Lactoferrin induces growth arrest and nuclear accumulation of Smad-2 in HeLa cells

Lactoferrin (Lf) is a multifunctional glycoprotein. Due to its anti-inflammatory and anti-cancer properties and the resulting therapeutical potential, lactoferrin is at present focus of a variety of research areas. The regulation of cell growth represents one of the prominent performances of lactoferrin. In this study we found lactoferrin to inhibit proliferation of the human epithelial cancer cell line HeLa. The extent of this growth inhibition was comparable to the one induced by the transforming-growth-factor-beta-1 (TGFβ1). In contrast to other cell lines where lactoferrin stimulates growth, lactoferrin failed to activate the MAP kinases ERK1/2 or p38 in HeLa cells. However, by immunocytochemistry and cell fractionation experiments, we found that lactoferrin is capable of activating the TGFβ/Smad-2 pathway. The nuclear accumulation of Smad-2 induced by Lf was comparable in magnitude to the one induced by TGFβ1.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.     

A satellite cell mitogen from crushed adult muscle

Single fiber-satellite cell units from skeletal muscle of adult rats were used to study the regulation of satellite cell proliferation. The satellite cells remained quiescent during culture in serum-containing medium but could be induced to enter the cell cycle by exposure to a saline extract of crushed adult muscle. The activity in the extract has a molecular weight greater than 30K and is heat and trypsin sensitive. The mitogenic activity does not result from transferrin. Little or no activity was obtained from crushed extracts of heterologous tissues. Proliferation of myogenic cells from rat embryos was also stimulated by the muscle mitogen but growth of muscle fibroblasts was not enhanced. The time response of satellite cell proliferation after exposure to the muscle mitogen showed that the cells enter DNA synthesis after a lag period of 18 hr and proliferate with a generation time of 12 hr. This confirms that satellite cells in adult muscle are in G0, or an extended G1. The mitogen is also effective in stimulating muscle growth and myoblast fusion in vivo when injected into 1-week-old rat pups. These experiments suggest that muscle regeneration is initiated by the release of an endogenous mitogen from traumatized muscle.     

A role for platelet-derived growth factor beta-receptor in a newborn rat model of endothelin-mediated pulmonary vascular remodeling

Newborn rats exposed to 60% O2 for 14 days develop endothelin (ET)-1-dependent pulmonary hypertension with vascular remodeling, characterized by increased smooth muscle cell (SMC) proliferation and medial thickening of pulmonary resistance arteries. Using immunohistochemistry and Western blot analyses, we examined the effect of exposure to 60% O2 on expression in the lung of receptors for the platelet-derived growth factors (PDGF), which are implicated in the pathogenesis of arterial smooth muscle hyperplasia. We observed a marked O2-induced upregulation of PDGF-alpha and -beta receptors (PDGF-alphaR and -betaR) on arterial smooth muscle. This led us to examine pulmonary vascular PDGF receptor expression in 60% O2-exposed rats given SB-217242, a combined ET receptor antagonist, which we found prevented the O2-induced upregulation of PDGF-betaR, but not PDGF-alphaR, on arterial smooth muscle. PDGF-BB, a major PDGF-betaR ligand, was found to be a potent in vitro inducer of hyperplasia and DNA synthesis in cultured pulmonary artery SMC from infant rats. A critical role for PDGF-betaR ligands in arterial SMC proliferation was confirmed in vivo using a truncated soluble PDGF-betaR intervention, which attenuated SMC proliferation induced by exposure to 60% O2. Collectively, these data are consistent with a major role for PDGF-betaR-mediated SMC proliferation, acting downstream of increased ET-1 in a newborn rat model of 60% O2-induced pulmonary hypertension.     

Lactoferrin promotes bone growth

We have demonstrated bovine or human lactoferrin to be an anabolic factor in skeletal tissue. In vitro, lactoferrin stimulates the proliferation of bone forming cells, osteoblasts, and cartilage cells at physiological concentrations (above 0.1 microg/ml). The magnitude of this effect exceeds that observed in response to other skeletal growth factors such as IGF-1 and TGFbeta. DNA synthesis is also stimulated in a bone organ culture system likely reflecting the proliferation of cells of the osteoblast lineage. Lactoferrin is also a potent osteoblast survival factor. In TUNEL and DNA fragmentation assays, lactoferrin decreased apoptosis, induced by serum withdrawal, by up to 70%. In addition, lactoferrin has powerful effects on bone resorbing cells, osteoclasts, decreasing osteoclast development at concentrations > 1 microg/ml in a murine bone marrow culture system. However, lactoferrin did not alter bone resorption in calvarial organ culture, suggesting that it does not influence mature osteoclast function. In vivo, local injection of lactoferrin in adult mice resulted in increased calvarial bone growth, with significant increases in bone area and dynamic histomorphometric indices of bone formation after only 5 injections. Taken together, these data demonstrate that the naturally-occurring glycoprotein lactoferrin is anabolic to bone in vivo, an effect which is consequent upon its potent proliferative and anti-apoptotic actions in osteoblasts, and its ability to inhibit osteoclastogenesis. Lactoferrin may therefore have a physiological role in bone growth, and a potential therapeutic role in osteoporosis.     

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 g/ml lactoferrin, 10M ethanolamine, 35 /ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.Abbreviations MoAb monoclonal antibody     

Early-age heat exposure affects skeletal muscle satellite cell proliferation and differentiation in chicks

Exposure of young chicks to thermal conditioning (TC; i.e., 37 degrees C for 24 h) resulted in significantly improved body and muscle growth at a later age. We hypothesized that TC causes an increase in satellite cell proliferation, necessary for further muscle hypertrophy. An immediate increase was observed in satellite cell DNA synthesis in culture and in vivo in response to TC of 3-day-old chicks to levels that were significantly higher than those of control chicks. This was accompanied by a marked induction of insulin-like growth factor-I (IFG-I), but not hepatocyte growth factor in the breast muscle. No significant difference between treatments in plasma IGF-I levels was observed. A marked elevation in muscle regulatory factors on day 5, followed by a decline in cell proliferation on day 6 together with continuous high levels of IGF-I in the TC chick muscle may indicate accelerated cell differentiation. These data suggest a central role for IGF-I in the immediate stimulation of satellite cell myogenic processes in response to heat exposure.     

Inhibition of rat smooth muscle proliferation by radiation after arterial injury: temporal characteristics in vivo and in vitro

Although several studies have suggested that inhibition of arterial narrowing by radiation after angioplasty is dependent on both time and dose, little is known regarding the temporal aspects of this effect and the mechanisms by which radiation affects the response of smooth muscle cells to injury. To determine the time course of inhibition of intimal hyperplasia by radiation, 135 rats were given single-fraction external gamma irradiation (1-10 Gy) to one carotid artery at intervals from 5 days before to 5 days after bilateral carotid artery balloon catheter injury, and intimal cross-sectional area was determined from histological sections at 20 days after injury. There was a prominent time- and dose-dependent inhibition of intimal hyperplasia by radiation when it was administered before or after balloon injury, with the greatest effect noted within 24 h before or after injury. To investigate the effect of radiation on smooth muscle cell growth (by cell counting) and proliferation, cell cycle kinetics (by BrdU incorporation), and cell killing (by clonogenic assay), smooth muscle cell cultures derived from rat aortic explants were seeded in equine plasma to induce quiescence, and radiation (2.5-10 Gy) was administered at various intervals before or after synchronous growth stimulation by 10% whole blood serum. A similar time and dose dependence was noted in growth kinetics, BrdU incorporation and cell killing for smooth muscle cells irradiated in vitro; in each case, the effect was most prominent for radiation administered in temporal proximity to stimulation with whole blood serum. By Western blot analysis, cultured smooth muscle cells showed a rapid time-dependent increase in Cdkn1a (formerly known as p21) protein expression, followed by a delayed increase in Tp53 (formerly known as p53) expression after irradiation. Activation of intracellular caspases, manifest by proteolytic poly(ADP-ribose) polymerase (PARP) cleavage, was not detected in smooth muscle cell cultures after irradiation. These observations suggest that radiation limits intimal hyperplasia in vivo by a transient, reversible process. Although apparent cytotoxic injury occurs in vitro, apoptosis of smooth muscle cells is not apparent. Both inhibition of proliferation of smooth muscle cells and cell cycle delay may contribute to inhibition of intimal hyperplasia in vivo by radiation.     

Lactoferrin and bone; structure-activity relationships.

The maintenance of the mechanical integrity of the skeleton depends on bone remodeling, the well-coordinated balance between bone formation by osteoblasts and bone resorption by osteoclasts. The coupled action of osteoblasts and osteoclasts is regulated by the action of many local and circulating hormones and factors as well as central regulation by a neurological mechanism. We have previously shown that lactoferrin can promote bone growth. At physiological concentrations, lactoferrin potently stimulates the proliferation and differentiation of primary osteoblasts and acts as a survival factor. Lactoferrin also affects osteoclasts, potently inhibiting their formation. In vivo, local injection of lactoferrin results in substantial increases in bone formation and bone area. In a critical bone-defect model in vivo, lactoferrin was also seen to promote bone growth. The mitogenic effect of lactoferrin in osteoblast-like cells is mediated mainly through low-density lipoprotein-receptor protein-1 (LRP1), a member of the low-density lipoprotein-receptor-related proteins that are primarily known as endocytic receptors; however, LRP1 is not necessary for the anti-apoptotic actions of lactoferrin. Lactoferrin also induces the activation of p42/44 mitogen-activated protein kinase (MAPK) signalling and the PI3-kinase-dependent phosphorylation of Akt in osteoblasts. In this study, we examined other properties of lactoferrin and the way they affect osteogenic activity. The degree of glycosylation, iron-binding, and the structure-activity relationships indicate that lactoferrin maintains osteogenic activity in deglycosylated, holo, and apo forms, and in with various small fragments of the molecule. These data suggest that lactoferrin signals through more than 1 membrane-bound receptor to produce its anabolic skeletal effects, and that it signals through diverse pathways. We conclude that lactoferrin might have a physiological role in bone growth and healing and a potential therapeutic role as an anabolic factor in osteoporosis.     

Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Regulation of satellite cells during skeletal muscle growth and development

Satellite cells are myogenic cells attributed with the role of postnatal growth and regeneration in skeletal muscle. Following proliferation and subsequent differentiation, these cells will fuse with one another or with the adjacent muscle fiber, thereby increasing myonuclei numbers for fiber growth and repair. The potential factors which could regulate this process are many, including exercise, trauma, passive stretch, innervation, and soluble growth factors. Three classes of growth factors in particular (fibroblast growth factor, insulin-like growth factor, and transforming growth factor-beta) have been studied extensively with respect to their effects on satellite cell proliferation and differentiation in culture. Fibroblast growth factor has been shown to stimulate proliferation but depress differentiation. Insulin-like growth factor stimulates both proliferation and differentiation, although the latter to a much greater degree. Transforming growth factor-beta slightly depresses proliferation but inhibits differentiation. When administered in combination, these factors can induce satellite cell activities in culture which mimic those typical of satellite cells found in vivo in growing, regenerating, or healthy mature muscle. Alterations in the concentrations of these growth factors in the muscle environment as well as alterations in the cell's sensitivity or responsiveness to these factors represent potential mechanisms for regulating satellite cell activity in situ.     

Lactoferrin as an effector molecule in the skeleton

Lactoferrin is a pleiotropic factor with potent antimicrobial and immunomodulatory activities. In recent years, studies have shown that lactoferrin also acts on the skeleton to promote bone growth. Lactoferrin stimulates the proliferation and differentiation of the bone forming cells, the osteoblasts, and acts as a survival factor for these cells. Lactoferrin also inhibits osteoclastogenesis, reducing the number of cells that can actively resorb bone, thus producing a greater overall increase in bone volume. In vivo, local injection of lactoferrin results in substantial increases in bone area, establishing lactoferrin as an effector molecule in the skeleton. Investigations of the mechanism of action of lactoferrin in bone cells showed that the mitogenic effect of lactoferrin in osteoblasts is mediated mainly through LRP1, a member of the low density lipoprotein receptor-related proteins. Lactoferrin induces activation of p42/44 MAPK signaling as well as PI3-kinase-dependent phosphorylation of Akt in osteoblasts. Differential gene expression studies indicated a possible role for the activation of IGF1, Ptgs2 and Nfatc1 in mediating the mitogenic activity of lactoferrin in osteoblasts. Lactoferrin is a positive regulator of bone with a possible physiological role in bone growth and healing. There is a growing interest in the potential use of lactoferrin for the improvement of bone health, and in a number of recent studies dietary lactoferrin supplementation improved bone mineral density and bone strength. Lactoferrin appears to be a promising candidate for the development of an anabolic therapeutic factor for osteoporosis.     

Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy

Skeletal muscles adapt to increasing workload by augmenting their fiber size, through mechanisms that are poorly understood. This study identifies the cytokine interleukin-6 (IL-6) as an essential regulator of satellite cell (muscle stem cell)-mediated hypertrophic muscle growth. IL-6 is locally and transiently produced by growing myofibers and associated satellite cells, and genetic loss of IL-6 blunted muscle hypertrophy in vivo. IL-6 deficiency abrogated satellite cell proliferation and myonuclear accretion in the preexisting myofiber by impairing STAT3 activation and expression of its target gene cyclin D1. The growth defect was indeed muscle cell intrinsic, since IL-6 loss also affected satellite cell behavior in vitro, in a STAT3-dependent manner. Myotube-produced IL-6 further stimulated cell proliferation in a paracrine fashion. These findings unveil a role for IL-6 in hypertrophic muscle growth and provide mechanistic evidence for the contribution of satellite cells to this process.     

Ovariectomy causes cell proliferation and matrix synthesis in the growth plate cartilage of the adult rat

The in vivo effects of ovariectomy in rats have been studied on cell proliferation and matrix synthesis in the growth plate cartilage by assessing immunohistochemically the levels of proliferating cell nuclear antigen and chondroitin sulphate proteoglycan(s). The serum levels of insulin-like growth factor-I and growth hormone were also measured by radioimmunoassay procedures. At 5 weeks after ovariectomy, the serum levels of the growth factor were significantly higher than those in sham-operated rats. In contrast, the level of growth hormone was lower. The nuclear staining of proliferating cell nuclear antigen was generally seen in the zone of proliferative chondrocytes from both groups of rats. Whereas almost all chondrocytes in the proliferative zone of ovariectomized rats expressed proliferating cell nuclear antigen immunoreactivity, fewer did so in that of the sham rats. Quantitative image analysis by ACAS 570 laser cytometry demonstrated that the n uclear antigen-positive sites in ovariectomized rats had significantly higher integrated values (staining intensity), areas and perimeters than those in sham rats. In addition, the number of chondroitin sulphate proteoglycan-immunoreactive cells in the proliferative chondrocytes was also higher in ovariectomized rats than in sham ones. These results suggest that ovariectomy significantly stimulates the cell proliferation and matrix synthesis in the growth plate cartilage, probably through the higher serum level of insulin-like growth factor-I.     

Srf-dependent paracrine signals produced by myofibers control satellite cell-mediated skeletal muscle hypertrophy

Adult skeletal muscles adapt their fiber size to workload. We show that serum response factor (Srf) is required for satellite cell-mediated hypertrophic muscle growth. Deletion of Srf from myofibers and not satellite cells blunts overload-induced hypertrophy, and impairs satellite cell proliferation and recruitment to pre-existing fibers. We reveal a gene network in which Srf within myofibers modulates interleukin-6 and cyclooxygenase-2/interleukin-4 expressions and therefore exerts a paracrine control of satellite cell functions. In Srf-deleted muscles, in vivo overexpression of interleukin-6 is sufficient to restore satellite cell proliferation but not satellite cell fusion and overall growth. In contrast cyclooxygenase-2/interleukin-4 overexpression rescue satellite cell recruitment and muscle growth without affecting satellite cell proliferation, identifying altered fusion as the limiting cellular event. These findings unravel a role for Srf in the translation of mechanical cues applied to myofibers into paracrine signals, which in turn will modulate satellite cell functions and support muscle growth.     

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.     

The low-density lipoprotein receptor-related protein 1 is a mitogenic receptor for lactoferrin in osteoblastic cells

Lactoferrin induces osteoblast proliferation and survival in vitro and is anabolic to bone in vivo. The molecular mechanisms by which lactoferrin exerts these biological actions are not known, but lactoferrin is known to bind to two members of the low-density lipoprotein receptor family, low- density lipoprotein receptor-related proteins 1 (LRP1) and 2 (LRP2). We have examined the role(s) of these receptors in the actions of lactoferrin on osteoblasts. We show that lactoferrin binds to cultured osteoblastic cells, and that LRP1 and LRP2 are expressed in several osteoblastic cell types. In primary rat osteoblastic cells, the LRP1/2 inhibitor receptor associated protein blocks endocytosis of lactoferrin and abrogates lactoferrin-induced p42/44 MAPK signaling and mitogenesis. Lactoferrin-induced mitogenesis is also inhibited by an antibody to LRP1. Lactoferrin also induces receptor associated protein-sensitive activation of p42/44 MAPK signaling and proliferation in osteoblastic human SaOS-2 cells, which express LRP1 but not LRP2. The mitogenic response of LRP1-null fibroblastic cells to lactoferrin is substantially reduced compared with that of cells expressing wild-type LRP1. The endocytic and signaling functions of LRP1 are independent of each other, because lactoferrin can activate mitogenic signaling in conditions in which endocytosis is inhibited. Taken together, these results 1) suggest that mitogenic signaling through LRP1 to p42/44 MAPKs contributes to the anabolic skeletal actions of lactoferrin; 2) demonstrate growth-promoting actions of a third LRP family member in osteoblasts; and 3) provide further evidence that LRP1 functions as a signaling receptor in addition to its recognized role in ligand endocytosis.     

IGF-I restores satellite cell proliferative potential in immobilized old skeletal muscle.

One of the key factors responsible for the age-associated reduction in muscle mass may be that satellite cell proliferation potential (number of doublings contained within each cell) could become rate limiting to old muscle regrowth. No studies have tested whether repeated cycles of atrophy-regrowth in aged animals deplete the remaining capacity of satellite cells to replicate or what measures can be taken to prevent this from happening. We hypothesized that there would be a pronounced loss of satellite cell proliferative potential in gastrocnemius muscles of aged rats (25- to 30-mo-old FBN rats) subjected to three cycles of atrophy by hindlimb immobilization (plaster casts) with intervening recovery periods. Our results indicated that there was a significant loss in gastrocnemius muscle mass and in the proliferative potential of the resident satellite cells after just one bout of immobilization. Neither the muscle mass nor the satellite cell proliferation potential recovered from their atrophied values after either the first 3-wk or later 9-wk recovery period. Remarkably, application of insulin-like growth factor I onto the atrophied gastrocnemius muscle for an additional 2 wk after this 9-wk recovery period rescued approximately 46% of the lost muscle mass and dramatically increased proliferation potential of the satellite cells from this muscle.     

Early treatment with fumagillin, an inhibitor of methionine aminopeptidase-2, prevents Pulmonary Hypertension in monocrotaline-injured rats

Pulmonary Hypertension (PH) is a pathophysiologic condition characterized by hypoxemia and right ventricular strain. Proliferation of fibroblasts, smooth muscle cells, and endothelial cells is central to the pathology of PH in animal models and in humans. Methionine aminopeptidase-2 (MetAP2) regulates proliferation in a variety of cell types including endothelial cells, smooth muscle cells, and fibroblasts. MetAP2 is inhibited irreversibly by the angiogenesis inhibitor fumagillin. We have previously found that inhibition of MetAP2 with fumagillin in bleomycin-injured mice decreased pulmonary fibrosis by selectively decreasing the proliferation of lung myofibroblasts. In this study, we investigated the role of fumagillin as a potential therapy in experimental PH. In vivo, treatment of rats with fumagillin early after monocrotaline injury prevented PH and right ventricular remodeling by decreasing the thickness of the medial layer of the pulmonary arteries. Treatment with fumagillin beginning two weeks after monocrotaline injury did not prevent PH but was associated with decreased right ventricular mass and decreased cardiomyocyte hypertrophy, suggesting a direct effect of fumagillin on right ventricular remodeling. Incubation of rat pulmonary artery smooth muscle cells (RPASMC) with fumagillin and MetAP2-targeting siRNA inhibited proliferation of RPASMC in vitro. Platelet-derived growth factor, a growth factor that is important in the pathogenesis of PH and stimulates proliferation of fibroblasts and smooth muscle cells, strongly increased expression of MetP2. By immunohistochemistry, we found that MetAP2 was expressed in the lesions of human pulmonary arterial hypertension. We propose that fumagillin may be an effective adjunctive therapy for treating PH in patients.