The smooth muscle cells (SMCs) of the arterial media play a predominant role
in functional and structural alterations of the arterial wall. The transition from
the &#8220;contractile&#8221; to the &#8220;synthetic&#8221; phenotype appears to be an early critical
event in the development of atherosclerotic disease. A number of observations
suggest that 1,25(OH)2D3 (calcitriol) is of importance in maintaining normal
cardiovascular function through its receptors in cardiac myocytes or aortal
SMCs. The present study has focused on the microtubular (MT) network reorganisation
after exposure to calcitriol. SMCs isolated by enzymatic digestion
from the aortal media of neonatal rats were cultured on glass cover slips.
1 &#956;M of 1,25(OH)2D3 was added to the culture medium every second day. The
cytoskeletal features of SMCs after calcitriol were visualised by the immunofluorescence
staining of &#945;-tubulin. The alterations in &#945;-tubulin expression and
the distribution of microtubules related to the activities of the vascular smooth
muscle cells, namely adhesion, migration, multilayer formation and cell division,
were observed. A spindle shape, decreased cell adhesion, low expression
of &#945;-tubulin and a longitudinally arranged microtubular network manifested
the high rate of SMC differentiation in the calcitriol-treated culture. A flat
stellate morphology, high expression of &#945;-tubulin and a radially distributed
three-dimensional microtubular network were observed in the SMCs of the
control culture. Destructive changes in the microtubular architecture which
altered the cellular shape were evident in SMCs undergoing apoptosis. Cells
with apoptotic features were more frequent in calcitriol-exposed culture. In
contrast to the regular SMC divisions observed in the control culture, some of
the mitotic cells exposed to calcitriol contained broader bipolar, multipolar or
disordered spindles.
These alterations in the SMCs&#8217; microtubular cytoskeleton after calcitriol treatment
were concomitant with changes in cell growth, differentiation and apoptosis,
and may suggest a similarity to atherosclerotic plaque formation

Bohdanowicz, Jerzy

Kubasik-Juraniec, Jolanta

Tukaj, Cecylia

English

Via Medica Journals

Folia Morphol. Vol. 63, No. 1, pp. 51–57Copyright © 2004 Via MedicaISSN 0015–5659www.fm.viamedica.plO R I G I N A L  A R T I C L E51The growth and differentiation of aortalsmooth muscle cells after calcitriol treatmentare associated with microtubule reorganisation— an in vitro studyCecylia Tukaj1, Jerzy Bohdanowicz2, Jolanta Kubasik-Juraniec11Laboratory of Electron Microscopy, Medical University, Gdańsk, Poland2Department of Genetics and Cytology, University, Gdańsk, Poland[Received 26 August 2003; Revised 5 November 2003; Accepted 5 November 2003]The smooth muscle cells (SMCs) of the arterial media play a predominant rolein functional and structural alterations of the arterial wall. The transition fromthe “contractile” to the “synthetic” phenotype appears to be an early criticalevent in the development of atherosclerotic disease. A number of observa-tions suggest that 1,25(OH)2D3 (calcitriol) is of importance in maintaining nor-mal cardiovascular function through its receptors in cardiac myocytes or aortalSMCs. The present study has focused on the microtubular (MT) network reor-ganisation after exposure to calcitriol. SMCs isolated by enzymatic digestionfrom the aortal media of neonatal rats were cultured on glass cover slips.1 mM of 1,25(OH)2D3  was added to the culture medium every second day. Thecytoskeletal features of SMCs after calcitriol were visualised by the immunoflu-orescence staining of a-tubulin. The alterations in a-tubulin expression andthe distribution of microtubules related to the activities of the vascular smoothmuscle cells, namely adhesion, migration, multilayer formation and cell divi-sion, were observed. A spindle shape, decreased cell adhesion, low expressionof a-tubulin and a longitudinally arranged microtubular network manifestedthe high rate of SMC differentiation in the calcitriol-treated culture. A flatstellate morphology, high expression of a-tubulin and a radially distributedthree-dimensional microtubular network were observed in the SMCs of thecontrol culture. Destructive changes in the microtubular architecture whichaltered the cellular shape were evident in SMCs undergoing apoptosis. Cellswith apoptotic features were more frequent in calcitriol-exposed culture. Incontrast to the regular SMC divisions observed in the control culture, some ofthe mitotic cells exposed to calcitriol contained broader bipolar, multipolar ordisordered spindles.These alterations in the SMCs’ microtubular cytoskeleton after calcitriol treat-ment were concomitant with changes in cell growth, differentiation and apop-tosis, and may suggest a similarity to atherosclerotic plaque formation.Key words: SMCs, 1,25(OH)2D3, cytoskeleton, tissue cultureAddress for correspondence: Cecylia Tukaj, PhD, Laboratory of Electron Microscopy, Medical University of Gdańsk, ul. Dębinki 1, 80–211 Gdańsk,Poland, tel: +48 58 349 15 00, e-mail: ctukaj@amedec.amg.gda.pl52Folia Morphol., 2004, Vol. 63, No. 1INTRODUCTIONIn the early stages of atherosclerosis, the SMCsare modified from the differentiated “contractile”phenotype to the immature “synthetic” one. Thisphenotypic modulation allows SMCs to migrate intothe intima, proliferate and secrete extracellular ma-trix components [1, 7]. This phenomenon, observednot only during atheromatous plaque formation butalso in primary cultures of SMCs, is associated withthe reorganisation of contractile and cytoskeletalproteins [13, 24, 28].One of the main cytoskeletal components, togeth-er with actin microfilaments and intermediate fila-ments, is a highly organised network of microtubulescomposed of a and b-tubulin heterodimers. A num-ber of studies in different cell types have shown thatthe microtubule cytoskeleton, which is very sensi-tive to differences in laboratory conditions, is essen-tial for the polarisation of the motile cells and thusplays an important role in regulating cell adhesion/migration [17, 23]. Dramatic microtubule rearrange-ments take place during cell division and differenti-ation [6]. Apoptosis occurs in response to variousstimuli under physiological and pathological circum-stances, whereas loss of cell volume and degrada-tion of the tubulin itself take place during the veryearly stages of apoptosis [8, 12]. It is not known howthe disruption of microtubules leads to more ad-vanced stages of apoptosis. It is well established,however, that, apart from its traditional role in cal-cium homeostasis, the active form of vitamin D3 —1,25-(OH)2cholecalciferol, has been shown to modu-late different cellular activities and participate in suchdiverse functions as cellular proliferation and differ-entiation [19]. Recent studies suggest that 1,25-(OH)2D3 plays an important role in the cardiovascularsystem through its receptors in the heart and vascu-lar smooth muscle cells. In particular, 1,25-(OH)2D3 hasbeen shown to modulate  growth, and increase calci-fication in smooth muscle cells [9, 11, 15, 16, 25, 26],although its effect on the microtubule network or-ganisation has not been investigated.MATERIAL AND METHODSThe investigation was carried out according tothe requirements of the Ethics Committee for Ani-mal Care of Poland.Isolation and culturing of cellsThe cytoskeletal protein expression of SMCs wasstudied in cultured cells obtained from the media ofthe aorta of neonatal Wistar rats. The rats were pur-chased from a single animal breeding company. Theaortas were immediately transferred into Eagle'sMinimum Essential Medium (MEM, Biomed, Poland)enriched with 10% foetal bovine serum (FBS, Sig-ma), 100 U/ml penicillin and 100 mg/ml streptomy-cin. After careful removal of the adventitia, the aor-tas were cut into 1 mm segments and digested for1 hour with a 0.3% solution of collagenase 1A (Sig-ma). The resulting cell suspension was filteredthrough 50 mm nylon mesh, centrifuged three times(5 min ¥ 100 g) and normally plated at a density of2 ¥ 105 viable cells/dish in plastic culture dishes oron glass coverslips. They were then incubated at 37oCin a humidified atmosphere of 5% CO2/95% air. Theculture medium was changed every second day, andeach time 1 mM of 1,25(OH)2D3 (a gift from Prof.Kutner) in 95% ethanol was added to the medium.The same amount of ethanol was added to one ofthe two control cultures and the final concentrationof ethanol both in the test and the control tissueculture medium was 0.1%. The second control cul-ture was maintained without calcitriol or alcohol.The cells were examined from 5 to 12 days after in-oculation, when they grew logarithmically in prima-ry culture.Examination of cell viabilityTrypan blue (Sigma) was added to the culture ofSMCs with the help of a haemocytometer, and thecells were assessed with an inverted microscope tocount living and dead cells. About 95% of cells ob-tained by this method excluded trypan blue.Immunocytochemical stainingThe analysis of the cytoskeleton of in vitro cul-tured SMCs was performed employing the directimmunofluorescence method. The cells attached tothe cover slips were washed with PBS, fixed by im-mersion in absolute methanol for 5 min at –20°C,and air-dried. Prior to incubation with antibodies,the specimens were washed with PBS and blockedfor 2 min in PBS containing 1% foetal bovine serum.The culture was incubated for 30 min at room tem-perature with the anti-a-tubulin monoclonal anti-body conjugated with FITC (Sigma, F2168) diluted1:200 in PBS.The cells were also stained with anti a-smoothmuscle actin monoclonal antibody conjugated withFITC (Sigma, F3777, 1:500). After gentle washing inPBS the cells were mounted in glycerol and exam-ined with a Nikon Eclipse 800 microscope equippedfor epifluorescence using the appropriate filter set.53Cecylia Tukaj et al., SMCs after calcitriol treatmentRESULTSSMCs were attached to the glass cover slips andspread out within 48 h after seeding in culture. 95%of the cells were positively identified as SMCs by theirreaction with antibodies against a-smooth muscleactin, which is specific to both contractile and syn-thetic phenotypes (Fig. 1).The proliferating cells that formed the monolay-er were large, well spread and polygonal in shapewith lamellipodia which appeared as flap-like struc-tures (Fig. 2). A clearly defined, three-dimensionalcage of microtubules surrounded the cell nucleus.MTs of the control cells radiated from the cen-trosome, while in the cell body they were sinuous(Fig. 4, 8). During enhanced proliferation a signifi-cant proportion of SMCs spontaneously convertedto a more mature phenotype (Fig. 6). In the laterstages of control culture multilayered regions ap-peared focally as mounds surrounded by a mono-layer (Fig. 8). After calcitriol treatment a large num-ber of loosely arranged SMCs exhibited morphologi-cal and functional characteristics consistent with a dif-ferentiated phenotype. The SMCs were spindle-shaped with a well-defined long axis (Fig. 3, 5). Re-duced cell spreading was associated with increasedcell migration and decreased cell adhesion (Fig. 7).In older culture calcitriol-treated SMCs clearlychanged their growth pattern forming more abun-dant multilayers (Fig. 9).Dramatic changes in microtubule distribution,which altered cellular shape, were evident in SMCsundergoing apoptosis. The microtubular cytoskele-ton was gradually disrupted, which correlated withalterations in the shape of apoptotic cells. The cellsrounded up and became less adhesive to the sub-stratum (Fig. 10, 11). The morphological featurescharacteristics of apoptosis were more frequent inthe cultures after calcitriol addition.Some of the mitotic cells of calcitriol-treatedasynchronous culture contained broader (polyp-loid?) bipolar, multipolar or disordered spindles(Fig. 14–17), whereas such abnormalities were notobserved during the cell divisions in control culture(Fig. 12, 13).DISCUSSION1,25-dihydroxycholecalciferol, the active formof vitamin D3, acts many physiological responsesin a variety of cell types [19]. Our previous results dem-onstrated that calcitriol promoted the initial rate ofphenotypic modulation, resulting in an increase in cellproliferation in log-phase [25, 26]. It is well establishedthat mature contractile SMCs in culture can undergophonotype modulation to the immature synthetictype, but in postconfluent primary culture they canspontaneously differentiate [2, 3, 18, 22, 24–27].In the present study novel direct evidence was pro-vided that noncontractile SMCs, after prolonged ex-posure to 1 mM of 1,25-(OH)2D3, developed quicklyinto a fully differentiated state. This high concentra-tion, causing significant responses in our experiments,was not comparable with the physiological circulat-ing level of calcitriol in the animal organism. Muchhigher local concentrations of 1,25-(OH)2D3 could,however, be reached under pathophysiological con-ditions [19]. A variety of cellular factors can modu-late microtubule dynamics and recent publicationshave proposed that SMC phenotypic states be dis-tinguished by alterations in the expression and dis-tribution of cytoskeletal proteins [4, 21, 28]. Immu-nofluorescence study using a monoclonal antibodyto a-tubulin indicated that calcitriol induced visiblemicrotubule reorganisation. Our data showed thatmicrotubule stability modulated by calcitriol alloweddramatic microtubule rearrangements concomitantwith changes in SMC proliferation, migration, celldivision and differentiation. The results are compa-rable with those obtained by Chitaley and Webb [5].The authors drew the conclusion that the microtu-bular network was expressed in increased amountsin the synthetic phenotype as long as the SMCs re-ceived a stimulus to growth, whereas the contrac-tile pathway was enhanced by microtubule depoly-merisation. The cytoskeleton takes on a more activerole in apoptosis, which occurs in response to vari-ous stimuli under physiological and pathological cir-cumstances [8, 10, 12, 14]. In our studies calcitriol-Figure 1. Identification of cultured cells as SMCs by positive re-action with anti a-smooth muscle actin monoclonal FITC conju-gated antibody. An abundant three-dimentional network of mi-crofilaments with stress fibres can be seen.54Folia Morphol., 2004, Vol. 63, No. 1Figure 2–9. SMCs in primary culture fixed and immunostained with anti-a-tubulin monoclonal FITC conjugated antibody; Fig. 2, 4, 6, 8— control culture; Fig. 3, 5, 7, 9 — calcitriol-treated culture; Fig. 2, 3 — SMCs viewed under Nomarski differential interference contrast;arrows point to lamellipodia.55Cecylia Tukaj et al., SMCs after calcitriol treatmentFigure 10–17. SMCs in primary culture fixed and immunostained with anti-a-tubulin monoclonal FITC conjugated antibody; Fig. 10 — cal-citriol treated culture; arrow points to SMC undergoing apoptosis; Fig. 11 — SMCs during apoptosis in calcitriol-treated culture; arrow-head points to SMC rounding-up and losing adhesion at an early stage of apoptosis; the asterisk marks apoptotic cell; Fig. 12, 13 — mito-ses in control culture; typical metaphasic and anaphasic cells, respectively; Fig. 14, 17 — abnormal spindles (broader bipolar, tripolar,multipolar and tetrapolar, respectively) in dividing cells from culture exposed to calcitriol.56Folia Morphol., 2004, Vol. 63, No. 1induced tubulin polymerisation took place, althoughthe microtubular network was gradually disrupted,which correlated with apoptotic morphology. Weobserved dramatic changes in microtubule disorgan-isation which altered cellular shape. Cells were round-ed up, shrank and became less adhesive to the sub-stratum. These characteristics were similar to thoseobserved during apoptosis following treatment withother drugs that do not interact directly with tubu-lin [8]. It is known that apoptosis takes place whenthe structural integrity of the cytoskeleton is com-promised and cell shape is altered. The mechanismsresponsible for cell shrinkage during this process are,however, poorly understood [12]. Our notions arealso in agreement with others which suggest thatadhesion-dependent changes in tubulin structuremay play a supporting role in both the induction andexecution of the apoptotic program [23]. We alsoobserved an increased incidence of atypical mitoticfigures of calcitriol-treated asynchronous culture.These abnormalities in mitotic spindles, organisedby multiple poles, can lead to chromosome segre-gation errors. Such defective mitoses are a commonfeature of human cancers but the mechanisms lead-ing to these abberations are still unclear [20].In conclusion, the findings indicate that microtu-bule alterations in aortal SMCs exposed to calcitriolin primary culture were concomitant with changesin cell growth, differentiation and apoptosis.ACKNOWLEDGEMENTS1,25(OH)2D3 (calcitriol) was kindly donated byProf. A. Kutner from the Pharmaceutical ResearchInstitute, Warszawa, Poland.This research was supported by grant (BW-989)from the Medical University of Gdańsk.REFERENCES1. Bochaton-Piallat ML, Ropraz P, Gabbiani F, Gabbiani G(1996) Phenotypic heterogeneity of rat arterial smoothmuscle cell clones. Implications for the developmentof experimental intimal thickening. Arterioscler ThrombVasc Biol, 16: 815–820.2. Campbell JH, Kocher O, Skalli O, Gabbiani G, Camp-bell GR (1989) Cytodifferentiation and expression ofalpha-smooth muscle actin mRNA and protein duringprimary culture of aortic smooth muscle cells: correla-tion with cell density and proliferative state. Arterio-sclerosis, 9: 633–643.3. Chamley-Campbell JH, Campbell GR, and Ross R(1979) The smooth muscle cell in culture. Physiol Rev,59: 1–61.4. Cassimeris L (1999) Accessory protein regulation ofmicrotubule dynamics throughout the cell cycle. CurrOpin Cell Biol, 11: 134–141.5. Chitaley K, Webb RC (2001) Microtubule depolymer-ization facilitates contraction of vascular smooth mus-cle via increased activation of RhoA/Rho-kinase. MedHypotheses, 56: 381–385.6. Heald R (2000) A dynamic duo of microtubule mo-dulators. Nat Cell Biol, 2: E11–E12.7. Hegele RA (1996) The pathogenesis of atherosclero-sis. Clin Chim Acta, 146: 21–38.8. Ireland CM, Pittman SM (1995) Tubulin alterations intaxol-induced apoptosis parallel those observed withother drugs. Biochem Pharmacol,  49: 1491–1499.9. Jono S, Nishizawa Y, Shioi A, Morii H (1998) 1,25-Dihy-droxyvitamin D3 increases in vitro vascular calcificationby modulating secretion of endogenous parathyroidhormone-related peptide. Circulation, 98: 1302–1306.10. Kockx MM, Knaapen MW (2000) The role of apoptosisin vascular disease. J Pathol, 190: 267–280.11. Koh E, Morimoto S, Fukuo K, Hironaka T, Onishi T, Ku-mahara Y (1988) 1,25-dihydroxyvitamin D3 binds spe-cifically to rat vascular smooth muscle cells and stimu-late their proliferation in vitro. Life Sci, 42: 315–323.12. Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V,Lepple-Wienhues A, Szabo I, Gulbins E (2000) Cell vol-ume in the regulation of cell proliferation and apop-totic cell death. Cell Physiol Biochem, 10: 417–428.13. Li Z, Cheng H, Lederer WJ, Froehlich J, Lakatta EG (1997)Enhanced proliferation and migration and altered cy-toskeletal proteins in early passage smooth muscle cellsfrom young and old rat aortic explants. Exp Mol Pathol,64: 1–11.14. Mayr M, Xu Q (2001) Smooth muscle cell apoptosis inarteriosclerosis. Exp Gerontol, 36: 968–687.15. Merke J, Hofmann W, Goldschmidt D, Ritz E (1987)Demonstration of 1,25(OH)2 vitamin D3 receptors andactions in vascular smooth muscle cells in vitro. CalcifTissue Int, 41: 112–114.16. Mitsuhashi T, Morris RC, Ives HE (1991) 1,25-dihydroxy-vitamin D3 modulates growth of vascular smoothmuscle cells. J Clin Invest, 87: 1889–1895.17. Nabi IR (1999) The polarization of the motile cell.J Cell Sci, 112: 1803–1811.18. Owens GK (1995) Regulation of differentiation of vascu-lar smooth muscle cells. Physiol Rev, 75: 487–517.19. Reichel H, Koeffler HP, Norman AW (1989) The role ofthe vitamin D endocrine system in health and disease.New Engl J Med, 320: 980–981.20. Sato N, Mizumoto K, Nakamura M, Maehara N, Mina-mishima Y, Nishio S, Nagai E, Tanaka M (2001) Corre-lation between centrosome abnormalities and chro-mosomal instability in human pancreatic cancer cells.Cancer Gen Cytogen, 126: 13–19.21. Skalli O, Bloom WS, Ropraz P, Azzarone B, Gabbiani G(1986) Cytoskeletal remodeling of rat aortic smoothmuscle cells in vitro: relationships to culture conditionsand analogies to in vivo situations. J Submicrosc Cy-tol, 18: 481–493.57Cecylia Tukaj et al., SMCs after calcitriol treatment22. Tao F, Chaudry S, Tolloczko B, Martin JG, Kelly SM(2003) Modulation of smooth muscle phenotype invitro by homologous cell substrate. Am J Physiol CellPhysiol, 284: C1531–C154.23. Thoumine O, Ott A (1996) Influence of adhesion andcytoskeletal integrity on fibroblast traction. Cell MotilCytoskeleton, 35: 269–280.24. Thyberg J, Hedin U, Sjölund M, Palmberg L, Bottger BA(1990) Regulation of differentiated properties and pro-liferation of arterial smooth muscle cells. Arterio-sclerosis, 10: 966–990.25. Tukaj C, Kubasik-Juraniec J, Kraszpulski M (2000)Morphological changes of aortal smooth muscle cellsexposed to calcitriol in culture. Med Sci Monit, 6:668–674.26. Tukaj C, Bohdanowicz J, Kubasik-Juraniec J (2000) Effectsof 1,25-dihydroxycholecalciferol on cytoskeleton organi-zation of aortal smooth muscle cells in primary culture. In:Norman AW, Bouillon R, Thomasset M (eds.). Vitamin DEndocrine System: structural, biological, genetic and clini-cal aspects. Univ. California, Riverside, pp. 431–434.27. Tukaj C, Bohdanowicz J, Kubasik-Juraniec J (2002)A scanning electron microscopic study of phenotypicplasticity and surface structural changes of aortal smoothmuscle cells in primary culture. Folia Morphol, 61:191–198.28. Worth NF, Rolfe BE, Song J, Campbell GR (2001) Vascularsmooth muscle cell phenotypic modulation in culture isassociated with reorganisation of contractile and cytos-keletal proteins. Cell Motil Cytoskeleton, 49: 130–145.

The growth and differentiation of aortal smooth muscle cells after calcitriol treatment are associated with microtubule reorganisation - an in vitro study

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The growth and differentiation of aortal smooth muscle cells after calcitriol treatment are associated with microtubule reorganisation - an in vitro study

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