Nucleocytoskeletal networks: flow sensing in atherosclerosis

Atherosclerosis, a disease of modern age, includes a multistep process initiated by increased lipoprotein accumulation leading to different stages of atheroma formation. In the so called "response to injury hypothesis" atherosclerosis is caused by a dysfunctional endothelium as a response to injury. Besides classical factors such as hyperlipidemia, also changes in intrinsic properties of the vasculature, such as the mechanosensing in endothelial cells, are increasingly recognized as important factors for disease development. Regions of arterial branching exposed to non-laminar blood flow alter the mechanosensing repertoire of endothelial cells leading to increased susceptibility to atheroma development. Endothelial cells of branched vascular regions are less elongated, exhibit altered cytoskeleton organization (VE-cadherin adherens junctions and actin cytoskeleton) and upregulate pro- inflammatory molecules such as ICAM-1 and VCAM-1. Thus, recent research has generated increasing interest in identifying molecules mediating this mechanosensing response with an ability to modulate atherogenic response of endothelial cells. This research project focuses on molecular mechanisms of mechanosensing by nucleoskeletal and cytoskeletal proteins in endothelial cells and molecular causes leading to its dysfunction by examining two model systems: plectin-deficient mice and Hutchinson-Gilford progeria syndrome (HGPS) mice. For both model systems we hypothesize that impaired cytoskeletal integrity, namely defective nucleo-cytoskeleton linkages in HGPS and a reduction in filament interconnections and filament anchorage in case of plectin deficiency, is responsible for altered cytoskeleton-mediated signal transmission which ultimately leads to an aberrant mechanosensing response to flow shear stress. Mutations in the cytoskeleton-associated protein plectin lead to epidermolysis bullosa simplex in conjunction with muscular dystrophy (EBS-MD). However, pronounced hemorrhagic blisters and increased bleedings of plectin patients and plectin-deficient mice indicate that the vascular system might also be affected by plectin-deficiency. Since plectin-deficient mice die shortly after birth and plectin patients have significantly reduced life span the analysis of vascular changes that typically develop in the second decade of life is missing. The conditional endothelial VE-Cre-plectinF/F mice that we intend to analyze in this study represent an ideal tool to analyze the impact plectin loss has on vascular tissue. HGPS is a very rare premature aging syndrome linked to mutations in the nucleoskeletal protein lamin A. HGPS symptoms include increased propensity to atherosclerosis and death in HGPS patients is most frequently due to cardiovascular problems. However, despite the obvious effects the mutant lamin (also termed progerin) exerts on the development of atherosclerosis relevant molecular properties of microvasculature cells in patients and mouse models have not been explored so far. We will use a conditional endothelial-specific progerin overexpressor mouse model to study the molecular defects, particularly mechanosensing impairment in endothelial cells. Importantly, since progerin expression is also linked to normal ageing, the expected results might also shed light on processes that occur upon normal aging leading to shear-stress insensitivity and increased susceptibility to atherosclerosis.

Atherosclerosis or hardening of arteries is mainly characterized by increased depositions of fat and extracellular matrix (collagen) eventually causing stroke or heart attack. It is the leading cause of death in the modern society. Atherosclerosis is mainly rooted in disturbed function of the innermost lining of blood vessels, the endothelium. Key features of endothelial dysfunction are atherogenic changes such as changes in the vascular barrier function, adhesive properties and reduction in bioavailability of vasodilators. Atherogenic changes are potentiated by blood flow disturbances occurring at regions of arterial branching or by endothelial intrinsic changes altering the ability of cells to sense forces exerted by blood flow over their surface (mechanosensing). Mechanosensing depends mainly on cells structural elements, cytoskeleton and partly also nucleoskeleton. Thus, recent research has generated increasing interest in identifying molecules mediating this mechanosensing response with an ability to modulate atherogenic response of endothelial cells. This research project focused on molecular mechanisms of mechanosensing by cytoskeletal and nucleoskeletal proteins in endothelial cells and causes leading to its dysfunction. In this respect two endothelium-restricted conditional mouse model systems were investigated: plectin-deficient- (P0) and Hutchinson-Gilford progeria syndrome (HGPS) mice, expressing mutated lamin A (progerin). For both model systems we hypothesized that impaired cyto-nucleoskeletal integrity, namely defective nucleo-cytoskeleton linkages in HGPS and a reduction in filament interconnections and filament anchorage in case of plectin deficiency is responsible for altered cyto-nucleoskeleton-mediated signal transmission which ultimately leads to an aberrant mechanosensing response to flow shear stress. In this research project we could successfully verify this hypothesis by showing impaired mechanoresponse and endothelial dysfunction in vitro and in vivo in both P0 and Prog-Tg systems providing novel molecular links for the respective endothelial dysfunction. As for plectin we could show that organization and cross-talk of cell contractile- and rope-like-filament systems, actin and vimentin, respectively, through this cytoskeletal linker protein plays an essential role for the organization and barrier properties of endothelial cell-cell junctions. On the other hand, progerin accumulation in endothelial cells disturbed nucleo-cytoskeletal coupling potentiating atherogenic changes through defective cell mechanoresponse and deregulation of factors promoting atherogenesis (Myocardin-Related Transcription Factor A). Atherogenic response could be verified in vivo showing increased collagen depositions around coronary arteries and dramatic heart enlargement in Prog-Tg animals. Our data show for the first time that mutations in plectin and progerin lead to endothelial dysfunction and in case of Prog-Tg mice provide molecular basis for cardiovascular pathology of HGPS in patients. These data unscramble molecular mechanisms through which structural intrinsic changes of endothelial cells potentiate atherosclerotic events providing insight into genetic predispositions to endothelial dysfunction and cardiovascular disease.