Murine models of vascular remodeling

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

Vascular remodeling, a common process in many vascular diseases, describes morphological changes within the vascular wall and adjacent perivascular structures. Inflammation, endothelial dysfunction, and vascular smooth muscle cell migration and proliferation are main factors triggering this process, which may lead to neointimal hyperplasia, medial dysplasia, perivascular thickening, or fibrosis and vascular calcification. Vascular remodeling also occurs after interventional procedures such as angioplasty and vascular stenting. Veins are frequently used in coronary artery bypass grafting, which may fail in the short term because of acute graft failure due to thrombosis or in the long term due to pathological vascular remodeling, a complex process that is poorly understood despite its clinical relevance for other diseases. Project 1: AD-HIES clinical features and signaling pathways that we identified as those affected in AD-HIES patient fibroblasts pointed to angiogenesis and extracellular matrix remodeling defects as factors contributing to AD-HIES pathologies and to HIF1a stabilizing drugs as possible treatment. Cell culture in vitro angiogenesis assays confirmed that AD-HIES fibroblasts have decreased ability to promote tube formation by HUVEC cells that could be corrected by treatment with HIF1a stabilizing drugs. To investigate AD-HIES abnormalities in vivo, we then used several mouse models of angiogenesis, tissue remodeling and wound healing. 1) Decreased ability of AD-HIES skin fibroblasts to support angiogenesis in mouse hind-limb ischemia (HLI) model: To investigate the ability of AD-HIES fibroblasts to support angiogenesis in the setting of tissue damage and remodeling, we used the murine model of hind-limb ischemia (HLI). Here, surgical ligation of the femoral artery stops blood supply to the hind-limb and stimulates angiogenesis in the calf muscles leading to increased capillary density. We tested the ability of AD-HIES fibroblasts to facilitate re-capillarization after femoral artery ligation in NSG mice. At baseline, NSG immunodeficient mice have decreased re-capillarization ability and require additional support with pro-angiogenic factors to be able to restore blood perfusion after femoral artery ligation. Injection of control fibroblasts into the calf muscle after femoral artery ligation stimulated angiogenesis, leading to restoration of blood perfusion due to increased capillary density. However, injection of AD-HIES skin fibroblasts failed to increase capillary density and did not restore blood perfusion. These results indicate that AD-HIES fibroblasts were not able to provide pro-angiogenic factors that were missing in immunodeficient mice due to lack of normal immune response initiated by injury, while normal skin fibroblasts were able to compensate this deficiency by secreting necessary pro-angiogenic factors. 2)Decreased ability of AD-HIES skin fibroblasts to support angiogenesis in iPSCs-derived teratoma model: We tested the pro-angiogenic potential of AD-HIES fibroblasts by studying their ability to promote the growth of teratomas derived from iPSCs after subcutaneous injection into NSG immunodeficient mice. Here, we used iPSCs generated from our control and AD-HIES skin fibroblasts cell lines. Compared to teratomas derived from control iPSCs, teratomas derived from AD-HIES iPSCs were small and avascular suggesting a deficiency in blood vessel formation and providing us a novel model to investigate angiogenic capacities of transplanted human cells (fibroblasts) into human tissue (teratoma). Despite being very small, AD-HIES iPSC-derived teratomas contained structures representing all three lineages indicating similar differentiation potential. Indeed, mixing of AD-HIES iPSCs with normal skin fibroblasts resulted in restoration of the teratoma growth rate accompanied by more efficient blood vessel formation, evidenced from increased proportion of CD31 positive cells. However, AD-HIES skin fibroblasts failed to provide such support for teratoma growth, consistent with the decreased ability of AD-HIES fibroblast to secrete pro-angiogenic factors. 3) Deficient angiogenesis and wound healing in AD-HIES mouse model: We found, that consistent with the AD-HIES angiogenesis defect identified in human fibroblasts, AD-HIES mice harboring loss-of-function STAT3 mutation have diminished ability to activate re-capillarization and restore blood perfusion after femoral artery ligation when compared to WT mice. Similar to deficient skin wound healing in AD-HIES patients, the healing in the mouse model was also delayed. 4) HIF1 stabilizers improve angiogenesis and wound healing in AD-HIES mouse model: We tested ability of HIF1 stabilizers to improve angiogenesis-related deficiencies in AD-HIES mice. Treatment by daily gavage with DMF improved re-capillarization after ligation of the femoral artery in the AD-HIES mice and restored growth of teratomas from AD-HIES-derived iPSCs in NSG mice. We used a splinted wound healing model to study skin wound healing in mice. We administered two HIF1 stabilizers, DMF and Daprodustat, to decrease off-target drugs effects. Topical treatment of wounds with both DMF and Daprodustat accelerated wound closure in AD-HIES mice. Immunohistochemical analysis demonstrated tissue-wide phosphorylation of Stat3 in the wound but not in undamaged skin, verifying importance of Stat3-dependent signaling in the wound healing process. Effectiveness of the topical treatment with DMF for increasing HIF1 level in granulation tissue was verified by immunostaining. These studies affirmed the fundamental role of STAT3-HIF1 signaling in the processes of ECM remodeling, angiogenesis, and wound healing in normal physiology, explained the aberrant wound healing and vascular structural abnormalities in AD-HIES. They also identify HIF1 as a potential treatment option for AD-HIES patients. In addition, the study provides in vivo clinical evidence of the importance of STAT3/HIF1 signaling in more common diseases in which tissue remodeling plays a major role, including restrictive lung disease, diabetes and atherosclerosis. We continue testing HIF1a stabilizing drugs for their efficiency in treating vascular abnormalities in AD-HIES mouse model. Project 2: Veins grafted into an arterial environment undergo complex vascular remodeling. EndMT is the process by which endothelial cells lose their cell-specific markers and morphology and acquire a mesenchymal cell-like phenotype. However, the role of EndMT and the mechanisms regulating cell phenotype adaptation during human vein graft remodeling are poorly understood. Using two independent endothelial lineage tracing systems, we demonstrated that, at 35 days after vein grafting, half the cells populating the neointima were of endothelial origin. These endothelial-derived cells had lost their endothelial phenotype and acquired smooth muscle cell-like properties, yet they failed to develop a fully mature smooth muscle cell phenotype. We detected early activation of the TGF-b-Smad2/3-Slug signaling pathway and confirmed that TGF-b-Smad2/3-Slug signaling pathway-mediated EndMT plays a pivotal role in regulating vein graft remodeling. We are further investigating BMP9/Smad1-mediated endothelial cell survival and its impact on acute vein graft patency and long-term outcome in vein graft remodeling due to the development of neointimal hyperplasia, but also in thrombosis formation leading to early graft failure. We hypothesize that activating the BMP9/Smad1 pathway positively regulates endothelial function to benefit murine vein grafting.