Obesity Downregulates MicroRNA-126 Inducing Capillary Rarefaction …downloads.hindawi.com/journals/omcl/2017/2415246.pdf · 2019. 7. 30. · ResearchArticle Obesity Downregulates

Research ArticleObesity Downregulates MicroRNA-126 InducingCapillary Rarefaction in Skeletal Muscle Effects ofAerobic Exercise Training

Joatildeo Lucas Penteado Gomes Tiago FernandesUrsula Paula Reno Soci Andreacute Casanova Silveira Diego Lopes Mendes BarrettiCarlos Eduardo Negratildeo and Edilamar Menezes Oliveira

School of Physical Education and Sport University of Sao Paulo Sao Paulo SP Brazil

Correspondence should be addressed to Edilamar Menezes Oliveira edilamaruspbr

Received 14 December 2016 Accepted 5 February 2017 Published 6 March 2017

Academic Editor Ryuichi Morishita

Copyright copy 2017 Joao Lucas Penteado Gomes et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

BackgroundWe investigated the effects of exercise training (ET) onmiR-126 levels and skeletalmuscle angiogenesis in obese Zuckerrats Results Zucker rats were randomly assigned to sedentary and swimming-trained groups lean sedentary (LS) and trained(LTR) obese sedentary (OB) and trained (OBTR) The OB group displayed capillary rarefaction compared with the LS group Incontrast ET increased the capillaryfiber ratio by 38 in the LTR group and normalized capillary rarefaction in the OBTR groupVEGF PI3K and eNOS levels were reduced in the skeletal muscle of the OB group ET normalized VEGF PI3K and eNOS levelsin OBTR contributing to vascular network homeostasis PI3KR2 inhibits PI3K a key mediator of the VEGF signaling pathwayObesity decreased miR-126 and increased PI3KR2 levels compared with the LS group However ET normalized miR-126 levelsin the OBTR group versus the LS group and decreased expression of PI3KR2 Conclusion Our findings show that obesity leads toskeletalmuscle capillary rarefaction which is regulated by decreasedmiR-126 levels and increased PI3KR2 Inversely ET normalizesmiR-126 levels and VEGF signaling and should be considered an important therapeutic strategy for vascular disorders

1 Introduction

Obesity is a chronic disease caused by an excess of bodyfat this leads to a number of systemic body changes thatpredispose to the development of other diseases such as dia-betes hypertension dyslipidemia and cancers The obesityepidemic has continues to increase and now affect about 13of the global population and is a major global public healthproblem [1ndash3]

Obesity causes deleteriousmorphological changes in ske-letal muscle for example capillary rarefaction which oftenoccurs in vascular diseases caused by inflammation and anabnormal lipid profile [4ndash6] In addition obesity alters thecontent of intramuscular fat and deregulates the expressionof several proteins related to angiogenesis and trophism [7ndash9] In contrast exercise training (ET) is an important non-pharmacological therapeutic strategy for the treatment of

chronic diseases ET promotes beneficial morphological cha-nges in many tissues and body systems such as an increasein capillary density increased diameter of muscle fibers anda reduction in intramuscular fat content [9ndash12] However theeffects of ET on the molecular mechanisms of obese skeletalmuscle capillary rarefaction are poorly understood

The microRNAs (miRs) have been widely studied asregulators of gene translation and thus protein content MiRsare small noncoding RNA molecules (containing about 17sim22 nucleotides) that exert functions such as RNA silencingand posttranscriptional regulation of gene expression Theseeffects occur bymiR coupling tomRNA in the 31015840 untranslatedseed region thereby preventing its translation [13ndash15]

MiR-126 controls angiogenesis and regulates the for-mation survival and maintenance of new vessels [16ndash18] Phosphatidylinositol 3-kinase regulatory subunit beta(PI3KR2) is one of the main targets of miR-126 and controls

HindawiOxidative Medicine and Cellular LongevityVolume 2017 Article ID 2415246 9 pageshttpsdoiorg10115520172415246

2 Oxidative Medicine and Cellular Longevity

the vascular endothelial growth factor (VEGF) signalingpathway via direct inhibition of phosphatidylinositol 3-kinase(PI3K 120572-110) Thus miR-126 inhibits PI3KR2 and promotesangiogenesis through an indirect increase in PI3K andVEGFThrough this process the expression of proteins in the nitricoxide synthase (eNOS) pathway is activated which alsoenhances angiogenesis Knockdown of miR-126 in animalsleads to impaired vascular formation due to a reductionin the migration of endothelial cells during the genesis ofvessels this compromises blood vessel integrity and leads tohemorrhage in the embryonic period [18]

ET regulates the expression of various miRs that regu-late biological processes related to adaptation [17 19] Theexpression of miR-126 increases in healthy trained rats [19]and is severely decreased in rats with chronic diseases suchas hypertension [17] However ET has been shown to restoremiR-126 levels in the skeletal muscle of hypertensive ratsand induces angiogenesis [17] However the effects of ET interms of controlling the expression of miRs in skeletal musclein the context of obesity need further investigation In thepresent study we found that reduced miR-126 levels inducedcapillary rarefaction in skeletal muscle by targeting PI3KR2in obese Zucker rats ET counteracted this effect by restoringthe expression of miR-126 and skeletal muscle capillarity inobese Zucker rats

2 Methods

21 Experimental Group Twenty-one Zucker rats were ran-domly divided into four groups sedentary lean (LS 119899 = 5)trained lean (LTR 119899 = 5) obese sedentary (OB 119899 = 5)and obese trained (OBTR 119899 = 6) The animals were housed3 to 5 per cage in a room heated between 22∘C and 24∘Cand were on an inverted light-dark cycle water and foodwere given ad libitum After the training protocol animalswere euthanized and the relevant tissues were dissectedand weighed All procedures were performed according tothe Ethical Principles of Animal Experimentation and theapproved by the Ethics Committee on Animal Use at theUniversity of Sao Paulo

22 Swimming Training Protocol Swimming training wasperformed according to the protocol developed by Medeirosand colleagues [20] in a swimming system with water at30ndash32∘C The training duration was 10 weeks with 5 weeklysessions with a progressive increase in time reaching 60min-utes andwith a progressive increase inworkload reaching 4of the body weight of the animal

23 Quantification of Intramuscular Lipids by Oil Red OSerial sections of skeletal muscle (soleus) were preparedat a thickness of 10 120583m Muscle sections were fixed in37 formaldehyde and stained with Oil Red O workingsolution (500mg of Oil Red O added to 100ml of aqueoussolution with 60 triethyl phosphate (Fluka) and 8ml ofdeionized water) The final product of this procedure is red-stained lipids The intramuscular fat content was analyzedin the soleus muscle in intact cellular membranes with acomputer-assisted morphometric system (Leica Quantimet

500 Cambridge UK) able tomeasure the redwavelength Foreach animal approximately 10 visual fields were analyzed

24 Oxygen Uptake Measurements Oxygen uptake (O2) was

measured by expired gas analysis during graded treadmillexercise training Gas analysis was performed using anoxygen and carbon oxide analyzer (Sable Systems SS3 FC-10a O

2CO2analyzer Las Vegas NV) O

2was calculated

using the measured flow through the metabolic chamber theexpired fraction of effluent oxygen and the fraction of oxygenin room air

25 Skeletal Muscle Oxidative Enzyme Activity Citrate syn-thase activity used as an index of aerobic ET was determinedspectrophotometrically in mixed soleus muscle [21] Theenzyme activitywasmeasured in soleusmuscle homogenatesand the amount of the complex resulting from acetyl-CoAand oxaloacetate was determined at 412 nm and 25∘C atan interval of 10min The solubilized protein extracts fromthe homogenates were quantified in duplicate using bovinealbumin as the standard Citrate synthase activity was thennormalized to the total protein content and reported innanomoles per milligram of protein per minute

26 Capillary-to-Fiber Ratio Twenty-four hours after the lastexercise training session sedentary and trained rats werekilled and the soleus muscle was immediately frozen inmelted isopentane and then stored in liquid nitrogen Frozenmuscle was cut into 10 120583m cross-sections from the proximalto the distal region using a cryostat (Micron HM505E ZeissWalldorf Germany) Muscle sections were then probed formyofibrillar ATPase activity with alkaline medium (myosinATPase pH 103) or acid medium preincubation (myosinATPase pH 46) The myosin ATPase reaction was used toidentify muscle fibers and capillaries The capillary-to-fiberratio was quantified using a 10 times 10 grid optically superim-posed on each of 10 nonoverlapping fields at times200 magnifi-cation distributed in a random manner using a computer-assisted morphometric system (Quantimet 500 Leica Cam-bridge UK) To calculate the capillary-to-fiber ratio the totalnumber of capillaries was divided by the total number offibers counted in the same field Only vessels with a diameterlt 10 120583m were counted All analyses were conducted by asingle observer (J Gomes) blinded to rat identity

27 Analysis of miR-126 miR-126 expression was measuredusing real-time PCR and the TaqMan MicroRNA Assay(Applied Biosystems CA USA) The 20120583l PCR reactionincluded 133120583l RT product 10 120583l TaqMan Universal PCRmaster mix II (2x) 767120583l nuclease-free water and 1 120583l ofprimers and probe mix from the TaqMan MicroRNA Assayfor miR-126 (INV 2228) The reactions were incubated in a96-well optical plate at 95∘C for 10min followed by 40 cyclesof 95∘C for 15 s and 60∘C for 1min Samples were normalizedby evaluating U6 expression Each sample was analyzedin triplicate Relative quantities of target gene expressionin sedentary rats versus trained rats were compared afternormalization to the values of the reference gene (ΔCT)Fold changes in miRNA expression were calculated using the

Oxidative Medicine and Cellular Longevity 3

differences in ΔCT values between the two samples (ΔΔCT)and the equation 2minusΔΔCT Results were expressed as ofcontrol

28 Immunoblotting The protein levels PI3KR2 PI3KVEGF and eNOS in the soleus muscle were analyzed byWestern blotting The frozen soleus muscle (100mg) washomogenized in cell lysis buffer containing 100mM Tris-HCl 50mM NaCl 1 Triton X-100 and a protease andphosphatase inhibitor cocktail (1 100 Sigma-Aldrich MOUSA) Soleus tissue debris was removed by centrifugationat 3000timesg 4∘C 10min Samples were loaded and subjectedto SDS-PAGE on polyacrylamide gels (6ndash15) dependingon the protein molecular weight After electrophoresis pro-teins were electrotransferred to a nitrocellulose membrane(BioRad Biosciences NJ USA) Equal loading of samples(30 120583g) and even transfer efficiency were monitored withthe use of 05 Ponceau S staining of the blot membraneThe blot membrane was then incubated in a blocking buffer(5 nonfat dry milk 10mM Tris-HCl (pH 76) 150mMNaCl and 01 Tween 20) for 2 h at room temperatureand then incubated overnight at 4∘C with rabbit anti-VEGF (Abcam Cambridge UK ab46154) anti-PI3K (AbcamCambridgeUK ab151549) anti-PI3KR2 (AbcamCambridgeUK ab62901) an anti-eNOS (Cell Signaling Tech MA USA9572) polyclonal antibodies and a mouse anti-GAPDHmonoclonal antibody (Abcam Cambridge UK ab9484)Binding of the primary antibody was detected by peroxidase-conjugated secondary antibodies and enhanced chemilumi-nescence reagent (Amersham Biosciences NJ USA) anddetection was performed in a digitalizing unit (ChemiDocBioRad CA USA) The bands were analyzed using Image Jsoftware (ImageJ Corporation based on NIH image) Skeletalmuscle GAPDH expression levels were used to normalizethe results which are expressed as a percentage of controlexpression

29 Statistical Analysis The results are represented as meanplusmn SEM Statistical analysis was performed using two-wayANOVA 119901 values lt 005 were accepted as statistically signifi-cant The Duncan post hoc test (STATISTICA softwareStatSoft Tulsa OK) was used for individual comparisonsbetween means when a significant change was observed withANOVA The Pearson coefficient of correlation was used toanalyze the correlations between parametric data

3 Results

31 Experimental Design All animals remained sedentaryfrom 0 to 12 weeks to allow obesity development and matu-ration After 12 weeks animals (LTR and OBTR groups) weresubmitted to the ET protocol for 10 weeks while the othergroups remained sedentary At 22 weeks all the physiologicalparameters (cardiac frequency blood pressure and VO

2)

weremeasured there were no differences in these parametersbetween groups All animals were killed after the last ETsession at 22 weeks All tissues were adequately collectedand the molecular and structural analyses were subsequentlyperformed (Figure 1(a))

32 Effectiveness of Aerobic Exercise Training To confirm theeffectiveness of ET we evaluated the oxygen consumptionpeak (VO

2max) At the end of the protocol the OB and

OBTR groups had lower oxygen uptake compared with theLS group However the OBTR group showed a higher VO

2

max comparedwith theOB group VO2maxwas significantly

higher in the LTR group compared with all other groups (LS655 plusmn 12 LTR 701 plusmn 38 OB 405 plusmn 215 and OBTR 553plusmn 13mLkgmin) (Figure 1(b)) We also evaluated oxidativecitrate synthase enzyme activity in the soleus muscle as anaerobic ET marker The results show that the OB group wasnot different from the LS group however the OBTR andLTR groups showed a significant increase in enzyme activitycompared with the LS and OB groups demonstrating theeffectiveness of the ET protocol (LS 67 plusmn 435 LTR 92 plusmn465 OB 57 plusmn 57 and OBTR 126 plusmn 948 nmolsdotminminus1sdotmgminus1)(Figure 1(c))

33 Effect of Exercise Training on Body Composition Theresults show that the OB group had a higher content ofintramuscular fat comparedwith all other groups In contrastET normalized the intramuscular fat content in the OBTRcompared to the LS group The LTR group was not differentfrom the LS group Thus the data indicate that ET preventedthe accumulation of intramuscular fat only in the OBTRgroup (LS 3793 plusmn 373 LTR 3455 plusmn 497 OB 5008 plusmn 289and OBTR 4214 plusmn 393) (Figure 2) In addition we havepreviously shown that body weight and visceral fat is greatlyincreased inOB group but ET is able to attenuate the increasein OBTR body weight and visceral fat [10]

34 Angiogenesis and VEGF Protein Expression Our datashow that the OB group presented severe capillary rarefac-tion showing 37 less capillarity compared to control Incontrast ET normalized capillary rarefaction in the OBTRgroup suggesting that increased muscle contractions mayincrease angiogenesis In addition the LTR group presented a38 increase in capillarity compared to LS (Figure 3(a)) (LS105 plusmn 008 LTR 144 plusmn 007 OB 065 plusmn 017 and OBTR 091plusmn 009)

VEGF is a potent angiogenic factor VEGFprotein expres-sion was decreased in the OB group compared to the LSgroup However ET normalized VEGF expression in theOBTR group toward control levels indicating the ET is aregulator of angiogenic factors in obesity Interestingly therewas no change in the expression of VEGF protein in the LTRgroup compared to control (Figure 3(b)) (LS 100 plusmn 125 LTR891 plusmn 637 OB 732 plusmn 466 and OBTR 875 plusmn 681)

35 Effect of Exercise Training and Obesity on miRNA-126Expression and Relation with Angiogenesis Our data showthat miR-126 expression was reduced in the OB groupcompared with all the other groups In contrast ET restoredmiR-126 expression in the OBTR group toward controllevels Furthermore ET was also effective in increasing theexpression of miR-126 in the LTR group (LS 100 plusmn 131 LTR140plusmn 107 OB 55plusmn 68 andOBTR 92plusmn 64) Indeed our datashow that there is a correlation between miR-126 expressionand the capillaryfiber ratio In this study an increase in the


0 12 22 weeks

Maturation obesity developmentand Exercise training for LTR and OBTR group

Physiologicalparameters

measurementmolecular and

structural analyses

(a)

LS LTR OB OBTR

lowastlowast

lowast

lowast

lowast

VO2

max

(mLmiddot

kgminus1middotm

inminus1)

0

10

20

30

40

50

60

70

80

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowastlowast

lowastlowast

0

20

40

60

80

100

120

140

160

Citr

ate s

ynth

ase a

ctiv

ity(n

molmiddotm

inminus1middotm

gmiddotpr

otei

nminus1)

(c)

Figure 1 (a) Presentation of experimental design (b) VO2max (mlsdotkgminus1sdotminminus1) and (c) citrate synthase activity (nmolsdotminminus1sdotmgsdotproteinminus1)

Groups LS lean sedentary LTR lean trained OB obese sedentary OBTR obese trained Data are reported as means plusmn SEM lowast119901 lt 005lowastlowast119901 lt 001

LS LTR OB OBTR

ND

0

10

20

30

40

50

60

in

tram

uscu

lar f

at

lowastlowast

lowast

lowastlowast

lowastlowast

Figure 2 Percentage of intramuscular fat Groups LS lean sedentary LTR lean trained OB obese sedentary OBTR obese trained Dataare reported as means plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 ND119901 gt 005


ND

LS LTR OB OBTR0

05

1

15

2

Capi

llary

fibe

r rat

io

lowastlowast

lowastlowastlowastlowast

lowast

lowast

(a)

ND

LS LTR OB OBTR

LTR

GAPDH 37 kDa

43 kDa

OBLS OBTR

0

20

40

60

80

100

120

VEG

F pr

otei

n le

vels

( o

f con

trol)

lowast

lowast lowast

(b)

LS LTR OB OBTR

200x

(c)

Figure 3 (a) Percentage of capillaryfiber ratio (b) VEGF protein levels (c) representative images of capillary density Groups LS leansedentary LTR lean trained OB obese sedentary OBTR obese trained Data are reported as means plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 ND119901 gt

005

expression of miR-126 was accompanied by increased capi-llarity (Figure 4(b))

36 Effect of Exercise Training and Obesity on PI3KR2 andthe PI3KeNOS Pathway PI3KR2 is a target gene of miR-126 PI3KR2 was increased in the OB group compared tothe LS group In contrast ET restored PI3KR2 in the OBTRgroup compared to the LS group demonstrating that ET isable to normalize the expression of this protein InterestinglyPI3KR2 levels were not different in the LTR group comparedto control (Figure 5(a)) (LS 100 plusmn 125 LTR 109 plusmn 381 OB1314 plusmn 763 and OBTR 1155 plusmn 707)

PI3K protein expression was decreased in the OB groupcompared to the LS group In contrast the OBTR grouppresented normal levels of this protein similar to the LSgroup (LS 100 plusmn 118 LSTR 105 plusmn 69 OB 802 plusmn 115 and

OBTR 1037plusmn 139)Thus there is a correlation between thesetwo proteins (PI3KR2 and PI3K) where increased expressionof PI3KR2 leads to a reduction in the expression of PI3K inobese Zucker rats ET has the opposite effect (Figure 5(b))

Our data show that the expression of eNOS protein wasreduced in the OB group compared to control while eNOSexpression was normalized in the OBTR group comparedto LS group Moreover the LTR group had much higherexpression of this protein compared to the LS group (LS 100plusmn 93 LTR 1721plusmn 221 OB 74plusmn 134 andOBTR 1466plusmn 224)(Figure 5(c))

4 Discussion

Obesity results in many deleterious changes in skeletalmuscle such as capillary rarefaction [9] Our study suggests


LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)

050 1 150 20Capillaryfiber ratio

R2= 05707

(b)

Figure 4 (a) Expression of miRNA-126 by real-time PCR and (b) positive correlation between miRNA-126 expression and capillaryndashfiberratio Groups LS lean sedentary LTR lean trained OB obese sedentary OBTR obese trained Data are reported asmeansplusmn SEM lowast119901 lt 005ND119901 gt 005

that long-term swimming training has a major influenceon muscle phenotype in obese Zucker rats Corroboratingwith the literature our study shows that ET is an importantnonpharmacological strategy to reduce microvascular rar-efaction induced by obesity The possible molecular factorsthat precede this impaired phenotype in obesity may includethe expression of miR-126 and proteins of the VEGF pathwayThe results of this study indicate that miR-126 is downreg-ulated in the skeletal muscle of obese rats and this changewas associated with capillary rarefaction Moreover we alsoshowed that ET normalizes the expression of miR-126 andreverses the capillary loss caused by obesity (Figure 6)

miR-126 is an endothelial specific miR (angiomiR) andits expression is closely linked to angiogenesis Expressionof miR126 is enriched in endothelial cells and endothelialprogenitor cells This miRNA is involved in the induction ofsignaling of the embryonic vasculogenesis being related toa differentiation of embryonic stem cells in endothelial cellsand endothelial progenitor cells promoting their maturation[22] Knockdown of miR-126 expression in animals results inan impairment in the formation and maintenance of bloodvessels and partial embryonic lethality [7]

The miR-126 contributes to vascular homeostasis inmature endothelial cells associated with maintaining vas-cular integrity and inhibiting proliferation and motility inthis case actins as an angiogenesis suppressor However incases of vascular lesion hypoxia or stress on the vessel wallthere is an increase in the expression of miR-126 whichactivates endothelial cells and endothelial progenitor cellswhich contributes to vascular healing and the formation ofnew vessels [22]

We were the first to show that sedentary obese animalspresent a decrease in miR-126 expression which contributesto microvascular rarefaction However in other chronicdiseases such as diabetes mellitus II it has previously beendemonstrated that there is also a decrease in the expressionof miR-126 in the circulation of rats and humans Moreoverthere is a correlation between the decrease of mir-126 andendothelial apoptosis It is important to note that a timecourse study in a type 2 diabetic rat model shows that thegradual decrease of miR-126 and other angiomiRs precedesendothelial apoptosis and decreased vascular integrity as wellas changes in the density of the microvasculature [23]

ET in turn increases blood flow causing a shear stress inthe vessel wall this becomes important because the hemo-dynamic changes lead to different expressions of microRNAsin the vascular system [24] The literature demonstrates thatonly a prolonged ET session in humans leads to increasedexpression of miR-126 in the circulation [25] Our previousstudies have shown that in healthy rats ET increases cardiacmiR-126 expression promoting cardiac angiogenesis [19]Other studies have shown that ET restores the expression ofmiR-126 in other diseases [17] Our evidence suggests thatmiR-126 also influences control of the microvasculature inthe skeletal muscle of obese Zucker rats Our study showsfor the first time that ET restores miR-126 expression inthe skeletal muscle of obese Zucker rats and is involved inexercise-induced angiogenesis

PI3KR2 is one of the validated targets of miR-126 andis an important factor in the regulation of angiogenesisPI3KR2 functions as an indirect negative regulator of VEGFby inhibiting the expression of PI3K The negative regulation


LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)

Figure 5 (a) PI3KR2 protein level (b) PI3K protein expression (c) eNOS protein expression and (d) representative blots of PI3KR2 PI3Kand eNOS Groups LS lean sedentary LTR lean trained OB obese sedentary OBTR obese trained Data are reported as means plusmn SEMlowast119901 lt 005 ND119901 gt 005

of VEGF by PI3KR2 occurs by the following mechanismVEGF induces phosphorylation of PI3K this then generatesa phosphorylation cascade that activates AKT which playsan important role in inducing eNOS signaling a fundamen-tal process in vessel growth and survival [6 17] PI3KR2inhibits PI3K by inhibiting that phosphorylation cascadeconsequently angiogenesis is inhibited [26 27]

We show here that obesity decreased miR-126 expressionwhich increased the protein content of its target genesDecreased miR-126 led to increases PI3KR2 protein expres-sion in obese rats However training restored the expressionof miR-126 and consequently normalized PI3KR2 expressionin obese rats

The present study also shows severe capillary rarefactionin sedentary obese animals Decreased vascularity leads toan inadequate supply of oxygen and nutrients to the tissuesand may even cause microvascular necrosis and apoptosis[4 14 28] However ET has been shown to be a potenttherapeutic agent for various vascular diseases It is knownthat ET increases microvascular network caliber and bloodvessels compliance [16 27 29]

There are several regulatory proteins involved in angio-genesis the principal mediators are VEGF and eNOS Recentfindings point to several angiogenic factors that may beupregulated by ETThese factors are influenced by increasingmechanical contraction and shear stress in vessels [17 19 27]


Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

Capillary rarefaction

Exercise training

Increased angiogenesis

Figure 6 Schematic presentation of swimming training effects onskeletal muscle angiogenesis by miRNA-126 Obesity reduces themiRNA-126 expression leading an increase of PI3KR2 blocking theVEGF pathway thereby contributing to the phenotype of capillaryrarefaction in skeletal muscle However ET increases miRNA-126expression improving the VEGF pathway promoting angiogenesis

We highlight VEGF and eNOS because the expressionof these proteins is closely related to ET [17 19] They areinvolved in the proliferation migration and differentiationof endothelial cells and the formation of new vessels [1617 19 27] An increase in eNOS expression leads to anincrease in the expression of nitric oxide (NO) These twofactors are critical to vasodilation thereby maintaining thestructure function and integrity of vessels [19 27] VEGFis an important regulator of angiogenesis by promoting theformation of new vessels from other existing vessels it is alsoan antiapoptotic factor that prevents capillary loss [17 27]There are other proteins that regulate the function of theseangiogenic factors such as PI3KR2 PI3K and AKT whichare also regulated by aerobic ET [19]

In the present study we found that obese animals had adeficiency in the expression of the above-mentioned proteinsleading to severe capillary rarefaction in skeletal muscle ETwas able to restore the impaired expression of these proteinsand thus partially restored the microvascular network

In conclusion miR-126 plays an important role in thecontrol of angiogenic pathways by targeting the expressionof PI3KR2 Obesity downregulates the expression of miR-126 which consequently prevents the expression of severalangiogenic proteins However aerobic exercise training isable to normalize the expression ofmiR-126 and consequentlyrestore the function of various proteins controlled by it

Competing Interests

The authors declare that they have no competing interests

Authorsrsquo Contributions

Joao Lucas Penteado Gomes Tiago Fernandes Ursula PaulaReno Soci and Edilamar Menezes Oliveira contributedto conception and design of research Joao Lucas Pen-teado Gomes Tiago Fernandes Andre Casanova SilveiraUrsula Paula Reno Soci and Diego Lopes Mendes Barrettiperformed experiments Joao Lucas Penteado Gomes andTiago Fernandes analyzed data Joao Lucas Penteado GomesTiago Fernandes Ursula Paula Reno Soci and Edilamar

Menezes Oliveira interpreted results of experiments JoaoLucas Penteado Gomes Tiago Fernandes Andre CasanovaSilveira Ursula Paula Reno Soci Carlos EduardoNegrao andEdilamar Menezes Oliveira edited and revised manuscript

Acknowledgments

This study was supported by the Research Support Founda-tion of Sao Paulo State (FAPESP-200918370-3 201050048-1201326615-1 and 201450673-4) and USPPRP-NAPmiR

References

[1] M Akbartabartoori M E J Lean and C R Hankey ldquoThe asso-ciations between current recommendation for physical activityand cardiovascular risks associated with obesityrdquo EuropeanJournal of Clinical Nutrition vol 62 no 1 pp 1ndash9 2008

[2] T Kelly W Yang C-S Chen K Reynolds and J He ldquoGlobalburden of obesity in 2005 and projections to 2030rdquo InternationalJournal of Obesity vol 32 no 9 pp 1431ndash1437 2008

[3] WHO Obesity Preventing and Managing the Global EpidemicReport of a WHO Consultation World Health OrganizationTechnical Report Series 894 World Health Organization 2000

[4] E M Conway ldquoAngiogenesis a link to thrombosis in athero-thrombotic diseaserdquo Pathophysiology of Haemostasis andThrombosis vol 33 no 5-6 pp 241ndash248 2003

[5] J C Frisbee ldquoRemodeling of the skeletal muscle microcircula-tion increases resistance to perfusion in obese Zucker ratsrdquoAmerican Journal of PhysiologymdashHeart and Circulatory Physi-ology vol 285 no 1 pp H104ndashH111 2003

[6] J C Frisbee J B Samora J Peterson and R Bryner ldquoExercisetraining blunts microvascular rarefaction in the metabolic syn-dromerdquoAmerican Journal of PhysiologymdashHeart and CirculatoryPhysiology vol 291 no 5 pp H2483ndashH2492 2006

[7] E Ferraro A M Giammarioli S Chiandotto I Spoletini andG Rosano ldquoExercise-induced skeletal muscle remodeling andmetabolic adaptation Redox signaling and role of autophagyrdquoAntioxidants amp Redox Signaling vol 21 pp 154ndash176 2014

[8] B H Goodpaster J He S Watkins and D E Kelley ldquoSkeletalmuscle lipid content and insulin resistance evidence for aparadox in endurance-trained athletesrdquo Journal of ClinicalEndocrinology and Metabolism vol 86 no 12 pp 5755ndash57612001

[9] C E Torgan J T Brozinick Jr G M Kastello and J L IvyldquoMusclemorphological and biochemical adaptations to trainingin obese Zucker ratsrdquo Journal of Applied Physiology vol 67 no5 pp 1807ndash1813 1989

[10] D LM Barretti F D CMagalhaes T Fernandes et al ldquoEffectsof aerobic exercise training on cardiac renin-angiotensin systemin an obese zucker rat strainrdquo PLOS ONE vol 7 no 10 ArticleID e46114 2012

[11] K SOhMKim J Lee et al ldquoLiver PPAR120572 andUCP2 are invol-ved in the regulation of obesity and lipid metabolism by swimtraining in genetically obese dbdb micerdquo Biochemical andBiophysical Research Communications vol 345 no 3 pp 1232ndash1239 2006

[12] J L Walberg P A Mole and J S Stern ldquoEffect of swimtraining on development of obesity in the genetically obese ratrdquoAmerican Journal of Physiology vol 242 no 3 pp R204ndashR2111982


[13] A S Ali S Ali A Ahmad B Bao P A Philip and F H SarkarldquoExpression of microRNAs potential molecular link betweenobesity diabetes and cancerrdquoObesity Reviews vol 12 no 12 pp1050ndash1062 2011

[14] N N Andersen and T Jess ldquoRisk of cardiovascular disease ininflammatory bowel diseaserdquo World Journal of GastrointestinalPathophysiology vol 5 pp 359ndash365 2014

[15] S Viboolvorakul and S Patumraj ldquoExercise training couldimprove age-related changes in cerebral blood flow and capil-lary vascularity through the upregulation of VEGF and eNOSrdquoBioMed Research International vol 2014 Article ID 230791 12pages 2014

[16] R Eisenstein ldquoAngiogenesis in arteries reviewrdquo PharmacologyampTherapeutics vol 49 no 1-2 pp 1ndash19 1991

[17] T Fernandes F C Magalhaes F R Roque M I Phillips andE M Oliveira ldquoExercise training prevents the microvascularrarefaction in hypertension balancing angiogenic and apoptoticfactors role of microRNAs-16 -21 and -126rdquoHypertension vol59 no 2 pp 513ndash520 2012

[18] J E Fish M M Santoro S U Morton et al ldquomiR-126 regulatesangiogenic signaling and vascular integrityrdquo DevelopmentalCell vol 15 no 2 pp 272ndash284 2008

[19] N D Da Silva T Fernandes U P R Soci A W A MonteiroM I Phillips and EMDeOliveira ldquoSwimming training in ratsincreases cardiac MicroRNA-126 expression and angiogenesisrdquoMedicine and Science in Sports and Exercise vol 44 no 8 pp1453ndash1462 2012

[20] A Medeiros E M Oliveira R Gianolla D E Casarini C ENegrao and P C Brum ldquoSwimming training increases cardiacvagal activity and induces cardiac hypertrophy in ratsrdquoBrazilianJournal of Medical and Biological Research vol 37 no 12 pp1909ndash1917 2004

[21] D X Scomparin S Grassiolli R M Gomes et al ldquoLow-inten-sity swimming training after weaning improves glucose andlipid homeostasis inMSG hypothalamic obesemicerdquo EndocrineResearch vol 36 no 2 pp 83ndash90 2011

[22] D A Chistiakov A N Orekhov and Y V Bobryshev ldquoThe roleof miR-126 in embryonic angiogenesis adult vascular home-ostasis and vascular repair and its alterations in atheroscleroticdiseaserdquo Journal of Molecular and Cellular Cardiology vol 97pp 47ndash55 2016

[23] S Rawal P E Munasinghe A Shindikar et al ldquoDown-regula-tion of proangiogenic microRNA-126 and microRNA-132 areearly modulators of diabetic cardiac microangiopathyrdquo Cardio-vascular Research vol 113 no 1 pp 90ndash101 2017

[24] P Neth M Nazari-Jahantigh A Schober and C WeberldquoMicroRNAs in flow-dependent vascular remodellingrdquo Cardio-vascular Research vol 99 no 2 pp 294ndash303 2013

[25] A L Baggish J Park P-K Min et al ldquoRapid upregulation andclearance of distinct circulating microRNAs after prolongedaerobic exerciserdquo Journal of Applied Physiology vol 116 no 5pp 522ndash531 2014

[26] N M Pandya N S Dhalla and D D Santani ldquoAngiogenesis-anew target for future therapyrdquo Vascular Pharmacology vol 44no 5 pp 265ndash274 2006

[27] B M Prior H T Yang and R L Terjung ldquoWhat makes vesselsgrow with exercise trainingrdquo Journal of Applied Physiology vol97 no 3 pp 1119ndash1128 2004

[28] A Behn and E Ur ldquoThe obesity epidemic and its cardiovascularconsequencesrdquoCurrent Opinion in Cardiology vol 21 no 4 pp353ndash360 2006

[29] M D Brown and O Hudlicka ldquoModulation of physiologicalangiogenesis in skeletal muscle by mechanical forces involve-ment of VEGF andmetalloproteinasesrdquoAngiogenesis vol 6 no1 pp 1ndash14 2003

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


the vascular endothelial growth factor (VEGF) signalingpathway via direct inhibition of phosphatidylinositol 3-kinase(PI3K 120572-110) Thus miR-126 inhibits PI3KR2 and promotesangiogenesis through an indirect increase in PI3K andVEGFThrough this process the expression of proteins in the nitricoxide synthase (eNOS) pathway is activated which alsoenhances angiogenesis Knockdown of miR-126 in animalsleads to impaired vascular formation due to a reductionin the migration of endothelial cells during the genesis ofvessels this compromises blood vessel integrity and leads tohemorrhage in the embryonic period [18]

ET regulates the expression of various miRs that regu-late biological processes related to adaptation [17 19] Theexpression of miR-126 increases in healthy trained rats [19]and is severely decreased in rats with chronic diseases suchas hypertension [17] However ET has been shown to restoremiR-126 levels in the skeletal muscle of hypertensive ratsand induces angiogenesis [17] However the effects of ET interms of controlling the expression of miRs in skeletal musclein the context of obesity need further investigation In thepresent study we found that reduced miR-126 levels inducedcapillary rarefaction in skeletal muscle by targeting PI3KR2in obese Zucker rats ET counteracted this effect by restoringthe expression of miR-126 and skeletal muscle capillarity inobese Zucker rats

2 Methods

21 Experimental Group Twenty-one Zucker rats were ran-domly divided into four groups sedentary lean (LS 119899 = 5)trained lean (LTR 119899 = 5) obese sedentary (OB 119899 = 5)and obese trained (OBTR 119899 = 6) The animals were housed3 to 5 per cage in a room heated between 22∘C and 24∘Cand were on an inverted light-dark cycle water and foodwere given ad libitum After the training protocol animalswere euthanized and the relevant tissues were dissectedand weighed All procedures were performed according tothe Ethical Principles of Animal Experimentation and theapproved by the Ethics Committee on Animal Use at theUniversity of Sao Paulo

22 Swimming Training Protocol Swimming training wasperformed according to the protocol developed by Medeirosand colleagues [20] in a swimming system with water at30ndash32∘C The training duration was 10 weeks with 5 weeklysessions with a progressive increase in time reaching 60min-utes andwith a progressive increase inworkload reaching 4of the body weight of the animal

23 Quantification of Intramuscular Lipids by Oil Red OSerial sections of skeletal muscle (soleus) were preparedat a thickness of 10 120583m Muscle sections were fixed in37 formaldehyde and stained with Oil Red O workingsolution (500mg of Oil Red O added to 100ml of aqueoussolution with 60 triethyl phosphate (Fluka) and 8ml ofdeionized water) The final product of this procedure is red-stained lipids The intramuscular fat content was analyzedin the soleus muscle in intact cellular membranes with acomputer-assisted morphometric system (Leica Quantimet

500 Cambridge UK) able tomeasure the redwavelength Foreach animal approximately 10 visual fields were analyzed

24 Oxygen Uptake Measurements Oxygen uptake (O2) was

measured by expired gas analysis during graded treadmillexercise training Gas analysis was performed using anoxygen and carbon oxide analyzer (Sable Systems SS3 FC-10a O

2CO2analyzer Las Vegas NV) O

2was calculated

using the measured flow through the metabolic chamber theexpired fraction of effluent oxygen and the fraction of oxygenin room air

25 Skeletal Muscle Oxidative Enzyme Activity Citrate syn-thase activity used as an index of aerobic ET was determinedspectrophotometrically in mixed soleus muscle [21] Theenzyme activitywasmeasured in soleusmuscle homogenatesand the amount of the complex resulting from acetyl-CoAand oxaloacetate was determined at 412 nm and 25∘C atan interval of 10min The solubilized protein extracts fromthe homogenates were quantified in duplicate using bovinealbumin as the standard Citrate synthase activity was thennormalized to the total protein content and reported innanomoles per milligram of protein per minute

26 Capillary-to-Fiber Ratio Twenty-four hours after the lastexercise training session sedentary and trained rats werekilled and the soleus muscle was immediately frozen inmelted isopentane and then stored in liquid nitrogen Frozenmuscle was cut into 10 120583m cross-sections from the proximalto the distal region using a cryostat (Micron HM505E ZeissWalldorf Germany) Muscle sections were then probed formyofibrillar ATPase activity with alkaline medium (myosinATPase pH 103) or acid medium preincubation (myosinATPase pH 46) The myosin ATPase reaction was used toidentify muscle fibers and capillaries The capillary-to-fiberratio was quantified using a 10 times 10 grid optically superim-posed on each of 10 nonoverlapping fields at times200 magnifi-cation distributed in a random manner using a computer-assisted morphometric system (Quantimet 500 Leica Cam-bridge UK) To calculate the capillary-to-fiber ratio the totalnumber of capillaries was divided by the total number offibers counted in the same field Only vessels with a diameterlt 10 120583m were counted All analyses were conducted by asingle observer (J Gomes) blinded to rat identity

27 Analysis of miR-126 miR-126 expression was measuredusing real-time PCR and the TaqMan MicroRNA Assay(Applied Biosystems CA USA) The 20120583l PCR reactionincluded 133120583l RT product 10 120583l TaqMan Universal PCRmaster mix II (2x) 767120583l nuclease-free water and 1 120583l ofprimers and probe mix from the TaqMan MicroRNA Assayfor miR-126 (INV 2228) The reactions were incubated in a96-well optical plate at 95∘C for 10min followed by 40 cyclesof 95∘C for 15 s and 60∘C for 1min Samples were normalizedby evaluating U6 expression Each sample was analyzedin triplicate Relative quantities of target gene expressionin sedentary rats versus trained rats were compared afternormalization to the values of the reference gene (ΔCT)Fold changes in miRNA expression were calculated using the





3 Results


2)





2








0 12 22 weeks




structural analyses

(a)

LS LTR OB OBTR

lowastlowast

lowast

lowast

lowast

VO2

max

(mLmiddot

kgminus1middotm

inminus1)

0

10

20

30

40

50

60

70

80

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowastlowast

lowastlowast

0

20

40

60

80

100

120

140

160

Citr

ate s

ynth

ase a

ctiv

ity(n

molmiddotm

inminus1middotm

gmiddotpr

otei

nminus1)

(c)



LS LTR OB OBTR

ND

0

10

20

30

40

50

60

in

tram

uscu

lar f

at

lowastlowast

lowast

lowastlowast

lowastlowast



ND

LS LTR OB OBTR0

05

1

15

2

Capi

llary

fibe

r rat

io

lowastlowast


lowast

lowast

(a)

ND

LS LTR OB OBTR

LTR

GAPDH 37 kDa

43 kDa

OBLS OBTR

0

20

40

60

80

100

120

VEG

F pr

otei

n le

vels

( o

f con

trol)

lowast

lowast lowast

(b)

LS LTR OB OBTR

200x

(c)


005






4 Discussion



LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)


R2= 05707

(b)









LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















3 Results


2)





2








0 12 22 weeks




structural analyses

(a)

LS LTR OB OBTR

lowastlowast

lowast

lowast

lowast

VO2

max

(mLmiddot

kgminus1middotm

inminus1)

0

10

20

30

40

50

60

70

80

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowastlowast

lowastlowast

0

20

40

60

80

100

120

140

160

Citr

ate s

ynth

ase a

ctiv

ity(n

molmiddotm

inminus1middotm

gmiddotpr

otei

nminus1)

(c)



LS LTR OB OBTR

ND

0

10

20

30

40

50

60

in

tram

uscu

lar f

at

lowastlowast

lowast

lowastlowast

lowastlowast



ND

LS LTR OB OBTR0

05

1

15

2

Capi

llary

fibe

r rat

io

lowastlowast


lowast

lowast

(a)

ND

LS LTR OB OBTR

LTR

GAPDH 37 kDa

43 kDa

OBLS OBTR

0

20

40

60

80

100

120

VEG

F pr

otei

n le

vels

( o

f con

trol)

lowast

lowast lowast

(b)

LS LTR OB OBTR

200x

(c)


005






4 Discussion



LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)


R2= 05707

(b)









LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0 12 22 weeks




structural analyses

(a)

LS LTR OB OBTR

lowastlowast

lowast

lowast

lowast

VO2

max

(mLmiddot

kgminus1middotm

inminus1)

0

10

20

30

40

50

60

70

80

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowastlowast

lowastlowast

0

20

40

60

80

100

120

140

160

Citr

ate s

ynth

ase a

ctiv

ity(n

molmiddotm

inminus1middotm

gmiddotpr

otei

nminus1)

(c)



LS LTR OB OBTR

ND

0

10

20

30

40

50

60

in

tram

uscu

lar f

at

lowastlowast

lowast

lowastlowast

lowastlowast



ND

LS LTR OB OBTR0

05

1

15

2

Capi

llary

fibe

r rat

io

lowastlowast


lowast

lowast

(a)

ND

LS LTR OB OBTR

LTR

GAPDH 37 kDa

43 kDa

OBLS OBTR

0

20

40

60

80

100

120

VEG

F pr

otei

n le

vels

( o

f con

trol)

lowast

lowast lowast

(b)

LS LTR OB OBTR

200x

(c)


005






4 Discussion



LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)


R2= 05707

(b)









LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















ND

LS LTR OB OBTR0

05

1

15

2

Capi

llary

fibe

r rat

io

lowastlowast


lowast

lowast

(a)

ND

LS LTR OB OBTR

LTR

GAPDH 37 kDa

43 kDa

OBLS OBTR

0

20

40

60

80

100

120

VEG

F pr

otei

n le

vels

( o

f con

trol)

lowast

lowast lowast

(b)

LS LTR OB OBTR

200x

(c)


005






4 Discussion



LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)


R2= 05707

(b)









LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

160

miR

NA-

126

expr

essio

n (

of c

ontro

l)lowast

lowastlowast

lowast

(a)

0

05

1

15

2

miR

NA-

126

expr

essio

n (

of c

ontro

l)


R2= 05707

(b)









LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















LS LTR OB OBTR

ND

0

20

40

60

80

100

120

140

PI3K

R2 p

rote

in le

vel (

o

f con

trol)

lowastlowast

lowast

(a)LS LTR OB OBTR

ND

lowast

lowast lowast

0

20

40

60

80

100

120

140

PI3K

pro

tein

leve

l (

of c

ontro

l)

(b)

LS LTR OB OBTR

lowast

lowastlowast

lowast

lowast

0

25

50

75

100

125

150

175

200

eNO

S pr

otei

n le

vel (

o

f con

trol)

(c)

PI3KR2

GAPDH

eNOS

PI3K

37 kDa

110 kDa

82 kDa

140 kDa

LTR OBLS OBTR

(d)







Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





Acknowledgments


References




































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Obesity

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS

miR-126

PI3KR2

VEGF

PI3K

AKT

eNOS


Exercise training






Competing Interests





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
















Documents

Obesity Downregulates MicroRNA-126 Inducing Capillary Rarefaction …downloads.hindawi.com/journals/omcl/2017/2415246.pdf · 2019. 7. 30. · ResearchArticle Obesity Downregulates