Cellular localization of cytomegalovirus nucleic acids in guinea pig salivary glands by in situ hybridization

In situ hybridization with guinea pig cytomegalovirus (CMV) DNA probes was used to detect guinea pig CMV nucleic acid sequences in salivary glands during the course of infection. Optimum conditions for detection of guinea pig CMV gene sequences at the cellular level were determined. The technique of nucleic acid hybridization was compared to antigen detection and histologic examination for cellular localization of guinea pig CMV during acute and chronic infection. Tissue sections fixed for 1 h in 4% paraformaldehyde demonstrated darker specific staining and contained significantly larger numbers of cells positive for guinea pig CMV nucleic acids and antigens than sections fixed longer and in other fixatives. The method of in situ hybridization detected more guinea pig CMV infected cells than did routine histological evaluation. Histologically normal salivary gland duct cells as well as cells bearing typical inclusions were found to contain guinea pig CMV nucleic acids. Guinea pig CMV nucleic acids were also detected although less frequently in cells outside of the ducts. These results suggest that in situ hybridization allows for the detection of histologically inapparent guinea pig CMV infections at the cellular level.     

Intimal thickening involves transdifferentiation of embryonic endothelial cells

Morphological studies have hypothesized different origins for the precursors of the vascular smooth muscle cells (SMCs). The intriguing possibility that intimal SMCs may arise from the endothelium has newly emerged. As a first step towards understanding of the possible mechanisms involved in the transdifferentiation of endothelium into smooth muscle cells, we characterized the in vivo phenotype of the cells located in the aortic wall (distal to the aortic arches). This was accomplished using advanced stages of chicken embryo development. Furthermore, we investigated whether the cells present at the intimal thickening derive from the endothelial cell transdifferentiation. Immunolabeling of serial cryosections suggested that mesenchymal cells observed in the intimal thickening may arise from the endothelium. These cells may persist either as non-muscle throughout the development or possibly convert to cells expressing smooth muscle alpha-actin (SM alpha-actin). To determine whether endothelial cells may actually transdifferentiate into mesenchymal cells, aortic explants from 14-day-old chicken embryos (stage 40) were used. We found that explanted endothelial cells lose their cobblestone-appearance and migrate toward cell-free area. Some of these cells maintain the vWf immunoreactivity, whereas other cells coordinately lose vWf and gain SM alpha-actin expression (transitional cells). Taken together these findings strongly support the possibility that embryonic aortic endothelial transdifferentiate into mesenchymal cells, some of which express SM alpha-actin. Since TGFbeta-3 is considered an essential factor during epithelial to mesenchymal transitions in earlier chicken heart development, we also investigated the distribution of this growth factor at day 14. Our observations indicated that the immunoreactivity for TGFbeta-3 in this stage may be associated with migrating mesenchymal cells and that this immunoreactivity appears to decrease as cell differentiation advances. Therefore, the present study provides evidence that could help to explain 1) the presence of cells displaying a phenotype reminiscent of fetal-like cells in the normal chicken aorta and in the intimal region of the human aorta; 2) the SM lineage diversity in the chicken embryo reported by others; 3) a subpopulation of immature cells in the subendothelial region of the main pulmonary arteries of fetal, neonatal and adult bovines; and 4) the presence of intimal cushions, intimal pads, eccentric and diffuse intimal thickening that are observed in mammalian and avian vessels at birth.     

Cytomegalovirus infection and viral-induced transformation of human endothelial cells

Human cytomegalovirus (CMV) has been associated with vascular pathology. In vivo, CMV is present in vessel wall cells during acute and chronic infections as well as in atherosclerotic lesions. CMV nucleic acids and proteins have also been detected within Kaposi's sarcoma lesions. Because of these associations, we studied the interaction of CMV with human endothelial cells with particular attention to its oncogenicity in this cell type. Our data demonstrate that human endothelial cells are permissive to viral replication but that the viral replication cycle is delayed compared with fibroblast cells. Persistent infections can result with minimal cytopathology. CMV can transform these cells to anchorage-independent growth, and noninfectious virus is still capable of inducing this transforming event. Our results demonstrate that productive or persistent CMV infection of endothelial cells and viral-induced transformation can occur, thus providing an in vitro correlate of in vivo events.     

Behaviour of chick embryo aortic cells obtained through nonenzymatic means cultured onto collagen gels

Summary In the present paper we used a method whereby some of the cellular events that take place in the aortic wall during chick embryo development can be studied in vitro. Collagen gels were utilized to culture endothelial cells obtained through nonenzymatic means from aortic explants isolated from 12- to 14-day-old chick embryos. These cells were characterized by morphological and immunocytochemical criteria. After 72 h, explanted endothelial cells from embryonic aorta formed a monolayer of polygonal cells, which gave rise to elongated cells that migrate into the collagen gel. These cells are similar to those of mesenchyme-like cells observed in vivo at the subendothelial region of 14-day-old chick embryonic aorta. In long-term cultures, some of these cells acquired features either of synthetic smooth muscle cell phenotype, or of fibroblast-like cells very similar to those found in developing aorta. These results indicate that the culture of explants of chick embryo aorta on three-dimensional collagen gel is a valuable system for studying some of the complex morphogenetic events that occur during the development of the aorta.     

Cytokinetic studies on the aortic endothelium and limb bud vascularization in avian embryos

Summary Cytokinetic studies on the aortic endothelium using the BrdU/anti-BrdU-method were carried out on 2.5– to 6-day chick and quail embryos. The mitotic activity of the aortic endothelium is related temporally to the age of the avian embryo and spatially to the embryonic region where the aorta originates. The mitotic activity of the aortic endothelium decreases with increasing age of the embryos. In the limb buds, however, the mitotic rate of the aortic endothelial cells increases independently of the age of the embryo. This increase in the mitotic activity of the aortic endothelium at the appropriate levels coincides with the vascularization of the outgrowing limb buds. We concluded therefore that the aortic endothelium probably supplies endothelial cells for the formation of limb vessels at this stage. Thus our results suggest that angiogenesis (sprouting of capillaries from pre-existing vessels) takes place during limb vascularization in avian embryos. On the other hand, immunohistochemical studies with QH-1 or MB-1 antibody show, beside a capillary network in the central core of the wing bud, individual immunolabelled cells of mesenchymal character within the primarily avascular subectodermal region from the onset of vascularization onwards. We suggest that these cells have partly to be regarded as endothelial precursor cells, which have differentiated in situ from the local limb mesenchyme, and which will contribute to the developing vascular plexus. This means that not only angiogenesis, but also vasculogenesis (in situ from mesenchymal precursors differentiated endothelial cells) appears to be involved in limb vessel formation.Supported by the Deutsche Forschungsgemeinschaft (Ch 44/9-1, Ch 44/9-2)This paper is dedicated to Prof. Dr. K.V. Hinrichsen on the occasion of his 65th birthday     

A study of the pathogenesis of equid herpesvirus-1 (EHV-1) abortion by DNA in-situ hybridization.

The polymerase chain reaction and DNA in-situ hybridization were used to study sections of uterine tissue collected from mares near the time of abortion due to equid herpesvirus-1 (EHV-1) infection. These techniques revealed viral nucleic acids in endothelial cells of endometrial arterioles, in accordance with previously published immunohistological data. In addition, however, they revealed nucleic acids in cellular debris within endometrial glands and diffusing across the placenta at sites of microcotyledonary infarction. Perivascular leucocytes were generally negative for viral DNA, despite marked perivascular cuffing. These data provided further support for the central role of the vascular endothelial cell in the pathogenesis of EHV-1 abortion and demonstrated direct transplacental spread of nucleic acids at sites of microcotyledonary infarction and across the endometrial glands in the vicinity of vascular lesions.     

Compartmentalized Culture of Perivascular Stroma and Endothelial Cells in a Microfluidic Model of the Human Endometrium

The endometrium is the inner lining of the uterus. Following specific cyclic hormonal stimulation, endometrial stromal fibroblasts (stroma) and vascular endothelial cells exhibit morphological and biochemical changes to support embryo implantation and regulate vascular function, respectively. Herein, we integrated a resin-based porous membrane in a dual chamber microfluidic device in polydimethylsiloxane that allows long term in vitro co-culture of human endometrial stromal and endothelial cells. This transparent, 2-μm porous membrane separates the two chambers, allows for the diffusion of small molecules and enables high resolution bright field and fluorescent imaging. Within our primary human co-culture model of stromal and endothelial cells, we simulated the temporal hormone changes occurring during an idealized 28-day menstrual cycle. We observed the successful differentiation of stroma into functional decidual cells, determined by morphology as well as biochemically as measured by increased production of prolactin. By controlling the microfluidic properties of the device, we additionally found that shear stress forces promoted cytoskeleton alignment and tight junction formation in the endothelial layer. Finally, we demonstrated that the endometrial perivascular stroma model was sustainable for up to 4 weeks, remained sensitive to steroids and is suitable for quantitative biochemical analysis. Future utilization of this device will allow the direct evaluation of paracrine and endocrine crosstalk between these two cell types as well as studies of immunological events associated with normal vs. disease-related endometrial microenvironments.     

Heterogeneous Susceptibility of Valve Endothelial Cells to Mesenchymal Transformation in Response to TNFα

Lack of understanding of the early mechanisms of aortic valve stenosis and calcification hinders the development of diagnostic and therapeutic intervention strategies. Inflammation is a known component of early aortic valve disease and can induce mesenchymal transformation in a subset of aortic valve endothelial cells. Here we present a three-dimensional culture system that allows transforming and non-transforming cells to be independently isolated and analyzed. We have used the system to identify and characterize the dynamic invasion and phenotypic transition of two distinct subsets of endothelial cells: those that invade and transform under TNFα treatment, and those that resist mesenchymal transformation and remain endothelial. We determine that non-transformed cells maintain control levels of endothelial genes VE-cadherin and eNOS, while transformed cells lose these endothelial characteristics and upregulate α-smooth muscle actin. Both subsets of cells have an inflammatory phenotype marked by increased ICAM-1, but transformed cells have increased MMP-9, Notch1, TGF-β, and BMP-4, while non-transformed cells do not. Transformed cells also have distinct effects on alignment of collagen fibers as they invade the hydrogel system, which is not found in control endothelial or interstitial valve cells. Understanding the role of transforming and non-transforming endothelial cells in valve disease will provide an important pathological link between early inflammation and later stages of disease. Discovery of the molecular signature of transformation-resistant endothelial cells could inform development of treatment strategies that promote survival of the valve endothelium.     

Remodeling of aortic smooth muscle during avian embryonic development

The dorsal aorta is the earliest formed intraembryonic blood vessel in vertebrates composed of an inner lining of endothelial cells (ECs) and a slightly later‐forming outer wall consisting of vascular smooth muscle cells (SMCs) and pericytes. We previously identified the sclerotome as the only somitic compartment contributing to aortic SMCs in the trunk of the avian embryo. However, we demonstrated that the first SMCs in the aortic floor are not of somitic origin and must be derived from a different source. Here, we show that the primary SMCs are a transient population of aortic wall cells originating from the splanchnic mesoderm. A model is presented suggesting that wall formation of the early dorsal aorta in chick is a two‐step process: The primary, transient SMCs in the aortic floor originate in the splanchnic mesoderm, whereas the secondary, definitive SMCs of the entire aortic wall originate in the sclerotome. Developmental Dynamics 238:624–631, 2009. © 2009 Wiley‐Liss, Inc.     

血管内皮细胞生长因子基因转染骨髓基质细胞的瞬时表达与稳定表达

目的探讨血管内皮细胞生长因子（VEGF）基因修饰的大鼠骨髓基质细胞（BMCS）的表达能力及表达活性。方法取6周龄SD大鼠BMCS传代培养后以3μl阳离子脂质体（Lipofectamine）：1μg pc DNA3．1-VEGF165的比例转染，通过RT—PCR技术、免疫组织化学S—P法检测转染细胞中外源性VEGF165基因的转录及瞬时表达和稳定表达，用血管内皮细胞（VEC）增殖试验测定转染细胞培养上清中VEGF的生物活性。结果VEGF基因转染的大鼠骨髓基质细胞可有效转录VEGF165，其分泌培养上清液中的表达产物可促进血管内皮细胞增殖，具有很强的生物活性。结论采用基因转染技术可将VEGF转染至BMCS中，并可有效表达具有生物活性的VEGF。     

Non-invasive pre-implantation genetic diagnosis of X-linked disorders

Pre-implantation genetic diagnosis (PGD) is a powerful clinical tool to identify embryos with or at risk of specific genetic diseases before implantation in utero after in vitro fertilization (IVF). PGD is performed on embryo biopsies that are obtained by aspiration of one or two cells from pre-implantation embryos at day 3 or day 5/6 of culture. However this is a traumatic method that cannot be avoided because non-invasive procedures to assess the genetic status of pre-implantation embryos are not available yet. We hypothesize that cell-free nucleic acids, which are released by embryos in the culture medium during the IVF procedure, could be used for genetic screening. To test our hypothesis we will focus first on X-linked disorders because these single-gene diseases due to the presence of defective genes on the X chromosome are dominant in males. Therefore the objective here is to discriminate between female (XX) and male (XY) embryos by detecting Y chromosome-specific sequences in cell-free nucleic acids. Using culture medium from embryos we are able to discriminate between male and female embryos. This opens new avenues for the development of a non-invasive PGD method.     

Cationic lipid-mediated transfection of bovine aortic endothelial cells inhibits their attachment

The need for a small-diameter vascular graft for coronary artery and peripheral vascular replacement is great and is projected to increase as the population ages. Synthetic small-diameter vascular grafts fail because of acute thrombosis or chronic intimal hyperplasia leading to restenosis. Endothelial cell seeding has been attempted with limited success in the femoral artery by Zilla and others. However, patency rates have not increased sufficiently to justify large clinical trials. Genetic engineering of endothelial cells before seeding has been proposed to encourage endothelial cell phenotypes that would predispose the graft to patency. In this study, we investigate the effect cationic lipid-mediated transfection of endothelial cells with respect to their attachment to a potential graft material, Fluoropassiv (Vascutek). Liposomal transfection was optimized for maximum gene expression. We report that transfection decreases the ability of bovine aortic endothelial cells to attach by approximately 100% as compared with nontransfected control over 18 h. Further, when placed under physiologic shear conditions, this difference is sustained. The effects of gene transfer on endothelial cell adhesion must be included as an important optimization criterion along with gene expression for engineered endothelial cell-seeding applications.     

Inducible expression of vascular cell adhesion molecule-1 by vascular smooth muscle cells in vitro and within rabbit atheroma.

Vascular cell adhesion molecule-1 (VCAM-1), a mononuclear leukocyte adhesion molecule, is expressed in cultured vascular endothelial cells activated by cytokines and is induced in rabbit aortic endothelium in vivo within 1 week after initiation of an atherogenic diet. We now demonstrate that vascular smooth muscle cells can also express VCAM-1 in rabbit atherosclerotic lesions in vivo and in response to cytokines in vitro. Immunohistochemical staining of aortas from rabbits fed a 0.3% cholesterol-containing diet revealed that a portion of smooth muscle cells within intimal foam cell-rich lesions expressed VCAM-1. The intimal VCAM-1-expressing cells localized predominantly in regions above the internal elastic lamina. These VCAM-1-positive cells had the typical spindle shape of smooth muscle cells but had reduced alpha-actin expression in comparison to normal medial smooth muscle cells, and did not bear markers for endothelium, macrophages, and T cells. In culture, rabbit aortic smooth muscle cells expressed VCAM-1 mRNA and protein in a time- and concentration-dependent fashion when exposed to interferon-gamma or Gram-negative bacterial lipopolysaccharide. Cultured human vascular smooth muscle cells also expressed VCAM-1 mRNA and protein in response to lipopolysaccharide, interferon-gamma, and interleukin-4. The monokines interleukin-1 alpha and tumor necrosis factor-alpha did not induce VCAM-1 expression in either rabbit or human vascular smooth muscle cells. Inducible VCAM-1 expression by vascular smooth muscle cells in vivo during hypercholesterolemia and in vitro in response to certain cytokines suggests a broader range of VCAM-1 functions in vascular biology than heretofore appreciated.     

Active participation of endothelial cells in inflammation

Leukocyte migration from the blood into tissues is vital for immune surveillance and inflammation. During this diapedesis of leukocytes, the leukocytes bind to endothelial cell adhesion molecules and then migrate across the vascular endothelium. Endothelial cell adhesion molecules and their counter-receptors on leukocytes generate intracellular signals. This review focuses on the active function of endothelial cells during leukocyte-endothelial cell interactions. We include a discussion of the "outside-in" signals in endothelial cells, which are stimulated by antibody cross-linking or leukocyte binding to platelet-endothelial cell adhesion molecule-1, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1. Some of these signals in endothelial cells have been demonstrated to actively participate in leukocyte migration. We suggest that some of the adhesion molecule signals, which have not been assigned a function, are consistent with signals that stimulate retraction of lateral junctions, stimulate endothelial cell basal surface adhesion, or induce gene expression.     

低氧对肺血管周细胞的影响

目的 研究低氧直接或通过内皮细胞的介导对体外培养的肺血管周细胞的能量代谢。细胞周期和血小板源性生长因子及其受体ｍＲＮＡ表达的影响。方法 应用 ＭＴＴ比色分析法,流式细胞术,核酸原位分子杂交及图像分析等方法进行定量分析。结果 低氧量直接或通过内皮细胞介导,促进肺血管周期由静止期进入ＤＮＡ合成期及有丝分裂期,且能促进周细胞ＰＤＧＦ及其受体ｍＲＮＡ的表达。     

Gene transfer by electroporation into hemogenic endothelium in the avian embryo

Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained by a population of self‐renewing multipotent hematopoietic stem cells (HSCs) throughout the life of an organism. Cells with definitive HSC properties appear in the mid‐gestation embryo as dense clusters of cells budding from the floor of the aorta, and that of the vitelline and umbilical arteries in the aorta‐gonads‐mesonephros region. Attempts to genetically modify the aortic floor from which these HSCs arise have been unsuccessful in the mouse, since the regulation of gene expression in the hemogenic endothelium is largely unknown. Here we report the implementation of gene transfer by electroporation into dorsal aortic endothelial cells in the chick embryo. This approach provides a quick and reproducible method of generating gain/loss‐of‐function models to investigate the function of genes involved in HSC birth. Developmental Dynamics 239:1748–1754, 2010. © 2010 Wiley‐Liss, Inc.     

Cytosolic nucleic acid sensors and innate immune regulation

During viral and bacterial infections, pathogen-derived cytosolic nucleic acids are recognized by the intracellular RNA sensors retinoic acid-inducible gene I and melanoma-differentiated gene 5 and intracellular DNA sensors, including cyclic-di-GMP-AMP synthase, absent in melanoma 2, interferon (IFN)–gamma inducible protein 16, polymerase III, and so on. Binding of intracellular nucleic acids to these sensors activates downstream signaling cascades, resulting in the production of type I IFNs and pro-inflammatory cytokines to induce appropriate systematic immune responses. While these sensors also recognize endogenous nucleic acids and activate immune responses, they can discriminate between self- and non-self-nucleic acids. However, dysfunction of these sensors or failure of regulatory mechanisms causes aberrant activation of immune response and autoimmune disorders. In this review, we focus on how intracellular immune sensors recognize exogenous nucleic acids and activate the innate immune system, and furthermore, how autoimmune diseases result from dysfunction of these sensors.