Ultrastructure of Zebra Fish Dorsal Aortic Cells

Expression of vascular smooth muscle cell (VSMC) markers such as serum response factor (SRF) is complicated in zebrafish because of the ill-defined histology of the dorsal aorta and the presence of perivascular pigment. We report the ultrastructure of aortic cells in 7-day, 1-month, and 3-month-old zebrafish and provide clear evidence for the presence of perivascular melanocytes harboring an abundance of melanin. In 7-day-old larvae, endothelial cells (EC) and synthetic mural cells that display little evidence of VSMC differentiation comprise the dorsal aorta. The latter mural cells appear to fully differentiate into VSMC by 1 month of age. In 3-month-old adult zebrafish, EC exhibit greater differentiation as evidenced by the accumulation of electron-dense bodies having a diameter of approximately 200 nm. Adult zebrafish aortae also exhibit at least one clear layer of VSMC with the characteristic array of membrane-associated dense plaques, myofilament bundles, and a basement membrane. Subjacent to VSMC are collagen-producing adventitial fibroblasts and melanocytes. These studies indicate that fully differentiated VSMC occur only after day 7 in zebrafish and that such cells are arranged in at least one lamellar unit circumscribing the endothelium. These findings provide new data about the timing and accumulation of VSMC around the zebrafish aorta, which will be useful in phenotyping mutant zebrafish that exhibit defects in blood circulation

Expression and Comparative Genomics of Two Serum Response Factor Genes in Zebrafish

Serum response factor (SRF) is a single copy, highly conserved transcription factor that governs the expression of hundreds of genes involved with actin cytoskeletal organization, cellular growth and signaling, neuronal circuitry and muscle differentiation. Zebrafish have emerged as a facile and inexpensive vertebrate model to delineate gene expression, regulation, and function, and yet the study of SRF in this animal has been virtually unexplored. Here, we report the existence of two srf genes in zebrafish, with partially overlapping patterns of expression in 3 and 7 day old developing animals. The mammalian ortholog (srf1) encodes for a 520 amino acid protein expressed in adult vascular and visceral smooth muscle cells, cardiac and skeletal muscle, as well as neuronal cells. The second zebrafish srf gene (srf2), encoding for a presumptive protein of only 314 amino acids, is transcribed at lower levels and appears to be less widely expressed across adult tissues. Both srf genes are induced by the SRF coactivator myocardin and attenuated with a short hairpin RNA to mammalian SRF. Promoter studies with srf1 reveal conserved CArG boxes that are the targets of SRF-myocardin in embryonic zebrafish cells. These results reveal that SRF was duplicated in the zebrafish genome and that its protein expression in all three muscle cell types is highly conserved across vertebrate animals suggesting an ancient code for transcriptional regulation of genes unique to muscle cell lineages

Retinoid-Induced Expression and Activity of an Immediate Early Tumor Suppressor Gene in Vascular Smooth Muscle Cells

Retinoids are used clinically to treat a number of hyper-proliferative disorders and have been shown in experimental animals to attenuate vascular occlusive diseases, presumably through nuclear receptors bound to retinoic acid response elements (RARE) located in target genes. Here, we show that natural or synthetic retinoids rapidly induce mRNA and protein expression of a specific isoform of A-Kinase Anchoring Protein 12 (AKAP12β) in cultured smooth muscle cells (SMC) as well as the intact vessel wall. Expression kinetics and actinomycin D studies indicate Akap12β is a retinoid-induced, immediate-early gene. Akap12β promoter analyses reveal a conserved RARE mildly induced with atRA in a region that exhibits hyper-acetylation. Immunofluorescence microscopy and protein kinase A (PKA) regulatory subunit overlay assays in SMC suggest a physical association between AKAP12β and PKA following retinoid treatment. Consistent with its designation as a tumor suppressor, inducible expression of AKAP12β attenuates SMC growth in vitro. Further, immunohistochemistry studies establish marked decreases in AKAP12 expression in experimentally-injured vessels of mice as well as atheromatous lesions in humans. Collectively, these results demonstrate a novel role for retinoids in the induction of an AKAP tumor suppressor that blocks vascular SMC growth thus providing new molecular insight into how retiniods may exert their anti-proliferative effects in the injured vessel wall

The Notch Ligand Jagged1 Regulates the Osteoblastic Lineage by Maintaining the Osteoprogenitor Pool

Notch signaling is critical for osteoblastic differentiation; however, the specific contribution of individual Notch ligands is unknown. Parathyroid hormone (PTH) regulates the Notch ligand Jagged1 in osteoblastic cells. To determine if osteolineage Jagged1 contributes to bone homeostasis, selective deletion of Jagged1 in osteolineage cells was achieved through the presence of Prx1 promoter-driven Cre recombinase expression, targeting mesenchymal stem cells (MSCs) and their progeny (PJag1 mice). PJag1 mice were viable and fertile and did not exhibit any skeletal abnormalities at 2 weeks of age. At 2 months of age, however, PJag1 mice had increased trabecular bone mass compared to wild-type (WT) littermates. Dynamic histomorphometric analysis showed increased osteoblastic activity and increased mineral apposition rate. Immunohistochemical analysis showed increased numbers of osteocalcin-positive mature osteoblasts in PJag1 mice. Also increased phenotypically defined Lin(-)/CD45(-)/CD31(-)/Sca1(-)/CD51(+) osteoblastic cells were measured by flow cytometric analysis. Surprisingly, phenotypically defined Lin(-)/CD45(-)/CD31(-)/Sca1(+)/CD51(+) MSCs were unchanged in PJag1 mice as measured by flow cytometric analysis. However, functional osteoprogenitor (OP) cell frequency, measured by Von Kossa(+) colony formation, was decreased, suggesting that osteolineage Jagged1 contributes to maintenance of the OP pool. The trabecular bone increases were not due to osteoclastic defects, because PJag1 mice had increased bone resorption. Because PTH increases osteoblastic Jagged1, we sought to understand if osteolineage Jagged1 modulates PTH-mediated bone anabolism. Intermittent PTH treatment resulted in a significantly greater increase in BV/TV in PJag1 hind limbs compared to WT. These findings demonstrate a critical role of osteolineage Jagged1 in bone homeostasis, where Jagged1 maintains the transition of OP to maturing osteoblasts. This novel role of Jagged1 not only identifies a regulatory loop maintaining appropriate populations of osteolineage cells, but also provides a novel approach to increase trabecular bone mass, particularly in combination with PTH, through modulation of Jagged1. (C) 2017 American Society for Bone and Mineral Research

GM-CSF drives myelopoiesis, recruitment and polarisation of tumour-associated macrophages in cholangiocarcinoma and systemic blockade facilitates antitumour immunity.

OBJECTIVE: Intrahepatic cholangiocarcinoma (iCCA) is rising in incidence, and at present, there are limited effective systemic therapies. iCCA tumours are infiltrated by stromal cells, with high prevalence of suppressive myeloid populations including tumour-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs). Here, we show that tumour-derived granulocyte-macrophage colony-stimulating factor (GM-CSF) and the host bone marrow is central for monopoiesis and potentiation of TAMs, and abrogation of this signalling axis facilitates antitumour immunity in a novel model of iCCA. METHODS: Blood and tumours were analysed from iCCA patients and controls. Treatment and correlative studies were performed in mice with autochthonous and established orthotopic iCCA tumours treated with anti-GM-CSF monoclonal antibody. RESULTS: Systemic elevation in circulating myeloid cells correlates with poor prognosis in patients with iCCA, and patients who undergo resection have a worse overall survival if tumours are more infiltrated with CD68 CONCLUSIONS: iCCA uses the GM-CSF-bone marrow axis to establish an immunosuppressive tumour microenvironment. Blockade of the GM-CSF axis promotes antitumour T cell immunity