pathogenesis of vasoproliferative ocular diseases, and antiangiogenic therapies have been available in the clinic and providing survival benefits to patients for nearly 20 years. Although hundreds of antiangiogenic drugs (AADs) have been developed, few of them are used in the clinic due to safety and efficacy limitations. The classical drugs in this category are mainly divided into two categories: antiangiogenic monoclonal antibodies (AA-MAs) and antiangiogenic tyrosine kinase inhibitors (AA-TKIs).

Anti-angiogenic monoclonal antibodies

AA-MAs are currently approved for clinical use in several countries, and three major drugs that target the VEGF–VEGFR pathway are in use in regions worldwide. Bevacizumab (BV), which was the first angiogenesis inhibitor to be approved for clinical use, is a humanized monoclonal antibody against VEGF that interferes with cancer angiogenesis by recognizing and blocking VEGF-A [162]. BV was originally approved for use in combination with chemotherapeutic agents for the treatment of metastatic colorectal cancer, and it is now used to treat a variety of cancers, including glioblastoma, renal cell carcinoma, ovarian cancer, cervical cancer, and non-small cell lung cancer [163]. In vivo studies have shown that BV can inhibit EC proliferation and migration by inhibiting VEGF pathway activation, triggering the regression of newly formed blood vessels, and facilitating the transport of chemotherapeutic agents by increasing perivascular cell coverage of tumors to normalize the vasculature [163], [164], [165]. BV in combination with chemotherapy has achieved significant results in antivascular therapy for various types of cancer; however, in recent years, it has gained widespread attention for its supraclinical applications in many ocular diseases. The use of BV for increasing PC coverage of PDR and DME and preventing the loosening of junctions between ECs has also been reported [166]. Aflibercept was approved by the U.S. Food and Drug Administration (FDA) in 2011 for the treatment of metastatic colorectal cancer (mCRC) and has subsequently been progressively applied to the treatment of other types of angiogenesis-associated tumors; this drug primarily targets VEGFA/B [167]. Similar to bevacizumab, the use of aflibercept in ophthalmic diseases has gained increasing acceptance in recent years. A recent study showed that aflibercept ameliorated peripapillary cell loss and increased the expression of endothelium-associated adhesions in mice with streptozotocin-induced diabetes. This drug led to the restoration of blood perfusion, suggesting that this anti-VEGF strategy modulates the crosstalk between ECs and PCs, playing an important role in restoring normal vascular function [168]. Ramucirumab, which targets VEGFR-2 [167], is also used in the clinic for treating cancer and related vasovagal ophthalmopathies, and it can be hypothesized that it not only acts as an antiangiogenic agent but also plays a role in the normalization of the vascular system by increasing PC coverage. However, there is a lack of experimental data to support this supposition.

Research on the inhibition of PDGF signaling by monoclonal antibodies has been slow, and research on antibodies that can block PDGF and PDGFR is still in the preclinical stage; no mature products are ready for clinical use. However, in terms of AA-TKIs, the use of PDGF signaling pathway-related drugs as well as VEGF signaling pathway-related drugs is growing.

Antiangiogenic tyrosine kinase inhibitors

Protein tyrosine kinases (PTKs) are enzymes that catalyze the phosphorylation of tyrosine residues in proteins and are among the major classes of signaling proteins that are involved in cellular signaling processes; PTKs are involved in the regulation of cell proliferation, differentiation, apoptosis, evolution, and metabolism, among other biological processes [169]. Among the major pathways discussed above, PDGF/PDGFR and VEGF/VEGFR signal through downstream effector tyrosine kinases; thus, regulation of signaling between ECs and PCs can be achieved by targeting the tyrosine kinase action sites of these pathways.

Unlike the anti-vascular regimen of AA-MAs combined with chemotherapy, AA-TKIs are usually applied alone to treat cancer. Below, we discuss several common AA-TKI formulations that are entering clinical use worldwide, as shown in Table 3.

Table 3.

Several current AA-TKIs approved for clinical use.

AA-TKIs	Targets	Mechanism of action	MVD/ PC coverage	Chemical Structure	Indications
Apatinib	VEGFR2	1. Raf/MEK/Erk pathway↓, the proliferation of ECs↓ 2. p38-MAPK pathway↓, the migration of ECs↓ 3. PI3K/AKT/mTOR pathway↓, the survival of ECs and vascular permeability↓ [204]	MVD↓ PC↑ [173]		1. STAD 2. GEJAC
Imatinib	BCR-Abl, PDGFR, c-Kit	1. PI3K-AKT-PKB/PKC pathway↓, cell proliferation↑, apoptosis↓ 2. RAS-RAF-MEK-ERK pathway↓, cell proliferation, differentiation, migratory, angiogenesis↓ [205]	MVD↓ PC↓ [170]		1CML 2Ph + ALL 3GIST
Axitinib	VEGFR1/2/3, PDGFRβ, c-Kit	1. PI3K-AKT-PKB/PKC pathway↓, cell proliferation↑, apoptosis↓ 2. RAS/PKC-RAF-MEK-ERK pathway↓, differentiation, cell proliferation, migratory, angiogenesis↓ 3. JAK-STAT ↓, cell proliferation and cell division↓ [206]	MVD↓↓ PC↓ [180]		1. RCC
Sunitinib	VEGFR2, PDGFRβ		MVD↓ PC↑ [171]		1. GIST 2. PAC 3. RCC
Sorafenib	VEGFR2, PDGFRβ, c-Kit		MVD↓ PC↑ [170]		1. HCC 2. RCC 3. TC
Lenvatinib	VEGFR1/2/3, PDGFRα/β, FGFR1	1. PI3K-AKT-PKB/PKC pathway↓, apoptosis↓, cell proliferation↑ 2. RAS/PKC-RAF-MEK-ERK pathway↓, differentiation, cell proliferation, migratory, angiogenesis↓ 3. PLCγ-IP3-Ca2 + pathway↓, regulation of vascular tone, cell proliferation, migration↓ 4. JAK-STAT ↓, cell proliferation and cell division↓ [207]	MVD↓ PC↑ [175]		1. EMC 2. HCC 3. RCC 4. TC
Nintedanib	VEGFR1/2/3, FGFR1/2/3, PDGFRα/β		MVD↓ PC↓ [177]		1. NSCLC
Pazopanib	VEGFR1/2/3, PDGFR, FGFR, c-Kit		MVD↓ PC↓ [178]		1. RCC 2. STS
Regorafenib	VEGFR1/2/3, PDGFRβ, FGFR, c-Kit		MVD↓↓ PC↓ [180]		1. mCRC 2. HCC 3. GIST

As of December 2023, there are a total of 77 TKIs that are FDA-approved or have entered clinical trials, of which the above nine drugs are supported by reports of relevant studies with therapeutic potential to target the EC–PC pathways. Abbreviation: microvessel density (MVD), stomach adenocarcinoma (STAD), gastroesophageal junction adenocarcinoma (GEJAC), chronic myeloid leukemia (CML), Ph+ acute lymphoblastic leukemia (Ph + ALL), malignant gastrointestinal stromal tumors (GIST), pancreatic cancer (PAC), hepatocellular carcinoma (HCC), renal cell carcinoma: (RCC), thyroid cancer (TC), endometrial carcinoma (EMC), non-small cell lung cancer (NSCLC), soft tissue sarcoma (STS), metastatic colorectal cancer (mCRC). [Images are from Drugbank [208], reprinted with permission. Copyright © 2023 OMx Personal Health Analytics, Inc.].

Alterations in PC coverage in tumor vessels after treatment with AA-TKIs have been observed in different clinical settings. Sorafenib is one of the most commonly used AA-TKIs, and imatinib is the first targeted drug to be approved for the treatment of cancer. Related studies have shown that both the density of ECs and the extent of PC coverage around the vascular system affect the ability of ECs to regulate tumor angiogenesis [170]. Another study demonstrated that treatment of renal cell carcinoma (RCC) with sunitinib, which targets the VEGF pathway, resulted in a significant reduction in primary tumor microvessel density and an increase in pericyte coverage [171], [172]. Other AA-TKIs, such as apatinib [173], [174], lenvatinib [175], [176], nintedanib [177], and pazopanib [178], have been reported to have similar effects and will not be discussed in detail here.

However, a significant proportion of cancer patients develop resistance to antiangiogenic therapy, resulting in ineffective control of disease progression [179] and limiting patient survival; this has limited the clinical application of antiangiogenic therapy. Therapeutic resistance is associated with the use of AA-TKIs for antivascular therapy and thus affects EC–PC crosstalk. One study showed that treating rectal cancer with regorafenib or axitinib significantly reduces the density of microvessels. However, this approach induces the formation of scaffolds with the basement membrane for tumor vascular recanalization by perivascular cells, including PCs and vascular smooth muscle cells. These changes affect the course of disease and prognosis [180]. In addition, PCs can influence the efficacy of AA-TKIs and AA-MAs through paracrine secretion, thus helping ECs escape drug attack and facilitating EC survival [181], [182], [183]. Overall, reducing resistance to antiangiogenic therapy by inhibiting the regulatory effects of PCs on ECs is a new research direction, and the development of related therapeutic strategies shows great promise for treatment applications.

Research progress on other drugs that may have potential therapeutic significance

Compared with the VEGF signaling pathway and the PDGF signaling pathway, for which several therapeutic drugs are already available for clinical use, most studies on drugs that target other pathways are still in the preclinical stage. However, the development of related drugs is highly important for the advancement of antivascular therapies, especially those targeting the pathways that connect ECs and PCs. A short summary of potential drugs that may exert therapeutic effects on EC–PC pathways is presented below.

AVID200 is a TGF-β ligand trap that is in development, and it constructed primarily by fusing the receptor ectodomain of TGF-β to IgG. Several ongoing phase I clinical trials have demonstrated that AVID200 has good antiproliferative and antifibrotic effects but does not completely negate the physiological functions of TGF-β [184]. Currently available inhibitors that target serine/threonine kinase inhibitors that are downstream of TGF-β include SB431542, SB505124, EW-7197 (vactosertib), GW788388, R-268712 and LY2157299 (galunisertib) [185]. However, most Alk inhibitors have not entered clinical trials due to their cardiovascular toxicity. Among the investigational drugs that target the Ang/Tie2 pathway, nesvacumab is a human (IgG1) monoclonal antibody that works by binding to Ang-2 and blocking its interaction with the Tie-2 receptor. Regeneron previously developed a combination therapy that includes nesvacumab and aflibercept in a single intravitreal injection for the treatment of nAMD and DME. This combination therapy has undergone phase II clinical trials [186]. Faricimab is a bispecific antibody for the treatment of eye diseases, specifically AMD and DR. Faricimab binds to VEGF-A and Ang-2 and reduces abnormal blood vessel growth and inflammation by simultaneously inhibiting both signaling molecules; however, it is still in development, and further clinical trials are needed to evaluate its efficacy, safety and long-term effects [187], [188]. Regarding the Notch signaling pathway, small molecule γ-secretase inhibitors (e.g., AL560, MRK-165, and nirogacestat) as well as antibody-based biologics that target Notch ligands or receptors (e.g., ABT-119, AMG 3, and rovalpituzumab tesirine (Rova-T)) are undergoing further investigation as drug candidates [189]. The immunomodulatory drug fingolimod (FTY720), which acts as an S1PR-targeting agent during therapy, is approved for the oral treatment of relapsing-remitting multiple sclerosis because of its efficacy and tolerability [190], but whether fingolimod can affect the S1P signaling pathway between ECs and PCs to promote angiogenesis awaits further experimental studies.

Future directions

In-depth study of cell-to-cell communication between ECs and PCs has made outstanding contributions to elucidating the pathogenic mechanisms of many diseases, focusing neurological disorders (disruption of the BBB and neurodegenerative diseases), retinal diseases, tumors, and vascular diseases themselves; however, research on diseases in other systems has been relatively superficial. Because microvessels are found in systems and organs throughout the body, there is still a considerable wealth of diseases that may be associated with disrupted intercellular communication between ECs and PCs. For example, bone is a microvessel-rich organ, and disruption of EC-PC interactions is likely to have great potential in femoral head necrosis, skeletal metabolic diseases, and skeletal antiaging. H-vessels, which form a link between blood circulation and osteogenesis, are likely to cause new research hotspots in the future. Both podocytes and mesangial cells are PC-like cells that play important roles in the functional architecture of the kidney, and they play important roles in renal angiogenesis, erythropoietin (EPO) production, and the regulation of renal blood flow. Additionally, renal PCs can be transformed into myofibroblasts; thus, these cells may play important roles in the progression of acute kidney injury (AKI) to chronic kidney disease (CKD). Further study of the specific functions and roles of different PC subpopulations and their connections with ECs may aid in the development of targeted therapies for diseases of various systems and organs.

In terms of drug research, although considerable progress has been made with these currently available antiangiogenic drugs, challenges remain. These challenges include overcoming drug resistance, improving therapeutic efficacy and identifying new antiangiogenic drugs. During cancer treatment, a significant proportion of patients have already developed resistance to antiangiogenic therapy. Currently, PCs can influence the efficacy of AA-TKIs and AA-MAs through paracrine secretion, thus helping ECs escape the effects of drugs and helping ECs survive. How to help ECs escape the effects of drugs by inhibiting the role of PCs will be a new direction for drug research and development. There are also reports showing that simple antivascular drug therapy may promote tumor invasion and metastasis, i.e., enhance intratumor hypoxia, thus increasing tumor resistance to chemotherapy and radiotherapy. This gave rise to the groundbreaking idea of “normalization of tumor vasculature”. Inadequate PC coverage of abnormal tumors prevents chemotherapeutic agents from reaching their targets. By combining antivascular drugs with antitumor drugs to ameliorate the imbalance of antiangiogenic and proangiogenic factors within a specific period, thus normalizing blood vessels and enabling chemotherapeutic agents to reach tumors to improve therapeutic efficacy, the study of antiangiogenic drugs to promote tumor blood vessel normalization will be a popular research topic. In addition, the existing antitumor angiogenic drug models are dominated by the application of monoclonal antibodies, VEGFR-targeting antibodies and fusion proteins. In addition to expanding the development of drugs that target Ang and other signaling pathways in this model, the search for natural antiangiogenic drugs, such as polyphenols, polysaccharides, alkaloids, terpenoids, and saponins, the determination of their bioavailability, dosage, and side effects, as well as their combination with artificial drugs, is also a broad area for future research.

Conclusion

Normal angiogenesis and development require precise interactions between ECs and PCs. During the last two decades, the understanding of the molecular mechanisms involved in the interactions of ECs and PCs has significantly increased. The paracrine signaling pathway, which involves signaling molecules such as VEGF, PDGF, Ang, NOTCH, TGF-β, and S1P and their corresponding receptors, is the most important component of EC-PC signaling crosstalk. These molecules regulate angiogenesis and remodeling and influence the development and outcome of related diseases.

We summarized the current progress in the study of these signaling molecules and detailed the pathogenic mechanisms by which the dysregulation of these signaling pathways leads to disease. Additionally, based on the targets of these pathways, we identified FDA-approved antiangiogenic drugs that have significant potential therapeutic effects on ECs and PCs. We provide the first comprehensive overview of advances in research on the role of EC-PC interactions in angiogenesis, from mechanism to disease to drug development, and we reveal the complexities and challenges that exist in the field today. We emphasize the importance of the EC-PC interactions in potential pathogenic mechanisms underlying diseases, such as femoral head necrosis and renal fibrosis. In addition, emerging trends and future research directions, including potential areas for the further exploration of mechanisms underlying resistance to antiangiogenic drugs and the development of targeted drugs, are provided. This review contributes to a deeper understanding of the role of EC-PC interactions in angiogenesis and may provide guidance for future research and practical applications in the field of angiogenesis.

CRediT authorship contribution statement

Gan Li: Conceptualization, Writing – original draft. Junjie Gao: Writing – review & editing. Peng Ding: Conceptualization, Supervision, Writing – review & editing. Youshui Gao: Conceptualization, Supervision, Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Biographies

Gan Li is a postgraduate researcher at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. His major research focus is related to mechanisms underlying vascular system-skeletal microenvironment coupling in skeletal diseases.

Junjie Gao is currently a professor at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. His research interests focus on the development of bone-brain axis regulation and basic and clinical studies on bone repair and regeneration.

Peng Ding is currently an orthopedic surgeon at the Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. His main research interests include the pathogenesis of bone metabolic diseases.

Youshui Gao is currently serving as a chief orthopedic surgeon at Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. His research interests include bone metabolism, intercellular coupling mechanisms, and novel biomaterials for bone tissue regeneration.