Early Contribution of Pericytes to Angiogenic Sprouting and Tube Formation

Immunostaining with endothelial and pericyte markers was used to evaluate the cellular composition of angiogenic sprouts in several types of tumors and in the developing retina. Confocal microscopy revealed that, in addition to conventional endothelial tubes heavily invested by pericytes, all tissues contained small populations of endothelium-free pericyte tubes in which nerve/glial antigen 2 (NG2) positive, platelet-derived growth factor beta (PDGF beta ) receptor-positive perivascular cells formed the lumen of the microvessel. Perfusion of tumor-bearing mice with FITC-dextran, followed by immunohistochemical staining of tumor vasculature, demonstrated direct apposition of pericytes to FITC-dextran in the lumen, confirming functional connection of the pericyte tube to the circulation. Transplantation of prostate and mammary tumor fragments into NG2-null mice led to the formation of tumor microvasculature that was invariably NG2-negative, demonstrating that pericytes associated with tumor microvessels are derived from the host rather than from the conversion of tumor cells to a pericyte phenotype. The existence of pericyte tubes reflects the early participation of pericytes in the process of angiogenic sprouting. The ability to study these precocious contributions of pericytes to neovascularization depends heavily on the use of NG2 and PDGF beta -receptor as reliable early markers for activated pericytes.     

Endosialin/TEM 1/CD248 is a pericyte marker of embryonic and tumor neovascularization

The formation of functional, mature blood vessels depends on the interaction between endothelial cells and pericytes. Commonality exists in the processes involved in vasculature development between tissues whether healthy or diseased. Endosialin/TEM 1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumorigenesis but not in normal mature vessels. Antibodies developed to human endosialin were used to investigate endosialin expression and function in human prenatal brain pericytes and pericytes residing in tumors. Anti-endosialin was capable of preventing pericyte tube formation in culture and inhibited migration. Brain pericytes in culture had higher levels of endosialin/TEM 1 than TEMs-2, -3, -4, -5, -7, and -8. Immunocytochemistry revealed that endosialin was present in the cytoplasmic body and in the elongated extensions essential to pericyte function. Transgenic mice engineered to express human endosialin bred on an immunocompromised background allowed the growth of human tumor xenografts. In human colon carcinoma Colo205 and HT29 xenografts grown in human endosialin-transgenic mice, endosialin expression was largely confined to NG2-expressing perivascular cells and not CD31-positive endothelial cells. Similar methods applied to human ovarian and colon tumors confirmed endosialin expression by pericytes. The data indicate that endosialin is strongly expressed by pericytes during periods of active angiogenesis during embryonic and tumor development. Anti-endosialin antibodies may have value in identifying vasculature in malignant tissues. With the appropriate agent, targeting endosialin may interfere with blood vessel growth during tumor development.     

Endosialin/TEM 1/CD248 is a pericyte marker of embryonic and tumor neovascularization

The formation of functional, mature blood vessels depends on the interaction between endothelial cells and pericytes. Commonality exists in the processes involved in vasculature development between tissues whether healthy or diseased. Endosialin/TEM 1 is a cell membrane protein that is expressed in blood vessels during embryogenesis and tumorigenesis but not in normal mature vessels. Antibodies developed to human endosialin were used to investigate endosialin expression and function in human prenatal brain pericytes and pericytes residing in tumors. Anti-endosialin was capable of preventing pericyte tube formation in culture and inhibited migration. Brain pericytes in culture had higher levels of endosialin/TEM 1 than TEMs-2, -3, -4, -5, -7, and -8. Immunocytochemistry revealed that endosialin was present in the cytoplasmic body and in the elongated extensions essential to pericyte function. Transgenic mice engineered to express human endosialin bred on an immunocompromised background allowed the growth of human tumor xenografts. In human colon carcinoma Colo205 and HT29 xenografts grown in human endosialin-transgenic mice, endosialin expression was largely confined to NG2-expressing perivascular cells and not CD31-positive endothelial cells. Similar methods applied to human ovarian and colon tumors confirmed endosialin expression by pericytes. The data indicate that endosialin is strongly expressed by pericytes during periods of active angiogenesis during embryonic and tumor development. Anti-endosialin antibodies may have value in identifying vasculature in malignant tissues. With the appropriate agent, targeting endosialin may interfere with blood vessel growth during tumor development.     

Pathological angiogenesis is reduced by targeting pericytes via the NG2 proteoglycan

The NG2 proteoglycan is expressed by nascent pericytes during the early stages of angiogenesis. To investigate the functional role of NG2 in neovascularization, we have compared pathological retinal and corneal angiogenesis in wild type and NG2 null mice. During ischemic retinal neovascularization, ectopic vessels protruding into the vitreous occur twice as frequently in wild type retinas as in NG2 null retinas. In the NG2 knock-out retina, proliferation of both pericytes and endothelial cells is significantly reduced, and the pericyte:endothelial cell ratio falls to 0.24 from the wild type value of 0.86. Similarly, bFGF-induced angiogenesis is reduced more than four-fold in the NG2 null cornea compared to that seen in the wild type retina. Significantly, NG2 antibody is effective in reducing angiogenesis in the wild type cornea, suggesting that the proteoglycan can be an effective target for anti-angiogenic therapy. These experiments therefore demonstrate both the functional importance of NG2 in pericyte development and the feasibility of using pericytes as anti-angiogenic targets.     

Analysis of relationships between pericytes and gas exchange capillaries in neonatal and mature bovine lungs

Neonatal and mature bovine lungs were examined ultrastructurally to quantitatively assess pericyte envelopment of gas exchange capillaries and proximities of pericyte margins to endothelial cell junctions. Pericytes were observed on 91% of the cross-sectioned capillary profiles examined, with 18 and 26% coverage in neonatal and mature lungs, respectively. Chi-square analysis indicated a random occurrence of endothelial cell junctions under pericytes; however, pericyte processes tended to end near endothelial cell junctions. In the neonatal and mature lungs, 38 and 40%, respectively, of all endothelial junctions were within 0.5 μm of the margins of pericyte processes; such distances covered only 16 and 17% of the circumferences of the capillary profiles examined. Thus, endothelial cell junctions were located near pericyte margins over twice as often as would occur in a random distribution. If pericytes are contractile cells, as recent research indicates, they may function as regulators of lymph formation by influencing the permeability of endothelial cell junctions in the gas exchange capillaries.     

The pericyte microenvironment during vascular development

Vascular pericytes provide critical contributions to the formation and integrity of the blood vessel wall within the microcirculation. Pericytes maintain vascular stability and homeostasis by promoting endothelial cell junctions and depositing extracellular matrix (ECM) components within the vascular basement membrane, among other vital functions. As their importance in sustaining microvessel health within various tissues and organs continues to emerge, so does their role in a number of pathological conditions including cancer, diabetic retinopathy, and neurological disorders. Here, we review vascular pericyte contributions to the development and remodeling of the microcirculation, with a focus on the local microenvironment during these processes. We discuss observations of their earliest involvement in vascular development and essential cues for their recruitment to the remodeling endothelium. Pericyte involvement in the angiogenic sprouting context is also considered with specific attention to crosstalk with endothelial cells such as through signaling regulation and ECM deposition. We also address specific aspects of the collective cell migration and dynamic interactions between pericytes and endothelial cells during angiogenic sprouting. Lastly, we discuss pericyte contributions to mechanisms underlying the transition from active vessel remodeling to the maturation and quiescence phase of vascular development.     

Pericytes promote selective vessel regression to regulate vascular patterning

Blood vessel networks form in a 2-step process of sprouting angiogenesis followed by selective branch regression and stabilization of remaining vessels. Pericytes are known to function in stabilizing blood vessels, but their role in vascular sprouting and selective vessel regression is poorly understood. The endosialin (CD248) receptor is expressed by pericytes associated with newly forming but not stable quiescent vessels. In the present study, we used the Endosialin(-/-) mouse as a means to uncover novel roles for pericytes during the process of vascular network formation. We demonstrate in a postnatal retina model that Endosialin(-/-) mice have normal vascular sprouting but are defective in selective vessel regression, leading to increased vessel density. Examination of the Endosialin(-/-) mouse tumor vasculature revealed an equivalent phenotype, indicating that pericytes perform a hitherto unidentified function to promote vessel destabilization and regression in vivo in both physiologic and pathologic angiogenesis. Mechanistically, Endosialin(-/-) mice have no defect in pericyte recruitment. Rather, endosialin binding to an endothelial associated, but not a pericyte associated, basement membrane component induces endothelial cell apoptosis and detachment. The results of the present study advance our understanding of pericyte biology and pericyte/endothelial cell cooperation during vascular patterning and have implications for the design of both pro- and antiangiogenic therapies.     

Identification of class III β-tubulin as a marker of angiogenic perivascular cells

A full understanding of the functional role for pericytes in microvascular network growth requires identifying the specific cell phenotypes involved in angiogenesis. The objective of this study was to evaluate class III β-tubulin expression along remodeling adult rat mesenteric microvascular networks. Mesenteric tissues were harvested from unstimulated adult male Wistar rats and at 2, 10 and 30 days post-compound 48/80 stimulation (n=4 per experimental group). Tissues were immunohistochemically labeled with antibodies for class III β-tubulin, NG2 and PECAM. In unstimulated microvascular networks, class III β-tubulin was nerve specific, and did not identify vascular cells along PECAM positive arterioles, venules, and capillaries. Two days post 48/80 stimuli, class III β-tubulin labeling of perivascular cells, including pericytes and smooth muscle cells, was observed along capillary sprouts, capillaries, venules, and arterioles in network regions characterized by increased vessel density and tortuosity. Pericyte identity along capillaries and capillary sprouts was confirmed by cell morphology and co-labeling with NG2. The percentage of vessels with class III β-tubulin positive labeling decreased at subsequent time points and temporally correlated with the time course of capillary sprouting. The results identify class III β-tubulin as a marker of angiogenic perivascular cells and suggest that specific pericyte phenotypes are associated with capillary sprouting.     

In vitro modeling of endothelial interaction with macrophages and pericytes demonstrates Notch signaling function in the vascular microenvironment

Angiogenesis is regulated by complex interactions between endothelial cells and support cells of the vascular microenvironment, such as tissue myeloid cells and vascular mural cells. Multicellular interactions during angiogenesis are difficult to study in animals and challenging in a reductive setting. We incorporated stromal cells into an established bead-based capillary sprouting assay to develop assays that faithfully reproduce major steps of vessel sprouting and maturation. We observed that macrophages enhance angiogenesis, increasing the number and length of endothelial sprouts, a property we have dubbed “angiotrophism.” We found that polarizing macrophages toward a pro-inflammatory profile further increased their angiotrophic stimulation of vessel sprouting, and this increase was dependent on macrophage Notch signaling. To study endothelial/pericyte interactions, we added vascular pericytes directly to the bead-bound endothelial monolayer. These pericytes formed close associations with the endothelial sprouts, causing increased sprout number and vessel caliber. We found that Jagged1 expression and Notch signaling are essential for the growth of both endothelial cells and pericytes and may function in their interaction. We observed that combining endothelial cells with both macrophages and pericytes in the same sprouting assay has multiplicative effects on sprouting. These results significantly improve bead-capillary sprouting assays and provide an enhanced method for modeling interactions between the endothelium and the vascular microenvironment. Achieving this in a reductive in vitro setting represents a significant step toward a better understanding of the cellular elements that contribute to the formation of mature vasculature.     

An intimate interplay between precocious,migrating pericytes and endothelial cells governs human fetal brain angiogenesis

In order to better understand the process of angiogenesis in the developing human brain, we have examined the spatial relationship and relative contributions of endothelial cells and pericytes, the two primary cell types involved in vessel growth, together with their relation with the vascular basement membrane. Pericytes were immunolocalized through use of the specific markers nerve/glial antigen 2 (NG2) proteoglycan, endosialin (CD248) and the platelet-derived growth factor receptor β (PDGFR-β), while endothelial cells were identified by the pan-endothelial marker CD31 and the blood brain barrier (BBB)-specific markers claudin-5 and glucose transporter isoform 1 (GLUT-1). The quantitative analysis demonstrates that microvessels of the fetal human telencephalon are characterized by a continuous layer of activated/angiogenic NG2 pericytes, which tightly invest endothelial cells and participate in the earliest stages of vessel growth. Immunolabelling with anti-active matrix metalloproteinase-2 (aMMP-2) and anti-collagen type IV antibodies revealed that aMMP-2 producing endothelial cells and pericytes are both associated with the vascular basement membrane during vessel sprouting. Detailed localization of the two vascular cell types during angiogenesis suggests that growing microvessels of the human telencephalon are formed by a pericyte-driven angiogenic process in which the endothelial cells are preceded and guided by migrating pericytes during organization of the growing vessel wall.     

Recent advances in pericyte biology--implications for health and disease.

This review highlights the contributions of recent pericyte research towards our understanding of normal and pathological functioning of microvessels. Pericytes are implicated in a variety of microvascular alterations, including wound healing, diabetes, inflammation, hypertension and neoplasia. They are capable of changing into other mesodermally derived cell types, including smooth muscle cells, osteoblasts and chondrocytes. The contractile properties of pericytes are being systematically examined in vitro; in addition to their tendency to contract spontaneously, pericytes can contract further in response to mediators of inflammation. In vivo studies indicate pericytes are concentrated near endothelial cell junctions along venules where they likely participate in inflammatory events. As agents are identified which modify pericyte responses to disease states, better therapeutic approaches will become possible.     

Differential arterial/venous expression of NG2 proteoglycan in perivascular cells along microvessels: identifying a venule-specific phenotype

OBJECTIVE: Similar to other vascular pericyte markers, including smooth muscle (SM) alpha-actin, desmin, and PDGF-beta-receptor, NG2 proteoglycan is not pericyte specific. Therefore, the use of NG2 as a pericyte marker, especially in cell lineage studies, in comparison to other nonspecific pericyte markers requires an understanding of how its expression varies spatially within a microvascular network. The objective of this study was to characterize NG2 expression along vessels within rat microvascular networks and compare this to SM alpha-actin expression. METHODS: Mesenteric tissue, subcutaneous tissue, spinotrapezius muscle, and gracilis muscle were harvested from 250-g, female, Sprague-Dawley rats and stained for NG2 and SM alpha-actin. The distribution of NG2 expression was evaluated in mesenteric networks (n = 28) with complementary observations in subcutaneous tissue and skeletal muscle. RESULTS: Perivascular cells, including mature smooth muscle cells (SMCs), immature SMCs, and pericytes, expressed NG2. Most importantly, NG2 expression was primarily confined to perivascular cells along arterioles and capillaries, and continuous expression was not observed along venules beyond the immediate postcapillary vessels. The differential expression of NG2 along the arteriolar side of microvascular networks was also observed in rat subcutaneous and skeletal muscle. CONCLUSIONS: The results indicate that NG2 is expressed by all perivascular cells along arterioles, and its absence denotes a venule-specific phenotype. These results identify for the first time a marker that differentiates venous smooth muscle and pericytes from other capillary- and arteriole-associated perivascular cells.     

TGFβ is required for the formation of capillary-like structures in three-dimensional cocultures of 10T1/2 and endothelial cells

New vessels form de novo (vasculogenesis) or from pre-existing vessels (angiogenesis) in a process that involves the interaction of endothelial cells (EC) and pericytes/smooth muscle cells (SMC). One basic component of this interaction is the endothelial-induced recruitment, proliferation and subsequent differentiation of pericytes and SMC. We have previously demonstrated that TGFβ induces the differentiation of C3H/10T1/2 (10T1/2) mesenchymal cells toward a SMC/pericyte lineage. The current study tests the hypothesis that TGFβ not only induces SMC differentiation but stabilizes capillary-like structures in a three-dimensional (3D) model of in vitro angiogenesis. 10T1/2 and EC in Matrigel™ were used to establish cocultures that form cord structures that are reminiscent of new capillaries in vivo. Cord formation is initiated within 2–3 h after plating and continues through 18 h after plating. In longer cocultures the cord structures disassemble and form aggregates. 10T1/2 expression of proteins associated with the SMC/pericyte lineage, such as smooth muscle α-actin (SMA) and NG2 proteoglycan, are upregulated in these 3D cocultures. Application of neutralizing reagents specific for TGFβ blocks cord formation and inhibits expression of SMA and NG2 in the 10T1/2 cells. We conclude that TGFβ mediates 10T1/2 differentiation to SMC/pericytes in the 3D cocultures and that association with differentiated mural cells is required for formation of capillary-like structures in Matrigel™. This revised version was published online in June 2006 with corrections to the Cover Date.     

A novel and simple method for culturing pericytes from mouse brain

Pericytes play critical roles in the development, maturation and remodeling of blood vessels, and in the central nervous system (CNS), evidence suggests that pericytes also regulate blood flow and form an integral part of the blood-brain barrier. The study of this important cell type has been hampered by the lack of any pericyte-specific marker and by the difficulty of culturing pericytes in adequate numbers to high purity. Here we present a novel yet simple approach to isolate and culture large numbers of pericytes from the mouse CNS that nevertheless leads to very pure pericyte cultures. In our method, vascular cells obtained from adult mice brains are cultured initially under conditions optimized for endothelial cells, but after two passages switched to a medium optimized for pericyte growth. After growing the cells for 1-2 additional passages we obtained a largely homogeneous population of cells that expressed the pericyte markers NG2, PDGFβ-receptor, and CD146, but were negative for markers of endothelial cells (CD31), microglia (Mac-1) and astrocytes (GFAP). Under these conditions, pericytes could be grown to high passage number, and were maintained highly pure and largely undifferentiated, as determined by antigen expression profile and low levels of α-SMA expression, a marker of pericyte differentiation. Furthermore, switching the cells from pericyte medium into DMEM containing 10% FBS promoted α-SMA expression, demonstrating that high passage pericytes could still differentiate. Thus, we provide an alternative approach to the culture of CNS pericytes that is easy to establish and provides large numbers of highly pure pericytes for extended periods of time. This system should provide others working in the pericyte field with a useful additional tool to study the behavior of this fascinating cell type.     

The EGFR inhibitor gefitinib suppresses recruitment of pericytes and bone marrow-derived perivascular cells into tumor vessels

Drugs that target EGFR have established anti-tumor effect and are used in the clinic. Here we addressed whether inhibition of EGFR tyrosine kinase activity by gefitinib in tumor microenvironment affected tumor angiogenesis or vasculogenesis. A syngeneic tumor model of mice with grafted GFP-labeled bone marrow cells was used to analyze the effects of gefitinib on different cellular components of tumor vasculature. To characterize tumor cell-independent stromal effects of EGFR targeting, the mice were injected with B16 melanoma cells not expressing significant quantities of EGFR, and treated with gefitinib for seven days, a period not sufficient for significant reduction in total tumor volume. Numbers of vessels as well as cell surface areas covered by markers of endothelial, pericyte and bone marrow-derived progenitor cells were quantified by image analysis of tumor sections. Quantitative analysis of immunohistochemical data demonstrated that gefitinib decreased the coverage of small CD31-positive vessels with NG2-positive pericytes, as well as reduced the recruitment of perivascular GFP-positive bone marrow-derived progenitor cells within the tumor tissue. These results suggest that inhibition of EGFR activity in tumors has vascular effects in the absence of direct effect on tumor cells. EGFR targeting may lead to suppressed mobilization of pericytes needed for vessel stabilization, as well as of bone marrow-derived perivascular progenitor cells. These findings introduce novel cellular mechanisms by which EGFR targeted drugs may suppress tumor growth.     

Inhibition of apelin expression switches endothelial cells from proliferative to mature state in pathological retinal angiogenesis

The recruitment of mural cells such as pericytes to patent vessels with an endothelial lumen is a key factor for the maturation of blood vessels and the prevention of hemorrhage in pathological angiogenesis. To date, our understanding of the specific trigger underlying the transition from cell growth to the maturation phase remains incomplete. Since rapid endothelial cell growth causes pericyte loss, we hypothesized that suppression of endothelial growth factors would both promote pericyte recruitment, in addition to inhibiting pathological angiogenesis. Here, we demonstrate that targeted knockdown of apelin in endothelial cells using siRNA induced the expression of monocyte chemoattractant protein-1 (MCP-1) through activation of Smad3, via suppression of the PI3K/Akt pathway. The conditioned medium of endothelial cells treated with apelin siRNA enhanced the migration of vascular smooth muscle cells, through MCP-1 and its receptor pathway. Moreover, in vivo delivery of siRNA targeting apelin, which causes exuberant endothelial cell proliferation and pathological angiogenesis through its receptor APJ, led to increased pericyte coverage and suppressed pathological angiogenesis in an oxygen-induced retinopathy model. These data demonstrate that apelin is not only a potent endothelial growth factor, but also restricts pericyte recruitment, establishing a new connection between endothelial cell proliferation signaling and a trigger of mural recruitment.     

A study of capillary pericyte viability on extracellular matrix produced by endothelial cells in high glucose

AIMS/HYPOTHESIS: Thickening of the basement membrane and selective loss of pericytes occur early in diabetic retinopathy. As we showed previously that pericyte adhesion is impaired on extracellular matrix produced by endothelial cells in high hexose concentrations, we aimed to verify if altered adhesion could influence pericyte viability and replication. METHODS: Conditioned extracellular matrices were obtained by growing human umbilical vein endothelial cells in media containing 28 mmol/l D-glucose, with or without the inhibitors of protein glycation thiamine or aminoguanidine, and D-galactose or L-glucose up to 28 mmol/l. Having removed the endothelium, bovine retinal pericytes were grown on these matrices and, in separate experiments, on laminin, fibronectin or type IV collagen. Pericyte viability and replication were measured by cell counts and DNA synthesis after 7 days, cell cycle traversal after 2 days and apoptosis after 18 h, 2 days and 7 days. RESULTS: Pericyte counts and DNA synthesis were reduced on matrices produced in high D-glucose and D-galactose, whilst matrix obtained in L-glucose reduced DNA synthesis but not counts. Both thiamine and aminoguanidine corrected reduced pericyte viability when added to high D-glucose. Cell cycle and apoptosis were not affected by growing pericytes on different conditioned matrices. Laminin, fibronectin and type IV collagen did not modify pericyte replication. CONCLUSIONS/INTERPRETATIONS: Reduced pericyte counts could depend on impaired initial adhesion to the extracellular matrix produced by endothelium in high hexose concentrations, rather than impaired replication or viability. Altered cell-matrix interactions might facilitate pericyte dropout in diabetic retinopathy, independently of the effects of high glucose on pericyte replication.     

Transforming growth factor beta1 induction of vascular endothelial growth factor receptor 1: mechanism of pericyte-induced vascular survival in vivo

Degeneration of vessels precedes and precipitates the devastating ischemia of many diseases, including retinopathy of prematurity and diabetic retinopathy. Ischemia then leads to proliferative retinopathy and blindness. Understanding the mechanisms of blood vessel degeneration is critical to prevention of these diseases. Vessel loss is associated with oxygen-induced suppression of vascular endothelial growth factor (VEGF) and with pericyte (vascular smooth muscle cell) dropout. The molecular mechanism of pericyte protection of the vasculature is unknown. We show that transforming growth factor beta1 (TGF-beta1)-expressing pericytes are specifically found on vessels resistant to oxygen-induced loss. TGF-beta1 potently induces VEGF receptor 1 (VEGFR-1) expression in endothelial cells and thereby prevents oxygen-induced vessel loss in vivo. Vessel survival is further stimulated with a VEGFR-1-specific ligand, placental growth factor 1. TGF-beta1 induction of VEGFR-1 in endothelial cells explains pericyte protection of vessels and the selective vulnerability of neonatal vessels to oxygen. These results implicate induction and activation of VEGFR-1 as critical targets to prevent vessel loss.     

Delta-like ligand 4-Notch signaling regulates bone marrow-derived pericyte/vascular smooth muscle cell formation

Delta-like ligand 4 (DLL4) is essential for the formation of mature vasculature. However, the role of DLL4-Notch signaling in pericyte/vascular smooth muscle cell (vSMC) development is poorly understood. We sought to determine whether DLL4-Notch signaling is involved in pericyte/vSMC formation in vitro and during vasculogenesis in vivo using 2 Ewing sarcoma mouse models. Inhibition of DLL4 with the antibody YW152F inhibited pericyte/vSMC marker expression by bone marrow (BM) cells in vitro. Conversely, transfection of 10T1/2 cells with the active domains of Notch receptors led to increased expression of pericyte/vSMC markers. Furthermore, the blood vessels of Ewing sarcoma tumors from mice treated with YW152F had reduced numbers of BM-derived pericytes/vSMCs, fewer open lumens, and were less functional than the vessels in tumors of control-treated mice. Tumor growth was also inhibited. These data demonstrate a specific role for DLL4 in the formation of BM-derived pericytes/vSMCs and indicate that DLL4 may be a novel therapeutic target for the inhibition of vasculogenesis.