Jonathan LaMarre

Thrombospondin-1 Inhibits VEGF Levels in the Ovary Directly by Binding and Internalization Via the Low Density Lipoprotein Receptor-Related Protein-1 (LRP-1)

VEGF is a potent pro‐angiogenic factor whose effects are opposed by a host of anti‐angiogenic proteins, including thrombospondin‐1 (TSP‐1). We have previously shown that VEGF has important extravascular roles in the ovary and that VEGF and TSP‐1 are inversely expressed throughout the ovarian cycle. To date, however, a causal interaction between TSP‐1 and VEGF has not been identified. Here, we show that TSP‐1 has a direct inhibitory effect on VEGF by binding the growth factor and internalizing it via LRP‐1. Mice lacking TSP‐1 are subfertile and exhibited ovarian hypervascularization and altered ovarian morphology. Treatment of ovarian cells with TSP‐1 decreased VEGF levels and rendered the cells more susceptible to TNFα‐induced apoptosis. Knockdown of TSP‐1, through RNA interference, resulted in overexpression of VEGF and reduced cytokine‐induced apoptosis. In conclusion, we demonstrate a direct inhibitory effect of TSP‐1 on VEGF in the ovary. TSP‐1s regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development. J. Cell. Physiol. 210: 807–818, 2007.

Thrombospondin and vascular endothelial growth factor are cyclically expressed in an inverse pattern during bovine ovarian follicle development

Abstract Angiogenesis does not normally occur in most adult tissues. However, in the ovary, there are cyclical vascular changes including angiogenesis that involve the interaction of numerous cytokines and growth factors. Angiogenic processes are regulated by a balance between pro- and antiangiogenic factors. The purpose of this study was to determine the expression of the antiangiogenic thrombospondin family and proangiogenic vascular endothelial growth factor (VEGF) in various sizes of healthy bovine follicles. Ovaries were collected from slaughterhouse animals and healthy follicles were sorted based on size (<0.5 cm, small; 0.5–1.0 cm, medium; >1.0 cm, large). Thrombospondin (TSP) protein levels were significantly higher in small follicles. Immunohistochemistry confirmed the granulosa layer as the primary area within the follicle involved in TSP generation and that small follicles had the highest proportion of immunopositive cells. TSP-1 and -2 mRNA levels were significantly higher in small follicles than either medium or large follicles. TSP colocalized with CD36 on granulosa cells (GC) in the follicle and in cultured cells. In contrast with TSP, VEGF expression increased during growth and development of the follicle. FSH stimulated GC expression of TSP, while LH had no effect. In summary, TSP-1 and -2 were coordinately expressed in the extravascular compartment of the ovary during early follicle development. VEGF was inversely expressed, with expression increasing as follicles developed. Regulated expression and localization of these proteins suggests that they may be involved in regulating growth and development of the follicle in a novel fashion.

α2‐Macroglobulin and the α2‐Macroglobulin Receptor/LRP A Growth Regulatory Axisa

The hypothesis that a2-macroglobulin ( a 2 M ) regulates the function of growth factors and cytokines in vivo is based on a number of distinct observations. First, a2M binds cytokines and these interactions are favored compared with other plasma proteincytokine interactions. Second, activated a2M, which has undergone conformational change due to reaction with primary amines or proteinases, targets cytokines to the surfaces of cells expressing the azM receptor (a2M receptorAow density lipoprotein receptor-related protein or a2MFULFW). Third, a2M regulates the activity of cytokines in vitro; the nature of the regulation is cell type-specific and may depend on whether the cell expresses a2MFULRP. Finally, cytokines modulate cellular expression of a2MR/LRP, thereby altering the ability of azM to affect the activity of other cytokines in the same cell type. These four principles constitute what we refer to as the a z M a2MR/LRP growth regulatory axis. In this report, we will present recent work related to each of these areas.

AU-rich elements and the control of gene expression through regulated mRNA stability.

Abstract The regulation of gene expression is a fundamental cellular process that is controlled at multiple levels. Abnormal regulation of gene expression has been directly implicated in the pathogenesis of some diseases of animals and humans and may contribute to the disease process in unrecognized ways in many others. Furthermore, novel treatment strategies for a number of different diseases may hinge upon our ability to exploit mechanisms that normally alter the expression of endogenous genes. While the study of gene regulation has traditionally focused on transcription as a major regulator of gene expression, it has recently become apparent that the post-transcriptional control of gene expression may play an equally important role. In particular, rapid, context-specific regulation of the stability of mRNA transcripts encoding highly active proteins, such as cytokines, growth factors, oncogenes and cell-cycle regulators, appears to play a key role in the control of these molecules and the processes they mediate. Many of the known regulatory pathways for mRNA stability involve proteins that interact with specific AU-rich elements in the 3′-untranslated region of the transcript. This review will address some important aspects of the normal regulation of mRNA stability and known or potential contributions of RNA stability regulation to health and disease.

Expression and Localization of Thrombospondin-1 and -2 and Their Cell-Surface Receptor, CD36, During Rat Follicular Development and Formation of the Corpus Luteum

Abstract Thrombospondin (TSP)-1 and -2 are extracellular matrix glycoproteins that are both antiangiogenic and important in regulating cellular development, differentiation, and function. To evaluate the expression of TSP in follicular and luteal development, ovarian cycles of Sprague-Dawley rats were synchronized and tissues collected daily at stages corresponding to the early antral, ovulatory, early luteal, and late luteal phases of the ovarian cycle. Immunohistochemistry and Western blot analyses demonstrated that TSP-1 protein and its receptor, CD36, were present in the early antral phase and were localized primarily to the granulosa cells of antral follicles. Both proteins were also present immediately after ovulation and were localized to the developing corpus luteum. Messenger RNA for TSP-1 showed a similar pattern, with expression at the early antral and ovulatory phases. Protein and mRNA expression for TSP-2 was relatively delayed compared to TSP-1, although TSP-2 also was expressed in granulosa cells. Both TSP-1 and -2 were increased in response to LH stimulation in vitro, whereas TSP-2 was suppressed by FSH. The temporal pattern of expression of TSP-1, -2, and CD36, which mirrors the active phases of angiogenesis in this experimental model, is compatible with a role for these proteins in the control of ovarian vascularization.

Transcriptional regulation of LDL receptor-related protein by IFN-gamma and the antagonistic activity of TGF-beta(1) in the RAW 264.7 macrophage-like cell line.

Low density lipoprotein receptor‐related protein (LRP) is a major receptor for multiple ligands, including chylomicron and VLDL remnants, bacterial toxins, viruses, proteinases, lipoprotein lipase, and activated α2‐macroglobulin (α2M). In this study, we used Northern blot analyses and nuclear run‐on experiments to demonstrate that interferon‐γ (IFN‐γ) causes a concentration‐dependent decrease in steady‐state LRP mRNA expression and gene transcription rate in RAW 264.7 cells. IFN‐γ also markedly increased expression of inducible nitric oxide synthase (NOS), as expected; however, the increase in nitric oxide was not responsible for the down‐regulation of LRP expression since the NOS inhibitor, N G‐monomethyl‐l‐arginine, did not preserve LRP expression in IFN‐γ‐treated cells. Trans‐forming growth factor‐β1 (TGF‐β1; 2.5 ng/mL) had no independent effect on LRP expression and did not modify the response to IFN‐γ when the two cytokines were added simultaneously to cultures. When TGF‐β1 was added 24 h prior to IFN‐γ, the extent of LRP down‐regulation was significantly reduced. Specific binding of the LRP ligand, activated 125I‐α2M, was decreased by 76 ± 5% in cells treated with 100 U/mL IFN‐γ, but only by 45 ± 7% in cells treated with 100 U/mL IFN‐γ after TGF‐β1‐pretreatment. The antagonistic activity of TGF‐β1 on the IFN‐γ response in RAW 264.7 cells did not result from a change in LRP mRNA stability or IFN‐γ receptor expression, as determined by Northern blot analyses 125I‐IFN‐γ binding experiments. The studies presented here suggest that the balance between IFN‐γ and TGF‐β1 may be critical in determining LRP expression at sites of infection and inflammation.

MicroRNA-140 Expression During Chondrogenic Differentiation of Equine Cord Blood-Derived Mesenchymal Stromal Cells

MicroRNAs are a class of short noncoding RNAs that are involved in various biological processes, including differentiation. MicroRNA-140 (miR-140) has been identified as a cartilage-specific microRNA with several targets involved in cartilage development and homeostasis. The aim of this study was to investigate the expression of miR-140 during chondrogenic differentiation of equine cord blood-derived mesenchymal stromal cells (eCB-MSCs). We demonstrate both that miR-140 is highly expressed in normal equine articular cartilage and that eCB-MSCs express significantly higher levels of this microRNA after 14 days of chondrogenic differentiation. Furthermore, miR-140 expression closely paralleled that of the cartilage-specific transcription factor Sox9, suggesting that miR-140 may be under the transcriptional regulation of Sox9 in these cells. The expression patterns of miR-140 targets the chemokine (CXC motif) ligand 12 (CXCL12), A disintegrin and metalloproteinase with thrombosponin motifs (ADAMTS)-5 and insulin growth factor binding protein 5 (IGFBP5) were also determined; however, only CXCL12 and ADAMTS-5 were repressed while miR-140 expression was upregulated. Together, these studies suggest that miR-140 is an important regulator of cartilage development and homeostasis in eCB-MSCs that may act, in part, through the regulation of CXCL12 and ADAMTS-5.

Reaction of α2-macroglobulin with plasmin increases binding of transforming growth factors-β1 and β2

The binding of125I-transforming growth factors-β1 and β2 (TGF-β1 and TGF-β2) to α2-macroglobulin(α2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-β and native or reacted forms of α2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native α2M with plasmin or methylamine markedly increased the binding of125I-TGF-β1 and125I-TGF-β2 to α2M. The α2M-plasmin/TGF-β complexes were minimally dissociated by heparin. Reaction of α2M with thrombin or trypsin reduced the binding of 125I-TGF-β1 and 125I-TGF-β2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-β2 and native or reacted forms of α2M were less dissociable by heparin than the equivalent complexes with TGF-β1. These studies demonstrate that the TGF-β-binding activity of α2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that α2M-plasmin preferentially binds TGFs-β suggest a mechanism by which α2M may regulate availability of TGFs-β to target cells in vivo.

Chronomics of Pressure Overload–Induced Cardiac Hypertrophy in Mice Reveals Altered Day/Night Gene Expression and Biomarkers of Heart Disease

There is critical demand in contemporary medicine for gene expression markers in all areas of human disease, for early detection of disease, classification, prognosis, and response to therapy. The integrity of circadian gene expression underlies cardiovascular health and disease; however time-of-day profiling in heart disease has never been examined. We hypothesized that a time-of-day chronomic approach using samples collected across 24-h cycles and analyzed by microarrays and bioinformatics advances contemporary approaches, because it includes sleep-time and/or wake-time molecular responses. As proof of concept, we demonstrate the value of this approach in cardiovascular disease using a murine Transverse Aortic Constriction (TAC) model of pressure overload–induced cardiac hypertrophy in mice. First, microarrays and a novel algorithm termed DeltaGene were used to identify time-of-day differences in gene expression in cardiac hypertrophy 8 wks post-TAC. The top 300 candidates were further analyzed using knowledge-based platforms, paring the list to 20 candidates, which were then validated by real-time polymerase chain reaction (RTPCR). Next, we tested whether the time-of-day gene expression profiles could be indicative of disease progression by comparing the 1- vs. 8-wk TAC. Lastly, since protein expression is functionally relevant, we monitored time-of-day cycling for the analogous cardiac proteins. This approach is generally applicable and can lead to new understanding of disease. (Author correspondence: tmartino@uoguelph.ca)

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface

BackgroundCommercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points.MethodsMesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections.ResultsAt gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50.ConclusionsWe provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.