Research Article Serum Caveolin-1 as a Novel Biomarker in ...downloads.hindawi.com/journals/bmri/2015/173970.pdf · Serum Caveolin-1 as a Novel Biomarker in Idiopathic Pulmonary Artery

Research ArticleSerum Caveolin-1 as a Novel Biomarker in IdiopathicPulmonary Artery Hypertension

Kuo-Yang Wang123 Mey-Fann Lee4 Hung-Chin Ho15 Kae-Woei Liang167 Chia-Chi Liu18

Wan-Jane Tsai1 and Wei-Wen Lin135

1Cardiovascular Center Taichung Veterans General Hospital Taichung Taiwan2School of Medicine Chung Shan Medical University Taichung Taiwan3School of Medicine China Medical University Taichung Taiwan4Department of Education and Research Taichung Veterans General Hospital Taichung Taiwan5Department of Life Science Tunghai University Taichung Taiwan6Institute of Clinical Medicine Cardiovascular Research Center Taiwan7Department of Medicine National Yang Ming University School of Medicine Taipei Taiwan8Department of Biomedical Engineering amp Environmental Sciences National Tsing Hua University Hsinchu Taiwan

Correspondence should be addressed to Wei-Wen Lin weinlinvghtcgovtw

Received 22 June 2015 Revised 16 August 2015 Accepted 18 August 2015

Academic Editor Renzhi Han

Copyright copy 2015 Kuo-Yang Wang et alThis is an open access article distributed under theCreativeCommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Pulmonary arterial hypertension (PAH) is a rare disease butwith significantmorbidity andhighmortalityThere is no specificway todiagnose PAHThus an easy used with good sensitivity and specificity biomarker of PAH is highly desirable to aid in the screeningdiagnosis and follow-up Caveolin-1 (Cav1) is the structural protein of caveolae and is highly expressed in type I pneumocytesLungs tissues from idiopathic PAH (IPAH) patients showed decreased expression of Cav1 in vascular endothelial cells Thereforewe developed a direct sandwich immunoassay for the determination of Cav1 in IAPH patientrsquos serum The result disclosed serumCav1 level was significantly lower in IPAH than control groups Using serum Cav1 1717 pgmL as a cutoff value the sensitivity was059 and the specificity was 10 There were two major findings in our results First serum Cav1 might be a novel biomarker in thediagnosis of IPAH with fare sensitivity and good specificity Second Cav1 might be used to make differential diagnosis betweenCOPD-PH and IPAH group

1 Introduction

Pulmonary arterial hypertension (PAH) is a rare disease butwith significant morbidity and high mortality Annual inci-dence is 1-2 cases per million people in the USA and it is 2ndash4times as common in women as in men [1 2] In untreatedpatients the median survival rate is only 28 years and the5-year survival rate is 34 [3] There is no specific way todiagnose PAH According to the American and Europeanclinical practice guidelines [4ndash6] the diagnosis involvessequences of steps and requires several invasive and nonin-vasive examinations Well experienced specialists are needed

to interpret the results andmanage these patients [7 8]Thussensitive and specific biomarkers of PAH are highly desirableto aid in the screening diagnosis and follow-up

Previous studies have suggested that atrial natriureticpeptide (ANP)N-terminal probrain natriuretic peptide (NT-proBNP) troponin and uric acid are potential biomarkersfor PAH [9ndash12] However these are not specific biomarkers ofthe pathology changed of the pulmonary artery hypertensionEndothelial cell dysfunction proliferation without apoptosisand vasoconstrictionmay play important roles in PAH there-fore vascular bed may be a good source of new biomarkers[13ndash15]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 173970 7 pageshttpdxdoiorg1011552015173970

2 BioMed Research International

Caveolae are 50ndash100 nm vesicular invaginations of thecell plasma membrane and caveolin-1 (Cav1) is the structuralprotein of caveolae and is highly expressed in adipocytesendothelial cells and type I pneumocytes Cav1minusminus miceexhibit pulmonary hypertension and right ventricle hyper-trophy [16ndash18] In monocrotaline-induced PH rat modelsCav1 deficiency is seen in lung tissue [19] Lungs tissues fromidiopathic PAH (IPAH) patients decreased expression of Cav1in vascular endothelial cells and also decreased in the totallung lysate [20 21] Furthermore Cav1 can be secreted intoserum and be detected [22]These results suggested that Cav1may play an important role in the pathogenesis of PAH andserum Cav1 level may be a good biomarker for diagnosis[23 24]

2 Materials and Methods

In the study age matched patients with normal left ventriclefunction were divided into 3 groups In Group (1) IPAHpatients (119899 = 21) definite diagnosis was made accordingto European Society of Cardiology (ECS) [4] and Ameri-can Heart Association (AHAACC) [5] guideline inclusioncriteria including mean pulmonary artery pressure (mPAP)ge 25mmHg pulmonary wedge pressure less or equal to15mmHg and pulmonary vascular resistance over 3 Woodunits measured by right heart catheterization In Group(2) chronic obstructive pulmonary disease with pulmonaryhypertension (COPD-PH) patients (119899 = 22) COPD werediagnosed by pulmonologist and estimated mean PAP ge25mmHg by echocardiography Group (3) (non-PAH group)includes healthy volunteers (119873 = 26) with mPAP less than15mmHg measured by echocardiography

Demographic data and clinical features of patientsincluded in this studywere summarized in Table 1 Accordingto Tahir et al reports [23 24] we developed a direct sandwichimmunoassay for the determination serum Cav1 level fromparticipants Serum hsCRP NT-proBNP and BMPR2 levelswere also measured by commercial ELISA kits This studywas approved by Local Ethical Committee in Taichung Veter-ans General Hospital Taichung Taiwan (number CE12022)Written informed consent was provided to all participants

21 Protocol for Serum Cav1 Assay Two commercial affinitypurified monoclonal mouse Cav1 antibodies and polyclonalrabbit Cav1 antibodies were chosen for a direct sandwichELISA The capture Cav1 antibody used was generated fromhuman recombinant Cav1 (RampD systems) and the detectionantibody was HRP-conjugated rabbit polyclonal antibodyraised against a peptide mapping at the NH

2

terminus ofhuman Cav1 (Santa Cruz Biotechnology) Costar microplatewells were coated with 100 120583L of Cav1 antibody (25mgL) inPBS (pH 74) and incubated overnight at 4∘C The wells werethen blocked with 300 120583L of PBS containing 05 BSA and005 vv Tween 20 for 2 hours at room temperature andwerewashed three timeswith PBS containing 05 vv Tween20 (PBST) Serum samples calibrators and controls wereadded (100 120583L) to the wells and incubated overnight at 4∘CThe wells were washed three times with 400 120583L of PBST and100 120583L of HRP-conjugated Cav1 (Santa Cruz Biotechnology)

I II III

M

(kD

a)

rCav1rCav1rCav1

100

72

55

40

35

25

15

Humanserum

Figure 1 Specificity of capture and detection antibodies to rCav-1and native Cav1 of human serum Recombinant Cav1 protein waspurified using affinity chromatography and analyzed by SDS-PAGE(I) and immunoblotting (II and III) In panel I the rCav-1 proteinmigrated as a single band and displayed gt95 purity by Coomassieblue staining The binding specificity of the capture and detectionantibodies to rCav-1 and human serum was demonstrated andshowed in panel II and panel III respectively Numbers on the leftindicate sizes of protein markers (lane M)

antibody diluted 1 2000 in PBST After incubation for 2 hrat room temperature the wells were washed three times withPBST and 100 120583L of 331015840551015840-tetramethylbenzidine substratesolution (Clinical Science Products Inc) was added andincubated for 30min at room temperature The reaction wasstopped by adding 100 120583L of 1NH

3

PO4

and the absorbancewas read at 450 nm with a microplate reader (TECANGrodig Austria) SerumCav1 levels weremeasured using lab-made recombinant pQE30-Cav 1 (a 101-amino acid region ofCav1 gene from GenBank accession number NM001753) asa standard A linear standard curve was constructed using aconcentration range (1219ndash780 pgmL) of recombinant Cav1in a parallel ELISA The levels of Cav1 in sera of 21 IPAHpatients 22 COPD-PH patients and 26 healthy controlsby the in-house ELISA were then measured The limit ofdetection of the sandwich ELISA was 1219 pgmL Any valuebelow the detective limitation of the assay referred to zeroIn Figure 1 Western blot data using capture and detectionantibodies that react to recombinant Cav1 proteins wasshowed

22 Enzyme-Linked Immunosorbent Assay for HsCRP NT-proBNP and BMPR2 Detection The serum levels of high-sensitivity C-reactive protein (hsCRP) NT-proBNP andbone morphogenetic protein type II receptor (BMPR2) weremeasured with enzyme-linked immunosorbent assay (ELISA)kits hsCRP (Cell Biolabs Inc San Diego CA) NT-proBNP(Invitrogen Corporation Camarillo CA) and BMPR2(MyBioSource San Diego CA) according to the manual

BioMed Research International 3

Non-PAH COPD-PH IPAH

Cav1

(pg

mL)

0

100

200

300

400

500p = 0014

p = 0047

(a)Non-PAH COPD-PH IPAH

NT-

proB

NP

(pg

mL)

0

1000

2000

3000

4000

5000

6000

p = 0000

p = 0000

(b)


hsCR

P (m

gdL

)

0

1

2

3

4

5

p = 0017

(c)Non-PAH COPD-PH IPAH

BMPR

2 (p

gm

L)

0

2

4

6

8

10

12

14

p = 0042

(d)

Figure 2 Serumbiomarker levels in PAHpatients and control subjects PAH pulmonary artery hypertension COPD-PH chronic obstructivepulmonary disease with pulmonary hypertension and IPAH idiopathic PAH

23 Statistical Analysis All data were expressed as mean plusmnstandard deviation One-way analysis of variance (ANOVA)was used to compare continuous variables among differentgroups and Mann-Whitney 119880 test was used to comparevariables between groups Optimal thresholds for survivalanalysis were identified using Receiver-Operated Character-istics (ROC) analysis Statistical analysis was performed usingSPSS 18 (SPSS Chicago IL USA)

3 Results

In Table 1 patients in IPAH group were younger than non-PAHvolunteer andCOPD-PHgroup but not significant (119901 =016) There were more female patients (119899 = 14) than malepatients (119899 = 6) in IPAH group The systolic blood pressurewas significantly lower than IPAH groups which may resultfrom right heart failure (119901 = 0002) The PA pressure wassignificantly different between IPAH COPD-PH and non-PAH group (119901 lt 0005)

In Figure 2(a) serum Cav1 level was significantly low inIPAH compared to non-PAH and COPD-PH group (7645 plusmn

3241 versus 14075plusmn5972 pgmL and 17357plusmn4275 pgmL119901 = 0014 and 119901 = 0047) But there was no significantdifference between non-PAH and COPD-PH NT-proBNP(Figure 2(b)) was significantly higher in IPAH and COPD-PH than normal group (93359plusmn21009 and 180638plusmn47407versus 83436 plusmn 2233 pgmL both 119901 lt 005) but there isno difference between IPAH and COPD-PH groups hsCRP(Figure 2(c)) was significantly higher in COPD-PH groupthan non-PAH group (102 plusmn 032 versus 020 plusmn 004mgmL119901 = 0017) but there is no difference between COPD-PH andIPAH group (102 plusmn 032 versus 037 plusmn 015mgmL 119901 gt 05)BMPR2 (Figure 2(d)) was higher in IPAHgroup thanCOPD-PH group (2235plusmn1560 versus 257plusmn099 pgmL119901 = 0019)but there is no significant difference between COPD-PH andnon-PAH group (257 plusmn 099 versus 641 plusmn 327 pgmL 119901 gt05)

In IPAH patients using serum Cav1 1717 pgmL as acutoff value (Table 2) the sensitivity was 059 the specificitywas 10 and area under ROC curve was 0816 (Figure 3(a))Using NT-proBNP 8925 pgmL as a cutoff value the sen-sitivity was 0889 the specificity was 0778 and area under


Table 1 Demographic data patients with pulmonary artery hypertension and healthy controls

Non-PAH (119899 = 27) COPD-PH (119899 = 20) IPAH (119899 = 20) 119901 value

Age yrs 5130 plusmn 1171 (64ndash38) 589 plusmn 1296 (75ndash39) 454 plusmn 1616 (78ndash18) 016Sex (malefemale) 225 164 614 0000Height cm 16194 plusmn 684 (182ndash152) 16063 plusmn 722 (1695ndash144) 15893 plusmn 753 (176ndash147) 0366Weight kg 6665 plusmn 1231 (865ndash495) 6728 plusmn 1250 (94ndash46) 6351 plusmn 1343 (98ndash46) 0578BMI kgm2 2545 plusmn 391 (3376ndash1829) 1616 plusmn 510 (3858ndash1710) 2515 plusmn 564 (427ndash1836) 0791History of DM 5 (185) 5 (250) 0 (0) 068History of HTN 8 (296) 10 (455) 4 (182) 0117PAP peak (mmHg) 1769 plusmn 448 (8ndash258) 4676 plusmn 1274 (35ndash726) 9637 plusmn 3076 (4730ndash169) 0000PAP mean (mmHg) 1236 plusmn 289 (63ndash1880) 3114 plusmn 793 (234ndash484) 5779 plusmn 1487 (2960ndash845) 0000SBP mmHg 13335 plusmn 1601 (170ndash104) 13280 plusmn 2224 (176ndash96) 11320 plusmn 2310 (175ndash86) 0002DBP mmHg 7977 plusmn 960 (101ndash60) 8180 plusmn 1687 (119ndash62) 7780 plusmn 1687 (128ndash54) 0683TC mgdL 17688 plusmn 3535 (236ndash92) 18335 plusmn 4446 (281ndash105) 14625 plusmn 3339 (188ndash103) 0078HDL C mgdL 416 plusmn 931 (59ndash29) 488 plusmn 385 (166ndash6) 524 plusmn 1484 (66ndash33) 0753TG mgdL 14288 plusmn 6920 (373ndash53) 12750 plusmn 9472 (458ndash23) 7988 plusmn 3328 (148ndash38) 0139Creatinine mgdL 108 plusmn 027 (2ndash07) 144 plusmn 075 (44ndash08) 088 plusmn 16 (12ndash06) 0515AC sugar mgdL 10823 plusmn 2636 (177ndash79) 11050 plusmn 4204 (235ndash49) 10875 plusmn 3478 (189ndash83) 0980Caveolin-1 pgmL 17357 plusmn 13518 (4722ndash40944) 16304 plusmn 14659 (5661ndash42554) 3381 plusmn 363 (18ndash235) 0029hsCRP mgdL 018 plusmn 023 (01ndash095) 102 plusmn 130 (013ndash438) 037 plusmn 062 (003ndash275) 0007NT-proBNP pgmL 5983 plusmn 6484 (40ndash336) 1426 plusmn 1231 (140ndash2790) 9336 plusmn 8913 (107ndash2120) 0004PAH pulmonary artery hypertension COPD-PH chronic obstructive pulmonary disease with pulmonary hypertension IPAH idiopathic PAH PAPpulmonary artery pressure SBP systolic blood pressure DBP diastolic blood pressure hsCRP high-sensitivity C-reactive protein and NT-proBNP N-terminal of the prohormone brain natriuretic peptide

Table 2 Sensitivity and specificity data for cutoff point of Cav1 andother biomarkers in IPAH patients

Biomarker Cutoff value Sensitivity SpecificityCav1 1717 pgmL 0588 1NT-proBNP 8925 pgmL 0889 0778hsCRP 027mgdL 0389 0852BMPR2 371 pgmL 1 0429

ROC curve was 089 (Figure 3(b)) Using hsCRP 027mgdLas a cutoff value the sensitivity was 039 the specificity was085 and area under ROC curve was 089 (Figure 3(c)) UsingBMPR2 371 pgmL as a cutoff value the sensitivity was 100the specificity was 043 and area under ROC curve was 078(Figure 3(d)) Linear regression analysis between Cav1 and6min walk test PAP and PVR was done but did not showgood correlation Data were not shown here

4 Discussion

There were two major findings in our results First serumCav1 might be a novel biomarker in the diagnosis of IPAHwith fare sensitivity and good specificity Second Cav1 mightbe used to make differential diagnosis between COPD-PHand IPAH group

Cav1 was highly expressed in vascular endothelial cellsbut less in smooth muscle cells The expression of Cav1 wasdecreased in the plexiform lesion from IAPH patientsrsquo lungtissue [20] The expression in the smooth muscle cell wasincreased and immunoblotting from whole lung preparedrevealed decreased expression of Cav1 [25 26] In this studywe further demonstrate that the serum Cav1 level in IPAHpatients was also decreased (Figure 2(a)) and the differencewas significant between IPAH COPD-PH and normal sub-jects By using serum Cav1 level 1717 pgmL as cutoff valuein the diagnosis of IPAH there were fare sensitivity (06) andgood specificity (10) (Figure 3(a))

In a small number of COPD with PH patients (meanPAP 295 plusmn 51mmHg) the intimal expression of Cav1 wasdecreased as compared with COPD patients without PAH(mean PAP 167 plusmn 27mmHg) [27] Our data did notshow significant difference between COPD-PH and normalsubjects but there was significant difference between COPD-PH and IPAH patients (Figure 2(a) 119901 = 0047) Althoughsmooth muscle proliferation with increasing Cav1 expressionwas noted in both COPD-PH and IPAH patients our resultssuggested the serum Cav1 level correlated with its expressionin endothelial cells but not the smooth muscle cells Our datasuggest that Cav1 may be potential biomarkers for elevatedPA pressure and could be used for differential diagnosis ofCOPD-PH and IPAH


Cav1

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0861p = 0001

1 minus specificity

(a)

NT-proBNP

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0869p = 0000

00 02 04 06 08 101 minus specificity

(b)

hsCRP


Sens

itivi

ty

00

02

04

06

08

10

AUC = 0511p = 0899

(c)


BMPR2

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0781p = 0131

(d)

Figure 3 Receiver operator curve analysis of Cav1 and other biomarkers in idiopathic pulmonary artery hypertension (IPAH) patients

NT-proBNP is secreted by the ventricles of the heart inresponse to excessive stretching of cardiomyocytes SerumNT-proBNP elevated in both left and right ventricle dysfunc-tion [28 29] In COPD patients with PAH and right heartfailure the NT-proBNP was also elevated [30] In our results(Figure 2(b)) NT-proBNP levels were significantly higher inbothCOPD-PAHand IPAHgroups than normal subjects butthere was no significant difference between the disease sub-jects hsCRP a nonspecific biomarker in response to differentpathogenesis of inflammation was higher in COPD patientsdue to chronic lung inflammation (Figure 2(c) 119901 = 0017)than normal group But there was no difference betweenCOPD-PH and IPAH groups Mutations in the BMPR2 generesulted in the development of familial primary pulmonaryhypertension but the role BMPR2 mutations play in thedevelopment of PH has not been clarified Cav1 and BMPR2were colocalized in both endothelial and smooth musclecell membrane [31 32] and Cav1 was suggested to regulate

BMPR2 downstream signaling In this study (Figure 2(d))serum BMPR2 level was not significantly different betweennormal subjects versus IPHA patients and normal subjectsversus COPD-PAH patients But the differences betweenIPAH and COPD with PAH were significantly different (119901 =0019) Further study may be indicated to elucidate therelation between Cav1 and BMPR2 in IPAH patients

Taken together our results demonstrated that reducedserumCav1 level may be a potential biomarker in IPAH diag-nosis and could be used for differential diagnosis of pul-monary artery hypertension patients between idiopathicpulmonary hypertension and COPD

5 Study Limitation

This is a small number cross-sectional study COPD-PH ismore frequent in males IPAH is more frequent in females Itis difficult to correct the match number of patients in gender


The IPAHpatients includedwere at different treatment statusincluding newly diagnosed IPAH without medication todouble or even triple drugs combined therapyThe functionalstatus of heart failure may also influence Cav1 serum levelTherefore we find only poor correlation between Cav1 leveland pulmonary artery pressure pulmonary vascular resis-tance and 6-minute wall test However our results suggestedserum Cav1 level might be used as an easy and convenientway for IAPH initial diagnosis Future studies are necessary toincludemore patients at different stages of disease to evaluateCav1 level in response to different treatment to predict theIPAH progression and long-term prognosis

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

This research was support by Grant nos TCVGH-1013104BTCVGH-1033101A from the Taichung Veterans General Hos-pital and Taichung Veterans General HospitalTunghai Uni-versity Joint Research Program (TCVGH-T1037802)

References

[1] A J Peacock ldquoTreatment of pulmonary hypertensionrdquo BritishMedical Journal vol 326 no 7394 pp 835ndash836 2003

[2] G E DrsquoAlonzo R J Barst SMAyres et al ldquoSurvival in patientswith primary pulmonary hypertension results from a nationalprospective registryrdquo Annals of Internal Medicine vol 115 no 5pp 343ndash349 1991

[3] D B Badesch G E Raskob CG Elliott et al ldquoPulmonary arte-rial hypertension baseline characteristics from the REVEALregistryrdquo Chest vol 137 no 2 pp 376ndash387 2010

[4] N Galie M M Hoeper M Humbert et al ldquoGuidelines for thediagnosis and treatment of pulmonary hypertensionrdquo EuropeanHeart Journal vol 30 pp 2493ndash2537 2009

[5] VVMcLaughlin S L Archer D B Badesch et al ldquoACCFAHA2009 expert consensus document on pulmonary hypertensiona report of the American College of Cardiology FoundationTask Force on Expert Consensus Documents and the Amer-ican Heart Association developed in collaboration with theAmerican College of Chest Physicians American ThoracicSociety Inc and the Pulmonary Hypertension AssociationrdquoCirculation vol 119 pp 2250ndash2294 2009

[6] M D McGoon R L Benza P Escribano-Subias et al ldquoPul-monary arterial hypertension epidemiology and registriesrdquoJournal of the American College of Cardiology vol 62 no 25supplement pp D51ndashD59 2013

[7] G Habib and A Torbicki ldquoThe role of echocardiography inthe diagnosis and management of patients with pulmonaryhypertensionrdquo European Respiratory Review vol 19 no 118 pp288ndash299 2010

[8] D B Badesch H C Champion M A Gomez Sanchez et alldquoDiagnosis and assessment of pulmonary arterial hyperten-sionrdquo Journal of the American College of Cardiology vol 54 no1 pp S55ndashS66 2009

[9] NNagaya T NishikimiMUematsu et al ldquoPlasma brain natri-uretic peptide as a prognostic indicator in patients with primarypulmonary hypertensionrdquo Circulation vol 102 no 8 pp 865ndash870 2000

[10] N Nagaya M Uematsu T Satoh et al ldquoSerum uric acid levelscorrelate with the severity and the mortality of primary pul-monary hypertensionrdquo American Journal of Respiratory andCritical Care Medicine vol 160 no 2 pp 487ndash492 1999

[11] J-L Cracowski andHH Leuchte ldquoThe potential of biomarkersin pulmonary arterial hypertensionrdquo The American Journal ofCardiology vol 110 no 6 pp S32ndashS38 2012

[12] S Rafeq A M Shah and I R Preston ldquoBiomarkers in pul-monary arterial hypertensionrdquo International Journal of ClinicalPractice vol 63 no 162 pp 36ndash41 2009

[13] M Humbert N W Morrell S L Archer et al ldquoCellular andmolecular pathobiology of pulmonary arterial hypertensionrdquoJournal of the American College of Cardiology vol 43 no 12supplement pp 13Sndash24S 2004

[14] S Sakao K Tatsumi and N F Voelkel ldquoEndothelial cells andpulmonary arterial hypertension apoptosis proliferationinteraction and transdifferentiationrdquo Respiratory Research vol10 article 95 2009

[15] M R Wilkins ldquoPulmonary hypertension the science behindthe disease spectrumrdquo European Respiratory Review vol 21 no123 pp 19ndash26 2012

[16] M Drab P Verkade M Elger et al ldquoLoss of caveolae vas-cular dysfunction and pulmonary defects in caveolin-1 gene-disruptedmicerdquo Science vol 293 no 5539 pp 2449ndash2452 2001

[17] L A Carver and J E Schnitzer ldquoCaveolae mining little cavesfor new cancer targetsrdquoNature Reviews Cancer vol 3 no 8 pp571ndash581 2003

[18] Y-Y Zhao Y Liu R-V Stan et al ldquoDefects in caveolin-1cause dilated cardiomyopathy and pulmonary hypertension inknockoutmicerdquo Proceedings of the National Academy of Sciencesof the United States of America vol 99 no 17 pp 11375ndash113802002

[19] J-F Jasmin I Mercier J Dupuis H B Tanowitz and MP Lisanti ldquoShort-term administration of a cell-permeablecaveolin-1 peptide prevents the development of monocrotaline-induced pulmonary hypertension and right ventricular hyper-trophyrdquo Circulation vol 114 no 9 pp 912ndash920 2006

[20] R O D Achcar Y Demura P R Rai et al ldquoLoss of caveolin andheme oxygenase expression in severe pulmonary hypertensionrdquoChest vol 129 no 3 pp 696ndash705 2006

[21] Y-Y Zhao Y D ZhaoM KMirza et al ldquoPersistent eNOS acti-vation secondary to caveolin-1 deficiency induces pulmonaryhypertension in mice and humans through PKG nitrationrdquoJournal of Clinical Investigation vol 119 no 7 pp 2009ndash20182009

[22] P Liu W-P Li T Machleidt and R G W Anderson ldquoIdentifi-cation of caveolin-1 in lipoprotein particles secreted by exocrinecellsrdquo Nature Cell Biology vol 1 no 6 pp 369ndash375 1999

[23] S A Tahir S Kurosaka R Tanimoto A A Goltsov S Parkand T C Thompson ldquoSerum caveolin-1 a biomarker of drugresponse and therapeutic target in prostate cancer modelsrdquoCancer Biology andTherapy vol 14 no 2 pp 117ndash126 2013

[24] S A Tahir C Ren T L Timme et al ldquoDevelopment of animmunoassay for serum caveolin-1 a novel biomarker forprostate cancerrdquo Clinical Cancer Research vol 9 part 1 no 10pp 3653ndash3659 2003


[25] H H Patel S Zhang F Murray et al ldquoIncreased smoothmuscle cell expression of caveolin-1 and caveolae contribute tothe pathophysiology of idiopathic pulmonary arterial hyperten-sionrdquoThe FASEB Journal vol 21 no 11 pp 2970ndash2979 2007

[26] R Mathew ldquoCell-specific dual role of caveolin-1 in pulmo-nary hypertensionrdquo Pulmonary Medicine vol 2011 Article ID573432 12 pages 2011

[27] L C Huber A Soltermann M Fischler et al ldquoCaveolin-1expression and hemodynamics in COPD patientsrdquo Open Res-piratory Medicine Journal vol 3 pp 73ndash78 2009

[28] J L Januzzi Jr S U Rehman A A Mohammed et al ldquoUse ofamino-terminal pro-B-type natriuretic peptide to guide outpa-tient therapy of patients with chronic left ventricular systolicdysfunctionrdquo Journal of the American College of Cardiology vol58 no 18 pp 1881ndash1889 2011

[29] N Nagaya T Nishikimi Y Okano et al ldquoPlasma brain natri-uretic peptide levels increase in proportion to the extent of rightventricular dysfunction in pulmonary hypertensionrdquo Journal ofthe American College of Cardiology vol 31 no 1 pp 202ndash2081998

[30] S Y Chi E Y Kim H J Ban et al ldquoPlasma N-terminal pro-brain natriuretic peptide a prognostic marker in patients withchronic obstructive pulmonary diseaserdquo Lung vol 190 no 3pp 271ndash276 2012

[31] J W Wertz and P M Bauer ldquoCaveolin-1 regulates BMPRIIlocalization and signaling in vascular smooth muscle cellsrdquoBiochemical and Biophysical Research Communications vol 375no 4 pp 557ndash561 2008

[32] M Ramos M W Lame H J Segall and D W Wilson ldquoTheBMP type II receptor is located in lipid rafts including caveolaeof pulmonary endothelium in vivo and in vitrordquo VascularPharmacology vol 44 no 1 pp 50ndash59 2006

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Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


Caveolae are 50ndash100 nm vesicular invaginations of thecell plasma membrane and caveolin-1 (Cav1) is the structuralprotein of caveolae and is highly expressed in adipocytesendothelial cells and type I pneumocytes Cav1minusminus miceexhibit pulmonary hypertension and right ventricle hyper-trophy [16ndash18] In monocrotaline-induced PH rat modelsCav1 deficiency is seen in lung tissue [19] Lungs tissues fromidiopathic PAH (IPAH) patients decreased expression of Cav1in vascular endothelial cells and also decreased in the totallung lysate [20 21] Furthermore Cav1 can be secreted intoserum and be detected [22]These results suggested that Cav1may play an important role in the pathogenesis of PAH andserum Cav1 level may be a good biomarker for diagnosis[23 24]

2 Materials and Methods

In the study age matched patients with normal left ventriclefunction were divided into 3 groups In Group (1) IPAHpatients (119899 = 21) definite diagnosis was made accordingto European Society of Cardiology (ECS) [4] and Ameri-can Heart Association (AHAACC) [5] guideline inclusioncriteria including mean pulmonary artery pressure (mPAP)ge 25mmHg pulmonary wedge pressure less or equal to15mmHg and pulmonary vascular resistance over 3 Woodunits measured by right heart catheterization In Group(2) chronic obstructive pulmonary disease with pulmonaryhypertension (COPD-PH) patients (119899 = 22) COPD werediagnosed by pulmonologist and estimated mean PAP ge25mmHg by echocardiography Group (3) (non-PAH group)includes healthy volunteers (119873 = 26) with mPAP less than15mmHg measured by echocardiography

Demographic data and clinical features of patientsincluded in this studywere summarized in Table 1 Accordingto Tahir et al reports [23 24] we developed a direct sandwichimmunoassay for the determination serum Cav1 level fromparticipants Serum hsCRP NT-proBNP and BMPR2 levelswere also measured by commercial ELISA kits This studywas approved by Local Ethical Committee in Taichung Veter-ans General Hospital Taichung Taiwan (number CE12022)Written informed consent was provided to all participants

21 Protocol for Serum Cav1 Assay Two commercial affinitypurified monoclonal mouse Cav1 antibodies and polyclonalrabbit Cav1 antibodies were chosen for a direct sandwichELISA The capture Cav1 antibody used was generated fromhuman recombinant Cav1 (RampD systems) and the detectionantibody was HRP-conjugated rabbit polyclonal antibodyraised against a peptide mapping at the NH

2

terminus ofhuman Cav1 (Santa Cruz Biotechnology) Costar microplatewells were coated with 100 120583L of Cav1 antibody (25mgL) inPBS (pH 74) and incubated overnight at 4∘C The wells werethen blocked with 300 120583L of PBS containing 05 BSA and005 vv Tween 20 for 2 hours at room temperature andwerewashed three timeswith PBS containing 05 vv Tween20 (PBST) Serum samples calibrators and controls wereadded (100 120583L) to the wells and incubated overnight at 4∘CThe wells were washed three times with 400 120583L of PBST and100 120583L of HRP-conjugated Cav1 (Santa Cruz Biotechnology)

I II III

M

(kD

a)

rCav1rCav1rCav1

100

72

55

40

35

25

15

Humanserum

Figure 1 Specificity of capture and detection antibodies to rCav-1and native Cav1 of human serum Recombinant Cav1 protein waspurified using affinity chromatography and analyzed by SDS-PAGE(I) and immunoblotting (II and III) In panel I the rCav-1 proteinmigrated as a single band and displayed gt95 purity by Coomassieblue staining The binding specificity of the capture and detectionantibodies to rCav-1 and human serum was demonstrated andshowed in panel II and panel III respectively Numbers on the leftindicate sizes of protein markers (lane M)

antibody diluted 1 2000 in PBST After incubation for 2 hrat room temperature the wells were washed three times withPBST and 100 120583L of 331015840551015840-tetramethylbenzidine substratesolution (Clinical Science Products Inc) was added andincubated for 30min at room temperature The reaction wasstopped by adding 100 120583L of 1NH

3

PO4

and the absorbancewas read at 450 nm with a microplate reader (TECANGrodig Austria) SerumCav1 levels weremeasured using lab-made recombinant pQE30-Cav 1 (a 101-amino acid region ofCav1 gene from GenBank accession number NM001753) asa standard A linear standard curve was constructed using aconcentration range (1219ndash780 pgmL) of recombinant Cav1in a parallel ELISA The levels of Cav1 in sera of 21 IPAHpatients 22 COPD-PH patients and 26 healthy controlsby the in-house ELISA were then measured The limit ofdetection of the sandwich ELISA was 1219 pgmL Any valuebelow the detective limitation of the assay referred to zeroIn Figure 1 Western blot data using capture and detectionantibodies that react to recombinant Cav1 proteins wasshowed

22 Enzyme-Linked Immunosorbent Assay for HsCRP NT-proBNP and BMPR2 Detection The serum levels of high-sensitivity C-reactive protein (hsCRP) NT-proBNP andbone morphogenetic protein type II receptor (BMPR2) weremeasured with enzyme-linked immunosorbent assay (ELISA)kits hsCRP (Cell Biolabs Inc San Diego CA) NT-proBNP(Invitrogen Corporation Camarillo CA) and BMPR2(MyBioSource San Diego CA) according to the manual



Cav1

(pg

mL)

0

100

200

300

400

500p = 0014

p = 0047


NT-

proB

NP

(pg

mL)

0

1000

2000

3000

4000

5000

6000

p = 0000

p = 0000

(b)


hsCR

P (m

gdL

)

0

1

2

3

4

5

p = 0017


BMPR

2 (p

gm

L)

0

2

4

6

8

10

12

14

p = 0042

(d)



3 Results












4 Discussion





Cav1

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0861p = 0001

1 minus specificity

(a)

NT-proBNP

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0869p = 0000


(b)

hsCRP


Sens

itivi

ty

00

02

04

06

08

10

AUC = 0511p = 0899

(c)


BMPR2

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0781p = 0131

(d)





5 Study Limitation






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















Cav1

(pg

mL)

0

100

200

300

400

500p = 0014

p = 0047


NT-

proB

NP

(pg

mL)

0

1000

2000

3000

4000

5000

6000

p = 0000

p = 0000

(b)


hsCR

P (m

gdL

)

0

1

2

3

4

5

p = 0017


BMPR

2 (p

gm

L)

0

2

4

6

8

10

12

14

p = 0042

(d)



3 Results












4 Discussion





Cav1

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0861p = 0001

1 minus specificity

(a)

NT-proBNP

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0869p = 0000


(b)

hsCRP


Sens

itivi

ty

00

02

04

06

08

10

AUC = 0511p = 0899

(c)


BMPR2

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0781p = 0131

(d)





5 Study Limitation






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























4 Discussion





Cav1

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0861p = 0001

1 minus specificity

(a)

NT-proBNP

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0869p = 0000


(b)

hsCRP


Sens

itivi

ty

00

02

04

06

08

10

AUC = 0511p = 0899

(c)


BMPR2

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0781p = 0131

(d)





5 Study Limitation






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cav1

00 02 04 06 08 10

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0861p = 0001

1 minus specificity

(a)

NT-proBNP

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0869p = 0000


(b)

hsCRP


Sens

itivi

ty

00

02

04

06

08

10

AUC = 0511p = 0899

(c)


BMPR2

Sens

itivi

ty

00

02

04

06

08

10

AUC = 0781p = 0131

(d)





5 Study Limitation






Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Acknowledgments


References







































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














