Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:1151-1166 DOI: 10.1159/000373940 Published online: February 06, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

Jin et al.: Gas6 Delays 29, the2014 Senescence Process in VSMCs Accepted: December 1421-9778/15/0353-1151$39.50/0 This is an Open Access article licensed under the terms of the Creative Commons AttributionNonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only.

Original Paper

Gas6 Delays Senescence in Vascular Smooth Muscle Cells through the PI3K/ Akt/FoxO Signaling Pathway Cheng-wei Jina Hui Wanga Yan-qing Chena Meng-xiong Tangb Guan-qi Fana Zhi-hao Wangc Li Lia Yun Zhanga Wei Zhanga Ming Zhonga Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Department of Cardiology, Qilu Hospital of Shandong University, Ji’nan, bDepartment of Emergency, Qilu Hospital of Shandong University, Ji’nan, cDepartment of Geriatrics Medicine, Qilu Hospital of Shandong University, Ji’nan, P.R. China a

Key Words Gas6 •*URZWKDUUHVWVSHFLÀFSURWHLQ• Axl • Cellular senescence • Cell cycle arrest Abstract Background/Aims: *URZWK DUUHVWVSHFLÀF SURWHLQ  *DV  LV D F\WRNLQH WKDW FDQ be synthesized by a variety of cell types and secreted into the extracellular matrix. Previous VWXGLHV KDYH FRQÀUPHG WKDW *DV LV LQYROYHG LQ FHUWDLQ SDWKRSK\VLRORJLFDO SURFHVVHV RI the cardiovascular system through binding to its receptor, Axl. In the present study, we investigated the role of Gas6 in cellular senescence and explored the mechanisms underlying its activity. Methods: We used vascular smooth muscle cells (VSMCs) to create two cellular senescence models, one for replicative senescence (RS) and one for induced senescence (IS), to test the hypothesis that Gas6 delays senescence. Results: Gas6-treated cells appear relatively younger compared with non-Gas6-treated cells. In particular, Gas6-treated cells GLVSOD\HG GHFUHDVHG VWDLQLQJ IRU 6$DŽ*DO IHZHU * SKDVH FHOOV DQG GHFUHDVHG OHYHOV RI p16INK4a and p21Cip1 expression; conversely, Gas6-treated cells displayed more S phase cells and VLJQLÀFDQWO\LQFUHDVHGSUROLIHUDWLRQLQGH[HV)XUWKHUPRUHLQERWKWKH,6DQG56PRGHOVZLWK Gas6 treatment, the levels of PI3K, p-Akt, and p-FoxO3a decreased following Axl inhibition by R428; similarly, the levels of p-Akt and p-FoxO3a also decreased following PI3K inhibition by LY294002. Conclusion: Gas6/Axl signaling is essential for delaying the cellular senescence process regulated by the PI3K/Akt/FoxO signaling pathway. Copyright © 2015 S. Karger AG, Basel

Ming Zhong

Department of Cardiology, Qilu Hospital of Shandong University, No.107, Wen Hua Xi Road, Ji’nan, 250012, P.R. (China) Tel. +86 531 8216 9339, Fax +86 531 8616 9356, E-Mail [email protected]

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:1151-1166 DOI: 10.1159/000373940 Published online: February 06, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

Jin et al.: Gas6 Delays the Senescence Process in VSMCs

Introduction

Aging is a phenomenon in which the functions, adaptability and resistance of an organism decrease over time [1]. With the global population aging at an accelerating pace, aging is becoming a primary focus of researchers worldwide. Accordingly, delaying many of the negative aspects of aging is vitally important for improving the human lifespan and quality of life. The aging of multiple organs can ultimately lead to diseases or even death, and the cardiovascular system is no exception [2-5]. Indeed, aging is one of the primary risk factors for cardiovascular diseases [6] because aging can alter cardiovascular metabolism, resulting ‹ ‡–ƒ„‘Ž‹… †‹•‘”†‡”• ‘ˆ –Š‡ ‡š–”ƒ…‡ŽŽ—Žƒ” ƒ–”‹šǡ ƒ„‘”ƒŽ ƒ’‘’–‘•‹• ƒ† ‹ϐŽƒƒ–‹‘ [7, 8]. These processes can affect the structure and function of the cardiovascular system, eventually leading to cardiovascular remodeling. The primary consequence of aging in blood vessels is a decrease in compliance and an increase in stiffness [9, 10]. In general, aging is caused by cell senescence [11, 12]. Cellular senescence, or the state of irreversible growth arrest, can be triggered by telomere shortening, the epigenetic derepression of the INK4a/ARF locus, and DNA damage, among others [11]. Vascular smooth muscle cells (VSMCs) are the primary cell type in the tunica media vasorum, and the status ‘ˆ–Š‡•‡…‡ŽŽ•…ƒ‹ϐŽ—‡…‡–Š‡•–”—…–—”‡ƒ†–Š‡ˆ—…–‹‘‘ˆ„Ž‘‘†˜‡••‡Ž•ǤŠ‡”‡ˆ‘”‡ǡ• are the most important factor to consider when studying vascular remodeling, particularly vascular stiffness [13]. Due to the limited life spans of somatic cells, vascular cells eventually enter into a state of irreversible growth arrest [14, 15]. During this process, the levels of negative regulators of the cell cycle, such as p53/p21 and p16, gradually increase with the number of cell divisions [16-22], leading to the stagnation of the cell cycle and the activation of cellular senescence [18, 23], which contributes to aging at both the tissue and individual levels [24, 25].

”‘™–Šƒ””‡•–Ǧ•’‡…‹ϐ‹…’”‘–‡‹͸ȋ ƒ•͸Ȍ‹•ƒ‡„‡”‘ˆ–Š‡˜‹–ƒ‹Ǧ†‡’‡†‡–’”‘–‡‹ family and is encoded by ‰”‘™–Šƒ””‡•–Ǧ•’‡…‹ϔ‹…‰‡‡ͼǡ™Š‹…Š™ƒ•ϐ‹”•–†‹•…‘˜‡”‡†‹ Ȁ͵͵ …‡ŽŽ•—†‡”•‡”—•–ƒ”˜ƒ–‹‘ȏʹ͸ȐǤŠ‡”‡…‡’–‘”ˆ‘” ƒ•͸ǡšŽǡ™ƒ•ϐ‹”•–†‹•…‘˜‡”‡†‹’”‹ƒ”› human myeloid leukemia cells [27] and was later shown to be a receptor tyrosine kinase [28]. Recent studies have shown that Gas6 can also activate two other receptors: Mer and Tyro3 (also called Sky). Although all three of these receptors are members of the TAM receptor family, their binding strengths with Gas6 and their biological effects are quite different [29]. —Ž–‹’Ž‡•–—†‹‡•Šƒ˜‡…‘ϐ‹”‡†–Šƒ––Š‡’”‹ƒ”›„‹‘Ž‘‰‹…ƒŽˆ—…–‹‘‘ˆ ƒ•͸„‹†‹‰–‘šŽ is to prevent cells from undergoing apoptosis [30]. In addition, Gas6 and Axl play roles in –Š‡’ƒ–Š‘Ž‘‰‹‡•‘ˆ˜ƒ•…—Žƒ”…ƒŽ…‹ϐ‹…ƒ–‹‘ȏ͵ͳȐǡ˜ƒ•…—Žƒ””‡‘†‡Ž‹‰ƒ†ƒ–Š‡”‘•…Ž‡”‘•‹•ȏ͵ʹȐǤ Therefore, the pathophysiological processes in blood vessels and VSMCs are likely mediated by Gas6 and Axl. Moreover, based on genetic polymorphism analyses, the ‰ƒ•ͼgene has been associated with stroke and acute coronary syndrome in humans [33-35]. Taken together, Gas6 and Axl play critical roles in cardiovascular diseases by regulating the survival and migration of vascular cells and various functions of circulating blood cells. Furthermore, Gas6 and Axl are closely associated with vascular remodeling, particularly in •Ǥ‘™‡˜‡”ǡ–Š‡”‘Ž‡‘ˆ–Š‡•‡‘Ž‡…—Ž‡•‹–Š‡…‘–‡š–‘ˆ˜ƒ•…—Žƒ”ƒ‰‹‰ƒ†Š‘™–Š‡› might affect vascular compliance and stiffness have not yet been addressed. In this study, we Š›’‘–Š‡•‹œ‡†–Šƒ– ƒ•͸ƒ†šŽ‹‰Š–‹ϐŽ—‡…‡ƒ‰‹‰ǡ–Š‡”‡„›‹ϐŽ—‡…‹‰–Š‡ƒ‰‹‰ ‘ˆ „Ž‘‘† ˜‡••‡Ž• ƒ• ƒ ™Š‘Ž‡Ǥ ‡”‡ ™‡ ‡•–ƒ„Ž‹•Š‡† –™‘ …‡ŽŽ—Žƒ” •‡‡•…‡…‡ ‘†‡Ž• „› …‡ŽŽ passaging serially and angiotensin II (Ang II) treatment, which were named the replicative senescence (RS) and the induced senescence (IS) respectively, in order to further tested this hypothesis and explored the signaling pathways downstream of Gas6/Axl that could provide ‹•‹‰Š–”‡‰ƒ”†‹‰–Š‡•’‡…‹ϐ‹…‡…Šƒ‹••‹˜‘Ž˜‡†‹–Š‹•’”‘…‡••Ǥ

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:1151-1166 DOI: 10.1159/000373940 Published online: February 06, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

Jin et al.: Gas6 Delays the Senescence Process in VSMCs

Materials and Methods ‡ƒ‰‡–•ƒ†–‹„‘†‹‡• Recombinant mouse Gas6 (Gas6) protein and the recombinant mouse Axl-Fc protein fragment (Axl-Fc) were purchased from R&D Systems (St. Paul, MN, USA). R428 (BGB324) was purchased from ‡ŽŽ‡…ȋ‘—•–‘ǡǡȌǤʹͻͶͲͲʹ™ƒ•’—”…Šƒ•‡†ˆ”‘‡”…ȋƒ”•–ƒ†–ǡ‡••‡ǡ ‡”ƒ›ȌǤ›’‡  …‘ŽŽƒ‰‡ƒ•‡ƒ†ƒ‰‹‘–‡•‹ ™‡”‡’—”…Šƒ•‡†ˆ”‘‹‰ƒǦŽ†”‹…Šȋ–Ǥ‘—‹•ǡǡȌǤƒŽŽ‹–‡”ˆ‡”‹‰ • ȋ•‹•Ȍ ƒ‰ƒ‹•– ‘š͵ƒ ƒ† –Š‡ ‡‰ƒ–‹˜‡ …‘–”‘Ž ™‡”‡ ’—”…Šƒ•‡† ˆ”‘ ‡‡Šƒ”ƒ ȋŠƒ‰Šƒ‹ǡ Š‹ƒȌǤ‹’‘ˆ‡…–ƒ‹‡™ ʹͲͲͲ™ƒ•’—”…Šƒ•‡†ˆ”‘ ˜‹–”‘‰‡ȋƒ”Ž•„ƒ†ǡǡȌǤ‡ŽŽ›…Ž‡ƒŽ›•‹•‹– ™ƒ• ’—”…Šƒ•‡† ˆ”‘ ‡…–‘Ǧ‹…‹•‘ ‹‘•…‹‡…‡• ȋƒ ‹‡‰‘ǡ ǡ ȌǤ  ‡ŽŽǦ‹‰Š–™ EdU DNA Cell ”‘Ž‹ˆ‡”ƒ–‹‘ ‹– ™ƒ• ’—”…Šƒ•‡† ˆ”‘ ‹„‘‹‘ ‡…Š‘Ž‘‰› ȋ —ƒ‰œŠ‘—ǡ —ƒ‰†‘‰ǡ Š‹ƒȌǤ Ȁ ͳʹ culture medium, fetal bovine serum (FBS) and 0.25% trypsin were purchased from Gibco (Grand Island, ǡ ȌǤ Š‡ ƒ–‹Ǧ–ǡ ƒ–‹Ǧ’Ǧ–ǡ ƒ† ƒ–‹Ǧ ͵Ǧ‹ƒ•‡ ’ͳͳͲȽ ƒ–‹„‘†‹‡•ǡ ƒ• ™‡ŽŽ ƒ• ƒ ǦȾǦ ƒŽ –ƒ‹‹‰ ‹–ǡ ™‡”‡ ’—”…Šƒ•‡† ˆ”‘ ‡ŽŽ ‹‰ƒŽ‹‰ ‡…Š‘Ž‘‰› ȋ‡˜‡”Ž›ǡ ǡ ȌǤ –Š‡” ƒ–‹„‘†‹‡• ”‡ˆ‡””‡† –‘ ‹ this paper were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All chemicals were of the highest purity grade. Cell culture Primary vascular smooth muscle cells (VSMCs) were extracted from the aortas of 8-10-week‘Ž† ͷ͹Ȁ͸  ™‹Ž†Ǧ–›’‡ ‹…‡ ’—”…Šƒ•‡† ˆ”‘ ‹–ƒŽ ‹˜‡”ǡ …Ǥ Š‹• •–—†› ™ƒ• ƒ’’”‘˜‡† „› –Š‡ ‹ƒŽ ƒƒ‰‡‡– —Ž‡• ‘ˆ –Š‡ Š‹‡•‡ ‹‹•–”› ‘ˆ ‡ƒŽ–Š ȋ‘…—‡– ‘Ǥ ͷͷǡ ʹͲͲͳȌ ƒ† –Š‡ •–‹–—–‹‘ƒŽ Animal Care and Use Committee of Shandong University. All animals were maintained with a regular diet ƒ† •Ž‡‡’ •…Š‡†—Ž‡ ƒ– –Š‡ ‡› ƒ„‘”ƒ–‘”› ‘ˆ ƒ”†‹‘˜ƒ•…—Žƒ” ‡‘†‡Ž‹‰ ƒ† —…–‹‘ ‡•‡ƒ”…Š ‹ ‹Ž— ‘•’‹–ƒŽ‘ˆŠƒ†‘‰‹˜‡”•‹–›ǤŠ‡ƒ‹ƒŽ•™‡”‡ƒ‡•–Š‡–‹œ‡†—•‹‰ƒ’Š‡‘„ƒ”„‹–ƒŽ•‘Ž—–‹‘†‡Ž‹˜‡”‡† ˜‹ƒ‹–”ƒ’‡”‹–‘‡ƒŽ‹Œ‡…–‹‘ƒ†–Š‡•ƒ…”‹ϐ‹…‡†„‡ˆ‘”‡‡š’‡”‹‡–ƒ–‹‘Ǥ”‹‡ϐŽ›ǡ–Š‡ƒ‘”–ƒ•™‡”‡‹…‡†ǡ and the VSMCs were prepared with type II collagenase. The tissues were digested in an enzyme solution for 60-90 minutes at 37°C, and the cell mass suspension was centrifuged at 1,000 rpm for 5 min. The cells were ”‡•—•’‡†‡†‹‡†‹—ƒ†–Š‡ƒ††‡†–‘ʹͷ……‡ŽŽ…—Ž–—”‡ϐŽƒ••ǤŠ‡‘—•‡˜ƒ•…—Žƒ”•‘‘–Š—•…Ž‡…‡ŽŽ Ž‹‡•ȋȌ™‡”‡‘„–ƒ‹‡†ˆ”‘–Š‡‡”‹…ƒ›’‡—Ž–—”‡‘ŽŽ‡…–‹‘ȋȌǤŽŽ…‡ŽŽ•™‡”‡ƒ‹–ƒ‹‡† ‹Ȁ ͳʹ…—Ž–—”‡‡†‹—•—’’Ž‡‡–‡†™‹–ŠͳͲΨ —†‡”ͷΨʹƒ†ͻͷΨŠ—‹†‹ϐ‹‡†ƒ‹”ƒ–͵͹ιǤ Š‡ǡ–Š‡…‡ŽŽ•™‡”‡—•‡†ˆ‘”™‡•–‡”„Ž‘––‹‰ƒƒŽ›•‹•ǡϐŽ‘™…›–‘‡–”›ƒ†…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘‡š’‡”‹‡–•Ǥ

•–ƒ„Ž‹•Š‡–ƒ†‹†‡–‹ϔ‹…ƒ–‹‘‘ˆ…‡ŽŽ•‡‡•…‡…‡‘†‡Ž• Primary VSMCs were serially passaged ten times to create a replicative senescence (RS) model. The cells passaged four times and treated with angiotensin II (Ang II) were used as an induced senescence (IS) ‘†‡ŽǤ ‹ƒŽŽ›ǡ™‡—•‡†ǦȾǦ‰ƒŽ•–ƒ‹‹‰ƒ†“—ƒ–‹ϐ‹‡†–Š‡Ž‡˜‡Ž•‘ˆ’ʹͳCip1 and p16 Ͷƒ protein expression by western blotting analysis to determine the degree of cell senescence. ‡•–‡”„Ž‘––‹‰ƒƒŽ›•‹• To extract protein, cells were harvested, washed with pre-cooled PBS solution three times for 5 min ƒ†–Š‡–”‡ƒ–‡†™‹–Š…‡ŽŽŽ›•‹•„—ˆˆ‡”ȋͷͲ”‹•ǡ’͹ǤͶǡͳͷͲƒŽǡͳΨ•‘†‹—†‡‘š›…Š‘Žƒ–‡ǡͲǤͳΨ ǡƒ†ͳΨ”‹–‘ǦͳͲͲȌ…‘–ƒ‹‹‰’”‘–‡ƒ•‡‹Š‹„‹–‘”…‘…–ƒ‹Žȋ‘…Š‡ǡƒ•‡Žǡ™‹–œ‡”Žƒ†ȌǤŠ‡ǡ–Š‡…‡ŽŽ lysates were centrifuged at 15,000 g for 10 min at 4°C. Protein concentrations were determined using a Thermo BCA protein assay kit (Pittsburgh PA, Pennsylvania, USA), and the protein samples were prepared by mixing 1 volume of the supernatants with 1/4 volume of the loading buffer, as previously described. Equal ’”‘–‡‹Ž‘ƒ†‹‰™ƒ•˜‡”‹ϐ‹‡†„›™‡•–‡”„Ž‘––‹‰ –‘†‡–‡…– ǤŠ‡•ƒ’Ž‡• ™‡”‡•‡’ƒ”ƒ–‡†‘ͳͲΨ Ǧ’‘Ž›ƒ…”›Žƒ‹†‡‰‡Ž•ƒ†–Š‡‡Ž‡…–”‘’Š‘”‡–‹…ƒŽŽ›–”ƒ•ˆ‡””‡†–‘’‘Ž›Ǧ˜‹›Ž‹†‡‡†‹ϐŽ—‘”‹†‡‡„”ƒ‡•Ǥ After incubation in blocking solution (5% non-fat milk) for 1.5 h, the membranes were incubated with primary antibodies diluted in blocking solution for 12-16 h at 4°C. Then, the membranes were washed with 1×TBST solution and treated with horseradish peroxidase-conjugated secondary antibodies for 1 h. ‡š–ǡ –Š‡ ‡„”ƒ‡• ™‡”‡ –”‡ƒ–‡† ™‹–Š  •‘Ž—–‹‘ ȋ‹ŽŽ‹’‘”‡ǡ ƒ”•–ƒ†–ǡ ‡”ƒ›Ȍ ƒ……‘”†‹‰ –‘ –Š‡ ƒ—ˆƒ…–—”‡”ǯ•‹•–”—…–‹‘•ƒ†–Š‡‹ƒ‰‡†—•‹‰ƒ ƒ‰‡—ƒ–™ͶͲͲͲ‹ƒ‰‡”ȋ ǡǡȌǤŠ‡ ‡ƒ‰”ƒ›˜ƒŽ—‡•‘ˆ‡ƒ…Š’”‘–‡‹„ƒ†™‡”‡‡ƒ•—”‡†„›Š‘–‘•Š‘’ͷ•‘ˆ–™ƒ”‡ȋ†‘„‡ǡƒ ‘•±ǡǡ

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:1151-1166 DOI: 10.1159/000373940 Published online: February 06, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

Jin et al.: Gas6 Delays the Senescence Process in VSMCs

ȌǤ‡Žƒ–‹˜‡„ƒ†‹–‡•‹–›™ƒ•–Š‡”ƒ–‹‘‘ˆ‰”ƒ›˜ƒŽ—‡‘ˆ‹–‡”‡•–’”‘–‡‹–‘–Šƒ–‘ˆ…‘””‡•’‘†‹‰ Ǥ As for the phosphorylated proteins, relative band intensity was the ratio of gray value of phosphorylated protein to that of total protein. ‡‡•…‡…‡Ǧƒ••‘…‹ƒ–‡†ȾǦ‰ƒŽƒ…–‘•‹†ƒ•‡•–ƒ‹‹‰ First, each group of cells was plated in 6-well plates. Then, the cells were washed with pre-cooled •‘Ž—–‹‘–Š”‡‡–‹‡•ˆ‘”ͷ‹‘ƒ†‡…‘Ž‘”‹œƒ–‹‘–ƒ„Ž‡ƒ†–Š‡ϐ‹š‡†™‹–ŠƒͶΨ’ƒ”ƒˆ‘”ƒŽ†‡Š›†‡ solution for 15 min at room temperature. All samples were treated as described in the instructions for the ǦȾǦ ƒŽ–ƒ‹‹‰‹–ǤŠ‡…‡ŽŽ•™‡”‡‹…—„ƒ–‡†ƒ–͵͹ι™‹–Š‘—–2–‘ƒ‹–ƒ‹ƒ’‘ˆ͸ǤͲǤ ‘”‡ƒ…Š‰”‘—’ǡ –Š”‡‡ϐ‹‡Ž†•‘ˆ…‡ŽŽ•™‡”‡•‡Ž‡…–‡†”ƒ†‘Ž›ƒ†’Š‘–‘‰”ƒ’Š‡†—•‹‰ƒ‹˜‡”–‡†’Šƒ•‡…‘–”ƒ•–‹…”‘•…‘’‡ ȋŽ›’—•ǡ‘›‘ǡ ƒ’ƒȌǤ‡ŽŽ•Šƒ†„Ž—‡Ǧ‰”‡‡‰”ƒ—Ž‡•‹–Š‡‹”…›–‘’Žƒ•™‡”‡”‡‰ƒ”†‡†ƒ•’‘•‹–‹˜‡•–ƒ‹‡† ‘ˆǦȾǦ‰ƒŽƒ†™‡—•‡† ƒ‰‡Ǧ”‘Ž—•͸ǤͲ•‘ˆ–™ƒ”‡ȋ‡–Š‡•†ƒǡǡȌ–‘…ƒŽ…—Žƒ–‡†–Š‡’‘•‹–‹˜‡•–ƒ‹‹‰ ‘ˆ…‡ŽŽ•ƒ……‘—–‡†ˆ‘”–Š‡’”‘’‘”–‹‘‘ˆƒŽŽ…‡ŽŽ•‹–Š‡•ƒ‡ϐ‹‡Ž†‘ˆ˜‹‡™Ǥ ‡ŽŽ…›…Ž‡ƒƒŽ›•‹•„›ϔŽ‘™…›–‘‡–”› Cells (1×104) were treated with 0.25% trypsin, centrifuged at 1,000 g for 5 min at 4°C, washed, and ”‡•—•’‡†‡†‹’”‡Ǧ…‘‘Ž‡†•‘Ž—–‹‘ǤŠ‡ǡ–Š‡ϐ‹š‡†…‡ŽŽ•™‡”‡‹…—„ƒ–‡†‹͹ͲΨ‡–Šƒ‘Žˆ‘”ͳʹǦʹͶŠƒ– ͶιǤŠ‡‡š–†ƒ›ǡ–Š‡…‡ŽŽ•™‡”‡’‡ŽŽ‡–‡†ǡ–”‡ƒ–‡†™‹–ŠͳͲͲƒ•‡ȋͲǤʹ‰Ȁ‹Ȍˆ‘”ͷ‹ƒ–”‘‘ –‡’‡”ƒ–—”‡ƒ†–Š‡”‡•—•’‡†‡†‹ͳ††2Ǥˆ–‡”•–ƒ‹‹‰™‹–ŠͶ‰Ȁ‘ˆ’”‘’‹†‹—‹‘†‹†‡ǡ–Š‡ …‘–‡–™ƒ•†‡–‡”‹‡†—•‹‰ƒ‡…–‘‹…•‘  Ž‘™›–‘‡–‡”›•–‡™‹–Š ‹˜ƒ•‘ˆ–™ƒ”‡ǡ ƒ†–Š‡…‡ŽŽ…›…Ž‡’”‘ϐ‹Ž‡™ƒ•ƒƒŽ›œ‡†—•‹‰‘† ‹–͵Ǥʹ•‘ˆ–™ƒ”‡ȋ‡”‹–›‘ˆ–™ƒ”‡‘—•‡ǡ‘’•ŠƒǡȌǤ ”‘Ž‹ˆ‡”ƒ–‹‘ƒ••ƒ› ‡—•‡†ͷǦ‡–Š›‹ŽǦʹԢǦ†‡‘š›—”‹†‹‡ȋ†Ȍǡ™Š‹…Š‹•ƒƒƒŽ‘‰‘ˆ–Š›‹†‹‡ǡ–‘†‡–‡…––Š‡’”‘Ž‹ˆ‡”ƒ–‹‘ levels of the cells. First, the cells were plated in 24-well plates, treated with complete medium containing ͷͲ Ɋ † •–ƒ‹‹‰ •‘Ž—–‹‘ǡ ƒ† ‹…—„ƒ–‡† ˆ‘” ʹ ŠǤ ŽŽ ’”‘…‡†—”‡• ™‡”‡ ’‡”ˆ‘”‡† ƒ……‘”†‹‰ –‘ –Š‡ ‹•–”—…–‹‘• ’”‘˜‹†‡† ‹ –Š‡ †  ‡ŽŽ ”‘Ž‹ˆ‡”ƒ–‹‘ ‹–Ǥ ƒ•‡”Ǧ•…ƒ‹‰ …‘ˆ‘…ƒŽ ‹…”‘•…‘’› ȋƒ”Ž ‡‹•• ‡ƒǡ„‡”‘…Š‡ǡ ‡”ƒ›Ȍƒ†–Š‡ƒ”Ž‡‹•••‘ˆ–™ƒ”‡•›•–‡™‡”‡—•‡†–‘…‘ŽŽ‡…–‹ƒ‰‡•Ǥ ‘” ‡ƒ…Š‰”‘—’ǡ–Š”‡‡ϐ‹‡Ž†•‘ˆ…‡ŽŽ•™‡”‡”ƒ†‘Ž›•‡Ž‡…–‡†ǡƒ†–Š‡…‡ŽŽ’”‘Ž‹ˆ‡”ƒ–‹‘Ž‡˜‡Ž™ƒ•ƒƒŽ›œ‡†—•‹‰ Image-Pro Plus 6.0 software (Bethesda, MD, USA). •‹Ǧ‡†‹ƒ–‡†‘…†‘™‡š’‡”‹‡–•  ‘š͵ƒ ȋͷԢǦ

 

 Ǧ͵ԢȌ ‘Ž‹‰‘”‹„‘—…Ž‡‘–‹†‡ •‹ ™ƒ• —•‡† –‘ ‹Š‹„‹– ‘š͵ƒ ‡š’”‡••‹‘ǡ ƒ† ƒ •…”ƒ„Ž‡† …‘–”‘Ž •‹ ȋͷԢǦ    Ǧ͵ԢȌ ™ƒ• —•‡† ƒ• ƒ negative control (NC). Plasmids or oligoribonucleotides were transfected into cells grown in a six-well plate —•‹‰‹’‘ˆ‡…–ƒ‹‡™ʹͲͲͲ–”ƒ•ˆ‡…–‹‘ƒ……‘”†‹‰–‘–Š‡ƒ—ˆƒ…–—”‡”ǯ•’”‘–‘…‘ŽǤ…‡ŽŽ•™‡”‡—•‡† for experimentation after 24-48 h of transfection. –ƒ–‹•–‹…ƒŽƒƒŽ›•‹• Š‡ †ƒ–ƒ ƒ”‡ ‡š’”‡••‡† ƒ• –Š‡ ‡ƒάǤ  ͳͻǤͲ •‘ˆ–™ƒ”‡ ȋŠ‹…ƒ‰‘ǡ ǡ Ȍ ™ƒ• —•‡† ˆ‘” –Š‡ statistical analyses. Differences between two groups were determined using independent samples, and –—†‡–ǯ•–Ǧ–‡•–ƒ†‘‡Ǧ™ƒ›™‡”‡—•‡†–‘…‘’ƒ”‡–Š‡”‡•—Ž–•ƒ‘‰–Š‡‰”‘—’•Ǥ–ƒ–‹•–‹…ƒŽƒƒŽ›•‡• were performed using the GraphPad Prism 5.01 software program (San Diego, CA, USA). Differences with  ˜ƒŽ—‡• Ž‡•• –Šƒ ͲǤͲͷ ™‡”‡ …‘•‹†‡”‡† •–ƒ–‹•–‹…ƒŽŽ› •‹‰‹ϐ‹…ƒ–Ǥ ‘’ƒ”‹•‘• –Šƒ– †‹† ‘– ”‡ƒ…Š –Š‹• •‹‰‹ϐ‹…ƒ…‡–Š”‡•Š‘Ž†™‡”‡‘–‘–‡†Ǥ

Results

Š‡‹†‡–‹ϔ‹…ƒ–‹‘‘ˆ…‡ŽŽ—Žƒ”•‡‡•…‡…‡‘†‡Ž• Š‡ –Š‡ ’”‹ƒ”› • ™‡”‡ ’ƒ••ƒ‰‡† –‡ –‹‡•ǡ ™‡ ˆ‘—† –Šƒ– –Š‡ ǦȾǦ‰ƒŽ ȋ•‡‡•…‡…‡Ǧƒ••‘…‹ƒ–‡†ȾǦ‰ƒŽƒ…–‘•‹†ƒ•‡Ȍ•–ƒ‹‹‰”ƒ–‡ƒ†–Š‡’ʹͳCip1 and p16 Ͷƒ expression Ž‡˜‡Ž• •‹‰‹ϐ‹…ƒ–Ž› ‹…”‡ƒ•‡† …‘’ƒ”‡† ™‹–Š ‡ƒ”Ž‹‡” ’ƒ••ƒ‰‡•Ǥ Š‡”‡ˆ‘”‡ǡ ™‡ —•‡† ͳͲthpassage cells as a replicative senescence model (RS) (Fig. 1A and 1B). In addition, we also

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Cellular Physiology and Biochemistry

Cell Physiol Biochem 2015;35:1151-1166 DOI: 10.1159/000373940 Published online: February 06, 2015

© 2015 S. Karger AG, Basel www.karger.com/cpb

Jin et al.: Gas6 Delays the Senescence Process in VSMCs

Fig. 1. •–ƒ„Ž‹•Š‹‰ ƒ† ˜‡”‹ˆ›‹‰ –Š‡ …‡ŽŽ—Žƒ” •‡‡•…‡…‡ ‘†‡Ž•Ǥ ȋ ƒ† Ȍ ‡•–‡” „Ž‘–• ƒ† ǦȾǦ‰ƒŽ stainings demonstrating that cells passaged ten times could serve as a model for replicative senescence (RS) (C, D and E) and that cells passaged four times and treated with Ang II (1×10-6‘ŽȀȌˆ‘”ͶͺŠ…‘—Ž†•‡”˜‡ as a model for induced senescence (IS). All the results shown are from representative experiments (n=3 in each case). The values are presented as the mean±SD. *P