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Title: 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells

Abstract

The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.

Authors:
 [1];  [2];  [2];  [2];  [3];  [4];  [5];  [2]
  1. Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Center, 6 Weizman Street 64239 Tel Aviv, and the Sackler Faculty of Medicine, Tel Aviv University (Israel). E-mail: gary_w@tasmc.health.gov.il
  2. Institute of Endocrinology, Metabolism and Hypertension, Tel Aviv Sourasky Medical Center, 6 Weizman Street 64239 Tel Aviv, and the Sackler Faculty of Medicine, Tel Aviv University (Israel)
  3. Department of Biological Regulation, Weizman Institute, Rehovot (Israel)
  4. EM Unit, Weizman Institute, Rehovot (Israel)
  5. Department of Chemical Engineering and Biotechnology, College of Judea and Sameria, Ariel (Israel)
Publication Date:
OSTI Identifier:
20991332
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 356; Journal Issue: 3; Other Information: DOI: 10.1016/j.bbrc.2007.03.012; PII: S0006-291X(07)00463-9; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ACTIN; ANGIOTENSIN; ARTERIES; CYCLOHEXIMIDE; ELECTRON MICROSCOPY; ENZYMES; FIBERS; HUMAN POPULATIONS; IN VITRO; IN VIVO; MUSCLES; PHOSPHORYLATION; SERINE; TYROSINE

Citation Formats

Weisinger, Gary, Limor, Rona, Marcus-Perlman, Yonit, Knoll, Esther, Kohen, Fortune, Schinder, Vera, Firer, Michael, and Stern, Naftali. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells. United States: N. p., 2007. Web. doi:10.1016/j.bbrc.2007.03.012.
Weisinger, Gary, Limor, Rona, Marcus-Perlman, Yonit, Knoll, Esther, Kohen, Fortune, Schinder, Vera, Firer, Michael, & Stern, Naftali. 12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells. United States. doi:10.1016/j.bbrc.2007.03.012.
Weisinger, Gary, Limor, Rona, Marcus-Perlman, Yonit, Knoll, Esther, Kohen, Fortune, Schinder, Vera, Firer, Michael, and Stern, Naftali. Fri . "12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells". United States. doi:10.1016/j.bbrc.2007.03.012.
@article{osti_20991332,
title = {12S-lipoxygenase protein associates with {alpha}-actin fibers in human umbilical artery vascular smooth muscle cells},
author = {Weisinger, Gary and Limor, Rona and Marcus-Perlman, Yonit and Knoll, Esther and Kohen, Fortune and Schinder, Vera and Firer, Michael and Stern, Naftali},
abstractNote = {The current study sets out to characterize the intracellular localization of the platelet-type 12S-lipoxygenase (12-LO), an enzyme involved in angiotensin-II induced signaling in vascular smooth muscle cells (VSMC). Immunohistochemical analysis of VSMC in vitro or human umbilical arteries in vivo showed a clear cytoplasmic localization. On immunogold electron microscopy, 12-LO was found primarily associated with cytoplasmic VSMC muscle fibrils. Upon angiotensin-II treatment of cultured VSMC, immunoprecipitated 12-LO was found bound to {alpha}-actin, a component of the cytoplasmic myofilaments. 12-LO/{alpha}-actin binding was blocked by VSMC pretreatment with the 12-LO inhibitors, baicalien or esculetine and the protein synthesis inhibitor, cycloheximide. Moreover, the binding of 12-LO to {alpha}-actin was not associated with 12-LO serine or tyrosine phosphorylation. These observations suggest a previously unrecognized angiotensin-II dependent protein interaction in VSMC through which 12-LO protein may be trafficked, for yet undiscovered purposes towards the much more abundantly expressed cytoskeletal protein {alpha}-actin.},
doi = {10.1016/j.bbrc.2007.03.012},
journal = {Biochemical and Biophysical Research Communications},
number = 3,
volume = 356,
place = {United States},
year = {Fri May 11 00:00:00 EDT 2007},
month = {Fri May 11 00:00:00 EDT 2007}
}
  • In vitro research on vascular tissue engineering has extensively used isolated primary human or animal smooth muscle cells (SMC). Research programs that lack such facilities tend towards commercially available primary cells sources. Here, we aim to evaluate the capacity of commercially available human SMC to maintain their contractile phenotype, and determine if dedifferentiation towards the synthetic phenotype occurs in response to conventional cell culture and passaging without any external biochemical or mechanical stimuli. Lower passage SMC adopted a contractile phenotype marked by a relatively slower proliferation rate, higher expression of proteins of the contractile apparatus and smoothelin, elongated morphology, andmore » reduced deposition of collagen types I and III. As the passage number increased, migratory capacity was enhanced, average cell speed, total distance and net distance travelled increased up to passage 8. Through the various assays, corroborative evidence pinpoints SMC at passage 7 as the transition point between the contractile and synthetic phenotypes, while passage 8 distinctly and consistently exhibited characteristics of synthetic phenotype. This knowledge is particularly useful in selecting SMC of appropriate passage number for the target vascular tissue engineering application, for example, a homeostatic vascular graft for blood vessel replacement versus recreating atherosclerotic blood vessel model in vitro. - Highlights: • Ability of human smooth muscle cells to alter phenotype in culture is evaluated. • Examined the effect of passaging human smooth muscle cells on phenotype. • Phenotype is assessed based on morphology, proliferation, markers, and migration. • Multi-resolution assessment methodology, single-cell and cell-population. • Lower and higher passages than P7 adopted a contractile and synthetic phenotype respectively.« less
  • During wound healing, fibroblasts deposit extracellular matrix that guides angiogenesis and supports the migration and proliferation of cells that eventually form the scar. They also promote wound closure via differentiation into α-smooth muscle actin (SMA)-expressing myofibroblasts, which cause wound contraction. Low oxygen tension typical of chronic nonhealing wounds inhibits fibroblast collagen production and differentiation. It has been suggested that hypoxic mesenchymal stromal cells (MSCs) secrete factors that promote wound healing in animal models; however, it is unclear whether these factors are equally effective on the target cells in a hypoxic wound environment. Here we investigated the impact of MSC-derived solublemore » factors on the function of fibroblasts cultured in hypoxic fibroblast-populated collagen lattices (FPCLs). Hypoxia alone significantly decreased FPCL contraction and α-SMA expression. MSC-conditioned medium restored hypoxic FPCL contraction and α-SMA expression to levels similar to normoxic FPCLs. (SB431542), an inhibitor of transforming growth factor-β{sub 1} (TGF-β{sub 1})-mediated signaling, blocked most of the MSC effect on FPCL contraction, while exogenous TGF-β{sub 1} at levels similar to that secreted by MSCs reproduced the MSC effect. These results suggest that TGF-β{sub 1} is a major paracrine signal secreted by MSCs that can restore fibroblast functions relevant to the wound healing process and that are impaired in hypoxia. - Highlights: • Fibroblasts were cultured in collagen lattices (FPCLs) as model contracting wounds. • Hypoxia decreased FPCL contraction and fibroblast α-smooth muscle actin expression. • Mesenchymal stromal cells (MSCs) restored function of hypoxic fibroblasts. • MSCs regulate fibroblast function mainly via secreted transforming growth factor-β{sub 1}.« less
  • Research highlights: {yields} Rosiglitazone rapidly (30 min) inhibited microsomal Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). {yields} After 4 h rosiglitazone exposure, the UPR transcription factor XBP-1 was activated. {yields} Within 24-72 h, UPR target genes were upregulated, enhancing ER Ca{sup 2+} sequestration. {yields} Replenishment of ER Ca{sup 2+} stores appeared to restore normal cell physiology. {yields} Monocyte/VSMC viability was not decreased during 2 weeks' rosiglitazone treatment. -- Abstract: Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPAR{gamma}-independent effects alongside its insulin-sensitising PPAR{gamma}-dependent effects, we hypothesisedmore » that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca{sup 2+}]{sub i} homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30 min) brought about concentration-dependent PPAR{gamma}-independent inhibition of Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). Fluo-3 fluorimetric data demonstrated in intact cells that 1 h treatment with 1 or 10 {mu}M rosiglitazone caused Ca{sup 2+} ions to leak into the cytoplasm. Gene expression analysis showed that within 4 h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca{sup 2+} depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72 h. After 72 h 1 or 10 {mu}M rosiglitazone treatment, microsomal Ca{sup 2+}ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca{sup 2+}]{sub i} returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca{sup 2+} sequestration within the ER. This appeared to be sufficient to replenish ER Ca{sup 2+} stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10 {mu}M rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes.« less
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