Ruolo del Metilgliossalenello sviluppo delle complicanze ... Nigro Cecilia - Ruolo del...0.1%....

Ruolo del Metilgliossale nello sviluppo delle

complicanze vascolari associate al diabete mellito

Cecilia Nigro

URT-Genomica del Diabete IEOS/CNRDiSMeT, Università di Napoli “Federico II”

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Glucose

Methylglyoxal

Hypoxia

Sources and Destiny of Methylglyoxal

AGEs

Glyoxalase 1

Glyoxalase 2

GSHS-D-LactoylGSH

D-Lactate

>99%ThreonineAcetone

Lipid

Glycatedptoteins

extracellularAGEs

RAGE

Oxidative stress

Pro-inflammatory state

+ NFkB

- Nrf2

GSHdepletion

Ageing

DicarbonylStress=

Pyruvate

L-Lactate

Glucose

Fructose6-diphosphate

G3P DHAP

0.1%

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MGO

Methylglyoxal & Diabetes

[McLellan et al. Clinical Science 1994]

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MGO

[Fleming T et al. Diabetologia 2012]

Even if normalized on glucose levels,intracellular MG is higer in diabetic patients

[Maessen DE et al. Diabetes Care 2015]

Post-prandial hyperglycaemia induces aglucose-similar trend of MGO increase

Methylglyoxal & Diabetes

HbA1c 7

HbA1c 8 to 7HbA1c 8

Healthy

[M. Lind et al. Diabetologia 2010]

HbA1c can only partly explain the latediabetic complications

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Methylglyoxal & Complications

Plasma MGO and MG-H1 are risk predictors for progression of diabetic kidney disease. [Jensen et al., Diabet Med 2016; Beisswenger et al., Diabetes Care 2013]

MG-H1 is independently associated with microvascular complication progression.[Genuth e al., Diabetes 2015]

Decreased Glo1 expression is a driver of coronary heart disease.[Makinen et al., PLOS genetics 2014]

Dicarbonyl stress in plasma likely contributes to CVD risk through induction of dyslipidaemia.[Rabbani N, et al. Diabetes 2011]

Inducer of Glo1 expression corrects insulin resistance and improves vascular function in overweight and obese subjects.[Xue et al., Diabetes 2016]

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Methylglyoxal in Experimental Models

Glo1 overexpression attenuates the life-shortening effect of glucose by preventingMG-modification of mitochondrial proteins in C. Elegans, and prevents the age-related endothelial dysfunction in rats.[Morcos et al., Aging Cell 2008; Jo-Watanabe et al., Aging Cell 2014]

MGO alters blood vessel formation in Zebrafish and via a RAGE-dependentmechanism in mice.[Jorgens et al., Diabetes 2015; Liu et al., PLoS ONE 2012]

Regulation of Glo1 plays a role in the prevention of early renal impairment inexperimental diabetes, but also independently of hyperglycaemia in ApoE-/- mice.[Geoffrion et al., Physiol Rep 2014]

Hyperglycaemia-induced impairment of endothelial-dependent vasorelaxation ismediated by increased intracellular MGO levels in a pathway dependent on oxidativestress.[Brouwers et al., Diabetologia 2010]

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IRS-1

PI3K

Akt

eNOS

KRAS

ERK 1/2

ECE-1

MGO

nucleus

miR-190a

ET-1

NO

endothelial cell

IR

P

KRAS mRNAAAA

Impaired vasodilation

X

Methylglyoxal impairs endothelial insulin-sensitivity

MGO exogenous exposure

Glo1 inhibitor

Endothelialinsulin-resistance

[Nigro C. et al., Diabetologia 2014;Nigro C. et al., Biochim Biophys Acta 2017]

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AIM

To investigate the effect of MGO on glucose homeostasis

and its role on the development of

vascular complications associated with diabetes

Glo1KD MAECsGlo1KD mice

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[Bierhaus A et al. Cell Metabolism 2012]

Glo1KD mice show thermal and mechanical hyperalgesia, a sign ofDiabetic Neuropathy.

[Giacco F et al. Diabetes 2014]

Glo1KD mice show alterations of kidney morphology (↑ mesangialfraction volume and GBM tickness), mimicking Diabetic Nephropathy.

Glo1KD mice

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Glo1KD mice

Characterization of Glo1KD mice

Glo1KD mice show a decreased tissue expression of glo1 and a 50% increase of serum MGO levels

0

500

1000

1500

2000

2500

WT Glo-/+

Seru

mM

GO

(nM

)

***

Glo1KD

******

***

0,0

0,2

0,4

0,6

0,8

1,0

1,2

Glo

1 ex

pres

sion

(nor

mal

ized

ratio

)

WT

Glo-/+

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Glo1KDWT

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Glo1KD mice

5 months 10 months

Variable wt mice Glo1kd mice Test T wt mice Glo1kd mice Test T

Body weight (g) 25.4±0.8 24.5±0.7 n.s. 32.6±1.5 35.1±1.8 n.s.

Fasting glycaemia(mg/dl) 105.9±5.3 97.1±3.5 n.s. 114.6±8.4 106.4±6.0 n.s

Fed glycaemia(mg/dl) 138.9±4.1 145.6±6.9 n.s. 143.6±3.1 137.3±4.4 n.s.

GTT (AUC) 8721±960 9921±849 n.s 12990±1595 23944±2300 p

Glo1KD mice

050

100150200250300350400450500

-15 0 15 30 45 60 75 90 105 120 135

Bloo

dgl

ucos

ele

vels

(mg/

dl)

Time (min)

**** ****

***

0

5000

10000

15000

20000

25000

30000

GTT

AU

C (m

g/dl

·min

)

***

10 months of age

wt mice Glo1KD mice

0

50

100

150

200

250

300

350

-15 0 15 30 45 60 75 90 105 120 135Blo

odgl

ucos

ele

vels

(mg/

dl)

Time (min)

0

5000

10000

15000

20000

25000

30000

GTT

AU

C (m

g/dl

·min

)

n.s.

5 months of age

wt mice Glo1KD mice

Metabolic characterization of Glo1KD mice

Glo1KD mice develop an age-dependent glucose intolerance

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Glo1KD mice

5 months 10 months

Variable wt mice Glo1kd mice Test T wt mice Glo1kd mice Test T

Body weight (g) 25.4±0.8 24.5±0.7 n.s. 32.6±1.5 35.1±1.8 n.s.

Fasting glycaemia(mg/dl) 105.9±5.3 97.1±3.5 n.s. 114.6±8.4 106.4±6.0 n.s

Fed glycaemia(mg/dl) 138.9±4.1 145.6±6.9 n.s. 143.6±3.1 137.3±4.4 n.s.

GTT (AUC) 8721±960 9921±849 n.s 12990±1595 23944±2300 p

Glo1KD mice

(*: KD mice vs WT mice; #: 3 and 30 minutes vs time 0)

0

1

2

3

4

5

6

0 5 10 15 20 25 30

Insu

linem

ia(n

g/m

l)

Time (min)

***

##

##

##

0

10

20

30

40

50

60

70

80

90

Insu

lin se

cret

ion

(AU

C)

*

WT mice Glo1KD mice

Metabolic characterization of Glo1KD mice

Glo1kd mice show an alteration of glucose-stimulated insulin secretion at 10 months of age

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Metabolic characterization of Glo1KD miceGlo1KD mice

0,00

0,50

1,00

1,50

2,00

2,50

3,00

TNF alpha

0,00

0,50

1,00

1,50

2,00

2,50

3,00

MCP1

**

0,00

0,50

1,00

1,50

2,00

2,50

3,00

IL-1beta

**

mRN

Ale

vels

(REU

)

WTmice

Glo1KDmice

WTmice

Glo1KDmice

WTmice

Glo1KDmice

WT Glo1KD

F4/80

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Glo1KD mice

5 months 10 months 18 months

Variable wt mice Glo1kd mice

wt mice Glo1kd mice wtmice

Glo1kd mice

Systolic blood pressure(mmHg)

124±3 147±4* 115±3 142±4* 144±3 159±4*

Vascular function of Glo1KD mice

Glo1KD mice show a higher systolic blood pressure,accompanied by an impaired endothelial-dependent vasodilation

and the development of left ventricular hypertrophy

Relative wall thickness (RWT)

Concentric hypertrophy

Eccentric hypertrophy

Vasodilation(% of residual vasoconstriction)

40±0.2 65±0.7* 23±0.2 43±0.3* n.s. n.s.

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Summary 1Glo1KD mice

Next question: Is there a cause-effect relationship between the metabolic and vascular phenotype?

Impairment of GLUCOSE

HOMEOSTASIS

Impairment ofVASCULAR FUNCTION

Glo1KD mice show an age-dependent impairment of glucose tolerance

Glucose intolerance is likely linked to the impairment of insulin secretion

and pancreatic dysfunction, rather than peripheral insulin-resistance

Glo1KD mice show an impaired endothelial-dependent vasodilation and

increased systolic blood pressure

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WT mice

Glo1KDmice

Effect of MGO accumulation on angiogenesis

Endothelial outgrowth from the aortic rings of Glo1KD mice is highly impaired compared to those from WT mice

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***

0,0

0,2

0,4

0,6

0,8

1,0

1,2

WT MAECs

Glo1KDMAECs

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

WT MAECs

Glo1KDMAECs

Intr

acel

lula

r M

ethy

lgly

oxal

(nm

ol m

g-1)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Glo

1 m

RN

Ale

vels

(RE

U)

0.0

0.5

1.0

1.5

2.0

2.5

3.5

3.0

***

Glo1KD MAECs

Characterization of Glo1KD MAECs

Glo1KD MAECs show a 50% decrease of Glo1 expression and a 5-fold increase of intracellular MGO accumulation

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0

1

2

3

4

5

6

7

8

9

10

11

0 12 24 36 48 60 72 84 96

Prol

ifera

tion

(fol

d ov

er ti

me

0)

****

**

WT MAECsGlo1KD MAECs

Time (h)

WT MAECs

Glo1KD MAECs

- FBS + FBS

Mig

ratio

n(f

old

incr

ease

ove

r ba

sal)

***

WT MAECs

Glo1KD MAECs

0

1

2

3

4

5

6

7

8

9

10

WT MAECs

Glo1KD MAECs

- FBS + FBS

Inva

sion

(fol

d in

crea

se o

ver

basa

l)

*

0

1

2

3

4

5

6

7

8

9

10

WT MAECs

Glo1KD MAECs

Glo1KD MAECs

Effect of MGO accumulation on angiogenesis in MAECs

Glo1KD MAECs show an impaired

proliferation, migration and

invasion ability,

compared to WT MAECs

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Glo1KD MAECs

Anti-angiogenic genes mRNA expression (REU) p value

HoxA5 1,87±0,37 0.00000003

HoxD10 1,20±0,92 n.s.

Pro-angiogenic genes mRNA expression (REU) p value

HoxA3 3,37±2,29 n.s.

HoxA9 2,19±1,4 n.s.

HoxB3 1,04±0,67 n.s.

HoxA5 involvement in MGO-mediated impairment of angiogenesis

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Hox

A5

mR

NA

leve

ls (R

EU

)***

0

10

20

30

40

50

60

70

80

90

100

Hox

A5/α-

Tub

ulin

(O.D

.)

*

HoxA5

α-Tubulin

2.5

2.0

1.5

1.0

0.5

0.0

Glo1KD MAECs


Both mRNA and protein levels of the anti-angiogenic HoxA5are 2-fold increased in Glo1KD MAECs

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Grafico1

11.873749602800.3702646218

wt

kd2

Foglio1

Pro-angiogeniciAnti-angiogenici

HoxB3 variante 1 e 2wtkd2HoxA5wtkd2

15.3.1710.3715.11.1612.10

11.3.1610.55292813.12.1611.51

14.7.1610.37453315.3.1712.01

15.3.1711.754.12.1512.03

11.3.1611.52526137242.38

14.7.1611.662970605620.710.8433749242

mean11.03928216329.711.50

dev.st00.671811483224.10.1611.60

SEM0.274265889530.11.1611.148698355

test.t0.888956201622.12.1611.26

27.1.1713.10

HoxD3 newwtkd26.2.1711.91

6.11.14121.6118.1.1613.40

17.10.1413.93esp silenz11.59

cp 3.2.16118.6311.82

cp 14.10.16113.7112.52

12.14

mean117.983333333315.53

dev.st03.98950289314.00

SEM02.3033405692mean11.874

test.t0.0018031364dev.st00.3702646218

SEM0.1026929292

test.t0.00000003B3D3A3A9A5D10

HoxA3wtkd2

17.316.727171322

22.3.1712.0373116199

116.5642387815HoxD10wtkd2

12.96561914417.310.632878297

22.3.1711.922966105

mean17.073585216810.1963727522

devst06.644093734812.0443846535

SEM3.3220468674mean11.1991504519

test.t0.1172695611devst00.9245822883

SEM0.4622911442

test.t0.6817

HoxA9 variante 1 e 2wtkd2

15.3.1711.7565193816

11.3.1613.8320602189

14.7.161

15.3.1710.9979499577

11.3.16

14.7.16

mean12.1955098527

dev.st01.4671672428

SEM0.8470694026

test.t0.1524326551

HoxD3 oldwtkd2

17.6.141

17.10.1410.51

21.10.1410.78

HoxB3 variante1wtkd2

15.3.1710.37

11.3.1610.552928

14.7.1610.374533

mean10.432487

dev.st00.1043295878

SEM0.0602347156

test.t0.0007074623

HoxB3 variante2wtkd2

15.3.1711.75

11.3.1611.5252613724

14.7.1611.6629706056

mean11.646077326

dev.st00.1133176944

SEM0.0654240013

test.t0.0005899848

HoxA9 variante1wtkd2

15.3.1711.7565193816

11.3.1613.8320602189

14.7.161

mean12.7942898003

dev.st01.4676290007

SEM1.0377704186

test.t

HoxA9 variante2wtkd2

15.3.1710.9979499577

11.3.16

14.7.16

mean10.9979499577

Foglio1

0.6718114832

3.989502893

6.6440937348

1.4671672428

0.3702646218

0.9245822883

kd2

00.3702646218

wt

kd2

**

*

***

**

0,0

0,5

1,0

1,5

2,0

2,5**

2.5

2.0

1.5

1.0

0.5

0.0

Hox

A5

mR

NA

exp

ress

ion

(RE

U)

Scramble 5nM - + -HoxA5 siRNA 5nM -- +

n.s.

Inva

sion

(fol

d in

crea

se o

ver

basa

l) *

+ + -

-- +

WT MAECs

Glo1KDMAECs

0

1

2

3

4

5

6

7

8

9

10

11

*

Mig

ratio

n (f

old

incr

ease

ove

r ba

sal)

*

+ + -

-- +

WT MAECs

Glo1KDMAECs

0

1

2

3

4

5

6

7

8

9

10

11

**

Glo1KD MAECs


HoxA5 silencing is able to improve both migration and invasion ability of Glo1KD MAECs

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WTMAECs

Glo1KDMAECs

NF-κB

-p65

mR

NA

leve

ls (R

EU

)

0,0

0,5

1,0

1,5

2,0

2,5

3,0***

0.0

0.5

1.0

1.5

2.0

2.5

3.0NF-κB-p65

GAPDH

cytosol

NF-κB

-p65

/GA

PDH

(O.D

.)

*

NF-κB-p65

Cyclin G1

nucleus

NF-κB

-p65

/Cyc

linG

1 (O

.D.)

*

Glo1KD MAECs

NF-κB-p65 role in the regulation of MGO-mediatedHoxA5 overexpression

Both mRNA and protein levels of NF-kB-p65are 2-fold increased in Glo1KD MAECs

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Grafico1

36.277935129462.68601506998.126591533123.7439387463

WT

KD2

p-Akt

Densitometria lastra del 3-7-15 cp 1-7-15nota: bande bianche

p-AktmeanODtubulinameanODpAkt/tubwt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50

wt -ins154,7141,986wt -ins219,80756,3653.5234631427/3/153.5273.5941.2517.99144.3816.62nota: stim vegf 150

wt +ins185,22332,495wt +ins207,60044,15873.588024826/26/1523.4952.1931.4818.7156.848.39

wt +VEGF150172,02519,297wt +VEGF150210,21946,77741.25317998166/26/1515.5362.3931.6028.1690.2522.60

kd -ins162,5769,848kd -ins218,18754,74517.98885742995/7/1514.3865.9413.8914.2591.6424.78

kd +ins210,24157,513kd +ins203,27639,834144.3816839886

kd +VEGF150160,5617,833kd +VEGF150210,57147,12916.6203399181mean14.1862.7234.7821.6297.1615.87

background152,728163,442dv.st10.0510.705.615.6744.187.13

0.020.17-insVEGF 50ng/ml

lastre del 26-6-15 cp 18-6 p100

p-AktmeanODTubulinameanODpAkt/tub

wt -ins181,77516,039wt -ins237,56368,28023.4900410076

wt +ins199,80134,065wt +ins234,55165,26852.1924986211

wt +VEGF50185,31019,574wt +VEGF50231,46162,17831.4805879893

kd -ins178,52012,784kd -ins237,62468,34118.7061939392

kd +ins203,90338,167kd +ins236,43367,15056.8384214445

kd +VEGF50171,3775,641kd +VEGF50236,48467,2018.3942203241

background165,736background169,283

lastre del 26-6-15 cp 24-6 p60


wt -ins176,62010,706wt -ins229,65968,92315.5332762648

wt +ins208,24842,334wt +ins228,59467,85862.3861593327

wt +VEGF50186,23520,321wt +VEGF50225,04664,31031.5985072306

kd -ins182,98117,067kd -ins221,35460,61828.1550034643

kd +ins219,01653,102kd +ins219,57658,84090.2481305235

kd +VEGF50180,01914,105kd +VEGF50223,15962,42322.5958380725


lastre del 13-5-15 cp 8-5

p-AktmeanODMANCA NORMALIZZATORE

wt -ins97,1497,367

wt +ins131,32041,538

wt +VEGF50100,46610,684

wt +VEGF15097,0397,257

kd -ins95,7095,927

kd +ins141,01951,237

kd +VEGF5094,4204,638

kd +VEGF15094,0214,239

background89,782

lastre del 7-5-15 cp 30-4 P100


wt -ins73,05011,771wt -ins146,94781,86614.38

wt +ins134,51673,237wt +ins176,153111,07265.94

wt +VEGF5078,20716,928wt +VEGF50186,950121,86913.89

kd -ins78,14816,869kd -ins183,419118,33814.25

kd +ins162,430101,151kd +ins175,455110,37491.64

kd +VEGF5073,48412,205kd +VEGF50114,32749,24624.78


lastre del 25-9-15

IKBαmeanODTubulinameanODIKBα/tubulinameandev.sttest.t

wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622

wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30

wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39

kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474

kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33

kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82


p-Akt

10.05161635495.6736980636

10.701721438144.1785639731

5.60847818227.1304694614

wt

kd

signaling pAKT

10.05161635495.6736980636

5.60847818227.1304694614

wt

kd

pAkt/tubulina (OD)

signaling eNOS

22.683367862232.4451951155

wt

kd

IKBa-NFkB

Densitometria 28.10.15 cp wt p25/kd2 p22-insVEGF 50ng/ml

p-AktmeanOD14.3.3meanODpAkt/14.3.3

wt p25 -176,35833,572wt p25 -191,06834,05798.5759168453wt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50

wt p25 +VEGF50181,72538,939wt p25 +VEGF50189,30232,291120.58777987677/3/156.7076.9412.927.8760.384.74nota: stim vegf 1507/3/151001148.8988903168117.5298069394901.66532258917/3/15100192.9469563869117.529806939470.7924584787

kd2 p22 -151,9759,189kd2 p22 -193,27336,26225.34057691256/26/1530.1874.3140.4722.3982.248.356/26/15100246.208369934174.1862488553272.45712563726/26/15100134.071546402974.186248855327.6542550773

kd2 p22 +VEGF50154,26611,480kd2 p22 +VEGF50193,07436,06331.83318082246/26/1517.7468.4437.5834.65104.9826.826/26/15100385.8082919831195.3163440198591.7988036876/26/15100211.8245924718195.3163440198151.1999742335

background142,786background157,0115/7/1516.3674.0114.1814.6197.3224.285/7/15100452.295768442489.2992749743594.777361807810/28/15100122.329858788925.706661143532.2930608623

14.3.310/28/1598.58120.5925.3431.8312/15/15100140.721001484724.666707594812.0123648366

Densitometria lastra del 3-7-15 cp 1-7-15nota: bande nere12/15/1589.65126.1622.1110.771/13/16100593.1357814234765.8291967053736.621379007

p-AktmeanODtubulinameanODpAkt/tub96.68100.1121.808.581/18/16100186.7782247738197.0159635832185.7824134586

wt -ins157,1243,440wt -ins212,98851,3686.69677620311/13/1620.74122.99158.80152.75-

wt +ins184,17530,491wt +ins201,25039,63076.939187484275.4868.69

wt +VEGF150159,6125,928wt +VEGF150207,49845,87812.9212258599138.78108.90mean100.00558.30119.08590.17mean100.00225.97200.04173.77

kd -ins157,2443,560kd -ins206,85145,2317.87070814261/18/1616.3530.5432.2230.38dv.st0.00402.9953.90256.90dv.st0.00165.37259.47256.77

kd +ins187,30833,624kd +ins217,30555,68560.38250875460.030.0060.030.426

kd +VEGF150155,7392,055kd +VEGF150204,96743,3474.740812513mean58.2072.2573.1823.4894.8518.440.2530.164

background153,684161,620dv.st40.683.3148.166.5211.5710.41

0.290.17

lastre del 26-6-15 cp 18-6 p100nota: in rosso esclusi esperimenti wt no risp a VEGF


wt -ins185,66718,099wt -ins226,02759,96430.1831098659

wt +ins209,45141,883wt +ins222,42356,36074.3133427963

wt +VEGF50189,38921,821wt +VEGF50219,98653,92340.4669621497

kd -ins180,54412,976kd -ins224,01357,95022.3917169974

kd +ins214,40846,840kd +ins223,02156,95882.2360335686

kd +VEGF50172,2804,712kd +VEGF50222,51556,4528.3469141926




wt -ins176,0819,811wt -ins222,00055,30617.7394857701

wt +ins203,86537,595wt +ins221,62554,93168.4404070561

wt +VEGF50185,34219,072wt +VEGF50217,44950,75537.5765934391

kd -ins182,55616,286kd -ins213,69847,00434.648115054wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50

kd +ins213,68247,412kd +ins211,85645,162104.98206456767/3/156.7076.947.8760.387/3/156.7012.927.874.74

kd +VEGF50179,40813,138kd +VEGF50215,67648,98226.82209791356/26/1530.1874.3122.3982.246/26/156.048.094.481.67

background166,270background166,6946/26/1517.7468.4434.65104.986/26/157.1015.0313.8610.73

5/7/1516.3674.0114.6197.3210/28/156.167.541.581.99

lastre del 13-5-15 cp 8-512/15/155.988.411.470.72

p-AktmeanOD1/13/166.9141.0052.9350.92

wt -ins97,4765,6901/18/165.4510.1810.7410.13

wt +ins140,53548,749

wt +VEGF50101,7709,984

wt +VEGF15097,8466,060mean17.7573.4319.8886.23mean6.3314.7413.2811.56

kd -ins96,2714,485dv.st9.643.5711.4919.65dv.st0.5911.9018.0817.82

kd +ins152,95761,1710.000.0010.040.430

kd +VEGF5094,9893,2030.3930.165

kd +VEGF15094,2292,443

background91,786



wt -ins77,72512,031wt -ins137,56873,52516.36

wt +ins140,32474,630wt +ins164,881100,83874.01

wt +VEGF5081,29715,603wt +VEGF50174,046110,00314.18

kd -ins81,30915,615kd -ins170,906106,86314.61

kd +ins162,26496,570kd +ins163,26899,22597.32

kd +VEGF5076,37010,676kd +VEGF50108,00743,96424.28


lastre del 15.12.15 cp del 3.12.15 (Potito) wtp19/kd2p17

p-AktmeanODActinameanODpAkt/actin

wt -1113,69431,230wt -1150,46634,83589.65

wt -2116,89134,427wt -2151,23935,60896.68

wt +VEGF50 1126,88344,419wt +VEGF50 1150,84035,209126.16

wt +VEGF50 2121,28538,821wt +VEGF50 2154,41138,780100.11

kd2 -192,77210,308kd2 -1162,24446,61322.11

kd2 -296,53114,067kd2 -2180,15464,52321.80

kd2 +VEGF50 188,0715,607kd2 +VEGF50 1167,69652,06510.77

kd2 +VEGF50 284,9332,469kd2 +VEGF50 2144,41728,7868.58


TubulinameanODpAkt/tub

wt -1200,53970,57444.25

wt -2215,51085,54540.24

wt +VEGF50 1216,74786,78251.18

wt +VEGF50 2218,93188,96643.64

kd2 -1213,24283,27712.38

kd2 -2213,29483,32916.88

kd2 +VEGF50 1215,84185,8766.53

kd2 +VEGF50 2208,25178,2863.15

background129,965

lasta del 13-1-16 cp del 17.12.15


wt -1102,57910,637wt -1149,25551,29720.74

wt +VEGF50 1157,64565,703wt +VEGF50 1151,37853,420122.99

wt -2146,85754,915wt -2170,71672,75875.48

wt +VEGF50 2127,43335,491wt +VEGF50 2149,62951,67168.69

kd2 -1167,57275,630kd2 -1145,58347,625158.80

kd2 +VEGF50 3187,56995,627kd2 +VEGF50 3160,56362,605152.75

wt -3168,06476,122wt -3152,80854,850138.78

wt +VEGF50 3147,39555,453wt +VEGF50 3148,87950,921108.90


lastra del 20.1.16 wb 18.1.16 cp vedi quaderno

p-AktmeanODTubulinameanODpAkt/tubulina

wt p20 -2182,78811,443wt p20 -2207,50869,97416.35

wt p20 +VEGF50 2192,43021,085wt p20 +VEGF50 2206,56569,03130.54

kd2 p18 -1192,30620,961kd2 p18 -1202,59365,05932.22

kd2 p18 +VEGF50 1190,17618,831kd2 p18 +VEGF50 1199,51661,98230.38


IKBa-NFkB

40.67599330046.5221844176

3.306600167811.5735223607

48.161030696710.4093376061

wt

kd

pAKT/Tub

NF-kB n-c

053.9033817395

402.9882219996256.9012048943

wt

kd2

HoxA5

0259.468648097

165.3739785067256.7718898938

wt

kd2

PARP

Densitometria 28.10.15 cp wt p25/kd2 p22

eNOSmeanOD14.3.3meanODeNOS/14.3.3

wt p25 -172,37030,350wt p25 -191,06834,05789.1153066917

wt p25 +VEGF50176,62634,606wt p25 +VEGF50189,30232,291107.1691802669

kd2 p22 -162,22020,200kd2 p22 -193,27336,26255.7056974243

kd2 p22 +VEGF50161,48219,462kd2 p22 +VEGF50193,07436,06353.9666694396


VEGFR2

Densitometria lastra del 21-10-15 cp del 19-10-15nota: bande nere

lastre del 25-9-15


wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622

wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30

wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39

kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474

kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33

kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82


lastra del 25-11-15lastra del 21-10

NF-kB p65meanOD14.3.3meanODNF-kB p65/14.3.3

wt -114,57936,699wt -144,06662,96558.28

wt +v50151,12973,249wt +v50138,67757,576127.22

kd -147,83569,955kd -158,98677,88589.82

kd +v50180,424102,544kd +v50179,32398,222104.40

kd2 -144,75166,871kd2 -174,70193,60071.44

kd2 +v50171,70393,823kd2 +v50151,83770,736132.64


-VEGF50

lastra del 21.10

IKB-αmeanODIKB-α/14.3.3

wt -138,70058,30592.60

wt +v50146,43066,035114.69

kd -162,14481,749104.96

kd +v50172,73892,34394.01

kd2 -167,39987,00492.95

kd2 +v50147,05666,66194.24

background80,395

VEGFR2

90.193494332384.866712302922.683367862232.4451951155

wt

kd

wt

kd

kd2

NF-kB p65

wt

kd

kd2

IKB-α

Densitometria lastra del 20-11-15 cp estrazione nucleo citosol: wtp17/kd2p15 (1x106 cell)+ wtp18/kdp18 (5x105 cell)nota: bande nerecitoplnuclSUMM31.748144877837.0358623023

NF-KbmeanODGAPDHmeanODNF-kB/GAPDHActinameanODNF-kB/ActinacitoplasmaNF-kB/GAPDH

wt p17 citopl130,44366,272wt p17 citopl191,99949,096134.98wt p17 citopl90,62918,472358.77citoplasmaNF-kB/GAPDH

kd2 p15 citopl171,805107,634kd2 p15 citopl196,12353,220202.24kd2 p15 citopl119,69647,539226.41WTKD2fold

wt p18 citopl168,943104,772wt p18 citopl182,82200.00wt p18 citopl118,86946,712224.29esp 20.11134.98202.241.4982960439WTKD2

kd p18 citopl169,506105,335kd p18 citopl175,08800.00kd p18 citopl132,44660,289174.72esp 13.1.1649.0880.761.6454767726esp 20.1130.993824443588.0407348343

background64,171background142,903background72,157esp.5.2.1659.48103.681.7432309455esp 16.1231.748144877837.0358623023

esp.10.2.16209.42269.311.2859996977esp 13.1.1649.0880.76

NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1esp 16.12222.2370141448259.2510361161.1665520126esp.5.2.1639.650459142769.1199073797

wt p17 nucleo87,45223,281wt p17 nucleo134,51656,72141.04mean81.1785629047128.8932870232esp.10.2.1629.917247183138.4735708332

kd2 p15 nucleo101,94337,772kd2 p15 nucleo121,29343,49886.84dev st46.882444060764.5456058631

wt p18 nucleo75,05310,882altra lastrawt p18 nucleo129,04051,24521.241.5877749299mean36.277935129462.6860150699

kd p18 nucleo74,73210,561kd p18 nucleo126,27448,47921.780.1793685306dev st8.126591533123.7439387463

background64,171background77,7951.7279377905

0.0232329513

nucleoNF-kB/CyclinG1


WTKD2fold

esp 20.1141.0486.842.1159844055WTKD2

esp 16.1279.8485.241.0675814633esp 20.1127.3657.8933333333

esp 13.1.16151.0826939471247.08973322551.6354602024esp 16.1279.8485.24

esp 5.2.1628.9738430584115.53557862753.9875821235lamin Besp 13.1.1675.5413469736123.5448666128

esp.10.2.16esp 5.2.1628.9738430584115.5355786275lamin B

esp.10.2.16

mean75.2353790321133.6765828756

dev st55.025803797376.8793600128mean52.930042288795.5536995557

1.7767782205dev st28.655490855830.0412797958

0.13127368911.8052828871

0.0429221995

Densitometria lastra del 16-12-15 cp estrazione nucleo citosol: wtp20/kd2 p19 25.11 /kd2 p20 1.12nota: kd2 errore CERII

NF-KbmeanODGAPDHmeanODNF-kB/GAPDH

wt p20 citopl172,08893,484wt p20 citopl190,74242,065222.24

kd2 p19 citopl140,63962,035kd2 p19 citopl158,6029,925625.04



NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1

wt p20 nucleo112,69634,612wt p20 nucleo144,31643,34979.84

kd2 p19 nucleo115,76937,685kd2 p19 nucleo147,31946,35281.30



Densitometria lastra del 13.1.16 cp estrazione nucleo citosol del 11.1.16 wtp19/kd2p18














NF-KbmeanODLamin BmeanODNF-kB/Lamin B




Densitometria lastra del 10.2.16 cp 8.2.16 estrazione nucleo citosol del wtp19/kd2p20






wt p19 nucleo168,89216,182wt p19 nucleo

kd2 p20 nucleo173,43320,723kd2 p20 nucleo

background152,710background

manca normalizzatore nucleo: Lamin B (lastra 17.2.16) troppo vicina a NFKB

wt p17

kd2 p15

NF-kB/Actin

wt p18

kd p18

wt p17

kd2 p15

NF-kB/Cyclin G1

wt p18

kd p18

wt

kd2

NF-kB/cyclG1

nucleo

wt

kd2

NF-kB/GAPDH

Citoplasma

55.025803797376.8793600128

wt

kd2

NF-kB/CyclG1

nucl 20.11/16.12

46.882444060764.5456058631

wt

kd2

NF-kB/GAPDH

citopl 20.11/16.12

8.126591533123.7439387463

wt

kd2

NF-kB/GAPDH

Citoplasma

28.655490855830.0412797958

NF-kB/CyclinG1

Nucleo

8.126591533123.7439387463

WT

KD2

28.655490855830.0412797958

WT

KD2

Densitometria lastra del 10.2.16 cp wtp29/kd2p27ntno fbsH2O2 1 mM

HoxA5meanODTubulinameanODHoxA5/Tubulina

WTp29 NT191,19229,444WTp29 NT193,80930,26397.29

WTp29 -FBS176,49214,744WTp29 -FBS196,89433,34844.21

WTp29 1mM H2O2201,42939,681WTp29 1mM H2O2196,69633,150119.70

KD2 p27 NT194,91833,170KD2 p27 NT198,75235,20694.22

KD2 p27 -FBS184,87523,127KD2 p27 -FBS201,85738,31160.37

KD2 p27 1mM H2O2190,52728,779KD2 p27 1mM H2O2198,86935,32381.47


Densitometria lastra 17.3.16 cp del 9.3/15.3/15.3.16


wt 9.3.16174,67032,439wt 9.3.16199,48757,23256.68

kd2 9.3.16168,10325,872kd2 9.3.16212,73270,47736.71

wt 15.3.16174,33632,105wt 15.3.16222,92480,66939.80

kd2 15.3.16162,44920,218kd2 15.3.16224,14181,88624.69

wt 15.3.16161,07018,839wt 15.3.16200,07157,81632.58

kd2 15.3.16157,51715,286kd2 15.3.16203,61461,35924.91


Densitometria lastra 17.3.16 cp del 15.2/22.2/26.2/29.2


wt 15.2.16177,71527,633wt 15.2.16182,42335,09478.74

wt 22.2.16170,40420,322wt 22.2.16187,10539,77651.09

wt 26.2.16172,95222,870wt 26.2.16181,64934,32066.64

wt 29.2.16200,54850,466wt 29.2.16188,65041,321122.13

kd2 15.2.16178,10028,018kd2 15.2.16188,68941,36067.74

kd2 22.2.16166,69516,613kd2 22.2.16197,00749,67833.44

kd2 26.2.16164,91914,837kd2 26.2.16190,93143,60234.03

kd2 29.2.16194,99044,908kd2 29.2.16172,27224,943180.04


Densitometria lastra 20.4.16 cp del 15.4.16


wt p17 1198,61841,726wt p17 1198,70544,50993.75

wt p17 2201,13044,238wt p17 2183,02128,825153.47

kd2 p19 1190,23533,343kd2 p19 1202,33348,13769.27

kd2 p19 2184,29327,401kd2 p19 2196,19041,99465.25

kd2 p19 3181,14724,255kd2 p19 3188,35034,15471.02




WT 6.4.16100,62413,499WT 6.4.16131,21919,84268.03

WT p19 NS1111,41624,291WT NS1135,41824,041101.04

WT p19 S1120,45533,330WT S1142,38231,005107.50meandev.sttest t

KD2 14.4.16114,28527,160KD2 14.4.16155,93044,55360.96wt92.1921.16915840950.0950304527

KD2 p22 NS1115,79128,666KD2 NS1147,03635,65980.39kd270.999.72899218

KD2 p22 S1117,37830,253KD2 S1153,62542,24871.61


nsns1s1

wt

kd2

wt

kd2

wt

kd2

wt

kd2

wt

kd2

Densitometria lastra 10.2.16 cp del 5.2.16NTNO FBSH2O2 1mM

PARPmeanODTubulinameanODPARP/Tubulina

WTp29 NT161,18514,933WTp29 NT193,80930,26349.34WT NTWT -FBSWT H2O2KD2 NTKD2 -FBSKD2 H2O2

WTp29 -FBS184,09937,847WTp29 -FBS196,89433,348113.4910.2.1649.3440835343113.491063931997.20965309224.333920354523.027328965627.71848370751mM H2O2

WTp29 1mM H2O2178,47732,225WTp29 1mM H2O2196,69633,15097.2112-18.2.1613.8809773647107.938595314119.37581274384.05015400291mM H2O2

KD2 p27 NT154,8198,567KD2 p27 NT198,75235,20624.3328.4.1614.2618.2422.229.949.407.41100uM H2O2

KD2 p27 -FBS155,0748,822KD2 p27 -FBS201,85738,31123.038.4.1615.636904208986.0782141944113.5084896238-2.167139376100uM H2O2

KD2 p27 1mM H2O2156,0439,791KD2 p27 1mM H2O2198,86935,32327.72


mean14.9552.1667.863.899.407.41

dev.st0.97322879347.970581526864.55251334598.561717336500

Densitometria lastra 17.3.16 cp del 12-18.2.16test.t


WT NT 12.2152,4464,630WT NT 12.2149,68233,35513.88

KD2 NT 12.2149,4861,670KD2 NT 12.2157,56041,2334.05

WT 1mM H2O2 O.N. 12.2153,3295,513WT 1mM H2O2 O.N. 12.2144,78028,45319.38

WT -FBS 12.2188,03540,219WT -FBS 12.2153,58837,261107.94

WT 2.5mM H2O2 5h 12.2155,7217,905WT 2.5mM H2O2 5h 12.2155,49939,17220.18

WT NT 18.2183,18835,372WT NT 18.2151,71335,38699.96

KD2 NT 18.2151,0013,185KD2 NT 18.2148,14131,81410.01

WT -FBS 18.2173,34025,524WT -FBS 18.2138,33822,011115.96

WT 1mM H2O2 5h 18.2156,8539,037WT 1mM H2O2 5h 18.2132,01915,69257.59

KD2 -FBS 18.2145,082-2,734KD2 -FBS 18.2124,4768,149-33.55


nb:normalizzatore no buono in ultimi pozzetti

Densitometria lastra 28.4.16 cp del 15.4.16stimolo con H2O2 100uM 48hstarvation 24h

acquisizione banda forma clivata


WT NT150,9918,015WT NT194,71256,20414.26

WT-FBS153,83110,855WT-FBS198,02859,52018.24

WT H2O2154,87311,897WT H2O2192,05653,54822.22

KD2 NT148,4485,472KD2 NT193,55355,0459.94

KD2-FBS148,7785,802KD2-FBS200,21161,7039.40

KD2 H2O2147,4544,478KD2 H2O2198,90060,3927.41


NTNO FBSH2O2 100µM



WT NT p26164,8586,732WT NT p26200,32543,05215.64

KD2 NTp25157,041-1,085KD2 NTp25207,33950,066-2.17

WT 100 Um H2O2 48h199,03940,913WT 100 Um H2O2 48h193,31736,044113.51

KD2 100 Um H2O2 48h160,2412,115KD2 100 Um H2O2 48h203,75146,4784.55

WT 5mM H2O2160,9692,843WT 5mM H2O2183,49226,21910.84

WT -FBS178,33220,206WT -FBS180,74723,47486.08


wt

kd2

wt

kd2

Densitometria lastra 28.4.16 cp delnss

VEGFR2meanODTubulinameanODVEGFR2/Tubulinamean

WT NS 1133,85583,448WT NS 1156,91750,229166.14

WT NS 2142,33391,926WT NS 2163,44156,753161.98164.06

WT S1105,05654,649WT S1150,48243,794124.79

WT S296,16245,755WT S2144,05337,365122.45123.62

KD2 NS1134,93384,526KD2 NS1151,50444,816188.61

KD2 NS2136,65286,245KD2 NS2153,07746,389185.92187.26

KD2 S1102,99952,592KD2 S1157,81651,128102.86

KD2 S295,26844,861KD2 S2170,95064,26269.8186.34


nota: NS=non starvate S=starvate

wt

kd2

Grafico1

52.930042288795.553699555728.655490855830.0412797958

WT

KD2

p-Akt

Densitometria lastra del 3-7-15 cp 1-7-15nota: bande bianche

p-AktmeanODtubulinameanODpAkt/tubwt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50

wt -ins154,7141,986wt -ins219,80756,3653.5234631427/3/153.5273.5941.2517.99144.3816.62nota: stim vegf 150

wt +ins185,22332,495wt +ins207,60044,15873.588024826/26/1523.4952.1931.4818.7156.848.39

wt +VEGF150172,02519,297wt +VEGF150210,21946,77741.25317998166/26/1515.5362.3931.6028.1690.2522.60

kd -ins162,5769,848kd -ins218,18754,74517.98885742995/7/1514.3865.9413.8914.2591.6424.78

kd +ins210,24157,513kd +ins203,27639,834144.3816839886

kd +VEGF150160,5617,833kd +VEGF150210,57147,12916.6203399181mean14.1862.7234.7821.6297.1615.87

background152,728163,442dv.st10.0510.705.615.6744.187.13

0.020.17-insVEGF 50ng/ml

lastre del 26-6-15 cp 18-6 p100


wt -ins181,77516,039wt -ins237,56368,28023.4900410076

wt +ins199,80134,065wt +ins234,55165,26852.1924986211

wt +VEGF50185,31019,574wt +VEGF50231,46162,17831.4805879893

kd -ins178,52012,784kd -ins237,62468,34118.7061939392

kd +ins203,90338,167kd +ins236,43367,15056.8384214445

kd +VEGF50171,3775,641kd +VEGF50236,48467,2018.3942203241




wt -ins176,62010,706wt -ins229,65968,92315.5332762648

wt +ins208,24842,334wt +ins228,59467,85862.3861593327

wt +VEGF50186,23520,321wt +VEGF50225,04664,31031.5985072306

kd -ins182,98117,067kd -ins221,35460,61828.1550034643

kd +ins219,01653,102kd +ins219,57658,84090.2481305235

kd +VEGF50180,01914,105kd +VEGF50223,15962,42322.5958380725


lastre del 13-5-15 cp 8-5

p-AktmeanODMANCA NORMALIZZATORE

wt -ins97,1497,367

wt +ins131,32041,538

wt +VEGF50100,46610,684

wt +VEGF15097,0397,257

kd -ins95,7095,927

kd +ins141,01951,237

kd +VEGF5094,4204,638

kd +VEGF15094,0214,239

background89,782



wt -ins73,05011,771wt -ins146,94781,86614.38

wt +ins134,51673,237wt +ins176,153111,07265.94

wt +VEGF5078,20716,928wt +VEGF50186,950121,86913.89

kd -ins78,14816,869kd -ins183,419118,33814.25

kd +ins162,430101,151kd +ins175,455110,37491.64

kd +VEGF5073,48412,205kd +VEGF50114,32749,24624.78


lastre del 25-9-15


wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622

wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30

wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39

kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474

kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33

kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82


p-Akt

10.05161635495.6736980636

10.701721438144.1785639731

5.60847818227.1304694614

wt

kd

signaling pAKT

10.05161635495.6736980636

5.60847818227.1304694614

wt

kd

pAkt/tubulina (OD)

signaling eNOS

22.683367862232.4451951155

wt

kd

IKBa-NFkB

Densitometria 28.10.15 cp wt p25/kd2 p22-insVEGF 50ng/ml


wt p25 -176,35833,572wt p25 -191,06834,05798.5759168453wt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50

wt p25 +VEGF50181,72538,939wt p25 +VEGF50189,30232,291120.58777987677/3/156.7076.9412.927.8760.384.74nota: stim vegf 1507/3/151001148.8988903168117.5298069394901.66532258917/3/15100192.9469563869117.529806939470.7924584787

kd2 p22 -151,9759,189kd2 p22 -193,27336,26225.34057691256/26/1530.1874.3140.4722.3982.248.356/26/15100246.208369934174.1862488553272.45712563726/26/15100134.071546402974.186248855327.6542550773

kd2 p22 +VEGF50154,26611,480kd2 p22 +VEGF50193,07436,06331.83318082246/26/1517.7468.4437.5834.65104.9826.826/26/15100385.8082919831195.3163440198591.7988036876/26/15100211.8245924718195.3163440198151.1999742335

background142,786background157,0115/7/1516.3674.0114.1814.6197.3224.285/7/15100452.295768442489.2992749743594.777361807810/28/15100122.329858788925.706661143532.2930608623

14.3.310/28/1598.58120.5925.3431.8312/15/15100140.721001484724.666707594812.0123648366

Densitometria lastra del 3-7-15 cp 1-7-15nota: bande nere12/15/1589.65126.1622.1110.771/13/16100593.1357814234765.8291967053736.621379007

p-AktmeanODtubulinameanODpAkt/tub96.68100.1121.808.581/18/16100186.7782247738197.0159635832185.7824134586

wt -ins157,1243,440wt -ins212,98851,3686.69677620311/13/1620.74122.99158.80152.75-

wt +ins184,17530,491wt +ins201,25039,63076.939187484275.4868.69

wt +VEGF150159,6125,928wt +VEGF150207,49845,87812.9212258599138.78108.90mean100.00558.30119.08590.17mean100.00225.97200.04173.77

kd -ins157,2443,560kd -ins206,85145,2317.87070814261/18/1616.3530.5432.2230.38dv.st0.00402.9953.90256.90dv.st0.00165.37259.47256.77

kd +ins187,30833,624kd +ins217,30555,68560.38250875460.030.0060.030.426

kd +VEGF150155,7392,055kd +VEGF150204,96743,3474.740812513mean58.2072.2573.1823.4894.8518.440.2530.164

background153,684161,620dv.st40.683.3148.166.5211.5710.41

0.290.17

lastre del 26-6-15 cp 18-6 p100nota: in rosso esclusi esperimenti wt no risp a VEGF


wt -ins185,66718,099wt -ins226,02759,96430.1831098659

wt +ins209,45141,883wt +ins222,42356,36074.3133427963

wt +VEGF50189,38921,821wt +VEGF50219,98653,92340.4669621497

kd -ins180,54412,976kd -ins224,01357,95022.3917169974

kd +ins214,40846,840kd +ins223,02156,95882.2360335686

kd +VEGF50172,2804,712kd +VEGF50222,51556,4528.3469141926




wt -ins176,0819,811wt -ins222,00055,30617.7394857701

wt +ins203,86537,595wt +ins221,62554,93168.4404070561

wt +VEGF50185,34219,072wt +VEGF50217,44950,75537.5765934391

kd -ins182,55616,286kd -ins213,69847,00434.648115054wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50

kd +ins213,68247,412kd +ins211,85645,162104.98206456767/3/156.7076.947.8760.387/3/156.7012.927.874.74

kd +VEGF50179,40813,138kd +VEGF50215,67648,98226.82209791356/26/1530.1874.3122.3982.246/26/156.048.094.481.67

background166,270background166,6946/26/1517.7468.4434.65104.986/26/157.1015.0313.8610.73

5/7/1516.3674.0114.6197.3210/28/156.167.541.581.99

lastre del 13-5-15 cp 8-512/15/155.988.411.470.72

p-AktmeanOD1/13/166.9141.0052.9350.92

wt -ins97,4765,6901/18/165.4510.1810.7410.13

wt +ins140,53548,749

wt +VEGF50101,7709,984

wt +VEGF15097,8466,060mean17.7573.4319.8886.23mean6.3314.7413.2811.56

kd -ins96,2714,485dv.st9.643.5711.4919.65dv.st0.5911.9018.0817.82

kd +ins152,95761,1710.000.0010.040.430

kd +VEGF5094,9893,2030.3930.165

kd +VEGF15094,2292,443

background91,786



wt -ins77,72512,031wt -ins137,56873,52516.36

wt +ins140,32474,630wt +ins164,881100,83874.01

wt +VEGF5081,29715,603wt +VEGF50174,046110,00314.18

kd -ins81,30915,615kd -ins170,906106,86314.61

kd +ins162,26496,570kd +ins163,26899,22597.32

kd +VEGF5076,37010,676kd +VEGF50108,00743,96424.28


lastre del 15.12.15 cp del 3.12.15 (Potito) wtp19/kd2p17

p-AktmeanODActinameanODpAkt/actin

wt -1113,69431,230wt -1150,46634,83589.65

wt -2116,89134,427wt -2151,23935,60896.68

wt +VEGF50 1126,88344,419wt +VEGF50 1150,84035,209126.16

wt +VEGF50 2121,28538,821wt +VEGF50 2154,41138,780100.11

kd2 -192,77210,308kd2 -1162,24446,61322.11

kd2 -296,53114,067kd2 -2180,15464,52321.80

kd2 +VEGF50 188,0715,607kd2 +VEGF50 1167,69652,06510.77

kd2 +VEGF50 284,9332,469kd2 +VEGF50 2144,41728,7868.58


TubulinameanODpAkt/tub

wt -1200,53970,57444.25

wt -2215,51085,54540.24

wt +VEGF50 1216,74786,78251.18

wt +VEGF50 2218,93188,96643.64

kd2 -1213,24283,27712.38

kd2 -2213,29483,32916.88

kd2 +VEGF50 1215,84185,8766.53

kd2 +VEGF50 2208,25178,2863.15

background129,965

lasta del 13-1-16 cp del 17.12.15


wt -1102,57910,637wt -1149,25551,29720.74

wt +VEGF50 1157,64565,703wt +VEGF50 1151,37853,420122.99

wt -2146,85754,915wt -2170,71672,75875.48

wt +VEGF50 2127,43335,491wt +VEGF50 2149,62951,67168.69

kd2 -1167,57275,630kd2 -1145,58347,625158.80

kd2 +VEGF50 3187,56995,627kd2 +VEGF50 3160,56362,605152.75

wt -3168,06476,122wt -3152,80854,850138.78

wt +VEGF50 3147,39555,453wt +VEGF50 3148,87950,921108.90


lastra del 20.1.16 wb 18.1.16 cp vedi quaderno

p-AktmeanODTubulinameanODpAkt/tubulina

wt p20 -2182,78811,443wt p20 -2207,50869,97416.35

wt p20 +VEGF50 2192,43021,085wt p20 +VEGF50 2206,56569,03130.54

kd2 p18 -1192,30620,961kd2 p18 -1202,59365,05932.22

kd2 p18 +VEGF50 1190,17618,831kd2 p18 +VEGF50 1199,51661,98230.38


IKBa-NFkB

40.67599330046.5221844176

3.306600167811.5735223607

48.161030696710.4093376061

wt

kd

pAKT/Tub

NF-kB n-c

053.9033817395

402.9882219996256.9012048943

wt

kd2

HoxA5

0259.468648097

165.3739785067256.7718898938

wt

kd2

PARP

Densitometria 28.10.15 cp wt p25/kd2 p22

eNOSmeanOD14.3.3meanODeNOS/14.3.3

wt p25 -172,37030,350wt p25 -191,06834,05789.1153066917

wt p25 +VEGF50176,62634,606wt p25 +VEGF50189,30232,291107.1691802669

kd2 p22 -162,22020,200kd2 p22 -193,27336,26255.7056974243

kd2 p22 +VEGF50161,48219,462kd2 p22 +VEGF50193,07436,06353.9666694396


VEGFR2

Densitometria lastra del 21-10-15 cp del 19-10-15nota: bande nere

lastre del 25-9-15


wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622

wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30

wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39

kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474

kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33

kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82


lastra del 25-11-15lastra del 21-10

NF-kB p65meanOD14.3.3meanODNF-kB p65/14.3.3

wt -114,57936,699wt -144,06662,96558.28

wt +v50151,12973,249wt +v50138,67757,576127.22

kd -147,83569,955kd -158,98677,88589.82

kd +v50180,424102,544kd +v50179,32398,222104.40

kd2 -144,75166,871kd2 -174,70193,60071.44

kd2 +v50171,70393,823kd2 +v50151,83770,736132.64


-VEGF50

lastra del 21.10

IKB-αmeanODIKB-α/14.3.3

wt -138,70058,30592.60

wt +v50146,43066,035114.69

kd -162,14481,749104.96

kd +v50172,73892,34394.01

kd2 -167,39987,00492.95

kd2 +v50147,05666,66194.24

background80,395

VEGFR2

90.193494332384.866712302922.683367862232.4451951155

wt

kd

wt

kd

kd2

NF-kB p65

wt

kd

kd2

IKB-α

Densitometria lastra del 20-11-15 cp estrazione nucleo citosol: wtp17/kd2p15 (1x106 cell)+ wtp18/kdp18 (5x105 cell)nota: bande nerecitoplnuclSUMM31.748144877837.0358623023

NF-KbmeanODGAPDHmeanODNF-kB/GAPDHActinameanODNF-kB/ActinacitoplasmaNF-kB/GAPDH

wt p17 citopl130,44366,272wt p17 citopl191,99949,096134.98wt p17 citopl90,62918,472358.77citoplasmaNF-kB/GAPDH

kd2 p15 citopl171,805107,634kd2 p15 citopl196,12353,220202.24kd2 p15 citopl119,69647,539226.41WTKD2fold

wt p18 citopl168,943104,772wt p18 citopl182,82200.00wt p18 citopl118,86946,712224.29esp 20.11134.98202.241.4982960439WTKD2

kd p18 citopl169,506105,335kd p18 citopl175,08800.00kd p18 citopl132,44660,289174.72esp 13.1.1649.0880.761.6454767726esp 20.1130.993824443588.0407348343

background64,171background142,903background72,157esp.5.2.1659.48103.681.7432309455esp 16.1231.748144877837.0358623023

esp.10.2.16209.42269.311.2859996977esp 13.1.1649.0880.76

NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1esp 16.12222.2370141448259.2510361161.1665520126esp.5.2.1639.650459142769.1199073797

wt p17 nucleo87,45223,281wt p17 nucleo134,51656,72141.04mean81.1785629047128.8932870232esp.10.2.1629.917247183138.4735708332

kd2 p15 nucleo101,94337,772kd2 p15 nucleo121,29343,49886.84dev st46.882444060764.5456058631

wt p18 nucleo75,05310,882altra lastrawt p18 nucleo129,04051,24521.241.5877749299mean36.277935129462.6860150699

kd p18 nucleo74,73210,561kd p18 nucleo126,27448,47921.780.1793685306dev st8.126591533123.7439387463

background64,171background77,7951.7279377905

0.0232329513



WTKD2fold

esp 20.1141.0486.842.1159844055WTKD2

esp 16.1279.8485.241.0675814633esp 20.1127.3657.8933333333

esp 13.1.16151.0826939471247.08973322551.6354602024esp 16.1279.8485.24

esp 5.2.1628.9738430584115.53557862753.9875821235lamin Besp 13.1.1675.5413469736123.5448666128

esp.10.2.16esp 5.2.1628.9738430584115.5355786275lamin B

esp.10.2.16

mean75.2353790321133.6765828756

dev st55.025803797376.8793600128mean52.930042288795.5536995557

1.7767782205dev st28.655490855830.0412797958

0.13127368911.8052828871

0.0429221995

Densitometria lastra del 16-12-15 cp estrazione nucleo citosol: wtp20/kd2 p19 25.11 /kd2 p20 1.12nota: kd2 errore CERII

























NF-KbmeanODLamin BmeanODNF-kB/Lamin B




Densitometria lastra del 10.2.16 cp 8.2.16 estrazione nucleo citosol del wtp19/kd2p20






wt p19 nucleo168,89216,182wt p19 nucleo

kd2 p20 nucleo173,43320,723kd2 p20 nucleo

background152,710background

manca normalizzatore nucleo: Lamin B (lastra 17.2.16) troppo vicina a NFKB

wt p17

kd2 p15

NF-kB/Actin

wt p18

kd p18

wt p17

kd2 p15

NF-kB/Cyclin G1

wt p18

kd p18

wt

kd2

NF-kB/cyclG1

nucleo

wt

kd2

NF-kB/GAPDH

Citoplasma

55.025803797376.8793600128

wt

kd2

NF-kB/CyclG1

nucl 20.11/16.12

46.882444060764.5456058631

wt

kd2

NF-kB/GAPDH

citopl 20.11/16.12

8.126591533123.7439387463

wt

kd2

NF-kB/GAPDH

Citoplasma

28.655490855830.0412797958

NF-kB/CyclinG1

Nucleo

8.126591533123.7439387463

WT

KD2

28.655490855830.0412797958

WT

KD2

Densitometria lastra del 10.2.16 cp wtp29/kd2p27ntno fbsH2O2 1 mM


WTp29 NT191,19229,444WTp29 NT193,80930,26397.29

WTp29 -FBS176,49214,744WTp29 -FBS196,89433,34844.21

WTp29 1mM H2O2201,42939,681WTp29 1mM H2O2196,69633,150119.70

KD2 p27 NT194,91833,170KD2 p27 NT198,75235,20694.22

KD2 p27 -FBS184,87523,127KD2 p27 -FBS201,85738,31160.37

KD2 p27 1mM H2O2190,52728,779KD2 p27 1mM H2O2198,86935,32381.47


Densitometria lastra 17.3.16 cp del 9.3/15.3/15.3.16


wt 9.3.16174,67032,439wt 9.3.16199,48757,23256.68

kd2 9.3.16168,10325,872kd2 9.3.16212,73270,47736.71

wt 15.3.16174,33632,105wt 15.3.16222,92480,66939.80

kd2 15.3.16162,44920,218kd2 15.3.16224,14181,88624.69

wt 15.3.16161,07018,839wt 15.3.16200,07157,81632.58

kd2 15.3.16157,51715,286kd2 15.3.16203,61461,35924.91


Densitometria lastra 17.3.16 cp del 15.2/22.2/26.2/29.2


wt 15.2.16177,71527,633wt 15.2.16182,42335,09478.74

wt 22.2.16170,40420,322wt 22.2.16187,10539,77651.09

wt 26.2.16172,95222,870wt 26.2.16181,64934,32066.64

wt 29.2.16200,54850,466wt 29.2.16188,65041,321122.13

kd2 15.2.16178,10028,018kd2 15.2.16188,68941,36067.74

kd2 22.2.16166,69516,613kd2 22.2.16197,00749,67833.44

kd2 26.2.16164,91914,837kd2 26.2.16190,93143,60234.03

kd2 29.2.16194,99044,908kd2 29.2.16172,27224,943180.04




wt p17 1198,61841,726wt p17 1198,70544,50993.75

wt p17 2201,13044,238wt p17 2183,02128,825153.47

kd2 p19 1190,23533,343kd2 p19 1202,33348,13769.27

kd2 p19 2184,29327,401kd2 p19 2196,19041,99465.25

kd2 p19 3181,14724,255kd2 p19 3188,35034,15471.02




WT 6.4.16100,62413,499WT 6.4.16131,21919,84268.03

WT p19 NS1111,41624,291WT NS1135,41824,041101.04

WT p19 S1120,45533,330WT S1142,38231,005107.50meandev.sttest t

KD2 14.4.16114,28527,160KD2 14.4.16155,93044,55360.96wt92.1921.16915840950.0950304527

KD2 p22 NS1115,79128,666KD2 NS1147,03635,65980.39kd270.999.72899218

KD2 p22 S1117,37830,253KD2 S1153,62542,24871.61


nsns1s1

wt

kd2

wt

kd2

wt

kd2

wt

kd2

wt

kd2

Densitometria lastra 10.2.16 cp del 5.2.16NTNO FBSH2O2 1mM


WTp29 NT161,18514,933WTp29 NT193,80930,26349.34WT NTWT -FBSWT H2O2KD2 NTKD2 -FBSKD2 H2O2

WTp29 -FBS184,09937,847WTp29 -FBS196,89433,348113.4910.2.1649.3440835343113.491063931997.20965309224.333920354523.027328965627.71848370751mM H2O2

WTp29 1mM H2O2178,47732,225WTp29 1mM H2O2196,69633,15097.2112-18.2.1613.8809773647107.938595314119.37581274384.05015400291mM H2O2

KD2 p27 NT154,8198,567KD2 p27 NT198,75235,20624.3328.4.1614.2618.2422.229.949.407.41100uM H2O2

KD2 p27 -FBS155,0748,822KD2 p27 -FBS201,85738,31123.038.4.1615.636904208986.0782141944113.5084896238-2.167139376100uM H2O2

KD2 p27 1mM H2O2156,0439,791KD2 p27 1mM H2O2198,86935,32327.72


mean14.9552.1667.863.899.407.41

dev.st0.97322879347.970581526864.55251334598.561717336500

Densitometria lastra 17.3.16 cp del 12-18.2.16test.t


WT NT 12.2152,4464,630WT NT 12.2149,68233,35513.88

KD2 NT 12.2149,4861,670KD2 NT 12.2157,56041,2334.05

WT 1mM H2O2 O.N. 12.2153,3295,513WT 1mM H2O2 O.N. 12.2144,78028,45319.38

WT -FBS 12.2188,03540,219WT -FBS 12.2153,58837,261107.94

WT 2.5mM H2O2 5h 12.2155,7217,905WT 2.5mM H2O2 5h 12.2155,49939,17220.18

WT NT 18.2183,18835,372WT NT 18.2151,71335,38699.96

KD2 NT 18.2151,0013,185KD2 NT 18.2148,14131,81410.01

WT -FBS 18.2173,34025,524WT -FBS 18.2138,33822,011115.96

WT 1mM H2O2 5h 18.2156,8539,037WT 1mM H2O2 5h 18.2132,01915,69257.59

KD2 -FBS 18.2145,082-2,734KD2 -FBS 18.2124,4768,149-33.55


nb:normalizzatore no buono in ultimi pozzetti

Densitometria lastra 28.4.16 cp del 15.4.16stimolo con H2O2 100uM 48hstarvation 24h

acquisizione banda forma clivata


WT NT150,9918,015WT NT194,71256,20414.26

WT-FBS153,83110,855WT-FBS198,02859,52018.24

WT H2O2154,87311,897WT H2O2192,05653,54822.22

KD2 NT148,4485,472KD2 NT193,55355,0459.94

KD2-FBS148,7785,802KD2-FBS200,21161,7039.40

KD2 H2O2147,4544,478KD2 H2O2198,90060,3927.41


NTNO FBSH2O2 100µM



WT NT p26164,8586,732WT NT p26200,32543,05215.64

KD2 NTp25157,041-1,085KD2 NTp25207,33950,066-2.17

WT 100 Um H2O2 48h199,03940,913WT 100 Um H2O2 48h193,31736,044113.51

KD2 100 Um H2O2 48h160,2412,115KD2 100 Um H2O2 48h203,75146,4784.55

WT 5mM H2O2160,9692,843WT 5mM H2O2183,49226,21910.84

WT -FBS178,33220,206WT -FBS180,74723,47486.08


wt

kd2

wt

kd2

Densitometria lastra 28.4.16 cp delnss

VEGFR2meanODTubulinameanODVEGFR2/Tubulinamean

WT NS 1133,85583,448WT NS 1156,91750,229166.14

WT NS 2142,33391,926WT NS 2163,44156,753161.98164.06

WT S1105,05654,649WT S1150,48243,794124.79

WT S296,16245,755WT S2144,05337,365122.45123.62

KD2 NS1134,93384,526KD2 NS1151,50444,816188.61

KD2 NS2136,65286,245KD2 NS2153,07746,389185.92187.26

KD2 S1102,99952,592KD2 S1157,81651,128102.86

KD2 S295,26844,861KD2 S2170,95064,26269.8186.34


nota: NS=non starvate S=starvate

wt

kd2

*

0

0,1

0,2

0,3

0,4

0,5

0,6

WTMAECs

Glo1 KDMAECs

IgG

NF-

kB-p

65 (%

ofi

nput

)

0.0

0.1

0.2

0.3

0.4

0.5

0.6

NF-κB-p65 binding site:5’-TGGGCTTTTCC-3’

EV

NF-kB-p65

pGL3 NF-kB-p65 bs

- - +

**

0

1

2

3

4

5

6

7

8

9

10

Luc

ifera

se A

ctiv

ity(F

iref

ly R

LU

/Ren

illa

RL

U)

WTMAECs

Glo1KDMAECs

JSH-23 60µM - - +

Hox

A5

mR

NA

leve

ls(R

EU

)

** **

0

0,5

1

1,5

2

2,5

3

0.0

0.5

1.5

1.0

2.0

2.5

3.0

+

***

Glo1KD MAECs

NF-κB-p65 role in the regulation of MGO-mediatedHoxA5 overexpression

NF-kB-p65 positively regulates HoxA5 expression

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Summary 2

Glo1KD MAECs

nucleus

cytosol

HoxA5 gene HoxA5 mRNA

HoxA5

Anti-angiogenic factors

Pro-angiogenic factors

Glo1 MG

↑ NFkB

IKBα

NFkB ↑ NFkB mRNA

p50 p65

IKBα

p50 p65

p50 p65

ENDOTHELIAL CELL

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Conclusion

Partial deletion of Glo 1, associated to endogenous MGO accumulation, promotes

glucose intolerance, β-cell and vascular dysfunction

in a context of normglycaemia

Further efforts in the development of pharmacological intervention to interfere with

these molecular events, that lead to dicarbonyl stress, are needed to provide

new therapeutic options aimed at preventing the onset and progression of

vascular complications in diabetes

→ MGO levels and Glo 1 activity themselves are drivers of

glucose homeostasis impairment and vascular defects

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Aknowledgments

Fundings

Dr Claudia Miele

Prof Francesco Beguinot

Prof Pietro Formisano

Prof Peter P Nawroth

Dr Thomas Fleming

SID/FO.DI.RI (2013-2015) and EFSD/Novo Nordisk (2015-1017) to C. Miele

Dr Michele Ciccarelli

EASD Study GroupReactive Metabolites in Diabetes

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0

20

40

60

80

100

120

140

160

Trig

lyce

rides

(mg/

dl)

0

20

40

60

80

100

120

Chol

este

rol (

mg/

dl)

WTmice

Glo1KDmice

WTmice

Glo1KDmice

**

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WT mice

Glo1KDmice

F4/80 staining

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Glo1KD mice

0

20

40

60

80

100

120

140

160

180

5 10 15

Syst

olic

blo

od p

ress

ure

(mm

Hg)

Age (months)

wt mice

Glo1kd mice

**

*

Glo1KD mice

WT mice

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Glo1KD mice

0

0,2

0,4

0,6

0,8

1

1,2

% o

fres

idua

lvas

ocon

stric

tion

*** **

1.2

1.0

0.8

0.6

0.4

0.2

0.0

Glo1KD mice at 10 months of age show a higher systolic blood pressure and impaired endothelial-dependent vasodilation

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Diapositiva numero 1Diapositiva numero 2Diapositiva numero 3Diapositiva numero 4Diapositiva numero 5Diapositiva numero 6Diapositiva numero 7Diapositiva numero 8Diapositiva numero 9Diapositiva numero 10Diapositiva numero 11Diapositiva numero 12Diapositiva numero 13Diapositiva numero 14Diapositiva numero 15Diapositiva numero 16Diapositiva numero 17Diapositiva numero 18Diapositiva numero 19Diapositiva numero 20Diapositiva numero 21Diapositiva numero 22Diapositiva numero 23Diapositiva numero 24Diapositiva numero 25Diapositiva numero 26Diapositiva numero 27Diapositiva numero 28Diapositiva numero 29Diapositiva numero 30Diapositiva numero 31Diapositiva numero 32

Ruolo del Metilgliossalenello sviluppo delle complicanze ... Nigro Cecilia - Ruolo del...0.1%....

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Transcript of Ruolo del Metilgliossalenello sviluppo delle complicanze ... Nigro Cecilia - Ruolo del...0.1%....