Studies on the regulation of human metallothionein gene expression

Bibliography: p. 171-181

Age-Associated Increase in Thrombogenicity and Its Correlation with von Willebrand Factor

Endothelial cells that cover the lumen of all blood vessels have the inherent capacity to express both pro and anticoagulant molecules. However, under normal physiological condition, they generally function to maintain a non-thrombogenic surface for unobstructed blood flow. In response to injury, certain stimuli, or as a result of dysfunction, endothelial cells release a highly adhesive procoagulant protein, von Willebrand factor (VWF), which plays a central role in formation of platelet aggregates and thrombus generation. Since VWF expression is highly restricted to endothelial cells, regulation of its levels is among the most important functions of endothelial cells for maintaining hemostasis. However, with aging, there is a significant increase in VWF levels, which is concomitant with a significant rise in thrombotic events. It is not yet clear why and how aging results in increased VWF levels. In this review, we have aimed to discuss the age-related increase in VWF, its potential mechanisms, and associated coagulopathies as probable consequences

Endothelial cell dysfunction in response to intracellular overexpression of amyloid precursor protein

Previous reports have shown that exposure of vascular endothelial and smooth muscle cells to exogenous amyloid beta (Aβ) peptide results in cell damage and toxicity via oxidative injury. In this study we demonstrate that overexpression of the amyloid precursor protein (APP) is toxic to bovine aortic endothelial cells but not to bovine aortic smooth muscle cells. Intracellular coexpression of the free radical scavenger proteins metallothionein or MnSOD abolished the toxic effect of APP overexpression in endothelial cells. Our results demonstrate that endothelial cells are specifically susceptible to intracellular overexpression of APP and free radical generation is the likely mechanism of cell damage due to APP overexpression

Von Willebrand factor promoter targets the expression of amyloid β protein precursor to brain vascular endothelial cells of transgenic mice

Dysfunction of brain vascular endothelial cells may be associated with the pathogenesis of several diseases including cerebral amyloid angiopathy, hemorrhagic stroke and Alzheimer disease. New model systems are necessary to examine the contribution of brain endothelial cells in these disorders. The Von Willebrand factor gene promoter fragment that spans sequences -487 to +247 targets the expression of LacZ marker gene in transgenic mice specifically to brain vascular endothelial cells. Transgenic mice have been prepared that express human amyloid β protein precursor protein (AβPP) isoforms 695 and 751 (wild-type and Dutch variant mutations) under the regulation of this VWF promoter sequence. These AβPP transgenes are specifically expressed in brain vascular endothelial cells. The VWF promoter is a valuable tool for targeting gene expression to brain vascular endothelial cells to provide a model to directly examine endothelial cell placement of genes and their contribution to cerebral vascular disease

Reprogramming of HUVECs into induced pluripotent stem cells (HiPSCs), generation and characterization of HiPSC-derived neurons and astrocytes.

Neurodegenerative diseases are characterized by chronic and progressive structural or functional loss of neurons. Limitations related to the animal models of these human diseases have impeded the development of effective drugs. This emphasizes the need to establish disease models using human-derived cells. The discovery of induced pluripotent stem cell (iPSC) technology has provided novel opportunities in disease modeling, drug development, screening, and the potential for "patient-matched" cellular therapies in neurodegenerative diseases. In this study, with the objective of establishing reliable tools to study neurodegenerative diseases, we reprogrammed human umbilical vein endothelial cells (HUVECs) into iPSCs (HiPSCs). Using a novel and direct approach, HiPSCs were differentiated into cells of central nervous system (CNS) lineage, including neuronal, astrocyte and glial cells, with high efficiency. HiPSCs expressed embryonic genes such as nanog, sox2 and Oct-3/4, and formed embryoid bodies that expressed markers of the 3 germ layers. Expression of endothelial-specific genes was not detected in HiPSCs at RNA or protein levels. HiPSC-derived neurons possess similar morphology but significantly longer neurites compared to primary human fetal neurons. These stem cell-derived neurons are susceptible to inflammatory cell-mediated neuronal injury. HiPSC-derived neurons express various amino acids that are important for normal function in the CNS. They have functional receptors for a variety of neurotransmitters such as glutamate and acetylcholine. HiPSC-derived astrocytes respond to ATP and acetylcholine by elevating cytosolic Ca2+ concentrations. In summary, this study presents a novel technique to generate differentiated and functional HiPSC-derived neurons and astrocytes. These cells are appropriate tools for studying the development of the nervous system, the pathophysiology of various neurodegenerative diseases and the development of potential drugs for their treatments

Microarray-based gene analysis.

<p>The graphs show cluster analysis of Row Z-score data of (A) pluripotent/embryonic, (B) endothelial, as well as (C) neuronal and glial genes expressed by HUVECs, HiPSCs, HiPSC-derived neurons (HiPSC-Ns), HFNs and samples obtained from previous publications as described in the Materials and Methods (hESCs, hESC-NSCs, hESC-SCNTs). Data are average values of 3 independent experiments.</p

Feeder-layer independent induced pluripotent stem cells generation from HUVECs (HiPSCs).

<p>(A) HUVECs were transduced with lentiviral vectors. Within 4 days (B) and a week (C) the endothelial cells were reduced in density but formed aggregates. (D) On day 16, immature colonies emerged. (E and F) Fully reprogrammed human embryonic stem cells (hESC)-like colonies were isolated after 21 days. Micrographs (G and I in low magnification) and (H and J in high magnification) show the morphology and quality of the colonies. Scale bars: A-F = 200 μm, G and I = 100 μm, H and J = 400 μm.</p

Differentiation of HiPSCs into neurons and astrocytes.

<p>Representative micrographs showing that differentiated HiPSCs acquired neuronal morphology with extending neuritic processes (A) in phase contrast, and became positive for (B) MAP-2 and (C) βIII-tubulin. (D) Primary human fetal cells differentiate into neurons (green) and GFAP-positive astrocytes (red). Similarly, HiPSCs differentiated not only to neurons but also to GFAP-positive astrocytes. The micrograph in (E) shows a HiPSC-derived mixed culture of neurons (green) and GFAP immune-stained astrocytes (red). (F) A micrograph showing few mature astrocytes positive for S100B. Blue is DAPI for nuclear staining. Scale bar: 200 μm.</p

Differentiation of HiPSCs to embryoid bodies (EB) and germ layers.

<p>The ability of HiPSCs to form EB was demonstrated by dispersing colonies to single cell suspensions and placing them in aggrewell plates (A). After 24 hours, homogenous EB were formed (B). EB were differentiated to three germ layers as described in the Materials and Methods. (C and D) Germ layer formation was demonstrated by RT-PCR (C, Pax6 for ectoderm, and D, VEGFR2 for mesoderm markers respectively). (E) The endoderm marker, alpha-fetoprotein (AFP), was detected by immunofluorescence. Scale bar: 200 μm. The experiments in panels C and D were repeated 3 times; P<0.001.</p

Characterization of HUVEC-derived iPSCs (HiPSCs).

<p>Gene expression was determined after 5 passages of the colonies. RT-PCR results (mRNA levels) showing endogenous embryonic (A) and endothelial (B) specific genes expressed by HUVECs and HiPSCs. Values in Figure B represent fold reduction compared to HUVECs. Data are average values of 4 HiPSC lines (N = at least 3 replicates; P<0.001). Non-detectable genes are denoted as n.d. At protein levels using immune-staining, representative micrographs show expression of embryonic markers-SSEA-4 and Oct 3/4 (C and D respectively), DAPI (E) and lack of expression of endothelial markers- PECAM (CD31) and VE-cadherin (F and G respectively) in HiPSC colonies. H is DAPI co-staining of panels F and G. Scale bar: 200 μm.</p