Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Ann Thorac Surg. 2016 Apr 16;101(6):2085–2096. doi: 10.1016/j.athoracsur.2016.01.026

AKT2 Promotes Bone Marrow Cell–Mediated Aortic Protection in Mice

Sili Zou 1,2,3, Pingping Ren 1,2, Lin Zhang 1,2, Alon R Azares 4, Sui Zhang 4, Joseph S Coselli 1,2,5,6, Ying H Shen 1,2,5,*, Scott A LeMaire 1,2,5,6,*
PMCID: PMC4877213  NIHMSID: NIHMS750876  PMID: 27090732

Abstract

Background

Insufficient aortic protection and repair may contribute to the development of aortic aneurysms and dissections (AAD). However, mechanisms of aortic protection and repair are poorly understood. We have shown that the multifunctional kinase AKT2 plays an important role in protecting the aortic wall. Here, we examined whether AKT2 protects against AAD by promoting bone marrow cell (BMC)–mediated aortic protection.

Methods

Irradiated wild-type mice received green fluorescent protein–expressing BMCs from wild-type mice or Akt2−/− mice, followed by challenge with angiotensin II infusion for 4 weeks. BMC recruitment, aortic destruction, and AAD development were compared between groups. The direct effects of wild-type and Akt2−/− BMCs on vascular smooth muscle cell survival were examined in co-culture experiments.

Results

After angiotensin II infusion, no (0/14) wild-type BMC recipients developed AAD; in contrast, 64% (9/14) of Akt2−/− BMC recipients developed AAD (P=0.002) with severe aortic destruction. Compared to aortas from challenged wild-type BMC recipients, aortas from challenged Akt2−/− BMC recipients showed significantly less BMC recruitment, NG2+ progenitor activation, and FSP-1+ fibroblast activation. Additionally, aortas from challenged Akt2−/− BMC recipients showed increased apoptosis and inflammation. In co-culture experiments, wild-type but not Akt2−/− BMCs prevented smooth muscle cells from undergoing oxidative stress–induced apoptosis.

Conclusions

Following aortic challenge, BMCs are recruited to the aortic wall and provide protection by activating progenitors and fibroblasts and by promoting aortic cell survival. Our findings indicate that AKT2 is involved in these processes and that defects in this pathway may promote progressive degeneration during AAD development.

Keywords: aortic, aortic dissection, aortic aneurysm, apoptosis, cell signaling, molecular biology

The pathogenesis of aortic aneurysms and dissections (AAD) is not completely understood. The aortic wall is constantly subjected to biologic insults and hemodynamic stress, which can cause aortic tissue injury. When tissue is injured, an interconnected healing program is triggered to quickly restore tissue homeostasis [1]. Insufficient tissue repair may lead to tissue degeneration. The process of tissue repair in response to aortic injury is poorly understood.

Bone marrow (BM) contains multiple cell populations and plays a dynamic role not only in inflammation but also in tissue protection, repair, and remodeling. In response to tissue injury, BM cells (BMCs) are recruited to the injured area and participate in tissue healing by clearing up the damaged cells [2], producing growth factors [3], differentiating into various tissue component cells, and replacing the damaged cells [5]. BMCs are important for cardiac [6–9] and vascular [10–13] tissue protection and repair. However, the roles and the regulation of BMCs in AAD are largely unknown.

The AKT signaling pathway is an important pathway that regulates a diverse range of cellular functions including metabolism, survival, and angiogenesis [14]. Aberrant AKT activation and AKT dysfunction are key mechanisms underlying many pathophysiologic conditions, including type-2 diabetes, cardiovascular diseases, and cancer [14]. We have recently reported that AKT2 has profound protective effects in the aorta [15]; however, the underlying mechanisms remain to be elucidated. In this study, we examined whether AKT2 plays a role in BM-mediated aortic homeostasis.

Material and Methods

Study Design

All animal procedures were approved by the Institutional Animal Care and Use Committee at Baylor College of Medicine and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health. Akt2−/− (B6.Cg-Akt2tm1.1Mbb/J, Jackson Laboratory, Bar Harbor, ME) mice were bred with green fluorescent protein (GFP) transgenic mice to generate Akt2−/− GFP+ mice. Lethally irradiated 8-week-old wildtype (WT) male C57BL/6 mice were transplanted with either WT GFP+ BMCs (WT BM transplant [BMT] mice) or Akt2−/− GFP+ BMCs (Akt2−/− BMT mice) (Fig 1A). After 4 weeks of recovery, WT BMT mice received either continuous saline infusion (unchallenged; n=7) or continuous 1000 ng/min/kg angiotensin II (AngII) infusion [15] (challenged; n=14) for 4 weeks. Akt2−/− BMT mice were also challenged with AngII infusion (n=14). At the end of the study, mice were euthanized and their aortas were harvested for diameter measurement, histologic analysis and immunofluorescence staining.

Fig 1. Increased susceptibility to aortic aneurysm and dissection (AAD) development in mice with Akt2-deficient bone marrow cells (BMCs).

Fig 1

Open in a new tab

(A) Schematic illustration of the study design. Wild-type (WT) mice were irradiated and were then administered green fluorescent protein (GFP+) BMCs from either WT mice (GFP+ WT BMCs) or Akt2−/− mice (GFP+ Akt2−/− BMCs). Four weeks later, the mice were either unchallenged with saline or challenged with continuous angiotensin II (AngII) infusion for 4 weeks. (B) The BM reconstitution rate in GFP+ chimeric mice was 98%. (C) Representative aortas show the development of AAD in challenged Akt2−/− bone marrow transplant (BMT) mice but not in challenged WT BMT mice. (D) Comparison of aortic diameters among unchallenged WT BMT mice, challenged WT BMT mice, and challenged Akt2−/− BMT mice. Asc=ascending, Desc=descending, SR=suprarenal, IR=infrarenal. (E) No (0/14) challenged WT BMT mice developed AAD, but 64% (9/14) of the challenged Akt2−/− BMT mice developed AAD.

Bone Marrow Transplantation

Donor BMCs were harvested from 8-week-old male GFP transgenic mice. Recipient mice of the same age were lethally irradiated (total dose, 10 Gy [1000 rad]) and then received 5 × 106 BM donor cells via tail-vein injection. Success of the BM transplantation was confirmed by the percentage of GFP+ cells in the peripheral blood from recipient mice 4 weeks after transplantation.

Criteria for Aortic Aneurysm and Dissection Development

Aortic aneurysm was defined as an aortic diameter 1.5x greater than that of the unchallenged mice [15]. Aortic dissection was defined as blood or thrombus observed in the medial layer or between the media and adventitia.

Immunofluorescence Studies

Frozen sections of the suprarenal abdominal aorta were stained with antibodies against CD68, FSP-1, or NG2. Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI). GFP+ cells and immunostained cells were quantified by using Image-Pro Plus 6.0 (Media Cybernetics, Rockville, MD). Cells within the thrombus in the false lumen were excluded. Cells were counted in 4 fields per section at a magnification of 400X. Four sections per mouse were analyzed (unchallenged WT BMT, n=7; challenged WT BMT, n=14; challenged Akt2−/− BMT, n=14).

Cell Co-culture

Aortic smooth muscle cells (VSMCs) from WT mice were cultured without BMCs, with WT BMCs, or with Akt2−/− BMCs, followed by treatment with hydrogen peroxide (300 μm) for 24 hours. VSMC apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) kit (Roche, Indianapolis, IN).

Statistical Analysis

Statistical analyses were performed by using the Statistical Package for the Social Sciences (Version 22, SPSS Inc., Chicago, IL). The distribution of the data was examined by using the Kolmogorov-Smirnov test. Aortic diameters were compared by using the Kruskal-Wallis test. Aortic medial thickness and immunostaining were compared by using one-way analysis of variance with the Bonferroni correction. Two-tailed P-values are reported.

Results

Mice with Akt2-Deficient BM are Susceptible to AAD Development

To determine the role of AKT2 in BM-mediated aortic protection, we examined the effects of BM-specific Akt2 deficiency on AAD formation in mice that received BMT (Fig 1A). The BM reconstitution in chimeric mice was 98% (Fig 1B). Aortas from AngII-challenged WT BMT mice were grossly normal, whereas aortas from challenged Akt2−/− BMT mice showed AAD formation (Fig 1C). Although aortic diameters in challenged WT BMT mice were slightly larger than those in unchallenged WT BMT mice, aortic diameters in challenged Akt2−/− BMT mice were significantly larger than those in challenged WT BMT mice for all segments (Fig 1D). Furthermore, whereas no (0/14) challenged WT BMT mice developed AAD, 64% (9/14) of the challenged Akt2−/− BMT mice developed AAD (Fig 1E, P=0.002).

We further examined aortic structure in these mice. To differentiate the responses of the BMCs to aortic challenge versus aortic damage, we separately examined the changes in non-diseased (without AAD) and diseased (with AAD) aortas. Compared with aortas from challenged WT BMT mice, non-diseased aortas from challenged Akt2−/− BMT mice showed significantly reduced medial thickness (Fig 2) but not significant aortic destruction (Fig 2A) or elastic fiber disruption (Fig 2B). Diseased aortas from challenged Akt2−/− BMT mice showed severe aortic damage (Fig 2A and 2B) and a compensational increase in medial thickness in relatively intact areas of the aorta (Fig 2A–C). Together, these findings suggest that mice with Akt2-deficient BM are susceptible to AAD development, indicating a critical role for AKT2 in BM-mediated aortic protection.

Fig 2. Increased aortic damage in mice with Akt2-deficient bone marrow (BM) cells.

Fig 2

Fig 2

Open in a new tab

Representative images of (A) hematoxylin and eosin (HE) staining and (B) elastic fiber staining in challenged wild-type (WT) BM transplant (BMT) mice, Akt2−/− BMT mice without (w/o) aneurysm and dissection (AAD), and two Akt2−/− BMT mice with AAD. Dotted boxes indicate the magnified region. (C) Quantitative analysis of aortic medial thickness in the groups of mice shown in (A–B)

Mice with Akt2-Deficient BM Have Reduced Recruitment of BMCs to the Aortic Wall

To determine the role of AKT2 in BMC activation, we first examined its role in aortic BMC recruitment. As shown in Figure 3A, whereas a few GFP+ cells were detected in the aortas of unchallenged WT BMT mice, a significant number of GFP+ cells were observed in the aortic wall of challenged WT BMT mice, indicating the induction of aortic BMC recruitment. However, the BMC recruitment was compromised in the aortic wall of challenged Akt2−/− BMT mice, regardless of whether AAD was present (Fig 3B). These findings suggest that AKT2 may play a role in BMC recruitment in response to aortic stress.

Fig 3. Reduced bone marrow (BM) cell recruitment in the aortic wall of mice with Akt2-deficient BM cells.

Fig 3

Fig 3

Fig 3

Open in a new tab

(A) Representative immunofluorescence images and (B) quantitative analysis showing the recruitment of BM-derived (GFP+) cells in challenged or unchallenged wild-type (WT) BM transplant (BMT) mice and challenged Akt2−/− BMT mice with or without (w/o) aortic aneurysm and dissection (AAD). DAPI = 4′,6-diamidino-2-phenylindole.

Mice with Akt2-deficient BM Have Reduced BMC-Derived Vascular Progenitor Cells in the Aortic Wall

NG2 is a proteoglycan expressed in immature cell types, including pericytes and vascular progenitor cells [16]. NG2+ cells can participate in vascular repair through growth factor secretion and differentiation into SMCs [16]. Therefore, we examined the role of AKT2 in regulating NG2+ cells by counting the number of NG2+ cells. In WT BMT mice, aortic challenge significantly increased the number of NG2+ cells in the intima, media, and adventitia of the aortic wall (Fig 4A). Double immunofluorescence staining showed that these NG2+ cells were both of resident (GFP−NG2+) and BM-derived (GFP+NG2+) origin (Fig 4A). However, compared with aortas from challenged WT BMT mice, both non-diseased and diseased aortas from challenged Akt2−/− BMT mice had significantly fewer NG2+ cells (Fig 4B), which was attributed to a reduction in BM-derived (Fig 4C) but not resident NG2+ cells (Fig 4D). The ratio of BM-derived NG2+ cells/total BM-derived cells was also reduced in challenged Akt2−/− BMT mice (Fig 4E), suggesting the impaired activation of NG2+ cells in BMCs. These findings suggest that AKT2 in BMCs plays a role in the activation of BM-derived NG2+ cells.

Fig 4. Reduced induction of bone marrow (BM) cell–derived pericytes/vascular progenitors in the aortic wall of mice with Akt2-deficient BM cells.

Fig 4

Fig 4

Fig 4

Fig 4

Fig 4

Open in a new tab

(A) Representative immunofluorescence images and (B–D) quantitative analysis showing the induction of total (NG2+) (B), BM-derived (GFP+NG2+) (C), and resident (GFP−NG2+) (D) pericytes in the aortic wall of challenged wild-type (WT) BM transplant (BMT) mice and challenged Akt2−/− BMT mice with or without (w/o) aortic aneurysm and dissection (AAD). (E) The activation of NG2+ cells in BM-derived cells (GFP+NG2+ cells/GFP+ cells) is also shown. DAPI = 4′,6-diamidino-2-phenylindole.

Mice with Akt2-Deficient BM Have Reduced Activation of Fibroblasts in the Aortic Wall

Fibroblasts are an important component of the aortic wall and play diverse roles in tissue repair, remodeling, and inflammation [17, 18]. Using FSP1 as a fibroblast marker [19], we found that aortic challenge in WT BMT mice significantly increased the number of aortic FSP1+ cells, with most in the aortic adventitia and a few in the aortic media (Fig 5A). Double immunofluorescence staining indicated that fibroblasts were both resident (GFP−FSP1+) and BM derived (GFP+FSP1+), suggesting the differentiation of BMCs into fibroblasts. However, aortas from challenged Akt2−/− BMT mice had significantly fewer total FSP1+ cells (Fig 5B) due to the reduction in the number of both BM-derived (Fig 5C) and resident (Fig 5D) fibroblasts. The reduction of BM-derived fibroblasts in Akt2−/− BMT mice was more significant in diseased aortas than in non-disease aortas (Fig 5C). The ratio of BM-derived fibroblasts/total BM-derived cells was reduced in the aortas of challenged Akt2−/− BMT mice with AAD (Fig 5E), suggesting the impaired activation of fibroblasts in BMCs. Together, these findings suggest that AKT2 in BMCs plays a role in the activation of BM-derived fibroblasts, as well as resident fibroblasts.

Fig 5. Reduced induction of fibroblasts in the aortic wall of mice with Akt2-deficient bone marrow (BM) cells.

Fig 5

Fig 5

Fig 5

Fig 5

Fig 5

Open in a new tab

(A) Representative immunofluorescence images and quantitative analysis (B–D) showing the induction of total (FSP1+) (B), BM-derived (GFP+FSP1+) (C), and resident (GFP−FSP1+) (D) fibroblasts in the aortic wall of challenged wild-type (WT) BM transplant (BMT) mice and Akt2−/− BMT mice with or without (w/o) aneurysm and dissection (AAD). (E) The activation of fibroblasts in BM-derived cells (GFP+ FSP1+ cells/GFP+ cells) is also shown. DAPI = 4′,6-diamidino-2-phenylindole.

Mice with Akt2-Deficient BM Have an Increased Number of Macrophages in the Aortic Wall

Macrophages have been shown to play a destructive role in the formation and progression of AAD [20–22]. Therefore, we examined the role of AKT2 in macrophage activation during AAD formation. As shown in Figure 6, aortic challenge in WT BMT mice increased macrophages in the aortic wall, mostly in the adventitial layer (Fig 6A). Double immunofluorescence staining indicated that these macrophages were both BM derived (GFP+CD68+) and resident (GFP−CD68+) (Fig 6A). Non-diseased aortas from Akt2−/− BMT mice showed a trend of slightly increased total (Fig 6B) and BM-derived (Fig 6C) macrophages when compared with the aortas from WT BMT mice, indicating that AKT2 inhibits challenge induced macrophage activation, independent of aortic damage. However, diseased aortas from challenged Akt2−/− BMT mice had even more BM-derived (Fig 6C) and resident (Fig 6D) macrophages, suggesting that, by preventing aortic injury, AKT2 may also indirectly inhibit aortic inflammation.

Fig 6. Increased induction of macrophages in the aortic wall of mice with Akt2-deficient bone marrow (BM) cells.

Fig 6

Fig 6

Fig 6

Open in a new tab

(A) Representative immunofluorescence images and quantitative analysis (B–D) showing the induction of total (CD68+) (B), BM-derived (GFP+CD68+) (C), and resident (GFP−CD68+) (D) macrophages in the aortic wall of challenged wild-type (WT) BM transplant (BMT) mice. DAPI = 4′,6-diamidino-2-phenylindole.

Mice with Akt2-Deficient BM Have More Apoptotic Cells in the Aortic Wall

To further understand the role of AKT2 in BM-mediated aortic cell survival, we examined aortic cell apoptosis in challenged Akt2−/− BMT mice. As shown in Figure 7A, few TUNEL+ apoptotic cells were detected in the aortas of challenged WT BMT mice. However, significantly more TUNEL+ cells were observed in the aortas of challenged Akt2−/− BMT mice than in those of challenged WT BMT mice (Fig 7A and 7B). These findings not only highlight the importance of BMCs in aortic protection, but also indicate the critical role of AKT2 in BM-mediated aortic cell survival.

Fig 7. Critical role of Akt2 in bone marrow cell (BMC)-mediated aortic cell survival.

Fig 7

Fig 7

Fig 7

Fig 7

Open in a new tab

Representative images (A) and quantitative data (B) from TUNEL analysis showing more apoptotic cells in the aortic wall of challenged Akt2−/− bone marrow transplant (BMT) mice than in that of challenged wild-type (WT) BMT mice. (C–D) Vascular smooth muscle cells (VSMCs) co-cultured with either WT BMCs or Akt2−/− BMCs were treated with hydrogen peroxide. The protective effect of WT BMCs on VSMCs was significantly diminished in VSMCs co-cultured with Akt2−/− BMCs. DAPI = 4′,6-diamidino-2-phenylindole.

Akt2 Plays a Critical Role in BM-Mediated VSMC Survival

Finally, using a co-culture system, we examined whether AKT2 plays a direct role in the BM-mediated protection of VSMCs. In cultured VSMCs, hydrogen peroxide triggered VSMC apoptosis (Fig 7C and 7D), which was significantly reduced by co-culturing VSMCs with WT BMCs (P<0.001). This protective effect was significantly diminished in VSMCs co-cultured with Akt2−/− BMCs (P<0.001 vs. VSMCs + WT BMCs), suggesting that AKT2 plays a critical role in BM-mediated VSMC survival.

Comment

The processes of aortic repair are poorly understood. We showed that AngII challenge in WT mice induced BMC recruitment and activation, as indicated by increase in BM-derived NG2+ progenitor cells, fibroblasts, and macrophages in aortic wall. WT BMCs also promoted aortic SMC survival. These findings highlight a critical role for BMCs in the response to aortic stress and reflect the dynamic role of BMCs in aortic inflammation and protection.

What, then, controls BMC activity in response to aortic injury? AKT is critically involved in vascular [23, 24] and cardiac [25] repair. We show here that, under aortic stress, mice with AKT2 deficiency in BM showed significant aortic destruction leading to aortic enlargement and AAD formation. Our study highlights a critical role of AKT2 in promoting BM-mediated aortic protection and enhances our understanding of AKT2’s protective role against AAD, which we described previously [15].

Our study sheds light on the role of AKT2 in regulating BMC functions, such as BMC recruitment and pericyte/VSMC progenitor activation during aortic stress. NG2+ cells serve as VSMC progenitor cells [16], secrete growth factors [26] and promote endothelial survival [27], thus playing critical roles in cardiovascular repair [16, 26]. In our study, significant activation of BM-derived and resident NG2+ cells was observed in the injured aorta, suggesting a potential role for these cells in aortic protection and repair. However, aortas from challenged Akt2−/− BMT mice had significantly fewer NG2+ cells, suggesting that AKT2 may play a critical role in BM-derived vascular progenitor activation in response to aortic injury. Recent studies have suggested that AKT controls the balance between proliferation and the lineage differentiation of cardiac [28] and hematopoietic [29, 30] progenitor cells. Further studies are required to define the role of AKT2 in regulating NG2 cell recruitment, proliferation, or lineage differentiation during aortic injury.

Fibroblasts are able to migrate, proliferate rapidly, and synthesize connective tissue components to participate in tissue repair [25]. The quick and profound activation of fibroblasts may increase aortic strength and prevent aortic rupture in the acute phase of aortic injury. In the aortic wall of challenged WT BMT mice, we observed a significant amount of fibroblasts near the dissection area. However, fibroblast activation was impaired in challenged Akt2−/− BMT mice, indicating a critical role of AKT2 in BM-mediated fibroblast activation in the setting of acute aortic injury. However, fibroblasts can also produce cytokines and induce vascular inflammation and aortic destabilization [17, 18]. It remains to be determined whether AKT2 also drives the inflammatory reaction or maladaptive fibrotic remodeling.

Macrophages play dynamic roles in tissue injury, repair and remodeling. Although macrophages are important for clearing damaged tissues, they are also involved in aortic destruction and AAD formation [14]. AKT has dynamic role in inflammation and has been shown to promote [31] and inhibit [32–37] inflammation. In this study, we found that the number of macrophages in the aortic wall was significantly higher in challenged Akt2−/− BMT mice than in challenged WT BMT mice, indicating that the overall effect of AKT2 in BMCs was the inhibition of inflammation. Our study also suggests that AKT2 inhibits aortic inflammation both directly and indirectly by preventing aortic injury. Interestingly, we observed resident (GFP−) macrophages in the aortic wall. These cells may be less sensitive and survive the radiation. They may be activated and proliferate [38] in response to aortic injury and contribute to aortic inflammation.

Finally, we showed that AKT2 plays a critical role in BMC-mediated aortic cell survival. The amount of aortic apoptotic cells was significantly higher in challenged Akt2−/− BMT mice than in challenged WT BMT mice. One of the principal mechanisms of BM-mediated protection is a paracrine effect, which can potentially affect various aspects of aortic wall including cell survival. Indeed, we found that co-culturing VSMCs with WT BMCs reduced hydrogen peroxide–induced VSMC apoptosis, supporting the paracrine effect of BMCs. However, this protective effect was significantly diminished when VSMCs were co-cultured with Akt2−/− BMCs, providing strong evidence for a critical role of AKT2 in the paracrine actions of BMCs. However, additional studies will be required to identify the paracrine factors that are regulated by AKT2 in BMCs in response to aortic injury.

In conclusion, AKT2 regulates BMC-mediated aortic protection in response to aortic challenge by promoting aortic BMC recruitment, activating vascular progenitors and fibroblasts, reducing inflammation, and preventing apoptosis. Defects in this pathway may impair aortic repair mechanisms and promote progressive degeneration during AAD development.

Abbreviations

AAD

aortic aneurysms and dissection

AngII

angiotensin II

BM

bone marrow

BMC

bone marrow cell

BMT

bone marrow transplant

DAPI

4′,6-diamidino-2-phenylindole

GFP

green fluorescent protein

VSMC

vascular smooth muscle cell

WT

wild type

TUNEL

terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature. 2008;453:314–21. doi: 10.1038/nature07039. [DOI] [PubMed] [Google Scholar]
  • 2.Mouquet F, Pfister O, Jain M, et al. Restoration of cardiac progenitor cells after myocardial infarction by self-proliferation and selective homing of bone marrow-derived stem cells. Circ Res. 2005;97:1090–2. doi: 10.1161/01.RES.0000194330.66545.f5. [DOI] [PubMed] [Google Scholar]
  • 3.Kinnaird T, Stabile E, Burnett MS, et al. Marrow-derived stromal cells express genes encoding a broad spectrum of arteriogenic cytokines and promote in vitro and in vivo arteriogenesis through paracrine mechanisms. Circ Res. 2004;94:678–85. doi: 10.1161/01.RES.0000118601.37875.AC. [DOI] [PubMed] [Google Scholar]
  • 5.Wang X, Hu Q, Nakamura Y, et al. The role of the sca-1+/CD31− cardiac progenitor cell population in postinfarction left ventricular remodeling. Stem Cells. 2006;24:1779–88. doi: 10.1634/stemcells.2005-0386. [DOI] [PubMed] [Google Scholar]
  • 8.Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004;364:141–8. doi: 10.1016/S0140-6736(04)16626-9. [DOI] [PubMed] [Google Scholar]
  • 9.Assmus B, Honold J, Schachinger V, et al. Transcoronary transplantation of progenitor cells after myocardial infarction. N Engl J Med. 2006;355:1222–32. doi: 10.1056/NEJMoa051779. [DOI] [PubMed] [Google Scholar]
  • 11.Majka SM, Jackson KA, Kienstra KA, Majesky MW, Goodell MA, Hirschi KK. Distinct progenitor populations in skeletal muscle are bone marrow derived and exhibit different cell fates during vascular regeneration. J Clin Invest. 2003;111:71–9. doi: 10.1172/JCI16157. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Au P, Tam J, Fukumura D, Jain RK. Bone marrow-derived mesenchymal stem cells facilitate engineering of long-lasting functional vasculature. Blood. 2008;111:4551–8. doi: 10.1182/blood-2007-10-118273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischaemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet. 2002;360:427–35. doi: 10.1016/S0140-6736(02)09670-8. [DOI] [PubMed] [Google Scholar]
  • 14.Manning BD, Cantley LC. AKT/PKB signaling: navigating downstream. Cell. 2007;129:1261–74. doi: 10.1016/j.cell.2007.06.009. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Shen YH, Zhang L, Ren P, et al. AKT2 confers protection against aortic aneurysms and dissections. Circ Res. 2013;112:618–32. doi: 10.1161/CIRCRESAHA.112.300735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Majesky MW, Dong XR, Regan JN, Hoglund VJ. Vascular smooth muscle progenitor cells: building and repairing blood vessels. Circ Res. 2011;108:365–77. doi: 10.1161/CIRCRESAHA.110.223800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Duan J, Gherghe C, Liu D, et al. Wnt1/betacatenin injury response activates the epicardium and cardiac fibroblasts to promote cardiac repair. EMBO J. 2012;31:429–42. doi: 10.1038/emboj.2011.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Tieu BC, Lee C, Sun H, et al. An adventitial IL-6/MCP1 amplification loop accelerates macrophage-mediated vascular inflammation leading to aortic dissection in mice. J Clin Invest. 2009;119:3637–51. doi: 10.1172/JCI38308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Osterreicher CH, Penz-Osterreicher M, Grivennikov SI, et al. Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver. Proc Natl Acad Sci U S A. 2011;108:308–13. doi: 10.1073/pnas.1017547108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Curci JA, Liao S, Huffman MD, Shapiro SD, Thompson RW. Expression and localization of macrophage elastase (matrix metalloproteinase-12) in abdominal aortic aneurysms. J Clin Invest. 1998;102:1900–10. doi: 10.1172/JCI2182. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Gong Y, Hart E, Shchurin A, Hoover-Plow J. Inflammatory macrophage migration requires MMP-9 activation by plasminogen in mice. J Clin Invest. 2008;118:3012–24. doi: 10.1172/JCI32750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Rateri DL, Howatt DA, Moorleghen JJ, Charnigo R, Cassis LA, Daugherty A. Prolonged infusion of angiotensin II in apoE(−/−) mice promotes macrophage recruitment with continued expansion of abdominal aortic aneurysm. Am J Pathol. 2011;179:1542–8. doi: 10.1016/j.ajpath.2011.05.049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Koide M, Ikeda K, Akakabe Y, et al. Apoptosis regulator through modulating IAP expression (ARIA) controls the PI3K/Akt pathway in endothelial and endothelial progenitor cells. Proc Natl Acad Sci U S A. 2011;108:9472–7. doi: 10.1073/pnas.1101296108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Han JK, Lee HS, Yang HM, et al. Peroxisome proliferator-activated receptor-delta agonist enhances vasculogenesis by regulating endothelial progenitor cells through genomic and nongenomic activations of the phosphatidylinositol 3-kinase/Akt pathway. Circulation. 2008;118:1021–33. doi: 10.1161/CIRCULATIONAHA.108.777169. [DOI] [PubMed] [Google Scholar]
  • 25.Mangi AA, Noiseux N, Kong D, et al. Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts. Nat Med. 2003;9:1195–201. doi: 10.1038/nm912. [DOI] [PubMed] [Google Scholar]
  • 26.Katare R, Riu F, Mitchell K, et al. Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Circ Res. 2011;109:894–906. doi: 10.1161/CIRCRESAHA.111.251546. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Franco M, Roswall P, Cortez E, Hanahan D, Pietras K. Pericytes promote endothelial cell survival through induction of autocrine VEGF-A signaling and Bcl-w expression. Blood. 2011;118:2906–17. doi: 10.1182/blood-2011-01-331694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Fischer KM, Din S, Gude N, et al. Cardiac progenitor cell commitment is inhibited by nuclear Akt expression. Circ Res. 2011;108:960–70. doi: 10.1161/CIRCRESAHA.110.237156. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kobayashi H, Butler JM, O’Donnell R, et al. Angiocrine factors from Akt-activated endothelial cells balance self-renewal and differentiation of haematopoietic stem cells. Nat Cell Biol. 2011;12:1046–56. doi: 10.1038/ncb2108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Juntilla MM, Patil VD, Calamito M, Joshi RP, Birnbaum MJ, Koretzky GA. AKT1 and AKT2 maintain hematopoietic stem cell function by regulating reactive oxygen species. Blood. 2010;115:4030–8. doi: 10.1182/blood-2009-09-241000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Calamito M, Juntilla MM, Thomas M, et al. Akt1 and Akt2 promote peripheral B-cell maturation and survival. Blood. 2010;115:4043–50. doi: 10.1182/blood-2009-09-241638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Guha M, Mackman N. The phosphatidylinositol 3-kinase-Akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells. J Biol Chem. 2002;277:32124–32. doi: 10.1074/jbc.M203298200. [DOI] [PubMed] [Google Scholar]
  • 33.Schabbauer G, Tencati M, Pedersen B, Pawlinski R, Mackman N. PI3K-Akt pathway suppresses coagulation and inflammation in endotoxemic mice. Arterioscler Thromb Vasc Biol. 2004;24:1963–9. doi: 10.1161/01.ATV.0000143096.15099.ce. [DOI] [PubMed] [Google Scholar]
  • 35.Zhang WJ, Wei H, Hagen T, Frei B. Alpha-lipoic acid attenuates LPS-induced inflammatory responses by activating the phosphoinositide 3-kinase/Akt signaling pathway. Proc Natl Acad Sci U S A. 2007;104:4077–82. doi: 10.1073/pnas.0700305104. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Carpintero R, Brandt KJ, Gruaz L, Molnarfi N, Lalive PH, Burger D. Glatiramer acetate triggers PI3Kdelta/Akt and MEK/ERK pathways to induce IL-1 receptor antagonist in human monocytes. Proc Natl Acad Sci U S A. 2010;107:17692–7. doi: 10.1073/pnas.1009443107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Jenkins SJ, Ruckerl D, Cook PC, et al. Local macrophage proliferation, rather than recruitment from the blood, is a signature of TH2 inflammation. Science. 2011;332:1284–8. doi: 10.1126/science.1204351. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES