Cardiovascular disease, most often due to atherosclerosis, is the leading cause of death both in the United States and throughout the world. During the past decades, medical therapy and life-style changes have decreased the prevalence of cardiovascular disease in the United States and other “Western” countries. However, this downward trend recently reversed in the United States, and the prevalence of cardiovascular disease continues to increase in other parts of the world. Targeted molecular interventions—that are based on a detailed understanding of the pathogenesis of cardiovascular disease—hold great promise for preventing and treating cardiovascular disease.
Overview of the Laboratory:
Our projects share a common goal: to discover the mechanisms that cause vascular disease and to translate these discoveries into novel therapies that prevent or reverse vascular disease. There are 3 major projects:
- Development of gene-transfer vectors capable of mediating long-term recombinant gene expression in blood vessels and use of these vectors to prevent or reverse atherosclerosis in arteries and vein grafts.
- Investigation of the role of transforming growth factor β (TGF-β) signaling in the development and prevention of aortic aneurysms.
- Investigation of the role of the urokinase plasminogen activator (uPA)/plasminogen system in the development of atherosclerosis and plaque rupture; defining the molecular mechanisms of atherosclerotic plaque rupture.
Approaches, Contributions, and Current Work:
We use techniques and approaches of molecular biology, biochemistry, genetics, virology, immunology, and whole-animal physiology.
Vascular gene therapy:
Since 1988,(1) our laboratory has worked on improving gene-transfer vectors and using these vectors to express genes in the blood vessel wall. Our early work clarified the promise of adenoviral vectors to express genes in injured and uninjured arteries and demonstrated their therapeutic efficacy in an animal model of neointimal growth.(2) We also identified important limitations of adenoviral vectors, including brevity of expression and proinflammatory effects.(3-7) More recently, we discovered that helper-dependent adenoviral (HDAd) vectors (which lack all viral genes) can express therapeutic genes stably in the artery wall, with minimal associated inflammation.(8, 9) Clinical applications of this gene-therapy approach include gene transfer to blood vessels that prevents or reverses atherosclerosis in arteries and in venous bypass grafts.
Several years ago, we found that HDAd-mediated expression of apolipoprotein (apo) A-I in arterial endothelium of cholesterol-fed rabbits prevents early atherosclerosis.(10) We then developed new animal models to allow more extensive preclinical testing of this gene therapy in both rabbit arteries and vein grafts.(11, 12) Using these new models, we found that an HDAd vector expressing apo A-I significantly retards development of atherosclerosis in rabbit carotid arteries for at least 6 months,(13) and appears to cause regression of established carotid atherosclerosis.(14)
Currently, we are completing development of a rabbit model of vein-graft atherosclerosis, which we will use to test HDAd-mediated gene therapy. We are also constructing and testing new transgene expression cassettes that we anticipate will drive durable high-level therapeutic transgene expression in endothelium.(15) (16) In addition, we are developing new approaches to atheroprotective gene therapy, based on exosome-mediated transfer of inhibitory RNAs and on upregulation of the cholesterol transporter ABCA1.
TGF-β signaling in vascular disease and development:
Transforming growth factor beta-1 (TGF-β1) is a pleiotropic cytokine that is expressed in the artery wall, circulates in plasma, and plays critical roles in vascular development, homeostasis, and disease. We use mouse models of gene transfer, germ-line transgenesis, gene knockout, and antibody-mediated inhibition to uncover the roles of TGF-β signaling in the blood vessel wall.
Our early work used a somatic gene-transfer approach to discover that increased TGF-β1 expression in the artery wall of rodents promotes intimal growth, and we identified the mechanisms through which TGF-β1 acts.(17-19) We then used germ-line transgenesis to discover that increased TGF-β1 expression in vascular smooth muscle cells (SMC) during embryogenesis disrupts vasculogenesis, and causes embryonic death.(20) To bypass this embryonic lethality and determine the role of TGF-β1 in adult vascular homeostasis and disease, we used the “tet” system to obtain regulated postnatal overexpression of TGF-β1 in mouse hearts, with secretion of TGF-β1 into the peripheral blood.(21) These experiments revealed that increased plasma TGF-β1 in adult mice retards atherosclerosis and slows aneurysm progression.(22) Therefore, these overexpression approaches revealed both pathogenic and protective effects of TGF-β1.
To discover the roles of normal levels of TGF-β signaling in vascular physiology, we then used the “Cre-Lox” system to delete the type II TGF-β receptor (TBRII; critical for all physiologic TGF-β signaling) in SMC during mouse development. Deletion of TBRII in SMC caused embryonic lethality during mid-gestation, revealing an essential role for TGF-β signaling in vascular morphogenesis, SMC differentiation, and matrix synthesis.(23, 24)
To bypass this embryonic lethality and uncover the role of physiologic TGF-β signaling in SMC of postnatal mice, we generated mice with inducible SMC-specific deletion of TBRII. These mice develop severe aortic pathology including intramural hematomas, penetrating aortic ulcers, aneurysmal dilation, and rare dissections.(25) Therefore, SMC TGF-β signaling is critical for maintenance of postnatal aortic homeostasis. We then tested the hypothesis that SMC TGF-β signaling also maintains aortic homeostasis in a mouse model of Marfan syndrome (MFS). This is an important question because a widely held model of MFS pathogenesis postulates that increased SMC TGF-β signaling causes MFS-associated aortopathy. We found that loss of SMC TGF-β signaling in MFS mice significantly exacerbated their aortopathy.(26) Therefore, SMC TGF-β signaling appears to be protective, not pathogenic, in MFS.
Currently, we continue to use mouse models to identify the pathways through which SMC TGF-β signaling protects the aorta and through which loss of physiologic SMC TGF-β signaling causes aortopathy. We anticipate that insights from these experiments will clarify roles of TGF-β signaling in vascular homeostasis, provide insights into the pathogenesis of aortic aneurysms—both genetically based and acquired—and reveal novel strategies for preserving vascular health.
The uPA/plasminogen system and vascular disease:
Urokinase-type plasminogen activator (uPA) is a serine protease that converts the zymogen plasminogen to plasmin (a broadly active protease). uPA circulates in plasma and is expressed by cells in the artery wall. We became interested in uPA because of its ability to act as a therapeutic agent by promoting lysis of occlusive intravascular thrombi. We first proposed using uPA (and the related enzyme tPA) for antithrombotic gene therapy delivered either directly to the vessel wall or from the surface of thrombogenic intravascular devices.(1, 27, 28) We then discovered that uPA—when expressed at increased levels in the artery wall of hyperlipidemic rabbits or mice—increases atherosclerosis and causes arterial constriction.(29, 30) More recently, we discovered that increased expression of uPA in mouse macrophages causes atherosclerotic plaque rupture.(31) These results suggest that increased uPA expression in the vessel wall contributes to the progression of vascular disease, and that uPA will not be useful for antithrombotic gene therapy.
Currently, our work is aimed at defining the mechanisms through which uPA accelerates atherosclerosis and causes plaque rupture and on testing whether pharmacologic inhibition of uPA and other proteases can prevent atherosclerosis.(32) Insights into the mechanisms of uPA-accelerated atherosclerosis and plaque rupture may reveal new strategies for preventing atherosclerosis and its complications.
- Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, and Anderson WF. Seeding of intravascular stents with genetically engineered endothelial cells. Circulation. 1989;80:1347–53.
- Rade JJ, Schulick AH, Virmani R, and Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointimal formation after arterial injury. Nat Med. 1996;2:293–8.
- Lee SW, Trapnell BC, Rade JJ, Virmani R, and Dichek DA. In vivo adenoviral vector-mediated gene transfer into balloon-injured rat carotid arteries. Circ Res. 1993;73:797–807.
- Schulick AH, Dong G, Newman KD, Virmani R, and Dichek DA. Endothelium-specific in vivo gene transfer. Circ Res. 1995;77:475–85.
- Schulick AH, Newman KD, Virmani R, and Dichek DA. In vivo gene transfer into injured carotid arteries. Optimization and evaluation of acute toxicity. Circulation. 1995;91:2407–14.
- Newman KD, Dunn PF, Owens JW, Schulick AH, Virmani R, Sukhova G, et al. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia. J Clin Invest. 1995;96:2955–65.
- Schulick AH, Vassalli G, Dunn PF, Dong G, Rade JJ, Zamarron C, et al. Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity. J Clin Invest. 1997;99:209–19.
- Wen S, Graf S, Massey PG, and Dichek DA. Improved vascular gene transfer with a helper-dependent adenoviral vector. Circulation. 2004;110:1484-91.
- Jiang B, Qian K, Du L, Luttrell I, Chitaley K, and Dichek DA. Helper-dependent adenovirus is superior to first-generation adenovirus for expressing transgenes in atherosclerosis-prone arteries. Arterioscler Thromb Vasc Biol. 2011;31:1317–25.
- Flynn R, Qian K, Tang C, Dronadula N, Buckler J, Jiang B, et al. Expression of apolipoprotein A-I in rabbit carotid endothelium protects against atherosclerosis. Mol Ther. 2011;19:1833–41.
- Du L, Zhang J, De Meyer GR, Flynn R, and Dichek DA. Improved animal models for testing gene therapy for atherosclerosis. Hum Gene Ther Methods. 2014;25:106–14.
- Du L, Zhang J, Clowes AW, and Dichek DA. Efficient gene transfer and durable transgene expression in grafted rabbit veins. Hum Gene Ther. 2015;26:47–58.
- Wacker BK, Dronadula N, Bi L, Stamatikos A, and Dichek DA. Apolipoprotein A-I vascular gene therapy provides durable protection from atherosclerosis in hyperlipidemic rabbits. Arterioscler Thromb Vasc Biol ( abstract; in press). 2017.
- Wacker BK, Dronadula N, Zhang J, and Dichek DA. Local Vascular Gene Therapy With Apolipoprotein A-I to Promote Regression of Atherosclerosis. Arterioscler Thromb Vasc Biol. 2017;37:316–27.
- Dronadula N, Du L, Flynn R, Buckler JM, Kho J, Jiang Z, et al. Construction of a novel expression cassette for increasing transgene expression in vivo in endothelial cells of large blood vessels. Gene Ther. 2011;18:501–8.
- Dronadula N, Wacker BK, Van Der Kwast R, Zhang J, and Dichek DA. Stable In Vivo Transgene Expression in Endothelial Cells with Helper-Dependent Adenovirus: Roles of Promoter and Interleukin-10. Hum Gene Ther. 2017;28(3):255-70.
- AH, Taylor AJ, Zuo W, Qiu C-B, Dong G, Woodward RN, et al. Overexpression of transforming growth factor b1 in arterial endothelium causes hyperplasia, apoptosis, and cartilaginous metaplasia. Proc Natl Acad Sci U S A. 1998;95:6983–8.
- Otsuka G, Agah R, Frutkin AD, Wight TN, and Dichek DA. Transforming growth factor beta 1 induces neointima formation through plasminogen activator inhibitor-1-dependent pathways. Arterioscler Thromb Vasc Biol. 2006;26:737–43.
- Otsuka G, Stempien-Otero A, Frutkin AD, and Dichek DA. Mechanisms of TGF-beta1–induced intimal growth: Plasminogen-independent activities of plasminogen activator inhibitor-1 and heterogeneous origin of intimal cells. Circ Res. 2007;100:1300–7.
- Agah R, Prasad KSS, Linnemann R, Firpo MT, Quertermous T, and Dichek DA. Cardiovascular overexpression of transforming growth factor-b1 causes abnormal yolk sac vasculogenesis and early embryonic death. Circ Res. 2000;86:1024–30.
- Lee S, Agah R, Xiao M, Frutkin AD, Kremen M, Shi H, et al. In vivo expression of a conditional TGF-beta1 transgene: no evidence for TGF-beta1 transgene expression in SM22alpha-tTA transgenic mice. J Mol Cell Cardiol. 2006;40:148–56.
- Frutkin AD, Otsuka G, Stempien-Otero A, Sesti C, Du L, Jaffe M, et al. TGF-beta1 limits plaque growth, stabilizes plaque structure, and prevents aortic dilation in Apolipoprotein E-null mice. Arterioscler Thromb Vasc Biol. 2009;29:1251–7.
- Frutkin AD, Shi H, Otsuka G, Leveen P, Karlsson S, and Dichek DA. A critical developmental role for tgfbr2 in myogenic cell lineages is revealed in mice expressing SM22-Cre, not SMMHC-Cre. J Mol Cell Cardiol. 2006;41:724–31.
- Jaffe M, Sesti C, Washington I, Du L, Dronadula N, Chin MT, et al. Transforming growth factor beta signaling in myogenic cells regulates vascular morphogenesis, differentiation, and matrix synthesis. Arterioscler Thromb Vasc Biol. 2012;32:e1–e11.
- Hu JH, Wei H, Jaffe M, Airhart N, Du L, Angelov SN, et al. Postnatal Deletion of the Type II Transforming Growth Factor-beta Receptor in Smooth Muscle Cells Causes Severe Aortopathy in Mice. Arterioscler Thromb Vasc Biol. 2015;35:2647–56.
- Wei H, Hu JH, Angelov SN, Fox K, Yan J, Enstrom R, et al. Aortopathy in a Mouse Model of Marfan Syndrome Is Not Mediated by Altered Transforming Growth Factor beta Signaling. J Am Heart Assoc. 2017;6(1).
- Dichek DA, Anderson J, Kelly AB, Hanson SR, and Harker LA. Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors. Circulation. 1996;93:301–9.
- Shayani V, Newman KD, and Dichek DA. Optimization of recombinant t-PA secretion from seeded vascular grafts. J Surg Res. 1994;57:495–504.
- Cozen AE, Moriwaki H, Kremen M, DeYoung MB, Dichek HL, Slezicki KI, et al. Macrophage-targeted overexpression of urokinase causes accelerated atherosclerosis, coronary artery occlusions, and premature death. Circulation. 2004;109:2129–35.
- Falkenberg M, Tom C, DeYoung MB, Wen S, Linnemann R, and Dichek DA. Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth. Proc Natl Acad Sci U S A. 2002;99:10665–70.
- Hu JH, Du L, Chu T, Otsuka G, Dronadula N, Jaffe M, et al. Overexpression of urokinase by plaque macrophages causes histologic features of plaque rupture and increases vascular MMP activity in aged apo E-Null mice. Circulation. 2010;121:1637–44.
- Hu JH, Touch P, Zhang J, Wei H, Liu S, Lund IK, et al. Reduction of mouse atherosclerosis by urokinase inhibition or with a limited-spectrum matrix metalloproteinase inhibitor. Cardiovasc Res. 2015;105:372–82.