Thymosin beta 4 - a novel regulator of low density lipoprotein receptor related protein 1 (LRP1) in vascular disease

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). Injury-induced growth factors stimulate a phenotypic switching of VSMCs, from their quiescent contractile state to a more active synthetic phenotype that proliferate and migrate. Chronic VSMC dedifferentiation leads to vascular thickening and stiffness, exacerbates inflammation and promotes atherosclerotic lesion development and susceptibility to abdominal aortic aneurysm (AAA). Inhibiting VSMC phenotypic transformation has thus been shown to attenuate progression of vascular disease. We previously identified Thymosin β4 (Tβ4) as a key regulator of embryonic VSMC differentiation. TMSB4X, encoding Tβ4, is the most abundant transcript in healthy and AAA aorta, therefore we hypothesised that Tβ4 may additionally function to maintain vasculature and protect against disease throughout postnatal life. We identified an interaction between Tβ4 and Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGFR? signalling which controls VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Adult Tβ4-null mice displayed aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. During development, Tβ4 functions in a paracrine manner, secreted from endothelial cells (ECs) to induce mesoderm to VSMC differentiation. To distinguish between cell-autonomous and paracrine roles for Tβ4, and simultaneously discern adult maintenance versus developmental requirements, we selectively induced deletion of Tβ4 from VSMCs or ECs at 3 weeks of age. Histological assessment of aortas at 12 weeks demonstrated that VSMC-specific Tβ4 knockdown recapitulated the global KO phenotype, revealing a postnatal requirement for Tβ4 to maintain healthy vasculature. In keeping with this, we confirmed predisposition of these mice to disease in models of atherosclerosis and AAA. Aneurysmal aorta and plaques of Tβ4KO were characterised by accelerated contractile-synthetic VSMC switching, elastin degradation and augmented PDGFR? signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with increased cell surface recycling of LRP1-PDGFR? complexes and reduced lysosomal targeting, suggesting that dysregulated endocytosis underlies enhanced phenotypic switching and proliferation. Given the VSMC differentiation, anti-inflammatory and anti-apoptotic roles of Tβ4, we sought to determine the vasculoprotective potential of exogenous Tβ4. In the AAA model, Tβ4 treatment significantly reduced aortic dilatation and rupture, and preserved VSMC phenotype and elastin integrity, associated with normalisation of PDGFR? signalling. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight that may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression