Final Report Summary - PVA DEVELOPMENT (IDENTIFYING AND CHARACTERIZING REGULATORS OF PRIMORDIAL VASCULAR ADVENTITIA DEVELOPMENT, ORGANIZATION AND FUNCTION)
Vascular development and function are complex processes of utmost importance to the organism involving numerous steps and cell types. Once a vessel forms, it goes through several processes such as stabilization, branching, remodeling and pruning and specialization. Many of these processes occur in parallel and the molecules involved in them, are in many cases shared and repeatedly used, making it hard to distinguish between their activities in the distinct processes. One of the tissues participating in these processes is the vascular adventitia, the outermost layer of the vessel mainly composed of fibroblasts and extra-cellular matrix (ECM) rich in collagens and elastins. Historically, it was mostly considered inert, essentially providing structural support to the vessel. Recently however, with the association of the adventitia to a growing number of diseases and the realization of ECM contribution to a multitude of processes, its activities have slowly become uncovered. The adventitia was shown to participate in a number of developmental and homeostatic processes such as regulation of vessel tone, remodelling and trafficking in and out of the vessel. Additionally, it plays key roles in replenishing the adjacent vascular tissues by accommodating progenitor cells. Despite this, the processes that regulate early adventitial development, organization and function are still unknown.
In this project we aimed to identify early events that occur during vascular development that affect vascular adventitia and the ECM surrounding the vessel. Previous analyses, have clearly demonstrated that alterations in the activity of the ECM remodeling enzyme, Lysyl oxidase (Lox), lead to vascular aneurysms in both humans and animal models. Therefore, in our work, we focused on the activities of this ECM remodeling enzyme family – the lysyl oxidases that includes Lox and Lox-Like 1-4. Using mouse genetics, ex vivo assays and biochemical experiments we aimed to test the roles this key enzyme family plays in vascular and adventitial development and maintenance in order to ultimately better understand the mechanisms they play in aortic aneurysms and human death.
We find that Lox and all other members of its family are expressed in the distinct layers of the vessel: endothelial cells, smooth muscle cells and adventitia. Since genetic ablation of all members of the family that will allow identification of their roles is technically impossible in mice, we took advantage of β-aminoproprionitrile (BAPN) which is an irreversible inhibitor of all enzymatic activities mediated by the lysyl oxidases. We use this inhibitor to block Lox activity both in vivo as well as in the ex vivo aortic ring assay that allows us to grow vessels in a highly controlled environment. These analyses demonstrated that Lox participates in endothelial sprouting and smooth muscle cell coating of the vessels. We have further conducted whole genome RNA-seq experiments and are currently analyzing the data in order to allow us to identify the specific pathways and genes acting downstream of Lox in vascular development and homeostasis.
We hope that our distinct approaches will grant us a better understanding of Lox activities as a regulator of vascular development and maintenance at large but more specifically on primordial vascular adventitia.
This re-integration grant has allowed me to set up a laboratory in the Technion's faculty of medicine. Nearly five years ago, upon my arrival to the Technion, my lab was empty, and now with the help of this grant, my lab is fully equipped for molecular biology, cell culture assays and dissecting facilities for extracting the necessary tissues in a safe environment. We have documenting equipment ranging from simple binoculars to a fluorescent microscope. My lab consists of 11 researchers altogether working on the distinct roles of lysyl oxidases in development and homeostasis.
More information on our research can be found in the lab website at: http://hasson.rappinst.com/
In this project we aimed to identify early events that occur during vascular development that affect vascular adventitia and the ECM surrounding the vessel. Previous analyses, have clearly demonstrated that alterations in the activity of the ECM remodeling enzyme, Lysyl oxidase (Lox), lead to vascular aneurysms in both humans and animal models. Therefore, in our work, we focused on the activities of this ECM remodeling enzyme family – the lysyl oxidases that includes Lox and Lox-Like 1-4. Using mouse genetics, ex vivo assays and biochemical experiments we aimed to test the roles this key enzyme family plays in vascular and adventitial development and maintenance in order to ultimately better understand the mechanisms they play in aortic aneurysms and human death.
We find that Lox and all other members of its family are expressed in the distinct layers of the vessel: endothelial cells, smooth muscle cells and adventitia. Since genetic ablation of all members of the family that will allow identification of their roles is technically impossible in mice, we took advantage of β-aminoproprionitrile (BAPN) which is an irreversible inhibitor of all enzymatic activities mediated by the lysyl oxidases. We use this inhibitor to block Lox activity both in vivo as well as in the ex vivo aortic ring assay that allows us to grow vessels in a highly controlled environment. These analyses demonstrated that Lox participates in endothelial sprouting and smooth muscle cell coating of the vessels. We have further conducted whole genome RNA-seq experiments and are currently analyzing the data in order to allow us to identify the specific pathways and genes acting downstream of Lox in vascular development and homeostasis.
We hope that our distinct approaches will grant us a better understanding of Lox activities as a regulator of vascular development and maintenance at large but more specifically on primordial vascular adventitia.
This re-integration grant has allowed me to set up a laboratory in the Technion's faculty of medicine. Nearly five years ago, upon my arrival to the Technion, my lab was empty, and now with the help of this grant, my lab is fully equipped for molecular biology, cell culture assays and dissecting facilities for extracting the necessary tissues in a safe environment. We have documenting equipment ranging from simple binoculars to a fluorescent microscope. My lab consists of 11 researchers altogether working on the distinct roles of lysyl oxidases in development and homeostasis.
More information on our research can be found in the lab website at: http://hasson.rappinst.com/