The incidence of intervertebral disk degeneration and foraminal stenosis in clinically normal Doberman Pinschers was high; cervical spinal cord compression may be present without concurrent clinical signs. A combination of static factors (ie, a relatively stenotic vertebral canal and wider intervertebral disks) distinguished CSM-affected dogs from clinically normal dogs and appears to be a key feature in the pathogenesis of CSM.

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-1's regulation of VEGF appears to be an important mediator of ovarian angiogenesis and follicle development.The growth of normal tissues and pathological structures such as tumors are dependent upon the formation of blood vessels for nutrient delivery and waste removal (Folkman, 1992). Growth of new vasculature is regulated by a balance between pro-and anti-angiogenic factors. A potent pro-angiogenic factor is vascular endothelial growth factor (VEGF), which is a heparin-binding glycoprotein secreted as a homodimer that stimulates endothelial cell proliferation and migration (Bernatchez et al., 2002;Castellon et al., 2002), promotes new vessel formation, increases vascular permeability (Ferrara, 2004), and acts as a survival factor for endothelial cells in vitro and in vivo (Gerber et al., 1998;Jia et al., 2004). In addition to its effects on endothelial cells, we recently reported that VEGF protects ovarian cells from apoptosis by signaling through VEGFR-2 expressed by these cells (Greenaway et al., 2004).The effects of pro-angiogenic factors are balanced by anti-angiogenic factors such as members of the thrombospondin (TSP) family, which consists of five proteins (TSP-1-5), of which TSP-1 and -2 share structural and functional homology (Bornstein, 1992; Adams and Lawler, 2004). TSP-1 is a secreted glycoprotein located in the extracellular matrix that has been shown to be a potent inhibitor of angiogenesis (Lawler, 2002; Armstrong and Bornstein, 2003;Wang et al., 2003;Cursiefen et al., 2004;Lawler and Detmar, 2004 (Detmar, 2000). In addition, a direct interaction of VEGF with TSP-1 and with a TSP-1 type I repeat domain of connective tissue growth factor has been reported (Gupta et al., 1999;Inoki et al., 2002). TSP-1 is also known to bind ligands and interact with the low-density lipoprotein receptorrelated protein (LRP-1), resulting in internalization and degradation of the protein (Mikhailenko et al., 1995;Emonard et al., 2004;Wang et al., 2004). LRP-1 is a member of the low-density lipoprotein receptor gene family, which also includes the LDL, VLDL, and apo...

Vascular endothelial growth factor (VEGF) is a potent mitogen and cytoprotective factor for vascular endothelial cells. Although VEGF is ubiquitously expressed, its role in nonvascular tissues is poorly understood. VEGF interacts with various cell surface receptors to mediate its cellular effects. It previously has been thought that the VEGF receptor Flk-1/KDR, its main signaling receptor, was expressed exclusively by endothelial cells. However, in the present study using bovine and rodent models, we demonstrate that VEGF and Flk-1/KDR are coexpressed in ovarian granulosa cells. VEGF and Flk-1/KDR mRNA and protein were both detectable in follicle tissue sections and in vitro cultured granulosa cells. Expression of both ligand and receptor increased in healthy follicles throughout follicular development. VEGF treatment of serum-starved and cytokine-exposed granulosa cells resulted in enhanced survival, and this cytoprotection was ameliorated when Flk-1/KDR signaling was inhibited. Reduced expression of Flk-1/KDR was also associated with the onset and progression of follicle atresia, suggesting involvement in follicular health in vivo. The results of this study demonstrate for the first time expression of Flk-1/KDR in ovarian granulosa cells and identify a novel extravascular role for VEGF and its receptor in ovarian function.

COX-1 and COX-2 were differentially sensitive to inhibition in vitro by NSAID. Meloxicam and tolfenamic acid were selective for COX-2. Effects of carprofen and ketoprofen approached equipotency against both isoenzymes. Selective COX-2 inhibitors are a new class of drugs with anti-inflammatory effects similar to conventional NSAID but with fewer adverse effects. Development of these agents for veterinary use would be facilitated by the convenience of using a canine cell line as a model system to screen COX-1 and COX-2 inhibitor activities in vitro.

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Radioiodinated transforming growth factor-,61 i1) bound to the plasma proteinase inhibitor, a2-macroglobulin (a2M), as determined by chromatography on Superose-6 and native polyacrylamide gel electrophoresis. When a2M conformational change was induced with methylamine, 1"I-TGF-jfl binding significantly increased. Intravenously injected 125I_ TGF-il cleared from the circulation of mice rapidly at first; however, intravascular radioactivity stabilized near 20% of the initial level. At necropsy, radioactivity was recovered predominantly in the liver (65%); however, the density of radioactivity (disintegrations per minute/g organ wt) was highest in the lungs. Markedly different results were obtained with purified 125I-TGF-fll-a2M-methylamine complex. Clearance of the complex occurred as a first-order process with a t11 of 4 min.Greater than 90% ofthe radioactivity was recovered in the liver.The clearance and distribution of 125I-TGF-81-a2M-methylamine were equivalent to those observed with '"I-a2M-methylamine and '"I-a2M-trypsin. The latter two radioligands clear via specific a2M receptors in the liver. Large molar excesses of a2M-trypsin or a2M-methylamine competed with 125I-TGFj01-a2M-methylamine for plasma clearance. Native a2M, which does not bind to the a2M receptor, did not compete. The receptor binding domain of a2M-methylamine was blocked by chemical modification or enzyme treatment. The resulting a2M preparations still bound 125I-TGF-fll; however, the complexes did not clear when injected intravenously in mice. The studies presented here demonstrate that a2M can mediate the plasma clearance of a growth factor via the a2M receptor system. We propose that a2M, the a2M receptor, and proteinases may function as a concerted system to regulate TGF-ftl activity and the activity of related factors in vivo. (J. Clin. Invest. 1991.

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.

BackgroundMesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies.Hypothesis and ObjectivesWe hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro.Results1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC.Conclusions/SignificanceNo significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.