Decreased vascular lesion formation in mice with inducible endothelial-specific expression of protein kinase Akt

Abstract

To determine whether endothelial Akt could affect vascular lesion formation, mutant mice with a constitutively active Akt transgene, which could be inducibly targeted to the vascular endothelium using the tet-off system (EC-Akt Tg mice), were generated. After withdrawal of doxycycline, EC-Akt Tg mice demonstrated increased endothelial-specific Akt activity and NO production. After blood flow cessation caused by carotid artery ligation, neointimal formation was attenuated in induced EC-Akt Tg mice compared with noninduced EC-Akt Tg mice and control littermates. To determine the role of eNOS in mediating these effects, mice were treated with N-nitro-L-arginine methyl ester (L-NAME). Neointimal formation was attenuated to a lesser extent in induced EC-Akt Tg mice treated with L-NAME, suggesting that some of the vascular protective effects were NO independent. Indeed, endothelial activation of Akt resulted in less EC apoptosis in ligated arteries. Immunostaining demonstrated decreased inflammatory and proliferative changes in induced EC-Akt Tg mice after vascular injury. These findings indicate that endothelial activation of Akt suppresses lesion formation via increased NO production, preservation of functional endothelial layer, and suppression of inflammatory and proliferative changes in the vascular wall. These results suggest that enhancing endothelial Akt activity alone could have therapeutic benefits after vascular injury.

Figure 1

Endothelial-specific expression of inducible myrAkt transgene in EC-Akt Tg mice. (A) Western blots in primary MLEC cultures. The expression of myrAkt transgene was determined by the expression of HA tag. ECs from EC-Akt Tg mice were divided into 2 groups, with and without myrAkt gene induction. Phosphorylated forms of Akt, GSK-3, or eNOS were determined in these ECs. Each blot is representative of 3 independent experiments. (B) Endothelial expression of myrAkt in vivo. Immunostaining in carotid artery, showing HA-tag expression in the endothelial layer of an induced EC-Akt Tg mouse. Scale bar: 50 μm. (C) Immunoblots showing phosphorylation of Akt in aortae from induced EC-Akt Tg mice (left) and in primary cultures of aortic SMCs isolated from induced EC-Akt Tg mice (MASMC, right).

Figure 2

Endothelial NO production and bioactivity in EC-Akt Tg mice. (A) NO production in MLECs from induced EC-Akt Tg mice. Nitrite (NO₂) was measured in ECs under serum-starved conditions with or without myrAkt transgene induction. Results are expressed as mean ± SEM. n = 8 in each group. **P < 0.01 versus cells without gene induction. (B) Force measurement in isolated mouse aorta. Endothelial-dependent contractions in response to L-NAME were determined. Results are expressed as mean ± SEM. n = 6 in each group. *P < 0.05 versus WT.

Figure 3

Photomicrographs of ligated mouse carotid arteries. (A) Photomicrographs of carotid arteries 4 weeks after ligation (H&E staining) in WT, single-Tg (tet-myrAkt), noninduced EC-Akt Tg, and induced EC-Akt Tg mice. Arrowheads indicate internal elastic lamina. (B) Photomicrographs of H&E staining of carotid arteries 4 weeks after ligation in WT and induced EC-Akt Tg mice treated with L-NAME. Scale bars: 100 μm.

Figure 4

Quantitative analysis of vascular lesions in ligated carotid arteries. Results are expressed as mean ± SEM. Number of animals in each group is as indicated. **P < 0.01 versus WT, single-Tg, and noninduced EC-Akt Tg. ^†P < 0.05 versus WT, single-Tg, and noninduced EC-Akt Tg. ^#P < 0.05 versus WT + L-NAME. ^§P < 0.05 versus EC-Akt Tg.

Figure 5

EC apoptosis. (A) Immunocytochemistry and immunoblotting for cleaved (activated) caspase-3. MLECs from induced and noninduced EC-Akt Tg mice were examined (upper panels). To induce apoptosis, a serum deprivation method for 24 hours was used. Positive staining for cleaved caspase-3 (green) is noted in cells without gene induction. Results are expressed as mean ± SEM. n = 3 in each group. *P < 0.05 versus ECs from EC-Akt Tg mice without induction. Counterstaining with Hoechst 33258 shows shrinkage of nuclei in some cells with cleaved caspase-3 expression (arrowheads). Expression of myrAkt was confirmed by detection of the HA tag (red) (lower left panel). Representative Western blots for cleaved and noncleaved caspase-3 in ECs with or without myrAkt gene induction are shown in the lower right panel. (B) Immunocytochemistry for cleaved (activated) caspase-3 in ECs isolated from aortae of induced and noninduced EC-Akt mice. A representative result from 3 separate experiments is shown. Cl. cas-3, cleaved caspase-3; UnCl. cas-3, uncleaved caspase-3. Magnification, _400.

Figure 6

Immunostaining for cleaved (activated) caspase-3 in ligated carotid arteries. (A) Double-immunofluorescent staining in ligated carotid arteries showing ECs positively stained for cleaved caspase-3 (C. cas-3; green). Staining for PECAM-1 (red) was used to detect and assess the integrity of the endothelial layer. Scale bar: 50 μm. Arrowheads indicate positively stained ECs. L, lumen; A, adventitia. (B) The number of positive-staining cells in 4 separate sections per animal is quantified. Results are expressed as mean ± SEM. n = 4 in each group. *P < 0.05 versus WT.

Figure 7

TUNEL staining in ligated carotid arteries. (A) Double-immunofluorescent staining to detect TUNEL-positive, apoptotic ECs in ligated arteries (green). Staining for PECAM-1 (red) was used to detect and assess the integrity of the endothelial layer. Scale bar: 50 μm. Arrowheads indicate apoptotic ECs. (B) The number of positive-staining cells in 4 separate sections per animal is quantified. Results are expressed as mean ± SEM. n = 4 in each group. *P < 0.05 versus WT.

Figure 8

Vascular permeability in EC-Akt Tg mice. (A) Incorporation of Evans blue dye in the vessel wall of sham-operated, WT, and induced EC-Akt Tg mice 1 week after carotid artery ligation. The blue area corresponds to area of increased vascular permeability. (B) Positively stained area of the carotid artery per animal is quantified. Results are expressed as mean ± SEM. n = 6 in each group. *P < 0.05 versus WT.

Figure 9

Vascular inflammation following carotid artery ligation. (A) Immunostaining of the vascular wall for leukocyte accumulation (CD45-positive cells), the expression of ICAM-1 and VCAM-1, and cellular proliferation in sham-operated, WT, and induced EC-Akt Tg mice after carotid artery ligation. Arrows indicate positively stained cells. Scale bar: 50 μm. (B) Quantification of cells that stained positively for CD45 antigen or PCNA. Results are expressed as mean ± SEM. n = 4 in each group. *P < 0.05 versus WT.