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Title: Increased circulating tissue inhibitor of metalloproteinase-2 is associated with resistant hypertension.
Short title: TIMP-2 and resistant hypertension.
Authors: Andrea R. SABBATINI a, PharmD, MSc; Natalia R. BARBARO a, PharmD, MSc; Ana Paula de FARIAa, PharmD, PhD; Rodrigo MODOLO a, MD, PhD; Alessandra Mileni V. RITTER a, PharmD, PhD; Claudio PINHO b, MD, PhD; Rivadavio Fernandes Batista AMORIM c, DDS, MSc, PhD ; Vanessa FONTANAa, PharmD, PhD; Heitor MORENO a, MD, PhD.
Affiliations:
a Laboratory of Cardiovascular Pharmacology, Faculty of Medical Sciences and Teaching Hospital University of Campinas (Unicamp), Campinas, SP, Brazil.
b Faculty of Medicine - Pontifical Catholic University of Campinas (Puccamp), Campinas, SP, Brazil.
c Laboratory of Neuromodulation & Center for Clinical Research Learning, Department of Physical Medicine and Rehabilitation (PM&R), Spaulding Rehabilitation Hospital, Harvard Medical School, Boston, USA.
Corresponding author:
Heitor Moreno, M.D., PhD.
Laboratory of Cardiovascular Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, SP, Brazil.
Phone: +55 19 3521 9538; email: HYPERLINK "mailto:hmoreno@uol.com.br" hmoreno@uol.com.br
Word count: 2873
Number of tables: 07
Number of figures: 02
ABSTRACT
Resistant hypertension (RH) is associated with organ damage and cardiovascular risk (CV). Evidences suggest the involvement of matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase 2 (TIMP-2) in hypertension and in CV remodeling. The aim of this study was assess the levels of MMP-2 and TIMP-2 in resistant hypertension and its relation with organ damage, including arterial stiffness and cardiac hypertrophy. MMP-2 and TIMP-2 levels were compared among 19 normotensive (NT), 116 non-resistant hypertensive (HT) and 116 resistant hypertensive (RH) subjects. MMP-2 levels showed no differences among NT, HT and RH groups, while TIMP-2 levels were higher in RH compared to HT and NT (90.0 (76.1-107.3) vs. 70.1 (57.7-88.3) vs.54.7 (40.9-58.1)ng/mL, p<0.01), respectively. MMP-2/TIMP-2 ratio was reduced in RH group compared to HT and NT subjects (2.7 (1.9-3.4) vs.3.3 (2.6-4.2) vs.4.9 (4.5-5.3), p<0.01), respectively. No associations were found between MMP-2 levels, TIMP-2 and MMP-2/TIMP-2 ratio with cardiac hypertrophy and arterial stiffness in resistant and non-resistant group. However TIMP-2 levels were associated with LVMI in total hypertensive group (r= 0.16, p=0.02). Finally, in a regression analysis reduced MMP-2/TIMP-2 ratio and increased TIMP-2 levels were independently associated with RH. The present findings provide evidence that TIMP-2 is associated with resistant hypertension and might be a possible biomarker for screening resistant hypertension subjects.
Keywords: Resistant Hypertension, Matrix Metalloproteinases 2, TissueInhibitorof Metalloproteinases 2, Organ Damage.
INTRODUCTION
Resistant hypertension (RH) is a multifactorial condition associated with mortality HYPERLINK \l "_ENREF_1" \o "Fatemi, 2016 #180" ADDIN EN.CITE Fatemi201618018018017Fatemi, O.Goa, C.Faselis, C.Kokkinos, P.Papademetriou, V.George Washington University Hospital, Washington, DC.
Georgetown University Hospital, Washington, DC.
Veterans Affairs Medical Center, Washington, DC.
Washington Hospital Center, Washington, DC.
George Washington University School of Medicine, Washington, DC.
Georgetown University School of Medicine, Washington, DC.
Washington DC VA Medical Center, Washington, DC.Improvement in All-Cause Mortality With Blood Pressure Control in a Group of US Veterans With Drug-Resistant HypertensionJ Clin Hypertens (Greenwich)33-91812015/10/072016Jan1751-7176 (Electronic)
1524-6175 (Linking)26440866http://www.ncbi.nlm.nih.gov/pubmed/2644086610.1111/jch.12672eng1 HYPERLINK \l "_ENREF_1" \o "Fatemi, 2016 #180" , greater risk of cardiovascular events HYPERLINK \l "_ENREF_2" \o "Daugherty, 2012 #3" ADDIN EN.CITE Daugherty201233317Daugherty, S. L.Powers, J. D.Magid, D. J.Tavel, H. M.Masoudi, F. A.Margolis, K. L.O'Connor, P. J.Selby, J. V.Ho, P. M.Division of Cardiology, University of Colorado-Denver, 12605 E 16th Ave., Aurora, CO 80045, USA. stacie.daugherty@ucdenver.eduIncidence and prognosis of resistant hypertension in hypertensive patientsCirculationCirculation1635-42125132012/03/02Cohort StudiesFemaleFollow-Up StudiesHumansHypertension/*diagnosis/*epidemiology/therapyIncidenceMaleMiddle AgedPrognosisRetrospective Studies2012Apr 31524-4539 (Electronic)
0009-7322 (Linking)22379110Comparative Study
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.http://www.ncbi.nlm.nih.gov/pubmed/22379110334363510.1161/CIRCULATIONAHA.111.068064eng2 and higher incidence of target organ damage ADDIN EN.CITE ADDIN EN.CITE.DATA 3-4. Target organ damage in RH is closely associated with the lack of blood pressure (BP) control and cardiovascular remodeling ADDIN EN.CITE ADDIN EN.CITE.DATA 3, 5. It requires the activation of intracellular pathways that lead to (i) proliferation, migration and apoptosis of cells and (ii) synthesis and degradation of extracellular matrix (ECM) proteins of cardiac and vascular tissues HYPERLINK \l "_ENREF_6" \o "Intengan, 2001 #28" ADDIN EN.CITE ADDIN EN.CITE.DATA 6-8.
Closely linked to the ECM reorganization, the matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases that degrade several proteins at cardiac and vascular tissues. The activity of the MMPs is affected by endogenous inhibitors, such as tissue inhibitors of metalloproteinases (TIMP) and alpha-2-macroglobulin (A2M), a non-specific inhibitor of circulating proteases. Therefore, the balance between MMPs and inhibitors are essential for the extracellular matrix remodeling in tissues HYPERLINK \l "_ENREF_9" \o "Nagase, 2006 #53" ADDIN EN.CITE Nagase200653535317Nagase, H.Visse, R.Murphy, G.Department of Matrix Biology, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, London W6 8LH, UK. h.nagase@imperial.ac.ukStructure and function of matrix metalloproteinases and TIMPsCardiovasc ResCardiovascular research562-736932006/01/13AnimalsCardiovascular Diseases/enzymologyCardiovascular System/*enzymologyExtracellular Matrix/*enzymologyHumansMatrix Metalloproteinase InhibitorsMatrix Metalloproteinases/*chemistryStructure-Activity RelationshipTissue Inhibitor of Metalloproteinases/*chemistry/metabolism2006Feb 150008-6363 (Print)
0008-6363 (Linking)16405877Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1640587710.1016/j.cardiores.2005.12.002eng9.
Gelatinases, such as MMP-2 and MMP-9, have been associated with hypertension and target organ damage ADDIN EN.CITE ADDIN EN.CITE.DATA 10-11. MMP-2 expression is up-regulated in cardiac cells in response to many elements such as angiotensin II, endothelin-1, pro-inflammatory cytokines, hormones and growth factors HYPERLINK \l "_ENREF_12" \o "Kandasamy, 2010 #169" ADDIN EN.CITE Kandasamy201016916916917Kandasamy, A. D.Chow, A. K.Ali, M. A.Schulz, R.Department of Pediatrics and Pharmacology, Cardiovascular Research Centre, 4-62 Heritage Medical Research Centre, University of Alberta, Edmonton, AB, Canada T6G 2S2.Matrix metalloproteinase-2 and myocardial oxidative stress injury: beyond the matrixCardiovasc ResCardiovascular research413-238532009/08/07AnimalsApoptosisCalpain/physiologyHumansMatrix Metalloproteinase 2/chemistry/genetics/*physiologyMyocardium/*metabolism*Oxidative StressPhosphorylationProtease Inhibitors/pharmacologyTissue Inhibitor of Metalloproteinase-1/physiology2010Feb 11755-3245 (Electronic)
0008-6363 (Linking)19656780Research Support, Non-U.S. Gov't
Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1965678010.1093/cvr/cvp268eng12. It has a role in cleaving ECM molecules, elastin and type IV collagen and digests interstitial collagen types I, II and II HYPERLINK \l "_ENREF_9" \o "Nagase, 2006 #53" ADDIN EN.CITE Nagase200653535317Nagase, H.Visse, R.Murphy, G.Department of Matrix Biology, Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, 1 Aspenlea Road, London W6 8LH, UK. h.nagase@imperial.ac.ukStructure and function of matrix metalloproteinases and TIMPsCardiovasc ResCardiovascular research562-736932006/01/13AnimalsCardiovascular Diseases/enzymologyCardiovascular System/*enzymologyExtracellular Matrix/*enzymologyHumansMatrix Metalloproteinase InhibitorsMatrix Metalloproteinases/*chemistryStructure-Activity RelationshipTissue Inhibitor of Metalloproteinases/*chemistry/metabolism2006Feb 150008-6363 (Print)
0008-6363 (Linking)16405877Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Reviewhttp://www.ncbi.nlm.nih.gov/pubmed/1640587710.1016/j.cardiores.2005.12.002eng9. In addition, MMP-2 is highly expressed in almost all cardiac cells and can be activated by oxidative stress HYPERLINK \l "_ENREF_13" \o "Viappiani, 2009 #178" ADDIN EN.CITE Viappiani200917817817817Viappiani, S.Nicolescu, A. C.Holt, A.Sawicki, G.Crawford, B. D.Leon, H.van Mulligen, T.Schulz, R.Cardiovascular Research Group, Departments of Pediatrics and Pharmacology, University of Alberta, Edmonton, AB T6G2S2, Canada.Activation and modulation of 72kDa matrix metalloproteinase-2 by peroxynitrite and glutathioneBiochem PharmacolBiochemical pharmacology826-347752008/12/03Amino Acid SequenceEnzyme ActivationGlutathione/*pharmacologyHumansMatrix Metalloproteinase 2/chemistry/*metabolismMolecular Sequence DataOxidative StressPeroxynitrous Acid/*pharmacologySpectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationTandem Mass Spectrometry2009Mar 11873-2968 (Electronic)
0006-2952 (Linking)19046943Research Support, Non-U.S. Gov'thttp://www.ncbi.nlm.nih.gov/pubmed/1904694310.1016/j.bcp.2008.11.004eng13. Therefore, MMP-2 is one of the most studied MMPs in essential hypertension. Nonetheless, the relation between MMP-2 and TIMP-2 levels in RH remains unclear. The aim of this study was assess the levels of MMP-2 and TIMP-2 in RH and its association with target-organ damage (arterial stiffness and cardiac hypertrophy).
METHODS
Subjects and Study Design
For this cross-sectional study, resistant hypertensive (RH) patients regularly followed at the Resistant Hypertension Clinic of the University of Campinas (Campinas, Brazil) and non-resistant hypertensives (HT), followed at the Hypertension Clinic of Valinhos (Brazil) were screened. The study was conducted at the aforementioned center, reviewed and approved by the IRB (IRB from the School of Medical Sciences of University of Campinas, Campinas-Brazil/ Approval number 188.161/2013) and conducted in accordance with the Declaration of Helsinki Principles. In addition, written informed consent was obtained from all participants.
Inclusion and exclusion criteria
The inclusion criteria were: i) Diagnosis of non-resistant or resistant hypertension HYPERLINK \l "_ENREF_14" \o "Mancia, 2013 #172" ADDIN EN.CITE ADDIN EN.CITE.DATA 14; ii) Age between 18-65 years; iii) Volunteer participation and informed consent signature; iv) Regular follow-up for at least six months; v) Previous proven adherence to non-pharmacological and pharmacological treatment; vi) Women in reproductive phase must be under use of contraception methods.
After 6-months of follow-up, patients were properly diagnosed as having RH or HT. RH was defined as BP that remained above the target pressure ( e"1 4 0 / 9 0 m m H g ) , d e s p i t e t h e u s e o f t h r e e o r m o r e d i f f e r e n t c l a s s e s o f a n t i h y p e r t e n s i v e d r u g s a t o p t i m a l d o s e s o r c o n t r o l l e d B P w i t h t h e u s e o f f o u r o r m o r e a n t i h y p e r t e n s i v e d r u g s H Y P E R L I N K \ l " _ E N R E F _ 1 5 " \ o " C a l h o u n , 2 0 0 8 # 1 2 " A D D I N E N . C I T E A D D I N E N .CITE.DATA 15. To identify true resistance to treatment and exclude white-coat hypertension, pseudoresistance, lack of medication adherence, and secondary forms of hypertension, RH subjects were submitted to drug therapy optimization and pill counts, office blood pressure (BP), ambulatory blood pressure monitoring (ABPM) measurements HYPERLINK \l "_ENREF_15" \o "Calhoun, 2008 #12" ADDIN EN.CITE ADDIN EN.CITE.DATA 15, imaging and biochemical exams. The HT subjects were defined as all hypertensive subjects without resistance to treatment HYPERLINK \l "_ENREF_14" \o "Mancia, 2013 #172" ADDIN EN.CITE ADDIN EN.CITE.DATA 14.
In regard to the exclusion criteria, the following point were taken into account: i) Medical history or clinical symptoms of heart failure (ejection fraction < 50%); ii) Presence of cardiomyopathy; iii) Valvular or pericardial heart diseases; iv) Diagnosis of cerebrovascular disease or peripheral vascular disease; v) Nephropathy; vi) Liver dysfunction; vii) Autoimmune diseases; viii) Pregnancy or declared intention to become pregnant.
All hypertensive volunteers were recruited consecutively and were divided into two groups: HT (n=116) and RH (n=116), according to the above-mentioned criteria (Figure 1). Nineteen normotensive subjects (NT) were randomly selected to be included as the control group (n=19).
Blood pressure measurements
To determine office systolic BP (SBP) and diastolic BP (DBP) a trained health professional performed at least three measurements with a 3-minute interval. Subjects were monitored in a seated position after a 10-minute rest, approximately at 8:00 a.m., using a digital BP monitor (OMRON Healthcare Inc., Bannockburn, IL, USA).
ABPM was used to exclude pseudoresistance hypertension through twenty-four hour BP monitoring by an automatic oscillometric device (Spacelabs 90207, Spacelabs Inc, Redmon, WA, USA). BP was measured automatically at a 20-min interval during 24-h period and subjects were instructed to keep their normal daily activities and take notes of their sleep period in a personal diary HYPERLINK \l "_ENREF_16" \o "Zakopoulos, 2001 #179" ADDIN EN.CITE Zakopoulos200117917917917Zakopoulos, N. A.Nanas, S. N.Lekakis, J. P.Vemmos, K. N.Kotsis, V. T.Pitiriga, V. C.Stamatelopoulos, S. F.Moulopoulos, S. D.Department of Clinical Therapeutics, Alexandra Hospital, University of Athens, National and Kapodestrial University, Athens, Greece.Reproducibility of ambulatory blood pressure measurements in essential hypertensionBlood Press MonitBlood pressure monitoring41-5612001/03/15AgedAnalysis of VarianceBlood PressureBlood Pressure Monitoring, Ambulatory/*standards/statistics & numerical dataCircadian Rhythm/physiologyFemaleHeart RateHumans*HypertensionMaleMiddle AgedReproducibility of Results2001Feb1359-5237 (Print)
1359-5237 (Linking)11248760http://www.ncbi.nlm.nih.gov/pubmed/11248760eng16.
Aortic pulse wave velocity measurement
Pulse wave velocity (PWV) measurements were obtained through Sphygmocor system (Artcor, Sidney, Australia) and used to estimate arterial stiffness. The pulse waves were obtained via transcutaneous using a tonometer at the right common carotid and femoral arteries. PWV was calculated as the ratio of the distance (meters) / t ( s e c o n d s ) b e t w e e n t h e f e m o r a l r e c o r d i n g s i t e a n d t h e s u p r a - s t e r n a l n o t c h m i n u s t h e d i s t a n c e f r o m t h e s u p r a - s t e r n a l n o t c h t o t h e c a r o t i d r e c o r d i n g s i t e H Y P E R L I N K \ l " _ E N R E F _ 1 7 " \ o " V a n B o r t e l , 2 0 1 2 # 5 8 " A D D I N E N . C I T E <