Placenta growth factor and sFlt-1 as biomarkers in ischemic heart disease and heart failure: a review.

Coronary heart disease (CHD) and heart failure (HF) produce significant morbidity/mortality but identifying new biomarkers could help in the management of each. In this article, we summarize the molecular regulation and biomarker potential of PIGF and sFlt-1 in CHD and HF. PlGF is elevated during ischemia and some studies have shown PlGF, sFlt-1 or PlGF:sFlt-1 ratio, when used in combination with standard biomarkers, strengthens predictions of outcomes. sFlt-1 and PlGF are elevated in HF with sFlt-1 as a stronger predictor of outcomes. Although promising, we discuss additional study criteria needed to confirm the clinical usefulness of PlGF or sFlt-1 in the detection and management of CHD or HF.

XIAP protein is induced by placenta growth factor (PLGF) and decreased during preeclampsia in trophoblast cells.

Increased trophoblast apoptosis has been implicated in pregnancies complicated by fetal growth restriction and preeclampsia (EC). We investigated placenta growth factor (PLGF) signaling during trophoblast apoptosis in culture and X-linked inhibitor of apoptosis protein (XIAP) and apoptosis inducible factor (AIF) in the preeclamptic placenta at term was determined. Primary trophoblasts were isolated and serum starved to induce apoptosis. Placenta growth factor was added and apoptosis markers were determined. Term preeclamptic placentae were homogenized and the levels of XIAP and XAF1 protein were assessed. In the absence of serum, primary cultures of term trophoblast showed a 5-fold increase in apoptosis as determined by annexin V binding. The increase in apoptosis induced by serum deprivation was caspase-independent and could be significantly reduced (p < 0.02) with the addition of 10 ng/ml rh PLGF to the media. In addition, PLGF mediated increased protein expression of the anti-apoptotic XIAP as well as decreased expression of the pro-apoptotic AIF in the primary trophoblast. In preeclamptic placenta, we determined the concomitant decrease in XIAP RNA as well as decreased expression of the phosphorylated XIAP protein. These results were coupled with increased levels of the pro-apoptotic protein XAF1. Our results suggest that PLGF protects trophoblast from caspase independent apoptosis in culture by increasing XIAP production and deceasing AIF. Also, our data suggests that decreased activation of XIAP and increased XAF1 could be factors associated with the increased placental apoptosis observed in the preeclamptic placenta at term.

Dysregulation of promyelocytic leukemia (PML) protein expression in preeclamptic placentae.

Promyelocytic leukemia (PML) protein is a nucleoprotein that can regulate a variety of cellular stress responses. The aim of this study was to determine qualitative and quantitative changes in PML expression in preeclamptic placentae. Immunoblot, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and immunohistochemistry techniques were used to determine PML gene expression and localization in normal (n = 6) and preeclamptic (n = 6) placentae and primary cells. Promyelocytic leukemia protein was immunolocalized within nuclei of villus mesenchyme, but largely absent in trophoblast nuclei, with a trend for increased PML reactivity in preeclamptic placenta. Immunoblot analyses of nuclear extracts confirmed relative increases (approximately 3-fold) of PML expression in preeclamptic placentae (P < .05). Conversely, less PML messenger RNA (mRNA; approximately 2-fold) was detected in preeclamptic versus normal placental samples. In vitro, PML expression could be increased by hypoxia in cultured endothelial cells but not trophoblast. Increased PML protein expression in preeclamptic villi suggests it could contribute to decreased vascularity and placental growth and/or function.

Hypoxia increases placenta growth factor expression in human myocardium and cultured neonatal rat cardiomyocytes.

BACKGROUND: Placenta growth factor (PlGF) plays an important role in pathologic angiogenesis and is believed to be an independent biomarker in patients with coronary artery disease. However, little is known regarding the regulation of PlGF expression in heart tissue. METHODS: We determined expression changes in PlGF and its receptor, VEGFR1, in normal and abnormal biopsies from human cardiac allografts and in cardiomyocytes cultured under hypoxia or cyclical stretch conditions. RESULTS: Human donor myocardium and biopsies from allografts without fibrin deposits expressed PlGF and VEGFR1 mRNA. Biopsies (n = 7) with myocardial fibrin, elevated serum cardiac troponin I titers (p < 0.03) and cellular infiltrates (p < 0.05) expressed 1.6-fold more PlGF mRNA than biopsies from allografts without fibrin (n = 11; p < 0.05). PlGF protein was localized in cardiomyocytes, extracellular matrix and some microvessels in areas with fibrin deposition. VEGFR1 mRNA expression was not different between groups. Cultured neonatal rat cardiomyocytes constitutively expressed PlGF/VEGFR1 under normoxia. PlGF expression was increased 3.88 +/- 0.62-fold after 12 hours (n = 6; p

Glial cell missing 1 regulates placental growth factor (PGF) gene transcription in human trophoblast.

Placental growth factor (PGF, previously known as PlGF) is prominently expressed by trophoblasts in human placenta, whereas most nontrophoblast cells express low levels of PGF mRNA under normal physiological conditions. We have shown that hypoxia decreases PGF expression in the trophoblast, but little is known about transcriptional regulation of PGF gene expression. We sought to determine promoter regions of the human PGF gene that contribute to its restricted high constitutive expression in the trophoblast. Overlapping putative promoter regions of human PGF gene encompassing 2-1.5 kb were cloned into reporter vectors and co-transfected into trophoblast and nontrophoblast cell lines. Promoter activity generated by a 2-1.5-kb clone was significantly higher in trophoblasts than in nontrophoblasts. Selective deletion mutants showed that a clone encompassing the PGF (2-828/++34) region generated promoter activity similar to the 2-1.5-kb region in the trophoblast. However, deletion of another 131 bp from this subclone (2-698/++34) resulted in significantly less promoter activity in the trophoblast. The (2-828/2-698) region significantly enhanced activity of a minimal promoter construct in trophoblast but not in nontrophoblast cells, suggesting that this region contributes to regulating PGF transcription in the trophoblast. Site-directed mutagenesis of a glial cell missing 1 (GCM1) motif in the 131-bp region significantly decreased enhancer activity in the trophoblast. Furthermore, overexpression of GCM1 significantly increased PGF 2-1.5-kb promoter activity and PGF mRNA expression in trophoblast and nontrophoblast cells. Forced overexpression of GCM1 restored PGF expression in the hypoxic trophoblast. These data support a functional role for GCM1 contributing to constitutively high trophoblast PGF expression and is the first direct evidence of an oxygen-responsive, trophoblast-specific transcription factor contributing to the regulation of PGF expression.

Angiogenesis in implantation.

PROBLEM: Implantation failure and early pregnancy loss are common following natural conceptions and they are particularly important clinical hurdles to overcome following assisted reproduction attempts. The importance of adequate vascular development and maintenance during implantation has recently become a major focus of investigation. MATERIALS AND METHODS: Review of current published literature was undertaken to summerize the cells and cell products that regulate tissue vascularity during implantation. RESULTS: Vascular development at the maternal fetal interface can be regulated by a number of different cell types; two principal candidates are trophoblast and natural killer cells. A wide range of soluble factors, some with well established angiogenic functions as well as other more novel factors, can contribute to vascular development and maintenance at the maternal-fetal interface. CONCLUSIONS: Robust vascular development occurs during implantation and early placentation of normal pregnancies. Studies to define the extent and mechanisms by which defects in vascularity contribute to human implantation failure and early miscarriage need to be undertaken.

Determinants of placental vascularity.

PROBLEM: Vascular growth during implantation and placentation is critical for successful gestation and it is thought that vascular insufficiencies during placentation contribute to a number of obstetrical complications. However, relatively little is known regarding the regulation of angiogenesis in the placenta. METHOD OF STUDY: We review literature concerning the potential significance of inadequate placental vascularity as a contributor to the obstetrical complications of spontaneous abortion, fetal growth restriction and preeclampsia. Gene expression assays were used to compare fluctuations of placenta growth factor (PlGF) and PlGF receptor expression in normal and preeclamptic trophoblast in vitro. RESULTS: Studies have shown that common obstetrical complications manifest altered placental vascularity. Both intrinsic defects (gene knockouts) and extrinsic factors (O(2) tension, cytokines, etc) may be responsible for the defects. Some of these factors have been shown to influence trophoblast vascular endothelial growth factor (VEGF)/PlGF expression suggesting this particular family of angiogenic proteins play an important role in placental angiogenesis. CONCLUSION: Placental vascularization reflects a complex interaction of regulatory factors. Understanding the regulation of vascular growth in the placenta will provide much needed insight into placenta-related vascular insufficiencies.

Altered expression of vascular endothelial growth factor and its receptors in normal saphenous vein and in arterialized and stenotic vein grafts.

BACKGROUND: Myointimal thickening is a major cause saphenous vein graft failure. The prominence of medial and adventitial microvessels in stenotic vein grafts and the known angiogenic effects of vascular endothelial growth factor (VEGF) lead us to investigate the expression of VEGF and its receptors in vein graft arterialization and stenosis. METHODS: Normal and arterialized vein graft segments were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) for expression of VEGF-R1 (flt), VEGF-R2 (KDR), and neuropilin-1. The cells expressing VEGF, VEGF-R1, VEGF-R2, and neuropilin-1 were identified in normal, stenotic, and arterialized vein graft segments by immunohistochemistry. RESULTS: Vascular endothelial growth factor, detected in the wall in endothelial cells and adventitial microvessels in normal vein, localized to smooth muscle cells, endothelial cells and adventitial microvessels in arterialized and stenotic vein. VEGF-R1 and VEGF-R2 were expressed infrequently on endothelial cells, macrophages, and smooth muscle cells in arterialized and stenotic vein. Neuropilin-1 was detected in all specimens. RT-PCR demonstrated significantly greater expression of neuropilin-1 in normal vein compared with arterialized vein (P <0.05). CONCLUSIONS: The differential expression of VEGF and its receptors in normal, arterialized, and stenotic vein grafts suggests that alterations in VEGF/VEGF-R2/neuropilin-1 interactions may be important determinants of the adaptive response of vein grafts to arterialization.

Evidence of a novel isoform of placenta growth factor (PlGF-4) expressed in human trophoblast and endothelial cells.

Placenta growth factor (PlGF), a homodimeric glycoprotein that is homologous to vascular endothelial growth factor (VEGF), is mitogenic to endothelial cells and protects trophoblast from apoptosis. Alternative splicing of mature mRNA gives rise to three known isoforms of PlGF. PlGF is expressed by human trophoblast during normal pregnancy, however, it is not known which isoforms are produced. We have utilized RT-PCR to characterize PlGF isoform expression in normal human trophoblast and umbilical vein endothelial cells (HUVEC). Our results show that PlGF-1, PlGF-2, and PlGF-3 isoforms are expressed by trophoblast and HUVECs. In addition, both cell types also express a novel variant of PlGF, tentatively termed PlGF-4, which has not been previously reported. PlGF-4 consists of the same sequence of PlGF-3, plus the heparin binding domain previously thought to be present only in PlGF-2. Presence of the heparin binding domain in PlGF-4 suggests that this variant would remain cell membrane-associated and thus could influence trophoblast and endothelial cells in an autocrine manner.

Expression and function of placenta growth factor: implications for abnormal placentation.

OBJECTIVE: Essential requirements for successful gestation include the coordinated growth and differentiation of the placenta and the development of a functional placental vasculature. However, relatively little is known about factors that are responsible for regulating these functions. One angiogenic growth factor that might be involved in regulating both vascular endothelial cell and trophoblast function is placental growth factor (PGF). METHODS: Current published reports were surveyed and our own work was reviewed to highlight the expression, function, and potential significance of PGF at the human maternal-fetal interface. RESULTS: PGF is highly expressed in trophoblasts during normal pregnancy, and its expression is significantly decreased in preeclampsia, an obstetric complication presumed to be associated with placental bed hypoxia and ischemia. In agreement with this, in vitro trophoblast expression of PGF can be down-regulated by low oxygen tension. The cognate receptor for PGF, fms-like tyrosine kinase receptor, is expressed on trophoblasts as well as vascular endothelial cells, suggesting that it has autocrine and paracrine functions. Accordingly, PGF can regulate proliferation in first trimester trophoblasts, apoptosis in term trophoblasts, and it can directly or indirectly regulate vascular growth, maturation, and permeability. CONCLUSIONS: Many obstetric complications, most notably preeclampsia, are associated with aberrant trophoblast function and inadequate or dysfunctional vasculature within the developing placenta. The ability of PGF to influence trophoblast and vascular endothelial cells provides clear impetus for further studies to investigate the biological and clinical significance of PGF in normal and abnormal human pregnancies.