Andrew J. Morris and Susan S. Smyth Christopher End, Christopher Vallant, Anping Dong, Kevin ...

17 Pages·2008·1 MB·English

Lysophosphatidic Acid Receptors 1 and 2 Play Roles in Regulation of Vascular
Injury Responses but Not Blood Pressure
Manikandan Panchatcharam, Sumitra Miriyala, Fanmuyi Yang, Mauricio Rojas,
Christopher End, Christopher Vallant, Anping Dong, Kevin Lynch, Jerold Chun,
Andrew J. Morris and Susan S. Smyth
Circulation Research 2008, 103:662-670: originally published online August 14, 2008
doi: 10.1161/CIRCRESAHA.108.180778
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Lysophosphatidic Acid Receptors 1 and 2 Play Roles in
Regulation of Vascular Injury Responses but Not
Blood Pressure
Manikandan Panchatcharam, Sumitra Miriyala, Fanmuyi Yang, Mauricio Rojas, Christopher End,
Christopher Vallant, Anping Dong, Kevin Lynch, Jerold Chun, Andrew J. Morris, Susan S. Smyth
Abstract—Phenotypicmodulationofvascularsmoothmusclecells(SMCs)isessentialforthedevelopmentofintimal
hyperplasia. Lysophosphatidic acid (LPA) is a serum component that can promote phenotypic modulation of
culturedSMCs,butanendogenousroleforthisbioactivelipidasaregulatorofSMCfunctioninvivohasnotbeen
established. Ligation injury of the carotid artery in mice increased levels in the vessel of both autotaxin, the
lysophospholipase D enzyme responsible for generation of extracellular LPA, and 2 LPA responsive G
protein–coupled receptors 1 (LPA1) and 2 (LPA2). LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice were partially protected from the
developmentofinjury-inducedneointimalhyperplasia,whereasLPA1(cid:1)/(cid:1)micedevelopedlargerneointimallesions
afterinjury.Growthinserum,LPA-inducedextracellularsignal-regulatedproteinkinaseactivation,andmigration
to LPA and serum were all attenuated in SMCs isolated from LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice. In contrast, LPA1(cid:1)/(cid:1) SMCs
exhibited enhanced migration resulting from an upregulation of LPA3. However, despite their involvement in
intimalhyperplasia,neitherLPA1norLPA2wasrequiredfordedifferentiationofSMCsfollowingvascularinjury
ordedifferentiationofisolatedSMCsinresponsetoLPAorseruminvitro.Similarly,neitherLPA1norLPA2was
required for LPA to elicit a transient increase in blood pressure following intravenous administration of LPA to
mice. These results identify a role for LPA1 and LPA2 in regulating SMC migratory responses in the context of
vascular injury but suggest that additional LPA receptor subtypes are required for other LPA-mediated effects in
the vasculature. (Circ Res. 2008;103:662-670.)
Key Words: arterial injury (cid:1) lipids (cid:1) lysophosphatidic acid (cid:1) vascular remodeling (cid:1) vascular smooth muscle cells
Phenotypic modulation of vascular smooth muscle cells important in health and disease.10 Prominent among the
(SMCs) occurs in response to vascular injury and is a pathwaysthatareactivatedbyLPAincludeRhoGTPasesand
criticalcomponentinthedevelopmentofatheroscleroticand extracellular signal-regulated protein kinase (ERK), which
restenoticlesions.1,2Changesintheextracellularenvironment areknowntoplayimportantrolesinvascularSMCfunction.
promote this response, which is characterized by alterations LPA has also been proposed to serve as an endogenous
inthedifferentiationstateofSMCsandintheiracquisitionof activator of peroxisome proliferator-activated receptor
the capacity to proliferate and migrate. Isolated vascular (PPAR)(cid:1),11 and there may be additional, as yet unidentified
SMCs from human and rodent species can be stimulated to receptor targets for LPA.
dedifferentiate, proliferate, and migrate by serum. The lipid LPA is produced in locations that position it to be a
mediatorlysophosphatidicacid(LPA)hasbeenproposedas1 pathophysiologic mediator of vascular cell function. LPA is
ofthefactorspresentinserumthatmaypromotephenotypic present in relatively low levels in plasma but is more
modulation of vascular SMCs.3–5 abundant in serum, where is it thought be derived at least in
LPA,thesimplestglycerophospholipid,promotesdediffer- part through processes that require platelet activation.12–18
entiation,3proliferation,6–8andmigration9ofisolatedvascu- Therefore, local concentrations of LPA may be increased
lar SMCs. Although the specific signaling systems involved along vessels at sites of platelet adhesion and thrombus
inSMCresponsesarenotknown,inothercellsystems,LPA formation. LPA is also found in abundance in the lipid-rich
actsthroughatleast5Gprotein–coupledreceptors,LPA1-5, coreofatheroscleroticplaque,whereismaybederivedfrom
tostimulateawidevarietyofintracellularsignalingpathways mildly oxidized LDL.19 Thus, LPA is present, or can be
OriginalreceivedNovember12,2007;resubmissionreceivedJune4,2008;revisedresubmissionreceivedJuly13,2008;acceptedJuly31,2008.
FromtheDivisionofCardiovascularMedicine(M.P.,S.M.,F.Y.,A.D.,A.J.M.,S.S.S.),TheGillHeartInstitute,UniversityofKentucky,Lexington;
Carolina Cardiovascular Biology Center (M.R., C.E., C.V., S.S.S.), University of North Carolina, Chapel Hill; Department of Pharmacology (K.L.),
UniversityofVirginia,Charlottesville;DepartmentofMolecularBiology(J.C.),TheScrippsResearchInstitute,SanDiego,Calif;andDepartmentof
VeteransAffairsMedicalCenter(S.S.S.),Lexington,Ky.
CorrespondencetoSusanS.Smyth,MD,PhD,TheGillHeartInstitute,326CharlesT.WethingtonBuilding,900SLimestoneSt,Lexington,KY
[email protected]
©2008AmericanHeartAssociation,Inc.
CirculationResearchisavailableathttp://circres.ahajournals.org DOI:10.1161/CIRCRESAHA.108.180778
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Panchatcharam et al Vascular Smooth Muscle Cell Responses to LPA 663
formed, in the settings associated with alterations in SMC Femoralarterydenudationinjurywasperformedandanalyzedat4
function. The lysophospholipase D autotaxin (ATX), which weeksaspreviouslydescribed.31,32
catalyzes the hydrolysis of lysophospholipid substrates, is
Isolation of SMCs
responsible for generation of biologically active LPA in
MouseaorticSMCswereobtainedfromthoracicaortasbyremoving
circulation. Mice that are heterozygous for the wild-type
theadventitiaandendotheliumbydigestionwithcollagenasetypeII
(WT)andnullATXallelehave50%normalcirculatingLPA (Worthington; 175 U/mL). The media were further digested in
levels,20,21 and mice that transgenically overexpress ATX in solution containing collagenase type II (175 U/mL) and elastase
the liver under control of the (cid:2)-antitrypsin promoter have (Sigma;0.5mg/mL),whichyielded(cid:5)100000cellsperaorta.Cells
weregrowninDMEMcontaining0.5ng/mLEGF,5(cid:3)g/mLinsulin,
elevated circulating levels of ATX and LPA.22 2 ng/mL basic fibroblast growth factor, 10% FBS, 100 U/mL
Exogenous administration of LPA to animals elicits re- penicillin,and100(cid:3)g/mLstreptomycinandincubatedat37°Cwith
sponses that are consistent with it serving as an endogenous 5% CO/95% air. SMC lineage was confirmed by the presence of
2
immunoreactivityfor(cid:2)-actin(Sigma)in(cid:2)99%ofthecells.Exper-
mediatorofvascularcellfunction.Forexample,intravenous
iments involving SMCs were performed by using cells with a
injectionofLPAelevatesarterialbloodpressureinrats,23and passagenumberof(cid:4)5.
local application causes cerebral vasoconstriction in pigs.24 An expanded Materials and Methods section is available in the
Moreover, local infusion of LPA in the rat common carotid onlinedatasupplementathttp://circres.ahajournals.org.
arteryinducesvascularremodelingbystimulatingneointimal
Statistical Analysis
formation.25Asimilarresponseisobservedinmiceandmay
be mediated by PPAR(cid:1).26 All results were expressed as mean(cid:6)SD. In vitro studies were
repeatedaminimumof3times,andresultswereanalyzedbyStudent
Untilrecently,alackofappropriateandspecifictoolshas t test or ANOVA. Statistical significance within strains was deter-
limitedourabilitytodefinepathophysiologicrolesofendog- mined using ANOVA with multiple pair-wise comparisons. Statis-
enous LPA in the vasculature. In particular, the most exten- tical analysis was performed using Sigma-STAT software version
3.5(SystatSoftwareInc).Aprobabilityvalueoflessthan0.05was
siveinvitrostudiesreportedtodatehaveusedratorhuman
consideredsignificant.
SMCs,whereasresponsesofmurineSMCstoLPAhavenot
beenexaminedindetail.AconcertedanalysisofmurineSMC Results
responsestoLPAinvitro,coupledwithaphenotypicanalysis Exposureofisolatedsmoothmusclecells3orintactvessels25
ofvascularinjuryresponsesofLPAreceptor–deficientmice, to exogenous LPA elicits SMC phenotypic modulation;
isrequiredtoprovidedefinitiveinsightsintotheroleofLPA however, the role of endogenous LPA in mediating SMC
in this important aspect of vascular function. In this report, responsesinthecontextofvascularinjuryisnotknown.The
weidentifyanupregulationofATXandLPAreceptors1and aimofthisstudywastodeterminewhetherLPAcontributes
2(LPA1andLPA2)followingvascularinjury.Weusemice to vascular injury responses in intact organisms. To accom-
deficientinLPA1and-2aloneandincombinationtodefine plish this aim, we used mice deficient in candidate LPA
rolesforthesereceptorsandLPAinthepathophysiologically receptorsandpharmacologicalantagonistsofthesereceptors
relevantvascularresponsesandinthephenotypicmodulation to attenuate normal LPA signaling in vascular SMCs and
of SMCs that occurs with vascular injury. determine the consequences on vascular and isolated cells
responses.
Materials and Methods
Upregulation of ATX and LPA Receptors
Mice
Following Vascular Injury
All procedures conformed to the recommendations of Guide for
Following ligation injury, we observed a time-dependent
theCareandUseofLaboratoryAnimals(DepartmentofHealth,
increase in vessel-associated levels of ATX protein that was
Education, and Welfare publication number NIH 78-23, 1996)
and were approved by the Institutional Animal Care and Use (cid:5)2.5-fold after 1 to 3 days (P(cid:7)0.05 at 1 day and 3 days
Committee. The production and initial characterization of mice versus uninjured; Figure 1A). ATX is a secreted lysophos-
deficient in LPA receptors 1 and 2 has previously been de- pholipaseDresponsibleforgenerationofbiologicallyactive
scribed.27,28 The mice were backcrossed for (cid:2)10 generations to extracellularLPA.IncreasingplasmalevelsofATXelevates
the BalbC background. Mice were housed in cages with HEPA-
circulatingLPAlevels22;therefore,extracellularLPAproduc-
filteredairinroomson12-hourlightcyclesandfedPurina5058
rodent chow ad libitum. Systolic blood pressure and heart rate tionalongthevesselwalllikelyincreasesfollowinginjury.In
weremeasuredfor5consecutivedaysinconsciousmiceusingthe many systems, LPA elicits cellular effects via G protein–
BloodPressureAnalysistailcuffsystem(HaterasSystems,Apex, coupledreceptorsignaling.Wethereforeassessedthelevelof
NC) daily after training for 1 week. Mean intraarterial pressure gene expression of the 5 known LPA receptors (LPA1-5)
was measured by placement of a 1.4 Fr Millar catheter in the
following injury and observed that expression of LPA1 and
carotid artery of isoflurane-anesthetized mice.
LPA2increasedfollowingcarotidinjuryby2.1-and6.2-fold,
Vascular Injury respectively (Figure 1B).
Atvariousintervalsaftercarotidsurgery,29,305mmofaortaproximal
to the suture was removed and processed for RNA analysis by LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) Mice Are Partially Protected From
qualitativePCR(qPCR)oranalysisofproteinmarkersbyimmuno- Development of Intimal Hyperplasia in Vascular
blotting. Neointimal formation along the length of the vessel was Injury Models, Whereas LPA1(cid:1)/(cid:1) Mice Exhibit
assessedat4weeksaftersurgeryusingcomputerassistedmorphom-
Positive Remodeling Characterized by an
etry as has been previously described.30 Digital images were taken
with a high performance digital camera (resolution 3840(cid:3)3072 Enhanced Neointimal Response
pixels)attachedtoaNikon80imicroscopewitha(cid:3)10(NA(cid:4)0.3)or Mice that are singly or doubly deficient in LPA1 and LPA2
(cid:3)20 (NA(cid:4)0.5) objective and analyzed with Metamorph software. (LPA1(cid:1)/(cid:1), LPA2(cid:1)/(cid:1), and LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice) are viable and
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664 Circulation Research September 12, 2008
Figure1.IncreasesinATX/
lysophospholipaseDandLPA
receptorexpressionfollowing
arterialinjury.Carotidarteries
wereisolatedfromcontrol,
uninjuredmice,oranimalsthat
underwentcarotidligation.A,
ATXexpressionwasdeter-
minedbyimmunoblottingand
wassignificantlyhigher
(P(cid:7)0.05byANOVA)at1and3
daysafterinjuryincomparison
touninjuredcontrollevels.B,
LPAreceptorexpressionwas
determinedbyquantitative
PCR.Resultsareexpressed
relativetouninjuredvesseland
presentedasmeans(cid:6)SDfrom
3separateexperiments.
fertile,27,28allowingustousethemtoinvestigatearoleforthese LPA2(cid:1)/(cid:1) or LPA1(cid:1)/(cid:1) mice, which displayed normal and
LPA signaling receptors in the regulation of vascular SMC enhanced neointima, respectively, LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) double
responses. Carotid ligation was performed in WT, LPA1(cid:1)/(cid:1), knockout were partially protected from the development of
LPA2(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)doubleknockoutmice.Fourweeks intimal hyperplasia with an (cid:5)4-fold lower intimal areas
after carotid ligation, neointima developed along the length of (P(cid:7)0.05; Figure 2B) and intima/media ratios after carotid
the carotid in WT mice (Figure 2A and 2B). The extent of ligation (Figure 2D). Medial areas were similar in all mice
intimal area and the intima/media ratio was similar in mice (Figure 2C).
lacking LPA2 (Figure 2B and 2D). In contrast, LPA1(cid:1)/(cid:1) mice To determine whether these results were model-specific,
displayed an (cid:5)3-fold enhanced intimal area (Figure 2B), an we denuded the endothelium of the mouse femoral artery to
increaseinintima/mediaratio(P(cid:7)0.017versusWT;Figure2D), elicitthedevelopmentofintimalhyperplasiaandobservedan
alargerlumenarea(Figure2E),andaresultingincreaseinthe enhanced injury response that was broadly similar to that
areawithintheexternalelasticlamina,consistentwithpositive observedinthecarotidarteryligationmodelinLPA1(cid:1)/(cid:1)mice
remodeling (ie, maintenance of luminal area) in the presence (see Figure I in the online data supplement). Additional
of heightened neointimal formation. Unlike either the experiments were not performed with the other strains of
Figure2.AlterationsinthedevelopmentofintimalhyperplasiainmicelackingLPAreceptors.A,RepresentativecombinedMasson
elastin–stainedsectionsofcarotidarteriesat4weeksaftersurgerytaken(cid:5)1.2mmfromthesiteofligationinWT,LPA1(cid:1)/(cid:1),
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)mice.BthroughE,Intimalarea(B),medialarea(C),intima/mediaratio(D),andluminalareas(E)inmicrometers
squaredalongthelengthofvesselsinWT(n(cid:4)8),LPA1(cid:1)/(cid:1)(n(cid:4)6),LPA2(cid:1)/(cid:1)(n(cid:4)6),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)(n(cid:4)6)mice.*P(cid:7)0.05incompar-
isontoWTbyANOVAwithmultiplepairwisecomparisons.
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Panchatcharam et al Vascular Smooth Muscle Cell Responses to LPA 665
Figure3.LPA1andLPA2arenotrequiredforinjury-inducedalterationsinproliferation,differentiation,orinflammationmarkers.Carotid
injuryincreasesERKactivityinvesselsfromWT,LPA1(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)mice(A),althoughresponsesintheLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)vessels
returntobaselinewithin3days.pERKindicatesphospho-ERK.B,InjuryupregulatesPCNAgeneexpressionanddownregulatesSMC
differentiationmarkersinWT,LPA1(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)vessels.HCindicatesheavychain.C,Upregulationofmyeloperoxidase(MPO)
andinterleukin-6(IL-6)occursfollowinginjuryandissimilarinWT,LPA1(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)mice.Resultsarepresentedasmean(cid:6)
SDfrom3separateexperimentsinvesselsharvestedat5daysafterinjury.
LPAreceptor–deficientmiceusingthisfemoralarterymodel tiation were unaltered by combined LPA1 and LPA2 defi-
because of a high incidence of acute arterial thrombosis in ciency, we considered the possibility that the attenuation of
WT Balb/C. injury induced intimal hyperplasia in LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice
Vascular injury elicits a characteristic sequence of events involves a requirement for these receptors as regulators of
that includes inflammatory cell recruitment and SMC dedif- SMCmigrationthat,asdiscussedbelow,weevaluatedusing
ferentiation, proliferation, and migration. To investigate the primary cultures of mouse SMCs.
mechanism(s) by which LPA receptors might regulate vas-
cularinjuryresponses,wemeasuredmarkersfortheseevents LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) Vascular SMCs Exhibit Attenuated
afterligation.Followinginjury,therewasanincreaseinERK Proliferative Responsiveness In Vivo and In Vitro
activation,asmeasuredbytheratioofphospho-ERKtototal AlthoughLPAresponsesofculturedSMCsfromhumansand
ERK,andtheincreaseinERKactivityreturnedtobaselineby rats have been investigated in detail, these types of studies
5daysinLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)miceandby7daysinLPA1(cid:1)/(cid:1)mice have not been reported with mouse SMCs. To identify the
(Figure3A).However,expressionoftheproliferationmarker mechanistic basis for vascular injury response phenotypes
PCNA was upregulated to similar extents in WT, LPA1(cid:1)/(cid:1) observed in the LPA receptor–deficient mice, we examined
and LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) vessels (Figure 3B), suggesting that cell proliferation, dedifferentiation, and migration responses to
proliferation following injury was similar in all genotypes. LPA of isolated mouse vascular SMCs. No significant dif-
Likewise, lack of LPA1 and LPA2 did not affect SMC ferencesingrowthofWTandLPA1(cid:1)/(cid:1)SMCsinserumwere
dedifferentiationfollowinginjurybecausedownregulationof observed,butLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCsgrewmoreslowly(Figure
SMC markers occurred in all genotypes of mice examined 4A). This difference appeared to be dependent on the LPA
(Figure 3B). Expression of interleukin-6 and the neutrophil- componentofserumbecausetherewasnodifferencebetween
and monocyte-derived inflammatory marker myeloperoxi- platelet-derived growth factor (PDGF)-induced growth of
dase increased following injury and was also unaffected by WTandLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCs(Figure4B).LPAregulatescell
theabsenceofLPA1andLPA2(Figure3C).Asdetectedby proliferation through ERK activation in many cell systems.
immunoblotting, vessel-associated CD68, myeloperoxidase, Therefore, we examined LPA-induced ERK responses in
and P-selectin, a marker of platelet activation, increased isolated SMCs. LPA treatment of isolated SMCs increased
following injury in all genotypes (see supplemental Figure ERK activation maximally at 5 to 10 minutes (Figure 4C).
II). Taken together, these results indicate that vascular in- Thisresponsewasconcentration-dependent,withmaximal
flammation,SMCdedifferentiation,andcellproliferationare activation being observed with 1 (cid:3)mol/L LPA (data not
similarinWT,LPA1(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)arteriesfollowing shown). A similar increase in LPA-induced ERK activity
injury. Because these markers of proliferation and differen- in SMCs cultured from LPA2(cid:1)/(cid:1) mice was observed (data not
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666 Circulation Research September 12, 2008
Figure4.GrowthandERKactivityisattenuatedinLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCs.GrowthcurvesforisolatedSMCsinserum(A)orinresponseto
20ng/mLPDGF(B)weregeneratedbycountingviablecellsattheindicatedtimepoints.LPA(1(cid:3)mol/L)inducesERKactivationinWT
cells,asmeasuredbyphospho-ERK/totalERKratios.C,LPA-inducedERKactivationisattenuatedinLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCs.Resultsare
presentedasmeans(cid:6)SDfrom3independentculturesofSMCsofeachgenotype.*P(cid:7)0.05incomparisontoWTbyANOVA.
shown).Incontrast,therewasan(cid:5)2-foldreductionintheinitial increase). In contrast, LPA1(cid:1)/(cid:1) cells were hypermigratory
burstofLPA-inducedERKactivationinLPA1(cid:1)/(cid:1)SMCsanda at baseline (7.9(cid:6)1.8-fold higher than WT cells; P(cid:4)0.003)
5-foldreductioninLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCs(Figure4C). and displayed a further exaggerated migration response to
LPA and serum (Figure 5).
To determine whether expression of an alternate LPA
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs Exhibit Decreased Migration receptormightberesponsibleforthehypermigratoryLPA
in Response to LPA, Whereas LPA1(cid:1)/(cid:1) SMCs response observed in the LPA1(cid:1)/(cid:1) cells, the assays were
Exhibit Enhanced Migration in Response to conducted in the presence of VPC32301, a selective
Upregulation of the LPA3 Receptor antagonistoftheLPA3receptor.33LPA3antagonismhada
Although the reduced proliferative responses observed in modest (cid:5)30% inhibition of WT migration (22978(cid:6)3507
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)SMCsinvitroareconsistentwiththeprotection versus 33 680(cid:6)6329 (cid:3)m2 in antagonist-treated and
of LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice from the formation of neointima in vehicle-treated cells, respectively) and inhibited LPA-
response to injury, the lack of a change in proliferation inducedERKactivationinWTcellsby10%to15%.LPA3
markers following injury in LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) vessels does not receptor antagonism reduced both baseline and LPA-
support the idea that protection from the development of induced migration in LPA1(cid:1)/(cid:1) cells to levels observed in
intimal hyperplasia is a direct result of attenuated cell WT cells (Figure 5) and inhibited LPA-induced ERK
proliferation. In many systems, LPA is a potent stimulus for activation in LPA1(cid:1)/(cid:1) cells by (cid:5)35%. We found that LPA3
migration; therefore, we compared migration responses to receptor expression was elevated 3.7(cid:6)0.2-fold in LPA1(cid:1)/(cid:1)
LPAinSMCsderivedfromWTandLPAreceptor–deficient but not LPA2(cid:1)/(cid:1) or LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) cells as compared to WT
mice (Figure 5). LPA, serum, and PDGF stimulated cells(seesupplementalTableI).Todeterminewhetherupregu-
4.7(cid:6)0.94-, 6.44(cid:6)0.046-, and 11.5(cid:6)0.79-fold increases, re- lation of LPA3 expression alone could account for the en-
spectively,inmigrationofSMCs(Figure5).Themigrationof hanced migration observed in LPA1(cid:1)/(cid:1) cells, LPA3 was
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) cells was lower at baseline (39(cid:6)1% that of overexpressed in WT SMCs using recombinant lentivirus
WT cells; P(cid:7)0.01), and the cells did not display any vectors, which resulted in enhanced migration toward LPA
increase in migration to LPA and serum (Figure 5), and serum phenocopying the behavior of LPA1(cid:1)/(cid:1) cells
althoughmigrationtoPDGFwaspreserved(10.5(cid:6)1.7-fold (Figure 5).
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Panchatcharam et al Vascular Smooth Muscle Cell Responses to LPA 667
Figure5.LPAreceptordeficiencyaltersSMCmigratoryresponses.SMCswerestainedwithDiff-Quikontheundersurfaceofamem-
branewitha5-(cid:3)mporefollowingmigrationtomediacontainingvehiclein0.5%FBSor1(cid:3)mol/LLPA(top).Bottomleft,Cellmigration
inWT,LPA2(cid:1)/(cid:1),LPA1(cid:8)/(cid:1)2(cid:1)/(cid:1),andLPA1(cid:1)/(cid:1)2(cid:1)/(cid:1)cellstoward1(cid:3)mol/LLPA,10%serum,or20ng/mLPDGF.Bottomright,Comparison
ofmigratoryresponsesofWTandLPA1(cid:1)/(cid:1)SMCs.EnhancedmigrationinLPA1(cid:1)/(cid:1)SMCsisattenuatedbytheLPA3antagonist
VPC32301(10(cid:3)mol/L)andoverexpressionofLPA3increasesWTmigration.Resultsfromatleast3separateexperimentsarereported
asmeans(cid:6)SDofsurfacecoveragebymigratedcellsinmicrometerssquared.ndindicatesnotdetermined.
LPA1 and LPA2 Are Not Required for Rho LPA1 and -2 Do Not Mediate Acute Blood
Activation or SMC Dedifferentiation Pressure Responses to LPA or Regulate Systemic
MigrationofSMCstoLPAwasattenuatedbyERKandRho Blood Pressure
kinaseinhibitors(seesupplementalFigureIII).Therefore,we The above results suggest that LPA regulates the vascular
examinedLPA-inducedRhoresponsesinWT,LPA1(cid:1)/(cid:1),and injuryresponsebymodulatingmigratorypropertiesofSMCs.
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs. In 2 of 3 independent SMC cultures, In other species, intravascular administration of LPA alters
LPA1(cid:1)/(cid:1) cells had higher active Rho at baseline, but no blood pressure, which likely results from direct effects on
differencesinLPA-inducedRhoactivationwereobservedin vascular smooth muscle cell contractility. We sought to
WT, LPA1(cid:1)/(cid:1), and LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs (Figure 6A). These determine whether the same receptors that regulate vascular
results suggest that lack of ERK activation, rather than SMC migration responses to LPA also contribute to the
changesRhoactivation,accountsforthereducedmigrationin effects of LPA on blood pressure. Intravenous infusion of
the LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice. BSA-conjugated LPA transiently increased mean arterial
BothLPAandRhohavebeenimplicatedasregulatorsof blood pressure in anesthetized mice, with 10 pmol of LPA
the differentiation state of vascular SMCs.3 However, our being the lowest dose that reproducibly increased mean
results following carotid ligation suggest that neither LPA arterialbloodpressureabovevehicleby17(cid:6)6mmHginWT
1 nor LPA2 is required for injury-induced SMC dediffer- mice.Asimilarresponsewasobservedinmicelackingeither
entiation in vivo. To determine whether either of these LPA1 or LPA2 or both, suggesting that neither receptor is
receptors plays role in dedifferentiation of isolated SMCs, responsible for acute increase in blood pressure elicited by
we examined expression patterns of the differentiation LPA (Table 1). Likewise, there was no difference in blood
marker SMC myosin heavy chain. Expression of SMC pressureorheartrateinconsciousWT,LPA1(cid:1)/(cid:1),LPA2(cid:1)/(cid:1),or
myosinheavychainwashighinconfluentSMCsdeprived LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) mice (Table 2).
ofserumfor72hours(Figure6B).Exposureofthesecells
to 10% serum or 1 (cid:3)mol/L LPA for 24 hours downregu- Discussion
lated SMC myosin heavy chain expression to a similar Inthisstudy,weprovidethefirstdemonstrationforarolefor
extent in WT, LPA1(cid:1)/(cid:1), and LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) cells (Figure theLPAsignalingnexusinendogenousregulationofpatho-
6B). Taken together, these results suggest that LPA1 and physiologic vascular responses. Specifically, we report that
LPA2arenotessentialforLPA-triggereddedifferentiation micedeficientin2definedLPAreceptors,LPA1andLPA2,
of SMCs in vitro. areprotectedfromintimalhyperplasiainresponsetovascular
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668 Circulation Research September 12, 2008
Figure6.NeitherLPA1norLPA2isrequiredforisolatedSMCdedifferentiation.A,ExpressionofSMC-specificmyosinheavychainin
culturedSMCsafterserumdeprivationorfollowingreexposuretoeither10%FCSor1(cid:3)mol/LLPAfor24hours.B,Rhoactivitywas
examined10minutesafterexposureofcellstoLPA(1(cid:3)mol/L)byRhotekinpull-downassay.Valuesrepresentedresultsobtainedfrom
3separateexperimentsandarereportedasafoldchangefrombaselineinWTcells(means(cid:6)SD).S1Pindicatessphingosine-1-phosphate.
injury through a mechanism that can primarily be accounted crosstalk between LPA receptors that has been reported in
forbyadecreaseintheabilityofLPAtopromotemigration other cell types including cardiomyocytes and embryonic
of vascular SMCs. Interestingly, we also found that mice fibroblasts. For example, LPA-induced ERK activity is
lacking LPA1 alone exhibited enhanced vascular injury normal in LPA2(cid:1)/(cid:1) SMCs, slightly reduced in LPA1(cid:1)/(cid:1) cells,
responses, which correlated with an upregulation of a third and nearly completely attenuated in LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs, indi-
LPA receptor, LPA3, and appeared to result from an en- cating that both LPA1 and LPA2 are required to produce
hancedLPA3-dependentvascularsmoothmusclecellmigra- maximal LPA activation of ERK, which likely accounts for
tory response. These findings suggest that LPA is a relevant the reduced proliferative responses in LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) cells in
regulatorofthemurinevascularinjuryresponseandfocus vitro. LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs display an attenuated migration
attention on possible sources of LPA at sites of vascular response to LPA, which may also related to the reduced
injury. We found that injury elevated vessel-associated ERK activity in response to LPA. These results also
levels of ATX, the lysophospholipase D responsible for underscore the importance of the LPA component to the
generationofextracellularLPA.Thismayreflectlocalized promigratory effects of serum on these cells, because
expressionofATXor,perhapsmorelikely,recruitmentof LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) SMCs display reduced migration to both
circulating ATX. Because ATX is responsible for gener- serum and LPA but not to PDGF.
ating circulating LPA and the ATX substrate lysophos- Our results also point to LPA-dependent vascular sig-
phatidylcholineisabundantinthecirculation,theincrease naling pathways that are not regulated by LPA1 or LPA2.
in vessel-associated ATX likely drives localized produc- Specifically, SMCs lacking both receptors appear to un-
tion of biologically active, extracellular LPA at the site of dergo essentially normal dedifferentiation in response to
injury, which in turn promotes SMC migration via LPA1 serum, LPA, or vascular injury. Because of the possibility
and LPA2. that localized production of LPA could promote vasocon-
Comparison of the in vivo injury and in vitro SMC striction at sites of vascular injury, we were particularly
responses from mice that are singly or doubly deficient in interested in evaluating the possible roles of LPA1 and
LPA1 and LPA2 identifies functional redundancy and LPA2 in blood pressure regulation. However, although
Table1. BloodPressureandHeartRateinWTMiceandMice Table2. IncreaseinMeanArterialPressureinResponseto
LackingLPA1and/orLPA2 IntravenousAdministrationofLPA
Genotype N SBP(mmHg) HeartRate(bpm) Genotype N ChangeinMAP(mmHg) Weight(g)
WT 6 124(cid:6)26 621(cid:6)38 WT 5 17(cid:6)6 29(cid:6)4
LPA1(cid:1)/(cid:1) 9 126(cid:6)12 621(cid:6)46 LPA1(cid:1)/(cid:1) 3 25(cid:6)8 22(cid:6)4
LPA2(cid:1)/(cid:1) 12 133(cid:6)11 623(cid:6)46 LPA2(cid:1)/(cid:1) 3 19(cid:6)8 23(cid:6)1
LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) 4 120(cid:6)24 588(cid:6)66 LPA1(cid:1)/(cid:1)2(cid:1)/(cid:1) 2 20(cid:6)2 29(cid:6)4
Values for systolic blood pressure (SBP) and heart rate are presented as Values for change in mean arterial pressure (MAP) and for weight are
means(cid:6)SD. There was not a statistically significant difference in blood presentedasmeans(cid:6)SD.Nostatisticallysignificantdifferencebetweengroups
pressure(P(cid:4)0.500)orheartrate(P(cid:4)0.621). wasobserved(P(cid:4)0.466).
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Panchatcharam et al Vascular Smooth Muscle Cell Responses to LPA 669
these receptors are clearly expressed and functional in 6. TokumuraA,IimoriM,NishiokaY,KitaharaM,SakashitaM,Tanaka
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Acknowledgments K,SakuK,TaguchiR,AraiH.Serumlysophosphatidicacidisproduced
through diverse phospholipase pathways. J Biol Chem. 2002;277:
We thank Jessica Caicedo, Kirk McNaughton, and Raina Tosheva 48737–48744.
forexcellenttechnicalassistance. 18. EichholtzT,JalinkK,FahrenfortI,MoolenaarWH.Thebioactivephos-
pholipid lysophosphatidic acid is released from activated platelets.
BiochemJ.1993;291:677–680.
Sources of Funding
19. SiessW,ZanglKJ,EsslerM,BauerM,BrandlR,CorrinthC,Bittman
ThisworkwassupportedbyNIHgrantsHL070304,HL078663,and R,TigyiG,AepfelbacherM.Lysophosphatidicacidmediatestherapid
HL074219(toS.S.S.);CA096496andGM050388(toA.J.M.);and activation of platelets and endothelial cells by mildly oxidized low
MH51699andNS048478(toJ.C.).S.S.S.receivedanAtorvastatin densitylipoproteinandaccumulatesinhumanatheroscleroticlesions.
ResearchAwardfromPfizer. ProcNatlAcadSciUSA.1999;96:6931–6936.
20. TanakaM,OkudairaS,KishiY,OhkawaR,IsekiS,OtaM,NojiS,
YatomiY,AokiJ,AraiH.Autotaxinstabilizesbloodvesselsandis
Disclosures
required for embryonic vasculature by producing lysophosphatidic
None. acid.JBiolChem.2006;281:25822–25830.
21. vanMeeterenLA,RuursP,StortelersC,BouwmanP,vanRooijenMA,
PradereJP,PettitTR,WakelamMJ,Saulnier-BlacheJS,MummeryCL,
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