Thymosin β4 protects against aortic aneurysm via endocytic regulation of growth factor signaling

Abstract

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic modulation which increases susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 (Tβ4) may function to maintain healthy vasculature throughout postnatal life. This was supported by the identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signaling and VSMC proliferation. LRP1 variants have been implicated by genome-wide association studies with risk of AAA and other arterial diseases. Tβ4-null mice displayed aortic VSMC and elastin defects, phenocopying LRP1 mutants, and their compromised vascular integrity predisposed to Angiotensin II-induced aneurysm formation. Aneurysmal vessels were characterised by enhanced VSMC phenotypic modulation and augmented platelet-derived growth factor (PDGF) receptor (PDGFR)β signaling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, associated with dysregulated endocytosis, with increased recycling and reduced lysosomal targeting of LRP1-PDGFRβ. Accordingly, the exacerbated aneurysmal phenotype in Tβ4-null mice was rescued upon treatment with the PDGFRβ antagonist, Imatinib. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health and provides insights into the mechanisms of growth factor-controlled VSMC phenotypic modulation underlying aortic disease progression.

Thymosin β4 preserves vascular smooth muscle phenotype in atherosclerosis via regulation of Low Density Lipoprotein Related Protein 1 (LRP1)

“…Indeed, a striking inverse relationship between Tmsb4x and Lum is apparent within the ModSMC cluster across the disease time course (Online Figure IC). These data strongly support a correlative relationship between high Tβ4 levels and maintenance of the contractile VSMC phenotype, consistent with its roles in promoting mural cell differentiation during development 16, 31, 32 , in vascular homeostasis postnatally and in contractile VSMC preservation in a murine AAA model 15 .…”

“…By western blotting, phosphorylated (activated) AKT and p42/p44 MAP kinase were significantly higher in aortas of Tβ4 -/Y ; ApoE -/- aortas, compared with Tβ4 +/Y ; ApoE -/- aortas, by immunofluorescence (Figure 6B). These data indicate that accelerated phenotypic modulation of VSMCs and exacerbated atherosclerosis in Tβ4 -/Y mice is associated with dysregulated LRP1-PDGFRβ pathway activity and point to a likely role for Tβ4 in controlling receptor turnover, as previously elucidated in isolated VSMCs 15 , to influence the progression and outcome of atherosclerotic disease.…”

“…Accelerated disease progression was characterized by augmented VSMC phenotypic modulation and underpinned by dysregulated PDGFRβ signaling and deleterious ECM remodelling, both of which may result from a failure of Tβ4 to functionally regulate LRP1 trafficking. Mechanistically, Tβ4 has been shown to bind the intracellular domain of LRP1, near NPxY domains that are necessary for signal transduction and endocytosis; accordingly, Tβ4 controls the balance between recycling and lysosomal targeting of LRP1-PDGFRβ complexes, to critically regulate VSMC sensitivity and responses to PDGF-BB 15 . Consistent with this, vascular defects in Tβ4 null mice closely phenocopy those reported with VSMC-specific loss of LRP1 13, 20, 33 , not only at baseline and in AAA 15 , but also, herein, in atherosclerotic disease.…”

“…Mechanistically, Tβ4 has been shown to bind the intracellular domain of LRP1, near NPxY domains that are necessary for signal transduction and endocytosis; accordingly, Tβ4 controls the balance between recycling and lysosomal targeting of LRP1-PDGFRβ complexes, to critically regulate VSMC sensitivity and responses to PDGF-BB 15 . Consistent with this, vascular defects in Tβ4 null mice closely phenocopy those reported with VSMC-specific loss of LRP1 13, 20, 33 , not only at baseline and in AAA 15 , but also, herein, in atherosclerotic disease. Thus, our findings further add support to the existence of a common regulatory mechanism through which Tβ4 and LRP1 act in vascular homeostasis to maintain vessel stability and protect against disease.…”

“…Following internalisation and transduction of signals to nuclear effectors, LRP1-PDGFRβ complexes may be recycled to the cell membrane or targeted for lysosomal degradation, to potentiate or attenuate downstream signalling activity, respectively 14 . We recently defined a key role for the small peptide Thymosin β4 (Tβ4) in regulating LRP1-PDGFRβ trafficking to control VSMC phenotype and protect against abdominal aortic aneurysm (AAA) 15 . This study demonstrated a postnatal VSMC-autonomous role for Tβ4 to maintain VSMC differentiation for vascular homeostasis beyond its developmental role to induce aortic VSMC differentiation 16 .…”