Colour-sidedness in Gloucester cattle is associated with a complex structural variant impacting regulatory elements downstream of KIT.

Colour-sidedness is a striking coat colour pattern found in a number of cattle breeds, typically characterised by a white stripe that extends along the back, head and belly of the animal. This dominant phenotype is caused by two related translocations (Cs6 and Cs29 ) that alter a region downstream of the KIT gene. Gloucester cattle are native to the UK and are known for an unusual colour-sided pattern that does not extend to the head. We carried out whole-genome sequencing of two Gloucester bulls as well as colour-sided Irish Moiled, British White and Pustertaler Sprinzen for comparison. We found that the Gloucester cattle also have a complex structural variant downstream of KIT, which overlaps the regions involved in Cs6 and Cs29 . All three alleles potentially disrupt a number of putative regulatory elements downstream of KIT. These results complement and expand on the recently published work focused on the Pinzgauer breed from Austria, a carrier of the same colour-sided pattern as seen in Gloucester cattle.

Prevalence of vitamin D deficiency and consequences for PTH reference values.

Reference values of PTH depend on vitamin D status of the reference population. This is often not described in package inserts. The aim of the present study was therefore to calculate assay specific PTH reference levels in EDTA plasma for the Architect (Abbott) in relation to 25-hydroxyvitamin D (25OHD) levels. The relation between PTH levels, 25OHD, BMI, age, gender and kidney function was determined in a cohort of older individuals from the Longitudinal Aging Study Amsterdam (LASA, n = 738, age 55-65 years) and in a cohort of healthy individuals from the Netherlands Study of Depression and Anxiety (NESDA, n = 633, 18-65 years). The LASA cohort is a representative sample of the Dutch older population. As expected, PTH reference values were significantly lower in 25OHD sufficient subjects (25OHD>50 nmol/L) than in 25OHD deficient and insufficient subjects. The 97.5th percentile of PTH in 25OHD sufficient subjects was 10 pmol/L (94.3 pg/mL), which was higher than the upper limit stated by the manufacturer (7.2 pmol/L or 68.3 pg/mL). The relation between vitamin D and PTH was independent of age, gender, BMI and kidney function. In conclusion, we have shown that it is important to establish PTH reference values in a local reference population taking 25OHD status into account.

Oral administration of GW788388, an inhibitor of TGF-beta type I and II receptor kinases, decreases renal fibrosis.

Progressive kidney fibrosis precedes end-stage renal failure in up to a third of patients with diabetes mellitus. Elevated intra-renal transforming growth factor-beta (TGF-beta) is thought to underlie disease progression by promoting deposition of extracellular matrix and epithelial-mesenchymal transition. GW788388 is a new TGF-beta type I receptor inhibitor with a much improved pharmacokinetic profile compared with SB431542. We studied its effect in vitro and found that it inhibited both the TGF-beta type I and type II receptor kinase activities, but not that of the related bone morphogenic protein type II receptor. Further, it blocked TGF-beta-induced Smad activation and target gene expression, while decreasing epithelial-mesenchymal transitions and fibrogenesis. Using db/db mice, which develop diabetic nephropathy, we found that GW788388 given orally for 5 weeks significantly reduced renal fibrosis and decreased the mRNA levels of key mediators of extracellular matrix deposition in kidneys. Our study shows that GW788388 is a potent and selective inhibitor of TGF-beta signalling in vitro and renal fibrosis in vivo.

Natural killer cells and CD4+ T-cells modulate collateral artery development.

OBJECTIVE: The immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. Here, we studied the role of lymphocytes in a murine model of hindlimb ischemia. METHODS AND RESULTS: Lymphocytes, detected with markers for NK1.1, CD3, and CD4, invaded the collateral vessel wall. Arteriogenesis was impaired in C57BL/6 mice depleted for Natural Killer (NK)-cells by anti-NK1.1 antibodies and in NK-cell-deficient transgenic mice. Arteriogenesis was, however, unaffected in J alpha281-knockout mice that lack NK1.1+ Natural Killer T (NKT)-cells, indicating that NK-cells, rather than NKT-cells, are involved in arteriogenesis. Furthermore, arteriogenesis was impaired in C57BL/6 mice depleted for CD4+ T-lymphocytes by anti-CD4 antibodies, and in major histocompatibility complex (MHC)-class-II-deficient mice that more selectively lack mature peripheral CD4+ T-lymphocytes. This impairment was even more profound in anti-NK1.1-treated MHC-class-II-deficient mice that lack both NK- and CD4+ T-lymphocytes. Finally, collateral growth was severely reduced in BALB/c as compared with C57BL/6 mice, 2 strains with different bias in immune responsiveness. CONCLUSIONS: These data show that both NK-cells and CD4+ T-cells modulate arteriogenesis. Promoting lymphocyte activation may represent a promising method to treat ischemic disease.

Inhibition of intimal hyperplasia by the tetracycline derived mmp inhibitor doxycycline in vein graft disease in vitro and in vivo.

AIMS: Intimal hyperplasia, characterised by smooth muscle cell migration and proliferation, requires extracellular matrix degradation which is mediated by matrix metalloproteinases (MMPs). In this study, the effect of tetracycline derived doxycycline, a specific MMP inhibitor of both activity and synthesis, on intimal hyperplasia in vitro and in vivo was assessed. METHODS AND RESULTS: Segments of human saphenous veins were cultured for 4 weeks in absence or presence of doxycycline (10microg/ml) (n=6). A 81% inhibition in intimal hyperplasia was observed in the doxycycline treated segments compared to controls. To assess the effect of doxycycline on intimal hyperplasia in vivo, perivascular cuffs were placed around femoral arteries in mice with or without doxycycline in the drinking water (3 mg/ml). In this in vivo model for intimal hyperplasia doxycycline significantly reduced (68%, n=6) intimal hyperplasia when compared to controls. In addition the effect of doxycycline on vein graft thickening was assessed in a murine venous interposition model. In this in vivo model vein graft thickening was reduced by 35 % in the doxycycline treated mice (3 mg/ml in drinking water). Furthermore, a reduction in vascular MMP expression was observed in these mice. CONCLUSION: Treatment with tetracycline derived doxycycline results in significant inhibition of intimal hyperplasia in vitro and in vivo and may be an effective strategy to prevent post interventional restenosis and vein graft disease.

Differentiation of murine preosteoblastic KS483 cells depends on autocrine bone morphogenetic protein signaling during all phases of osteoblast formation.

In this study, we examine the role of bone morphogenetic protein (BMP) signaling during differentiation of the murine preosteoblastic KS483 cell line, which formed alkaline phosphatase (ALP)-positive and mineralized nodules during a 3 week culture period. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) demonstrated the presence of various BMPs (BMP-2, -3, -4, -6, -7, and -8A and -8B), BMP type I and II receptors (ALK2, ALK3, ALK4, BMPR-II, and ActR-IIA and -IIB), BMP antagonists (DAN, gremlin, chordin, cerberus, noggin, and tsg), and Smads 1-8. mRNA expression of these genes did not change during differentiation, except for BMP-3, BMP-8a, and noggin. BMP-3 increased gradually, particularly in the matrix formation phase; BMP-8a was induced from the onset of matrix maturation and mineralization, in parallel to the expression of osteocalcin; and noggin tended to decline during the mineralization phase. Treatment of KS483 cells with the BMP antagonists noggin or soluble truncated BMPR-IA, either continuously or during distinct periods of osteoblast differentiation; that is, matrix formation or matrix maturation and mineralization phase, decreased ALP-positive and mineralized nodule area independent of the phase of osteoblast differentiation. Notably, the antagonists inhibited mineralization of already existing nodules. Similarly, BMP-4 stimulated differentiation not only at the beginning of the culture period, but also at late stages of differentiation. These data indicate that autocrine BMP signaling is involved in KS483 osteoblastic differentiation not only during the early phase of differentiation, but also during matrix maturation and mineralization. The different expression patterns of components of BMP signaling in the KS483 cells suggest distinct functions of individual BMPs during osteoblast differentiation. In summary, our data suggest that BMP activity is required not only for initiation of osteoblast differentiation and further development of early osteoblasts, but is also involved in late-stage osteoblast differentiation and matrix mineralization.

Monitoring metastatic behavior of human tumor cells in mice with species-specific polymerase chain reaction: elevated expression of angiogenesis and bone resorption stimulators by breast cancer in bone metastases.

Tumor-stroma interactions are of primary importance in determining the pathogenesis of metastasis. Here, we describe the application of sensitive competitive polymerase chain reaction (PCR) techniques for detection and quantitation of human breast cancer cells (MDA-MB-231) in an in vivo mouse model of experimental metastasis. Human-specific oligonucleotide primers in competitive PCR reactions were used to quantify the amount of MDA-MB-231 cells per tissue per organ. Using this species-specific (semi)quantitative PCR approach, gene expression patterns of (human) tumor cells or (mouse) stromal cells in metastatic lesions in the skeleton or soft tissues were investigated and compared. In all metastatic lesions, MDA-MB-231 cells express angiogenic factors (vascular endothelial growth factors [VEGFs]; VEGF-A, -B, and -C) and bone-acting cytokines (parathyroid hormone-related protein [PTHrP] and macrophage colony-stimulating factor [M-CSF]). In these metastases, PECAM-1-positive blood vessels and stromal cells of mouse origin are detected. The latter express angiogenic factors and markers of sprouting vessels (VEGF receptors flt-1/flk - 1/flk-4 and CD31/PECAM-1). Strikingly, steady-state messenger RNA (mRNA) levels of VEGF-A and -B and the major bone resorption stimulators PTHrP and M-CSF by tumor cells were elevated significantly in bone versus soft tissues (p < or = 0.05, p < or = 0.0001, p < or = 0.001, and p < or = 0.05, respectively), indicating tissue-specific expression of these tumor progression factors. In conclusion, MDA-MB-231 breast cancer cells express a variety of factors in vivo that have been implicated in metastatic bone disease and that correlate with poor survival of patients with breast cancer. We hypothesize that the observed up-regulated expression of angiogenic and bone-resorbing factors by the breast cancer cells in the skeleton underlie the clinically observed osteotropism of breast cancer cells and pathogenesis of osteolytic bone metastases. The application of the species-specific competitive PCR-based assay in vivo can provide new information concerning the involvement of gene families in tumor progression and metastatic disease and greatly facilitates the study of tumor-stroma interactions in cancer invasion and metastasis.

Effect of angiogenic and antiangiogenic compounds on the outgrowth of capillary structures from fetal mouse bone explants.

Fetal mouse metatarsals are well-known models to study cartilage differentiation and osteoclastic resorption. We show here the outgrowth of PECAM-1 positive tubelike structures from the bone rudiments. This feature can be used to study angiogenesis in vitro. The area of outgrowth significantly increased with culture time, as shown by computerized image analysis of PECAM-1 positive tubelike structures. Treatment with recombinant human vascular endothelial growth factor (rhVEGF-A) stimulated the formation of tubelike structures. Treatment of explants with the angiogenesis inhibitor endostatin, the chemokine IP-10, and the thalidomide derivative phatolyl glutamic acid (PG-acid) resulted in an inhibition of the formation of PECAM-1 positive tubelike structures of 48.8% (+/- 4%), 50.2% (+/- 12%), and 80.8% (+/- 3%), respectively. Outgrowth of tubelike structures was partly dependent on endogenous VEGF-A because treatment with anti-mVEGF-A and truncated VEGF receptor 1 (soluble fms-like tyrosine kinase 1, sFIt1) strongly inhibited the formation of tubelike structures 74% (+/- 4%) and 38% (+/- 5%), respectively. Neither onset of tube formation nor total area of tubelike structures were changed when metatarsals were cultured on a fibrin gel or collagen type I gel. Tube formation required activation of matrix metalloproteinases because treatment of the bones with an inhibitor of matrix metalloproteinases completely inhibited migration and tube formation, whereas treatment with an inhibitor of plasmin had no effect. In conclusion, we describe a new in vitro model to study angiogenesis that can be used to test the angiogenic or antiangiogenic potential of novel test compounds that also combines the multicellularity of in vivo assays with the accessibility and flexibility of in vitro assays.

Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro.

Two mRNAs are transcribed from the extracellular matrix protein 1 gene (Ecm1): Ecm1a and an alternatively spliced Ecm1b. We studied Ecm1 mRNA expression and localization during endochondral bone formation and investigated the effect of recombinant human (rh) Ecm1a protein on organ cultures of embryonic mouse metatarsals. Of the two transcripts, Ecm1a mRNA was predominantly expressed in fetal metacarpals from day 16 to 19 after gestation. Ecm1 expression was not found in 16- and 17-day-old metatarsals of which the perichondrium was removed. In situ hybridization and immunohistochemistry demonstrated Ecm1 expression in the connective tissues surrounding the developing bones, but not in the cartilage. Biological effects of rhEcm1a protein on fetal metatarsal cultures were biphasic: at low concentrations, Ecm1a stimulated alkaline phosphatase activity and had no effect on mineralization, whereas at higher concentrations, Ecm1a dose dependently inhibited alkaline phosphatase activity and mineralization. These results suggest that Ecm1a acts as a novel negative regulator of endochondral bone formation.

Accelerated atherosclerosis by placement of a perivascular cuff and a cholesterol-rich diet in ApoE*3Leiden transgenic mice.

Intimal hyperplasia after vascular injury is usually studied in animal models with healthy, normocholesterolemic animals. Here, we assess the effect of diet-induced hypercholesterolemia on the induction of intimal hyperplasia in ApoE*3Leiden mice. A nonconstrictive polyethylene cuff was placed around the femoral artery of ApoE3*Leiden mice fed a highly cholesterol-rich diet, a mildly cholesterol-rich diet, or a chow diet for 4 weeks. Diets were continued after cuff placement until euthanization. At several time points (1 to 14 days), mice were euthanized and the intimal hyperplasia in the cuffed arteries was analyzed. In mice fed a chow diet, a 2- to 4-cell-layer-thick intima, predominantly consisting of alpha smooth muscle cell actin-positive cells, was observed after 14 days. A mildly cholesterol-rich diet (mean plasma-cholesterol level, 10.5 mmol/L) resulted in a 2.7-fold increase of total intimal area, and a highly cholesterol-rich diet (mean plasma cholesterol level 28. 6 mmol/L), in a 7.8-fold increase. In the high-cholesterol group, the intima consisted predominantly of lipid-loaded foam cells and alpha smooth muscle cell actin-positive cells. Foam cell accumulation could be observed by as early as 3 days, resulting in a near-total occlusion of the lumen after 14 days. Hypercholesterolemia resulted in a rapid, cholesterol-dependent induction of foam cell-rich intimal hyperplasia in cuffed femoral arteries of ApoE*3Leiden mice. In conclusion, the present data show that the combination of a local (cuff placement) and a systemic (hypercholesterolemic) risk factor of atherosclerosis results in a rapid induction (within 14 days) of atherosclerotic-like lesions in ApoE*3Leiden mice.

Expression of vascular endothelial growth factors and their receptors during osteoblast differentiation.

Endochondral bone formation is regulated by systemically and locally acting growth factors. A role for vascular endothelial growth factor (VEGF) in this process has recently been proposed, because inactivation of VEGF inhibits endochondral bone formation via inhibition of angiogenesis. Despite the known effect of VEGF as specific endothelial growth factor, its effects on osteoblast differentiation have not been studied. We, therefore, examined the expression of VEGF-A, -B, -C, and -D and their receptors in a model of osteoblast differentiation using the mouse preosteoblast-like cell line KS483. Early in differentiation, KS483 cells express low levels VEGF-A, -B, and -D messenger RNA, whereas during mineralization, KS483 cells express high levels. In addition, expression of the VEGF receptors, VEGFR1, VEGFR2, and VEGF165R/neuropilin, coincided with expression of their ligands, being maximally expressed during mineralization. VEGF-A production during osteoblast differentiation was stimulated by insulin-like growth factor I that enhances osteoblast differentiation and was inhibited by PTH-related peptide that inhibits osteoblast differentiation. Furthermore, continuous treatment of KS483 cells with recombinant human VEGF-A stimulated nodule formation. Although treatment of KS483 cells with soluble FLT1, an agent that blocks binding of VEGF-A and -B to VEGFR1, did not inhibit nodule formation, this observation does not exclude involvement of VEGFR2 in the regulation of osteoblast differentiation. As it is known that VEGF-A, -C, and -D can act through activation of VEGFR2, other isoforms might compensate for VEGF-A loss. The expression pattern of VEGFs and their receptors shown here suggests that VEGFs play an important role in the regulation of bone remodeling by attracting endothelial cells and osteoclasts and by stimulating osteoblast differentiation.

Tonsil stromal-cell lines expressing FDC-like properties: isolation, characterization, and interaction with B lymphocytes.

The microenvironment of secondary lymphoid organs consists of two major populations of cells, the lymphoid cells and a population of stromal cells that contribute to both tissue architecture and function. Interactions of both populations are essential for the development and control of humoral immune responses. In this study, stromal-cell preparations were obtained by a multistage process. This involved culturing 300-400-microm slices of human tonsil for 6-8 days at 25 degrees C, trypsin digestion of the residual explant, followed by CD45-positive-cell depletion using magnetic beads, and a final period of culture for 4 days to remove remaining nonadherent cells. Phenotyping with a panel of monoclonal antibodies revealed that the cells express HLA-DR, CD54 (ICAM-1), CD44, but no CD45 nor a range of other markers for epithelial and endothelial cells. Immunoassays of supernatants from stromal cells revealed that IL-6 was produced constitutively, and its production was increased by treatment with TNF-alpha and IFN-gamma. In contrast IL-1, IL-2, IL-4, IL-7, IL-8, IL-10, IL-12, TNF-alpha, and IFNgamma were not produced. Functional tests showed that these cells express follicular dendritic cell-like properties. Coculturing of tonsilar B cells with stromal cells resulted in enhanced proliferation and also led to increased production of immunoglobulins and IL-6, suggesting crucial signaling between these populations.