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AKT1 Overexpression in Endothelial Cells Leads to the Development of Cutaneous Vascular Malformations In Vivo FREE

Betsy Perry, MD; Jacqueline Banyard, PhD; Elizabeth R. McLaughlin, MD; Randy Watnick, PhD; Allie Sohn, BS; David N. Brindley, PhD; Toshiyuki Obata, PhD; Lewis C. Cantley, PhD; Cynthia Cohen, MD; Jack L. Arbiser, MD, PhD
[+] Author Affiliations

Author Affiliations: Department of Dermatology, Emory University School of Medicine, Atlanta, Ga (Drs Perry, McLaughlin, Cohen, and Arbiser and Ms Sohn); Vascular Biology Program, Department of Surgery, Children's Hospital Boston (Drs Banyard and Watnick), and Division of Signal Transduction, Department of Medicine, Beth Israel Deaconess Medical Center (Drs Obata and Cantley), Harvard Medical School, Boston, Mass; Whitehead Institute for Biomedical Research and Massachusetts Institute of Technology (Drs Obata and Cantley), Cambridge; and Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton (Dr Brindley).


Arch Dermatol. 2007;143(4):504-506. doi:10.1001/archderm.143.4.504.
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Background  Vascular malformations are clinical disorders in which endothelial cells fail to remodel and/or undergo programmed cell death, leading to abnormal persistence of blood vessels. The abnormal persistence of vessels makes therapy difficult because these lesions are resistant to interventions that are effective against hemangiomas. Akt1 is a serine-threonine protein kinase, which is a key mediator of resistance to programmed cell death. Our objective was to determine whether sustained activation of Akt1 could lead to vascular malformation in mice.

Observations  We examined the effect of constitutive activation of Akt1 in murine endothelial cells (MS1 cells). Overexpression of active AKT1 in MS1 cells led to the development of vascular malformations, characterized by wide endothelial lumens and minimal investment of smooth muscle surrounding the vessels. The histologic features of these vascular malformations is distinct from ras-transformed MS1 cells (angiosarcoma) and suggest that differing signal abnormalities give rise to human vascular malformations vs malignant vascular tumors.

Conclusions  Inhibition of Akt signaling may be useful in the treatment of vascular malformations. Examination of problematic hemangiomas and vascular malformations for the presence of activated Akt or downstream targets of Akt, such as mammalian target of rapamycin (mTOR), may predict response to treatment with Akt inhibitors or rapamycin. This study provides a potential rationale for the systemic and topical use of these inhibitors for vascular malformations and hemangiomas.

Figures in this Article

A wide range of endothelial neoplasms occurs in humans. They can be classified into 3 categories based on their clinical behavior.1 These lesions include hemangiomas of childhood, which grow rapidly and regress. The second most common vascular lesion is a vascular malformation, which starts in childhood and grows progressively with the patient and never regresses. The least common vascular lesion is angiosarcoma, which is a highly malignant tumor derived from endothelial cells. Angiosarcomas have a great propensity for local invasion and distant metastasis, leading to death. Angiosarcoma cells have been demonstrated to have mutations in the tumor suppressor gene p53 and/or oncogenic H-ras.24 Mouse models of hemangiomas and angiosarcomas exist, but, to our knowledge, we describe the first example of a transplantable vascular malformation.

The akt1 oncogene has been implicated in a variety of malignant neoplasms, usually in conjunction with complex oncogenes such as ras, which activate Akt1 as well as numerous other pathways.5 In previous studies of immortalized and H-ras–transformed endothelial cells, we have found that overexpression of oncogenic H-ras causes increased angiogenesis and malignant transformation of endothelial cells to angiosarcoma.5 Because Akt1 has been implicated as a major downstream effector of ras activity, we examined the effect of expression of active Akt1 alone in immortalized, poorly tumorigenic MS1 cells. Overexpression of Akt in immortalized endothelium led to the development of vascular malformations in vivo, which were histologically distinct from angiosarcomas derived from the same endothelial cell line. To our knowledge, this is the first murine model of transplantable vascular malformations, and provides a rationale for Akt inhibition in the treatment of vascular malformations.

CELL LINES

MS1 cells are murine endothelial cells immortalized by infection with an ecotropic retrovirus encoding SV40 large T antigen. When injected subcutaneously into nude mice, these cells form benign hemangiomas.5 Subsequent efforts to transduce MS1 cells with a myristoylated Akt retroviral supernatant were accomplished with 293T cells by cotransfection with pBabepuroHA-myrAkt (or control pBabepuro) and pCLEco, which encodes gag, pol, and env proteins specifying the ecotropic receptor, using a FuGene 6 transfection reagent (Roche Applied Science, Indianapolis, Ind). Thirty-six hours after transfection, the 293T medium was collected and filtered through a 0.45-μm syringe filter onto the MS1 cells, and protamine sulfate was added to a concentration of 0.6 mg/mL. After 3 hours, the medium was replaced with Dulbecco modified Eagle medium plus 5% fetal calf serum. Thirty-six hours after infection, the cells were selected with 1-μg/mL puromycin for 3 days, at which time 100% of the noninfected cells were dead.

WESTERN BLOT ANALYSIS

Cells were grown on 10-cm dishes and allowed to reach approximately 80% confluence before protein isolation. Sample aliquots normalized for protein content were size fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were transferred to a polyvinylidene fluoride microporous membrane. The blots were incubated in blocking solution (phosphate-buffered saline with 2% [weight-volume ratio] bovine serum albumin) for 1 hour at room temperature. The blots were then incubated overnight in sheep polyclonal anti-Akt (Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal antiphosphorylated Akt (Ser 473; Cell Signaling Technology, Beverly, Mass), monoclonal anti-ras antibody (Transduction Laboratories, Lexington, Ky), or monoclonal antiactin antibody (Chemicon International Inc, Temecula, Calif). Blots were washed 3 times with phosphate-buffered saline with 0.05% Tween 20 and were then incubated 1 hour at room temperature in secondary antibody solution consisting of a 1:16 000 dilution of horseradish peroxidase–conjugated anti–rabbit IgG (Amersham Pharmacia Biotech, Piscataway, NJ) or 1:10 000 dilution of horseradish peroxidase–conjugated anti–mouse IgG (Amersham Pharmacia Biotech) and washed 3 times with phosphate-buffered saline with 0.05% Tween 20. Finally, the blots were developed using enhanced chemiluminescence.

IN VIVO TUMORIGENESIS STUDIES

One million MS1 or MS1 m-Akt cells were injected subcutaneously into nude mice as described in our previous studies.5 MS1-derived cells were allowed to remain in nude mice up to 2 months prior to killing. Tumor tissue was fixed in formalin and stained with hematoxylin-eosin.

CONSTITUTIVELY ACTIVE Akt EXPRESSION LEADS TO THE DEVELOPMENT OF VASCULAR MALFORMATIONS

Introduction of AKT1 into endothelial cells leads to a high-level expression of activated Akt (Figure 1). Akt is also known to cause phosphorylation of another transcription factor, FKHRL-1, which is accomplished in our cells by Akt introduction (Figure 1). To determine the effect of Akt activation on in vivo transformation, 1 million cells were injected subcutaneously into the right flank of 3 male nude mice. Long-term implantation (1 month) of MS1 m-Akt cells in nude mice led to the development of progressive vascular malformations (Figure 2B). Histological examination of the tumors from mice injected with MS1 m-Akt cells showed a vascular malformation with wide lumens and minimal investment by pericytes and/or smooth muscle, reminiscent of human vascular malformations (Figure 2). Note the contrast in histologic features to SVR angiosarcomas, which are highly cellular and do not possess large lumens (Figure 2).

Place holder to copy figure label and caption
Figure 1.

Western blot analysis of total and phosphorylated Akt (pAkt) in MS1, SVR, and MS1 m-Akt cells. Cell lysates isolated from MS1 and MS1 m-Akt cells were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies to pAkt, total Akt, actin, and phosphorylated FKHRL-1(P-FKHRL-1), a target of activated Akt, showing that the Akt is biologically active. Actin serves as a loading control.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Gross and microscopic views of Akt-induced vascular malformation. A, Representative mouse injected with MS1 m-AKT cells subcutaneously. Histologic features of MS1 m-Akt vascular malformation (B) and SVR angiosarcoma (C) (hematoxylin-eosin, original magnification ×40). Note the contrasting histologic features of wide vascular lumens with minimal smooth muscle investment in Akt overexpression cells compared with the highly cellular morphologic features of the SVR angiosarcoma.

Graphic Jump Location

We have previously shown that ras transformation increased angiogenic activity in immortalized endothelial cells and induces Akt as well as other signaling pathways.46 Akt is a well-studied downstream mediator of ras activity, and Akt has been implicated in transformation and protection from apoptosis.79 We studied the effect of Akt overexpression alone to determine whether Akt could confer the ability of endothelial cells to persist in vivo. Surprisingly, we found that constitutive overexpression of Akt led to the development of lesions that closely resemble vascular malformations in that they have wide lumens with small amounts of smooth muscle investment.

Recent data have shown that too much Akt can perturb angiogenesis. Targeting Akt to endothelial cells of transgenic mice leads to perinatal lethality owing to the development of vascular malformations.10 Interestingly, in the mice that have the induced akt transgene, total Akt levels remain the same, even though induction of Akt by the withdrawal of doxycycline leads to increased phosphorylation of Akt.9 This may be due to a feedback suppression of endogenous Akt expression by Akt induction. Our model has the advantage that it can be transplanted to nude mice and thus be used to evaluate potential therapies for vascular malformations.

Our findings provide a rationale for the use of Akt inhibitors and drugs that work downstream of Akt, such as rapamycin,11 for the treatment of vascular malformations. Akt inhibitors in clinical trial include perifosine and miltefosine, drugs originally developed for the treatment of leishmaniasis.12,13 Another protein, mammalian target of rapamycin (mTOR), and downstream activators of mTOR, such as S6 kinase, can be detected in paraffin sections by immunohistochemical analysis.14 Rapamycin has already been used for the treatment of other vascular neoplasms, including Kaposi sarcoma, as well as dermatomyositis, and is well tolerated by patients.15,16 Our findings suggest that vascular malformations that demonstrate activation of the Akt pathway, including mTOR and S6 kinase, may be treated with currently available drugs such as rapamycin and novel drugs such as Akt inhibitors.

Correspondence: Jack L. Arbiser, MD, PhD, Department of Dermatology, Emory University School of Medicine, WMB 5309, 1639 Pierce Dr, Atlanta, GA 30322 (jarbise@emory.edu).

Financial Disclosure: None reported.

Accepted for Publication: August 14, 2006.

Author Contributions:Study concept and design: Cohen and Arbiser. Acquisition of data: Perry, Banyard, McLaughlin, Watnick, Sohn, Brindley, and Cohen. Analysis and interpretation of data: Perry, Banyard, Brindley, and Obata. Drafting of the manuscript: Perry, Banyard, McLaughlin, Le Good, Cohen, and Arbiser. Critical revision of the manuscript for important intellectual content: Perry, Banyard, Watnick, Sohn, Brindley, Obata, and Cantley. Obtained funding: Arbiser. Administrative, technical, and material support: Perry, McLaughlin, Watnick, Brindley, Obata, Cohen, and Arbiser. Study supervision: Arbiser.

Funding/Support: Dr Arbiser was supported by grants RO1 AR47901, P30 AR42687, and Emory Skin Disease Research Core Center Grant P30 AR42687 from the National Institutes of Health, as well as by a VA Merit Award.

Acknowledgment: We thank J. Ann Le Good, PhD, Mary Hendrix, PhD, and Elisabeth Seftor, PhD, for their critical evaluation of this manuscript and J. Dewald, BS, for performing Western blot analysis.

Chiller  KGFrieden  IJArbiser  JL Molecular pathogenesis of vascular anomalies: classification into three categories based upon clinical and biochemical characteristics. Lymphat Res Biol 2003;1267- 281
PubMed
Hollstein  MMarion  MJLehman  T  et al.  p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride–exposed factory workers. Carcinogenesis 1994;151- 3
PubMed
Froment  OBoivin  SBarbin  ABancel  BTrepo  CMarion  MJ Mutagenesis of ras proto-oncogenes in rat liver tumors induced by vinyl chloride. Cancer Res 1994;545340- 5345
PubMed
Zietz  CRossle  MHaas  C  et al.  MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 1998;1531425- 1433
PubMed
Arbiser  JLMoses  MAFernandez  CA  et al.  Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci U S A 1997;94861- 866
PubMed
LaMontagne  KR  JrMoses  MAWiederschain  D  et al.  Inhibition of MAP kinase causes morphological reversion and dissociation between soft agar growth and in vivo tumorigenesis in angiosarcoma cells. Am J Pathol 2000;1571937- 1945
PubMed
Papapetropoulos  AFulton  DMahboubi  K  et al.  Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 2000;2759102- 9105
PubMed
Songyang  ZBaltimore  DCantley  LCKaplan  DRFranke  TF Interleukin 3-dependent survival by the Akt protein kinase. Proc Natl Acad Sci U S A 1997;9411345- 11350
PubMed
Bellacosa  ATesta  JRStaal  SPTsichlis  PN A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 1991;254274- 277
PubMed
Sun  JFPhung  TShiojima  I  et al.  Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 2005;102128- 133
PubMed
Sarbassov  DDAli  SMSengupta  S  et al.  Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006;22159- 168
PubMed
Ruiter  GAZerp  SFBartelink  Hvan Blitterswijk  WJVerheij  M Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol 3-kinase-Akt/PKB survival pathway. Anticancer Drugs 2003;14167- 173
PubMed
Belay  ADAsafa  YMesure  JDavidson  RN Successful miltefosine treatment of post-kala-azar dermal leishmaniasis occurring during antiretroviral therapy. Ann Trop Med Parasitol 2006;100223- 227
PubMed
El-Hashemite  NZhang  HHenske  EPKwiatkowski  DJ Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma. Lancet 2003;3611348- 1349
PubMed
Stallone  GSchena  AInfante  B  et al.  Sirolimus for Kaposi's sarcoma in renal-transplant recipients. N Engl J Med 2005;3521317- 1323
PubMed
Nadiminti  UArbiser  JL Rapamycin (sirolimus) as a steroid-sparing agent in dermatomyositis. J Am Acad Dermatol 2005;52 ((suppl 1)) 17- 19
PubMed

Figures

Place holder to copy figure label and caption
Figure 1.

Western blot analysis of total and phosphorylated Akt (pAkt) in MS1, SVR, and MS1 m-Akt cells. Cell lysates isolated from MS1 and MS1 m-Akt cells were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis, followed by immunoblotting with antibodies to pAkt, total Akt, actin, and phosphorylated FKHRL-1(P-FKHRL-1), a target of activated Akt, showing that the Akt is biologically active. Actin serves as a loading control.

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Gross and microscopic views of Akt-induced vascular malformation. A, Representative mouse injected with MS1 m-AKT cells subcutaneously. Histologic features of MS1 m-Akt vascular malformation (B) and SVR angiosarcoma (C) (hematoxylin-eosin, original magnification ×40). Note the contrasting histologic features of wide vascular lumens with minimal smooth muscle investment in Akt overexpression cells compared with the highly cellular morphologic features of the SVR angiosarcoma.

Graphic Jump Location

Tables

References

Chiller  KGFrieden  IJArbiser  JL Molecular pathogenesis of vascular anomalies: classification into three categories based upon clinical and biochemical characteristics. Lymphat Res Biol 2003;1267- 281
PubMed
Hollstein  MMarion  MJLehman  T  et al.  p53 mutations at A:T base pairs in angiosarcomas of vinyl chloride–exposed factory workers. Carcinogenesis 1994;151- 3
PubMed
Froment  OBoivin  SBarbin  ABancel  BTrepo  CMarion  MJ Mutagenesis of ras proto-oncogenes in rat liver tumors induced by vinyl chloride. Cancer Res 1994;545340- 5345
PubMed
Zietz  CRossle  MHaas  C  et al.  MDM-2 oncoprotein overexpression, p53 gene mutation, and VEGF up-regulation in angiosarcomas. Am J Pathol 1998;1531425- 1433
PubMed
Arbiser  JLMoses  MAFernandez  CA  et al.  Oncogenic H-ras stimulates tumor angiogenesis by two distinct pathways. Proc Natl Acad Sci U S A 1997;94861- 866
PubMed
LaMontagne  KR  JrMoses  MAWiederschain  D  et al.  Inhibition of MAP kinase causes morphological reversion and dissociation between soft agar growth and in vivo tumorigenesis in angiosarcoma cells. Am J Pathol 2000;1571937- 1945
PubMed
Papapetropoulos  AFulton  DMahboubi  K  et al.  Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway. J Biol Chem 2000;2759102- 9105
PubMed
Songyang  ZBaltimore  DCantley  LCKaplan  DRFranke  TF Interleukin 3-dependent survival by the Akt protein kinase. Proc Natl Acad Sci U S A 1997;9411345- 11350
PubMed
Bellacosa  ATesta  JRStaal  SPTsichlis  PN A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. Science 1991;254274- 277
PubMed
Sun  JFPhung  TShiojima  I  et al.  Microvascular patterning is controlled by fine-tuning the Akt signal. Proc Natl Acad Sci U S A 2005;102128- 133
PubMed
Sarbassov  DDAli  SMSengupta  S  et al.  Prolonged rapamycin treatment inhibits mTORC2 assembly and Akt/PKB. Mol Cell 2006;22159- 168
PubMed
Ruiter  GAZerp  SFBartelink  Hvan Blitterswijk  WJVerheij  M Anti-cancer alkyl-lysophospholipids inhibit the phosphatidylinositol 3-kinase-Akt/PKB survival pathway. Anticancer Drugs 2003;14167- 173
PubMed
Belay  ADAsafa  YMesure  JDavidson  RN Successful miltefosine treatment of post-kala-azar dermal leishmaniasis occurring during antiretroviral therapy. Ann Trop Med Parasitol 2006;100223- 227
PubMed
El-Hashemite  NZhang  HHenske  EPKwiatkowski  DJ Mutation in TSC2 and activation of mammalian target of rapamycin signalling pathway in renal angiomyolipoma. Lancet 2003;3611348- 1349
PubMed
Stallone  GSchena  AInfante  B  et al.  Sirolimus for Kaposi's sarcoma in renal-transplant recipients. N Engl J Med 2005;3521317- 1323
PubMed
Nadiminti  UArbiser  JL Rapamycin (sirolimus) as a steroid-sparing agent in dermatomyositis. J Am Acad Dermatol 2005;52 ((suppl 1)) 17- 19
PubMed

Correspondence

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