title: Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy.
abstract: OBJECTIVE
  Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis.
  
  
  METHODS
  We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM).
  
  
  RESULTS
  IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1 (compared to untreated Marfan aorta: Ad.hTIMP-1 p = 0.902; control Ad.β-Gal. p = 0.165). The virus-untreated and not transplanted mgR/mgR aorta revealed a significant increase of albumin diffusion through the endothelial barrier (p = 0.037). TEM analysis of adenovirus-exposed mgR/mgR aortas displayed disruption of the basement membrane and basolateral space.
  
  
  CONCLUSIONS
  Murine Marfan aortic grafts developed severe inflammation after adenoviral contact. We demonstrated that fibrillin-1 deficiency is associated with relevant dysfunction of the endothelial barrier that enables adenovirus to induce vessel-harming inflammation. Endothelial dysfunction may play a pivotal role in the development of the vascular phenotype of Marfan syndrome.
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Philipp Christian
authors: PC
authors: Seppelt
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Simon
authors: S
authors: Schwill
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Alexander
authors: A
authors: Weymann
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Rawa
authors: R
authors: Arif
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Antje
authors: A
authors: Weber
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Marcin
authors: M
authors: Zaradzki
authors: German Cancer Research Center, Division of Molecular Genetics, Heidelberg, Germany.
authors: Karsten
authors: K
authors: Richter
authors: Heart and Diabetes Center NRW, Clinic for Thoracic and Cardiovascular Surgery, Bad Oeynhausen, Germany.
authors: Stephan
authors: S
authors: Ensminger
authors: Institute for Medical Genetics and Human Genetics, Charité University Hospital, Berlin, Germany.|Max-Planck Institute for Molecular Genetics and Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
authors: Peter Nicholas
authors: PN
authors: Robinson
authors: Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.
authors: Andreas H
authors: AH
authors: Wagner
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Matthias
authors: M
authors: Karck
authors: Department of Cardiac Surgery, University Hospital Heidelberg, Heidelberg, Germany.
authors: Klaus
authors: K
authors: Kallenbach
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000276:immunology; D000818:Animals; D001011:Aorta / Q000473:pathology / Q000637:transplantation*; D002478:Cells, Cultured; D004549:Elastin / Q000502:physiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000502:physiology; D005260:Female; D000071838:Fibrillin-1; D000071837:Fibrillins; D015316:Genetic Therapy / Q000379:methods*; D007249:Inflammation / Q000276:immunology; D008382:Marfan Syndrome / Q000503:physiopathology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008840:Microfilament Proteins / Q000235:genetics*; D058426:Neointima / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology*; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics; D054392:Adipokines
chemical_list: D004549:Elastin; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1; D001616:beta-Galactosidase; C000605722:FBN1 protein, human; D054392:Adipokines; C000605724:Fbn1 protein, mouse; C560822:TIMP1 protein, human