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Profilin acts downstream of LDL to mediate diabetic endothelial cell dysfunction ¹

GIULIO ROMEO^*, JOHN V. FRANGIONI and ANDRIUS KAZLAUSKAS^*,2

^* Schepens Eye Research Institute and Department Of Ophthalmology, and
Beth Israel Deaconess Center, Harvard Medical School, Boston, Massachusetts, USA

² Correspondence: Schepens Eye Research Institute, Harvard Medical School, 20 Staniford St., Boston, MA 02114, USA. E-mail: Kazlauskas{at}vision.eri.harvard.edu

SPECIFIC AIMS

The changes occurring on the surface of endothelial cells (EC) in diabetes have not been fully elucidated in vivo. it is unclear whether such changes may play a role in diabetic endothelial dysfunction (ED), which precedes and may contribute to the structural vascular abnormalities. The aims of this study were to 1) determine whether changes occur on the luminal surface of the diabetic endothelium in vivo and 2) test their role in diabetic ED. We have applied the unbiased approach of peptide phage display library to identify EC surface targets in the intact aorta and retina of diabetic rats.

PRINCIPAL FINDINGS

1. Profilin-1 is a binding partner for a diabetic aorta-specific phage clone and is increased in the diabetic aorta endothelium
A high-complexity peptide phage display library was screened in the intact aorta and retina EC of 5 month diabetic rats ex vivo. Phage clones isolated after the last round of selection were further tested for their ability to bind the EC in the respective tissue sections by immunofluorescence. Several phage clones (each bearing a different peptide) were found to recognize both the diabetic and the nondiabetic endothelium. However, 50% of the phage clones were able to bind only diabetic sections. We reasoned that the isolation of the cognate binding partner(s) on the EC for the diabetic-specific clones would provide new insight into the molecular bases of diabetic ED. We focused on the diabetic aorta-specific clone 45. Using a novel filter retention assay, we purified profilin-1 as a binding partner for clone 45 and showed they can interact directly in vitro. Western blot analysis showed that profilin-1 levels were significantly increased in the aorta EC of diabetic rats (2.1±0.32-fold over control; n=5; P<0.05) and diabetic individuals (1.95±0.37-fold over nondiabetic specimens; n=3; P<0.05). Immunofluorescence analysis in rat aorta sections confirmed that profilin was increased in diabetic specimens with a preferential staining of the endothelial layer. We have shown that the diabetic endothelial surface displays a specific repertoire of moieties and that a binding partner for one of these, profilin-1, is increased in the diabetic aorta.

2. Profilin-1 mediates endothelial dysfunction
We extended this finding to profilin-1 function in diabetic vascular disease. Profilin overexpression in rat aortic EC (RAEC) increased cell death by apoptosis (12.5±3.3% vs. 5±2.1%; n=4; P<0.05) compared with vector-RAEC. Then we assessed whether profilin-1 overexpression could alter the levels of ICAM-1, a key player in leukocyte adhesion and inflammation. ICAM-1 was significantly up-regulated in profilin-RAEC (2.2±0.14-fold over vector-RAEC; n=6; P=0.03). Likewise, ICAM-1 levels were elevated in the diabetic aorta EC in vivo (1.85±0.9-fold over control; n=8; P<0.05). Diabetic individuals show an impaired nitric oxide (NO) -mediated vasodilation. The vasodilator-stimulated phosphoprotein (VASP) interacts with profilin and can be used as an indicator of nitric oxide signaling (phosphorylation at Ser239). We evaluated whether profilin-1 overexpression could correlate with VASP phosphorylation in vitro and in vivo. In profilin-RAEC, phospho-VASP was dramatically decreased (0.32±0.11-fold over vector-RAEC; n=4; P<0.01) whereas total VASP levels were increased. Phospho-VASP was also significantly decreased in the diabetic aorta (0.52±0.13-fold over control; n=7; P<0.05) whereas VASP levels were comparable. Immunofluorescence analysis for phospho-VASP showed a marked decrease in the endothelial layer of diabetic rat aorta. Conversely, staining for total VASP was similar in control and diabetic sections. In summary, profilin modulates three indicators of ED: 1) increased apoptosis, 2) ICAM-1 up-regulation, and 3) a decrease in VASP phosphorylation. The last two abnormalities were recapitulated in the diabetic aorta in vivo concomitant to the aforementioned profilin increase.

3. Profilin-1 expression is increased by LDL and in the atherosclerotic plaque
We investigated the mechanisms responsible for the diabetes-mediated profilin increase. We found that oxidized LDL and, to a lesser extent, native LDL were able to up-regulate profilin protein (Fig. 1 a) and RNA levels (Fig. 1b ) in RAEC. We further examined which component(s) of the oxLDL particle was sufficient to reproduce the profilin increase. Treatment with oxysterols (7-keto- and 7ß-hydroxycholesterol), but not native cholesterol, elevated profilin (Fig. 1c ). Exposure to high (30 mM) glucose or increasing concentration of free fatty acids, both hallmarks of type 2 diabetes, exerted no effect on profilin levels (Fig. 1d ). These results suggest a critical role for LDL/oxysterols in the diabetes-mediated profilin-1 elevation.

View larger version (56K): [in this window] [in a new window]   Figure 1. LDL and oxysterols, but not high glucose, can up-regulate profilin-1 in RAEC. Immunoblot analysis shows a dose-dependent increase in profilin levels upon stimulation with native or oxidized LDL, along with ICAM-1 up-regulation (a) and a parallel up-regulation of profilin transcript (b) (c)Immunoblot analysis shows that oxysterols (7ß-hydroxy- and 7-ketocholesterol), but not cholesterol, can elevate profilin levels. Treatment with 7-ketocholesterol determined a parallel increase in ICAM-1. Caveolin-1, known to be regulated by sterols, was used as positive control. d) In contrast, RAEC exposed to high glucose (HG, 30mM) for 2 wk or to increasing concentrations of free fatty acids (FFA) did not show any profilin elevation. The relative molecular mass (kDa) of the proteins is indicated on the left.

Circulating levels of LDL are a major risk factors for atherosclerosis. ED herald and possibly underlie atherosclerotic plaque formation and progression. Thus, we tested the expression of profilin in sections cut from the aortic sinus of apoE null mice, a validated model of atherosclerosis that shows elevated levels of LDL. Profilin-1 expression was dramatically up-regulated in atherosclerotic lesions compared with the adjacent lesion-free, Oil Red O-negative areas (Fig. 2 a). Double labeling with the endothelial marker PECAM-1 and the monocyte marker F4/80 showed that profilin was elevated in these two cell types within the plaque (Fig. 2b and c , respectively). These results suggest a role for profilin-1 in atherosclerosis and are consistent with the finding of LDL as a mediator of profilin-1 increase.

View larger version (62K): [in this window] [in a new window]   Figure 2. Profilin-1 expression is increased in atherosclerotic lesions. a) Immunofluorescence analysis for profilin (red; left panel) in sections from the aortic sinus of apoE null mice shows increased staining in atherosclerotic lesions (arrow) compared with lesion-free adjacent areas. Lesions were identified by Oil Red O staining, followed by counterstain with hematoxylin/light green (middle panel). Negative control (right panel) was obtained using nonimmune rabbit IgG on a consecutive section. EC, endothelium: Adv., tunica adventitia. Scale bar: 12 µm. b) Immunohistochemistry analysis shows that profilin (brown) is highly expressed in EC within lesions (arrows) as evaluated by double-labeling staining with the EC marker PECAM (red). Inset shows a detail of a double-stained EC (arrow) and vasa vasorum of the aorta (small arrows) positive only for PECAM. Scale bar: 45 µm. c) Immunohistochemistry analysis demonstrates that profilin expression (brown) is increased in macrophage (arrow) as evaluated by staining with the marker F4/80 (red). Scale bar: 12 µm.

CONCLUSIONS AND SIGNIFICANCE

We have successfully applied our integrated strategy to identify surface targets associated with diabetes and test their role in diabetic vascular disease. The selection of peptides specific for the endothelium of diabetic retina and aorta proves our working hypothesis that diabetes changes the surface of the endothelium. We extended the identification of diabetes-specific peptides by isolating their counterpart on the endothelium and found that profilin-1 is a binding partner for a diabetic aorta-specific peptide. We have begun to test the role of profilin in diabetic EC dysfunction. We demonstrated that profilin levels are increased in the aorta endothelium in both human and experimental diabetes. Profilin overexpression in primary aorta EC was able to trigger a maladaptive program characterized by increased apoptosis, ICAM-1 up-regulation, and decreased VASP phosphorylation. The last two alterations were recapitulated in the diabetic aorta in vivo. We also found that LDL/cholesterol signaling, but not high glucose, regulates profilin, which in turn is required for LDL-mediated ICAM-1 up-regulation. Finally, profilin was markedly increased in EC and macrophages within atherosclerotic lesions of apoE null mice. Thus, we propose a new model whereby profilin contributes to diabetic macrovascular disease as an effector of a metabolic pathway downstream of LDL/oxysterols.

View larger version (11K): [in this window] [in a new window]   Figure 3. Profilin-1 mediates LDL-induced endothelial dysfunction. LDL alone, and possibly within the diabetic milieu, up-regulates profilin, which in turn mediates endothelial dysfunction leading to accelerated atherogenesis.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0841fje; doi: 10.1096/fj.03-0841fje

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