Connective Tissue Mediated Vascular Disease

National Heart, Lung, And Blood Institute

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Abstract

To learn more about the mechanism of Lox mediated disease, we chose to study a mouse point mutation in Loxb2b370.2Clo (c.G854T; p.C285F) that falls within the suspected copper binding domain of the molecule. We show that Lox mRNA is present to the same degree in the Lox+/ c.G854T as in WT but, enzymatic activity is reduced. Structural studies suggest that the loss of the Cys changes the structure of the copper binding domain, but that the protein is still secreted and able to bind elastin. Pressure-diameter testing reveals a narrower aorta in 3 month C57Bl/6 Lox+/- males relative to C57Bl/6 WT, but dilates with age faster than a WT. Lox+/c.G854T females do not progress as quickly. When crossed into a hypertensive background (C57Bl/6; 129x1/J), obvious aortic dilation can be noted as early as 3 months in Lox+/ c.G854T males. In females, however, there is no dilatory effect in nulliparous females. Previously pregnant females, however, do show dilation, however. Castration of C57Bl/6 Lox+/ c.G854T males reverts their aortic progression to that of a nulliparous female. Crosslink analysis reveals increased free lysine in elastin and an altered pattern of desmosine and other elastin crosslinks. Age and hypertension exaggerate these effects. When studied by two photon imaging, Lox+/ c.G854T mutants reveal decreased ruffling of their elastic lamellae. Lox+/ c.G854T mice also show increase numbers of holes in their lamellae relative to WT, a phenotype that becomes more pronounced with age or increased blood pressure. Ultrastructural imaging of the vessel wall using Fib-SEM shows in detail the structural derangements that occur in the Lox mutants. Likewise, aortas from Lox+/ c.G854T mice exposed to elastase dilate faster suggesting that the elastin and collagen produced in these mutants is structurally inferior making it increasingly susceptible to proteolytic damage. Taken together these findings better describe the mechanism by which single missense variants in the copper binding domain for Lox cause hereditary aortic aneurysm.