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.     

Cultured retinal capillary pericytes die by apoptosis after an abrupt fluctuation from high to low glucose levels: a comparative study with retinal capillary endothelial cells

Summary A number of clinical observations concerning cases of glycemic fluctuation have prompted us to study whether or not a rapid change in blood glucose concentration can aggravate retinal microvascular pathology during the early stage of diabetic retinopathy. We conducted a comparative study of retinal capillary pericytes and endothelial cells in vitro. Both types of cells, either in single culture or in co-culture, were initially incubated in medium with high glucose (20–40 mmol/l), followed by a rapid reduction of glucose to 3.5, 1, or 0.5 mmol/l. This type of reduction of extracellular glucose resulted in depletion of intracellular glucose, occurring much faster in pericytes than in endothelial cells. The abrupt reduction in glucose caused pericyte cell shrinkage and nuclear condensation associated with DNA fragmentation, followed by loss of cell viability. All of these pericyte changes are apoptosis-like characteristics. This apoptotic process was prevented by the addition of cycloheximide, a protein synthesis inhibitor, or by platelet-derived growth factor BB, which is a known competent factor for pericyte growth. In analysis of signalling pathways during the abrupt fluctuation of glucose, the occurrence of pericyte apoptosis was an intracellular calcium-dependent, protein kinase C and protein kinase A mediated, and poly (ADP-ribose) synthetase-dependent process. Interestingly, a larger degree of DNA fragmentation was observed with a higher magnitude and a longer duration of pre-existing hyperglycaemia. These results suggest that the magnitude and duration of pre-existing hyperglycaemia prime the apoptotic responsiveness of pericytes. Retinal capillary endothelial cells, after an identical glucose fluctuation treatment did not undergo an apoptotic process.Abbreviations RCEC Retinal capillary endothelial cells - IDDM insulin-dependent diabetes mellitus - FCS fetal calf serum - DMEM Dulbecco's modified Eagle's medium - PDGF platelet-derived growth factor - PKC protein kinase C - PKA protein kinase A - PMA phorbol 12-myristate 13-acetate     

Epidermal growth factor receptor at endothelial cell and pericyte interdigitation in human granulation tissue.

Angiogenic immature capillaries in human granulation tissue possess many cytoplasmic interdigitations between endothelial cells and pericytes (CIDEP). Epidermal growth factor (EGF) is a potent mitogenic polypeptide which accelerates angiogenesis in vivo and in vitro. We have recently demonstrated in immature capillaries that EGF is present at the CIDEP and proposed that the CIDEP may be involved in a signaling pathway for EGF (Wakui et al., 1990c. Microvasc. Res. 40, 285-291). This study follows that previous report. In the present study, I have investigated the ultrastructural localization of the EGF receptor (EGF-r) at CIDEP. Immunoreactivity for the EGF-r in immature capillaries was located strictly at the CIDEP in large numbers, at the coated pits and vesicles of endothelial cells, and at some lysosome-like structures of the endothelial cells and the pericytes. On the other hand, immunoreactivity for EGF-r was absent in mature capillaries. At the CIDEP, EGF-r immunoreactivity was present at the cell membrane and a few cytoplasmic elements at the tip of pericyte cytoplasmic projections, but it was completely absent at the corresponding endothelial membrane indentation. The present results support our proposed hypothesis that the CIDEP in immature capillaries act as a pathway for EGF transportation from the endothelial cell to the pericyte by a receptor-mediated process.     

Pigment epithelium-derived factor (PEDF) promotes growth of pericytes through autocrine production of platelet-derived growth factor-B

Microvessels are composed of two types of cells, endothelial cells and pericytes. Pericyte loss or dysfunction participates in various types of disorders, including diabetic retinopathy. Recently, decreased levels of pigment epithelium-derived factor (PEDF) in the eye have been found to predict progression of diabetic retinopathy. However, the effect of PEDF on pericyte growth remains to be unknown. In this study, we investigated whether or how PEDF could stimulate proliferation of cultured retinal pericytes. PEDF stimulated DNA synthesis in pericytes in a dose-dependent manner. PEDF up-regulated pericyte mRNA levels of platelet-derived growth factor-B (PDGF-B). Down-regulation of PDGF-B gene expression by small interfering RNAs completely inhibited the PEDF-induced DNA synthesis in pericytes. Furthermore, PEDF increased protein kinase C (PKC) activity in pericytes and staurosporine, a potent cell-permeable inhibitor of PKC, completely blocked the PDGF-B gene induction and subsequent increase in DNA synthesis in PEDF-exposed pericytes. These results demonstrate that PEDF promotes the growth of cultured pericytes possibly through autocrine production of PDGF-B via PKC activation. Our present study suggests that PEDF could act as a mitogen or survival factor for pericytes, thereby being involved in the maintenance of retinal microvascular homeostasis.     

Isolation and characterization of ovine luteal pericytes and effects of nitric oxide on pericyte expression of angiogenic factors

We have demonstrated that vascular endothelial growth factor (VEGF) is expressed in capillary pericytes of the developing corpus luteum (CL) and others have shown that basic fibroblast growth factor (FGF2) and angiopoietins (ANGPT) are present in the CL. VEGF and FGF2 target endothelial cells to initiate angiogenesis and stimulate nitric oxide (NO) production. Conversely, NO may increase VEGF expression by vascular smooth muscle cells and pericytes. To investigate the relationship between these angiogenic factors and NO in the CL, microvascular pericytes and endothelial cells were isolated from CL collected from superovulated ewes (n = 5) on d 9 of the estrous cycle. Pericytes were identified by their morphology in culture and by immunofluorescent staining for smooth muscle cell actin. Pericytes were incubated with or without varying doses of the NO-donor DETA-NO for 8 h. Then, total cellular RNA was extracted from the cells and evaluated for expression of mRNA for VEGF, FGF2, ANGPT1, ANGPT2, and NO receptor, guanylate cyclase 1, soluble beta3 (GUCY1B3), using real-time quantitative RT-PCR. NO caused a dose-dependent increase in VEGF (p < 0.001), FGF2 (p < 0.001), ANGPT2 (p < 0.06), and GUCY1B3 (p < 0.03) mRNA expression. Expression of mRNA for ANGPT1 in luteal pericytes was not affected by the NO treatment. These data provide further evidence of the role of the luteal pericyte and NO in angiogenic factor expression, and of the potential interactions of pericytes with endothelial cells via NO production.     

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.     

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.     

A new ex vivo model to study venous angiogenesis and arterio-venous anastomosis formation

Explants of rat inferior vena cava embedded in collagen gel and cultured under serum-free conditions produced microvascular outgrowths composed of endothelial cells and pericytes. Exogenous vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) dose-dependently stimulated angiogenesis and induced the formation of complex networks of highly branched microvessels. VEGF and the VEGF/bFGF combination also promoted pericyte recruitment. Medium conditioned by untreated vena cava cultures contained endogenous VEGF, and a blocking antibody against VEGF significantly reduced the spontaneous angiogenic response of the explants. Vena cava explants exhibited a greater capacity to form neovessels than aortic rings when tested in parallel cultures from the same animal. When compared with aorta-derived microvessels, neovessels of vena cava origin were longer and had fewer pericytes. Vena cava-aorta cocultures produced extensive anastomosing networks of microvessels, which were primarily contributed by the venous explants. Because of its florid angiogenesis and exquisite sensitivity to angiogenic factor stimulation, the vena cava model may provide novel insights into the regulation of the angiogenic process, which typically initiates from the venous side of the vascular bed. Combined with the aortic ring model, this new assay may also enhance our understanding of the mechanisms of anastomosis formation between the arterial and the venous circulations.     

NG2 proteoglycan promotes tumor vascularization via integrin-dependent effects on pericyte function

The NG2 proteoglycan stimulates the proliferation and migration of various immature cell types, including pericytes. However, the role of NG2 in mediating pericyte/endothelial cell interaction has been less clear. In this study, we show that pericyte-specific NG2 ablation causes several structural deficits in blood vessels in intracranial B16F10 melanomas, including decreased pericyte ensheathment of endothelial cells, diminished formation of endothelial junctions, and reduced assembly of the vascular basal lamina. These deficits result in decreased tumor vessel patency, increased vessel leakiness, and increased intratumoral hypoxia. NG2-dependent mechanisms of pericyte interaction with endothelial cells are further explored in pericyte/endothelial cell co-cultures. siRNA-mediated NG2 knockdown in pericytes leads to reduced formation of pericyte/endothelial networks, reduced formation of ZO-1 positive endothelial cell junctions, and increased permeability of endothelial cell monolayers. We also show that NG2 knockdown results in loss of β1 integrin activation in endothelial cells, revealing a mechanism for NG2-dependent cross talk between pericytes and endothelial cells.     

Pericytes and vessel maturation during tumor angiogenesis and metastasis

Despite promising results in preclinical and clinical studies, the therapeutic efficacy of antiangiogenic therapies has been restricted by a narrow focus on inhibiting the growth of endothelial cells. Other cell types in the tumor stroma are also critical to the progression of cancer, including mural cells. Mural cells are vascular support cells that range in phenotype from pericytes to vascular smooth muscle cells. Although the role of pericytes and pericyte‐like cells in the pathophysiology of cancer is still unclear, evidence indicates that aberrations in pericyte–endothelial cell signaling networks could contribute to tumor angiogenesis and metastasis. The purpose of this review is to evaluate critically recent evidence on the role of pericytes in tumor biology and discuss potential therapeutic targets for anticancer intervention. Am. J. Hematol. 85:593–598, 2010. © 2010 Wiley‐Liss, Inc.     

Soluble receptor-mediated selective inhibition of VEGFR and PDGFRbeta signaling during physiologic and tumor angiogenesis

The simultaneous targeting of both endothelial cells and pericytes via inhibition of VEGF receptor (VEGFR) and PDGFbeta receptor (PDGFRbeta) signaling, respectively, has been proposed to enhance the efficacy of antiangiogenic tumor therapy. Clinical and preclinical modeling of combined VEGFR and PDGFRbeta signaling inhibition, however, has used small molecule kinase inhibitors with inherently broad substrate specificities, precluding detailed examination of this hypothesis. Here, adenoviral expression of a soluble VEGFR2/Flk1 ectodomain (Ad Flk1-Fc) in combination with a soluble ectodomain of PDGFRbeta (Ad sPDGFRbeta) allowed highly selective inhibition of these pathways. The activity of Ad sPDGFRbeta was validated in vitro against PDGF-BB and in vivo with near-complete blockade of pericyte recruitment in the angiogenic corpus luteum, resulting in prominent hemorrhage, thus demonstrating an essential function for PDGF signaling during ovarian angiogenesis. Combination therapy with Ad PDGFRbeta and submaximal doses of Ad Flk1-Fc produced modest additive antitumor effects; however, no additivity was observed with maximal VEGF inhibition in numerous s.c. models. Notably, VEGF inhibition via Ad Flk1-Fc was sufficient to strongly suppress tumor endothelial and pericyte content as well as intratumoral PDGF-B mRNA, obscuring additive Ad sPDGFRbeta effects on pericytes or tumor volume. These studies using highly specific soluble receptors suggest that additivity between VEGFR and PDGFRbeta inhibition depends on the strength of VEGF blockade and appears minimal under conditions of maximal VEGF antagonism.     

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.     

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.     

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.     

The CNS pericyte response to low oxygen: early synthesis of cyclopentenone prostaglandins of the J-series

Exposure to low oxygen induces adaptive changes at the microvascular level that are beneficial to cell survival. These adaptive changes involve complex signaling mechanisms between the vascular endothelial cell and the pericyte, and are important to the maintenance of vascular homeostasis and hemostasis. We have investigated the early response of the central nervous system (CNS) microvascular pericyte to low oxygen. In vitro exposure of primary rat CNS pericytes to low oxygen induced the rapid synthesis and release of the cyclopentenone prostaglandin (PG) PGD2 and PGJ2 within 15-30 min following hypoxic stress signal. Hypoxia-induced release of PGD2/PGJ2 was COX-1 dependent and did not involve COX-2. The exogenous addition of 15-deoxyDelta(12,14) PGJ2 to pericytes under normoxic conditions increased glut-1 protein in the absence of hypoxia. PGD2 and PGJ2 may be early signaling molecules in the pericyte stress response.     

The Semaphorin 4D-Plexin-B1-RhoA signaling axis recruits pericytes and regulates vascular permeability through endothelial production of PDGF-B and ANGPTL4

Semaphorin 4D (SEMA4D) is a member of a family of transmembrane and secreted proteins that have been shown to act through its receptor Plexin-B1 to regulate axon growth cone guidance, lymphocyte activation, and bone density. SEMA4D is also overexpressed by some malignancies and plays a role in tumor-induced angiogenesis similar to vascular endothelial growth factor (VEGF), a protein that has been targeted as part of some cancer therapies. In an attempt to examine the different effects on tumor growth and vascularity for these two pro-angiogenic factors, we previously noted that while inhibition of both VEGF and SEMA4D restricted tumor vascularity and size, vessels forming under conditions of VEGF blockade retained their association with pericytes while those arising in a background of SEMA4D/Plexin-B1 deficiency did not, an intriguing finding considering that alteration in pericyte association with endothelial cells is an emerging aspect of anti-angiogenic intervention in the treatment of cancer. Here we show through array analysis, immunoblots, migration and co-culture assays and VE-cadherin immunohistochemistry that SEMA4D production by head and neck carcinoma tumor cells induces expression of platelet-derived growth factor-B and angiopoietin-like protein 4 from endothelial cells in a Plexin-B1/Rho-dependent manner, thereby influencing proliferation and differentiation of pericytes and vascular permeability, whereas VEGF lacks these effects. These results partly explain the differences observed between SEMA4D and VEGF in pathological angiogenesis and suggest that targeting SEMA4D function along with VEGF could represent a novel anti-angiogenic therapeutic strategy for the treatment of solid tumors.     

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.     

Expression of placenta growth factor is regulated by both VEGF and hyperglycaemia via VEGFR-2

Placenta growth factor (PlGF) has been implicated in both physiological and pathological angiogenesis; however, little is known about what regulates its expression. In this study, retinal microvascular endothelial cells and pericytes were exposed to varying concentrations of VEGF and glucose and PlGF expression measured by RT-PCR and Western blotting. Both PlGF mRNA and protein were observed in unstimulated microvascular endothelial cells with only weak expression in pericytes. In endothelial cells, VEGF (100 ng/ml) and glucose (15 mM) induced an increase in expression of PlGF at both the mRNA and protein level while no effect was observed for pericytes. The increase in PlGF expression could be totally abolished by blocking VEGFR-2, and in the case of glucose by neutralising VEGF. VEGF-stimulated PlGF expression was largely inhibited by PD 98059, an inhibitor of mitogen-activated protein kinase (MAPK) and partially by GF 109203X, an inhibitor of protein kinase C (PKC), indicating that VEGF up-regulates PlGF expression via the MAPK signalling pathway and partially through PKC. Taken together, our findings suggest that VEGF orchestrates the contribution of PlGF in angiogenesis via more than one intracellular pathway and that hyperglycaemia, as occurs in diabetes, is an important regulator of PlGF expression via VEGF up-regulation.