A Sticky Situation A mouse model of diabetes-accelerated atherosclerosis exhibits elevated endothelial cell VCAM1, ICAM1 and CCL2 expression: Possible role for hyperglycemia in combination with inflammation

Dilreet Rai1, Jenny E. Kanter2, Karin E. Bornfeldt 2

1UMKC School of Medicine, 2University of Washington School of Medicine

INTRODUCTION SUMMARY AND CONCLUSIONMETHODS

• Diabetes results in an increased risk of cardiovascular disease mediated by increased atherosclerosis

• Myeloid cell recruitment is critical for development of atherosclerotic lesions1

• Binding and recruitment of monocytes to the endothelium is mediated by adhesion molecules and chemokines including Vascular Cell Endothelial Molecule-1 (VCAM1), Intercellular Adhesion Molecule-1(ICAM1) and Chemokine (C-C motif) Ligand 2 (CCL2).

• Preliminary data show primary aortic endothelial cells from mice with diabetes express higher levels of adhesion molecules

FUTURE DIRECTIONSHYPOTHESES

The effect of diabetes on the endothelium is mediated by a combination of elevated glucose and inflammatory mediators

Hyperglycemia in combination with inflammation stimulates adhesion by increasing endothelial expression of VCAM1, ICAM1 as well as the chemokine CCL2

• Diabetes stimulates increased expression of Vcam1, Icam1 and Ccl2 mRNA in aortic endothelial cells in a mouse model of diabetes-accelerated atherosclerosis when compared with non-diabetic controls.

• VCAM1 is expressed in atherosclerotic lesions.

• TNF-α stimulates the expression of Vcam1, Icam1 and Ccl2 mRNA under normal glucose conditions.

• High glucose enhances the effect of TNF-α.

• These findings suggest that the impact of diabetes on the endothelium may be driven by combination of hyperglycemia and an increased inflammatory environment in diabetes.

• While the results show that endothelial cells stimulated in higher concentrations of glucose with TNF-α have increased adhesion molecules, it would be interesting to further explore if there is an increased monocyte adhesion in similar conditions.

• The potential increase in monocyte adhesion will also be tested under flow conditions, more closely mimicking in vivo physiology.

Diabetes accelerates early fatty streak lesions

Diabetes accelerates more advanced lesions

Endothelial changes are likely to play an important role. In addition, monocytes from people with type 1 diabetes have been

shown to be more pro-inflammatory3

5.5 mM glucose+19.5mM mannitol

5.5 mM glucose+19.5mM mannitol+20ng/ml TNFα

20 mM glucose+5mM mannitol

20 mM glucose+5mM mannitol + 20ng/ml TNFα

Stimulate 4 hours

Does aortic endothelium from STZ diabetic mice have increased expression of Icam1, Vcam1 and Ccl2?

Isolate aortic mRNA using Qiazol

*P=0.05

**

*

A. Blood glucose levels in STZ diabetic and non-diabetic mice are shown. B. VCAM1 immunoreactivity from lesions in brachiocephalic artery in non-diabetic, STZ diabetic mouse and IgG control are shown. C. mRNA levels of Vcam1, Icam1 and Ccl2 in STZ diabetic and non-diabetic mice are expressed as fold-over non-diabetic control. N=3-4, * p<0.05, t-test.

Isolation of Primary Endothelial Cells

Biotin conjugated anti CD 102/ICAM2 antibody + Biotin Selection cocktail+ magnetic particles

Cells expressing CD102/ICAM2

Anti-ICAM2/CD102 antibody-conjugated magnetic beads

Digestion medium: RPMI1640 + 2mg/mL Collagenase Type 1 + 0.25% Bovine Serum Albumin

Culture in collagen coated T175cm2 flask in Growth Media containing VEGF and ECGS until confluent (4-7 days)

LDLR-/-

STZ is a β-cell toxin taken up via GLUT2. 50mg/kg for 5 days Non-diabetic Diabetic

Insulin Immunohistochemistry (pancreas)

qPCR analysis of Vcam1, Icam1 and Ccl2

Does elevated glucose stimulate expression of Icam1, Vcam1 and Ccl2 in primary endothelial cells?

Purification of Primary Endothelial Cells

Stimulation of Primary Endothelial Cells

Diabetes increases endothelial Vcam1, Icam1 and Ccl2

A. Blood glucose B. VCAM1 immunohistochemistry

C. Vcam1, Icam1 and Ccl2 mRNA expression

RESULTS

Glucose augments the expression of Vcam1, Icam1 and Ccl2 in primary mouse endothelial cells

Primary endothelial cells were stimulated with 5.5mM (NG: Normal Glucose) or 20mM (HG: High Glucose) for 4 hours in the absence or presence of 20ng/mL TNF-α. RNA was extracted, reverse transcribed and the expression of mRNA was quantified using qPCR. A. Vcam1 , B. Icam1 and C. Ccl2 mRNA levels are expressed as fold-over NG, without TNF-α control. N=5-6, * p<0.05, **p< 0.01, ANOVA

A. Vcam1 mRNA B. Icam1 mRNA

C. Ccl2 mRNA

Diabetes is likely to accelerate atherosclerosis by augmentingmonocyte adhesion to the endothelium2

control

Non-diabetic Diabetic

******

1. Tethering and rolling2. Adhesion3. Transmigration

Lipid loaded cellsLumen

Artery wall

Lesion

Macrophages

Monocytes

REFERENCES1Kanter JE, Bornfeldt KE (2012): Inflammation and diabetes-accelerated atherosclerosis: myeloid cell mediators. Trends Endocrinol Metab. 24,137-44.

2Nishizawa T, Kanter JE, Kramer F, Barnhart S, Shen X, Vivekanandan-Giri A, Wall VZ, Kowitz J, Devaraj S, O’Brien KD, Pennathur S, Tang J, Miyaoka RS, Raines EW, Bornfeldt KE (2014): Testing the role of myeloid cell glucose flux in inflammation and atherosclerosis. Cell Rep. 7, 356-65.

3Kanter JE, Averill MM, Leboeuf RC, Bornfeldt KE (2008): Diabetes-accelerated atherosclerosis and inflammation. Circ Res. 103, e116-7.