Sensing Fluid-Shear Stress in the Endothelial System with a Special Emphasis on the Primary Cilium

Fluid shear stress (FSS) is able to generate phenotypic changes in the cells in direct contact with the strain force. In order to detect and transduce FSS into intracellular pathways, biological systems use a specific set of sensors, called mechanosensors. The process involves the conversion of the mechanical force into a chemical or electrical signal. Primary cilium is a non-motile organelle that emanates from the cell surface of most mammalian cell types that act as a mechanosensor. Increasing evidence suggests that primary cilia are key coordinators of signaling pathways in tissue homeostasis and when defective may cause human diseases and developmental disorders. Here, we will describe the endothelial primary cilium as a mechanotransductory organelle sensing FSS. To fulfill this function, primary cilium requires the localization of mechanoproteins, polycystin-1 and -2, in their membrane and the structural gene product, polaris. Physiologically, deflection of primary cilium increases the intracellular calcium concentration triggering a signaling pathway that leads to nitric oxide (NO) formation and vasodilation. Additionally, ciliopathies, such as polycystic kidney disease and atherosclerosis, will also be discussed. We also analyze available information regarding a trio of membrane receptors involved in FSS sensing and transducing such as vascular endothelial growth factor receptors (VEGFRs) and its coreceptor neuropilin (NRP), as well as purinergic receptors (P2Y2). Whether or not they modulate, the primary cilium role in sensing FSS is poorly understood. This chapter highlights the main relevance of primary cilium in sensing blood flow, although exact mechanisms are not fully known yet

Drosophila photoreceptor cells exploited for the production of eukaryotic membrane proteins: receptors, transporters and channels.

BACKGROUND: Membrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies. PRINCIPAL FINDINGS: We describe the homologous and heterologous expression of 10 different targets from the three major MP classes--G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2-0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter--both involved in synaptic transmission--showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies. SIGNIFICANCE: We demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies

A light- and electron-microscopic study of enzymes in the embryonic shell-forming tissue of the freshwater snail, Biomphalaria glabrata

The mode of formation of the molluscan exoskeleton is still poorly understood, but studies on adult snails indicate that enzymes involved in vertebrate bone formation also participate in mollusc shell formation. The enzymes peroxidase, alkaline phosphatase, and acid phosphatase are expressed in a constant pattern and help to identify the different zones of the adult shell-forming tissue. The present study evaluates whether the expression of these enzymes is also a tool for the identification of the developing zones of the embryonic shell-forming tissue. Thus, we analyzed the temporal and spatial activity of the above-mentioned enzymes and of tartrate-resistant acid phosphatase in the shell forming tissues in Biomphalaria glabrata. Embryos of different age groups and adults were studied; alkaline phosphatase activity was seen in very young embryos in the shell field invagination prior to the secretion of any shell material, while peroxidase activity was present from the start of the periostracum production. Acid phosphatase, found in considerable amounts in yolk granules and albumen cells, appeared in the embryonic shell-forming tissue in relatively few Golgi stacks. Tartrate-resistant phosphatase was not present in embryos, but was found in adults in the same zone of the mantle edge as acid phosphatase. Using the enzymes as cell markers, the differentiation of the embryonic shell-forming tissue to the different zones of the adult mantle edge could clearly be followed

Gestational diabetes mellitus is associated with increased pro-migratory activation of vascular endothelial growth factor receptor 2 and reduced expression of vascular endothelial growth factor receptor 1

<div><p>Placentas from gestational diabetes mellitus (GDM) are often hypervascularized; however, participation of vascular endothelial growth factor (VEGF) and its receptors in this placental adaptation is unclear. We aimed to test whether changes in phosphorylation of tyrosine 951 or tyrosine 1175 (pY951 or pY1175) of the vascular endothelial growth factor receptor 2 (KDR) are associated with the proangiogenic state observed in placentas from GDM. We obtained placental samples from women with normal pregnancies (n = 24) or GDM (n = 18). We measured the relative expression of markers for endothelial cell number (CD31, CD34), VEGF, vascular endothelial growth factor receptor 1 (Flt-1), KDR, pY951 and pY1175 of KDR in placental homogenate. Immunohistochemistry of placental blood vessels were performed using CD34. Proliferation and migration of human umbilical vein endothelial cells (HUVEC) obtained from normal pregnancy and GDM were determined in absence or presence of conditioned medium (CM) harvested from GDM or normoglycemic HUVEC cultures. GDM was associated with more CD31 and CD34 protein compared to normal pregnancy. High number, but reduced area of placental blood vessels was found in GDM. Reduced Flt-1 levels (mRNA and protein) are associated with reduced KDR mRNA, but higher KDR protein levels in placentas from GDM. No significant changes in Y951-or Y1175-phosphorylation of KDR in placentas from GDM were found. GDM did not alter proliferation of HUVECs, but enhanced migration. Conditioned medium harvested from GDM HUVEC cultures enhanced KDR protein amount, tube formation capacity and cell migration in HUVEC isolated from normoglycemic pregnancies. The data indicate that GDM is associated with reduced expression of Flt-1 but high pro-migratory activation of KDR reflecting a proangiogenic state in GDM.</p></div

Total KDR and tyrosine 951-and tyrosine 1175 phosphorylation of KDR.

<p><b>A)</b> Representative image of PCR for KDR and 18S in placental homogenates from normal (Normal, white bar) and gestational diabetic pregnancies (GDM, hatched bar). In the bottom panel is presented densitometic analysis of mRNA KDR/18S. <b>B)</b> Representative images of total KDR (170kDa) and tyrosine 951 phosphorylated (pY951-KDR) (70 kDa) and β-actin (43 kDa) in placenta homogenates. Chart represents densitometry of KDR/β-actin ratio and pY951-KDR/KDR. <b>C)</b> Optical units at 450 nm were used for estimating the relative amount of KDR phosphorylated in the Y1175 residue. Significant differences between groups are indicated with respective P value. In A and C, n = 8 in each group. In B, n = 18 in GDM, n = 20, for total KDR; while 8 per group for Y951. P-value is indicated in each graph. Outliners are indicated in gray.</p

Placental CD31 and CD34 protein.

<p>Protein levels of CD31 and CD34 in total placental homogenates of normal pregnancy (NP, white bar) and gestational diabetes mellitus (GDM, hatched bar) were determined by western blot. <b>A)</b> Representative western blot for CD31 (55 kDa), CD34 (80 kDa) and β-actin (43 kDa). <b>B)</b> Densitometric analysis of CD31/ β-actin ratio (left Y-axis) and CD34/ β-actin ratio (right Y-axis). <b>C)</b> Representative images of CD34 staining in placentas from Normal and GDM. <b>D)</b> Number of vessels per villi. <b>E)</b> Mean blood vessel area (MVA)/mean chorionic villi area (MCVA) ratio. In B, n = 6–12 in each group. In D and E, n = 4–5 in each group. Magnification 40X. P-value is indicated in each graph.</p

Primers used for RT-PCR.

<p>Primers used for RT-PCR.</p

Characteristics of women included in the study.

<p>Characteristics of women included in the study.</p

Working model.

<p>Results of this study suggest that placentas from gestational diabetes mellitus are in a proangiogenic state characterized by elevated endothelial cell markers, ie. more endothelial cells, and higher KDR, resulting in enhanced migration in GDM cells than cells from normal control pregnancies. Additional alteration/compensatory mechanisms include reduction in the mRNA and protein levels of Flt-1, reduced mRNA levels of KDR, and no changes in VEGF, or cell proliferation. Changes observed in GDM are shown in red and black arrows.</p