This study investigated how metabolic dysfunction induced by a Western diet affects levels of placenta growth factor (PLGF) in mouse skeletal muscle. Mice were fed either a control or Western diet for 6 months to induce varying degrees of metabolic dysfunction. Skeletal muscle PLGF levels were assessed at baseline and after femoral artery occlusion. The results showed that the Western diet reduced baseline PLGF levels in skeletal muscle across all mouse models. After occlusion, PLGF upregulation was inhibited in Western diet-fed mice compared to controls. This suggests that Western diet-induced metabolic dysfunction impairs the normal upregulation of PLGF in response to arterial occlusion. As PLGF is important for arteriogenesis, this phenomenon may contribute to the reduced ability

1. Abnormal Regulation of Placenta Growth Factor in Skeletal Muscle of Western-Diet
Fed Mice
Asitha T Silva1, Mark Payton2, Pamela G Lloyd1
1Department of Physiological Sciences,2 Department of Statistics, Oklahoma State University, Stillwater OK 74078
Funding : NIH R01 HL084494 (PL)
• We conclude that in skeletal muscle, Western diet-induced metabolic
dysfunction both reduces baseline levels of PLGF and inhibits its
upregulation by the physiological stimulus of occlusion. This phenomenon
may contribute to the poor arteriogenic response in diabetes.
• Arteriogenesis (collateral artery remodeling) is a vital adaptation of the vasculature in
response to peripheral artery disease (PAD)which serves to decrease ischemic injury
and averse limb amputations.
• Diabetics are prone to develop PAD. However, arteriogenesis is reduced in diabetics by
an undefined mechanism.
• Placenta growth factor (PLGF) is a key arteriogenic factor. Therefore, we hypothesized
that skeletal muscle PLGF expression and/or regulation could be abnormal in diabetic
PAD contributing to impaired arteriogenesis in diabetes..
Introduction
• We tested 4 mouse models exhibiting varying degrees of metabolic dysfunction
due to long term consumsion of western diet (WD)or control diet (CD)
 Hyperglycemic + hypercholesterolemic- WD fed C57BL/6J (C57-WD; n=58)
 Moderately hyperlipidemic-CD fed ApoE-/- (ApoE-CD; n=42)
 Extremely hyperlipidemic – WD fed ApoE-/- (ApoE-WD; n=45)
 Normoglycemic + normolipidemic control – CD fed C57BL/6J (C57-CD; n=52)
• Metabolic phenotype was confirmed by measuring plasma cholesterol,
triglycerides, insulin, and isoprostane; and by intraperitoneal glucose tolerance
testing (IPGTT).
• After 6 mo on the diets, skeletal muscle PLGF was assessed under baseline
conditions (n=58) and 3-28 d post-femoral artery occlusion (dpo; n=144).
• We performed gradual femoral artery (FA) occlusion method using ameroid
constrictors that enable to better mimic clinically relevant long tem arterial
occlusion . A small incision is made near the groin and FA is exposed from the
vein and the nerve.
• Upon placing the constrictor on FA followed by closing the wound mice were
returned to the diet program for 3-28 days before scarifying to isolate medial
thigh skeletal muscles.
• Skeletal muscle PLGF, VEGF, and PLGF receptor (VEGFR1) protein levels were
measured by ELISA. Data represents combined for genders.
Methods
Results
• We conclude that in skeletal muscle, Western diet-induced metabolic dysfunction
both reduces baseline levels of PLGF and inhibits its upregulation by the
physiological stimulus of occlusion. This phenomenon may contribute to the poor
arteriogenic response in diabetes.
Discussion and Conclusions
C57BL6/J ApoE-/-
Male Female Male Female P value
CD WD CD WD CD WD CD WD
Total cholesterol 104.94 ± 8.43† 338.30 ± 10.92*†‡ 77.61 ± 9.70† 260.09 ± 19.26*†‡ 614.20 ± 9.27† 1207.00 ± 16.70*† 604.13 ± 18.97† 1242.73 ± 18.24*†
*†‡<0.0001
Plasma isoprostane 84.17 ± 10.90† 298.40 ± 13.09*†‡ 60.90 ± 5.85† 194.93 ± 15.20*†‡ 275.13 ± 21.39† 492.70 ± 17.58*†‡ 302.88 ± 26.73† 574.28 ± 50.10*†‡
*†<0.0001
‡<0.05
Plasma triglycerides 94.38 ± 9.40† 92.75 ± 10.74† 103.81 ± 11.23† 133.79 ± 12.27† 204.72 ± 35.96†‡ 581.72 ± 15.94*†‡ 365.31 ± 18.57†‡ 733.31 ± 85.90*†‡
*<0.0001 †<0.05 ‡<0.01
Fasting glucose wk0 137.33 ± 10.64 136.60 ± 6.72 117.50 ± 6.62* 121.43 ± 7.60 135.83 ± 5.06 136.67 ± 10.11 125.33 ± 4.14 128.67 ± 7.20
‡<0.05 *†NS
Fasting glucose
wk23
131.67 ± 3.00† 193.80 ± 3.63*† 121.00 ± 2.23† 194.71 ± 5.09* 155.17 ± 4.83† 175.33 ± 4.94*† 164.50 ± 4.09† 181.67 ± 6.24
*†<0.05 ‡NS
Plasma insulin 1.36 ± 0.49 7.33 ± 0.43*† 1.61 ± 0.38 6.93 ± 0.78*† 0.54 ± 0.02 5.54 ± 0.45*† 0.79 ± 0.06 5.15 ± 0.33*†
*<0.0001 †<0.001 ‡NS
Area under the curve 26852 ± 1126‡ 39959 ± 742*†‡ 23003 ± 890‡ 44731 ± 2317*†‡ 23524 ± 1344 29876 ± 1161*† 24654 ± 1078 27245 ± 2806†
*<0.01 †<0.0001 ‡<0.05
HOMA-IR index 10.74 ± 1.01 60.31 ± 1.81* 10.29 ± 1.17 65.40 ± 7.94*† 4.51 ± 0.22 53.28 ± 4.34* 4.50 ± 0.30 42.66 ± 4.92*†
*†<0.0001 ‡NS
Values are mean ± SEM. * Diet effect by strain/gender; † strain effect by gender/diet; ‡ gender effect by strain/diet (3-way ANOVA followed by post-hoc testing using SAS software. Analysis performed by Dr. Mark Payton, Professor & Head, Dept.
of Statistics, Oklahoma State University).
0
5
10
15
20
25
30
35
40
0 5 10 15 20 25 30
MCP-1Ratio
Days post surgery
0.75
1.5
2.25
3
3.75
4.5
5.25
0 5 10 15 20 25 30
VEGFratio
Days post surgery
0.75
1.25
1.75
2.25
2.75
0 5 10 15 20 25 30
PLGFratio
Days post surgery
C57BL/6J Control diet
C57BL/6J Western diet
ApoE-/- Control diet
ApoE-/- Western diet
0.75
1.25
1.75
2.25
2.75
0 5 10 15 20 25 30
VEGFR1ratio
Days post surgery
+ Glucose
Insulin
X HOMA IR
PLGFpg/mgtotalprotein
r2 = 0.17
r2 = 0.42
r2 = 0.43
Cholesterol
8-Isoprostane
Plasma level of the metabolite
r2 = 0.001
r2 = 0.01
Plasma level of the metabolite
PLGFpg/mgtotalprotein
Figure 1 (above): Skeletal muscle PLGF protein was decreased in all WD fed
groups at baseline (p<0.005), with no effect of gender or strain. Figure 7 (below
left): Regression analysis of PLGF protein vs metabolic parameters revealed a
modest correlation with plasma insulin (r2=0.44, p<0.0001), (below right)but not
with plasma cholesterol and plasma isoprostane.
Figure 3 (left): Ameroid constrictor dimensions before placing on femoral artery. (center)
Isolation of femoral artery from nuro-vascular bundle. (Right)Placement of the constrictor on the
femoral artery close to the abdominal wall. Constrictor get fully close approximately in a week.
0
20
40
60
80
100
120
C57BL/6J C57BL/6J ApoE-/- ApoE-/-
VEGF,pg/mgtotalprotein
♀♂ ♀♂
Light bars: Control diet
Dark bars: High-fat diet
VEGF-A protein
Medial thigh
* p<0.0001 for diet by strain/gender
† p<0.0001 for strain by diet/gender
P=NS for gender by diet/strain
†
†
†
†
*
*
*
*
0
100
200
300
400
500
600
700
800
900
1000
C57BL/6J C57BL/6J ApoE-/- ApoE-/-
VEGFR1,pg/mgtotalprotein
♀♂ ♀♂
Light bars: Control diet
Dark bars: High-fat diet
VEGFR1 protein
Medial thigh
* p<0.05 for diet by strain/gender
† p<0.05 for strain by diet/gender
‡ p<0.01 for gender by diet/strain
‡
‡
*
*
†
†
†
†
0
5
10
15
20
25
30
35
40
45
50
C57BL/6J C57BL/6J ApoE-/- ApoE-/-
PLGF,pg/mgtotalprotein
♀♂ ♀♂
PLGF protein
Medial thigh
Light bars: Control diet
Dark bars: High-fat diet
* p<0.001 for diet by strain/gender
† p<0.05 for strain by diet/gender
‡ p<0.01 for gender by diet/strain
‡
*
* *
†
†
†
†
Figure 2 : (Left)Skeletal muscle VEGF increase in C57-WD in both genders . In contrast ,ApoE-WD
decreases VEGF levels. There are no gender difference s within each strain.(right) VEGF1 shows
gender differences in both strains.
Figure 4 :Effect of WD on PLGF, VEGF, VEGFR1 and MCP-1 expression regulation 3-28 days
following femoral artery occlusion Graphs represents the ratio of each proteins between
occluded leg and non occluded leg. (above left): PLGF expression inclined to maximum
within 3-10 days post ligation in C57-CD. How ever, all the other groups failed to elicit a
similar response. C57-WD showed delayed and reduced PLGF response. (above right and
below left) Similar behavior was seen for VEGF and VEGFR1 where the peak expression seen
in C57-CD expressions were absent in other test groups. (below right)MCP-1 proteins were
elevated early in C57BL/6J stain but not in ApoE-/-.