Pigment epithelium-derived factor (PEDF)-induced apoptosis and inhibition of vascular endothelial growth factor (VEGF) expression in MG63 human osteosarcoma cells

Pigment epithelium-derived factor (PEDF) has been shown to be the most potent inhibitor of angiogenesis in the mammalian eye, thus suggesting that loss of PEDF is involved in angiogenic eye diseases such as proliferative diabetic retinopathy. Angiogenesis is required for tumor growth and progression as well. We, along with others, have recently found that PEDF could inhibit growth of melanoma and hepatocellular carcinoma in nude mice through its anti-angiogenic effects on tumor endothelial cells. However, the possibility of the direct effect of PEDF on tumor cells has remained. In this study, we investigated the effects of PEDF on growth and vascular endothelial growth factor (VEGF) expression in MG63 human cultured osteosarcoma cells. PEDF decreased viable cell number as well as DNA synthesis in MG63 cells in a dose-dependent manner. Furthermore, PEDF was found to increase caspase-3/7 activity and to subsequently induce apoptotic cell death in MG63 cells. PEDF also inhibited VEGF expression in MG63 cells at both mRNA and protein levels. Our present study provides novel beneficial aspects of PEDF on osteosarcoma cells; one is induction of apoptotic cell death of tumor cells, and the other is the suppression of VEGF expression, which would lead to inhibition of tumor angiogenesis. PEDF therefore might be a promising therapeutic agent for treatment of patients with osteosarcoma.     

Pigment epithelium-derived factor plays an inhibitory role in proliferation and migration of HaCaT cells

The normal vasculature is maintained by a balance between angiogenic factors and anti-angiogenic factors. Recent studies have shown that pigment epithelium-derived factor (PEDF) can induce differentiation and inhibit angiogenesis of tumors. This study was designed to investigate the expression of PEDF and its roles in proliferation, adhesion and migration of HaCaT cells, a human keratinocyte cell line. Our results have shown that PEDF is expressed in HaCaT cells at both mRNA and protein levels determined by RT-PCR and Western blot, separately. PEDF signal mainly localizes in the cytoplasm of HaCaT cell, as determined by immunofluorescence. Furthermore, expression of PEDF is decreased by 50 ng/ml of VEGF₁₆₅. Proliferation and migration of HaCaT cells are decreased by PEDF, while adhesion of HaCaT cells is upregulated approximately by 29%. PEDF also induce the S phase accumulation of HaCaT cells. In addition, phosphorylation of ERK1/2, not JNK and p38, is decreased by PEDF. These results indicate that PEDF may play an inhibitory role on growth and migration of HaCaT cells through dephosphorylation of ERK1/2.     

Vascular endothelial growth factor upregulates pigment epithelium-derived factor expression via VEGFR-1 in human retinal pigment epithelial cells

We previously demonstrated that differentiated retinal pigment epithelial (RPE) cells express high levels of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), and a critical balance between VEGF and PEDF is important to prevent the development of choroidal neovascularization. We report here that VEGF secreted by RPE cells upregulates PEDF expression via VEGFR-1 in an autocrine manner. PEDF mRNA and protein expression was downregulated by neutralizing antibody against VEGF in differentiated human RPE cells. VEGFR-1 neutralization decreased PEDF mRNA and protein expression whereas anti-VEGFR-2 antibody had no effect. Addition of placenta growth factor (PlGF) restored PEDF expression in the presence of anti-VEGF antibody. These results demonstrate a regulatory interaction between angiogenesis stimulators and inhibitors to maintain homeostasis in normal human retina.     

The ratio of VEGF/PEDF expression in bone marrow mesenchymal stem cells regulates neovascularization

Angiogenesis, or neovascularization, is a finely balanced process controlled by pro- and anti-angiogenic factors. Vascular endothelial growth factor (VEGF) is a major pro-angiogenic factor, whereas pigment epithelial-derived factor (PEDF) is the most potent natural angiogenesis inhibitor. In this study, the regulatory role of bone marrow stromal cells (BMSCs) during angiogenesis was assessed by the endothelial differentiation potential, VEGF/PEDF production and responses to pro-angiogenic and hypoxic conditions. The in vivo regulation of blood vessel formation by BMSCs was also explored in a SCID mouse model. Results showed that PEDF was expressed more prominently in BMSCs compared to VEGF. This contrasted with human umbilical vein endothelial cells (HUVECs) where the expression of VEGF was higher than that of PEDF. The ratio of VEGF/PEDF gene expression in BMSCs increased when VEGF concentration reached 40ng/ml in the culture medium, but decreased at 80ng/ml. Under CoCl(2)-induced hypoxic conditions, the VEGF/PEDF ratio of BMSCs increased significantly in both normal and angiogenic culture media. There was no expression of endothelial cell markers in BMSCs cultured in either pro-angiogenic or hypoxia culture conditions when compared with HUVECs. The in vivo study showed that VEGF/PEDF expression closely correlated with the degree of neovascularization, and that hypoxia significantly induced pro-angiogenic activity in BMSCs. These results indicate that, rather than being progenitors of endothelial cells, BMSCs play an important role in regulating the neovascularization process, and that the ratio of VEGF and PEDF may, in effect, be an indicator of the pro- or anti-angiogenic activities of BMSCs.     

PEDF: anti-angiogenic guardian of ocular function

Sight-threatening eye diseases can be caused and exacerbated by the aberrant growth of new blood vessels. Recent work indicates that this neovascularization not only is a response to a rise in the local concentration of molecules that induce such angiogenesis but also requires a fall in the levels of endogenous molecules that inhibit angiogenesis. One of the most potent of these endogenous inhibitors is pigment epithelium-derived factor (PEDF), which serves as a survival factor for neuronal components of the eye as well as an essential inhibitor of the growth of ocular blood vessels. Its anti-angiogenic activity is selective in that it is effective against newly forming vessels but spares existing ones, and it is reversible. The molecular basis for this delicate control of endothelial cells is beginning to be understood and strategies to test the ability of PEDF to ameliorate or prevent vessel damage in the eye are developing rapidly.     

Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism

Background

Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.

Methodology

CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68) of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7), samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.

Principal Findings

Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.

Conclusions

This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.     

Ischemia/reperfusion in rat heart induces leptin and leptin receptor gene expression

Concentrations of leptin, an adipocyte-derived hormone, are elevated in obesity. Recently, leptin has been shown to participate in multiple biological actions including inflammation, reproduction, and angiogenesis. Leptin has also been documented as a critical component in the process of wound healing; however, leptin involvement in cardiovascular disease is poorly understood. We examined the expression of leptin (ob) and leptin receptor (ob-R) genes in the rat heart following ischemia/reperfusion, which was induced by coronary artery ligation, and mRNA was obtained from hearts 0.5 to 36 h after initiating reperfusion. Expressions of ob and ob-R mRNA were examined by real-time quantitative RT-PCR and immunohistochemistry. The ob and ob-Ra mRNA and protein expressions were significantly increased (p<0.01) and ob-Rb mRNA was significantly decreased (p<0.01) in hearts after 8 h of reperfusion. Furthermore, ob and ob-R proteins were expressed in injured myocytes where inflammatory cells infiltrated. In contrast, those expressions were not influenced in hearts after 8 h of ischemia stress only. To determine the functional effects of leptin on the ischemic/reperfused heart, rats were treated with anti-leptin antibodies prior to ischemia/reperfusion; however, this treatment did not affect the elevation of mRNA expression levels of inflammatory markers such as TNF-alpha and IL-1beta in ischemic hearts. Our results demonstrated for the first time that ischemia/reperfusion induced leptin and leptin receptor gene expression in the rat heart. This study helps to elucidate the mechanisms behind the onset and development of ischemic heart disease concomitant with obesity.     

The ATP-binding cassette transporter ABCG2 (BCRP), a marker for side population stem cells, is expressed in human heart.

Efforts to improve severely impaired myocardial function include transplantation of autologous hematopoietic side population (SP) stem cells. The transmembrane ABC-type (ATP binding cassette) half-transporter ABCG2 (BCRP) serves as a marker protein for SP cell selection. We have recently shown that other ABC transport proteins such as ABCB1 and ABCC5 are differentially expressed in normal and diseased human heart. Here we investigated localization and individual ABCG2 expression in 15 ventricular (including 10 cardiomyopathic) and 51 auricular heart tissue samples using immunohistochemistry, confocal laser scanning fluorescence microscopy, and real-time RT-PCR. Individual genotypes were assigned using PCR-restriction fragment length polymorphism (RFLP) analysis and subsequently correlated to ABCG2 mRNA levels. ABCG2 was localized in endothelial cells of capillaries and arterioles of all samples. Ventricular samples from cardiomyopathic hearts exhibited significantly increased levels of ABCG2 mRNA (ABCG2/18S rRNA: 1.08 +/- 0.30 x 10(-7); p=0.028 (dilative cardiomyopathy) and 1.16 +/- 0.46 x 10(-7); p=0.009 (ischemic cardiomyopathy) compared with 0.44 +/- 0.26 x 10(-7) in nonfailing hearts). The individual haplotypes were not associated with altered mRNA expression. ABCG2 is variably expressed in endothelial cells of human heart, where it may function as a protective barrier against cardiotoxic drugs such as anthracyclines or mitoxantrone. ABCG2 expression is induced in dilative and ischemic cardiomyopathies.     

Expression of pigment-epithelium-derived factor during kidney development and aging

Inhibitors and stimulators of endothelial cell growth are essential for the coordination of blood vessel formation during organ growth and development. In the adult kidney, one of the major inhibitors of angiogenesis is pigment-epithelium-derived factor (PEDF). We have analyzed the expression and distribution of PEDF during various stages of renal development and aging with particular emphasis on the formation of functional glomeruli. We show that PEDF gene expression and protein levels in the kidney significantly increase with age. We have detected PEDF in the mesenchyme and endothelial cells at all developmental stages studied, in all regions of the nephrogenic zone in which the formation of new blood vessels is associated with the development of nephrons and collecting ducts, and in mature podocytes in the adult kidney. Our results are the first to suggest that PEDF is important in early renal postnatal development, that it could be relevant to the maturation of glomerular function and the filtration barrier formed by these cells, and that it may serve as an anti-angiogenic modulator during kidney development. Ana Luisa Pina and Marion Kubitza contributed equally to this work.     

Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis

Pigment epithelium-derived factor (PEDF), a recently defined retinal trophic factor and anti-angiogenic factor for the eye, is also present in the CNS and is a motor neuron protectant. We asked whether PEDF levels in CSF are altered in patients with amyotrophic lateral sclerosis (ALS). Pigment epithelium-derived factor protein was detected by quantitative western blot analysis with a PEDF-specific antiserum. Levels of PEDF in CSF, expressed as a ratio to total CSF protein, were significantly elevated 3.4-fold in 15 patients with ALS compared with neurologic disease controls (p < 0.0003). This increase does not seem likely to reflect up-regulation of PEDF synthesis in muscle in response to denervation, as CSF PEDF was not elevated in severe denervating diseases other than ALS. Nor does the increase represent some non-specific release in neurodegeneration, as CSF PEDF was not elevated in other neurodegenerative diseases. While the mechanism of this presumably reactive increase is not known, the distinctive, surprisingly elevated level of PEDF in the CSF may be an autoprotective reaction in ALS.     

The Ku protein complex interacts with YY1, is up-regulated in human heart failure, and represses alpha myosin heavy-chain gene expression

Human heart failure is accompanied by repression of genes such as alpha myosin heavy chain (alphaMyHC) and SERCA2A and the induction of fetal genes such as betaMyHC and atrial natriuretic factor. It seems likely that changes in MyHC isoforms contribute to the poor contractility seen in heart failure, because small changes in isoform composition can have a major effect on the contractility of cardiac myocytes and the heart. Our laboratory has recently shown that YY1 protein levels are increased in human heart failure and that YY1 represses the activity of the human alphaMyHC promoter. We have now identified a region of the alphaMyHC promoter that binds a factor whose expression is increased sixfold in failing human hearts. Through peptide mass spectrometry, we identified this binding activity to be a heterodimer of Ku70 and Ku80. Expression of Ku represses the human alphaMyHC promoter in neonatal rat ventricular myocytes. Moreover, overexpression of Ku70/80 decreases alphaMyHC mRNA expression and increases skeletal alpha-actin. Interestingly, YY1 interacts with Ku70 and Ku80 in HeLa cells. Together, YY1, Ku70, and Ku80 repress the alphaMyHC promoter to an extent that is greater than that with YY1 or Ku70/80 alone. Our results suggest that Ku is an important factor in the repression of the human alphaMyHC promoter during heart failure.     

PEDF: a potential molecular therapeutic target with multiple anti-cancer activities

Pigment epithelium-derived factor (PEDF) is an endogenously produced protein that is widely expressed throughout the human body, and exhibits multiple and varied biological activities. Already established as a potent anti-angiogenic molecule, PEDF has recently shown promise as a potential anti-tumour agent, causing both direct and indirect tumour suppression. Here, we explore the unique anti-tumour properties of PEDF and discuss its role as an effective anti-angiogenic, anti-proliferative and pro-differentiation factor. We also discuss the prospects for PEDF therapy and the need for a closer evaluation of issues such as delivery, stability and potential toxicity.     

Moderate levels of ethanol induce expression of vascular endothelial growth factor and stimulate angiogenesis

Alcohol abuse has a negative impact on human health; however, epidemiological studies show that moderate consumption of ethanol (EtOH) reduces the risk of coronary heart disease, sudden cardiac death, and ischemic stroke. The mechanisms for these reductions in cardiovascular disease are not well established. Using cultured coronary artery vascular smooth muscle cells, we found that moderate levels of EtOH (10 and 20 mM) caused dose-related increases in both vascular endothelial growth factor (VEGF) mRNA (Northern blot) expression (1.9- and 2.6-fold) and VEGF protein (ELISA) expression (19 and 68%) compared with control (P < 0.05). EtOH at 0.25 g. kg(-1). day(-1) (7 days) increased VEGF mRNA expression by 1.48-fold over control, and increased vessel length density from 3.9 +/- 0.7 (control) to 6.0 +/- 0.3 mm/mm(2) (P < 0.05) in chick chorioallantoic membrane (CAM). We conclude that moderate levels of ethanol can induce VEGF expression and stimulate angiogenesis in chick CAM. Therefore, the results provide a theoretical basis for speculating that the cardiovascular-protective effects of moderate alcohol consumption may be partly mediated through VEGF-induced angiogenesis.     

Expression and localization of P-glycoprotein in human heart: effects of cardiomyopathy.

ABC-type transport proteins, such as P-glycoprotein (P-gp), modify intracellular concentrations of many substrate compounds. They serve as functional barriers against entry of xenobiotics (e.g., in the gut or the blood-brain barrier) or contribute to drug excretion. Expression of transport proteins in the heart could be an important factor modifying cardiac concentrations of drugs known to be transported by P-gp (e.g., beta-blockers, cardiac glycosides, doxorubicin). We therefore investigated the expression and localization of P-gp in human heart. Samples from 15 human hearts (left ventricle; five non-failing, five dilated cardiomyopathy, and five ischemic cardiomyopathy) were analyzed for expression of P-gp using real-time RT-PCR, immunohistochemistry, and in situ hybridization. Immunohistochemistry revealed expression of P-gp in endothelium of both arterioles and capillaries of all heart samples. Although P-gp mRNA was detected in all samples, its expression level was significantly reduced in patients with dilated cardiomyopathy. We describe variable expression of P-gp in human heart and its localization in the endothelial wall. Thus, intracardiac concentrations of various compounds may be modified, depending on the individual P-gp level.     

Age-related changes of bradykinin B1 and B2 receptors in rat heart

Aging is a major risk factor for the development of vascular diseases, such as hypertension and atherosclerosis, that leads to end organ damage and especially heart failure. Bradykinin has been demonstrated to have a cardioprotective role by affecting metabolic processes and tissue perfusion under conditions of myocardial ischemia. Its actions are exerted via the bradykinin B1- and B2-type receptors (B1Rs and B2Rs), but the functional status of these receptors during the aging process is poorly understood. This study aims to investigate whether changes in B1R and B2R gene and protein expression in rat heart are associated with the age-related alterations of cardiac structure and function. Using real-time PCR, we found that B1R mRNA expression increased 2.9-fold in hearts of older rats (24 mo of age) compared with younger rats (3 mo of age), whereas B2R gene expression remained unchanged. Western blot analysis showed that expression of B2R at the protein level is approximately twofold higher in young rats compared with old rats, whereas the B1R protein is approximately twofold higher in old rats compared with young rats. The present results provide clear functional and molecular evidence that indicate age-related changes of bradykinin B1Rs and B2Rs in heart. Because the cardioprotective actions of bradykinin are physiologically mediated via the B2Rs, whereas the B1Rs become induced by tissue damage, these results suggest that age-related decreases in B2R protein levels may leave the heart vulnerable to ischemic damage, and increases in B1R expression and activity may represent a compensatory reaction in aging hearts.     

Low over-expression of TNFalpha in the mouse heart increases contractile performance via TNFR1

TNFalpha is a cytokine wit pleiotropic functions in many organs. In the heart increased TNFalpha levels are not only associated with heart failure, but also, paradoxically, with protection from ischemic damage. To test whether the protective role of TNFalpha in the heart is concentration-dependent, we studied two mouse heart models with low (two- to threefold) over-expression of endogenous TNFalpha: mice deficient in a translational repressor of TNFalpha mRNA, TIA-1(-/-), and mice over-expressing human TNFalpha. Hearts lacking TIA-1 were characterized for their endogenous TNFalpha over-expression during normal Langendorff perfusion. To define which TNFalpha receptor mediates cardiac protection, we also used mice lacking the TNFR1 receptor. Contractile function was assessed in isolated hearts perfused in the isovolumic Langendorff mode during and following global no-flow ischemic stress and in response to varying extracellular [Ca(2+)] to determine their contractile response and Ca(2+) sensitivity. All hearts with low over-expression of TNFalpha, independent of human or murine origin, have improved contractile performance and increased Ca(2+) sensitivity (by 0.2-0.26 pCa). Hearts lacking TNFR1 have contractile performance equal to wild type hearts. Recovery from ischemia was greater in TIA-1(-/-) and was diminished in TNFR1(-/-). Better contractile function in TNFalpha over-expressing hearts is not due to improved cardiac energetics assessed as [ATP] and glucose uptake or to differences in expression of SERCA2a or calmodulin. We suggest that low levels of TNFalpha increase the Ca(2+) sensitivity of the heart via a TNFR1-mediated mechanism.     

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might represent a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.