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    Transforming Growth Factor-/Jl GeneActivation and Growth of SmoothMuscle From Hypertensive Rats

    Alex Agrotis, John Saltis, Alex Bobik

    Abstract Cultured vascular smooth muscle cells derivedfrom the spontaneously hypertensive rat (SHR) are known toreplicate more rapidly than cells from the normotensiveWistar-Kyoto (WKY) rat. In this study we compared theresponses of vascular smooth muscle cells from the two strainsto transforming growth factor-01 (TGF-/31) and evaluated itspotential to account for the different growth properties ofthese cells in response to a number of vascular-derived growthfactors. TGF-/31 potentiated the proliferative effects of epider-mal growth factor, basic fibroblast growth factor, or thedifferent isoforms of platelet-derived growth factor on vascularsmooth muscle cells from SHR but inhibited growth factor-stimulated proliferation of vascular smooth muscle cells fromWKY rats. These differential effects of TGF-01 on prolifera-tion could not be attributed to alterations in the expression ofthe type I, II, or III TGF-/3 receptors but appeared more

    Vascular hypertrophy is a major contributor tothe increase in vascular resistance in sponta-neously hypertensive rats (SHR).1 In theseanimals hypertrophy of the resistance vessels is, at leastin part, attributed to a greater number of smooth musclecells within the vessel wall.2 It has been suggested thatdifferences between the ability of vascular smooth mus-cle cells (VSMC) from SHR and normotensive Wistar-Kyoto (WKY) rats to respond to the mitogenic influ-ence of vascular-derived growth factors could accountfor this "proliferative hypertrophy" in genetic hyperten-sion.36 One of these vascular-derived growth factors,transforming growth factor-^1 (TGF-/31), is a multi-functional protein that regulates the growth and differ-entiation of a wide variety of cells in culture.78 InVSMC TGF-/31 can induce both cellular hypertrophyand polyploidy as well as stimulate and/or inhibit pro-liferation.9'10 For example, TGF-/31 has been shown tostimulate the proliferation of both sparse and densecultures of human smooth muscle cells." Also, TGF-/31inhibits serum-stimulated proliferation of rat VSMC,seeded at low densities; at high densities the oppositeeffect on proliferation is observed.1013 These opposingeffects of TGF-01 are elicited after its interaction withmultiple receptors on the surface of smooth musclecells.7

    Received May 13, 1993; accepted in revised form February 9,1994.

    From the Baker Medical Research Institute and Alfred Hospi-tal, Prahran, Victoria, Australia.

    Correspondence to Dr Alex Agrotis, Baker Medical ResearchInstitute, Commercial Rd, Prahran, 3181 Victoria, Australia.

    related to the ability of cells to autoinduce the TGF-/31 gene.TGF-/31 caused a time-dependent increase in its own mRNAlevels in vascular smooth muscle cells of WKY rats butattenuated levels in vascular smooth muscle cells of SHR. Thiseffect was specific to TGF-01 autoinduction since similarelevations in TGF-01 mRNA levels were observed whenvascular smooth muscle cells from the two rat strains wereexposed to phorbol myristate acetate, basic fibroblast growthfactor, or platelet-derived growth factor-BB. These data sug-gest that the production of TGF-01 may contribute to thedifferent growth properties of vascular smooth muscle cellsfrom SHR and WKY rats through alterations in TGF-/31signaling systems. (Hypertension. 1994;23:593-599.)

    Key Words transforming growth factor-/3 muscle,smooth, vascular protein-tyrosine kinase proliferation rats, inbred SHR

    Several lines of evidence suggest that TGF-/31 mayplay an important role in the development of hyperten-sion. Initially, Sarzani et al14 demonstrated elevatedTGF-/31 mRNA expression in the aorta of rats whosehypertension was induced with deoxycorticosterone andsalt. Others have shown increased expression ofTGF-/31 mRNA levels in the aorta of SHR and WKYrats as their age increases from 5 to 40 weeks.15 ElevatedmRNA levels encoding TGF-/31 have also been re-ported in VSMC from SHR, compared with those fromWKY rats, cultured in the presence of serum.16 Morerecently it has been suggested that increased TGF-/31expression and activation is associated with augmentedproliferation of VSMC from SHR.17 We have previouslyshown that TGF-/31 interacts with tyrosine kinase-activating growth factors such as basic fibroblast growthfactor (bFGF), platelet-derived growth factor (PDGF),or epidermal growth factor (EGF) to potentiate theirmitogenic activity in VSMC from the SHR.18

    The aim of the present study was twofold: (1) toinvestigate whether the ability of TGF-^1 to potentiatethe effects of tyrosine kinase-activating growth factorswas unique to VSMC from SHR and (2) to examine therelation between TGF-/31 and growth factor-stimulatedproliferation of VSMC from SHR and WKY rats. Ourresults indicate that TGF-/31 inhibits growth factor-stimulated proliferation of VSMC from normotensiveWKY rats, whereas under identical conditions there ispotentiation of proliferation in VSMC from SHR.These differential effects appear to be due to alterationsin intracellular signaling mechanisms stimulated byTGF-/31 and involved in the regulation of proliferation.In addition, the production of endogenous TGF-/31 by

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  • 594 Hypertension Vol 23, No 5 May 1994

    cells after stimulation with growth factors may accountfor the well-known differences in the growth propertiesof VSMC from the two rat strains.

    MethodsAnimals

    Male SHR and WKY rats (weight, 250 to 300 g) were bredat the Baker Medical Research Institute from stock originallysupplied by Y. Yamori. Immediately before excising the aortasfor dispersion into single cells (see "Methods"), the animalswere deeply anesthetized with halothane. This procedure wasapproved by the Baker Institute-Alfred Hospital AnimalsExperimentation Committee and conformed to the guidelinesof the Australian National Health and Medical ResearchCouncil.

    MaterialsFetal calf serum (FCS), penicillin G, and Dulbecco's phos-

    phate-buffered saline (PBS) were purchased from Common-wealth Serum Laboratories. We obtained Dulbecco's modifiedEagle's medium (DMEM) from FLOW Laboratories. Tissueculture dishes were purchased from Sterilin Ltd. Collagenasetype-I (C-0130), elastase (E-0258), EGF, phenylmethylsulfonylfluoride (PMSF), and leupeptin were obtained from SigmaChemical Co. Porcine transforming growth factor-/31 was ob-tained from British Bio-Technology Ltd. Human [123I]TGF-/31was purchased from Dupont Australia Ltd. bFGF and methyl-pHJthymidine were obtained from Amersham. PDGF-AA andPDGF-AB, bovine serum albumin (BSA), and the randompriming DNA labeling kit were obtained from Boehringer-Mannheim. PDGF-BB was obtained from Zymogenetics. A ratTGF-/31 cDNA probe consisting of a 1-kb fragment spanning themajor coding region of the TGF-01 precursor (Qian et al19) wasprovided by Dr A. Roberts, Laboratory of Chemoprevention,National Cancer Institute, National Institutes of Health, Be-thesda, Md. A 30-base oligonucleotide that hybridizes to the 18srRNA species was provided by Dr Z. Krozowski, MolecularHypertension Laboratory, Baker Medical Research Institute,Melbourne, Australia.

    Isolation and Culture of AorticSmooth Muscle Cells

    Primary cultures of VSMC were prepared by enzyme dis-persion of aortic media from 12- to 14-week-old SHR andWKY rats as previously described.6 Examination of the cul-tures by phase-contrast microscopy indicated a "hills andvalleys" pattern at confluency, a well-known characteristic ofVSMC in culture. The identity of smooth muscle cells in ourcultures was verified by immunocytochemical analysis usingantibodies for smooth muscle myosin.20 These "primary"smooth muscle cells were subsequently passaged every weekand grown in 10% FCS/DMEM using 90-mm tissue culturedishes. Cells between primary and fourth passages were usedin all experiments.

    ['HJThymidine Incorporationand Cell Proliferation

    Cells were grown to confluency (1 to 2x 103 cells) in 24-welltissue culture dishes. At this time the medium was replacedwith 1.0 mL of DMEM, and cells were incubated for anadditional 48 to 72 hours. After 24 hours of serum deprivation,in excess of 86% of the VSMC were present in Go/G, assessedwith a fluorescent-activated cell sorter, the remainder werepresent in the S phase and G2-M phase.

    21 Growth factors, EGF(50 ng/mL), and/or TGF-/31 (1 ng/mL) (dissolved in DMEMcontaining 1% BSA) were then added to the quiescent cells, ina total volume of 0.5 mL of DMEM alone. At the indicatedtimes the medium was aspirated, and the cells were washedonce with 1.0 mL of DMEM and then incubated for anadditional 2 hours in 1.0 mL DMEM containing methyl-

    [3H]thymidine (1 /xCi/mL). At the end of this period, themedium was removed and cells washed three times with 1.0ml, of ice-cold Dulbecco's PBS before the addition of 0.5 mLof ice-cold 10% trichloroacetic acid for 15 to 30 minutes. Afteran additional wash with 10% trichloroacetic acid, the cellswere solubilized in 1 mol/L NaOH, neutralized with 1 mol/LHC1, and radioactivity was then determined by scintillationspectrometry.

    In the proliferation studies VSMC (^lxlO4) were platedinto 24-well tissue culture dishes in 1.0 mL of 10% FCS/DMEM. The next day the medium was replaced with 1.0 mLof DMEM, and cells were cultured for an additional 24 hours.Growth factors were then added (TGF-01 [1 ng/mL], EGF [50ng/mL], bFGF [25 ng/mL], PDGF-AA [400 ng/mL],PDGF-AB [200 ng/mL], and PDGF-BB [200 ng/mL]) in 0.5mL of DMEM containing insulin and transferrin (4%Monomed A, Commonwealth Serum Laboratories). Thegrowth factor-containing medium was replaced every 2 days,and cell number was then determined at indicated times usinga Coulter counter. We have pre