CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef.

The human immunodeficiency virus type 1 (HIV-1) genes vpu, env, and nef have all been implicated in modulating the levels of cell surface CD4 on infected cells. To quantitatively assess the relative contribution of each gene product to the regulation of CD4 during HIV infection of Jurkat T cells and peripheral blood mononuclear cells, we have developed an infectious HIV reporter system which expresses different combinations of these genes. To distinguish infected cells in the early or late stages of infection from uninfected cells, these viruses were designed to express human placental alkaline phosphatase with the kinetics of either early or late viral genes. Flow cytometry to detect placental alkaline phosphatase and CD4 in infected cells showed that vpu, env, and nef are independently capable of down-modulation of CD4. As predicted by their respective expression patterns, nef down-modulated CD4 rapidly during the early phase of virus infection whereas vpu and env functioned late in the infection. In both Jurkat cells and peripheral blood mononuclear cells, a combination of the three genes was more efficient than any one or two genes, demonstrating that all three genes are required to achieve maximal CD4 down-modulation. In primary cells, down-modulation of CD4 was less efficient than in Jurkat cells and there was a stronger dependence on nef function for reducing cell surface CD4. HIV therefore has three genes that are able to independently down-modulate CD4; together, they can eliminate the bulk of cell surface CD4.     

Intravital two-photon microscopy of lymphatic vessel development and function using a transgenic Prox1 promoter-directed mOrange2 reporter mouse

Lymphatic vessels, the second vascular system of higher vertebrates, are indispensable for fluid tissue homoeostasis, dietary fat resorption and immune surveillance. Not only are lymphatic vessels formed during fetal development, when the lymphatic endothelium differentiates and separates from blood endothelial cells, but also lymphangiogenesis occurs during adult life under conditions of inflammation, wound healing and tumour formation. Under all of these conditions, haemopoietic cells can exert instructive influences on lymph vessel growth and are essential for the vital separation of blood and lymphatic vessels. LECs (lymphatic endothelial cells) are characterized by expression of a number of unique genes that distinguish them from blood endothelium and can be utilized to drive reporter genes in a lymph endothelial-specific fashion. In the present paper, we describe the Prox1 (prospero homeobox protein 1) promoter-driven expression of the fluorescent protein mOrange2, which allows the specific intravital visualization of lymph vessel growth and behaviour during mouse fetal development and in adult mice.     

Angiogenesis during human extraembryonic development involves the spatiotemporal control of PDGF ligand and receptor gene expression.

We have examined the role of platelet-derived growth factor (PDGF) ligand and receptor genes in the angiogenic process of the developing human placenta. In situ hybridization analysis of first trimester placentae showed that most microcapillary endothelial cells coexpress the PDGF-B and PDGF beta-receptor genes. This observation indicates that PDGF-B may participate in placental angiogenesis by forming autostimulatory loops in capillary endothelial cells to promote cell proliferation. Endothelial cells of macro blood vessels maintained high PDGF-B expression, whereas PDGF beta-receptor mRNA was not detectable. In contrast, PDGF beta-receptor mRNA was readily detectable in fibroblast-like cells and smooth muscle cells in the surrounding intima of intermediate and macro blood vessels. Taken together, these data suggest that the PDGF-B signalling pathway appears to switch from an autocrine to a paracrine mechanism to stimulate growth of surrounding PDGF beta-receptor-positive mesenchymal stromal cells. Smooth muscle cells of the blood vessel intima also expressed the PDGF-A gene, the protein product of which is presumably targeted to the fibroblast-like cells of the mesenchymal stroma as these cells were the only ones expressing the PDGF alpha-receptor. PDGF-A expression was also detected in columnar cytotrophoblasts where it may have a potential role in stimulating mesenchymal cell growth at the base of the growing placental villi. We discuss the possibility that the regulation of the PDGF-B and beta-receptor gene expression might represent the potential targets for primary angiogenic factors.     

Adult and embryonic blood and endothelium derive from distinct precursor populations which are differentially programmed by BMP in Xenopus

Blood and blood vessels develop in close association in vertebrate embryos and loss-of-function mutations suggest common genetic regulation. By the criteria of co-expression of blood and endothelial genes, and lineage tracing of progeny, we locate two distinct populations of progenitors for blood and endothelial cells in developing Xenopus embryos. The first population is located immediately posterior to the cement gland during neurula stages and gives rise to embryonic blood and vitelline veins in the anterior ventral blood island (aVBI), and to the endocardium of the heart. The second population resides in the dorsal lateral plate mesoderm, and contains precursors of adult blood stem cells and the major vessels. Both populations differentiate into endothelial cells in situ but migrate to new locations to differentiate into blood, suggesting that their micro-environments are unsuitable for haematopoietic differentiation. Both require BMP for their formation, even the Spemann organiser-derived aVBI, but individual genes are affected differentially. Thus, in the embryonic population, expression of the blood genes, SCL and GATA2, depend on BMP signalling while expression of the endothelial gene, Xfli1, does not. By contrast, Xfli1 expression in the adult, DLP population does require BMP. These results indicate that both adult and the anterior component of embryonic blood in Xenopus embryos derive from populations of progenitors that also give rise to endothelial cells. However, the two populations give rise to distinct regions of the vasculature and are programmed differentially by BMP.     

Dicer及其miRNAs是血管平滑肌细胞分化和增殖所必需的基因和调节因子

为了研究Dicer及其所产生的miRNAs在血管平滑肌细胞(VSMC)中的作用,本研究采用条件性基因打靶法敲除了小鼠VSMC中的Dicer外显子23,通过采用连续性剖检妊娠小鼠法、病理组织学、免疫荧光、PCR、Western blot和实时PCR等技术对条件性敲除Dicer(Dicer c KO)胚胎的血管病变和VSMC中的Dicer、miRNAs和信号转导通路蛋白变化进行了详细研究.结果发现,在培育条件性敲除Dicer小鼠的过程可产生三种不同基因型小鼠,即野生型、杂合型和纯合型(Dicer c KO)小鼠.其中野生型和杂合型小鼠出生后无明显临床异常,而Dicer c KO小鼠却死于腹中而不能出生.Dicer c KO胚胎在胚胎发育的第12.5天(E12.5)就出现发育迟滞变化,在E14.5,皮肤、骨骼肌和肝脏的血管极度扩张、血液淤滞和广泛的弥漫性出血,在E15.5死亡.Dicer c KO胚胎血管壁的病变于E13.5即出现,主要表现为血管中膜的VSMC排列不整,增生减少;E14.5血管壁变薄、塌陷,管腔不规则,细胞增生明显减少;E15.5血管壁的结构完全破坏,细胞增生停止,血管壁的屏障作用破坏,通透性增强,向外渗血.在胚胎发育的E14.5,VSMC标志性基因的表达明显下调,VSMC中大部分受检miRNAs的表达也明显降低,磷酸化的信号转导通路蛋白,即细胞外信号调节激酶和蛋白激酶明显衰减.研究证明,Dicer是血管发育所必需的基因,它可通过控制mi RNA产生和成熟来调节VSMC标志性基因的表达,借以促进VSMC的增殖与分化,保障血管壁结构的完整.     

Expression of stathmin family genes in the murine uterus during early pregnancy

Stathmin, a cytosolic phosphoprotein that regulates microtubule dynamics during cell-cycle progression, is abundantly expressed at embryo implantation sites in rats. Here, we characterized the expression of stathmin and its family genes in the murine uterus during the peri-implantation period. Stathmin protein was expressed in the glandular and luminal epithelium, blood vessels, and stromal cells on day 3 of pregnancy. On the day of implantation (day 5), stathmin was mainly localized in blood vessels in the endometrium. On day 7, intense stathmin expression was limited to capillary vessels and secondary decidual cells. Stathmin expression was higher at implantation sites than at uterine segments between implantation sites and increased during oil-induced decidualization. Although the artificially-induced deciduoma weights and number of implantation sites were similar between stathmin-knockout (KO) and wild-type (WT) mice, the stathmin-KO mice had fewer newborn pups (reduced by 30%). The expression of alkaline phosphatase, desmin, and cyclin D3 was attenuated in decidual zones of stathmin-KO mice. Messenger RNA level of the stathmin family gene, SCG10, was high at the time of decidualization in WT and stathmin-KO mice. In contrast, the others of stathmin family members, SCLIP and RB3 were highly expressed in stathmin-KO mice compared to WT mice. These results suggest that stathmin and stathmin family genes are expressed in the murine endometrium with enhanced expression in the implantation or the decidualization process.     

Cell and viral regulatory elements enhance the expression and function of a human immunodeficiency virus inhibitory gene.

Regulated expression of recombinant genes in CD4+ cells is an important objective for gene therapy of AIDS, as these cells represent the principal target for viral replication of human immunodeficiency virus (HIV). We report here that specific combinations of CD4 cell-specific and viral regulatory elements can enhance expression of an antiviral gene product. Different viral regulatory elements were incorporated into a previously reported CD4 locus control region to increase the expression of reporter genes in T and monocytic cell lines. The CD4-specific regulatory elements were included to enhance expression in CD4 cells, and viral regulatory regions, including the cytomegalovirus immediate-early (CMV IE) upstream enhancer, which contains the kappa B and Ap1 regulatory elements and a Tat-responsive element of the HIV type 1 long terminal repeat, were used to increase gene expression and modulate its activity in response to viral infection. In transient transfection assays, this vector was 100- to 1,000-fold more active than the original CD4 regulatory elements alone. Expression of an inhibitory form of the Rev protein, Rev M10, was more effective than previously described vectors and protected against productive viral replication in CD4+ peripheral blood mononuclear cells. The combination of CD4 lineage-specific and viral regulatory elements will facilitate the development of more effective antiviral genetic strategies for AIDS.     

Tumor microenvironment abnormalities: causes, consequences, and strategies to normalize

A solid tumor is an organ-like entity comprised of neoplastic cells and non-transformed host stromal cells embedded in an extracellular matrix. The expression of various genes is influenced by interactions among these cells, surrounding matrix, and their local physical and biochemical microenvironment. The products encoded by these genes, in turn, control the pathophysiological characteristics of the tumor, and give rise to the abnormal organization, structure, and function of tumor blood vessels. These abnormalities contribute to heterogeneous blood flow, vascular permeability, and microenvironment. Proliferating tumor cells produce solid stress which compresses blood and lymphatic vessels. As a result of vessel leakiness and lack of functional lymphatics, interstitial fluid pressure is significantly elevated in solid tumors. Each of these abnormalities forms a physiological barrier to the delivery of therapeutic agents to tumors. Furthermore, the metabolic microenvironment in tumors such as hypoxia and acidosis hinder the efficacy of anti-tumor treatments such as radiation therapy and chemotherapy. A judicious application of anti-angiogenic therapy has the potential to overcome these problems by normalizing the tumor vessels and making them more efficient for delivery of oxygen and drugs. Combined anti-angiogenic and conventional therapies have shown promise in the clinic.     

Viral suppression and immune restoration in the gastrointestinal mucosa of human immunodeficiency virus type 1-infected patients initiating therapy during primary or chronic infection

Although the gut-associated lymphoid tissue (GALT) is an important early site for human immunodeficiency virus (HIV) replication and severe CD4+ T-cell depletion, our understanding is limited about the restoration of the gut mucosal immune system during highly active antiretroviral therapy (HAART). We evaluated the kinetics of viral suppression, CD4+ T-cell restoration, gene expression, and HIV-specific CD8+ T-cell responses in longitudinal gastrointestinal biopsy and peripheral blood samples from patients initiating HAART during primary HIV infection (PHI) or chronic HIV infection (CHI) using flow cytometry, real-time PCR, and DNA microarray analysis. Viral suppression was more effective in GALT of PHI patients than CHI patients during HAART. Mucosal CD4+ T-cell restoration was delayed compared to peripheral blood and independent of the time of HAART initiation. Immunophenotypic analysis showed that repopulating mucosal CD4+ T cells were predominantly of a memory phenotype and expressed CD11 alpha, alpha(E)beta 7, CCR5, and CXCR4. Incomplete suppression of viral replication in GALT during HAART correlated with increased HIV-specific CD8+ T-cell responses. DNA microarray analysis revealed that genes involved in inflammation and cell activation were up regulated in patients who did not replenish mucosal CD4+ T cells efficiently, while expression of genes involved in growth and repair was increased in patients with efficient mucosal CD4+ T-cell restoration. Our findings suggest that the discordance in CD4+ T-cell restoration between GALT and peripheral blood during therapy can be attributed to the incomplete viral suppression and increased immune activation and inflammation that may prevent restoration of CD4+ T cells and the gut microenvironment.     

The induction of neovascularization in chorioallantoic membrane of chicken embryos transfected by a recombinant plasmid containing human angiogenin gene

A method was elaborated to evaluate the biological activity of expression products of gene in the plasmid vectors, which are crucial for the synthesis of growth factor of blood vessels. It was proven as possible that the chrioallantonic membrane (CAM) of chicken's embryos could be transfected by recombinant plasmids containing both the reporter and target genes. The efficiency of CAM transfection was assessed by a plasmid carrying the reporter gene of green fluorescent protein (GFP). Finally, it was demonstrated that, at an infiltration of the recombinant plasmid containing the human angiogenine gene, its expression products induce the neovascularization in the CAM cells of chicken's embryos and stimulate an accretion in vessels of the 1st, 2nd and 3d orders.     

Temporal fluctuations in HIV quasispecies in vivo are not reflected by sequential HIV isolations

A genetic study has been made of the HIV tat gene from sequential HIV-1 isolates and the corresponding infected peripheral blood mononuclear cells. DNA was amplified by polymerase chain reaction (PCR) and cloned into a eukaryotic expression vector. Twenty clones were sequenced from each sample. Comparing the sequential HIV isolates, abrupt differences were seen between the major forms of each isolate. These progressive changes were not reflected at all among the in vitro samples. The fluctuation in the quasispecies in vivo may suggest a much more dynamic role for latently infected mononuclear cells. High frequencies of functionally defective tat genes were identified. Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult.     

Expression of genes involved in vascular development and angiogenesis in endothelial cells of adult lung

Profiling gene expression in endothelial cells advances the understanding of normal vascular physiology and disease processes involving angiogenesis. However, endothelial cell purification has been challenging because of the difficulty of isolating cells and their low abundance. Here we examine gene expression in endothelial cells freshly isolated from lung capillaries after in vivo labeling with fluorescent cationic liposomes and purification by fluorescence-activated cell sorting (FACS). Of the 39,000 genes and expressed sequence tags evaluated on custom oligonucleotide arrays, 555 were enriched in endothelial cell fraction. These included familiar endothelial cell-associated genes such as VEGF, VEGF receptor (VEGFR)-1, VEGFR-2, angiopoietin-2, Tie1, Tie2, Edg1 receptor, VE-cadherin, claudin 5, connexin37, CD31, and CD34. Also enriched were genes in semaphorin/neuropilin (Sema3c and Nrp1), ephrin/Eph (ephrin A1, B1, B2, and EphB4), delta/notch (Hey1, Jagged 2, Notch 1, Notch 4, Numb, and Siah1b), and Wingless (Frizzled-4 and Tle1) signaling pathways involved in vascular development and angiogenesis. Expression of representative genes in alveolar capillary endothelial cells was verified by immunohistochemistry. Such expression reflects features that endothelial cells of normal lung capillaries have in common with embryonic and growing blood vessels. About half of the enriched genes, including exostosin 2, lipocalin 7, phospholipid scramblase 2, pleckstrin 2, protocadherin 1, Ryk, scube 1, serpinh1, SNF-related kinase, and several tetraspanins, had little or no previous association with endothelial cells. This approach can readily be used to profile genes expressed in blood vessels in tumors, chronic inflammation, and other sites in which endothelial cells avidly take up cationic liposomes.     

A novel <Emphasis Type="Italic">Flk1</Emphasis>-TVA transgenic mouse model for gene delivery to angiogenic vasculature

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.