Skip to main content
Key Specialties
Cardiology Diabetology Endocrinology Gastroenterology Geriatrics and Gerontology Gynecology and Obstetrics Hematology Infectious Disease Internal Medicine Nephrology Neurology Oncology Pediatrics Respiratory Medicine Rheumatology Surgery
Congress Coverage
2024 ACC Congress 2023 ESMO Congress 2023 EASD Congress 2023 ERS Congress 2023 ESC Congress 2023 EHA Congress 2023 ASCO Annual Meeting
Clinical Collections
Advances in Alzheimer’s

ACC 2024 Congress

Catch up on all the top stories and latest evidence in cardiology research with our ACC 2024 Congress hub page.
ADVANCED SEARCH
Log in
Top
Cardiovascular Drugs and Therapy
Published in: Cardiovascular Drugs and Therapy 3/2016

01-06-2016 | ORIGINAL ARTICLE

High-Throughput RNAi Screening Identifies a Role for the Osteopontin Pathway in Proliferation and Migration of Human Aortic Smooth Muscle Cells

Authors: Lei Zhang, Holger Erfle, Nathalie Harder, Jürgen Beneke, Nina Beil, Ruben Bulkescher, Karl Rohr, Michael Keese

Published in: Cardiovascular Drugs and Therapy | Issue 3/2016

Login to get access

Abstract

Purpose

Understanding of the mechanisms of vascular smooth muscle cells (VSMCs) phenotypic regulation is critically important to identify novel candidates for future therapeutic intervention. While HTS approaches have recently been used to identify novel regulators in many cell lines, such as cancer cells and hematopoietic stem cells, no studies have so far systematically investigated the effect of gene inactivation on VSMCs with respect to cell survival and growth response.

Methods and Results

257 out of 2000 genes tested resulted in an inhibition of cell proliferation in HaoSMCs. After pathway analysis, 38 significant genes were selected for further study. 23 genes were confirmed to inhibit proliferation, and 13 genes found to induce apoptosis in the synthetic phenotype. 11 genes led to an aberrant nuclear phenotype indicating a central role in cell mitosis. 4 genes affected the cell migration in synthetic HaoSMCs. Using computational biological network analysis, 11 genes were identified to have an indirect or direct interaction with the Osteopontin pathway. For 10 of those genes, levels of proteins downstream of the Osteopontin pathway were found to be down-regulated, using RNAi methodology.

Conclusions

A phenotypic high-throughput siRNA screen could be applied to identify genes relevant for the cell biology of HaoSMCs. Novel genes were identified which play a role in proliferation, apoptosis, mitosis and migration of HaoSMCs. These may represent potential drug candidates in the future.
Appendix
Available only for authorised users
Literature
1.
Piotin M, Spelle L, Martin JB, Weill A, Rancurel G, Ross IB, et al. Percutaneous transluminal angioplasty and stenting of the proximal vertebral artery for symptomatic stenosis. AJNR Am J Neuroradiol. 2000;21:727–31.PubMed
2.
Linni K, Aspalter M, Ugurluoglu A, Holzenbein T. Proximal common carotid artery lesions: endovascular and open repair. Eur J Vasc Endovasc Surg. 2011;41:728–34.CrossRefPubMed
3.
Wu R, Yao C, Wang S, Xu X, Wang M, Li Z. Percutaneous transluminal angioplasty versus primary stenting in infrapopliteal arterial disease: a meta-analysis of randomized trials. J Vasc Surg. 2014;59:1711–20.CrossRefPubMed
4.
Rajagopal V, Rockson SG. Coronary restenosis: a review of mechanisms and management. Am J Med. 2003;115:547–53.CrossRefPubMed
5.
Terada T, Tsuura M, Masuo O, Matsumoto H, Yamaga H, Yokote H, et al. Treatment of restenosis after percutaneous transluminal angioplasty for internal carotid artery stenosis. Neuroradiology. 2000;42:296–301.CrossRefPubMed
6.
7.
Campbell GR, Campbell JH. The phenotypes of smooth muscle expressed in human atheroma. Ann N Y Acad Sci. 1990;598:143–58.CrossRefPubMed
8.
Shi N, Chen SY. Mechanisms simultaneously regulate smooth muscle proliferation and differentiation. J Biol Res. 2014;28:40–6.
9.
Rudijanto A. The role of vascular smooth muscle cells on the pathogenesis of atherosclerosis. Acta Med Indones. 2007;39:86–93.PubMed
10.
Rensen SS, Doevendans PA, van Eys GJ. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Neth Hear J. 2007;15:100–8.CrossRef
11.
Rzucidlo EM, Martin KA, Powell RJ. Regulation of vascular smooth muscle cell differentiation. Journal of vascular surgery. 2007; 45 Suppl A:A25–A32.
12.
Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev. 2004;84:767–801.CrossRefPubMed
13.
Ovcharenko D, Jarvis R, Hunicke-Smith S, Kelnar K, Brown D. High-throughput RNAi screening in vitro: from cell lines to primary cells. RNA. 2005;11:985–93.CrossRefPubMedPubMedCentral
14.
Kiefer J, Yin HH, Que QQ, Mousses S. High-throughput siRNA screening as a method of perturbation of biological systems and identification of targeted pathways coupled with compound screening. Methods Mol Biol. 2009;563:275–87.CrossRefPubMed
15.
Sims D, Mendes-Pereira AM, Frankum J, Burgess D, Cerone MA, Lombardelli C, et al. High-throughput RNA interference screening using pooled shRNA libraries and next generation sequencing. Genome Biol. 2011;12:R104.CrossRefPubMedPubMedCentral
16.
Erfle H, Neumann B, Liebel U, Rogers P, Held M, Walter T, et al. Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc. 2007;2:392–9.CrossRefPubMed
17.
Erfle H, Pashayeva K, Harder N, Zhang L, Rohr K, Schadendorf D, et al. Targeting mitosis-regulating genes in cisplatin-sensitive and -resistant melanoma cells: A live-cell RNAi screen displays differential nucleus-derived phenotypes. Biom J. 2015;10:1467–77.
18.
Held MA, Langdon CG, Platt JT, Graham-Steed T, Liu Z, Chakraborty A, et al. Genotype-selective combination therapies for melanoma identified by high-throughput drug screening. Cancer Discov. 2013;3:52–67.CrossRefPubMed
19.
Bushway PJ, Mercola M. High-throughput screening for modulators of stem cell differentiation. Methods Enzymol. 2006;414:300–16.CrossRefPubMed
20.
Poliseno L, Cecchettini A, Mariani L, Evangelista M, Ricci F, Giorgi F, et al. Resting smooth muscle cells as a model for studying vascular cell activation. Tissue Cell. 2006;38:111–20.CrossRefPubMed
21.
Han M, Wen JK, Zheng B, Cheng Y, Zhang C. Serum deprivation results in redifferentiation of human umbilical vascular smooth muscle cells. Am J Physiol Cell Physiol. 2006;291:C50–8.CrossRefPubMed
22.
Zhang QJ, Goddard M, Shanahan C, Shapiro L, Bennett M. Differential gene expression in vascular smooth muscle cells in primary atherosclerosis and in stent stenosis in humans. Arterioscler Thromb Vasc Biol. 2002;22:2030–6.CrossRefPubMed
23.
Cecchettini A, Rocchiccioli S, Boccardi C, Citti L. Vascular smooth-muscle-cell activation: proteomics point of view. Int Rev Cell Mol Biol. 2011;288:43–99.CrossRefPubMed
24.
Kaplan-Albuquerque N, Bogaert YE, Van Putten V, Weiser-Evans MC, Nemenoff RA. Patterns of gene expression differentially regulated by platelet-derived growth factor and hypertrophic stimuli in vascular smooth muscle cells: markers for phenotypic modulation and response to injury. J Biol Chem. 2005;280:19966–76.CrossRefPubMed
25.
Erfle H, Neumann B, Rogers P, Bulkescher J, Ellenberg J, Pepperkok R. Work flow for multiplexing siRNA assays by solid-phase reverse transfection in multiwell plates. J Biomol Screen. 2008;13:575–80.CrossRefPubMed
26.
Harder N, Mora-Bermudez F, Godinez WJ, Wunsche A, Eils R, Ellenberg J, et al. Automatic analysis of dividing cells in live cell movies to detect mitotic delays and correlate phenotypes in time. Genome Res. 2009;19:2113–24.CrossRefPubMedPubMedCentral
27.
Harder N, Batra R, Diessl N, Gogolin S, Eils R, Westermann F, et al. Large-scale tracking and classification for automatic analysis of cell migration and proliferation, and experimental optimization of high-throughput screens of neuroblastoma cells. Cytometry Part A: the journal of the International Society for Analytical. Cytology. 2015;87:524–40.
28.
Hao H, Gabbiani G, Bochaton-Piallat ML. Arterial smooth muscle cell heterogeneity: implications for atherosclerosis and restenosis development. Arterioscler Thromb Vasc Biol. 2003;23:1510–20.CrossRefPubMed
29.
Purschke M, Rubio N, Held KD, Redmond RW. Phototoxicity of Hoechst 33342 in time-lapse fluorescence microscopy. Photochem Photobiol Sci. 2010;9:1634–9.CrossRefPubMed
30.
Nakano-Kurimoto R, Ikeda K, Uraoka M, Nakagawa Y, Yutaka K, Koide M, et al. Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Am J Physiol Heart Circ Physiol. 2009;297:H1673–84.CrossRefPubMed
31.
Vantler M, Caglayan E, Zimmermann WH, Baumer AT, Rosenkranz S. Systematic evaluation of anti-apoptotic growth factor signaling in vascular smooth muscle cells. only phosphatidylinositol 3′-kinase is important. J Biol Chem. 2005;280:14168–76.CrossRefPubMed
32.
Goel SA, Guo LW, Wang B, Guo S, Roenneburg D, Ananiev GE, et al. High-throughput screening identifies idarubicin as a preferential inhibitor of smooth muscle versus endothelial cell proliferation. PLoS ONE. 2014;9:e89349.CrossRefPubMedPubMedCentral
33.
Raines EW. The extracellular matrix can regulate vascular cell migration, proliferation, and survival: relationships to vascular disease. Int J Exp Pathol. 2000;81:173–82.CrossRefPubMedPubMedCentral
34.
Mill C, George SJ. Wnt signalling in smooth muscle cells and its role in cardiovascular disorders. Cardiovasc Res. 2012;95:233–40.CrossRefPubMed
35.
Evensen L, Micklem DR, Link W, Lorens JB. A novel imaging-based high-throughput screening approach to anti-angiogenic drug discovery. Cytometry A. 2010;77:41–51.PubMed
36.
Doring Y, Noels H, Weber C. The use of high-throughput technologies to investigate vascular inflammation and atherosclerosis. Arterioscler Thromb Vasc Biol. 2012;32:182–95.CrossRefPubMed
37.
Ishigaki T, Imanaka-Yoshida K, Shimojo N, Matsushima S, Taki W, Yoshida T. Tenascin-C enhances crosstalk signaling of integrin alphavbeta3/PDGFR-beta complex by SRC recruitment promoting PDGF-induced proliferation and migration in smooth muscle cells. J Cell Physiol. 2011;226:2617–24.CrossRefPubMed
38.
Beier I, Dusing R, Vetter H, Schmitz U. Epidermal growth factor stimulates Rac1 and p21-activated kinase in vascular smooth muscle cells. Atherosclerosis. 2008;196:92–7.CrossRefPubMed
39.
Lv L, Zhang J, Huang X, Zhao Y, Zhou Z, Zhang H. Lentivirus-mediated RNA interference targeting STAT4 inhibits the proliferation of vascular smooth muscle cells. Arch Med Res. 2008;39:582–9.CrossRefPubMed
40.
41.
Halvorsen B, Smedbakken LM, Michelsen AE, Skjelland M, Bjerkeli V, Sagen EL, et al. Activated platelets promote increased monocyte expression of CXCR5 through prostaglandin E2-related mechanisms and enhance the anti-inflammatory effects of CXCL13. Atherosclerosis. 2014;234:352–9.CrossRefPubMed
42.
Smedbakken LM, Halvorsen B, Daissormont I, Ranheim T, Michelsen AE, Skjelland M, et al. Increased levels of the homeostatic chemokine CXCL13 in human atherosclerosis - potential role in plaque stabilization. Atherosclerosis. 2012;224:266–73.CrossRefPubMed
43.
Grabner R, Lotzer K, Dopping S, Hildner M, Radke D, Beer M, et al. Lymphotoxin beta receptor signaling promotes tertiary lymphoid organogenesis in the aorta adventitia of aged ApoE−/− mice. J Exp Med. 2009;206:233–48.CrossRefPubMedPubMedCentral
44.
Oudit GY, Penninger JM. Cardiac regulation by phosphoinositide 3-kinases and PTEN. Cardiovasc Res. 2009;82:250–60.CrossRefPubMed
45.
Oudit GY, Crackower MA, Eriksson U, Sarao R, Kozieradzki I, Sasaki T, et al. Phosphoinositide 3-kinase gamma-deficient mice are protected from isoproterenol-induced heart failure. Circulation. 2003;108:2147–52.CrossRefPubMed
46.
Bacqueville D, Deleris P, Mendre C, Pieraggi MT, Chap H, Guillon G, et al. Characterization of a G protein-activated phosphoinositide 3-kinase in vascular smooth muscle cell nuclei. J Biol Chem. 2001;276:22170–6.CrossRefPubMed
47.
Vecchione C, Patrucco E, Marino G, Barberis L, Poulet R, Aretini A, et al. Protection from angiotensin II-mediated vasculotoxic and hypertensive response in mice lacking PI3Kgamma. J Exp Med. 2005;201:1217–28.CrossRefPubMedPubMedCentral
48.
Macrez N, Mironneau C, Carricaburu V, Quignard JF, Babich A, Czupalla C, et al. Phosphoinositide 3-kinase isoforms selectively couple receptors to vascular L-type Ca(2+) channels. Circ Res. 2001;89:692–9.CrossRefPubMed
49.
Naga Prasad SV, Esposito G, Mao L, Koch WJ, Rockman HA. Gbetagamma-dependent phosphoinositide 3-kinase activation in hearts with in vivo pressure overload hypertrophy. J Biol Chem. 2000;275:4693–8.CrossRefPubMed
50.
Li Y, Song YH, Mohler J, Delafontaine P. ANG II induces apoptosis of human vascular smooth muscle via extrinsic pathway involving inhibition of Akt phosphorylation and increased FasL expression. Am J Physiol Heart Circ Physiol. 2006;290:H2116–23.CrossRefPubMed
51.
Xie CW, Wang WY, Yang F, Wu M, Mei YD. RUVBL2 is a novel repressor of ARF transcription. FEBS Lett. 2012;586:435–41.CrossRefPubMed
52.
Zerrouqi A, Pyrzynska B, Febbraio M, Brat DJ, Van Meir EG. P14ARF inhibits human glioblastoma-induced angiogenesis by upregulating the expression of TIMP3. J Clin Invest. 2012;122:1283–95.CrossRefPubMedPubMedCentral
53.
Neumann B, Walter T, Heriche JK, Bulkescher J, Erfle H, Conrad C, et al. Phenotypic profiling of the human genome by time-lapse microscopy reveals cell division genes. Nature. 2010;464:721–7.CrossRefPubMedPubMedCentral
54.
Sumara I, Gimenez-Abian JF, Gerlich D, Hirota T, Kraft C, de la Torre C, et al. Roles of polo-like kinase 1 in the assembly of functional mitotic spindles. Curr Biol. 2004;14:1712–22.CrossRefPubMed
55.
Gruss OJ, Wittmann M, Yokoyama H, Pepperkok R, Kufer T, Sillje H, et al. Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells. Nat Cell Biol. 2002;4:871–9.CrossRefPubMed
56.
Zhu C, Zhao J, Bibikova M, Leverson JD, Bossy-Wetzel E, Fan JB, et al. Functional analysis of human microtubule-based motor proteins, the kinesins and dyneins, in mitosis/cytokinesis using RNA interference. Mol Biol Cell. 2005;16:3187–99.CrossRefPubMedPubMedCentral
57.
58.
Mazzone A, Parri MS, Giannessi D, Ravani M, Vaghetti M, Altieri P, et al. Osteopontin plasma levels and accelerated atherosclerosis in patients with CAD undergoing PCI: a prospective clinical study. Coron Artery Dis. 2011;22:179–87.CrossRefPubMed
59.
Han M, Wen JK, Zheng B, Liu Z, Chen Y. Blockade of integrin beta3-FAK signaling pathway activated by osteopontin inhibits neointimal formation after balloon injury. Cardiovasc Pathol. 2007;16:283–90.CrossRefPubMed
60.
Jones PL, Crack J, Rabinovitch M. Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the alpha v beta 3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol. 1997;139:279–93.CrossRefPubMedPubMedCentral
61.
Lamkin TJ, Chin V, Yen A. All-trans retinoic acid induces p62DOK1 and p56DOK2 expression which enhances induced differentiation and G0 arrest of HL-60 leukemia cells. Am J Hematol. 2006;81:603–15.CrossRefPubMed
62.
Holness W, Santore TA, Brown GP, Fallon JT, Taubman MB, Iyengar R. Expression of Q227L-Galpha(s) inhibits intimal vessel wall hyperplasia after balloon injury. Proc Natl Acad Sci U S A. 2001;98:1288–93.CrossRefPubMedPubMedCentral
63.
Vichalkovski A, Gresko E, Hess D, Restuccia DF, Hemmings BA. PKB/AKT phosphorylation of the transcription factor Twist-1 at Ser42 inhibits p 53 activity in response to DNA damage. Oncogene. 2010;29:3554–65.CrossRefPubMed
64.
Low-Marchelli JM, Ardi VC, Vizcarra EA, van Rooijen N, Quigley JP, Yang J. Twist1 induces CCL2 and Recruits macrophages to promote angiogenesis. Cancer Res. 2013;73:662–71.CrossRefPubMedPubMedCentral
65.
Rodrigues CO, Nerlick ST, White EL, Cleveland JL, King ML. A Myc-Slug (Snail2)/Twist regulatory circuit directs vascular development. Development. 2008;135:1903–11.CrossRefPubMedPubMedCentral
66.
Moren A, Raja E, Heldin CH, Moustakas A. Negative regulation of TGFbeta signaling by the kinase LKB1 and the scaffolding protein LIP1. J Biol Chem. 2011;286:341–53.CrossRefPubMed
67.
Hayashi M, Nimura K, Kashiwagi K, Harada T, Takaoka K, Kato H, et al. Comparative roles of Twist-1 and Id1 in transcriptional regulation by BMP signaling. J Cell Sci. 2007;120:1350–7.CrossRefPubMed
68.
Zuo HJ, Lin JY, Liu ZY, Liu WF, Liu T, Yang J, et al. Activation of the ERK signaling pathway is involved in CD151-induced angiogenic effects on the formation of CD151-integrin complexes. Acta Pharmacol Sin. 2010;31:805–12.CrossRefPubMedPubMedCentral
Metadata
Title
High-Throughput RNAi Screening Identifies a Role for the Osteopontin Pathway in Proliferation and Migration of Human Aortic Smooth Muscle Cells
Authors
Lei Zhang
Holger Erfle
Nathalie Harder
Jürgen Beneke
Nina Beil
Ruben Bulkescher
Karl Rohr
Michael Keese
Publication date
01-06-2016
Publisher
Springer US
Published in
Cardiovascular Drugs and Therapy / Issue 3/2016
Print ISSN: 0920-3206
Electronic ISSN: 1573-7241
DOI
https://doi.org/10.1007/s10557-016-6663-4

Other articles of this Issue 3/2016

Cardiovascular Drugs and Therapy 3/2016 Go to the issue

EDITORIAL

GSK-3 Inhibitors: Anti-Diabetic Treatment Associated with Cardiac Risk?

ORIGINAL ARTICLE

Advanced Interfere Treatment of Diabetic Cardiomyopathy Rats by aFGF-Loaded Heparin-Modified Microbubbles and UTMD Technique

REVIEW ARTICLE

Which long-term antiplatelet regimen for patients with acute coronary syndromes?

ORIGINAL ARTICLE

Paclitaxel-Coated Balloons: Investigation of Drug Transfer in Healthy and Atherosclerotic Arteries – First Experimental Results in Rabbits at Low Inflation Pressure

ORIGINAL ARTICLE

Remote Postconditioning Induced by Trauma Protects the Mouse Heart against Ischemia Reperfusion Injury. Involvement of the Neural Pathway and Molecular Mechanisms

ORIGINAL ARTICLE

Efficacy and Safety of the PCSK9 Inhibitor Evolocumab in Patients with Mixed Hyperlipidemia

ACC 2024 Congress Coverage

Cardiology Video

Highlights from the ACC 2024 Congress

Watch now

Watch official videos from the ACC congress summarizing key highlights from the last year in heart failure, cardiomyopathies, valvular disease, pulmonary vascular disease, and pediatric cardiology.

Semaglutide benefits people with type 2 diabetes and obesity-related heart failure

16-04-2024 Type 2 Diabetes News

Incentivised to exercise

11-04-2024 Lifestyle Modifications News

New intervention for STEMI-related cardiogenic shock

10-04-2024 Myocardial Infarction News

» View more highlights on the ACC 2024 congress hub page

Image Credits