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Supplemental Materials

"Regional determinants of arterial endothelial phenotype dominate the impact of gender or short-term exposure to a high-fat diet", Passerini et al., Biochem Biophys Res Commun., 2005



Supplementary Methods (PDF)

Supplementary Figure 1: Endothelial cell sample characterization

A. Nuclear staining en face  with Hoechst 33258. EC displayed alignment and morphology characteristic of their local hemodynamic environment. B. H & E stained histology sections. Histology was consistent with that of normal vessels and there was no evidence of pathology associated with either flow region.  Staining for oil red-O (not shown) was negative. C. Staining with EC-specific PECAM-1 (green) and SMC-specific a -actin (red) antibodies. Pure endothelial populations (>99%) were rapidly and routinely isolated.

 

 

 

 

Table II (Online Version): Differentially Expressed Genes by Region within Animal Group

 

 

ALL (n=5)

FDR = 10%

HCM (n=5)

FDR = 15%

NM (n=5)

FDR = 15%

NF (n=5)

FDR = 15%

DF-A vs UF-A

1475

364

259

220

DF-A vs UF-C

1842

604

48

506

UF-A vs UF-C

1158

130

16

241

 

DF-A = disturbed flow region (aortic arch), UF-A = undisturbed flow region (descending thoracic aorta), UF-C = undisturbed flow region (common carotid), ALL = across all animals, HCM = high cholesterol males, NM = normal males, NF = normal females, FDR = false discovery rate

 

 

 

Table III (Online Version): Differentially Expressed Genes by Risk Factor (Gender or Diet) within Region

 

 

DF-A (n=5)

FDR = 50%

UF-A (n=5)

FDR = 50%

UF-C (n=5)

FDR = 50%

HCM vs NM

0

0

0

NM vs NF

2

4

10

 

DF-A = disturbed flow region (aortic arch), UF-A = undisturbed flow region (descending thoracic aorta), UF-C = undisturbed flow region (common carotid), HCM = high cholesterol males, NM = normal males, NF = normal females, FDR = false discovery rate

 

 

 

Supplementary Figure 2: Venn diagram showing the distribution of differentially expressed genes (DF-A vs UF-A) identified by PaGE analysis (FDR = 15%, confidence ≥ 85% within each animal subgroup (n=5)

 

 

 

HCM = high cholesterol males, NM = normal males, NF = normal females

 

 

 

Table IV (Online Version): Differentially expressed genes (DF-A vs UF-A) by biological classification

 

Biological Classification

# Genes Represented

# Differentially Expressed (DF-A/UF-A)

ALL (n=15) FDR=10%

HCM (n=5) FDR=15%

NM (n=5) FDR=15%

NF (n=5) FDR=15%

All genes

12684

1475

364

259

220

Adhesion

498

69

18

20

17

Apoptosis

305

32

6

9

2

Cell Cycle

326

34

6

6

6

Coagulation

75

7

3

1

1

Complement

55

15

6

4

4

Extracellular Matrix

181

32

10

9

8

Growth Factor

263

46

19

17

15

Immune Response

321

48

18

12

9

Inflammation

169

19

6

2

4

Integrin

108

11

5

4

2

Lipid/Cholesterol/Fatty Acid Metabolism

337

37

14

10

3

NF-kB

17

1

0

0

0

Oxidative Mechanisms

176

22

7

4

6

Oxidative Stress

20

5

1

0

0

Proliferation

337

39

7

7

7

Signal Transduction

924

116

34

22

20

TNF

81

9

2

2

1

Transcription Factor

759

83

31

19

28

 

DF-A = disturbed flow region (aortic arch), UF-A = undisturbed flow region (descending thoracic aorta), ALL = across all animals, HCM = high cholesterol males, NM = normal males, NF = normal females, FDR = false discovery rate

 

 

 

Supplementary Table I: QRT-PCR Results

 

Gene

Median Expression Ratio (DF-A/UF-A) + SD

HCM

NM

NF

VCAM

0.50 + 0.32

1.02 + 0.42

0.62 + 0.88

eNOS

0.63 + 0.42

0.49 + 0.08

0.59 + 0.21

E-selectin

0.43 + 0.24

0.49 + 0.16

0.46 + 0.65

GPX

0.64 + 0.24

0.96 + 0.22

0.72 + 0.14

HO

1.19 + 1.99

0.85 + 0.41

1.42 + 0.57

 

* indicates significantly different from 1.00

HCM = high cholesterol males, NM = normal males, NF = normal females

 

 

 

Supplementary Table II: Over-represented biological categories identified by EASE analysis

 

DF-A vs UF-A

ALL

(n=15) FDR=10%

HCM

(n=5) FDR=15%

NM

(n=5) FDR=15%

NF

(n=5) FDR=15%

 

DF-A = disturbed flow region (aortic arch), UF-A = undisturbed flow region (descending thoracic aorta), ALL = across all animals, HCM = high cholesterol males, NM = normal males, NF = normal females

 

 

 

Supplementary Table III: Identification of Biological Themes using Ingenuity's Pathways Analysis

 

DF-A vs UF-A

Network Summary

Top Ranked Networks

High Level Functions

Canonical Pathways

ALL

1

2

3

4

5

ALL

ALL

HCM

1

2

3

4

5

HCM

HCM

NM

1

2

3

4

5

NM

NM

NF

1

2

3

4

5

NF

NF

 

DF-A = disturbed flow region (aortic arch), UF-A = undisturbed flow region (descending thoracic aorta), ALL = across all animals, HCM = high cholesterol males, NM = normal males, NF = normal females

 

 

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