Endothelial cells activate the expression of progranulin during angiogenesis. Here we discuss methods to investigate progranulin activity on endothelial cells in vitro and on aortic explants. We then discuss methods to generate transgenic mice in which progranulin expression is targeted to endothelial cells. These mice can be used to study the influence of progranulin on angiogenesis during development in vivo. The transgenic strategy summarized here could be readily adapted to investigate the roles of progranulin in other cell types and tissues by use of appropriate targeting vectors to drive the expression of progranulin in the cell type of choice.

Objective To evaluate the horizontal mattress technique in microvascular anastomosis for size-mismatched vessels. Methods The present study involved cadaveric simulation using size-mismatched (1.5:1) Thiel-embalmed cadaveric arteries. The authors performed horizontal mattress anastomoses using 9-0 nylon suture and recorded the procedure. Vessel patency was evaluated by saline infusion. Vessels were cut open and photographed; histological slides were prepared and stained with hematoxylin and eosin. Results Four anastomoses were performed. Vessels were found to be patent in all cases, with grade 0 leaks. Intima-on-intima apposition with no intraluminal exposure of muscularis nor adventitia were observed. Conclusion The present cadaveric study supports the technical feasibility of using horizontal mattress sutures in size-mismatched end-to-end anastomoses.

1Plastic and Reconstructive Surgery Service; 2Otolaryngology-Head and Neck Surgery Service, Notre-Dame Hospital, University of Montréal Hospital Center (CHUM); 3Department of Anatomy, McGill University, Montreal, Quebec Correspondence: Dr Michel Alain Danino, Plastic Surgery Department, Centre Hospitalier Universite de Montreal, 1560 rue de sherbrooke est, Montreal, Quebec H2L 4M1. Telephone 514-890-8000 ext 26808, e-mail daninoalain@gmail.com The patency of the microvascular anastomosis is paramount for flap survival in microsurgery (1). In addition to endothelial dysfunction and hypercoagulability, flow turbulence is an essential part of Virchow’s triad and must be minimized to prevent anastomotic thrombosis (2). Unfortunately, microsurgeons occasionally encounter cases in which mismatched vessels must be anastomosed, which is a situation at high risk for flow turbulence and thrombosis (1). Mismatch, defined as a vessel diameter ratio of ≥1.5:1, is typically encountered in 33% of arterial and 50% of venous anastomoses (3). Multiple, technically demanding techniques to overcome this problem have been described including coupling devices, forced dilation of the smaller vessel, oblique cuts, fish-mouth incisions, end-to-side anastomoses and interpositional grafts (1). We describe a simple technique using horizontal mattress (HM) sutures based on our previously published work in size-appropriate arteries (4). We believe this can overcome the problem of size mismatched arteries without excessive manipulation of the vessels in an end-to-end fashion.

Purpose: To assess the utility of the Thiel arterial model in microsurgical research, we compared interrupted horizontal mattress (HM) sutures to simple interrupted (SI) sutures in human vessels.Methods: A microsurgical set-up using an operating microscope and Thiel-embalmed arteries was used to practice ten SI and HM anastomoses. Vessel patency, leak and stricture were evaluated using angiography, and vessel wall architecture was evaluated using light microscopy and scanning electron microscopy (SEM). The technique speed was also assessed.Results: We have successfully evaluated all outcomes. All anastomoses were patent. The stricture rate was higher with HM than with SI (60% vs. 35% surface area reduction). Three minor leaks occurred with HM sutures versus one with SI sutures. Edges were evenly everted without any intimal flaps with HM compared to SI. The anastomoses were performed faster using HM than SI sutures (7:58 min vs. 12:41 min, respectively).Conclusion: This is the first study to evaluate the feasibility of a Thiel-embalmed artery model for research purposes. The HM microvascular suture is a promising technique that requires further in vivo validation. (C) 2014 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

Progranulin is a secreted glycoprotein that regulates cell proliferation, migration and survival. It has roles in development, tumorigenesis, wound healing, neurodegeneration and inflammation. Endothelia in tumors, wounds and placenta express elevated levels of progranulin. In culture, progranulin activates endothelial proliferation and migration. This suggested that progranulin might regulate angiogenesis. It was, however, unclear how elevated endothelial progranulin levels influence vascular growth in vivo. To address this issue, we generated mice with progranulin expression targeted specifically to developing endothelial cells using a Tie2-promoter/enhancer construct. Three Tie2-Grn mouse lines were generated with varying Tie2-Grn copy number, and were called GrnLo, GrnMid, and GrnHi. All three lines showed increased mortality that correlates with Tie2-Grn copy number, with greatest mortality and lowest germline transmission in the GrnHi line. Death of the transgenic animals occurred around birth, and continued for three days after birth. Those that survived beyond day 3 survived into adulthood. Transgenic neonates that died showed vascular abnormalities of varying severity. Some exhibited bleeding into body cavities such as the pericardial space. Smaller localized hemorrhages were seen in many organs. Blood vessels were often dilated and thin-walled. To establish the development of these abnormalities, we examined mice at early (E10.5-14.5) and later (E15.5-17.5) developmental phases. Early events during vasculogenesis appear unaffected by Tie2-Grn as apparently normal primary vasculature had been established at E10.5. The earliest onset of vascular abnormality was at E15.5, with focal cerebral hemorrhage and enlarged vessels in various organs. Aberrant Tie2-Grn positive vessels showed thinning of the basement membrane and reduced investiture with mural cells. We conclude that progranulin promotes exaggerated vessel growth in vivo, with subsequent effects in the formation of the mural cell layer and weakening of vessel integrity. These results demonstrate that overexpression of progranulin in endothelial cells influences normal angiogenesis in vivo.

Abstract Progranulin (also called PC cell-derived growth factor, acrogranin or proepithelin), is a secreted glycoprotein with growth factor-like activities. It is often highly over-expressed in tumors including cancers of the breast, ovary, endometrium, kidney, prostate, bladder and liver. It stimulates cellular proliferation, migration and survival and increases the growth of cancers in mice. Recent work suggests that progranulin has, in addition, potentially important roles in fibroblast activation during the formation of tumor stroma. An elevated progranulin level has been observed in endothelial cells in some cancers including ovarian tumors, esophageal carcinomas and gliomas. Since progranulin is not expressed in normal quiescent endothelia, its presence in endothelial cells from cancers prompted us to propose that progranulin may have a novel role in tumor angiogenesis. It is, however, unknown at present, how, if at all, progranulin modulates angiogenesis in vivo. To address this question we created transgenic models in which progranulin expression is specifically targeted to endothelial cells under the control of the Tie2 promoter. Three independent transgenic lines were obtained, with lower (Tie2-GrnLo), intermediate (Tie2-GrnMid) and higher (Tie2-GrnHi) transgene copy number. Increased mortality was observed in heterozygotic crosses from all three transgenic lines, with the highest mortality and lowest germline transmission ratio in the Tie2-GrnHi line; at three weeks after birth the germline transmission ratios of Tie2-Grn positive mice were 26.01%, 19.61% and 4.72% for PgrnLo, PgrnMid and PgrnHi respectively. Premature deaths occurred during the perinatal period after embryonic day 17.5 and before post-natal day 3. Importantly, we observed an array of vascular abnormalities in Tie2-Grn transgenic neonates. These include extensive bleeding into body cavities such as the pericardial space as well as smaller localized hemorrhages in multiple organs. The abnormal blood vessels had dramatically enlarged diameters with irregular edges; several had an incomplete basement membranes and, typically, the perivascular cell coverage was reduced or absent when compared to Tie2-Grn negative litter mates. To establish the developmental timing of vascular disruption, we examined Tie2-GrnHi mice from embryonic day 10.5 to fetal age 17.5. An apparently normal primitive vasculature was established at E10.5 in Tie2-Grn positive embryos suggesting that vasculogenesis proceeded normally. The earliest onset of vascular impairment was observed at E15.5, after vasculogenesis has occurred but during active angiogenesis. As with neonates, from E15.5 and later, vessels became enlarged and perivascular cell investiture was reduced contributing to loss of structural integrity and causing the blood vessels to be prone to hemorrhaging. Given the vessel dilation in Tie2-Grn positive late-stage fetuses and neonates we conclude that progranulin is a novel regulator of angiogenesis. The elevated expression of progranulin by endothelial cells is observed in some cancers in vivo, and, as we report here, in transgenic mice results in the development of enlarged, disorganized, thin-walled vessels that resemble to some extent the disorganized blood vessels in tumors. We suggest, therefore, that progranulin may be significant in regulating tumor angiogenesis. Citation Format: Huishi Toh, Ming Cao, Yonghua Zhang, Eugene Daniels, Andrew Bateman. A novel role for the secreted growth factor progranulin in angiogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B91.

Loop-tail (Lp) mice show a very severe neural tube defect, craniorachischisis, which is caused by mis-sense mutations in the Vangl2 gene. The membrane protein Vangl2 belongs to a highly conserved group of proteins that regulate planar polarity in certain epithelia, and that are also important for convergent extension movements during gastrulation and neurulation. A specific anti-Vangl2 antiserum was produced and used to examine the tissue, cell type, and sub-cellular localization of Vangl2 during embryogenesis. Vangl2 protein is expressed at high levels in the neural tube and shows a dynamic expression profile during neurulation. After neural tube closure, robust Vangl2 staining is detected in several neural and neurosensory tissues, including cerebral cortex, dorsal root ganglia, olfactory epithelium, retina, mechanosensory hair cells of the cochlea, and optic nerve. Vangl2 is also expressed during organogenesis in a number of tubular epithelia, including the bronchial tree, intestinal crypt/villus axis, and renal tubular segments derived from ureteric bud and from metanephric mesenchyme. Examination of Vangl2 localization in the neural tubes and cochleas of the normal and Lp/Lp embryos shows disruption of normal membrane localization of Vangl2 in independent alleles at Lp (Lp, Lp(m1Jus)) as well as overall decrease in the expression level.

PTP (protein-tyrosine phosphatase)-PEST is a ubiquitously expressed cellular regulator of integrin signalling. It has been shown to bind several molecules such as Shc, paxillin and Grb2, that are involved downstream of FAK (focal adhesion kinase) pathway. Through its specific association to p130cas and further dephosphorylation, PTP-PEST plays a critical role in cell-matrix interactions, which are essential during embryogenesis. We report here that ablation of the gene leads to early embryonic lethality, correlating well with the high expression of the protein during embryonic development. We observed an increased level of tyrosine phosphorylation of p130cas protein in E9.5 PTP-PEST−/− embryos, a first evidence of biochemical defect leading to abnormal growth and development. Analysis of null mutant embryos revealed that they reach gastrulation, initiate yolk sac formation, but fail to progress through normal subsequent developmental events. E9.5–10.5 PTP-PEST−/− embryos had morphological abnormalities such as defective embryo turning, improper somitogenesis and vasculogenesis, impaired liver development, accompanied by degeneration in both neuroepithelium and somatic epithelia. Moreover, in embryos surviving until E10.5, the caudal region was truncated, with severe mesenchyme deficiency and no successful liver formation. Defects in embryonic mesenchyme as well as subsequent failure of proper vascularization, liver development and somatogenesis, seemed likely to induce lethality at this stage of development, and these results confirm that PTP-PEST plays an essential function in early embryogenesis.

ABSTRACT The effect of a deficiency in the C5 component of complement on the pathophyisology of infection with the fungal pathogen Candida albicans was studied by using the A/J inbred mouse strain and the BcA17 congenic mouse strain. Acute infection caused by intravenous injection of C. albicans blastospores is associated with rapid fungal replication in the heart, brain, and, in particular, kidneys of C5-deficient mice. Histological studies and analysis of markers for tissue damage indicated that the heart is the organ that is most affected and that it ultimately fails in C5-deficient mice. In A/J and BcA17 mice, tissue damage is associated with (i) cellular infiltration in the heart, which is not seen in the kidney despite the higher fungal load in the latter organ, and (ii) a very strong inflammatory response, including elevated levels of many cytokines and chemokines. This results in cardiomyopathy, which is associated with elevated levels of creatine kinase and cardiac troponin I in the circulation. Damage to the cardiac muscle is associated with metabolic changes, including hypoglycemia, decreased lipid utilization resulting in elevated levels of cardiac triglycerides, and unproductive glucose utilization linked to a dramatic increase in the level of pyruvate dehydrogenase kinase 4 (Pdk4), a negative regulator of the pyruvate dehydrogenase complex.

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.

The PREP, MEIS, and PBX families are mammalian members of the TALE (three amino acid loop extension) class of homeodomain-containing transcription factors. These factors have been implicated in cooperative DNA binding with the HOX class of homeoproteins, but PREP and MEIS interact with PBX in apparently non-HOX-dependent cooperative DNA binding as well. PREP, MEIS, and PBX have all been reported to reside in the cytoplasm in one or more tissues of the developing vertebrate embryo. In the case of PBX, cytoplasmic localization is due to the modulation of nuclear localization signals, nuclear export sequences, and interaction with a cytoplasmic anchoring factor, non-muscle myosin heavy chain II B. Here we report that murine PREP2 exists in multiple isoforms distinguished by interaction with affinity-purified antibodies raised to N- and C-terminal epitopes and by nuclear versus cytoplasmic localization. Alternative splicing gives rise to some of these PREP2 isoforms, including a 25-kDa variant lacking the C-terminal half of the protein and homeodomain and having the potential to act as dominant-negative. We further show that cytoplasmic localization is due to the concerted action of nuclear export, as evidenced by sensitivity to leptomycin B, and cytoplasmic retention by the actin and microtubule cytoskeletons. Cytoplasmic PREP2 colocalizes with both the actin and microtubule cytoskeletons and coimmunoprecipitates with actin and tubulin. Importantly, disruption of either cytoskeletal system redirects cytoplasmic PREP2 to the nucleus. We suggest that transcriptional regulation by PREP2 is modulated through the subcellular distribution of multiple isoforms and by interaction with two distinct cytoskeletal systems.

The growth factor progranulin (acrogranin/PC‐derived growth factor/granulin‐epithelin precursor) promotes onset of blastocyst cavitation and is required for neonatal hypothalamic sexual differentiation. Little is known, however, of the range of developmental processes in which it is involved. We used in situ hybridization to investigate progranulin expression in murine embryos. Progranulin mRNA is expressed in maternal and embryonic components during early establishment of pregnancy. Abundant expression is observed in the early decidualizing uterine stroma and glands. In the embryo, the trophoblast giant cells at the interface of placental exchange sites (both choriovitelline and chorioallantoic placenta) show strong expression. The gastrulating epiblast and mesenchyme (intraembryonic and extraembryonic mesenchyme) all revealed activity. The allantois and yolk sac mesenchyme (site of early hemopoiesis) were positive, as were later phases of active vessel formation (pia mater of brain, epicardium of the heart). In the urogenital system, it was expressed in Sertoli cells and in kidney tubules. It was highly expressed in proliferating epidermal cells. During epidermal appendage formation, the early epithelial bud was positive, but the forming duct and differentiating adjacent mesenchyme was negative. It is widely distributed during central nervous system development and the peripheral nervous system (dorsal root ganglia and sympathetic ganglia). Based on the pattern of progranulin gene expression, we propose proliferative and developmental roles for progranulin in establishing pregnancy, during gastrulation, and during embryonic development of the epidermis, nervous system, blood vessel, formation, and spermatogenesis. Developmental Dynamics 593–599, 2003. © 2003 Wiley‐Liss, Inc.

Human WDR9 has been mapped to chromosome 21, within one of the Down syndrome (DS) critical regions. Here, we study the expression pattern of the murine Wdr9 gene and its protein product. We show that Wdr9 is broadly expressed in the mouse embryo by means of in situ hybridization and immunohistochemistry. Wdr9 expression levels are dynamic during embryonic development as revealed by Northern blot analysis. We further show that WDR9 is a nuclear protein associated with BRG1, a SWI/SNF complex component. We also demonstrate that a polyglutamine‐containing region of the protein functions as a transcriptional activation domain. We propose that WDR9 is a transcriptional regulator involved in chromatin remodeling through the action of two bromodomains and contacts to the SWI/SNF complex. These results may provide a molecular basis for the association of WDR9 with DS. Developmental Dynamics 227:608–614, 2003. © 2003 Wiley‐Liss, Inc.

We describe Prep2, a new murine homeobox‐containing gene closely related to Prep1. The PREP2 protein belongs to the three amino acid loop extension (TALE) superclass of homeodomain‐containing proteins and encodes a polypeptide of 462 residues. As for PREP1, PREP2 binds an appropriate site on DNA as a heterodimer with PBX1A. Northern analysis, immunoblotting, immunohistochemistry, and in situ hybridization show widespread Prep2 expression during organogenesis and in the adult. The data suggest that Prep2 functions to varying degrees in a broad array of tissues and developmental processes. © 2002 Wiley‐Liss, Inc.

Biliary glycoproteins are members of the carcinoembryonic antigen (CEA) family and behave as cell adhesion molecules. The mouse genome contains two very similar Bgp genes, Bgp1 and Bgp2, whereas the human and rat genomes contain only one BGP gene. A Bgp2 isoform was previously identified as an alternative receptor for the mouse coronavirus mouse hepatitis virus. This isoform consists of two extracellular immunoglobulin domains, a transmembrane domain and a cytoplasmic tail of five amino acids. In this report, we have examined whether the Bgp2 gene can express other isoforms in different mouse tissues. We found only one other isoform, which has a long cytoplasmic tail of 73 amino acids. The long cytodomain of the Bgp2 protein is highly similar to that of the Bgp1/4L isoform. The Bgp2 protein is expressed in low amounts in kidney and in a rectal carcinoma cell line. Antibodies specific to Bgp2 detected a 42-kDa protein, which is expressed at the cell surface of these samples. Bgp2 was found by immunocytochemistry in smooth muscle layers of the kidney, the uterus, in gut mononuclear cells and in the crypt epithelia of intestinal tissues. Transfection studies showed that, in contrast with Bgp1, the Bgp2 glycoprotein was not directly involved in intercellular adhesion. However, this protein is found in the proliferative compartment of the intestinal crypts and in cells involved in immune recognition. This suggests that the Bgp2 protein represents a distinctive member of the CEA family; its unusual expression patterns in mouse tissues and the unique functions it may be fulfilling may provide novel clues about the multiple functions mediated by a common BGP protein in humans and rats.

Biliary glycoprotein (Bgp) is a member of the immunoglobulin superfamily and the carcinoembryonic antigen family. Previous studies have shown that Bgp functions as an intercellular adhesion molecule and a canalicular bile salt transporter. Moreover, we and others demonstrated that Bgp can inhibit colonic and prostatic tumor cell growth in vivo , through a mechanism which depends on sequences present in its cytoplasmic domain. In this study, we have examined the possibility that the cytoplasmic domain of Bgp can interact with signal transduction molecules. We showed that tyrosine phosphorylated Bgp, expressed in mouse colon carcinoma CT51 cells, could reversibly associate with protein tyrosine phosphatase SHP-1. Mutation of either of two tyrosine residues present in the cytoplasmic domain of Bgp abrogated SHP-1 binding, suggesting that this association was mediated by both tyrosine residues. Similarly, we noted that either of the two SH2 domains of SHP-1 could bind tyrosine phosphorylated Bgp in vitro . It is therefore conceivable that some of the functions of Bgp are mediated through its ability to induce intracellular protein tyrosine dephosphorylation.

Rodent biliary glycoprotein (Bgp), also known as C-CAM, has recently been shown to function as a tumor suppressor in colon, prostate, and bladder cancers. This glycoprotein is a member of the carcinoembryonic antigen family and is one of the only proteins in this family to encode either a long (71-73 amino acids) or short (10 amino acids) cytoplasmic domain. We and others have shown that the growth-inhibitory properties of Bgp depend upon the expression of its long cytoplasmic domain. However, the two Bgp isoforms normally coexist in most cell types surveyed; the longer variant is usually present in lower amounts than the shorter one. In this study, we have examined the in vitro and in vivo growth properties of both mouse Bgp variants separately and in combination. To determine the physiologically relevant expression levels and ratios of the two Bgp variants, we have quantified the amount of the longer variant in normal colonic epithelial cells and showed that it constitutes 15-20% of total Bgp expressed in this tissue. To mimic the in vivo situation, we have generated double transfectant cell lines expressing the longer and shorter Bgp isoforms coordinately in tumorigenic CT51 mouse colonic carcinoma cells and demonstrated that the longer Bgp isoform exhibits a dominant tumor growth inhibition phenotype over that of the shorter variant within physiological levels of expression of Bgp. Unexpectedly, significant overexpression of the longer Bgp isoform alone led to reversal of the tumor inhibition phenotype. These results, therefore, suggest that there may be a limiting threshold of Bgp expression or Bgp-associating proteins mediating the tumor inhibition phenotype.

Biliary glycoprotein (Bgp1), a carcinoembryonic antigen-related family member of the immunoglobulin superfamily, is involved in normal and neoplastic events. Analysis of Bgp1 expression throughout post-implantation mouse embryogenesis using reverse transcription-polymerase chain reactions, immunostaining with anti-Bgp1 monoclonal antibodies, and in situ hybridization with specific Bgp1 cDNA fragments revealed that Bgp1 may be involved in a number of specific embryonic processes. Immunoblot analysis of Bgp1 deletion mutant proteins indicated that distinguishable epitopes of the molecule were preferentially identified by the three Bgp1 antibodies used in this study. This distinction is supported by our immunolocalization studies during mouse embryogenesis in which the three antibodies revealed specific patterns of Bgp1 expression. Bgp1 is not expressed in early post-implantation embryos (7.5 dpc), but is found in the placenta and extra-embryonic tissues (decidual endothelial cells, giant trophoblasts, yolk sac visceral endoderm, and endometrial glands) at this time. The primitive gut epithelium and surface ectoderm were the first embryonic tissues to express Bgp1. Significant Bgp1 expression was also observed later during epithelial-mesenchymal interactions (skin, meninges, lung, kidney, salivary glands, pancreas). A unique epitope of Bgp1, detectable by the monoclonal antibody CC1, was also associated with mesenchymal expression and was prominent during myogenesis (secondary myotube formation) at sites of terminal differentiation. These studies suggest multiple roles for isoforms and glycoforms of the Bgp1 proteins localized in specific sites during prenatal development. © 1996 Wiley-Liss, Inc.