Regulation of Flt 1 Splicing by Fibronectin and Integrin Signaling During Aging

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Abstract Risk factors for cognitive impairment and dementia target the vasculature, a condition known as Vascular contributions to Cognitive Impairment & Dementia (VCID). However, the molecular mechanisms that result in reduced vascular function, density and perfusion in disease are poorly understood. Recent work has focused on vascular endothelial growth factor (VEGF), which signals through VEGF receptor 2, and is a major contributor to endothelial cell survival and vessel maintenance. VEGF bioavailability declines with age, leading to vascular dysfunction and reduced vessel density in the brain and other organs. This effect is due to increased levels of a VEGF antagonist, an alternatively spliced soluble form of the decoy receptor VEGFR1, termed soluble Flt1 (sFlt1), primarily expressed in the endothelium. Interestingly, the above risk factors for vascular disease – age, diabetes, hypertension and vascular injury – are linked to increased subendothelial accumulation of the extracellular matrix protein fibronectin (FN) in vascular basement membrane. FN contributes to vessel repair and integrity but in disease settings can drive inflammation and dysfunction. Preliminary data show that, under conditions of high substrate stiffness, cell adhesion to FN through integrin α5β1 induces alternative splicing of the Flt1 transcript to increase sFlt1 production These findings lead us to propose the novel hypothesis that FN accumulation and vessel stiffening with age drives increased production of sFlt1, which mediates vascular rarefaction in the brain and impairs cognitive function. In the first Aim, we will determine how mechanical strain on integrin α5β1 leads to Flt1 splicing and premature polyadenylation, identifying the RNA-binding proteins (RBPs) responsible for the change in splicing and elucidating mechanisms of regulation. In the second Aim, we will test the above hypothesis in mouse models and identify therapeutic targets whose blockade prevents sFlt1 production and protects from cognitive impairment. Together, the completion of these aims will provide new molecular insights into VCID by linking the increased vascular accumulation of FN to the production of sFlt1, determining molecular mechanisms, and identifying ways to inhibit this pathway to limit vascular rarefaction and cognitive decline associated with VCID risk factors.