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Thymosin β4 mediates vascular protection via regulation of Low Density Lipoprotein Related Protein 1 (LRP1)

Vascular stability and tone are maintained by contractile smooth muscle cells (VSMCs). However, injury-induced growth factors stimulate a contractile-synthetic phenotypic switch which promotes atherosclerosis and susceptibility to abdominal aortic aneurysm (AAA). As a regulator of embryonic VSMC differentiation, we hypothesised that Thymosin β4 (Tβ4) may additionally function to maintain healthy vasculature and protect against disease throughout postnatal life. This was supported by identification of an interaction with Low density lipoprotein receptor related protein 1 (LRP1), an endocytic regulator of PDGF-BB signalling and VSMC proliferation. LRP1 variants have been identified by GWAS as major risk loci for AAA and coronary artery disease. Tβ4-null mice display aortic VSMC and elastin defects, phenocopying LRP1 mutants and suggesting compromised vascular integrity. We confirmed predisposition to disease in models of atherosclerosis and AAA. Diseased vessels and plaques were characterised by accelerated contractile-synthetic VSMC switching and augmented PDGFRβ signalling. In vitro, enhanced sensitivity to PDGF-BB, upon loss of Tβ4, coincided with dysregulated endocytosis, leading to increased recycling of LRP1-PDGFRβ and reduced lysosomal targeting. Our study identifies Tβ4 as a key regulator of LRP1 for maintaining vascular health, providing insight which may reveal useful therapeutic targets for modulation of VSMC phenotypic switching and disease progression.

irregular VSMC morphology, with a shift towards expression of synthetic markers (Figure 2,. The recapitulation of the global knockout phenotype suggests that reduction in Tβ4 levels through 8 weeks of postnatal life impairs vascular stability and defines a VSMC-autonomous, protective role in adult vessel homeostasis.

Tβ4 interacts with the vasculoprotective endocytic receptor, LRP1
To gain insight into the possible mechanisms by which Tβ4 maintains vascular health, we performed a yeast two hybrid screen to identify putative binding partners from an E11.5 murine embryonic library. A leading candidate was Low density lipoprotein receptor related protein 1 (LRP1), associated in human (12)(13)(14) and animal studies (15,16) with protection against AAA and atherosclerosis. Two independent clones expressing regions of the intracellular domain of LRP1 were identified among the "prey" plasmids after stringent selection. Although we could not reproducibly co-immunoprecipitate Tβ4-LRP1 from murine aorta or MOVAS-1, a murine aortic VSMC line, we validated close association of the proteins (<40nm) by proximity ligation assay (PLA), consistent with the possibility of a weak or transient interaction, or an indirect association via a complex. Foci of Tβ4-LRP1 PLA signals were detected within medial VSMCs of murine aorta, at baseline ( Figure 3A), as well as in disease (not shown). Moreover, conservation across species and clinical relevance was confirmed by detection of PLA signals in human VSMCs, both in aneurysmal aorta from AAA patients enrolled in the Oxford Abdominal Aortic Aneurysm Study (OxAAA study(26)); Figure 3B) and healthy vessels (matched omental artery samples from the same patients). Specificity for the PLA was ensured by lack of signal in -/Y Tβ4 null aortas ( Figure   3A) and with omission of the LRP1 antibody ( Figure 3B). While PLA signals were more abundant in omental arteries than AAA samples, inferences about disease relationship cannot be made, due to the small sample size and potentially compromised tissue integrity in the diseased vessels.
Nevertheless, these observations collectively support a potential role for Tβ4 in regulating VSMC function via LRP1. We examined localisation of Tβ4-LRP1 foci more closely in primary murine aortic VSMCs. By immunofluorescence, LRP1 localised to punctate structures, consistent with its known incorporation into endocytic vesicles ( Figure 3C). While Tβ4 was distributed throughout muscle-specific Lrp1 mutant (Myh11 Cre ; Lrp1 fl/fl ; ApoE -/-; tamoxifen-induced at 3 weeks of age).
Increased plaque size did not correlate with an increase in weight gain or in cholesterol levels in Tβ4 -/Y ; ApoE -/mice, compared with Tβ4 +/Y ; ApoE -/-, although mice of both genotypes weighed more than Myh11 Cre ; Lrp1 fl/fl ; ApoE -/-, both at baseline and after high fat feeding (Supplemental Figure 3). We tracked VSMC phenotype over the course of WD feeding and, after 7 weeks, the medial layer VSMCs of Tβ4 -/Y ; ApoE -/mice displayed a pronounced alteration in morphology, with loss of their characteristic elongated shape and acquisition of a smaller, more rounded appearance( Figure   4D). This was accompanied by a shift in the ratio of synthetic (L-caldesmon and vimentin increased) over contractile markers (αSMA, Sm22α and Myh11 decreased), determined by immunofluorescence and qRT-PCR ( Figure 4D and E, respectively). Tβ4 +/Y ; ApoE -/-VSMCs underwent a similar shift, only this occurred more gradually, becoming noticeable from 9 weeks' WD, once plaques were already established ( Figure 4D). We measured fibrous cap thickness, relative to lesion thickness, after 12 weeks and, whilst this was comparable between genotypes ( Figure 4, D, F), the caps of Tβ4 -/Y ; ApoE -/plaques were composed of cells expressing exclusively synthetic markers, such as L-caldesmon ( Figure 4D). In comparison, control Tβ4 +/Y ; ApoE -/cap cells retained higher levels of contractile markers, such as αSMA ( Figure 4D). Consistent with dedifferentiation and acquisition of a synthetic phenotype, medial VSMCs were significantly more proliferative, as determined by increased EdU incorporation between weeks 5 and 7 of WD feeding ( Figure 4G); in contrast, cells within the plaque showed no difference in proliferation between genotypes (Supplemental Figure 4A; quantified in Figure 4G). These cells, confirmed to be mostly macrophages/foam cells, based on CD68 expression (Supplemental Figure 4B), were present in similar number in Tβ4 -/Y ; ApoE -/and Tβ4 +/Y ; ApoE -/plaques, with plaque composition shown to be quantitatively indistinguishable, in terms of necrotic core and area occupied by macrophages (Supplemental Figure 4C). These data, along with comparable expression of pro-inflammatory cytokines, Il1b and Tnfα in descending aorta biopsies (Supplemental Figure 4D-E), suggest that altered inflammatory response are not primarily responsible for the altered medial layer phenotypes observed. Of note, a significantly higher proportion of Tβ4 -/Y ; ApoE -/medial VSMCs (3weeks' WD) and plaque cells (9 weeks' WD) were apoptotic, as revealed by TUNEL staining ( Figure 4H), compared with Tβ4 +/Y ; ApoE -/-.
Collectively, the preponderance of apoptotic and synthetic VSMCs, relative to the number of contractile VSMCs, suggests that, despite having thick fibrous caps, plaques in Tβ4 -/Y ; ApoE -/mice may be more unstable than those in Tβ4 +/Y ; ApoE -/mice. Finally, a striking degeneration of the elastin lamellae was apparent in Tβ4 -/Y ; ApoE -/aortas, with more elastin breaks per section than in Tβ4 +/Y ; ApoE -/aortas and a higher elastin damage score ( Figure 4I-J). By these parameters, elastin degeneration in Tβ4 -/Y ; ApoE -/aortas was comparable with, and in some cases more severe than, Myh11 Cre ; Lrp1 fl/fl ; ApoE -/aortas ( Figure 4I-J).
Whilst our study examined male mice in WD cohorts, the unanticipated observation of hindlimb paralysis, an indication of possible aortic thrombosis, aneurysm or rupture, prompted us to investigate a cohort of regular chow-fed 8 month old female Tβ4 -/-; ApoE -/and age-matched Tβ4 +/+ ; ApoE -/mice. Even without a cholesterol-rich diet, aortas of Tβ4 -/-; ApoE -/mice were aneurysmal (>1.5 fold dilated), compared with Tβ4 +/+ ; ApoE -/-, contained large atherosclerotic plaques and exhibited severe degeneration of the underlying elastin layers (Supplemental figure   5). in view of the increasingly recognised sexual dimorphism in vascular disease, both in patients and in experimental studies (29), we compared a second cohort of female mice, this time with WD over an equivalent time course to that used in male cohorts, and determined a significant increase in aortic arch plaque size in Tβ4 -/-; ApoE -/-, compared with Tβ4 +/+ ; ApoE -/mice (Supplemental figure 6). Interestingly, in female mice, as compared to male mice, increased plaque size did correlate with increased weight gain in Tβ4 -/-; ApoE -/mice, compared with Tβ4 +/+ ; ApoE -/-. Taken together, these studies suggest that atherogenesis is accelerated in Tβ4 null mice, regardless of gender, and is underpinned by an advanced VSMC contractile-synthetic phenotype shift, which closely recapitulates the phenotype associated with loss of LRP1.
We next determined whether loss of Tβ4 similarly predisposed to abdominal aneurysm, using the well-characterised model of Angiotensin II infusion (Ang II; 1mg/kg/day), via a subcutaneously implanted osmotic mini pump for up to 10 days. Analysis of whole mount aortas revealed an increased susceptibility of Tβ4 -/Y mice to aneurysm, compared with Tβ4 +/Y controls ( Figure 5A).
Phenotypes ranged from more pronounced ascending and descending aortic aneurysm (defined as >1.5-fold dilatation; mild) to abdominal aortic rupture, haematoma formation and death in <5 days (severe) (quantified in Figure 5B; examples of ruptures shown in Supplemental Figure 7).
Although rare (<10%), dissections were detectable in Tβ4 -/Y as blood tracking into the adventitial matrix or between medial and adventitial layers; Supplemental Figure 7, death at day 8). Given the prominent role of inflammation in driving aneurysm progression with AngII treatment and numerous anti-inflammatory roles ascribed to Tβ4(23,24,30), we also investigated susceptibility in tamoxifen-dosed Myh11 CreERT2 ; Hprt Tβ4shRNA knockdown mice, alongside Myh11 CreERT2 ; Hprt +/+ controls ( Figure 5A) and Myh11 CreERT2 ; Lrp1 fl/fl mice, in order to avoid disrupting Tβ4-LRP1 function in immune cells. Similar to global Tβ4 knockouts, VSMC-specific Tβ4 knockdown mice displayed an increased incidence of rupture (Supplemental Figure 7), as well as aortic aneurysms and a higher mortality rate over the 10 day time course (incidence rates quantified in Figure 5B and representative ruptures shown histologically in Supplemental Figure 7). Mean aortic diameter, measured on histological sections, was increased by 1.6-fold in Tβ4 -/Y mice and 1.8fold in Myh11 CreERT2 ; Hprt Tβ4shRNA mice, compared with their respective AngII-treated controls. This days' AngII). The prominent morphological alterations were accompanied by increased expression of synthetic maker, L-caldesmon ( Figure 5F). After 10 days' infusion, a further phenotype shift was observed, along with a greater VSMC loss, in Tβ4 -/Y , Myh11 CreERT2 ; Hprt Tβ4shRNA and Myh11 Cre ; Lrp1 fl/fl aortas, compared with controls ( Figure 5G). Collectively, these data demonstrate that loss of Tβ4 predisposes to aortic aneurysm, in the same way as loss of LRP1. Accelerated disease progression in mutants appeared not to relate to exacerbated inflammation, but rather to the more rapid VSMC phenotypic switch and degeneration of the elastin lamellae.
Signalling responses of VSMCs were more closely interrogated in vitro in primary VSMC cultures established from the descending aortas of Tβ4 +/Y and Tβ4 -/Y mice. While some degree of VSMC dedifferentiation and contractile-synthetic phenotypic switching is inherent upon culture in high serum-containing medium, Tβ4 +/Y VSMCs typically exhibited a characteristic spindle-like morphology, whereas Tβ4 -/Y VSMCs were typically more rhomboid ( Figure 6C). Although synthetic markers, such as L-caldesmon were expressed at comparable levels in the majority of VSMCs, expression of contractile VSMC markers, including αSMA and SM-MHC, was reduced in Tβ4 -/Y VSMCs. To compare signalling responses, serum-starved VSMCs were treated with 20ng/ml PDGF-BB over a 60 minute time course, prior to fixation, for analysis of pathway components by immunofluorescence. Phosphorylation (activation) of PDGFRβ, p42/p44 MAPK and JNK were strongly induced within 10 minutes of treatment ( Figure 6, D-F). In Tβ4 +/Y VSMCs, phosphorylation of pathway components diminished by 30 minutes and returned to near-baseline by 60 minutes. This contrasted with Tβ4 -/Y VSMCs, in which phosphorylation remained high (PDGFRβ) or further increased (ERK, JNK) between 10 and 30 minutes and remained significantly higher (close to maximal Tβ4 +/Y levels) even at 60 minutes. These data reveal that PDGFRβ pathway activity is both enhanced in magnitude and more sustained in duration in Tβ4 -/Y , compared with Tβ4 +/Y VSMCs, in response to the same dose of PDGF-BB. We, therefore, hypothesised that VSMCs lacking Tβ4 would be more sensitive to PDGF-BB-stimulated cellular responses, such as proliferation and migration. Indeed, over a range of tested PDGF-BB doses (2ng/ml -50ng/ml), proliferation rate was significantly greater in Tβ4 -/Y than in Tβ4 +/Y VSMCs ( Figure 6G). Of note, although only nuclear P-ERK signals were quantified above, a striking accumulation of perinuclear P-ERK was also apparent in Tβ4 -/Y , but not Tβ4 +/Y , VSMCs ( Figure   6E); association of Tyrosine phosphorylated ERK with Golgi occurs during the G2/M phase of the cell cycle (35), further evidence of enhanced proliferation in Tβ4 -/Y VSMCs. By contrast, increased sensitivity to PDGF-BB did not correlate with enhanced migration, as assessed by scratch wound assay (Supplemental Figure 9); in fact, Tβ4 -/Y VSMCs migrated significantly more slowly than Tβ4 +/Y VSMCs. As this result was unexpected, we additionally compared migration rates in Myh11 Cre ; Lrp1 fl/fl VSMCs, which are similarly known to be hypersensitive to PDGF-BB, and found their migration to be likewise reduced compared with controls.
Western blotting confirmed a similarly enhanced and more sustained activation of components of the PDGFRβ pathway in serum-starved, Tβ4 siRNA-treated MOVAS-1, with addition of 20ng/ml PDGF-BB ( Figure 7A). Y1021-phosphorylated PDGFRβ and S473 phosphorylated Akt were significantly enhanced, although phosphorylated p42/p44 was unaffected in knocked down MOVAS-1 ( Figure 7B). Total PDGFRβ levels declined steadily over the 60 minutes, consistent with lysosomal targeting and degradation and decline was notably more gradual in Tβ4 knockdown cells, albeit this was not significant ( Figure 7B). In parallel, we performed surface biotinylation assays to measure levels of LRP1 and PDGFRβ at the cell surface over the same   (13) and coronary artery disease (14). Tβ4 is the most abundant transcript in healthy and AAA aorta (5), yet its roles in vascular protection and regulation of LRP1-mediated growth factor signalling had not been recognised. Our study provides a mechanism by which Tβ4 controls LRP1-mediated VSMC responses to protect against vascular disease.  (46), in cell migration. Whether LRP1 promotes or inhibits migration of VSMCs appears to be dependent on extracellular matrix and ligand-binding cues (47), as discussed in (48). Moreover, there are likely distinct paracrine stimulatory roles for Tβ4-LRP1 as well as direct effects upon remodeling of the actin cytoskeleton (49) (50).
PDGF-BB, secreted from infiltrating macrophages during the initiating phases of aortic disease, potently drives VSMC phenotypic switching. The vasculoprotective effects of Tβ4 that we report appear to be mediated, at least in part, via LRP1-regulated growth factor signalling, specifically to control the sensitivity of VSMCs to PDGF-BB and potentially other ligands that are regulated by LRP1 (51). Tβ4 was found to alter cellular responses to PDGF-BB by shifting the balance between receptor degradation and recycling. Further research is required to elucidate the precise mechanism(s) by which Tβ4 controls receptor trafficking and to determine whether this is limited to selected receptors, such as LRP1, or a generic regulation of endocytosis. De novo actin filament assembly is essential for endocytosis and, as an actin monomer-sequestering peptide, Tβ4 displays a biphasic effect: stimulating receptor-mediated endocytosis at low concentrations (<0.5µM) but inhibiting the process at high concentrations (10µM), consistent with actin depolymerization induced with high doses (52). More specifically, Tβ4-mediated, membraneinduced actin polymerization was shown to be required for fusion of late endosomes and phagosomes/lysosomes, but not for fusion of early endosomes, which can proceed without actin polymerization (53). Reduced lysosomal targeting of LRP1-PDGFRβ in our experiments is consistent with a defect in late endosome-lysosome fusion but seems an unlikely explanation for the increased partitioning to recycling endosomes. Recycling of signalling receptors occurs via a selective and carefully-regulated mechanism, which also requires actin polymerization, namely 'Actin-Stabilized Sequence-dependent Recycling Tubule (ASSERT)' scaffold formation (54), although a direct role for Tβ4 in this process has not been investigated. Thus, although there is support for a fundamental role for Tβ4 in endocytosis, given the remarkable similarity in developmental and disease phenotypes of Tβ4 and LRP1 mutants, a more selective role in regulation of LRP1-PDGFRβ by Tβ4 remains a possibility, supported by our identification of a physical interaction of LRP1-Tβ4 from a yeast two hybrid screen. While we failed to reproducibly validate the interaction by immunoprecipitation, GST pulldown and surface plasmon resonance (SPR), we cannot actually exclude a direct interaction, as there are technical complications with each of these approaches. The cytoplasmic domain of LRP1 interacts with multiple adaptor proteins and signalling proteins; while the 5kDa Tβ4 may participate, the complex may be too 'congested' to permit simultaneous binding of an anti-Tβ4 antibody, given the small size of the peptide. While pulldown and SPR methodologies obviate the need for antibody binding, they do not recreate the precise cellular context under which interaction potentially occurs, and which may be carefully regulated. Alternatively, an interaction may go undetected if it is transient and/or weak. A physical interaction is frequently inferred by proximity ligation assay, which requires proteins to be within 40nm and, in this regard, our data are supportive, although we cautiously conclude proximity, rather than a definitive protein-protein interaction. It is worth highlighting the functionally similar stabilin-2, an endocytic scavenger receptor which, like LRP1, internalizes coagulation factor VIII and LDL (55), amongst its many ligands, and contains NPxY motifs on its intracellular domain (56). Stabilin-2 was identified by yeast two hybrid (57) and validated by biolayer interferometry (58) to be a "weak and fuzzy", yet specific, interactor of Tβ4, thus there is precedent for direct interaction with structurally and functionally similar receptors. Whether or not the proteins physically interact, we have validated an important functional interaction for Tβ4-controlled LRP1-PDGFRβ trafficking. Given the disease relevance of LRP1, not just in aortic disease but also in the pathogenesis of Alzheimer's disease (59), further investigation into its internalization mechanism is warranted. Prevalence of aortic aneurysm is 5% amongst the elderly and treatment involves a high risk surgical procedure with no pharmacological therapeutic options. That Tβ4 can regulate endocytosis, even when administered exogenously (52), raises the possibility of developing novel strategies to maintain differentiated VSMC phenotype and treat aortic disease. In vivo blood pressure measurement. One day after the MRI scan (exactly as previously described (61)), animals were anaesthetised with isoflurane (4%) and placed in supine position onto a heating plate with feedback control (Physitemp Instruments, NJ). Animals were kept at 37±1°C while oxygen and anaesthetics (1-2% isoflurane) were supplied via a nose cone (1 L/min). Body temperature and heart rate were recorded for the whole experiment using PowerLab with Chart 5 (ADInstruments, UK). A T-shaped middle-neck incision from mandible to the sternum was made. Blunt dissection was used to expose the left common carotid artery. A 5.0 Mersilk (Ethicon, NJ) suture was used to tie off the distal end of the artery while a micro vascular clip was used to occlude the proximal end of the exposed artery. A small incision near the distal end of the artery was made and a fluid filled tube (heparin; 100 U/mL diluted in 0.9% saline) with an inner diameter of 0.28 mm (Critchley Electrical Products Pty Ltd, Australia) was introduced into the artery and fixed in place with a suture. Following that, the micro vascular clip was removed and the arterial pressure was recorded using the MLT844 pressure transducer (ADInstruments, UK). After stabilisation of the signal for about 5-10 min an average of 1000 heart beats was used for analysis.

Animal models.
Atherosclerosis model.  week old) mice, on the Apoe -/background, were fed on Western diet (21.4% fat; 0.2% cholesterol; Special Diets Services, Essex, UK) for up to 12 weeks. Mice were weighed weekly and, at harvest, serum collected for cholesterol analysis (by Charles River Laboratories). EdU (5-ethynyl-2'-deoxyuridine), prepared in 0.9% saline, was administered at a dose of 150mg/kg by intraperitoneal injection (4 doses over the 10 days prior to harvest).
Aneurysm model. 8-12 week old adult mice were infused with 1mg/kg/day Angiotensin II (Sigma) by subcutaneous implantation of an osmotic mini pump (Alzet) for 10 days. Animals were regularly monitored and weighed post-surgery until harvest.

Human tissue sampling
Subjects undergoing open abdominal aortic aortic aneurysm repair were prospectively recruited from the Oxford Abdominal Aortic Aneurysm (OxAAA) study. The study was approved by the Oxford regional ethics committee (Ethics Reference: 13/SC/0250). All subjects gave written informed consent prior to the study procedure. Baseline characteristics of each participant were recorded. During surgery to repair the AAA, a wedge of abdominal omentum containing a segment of omental artery was identified and biopsied en bloc. Isolation of omental artery was performed immediately in the operating theatre. The omental artery segment was cleared of perivascular tissue and snapped frozen. Prior to incision of the aortic aneurysm, a marker pen was used to denote the cross section of maximal dilatation according visual inspection. A longitudinal strip of the aneurysm wall along the incision was then excised. The aneurysm tissue was stripped off the peri-vascular tissue and mural thrombus. The tissue at the maximal dilatation was isolated, divided into smaller segments, and snap frozen for subsequent analysis. ab198337). AlexaFluor-conjugated secondary antibodies raised against Rat, Rabbit, Mouse or Armenian Hamster IgG (Invitrogen) were used at 1:200.
Immunoblotting. SiRNA-treated and PDGFβ-stimulated cells were lysed in RIPA buffer (50 mM Tris-HCl pH7.5, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, PhosSTOP and cOmplete protease inhibitor cocktail (Roche)). Western blots were performed using a standard protocol. Proteins were transferred by semi-dry transfer onto PVDF membrane. After blocking, membranes were probed with primary antibodies overnight. A modified protocol was used for Thymosin β4 Western blotting (63). Proteins were separated on Tris-Tricine gels and cross-linked with 10% glutaraldehyde, before wet overnight transfer onto nitrocellulose membrane. Membranes were crosslinked with UV light (254 nm, 10 min) before blocking and overnight antibody probing. Antibodies used were Rabbit anti-phospho-PDGFRβ (Tyr1021) Green (Sigma) following manufacture's protocol. Following Duolink® protocol, cells were costained for smooth muscle markers or endocytic compartments using the antibodies described above. Colocalisation was determined, using the ImageJ plug-in JACOP 2.0(37). washed frozen and processed for DNA-based assay, as described (65), and background subtracted using a plate which had been incubated with medium but no cells.
Smooth muscle migration assays. Primary aortic VSMCs were cultured as a confluent monolayer in 12-well plates. After overnight serum starvation, scratch wounds were introduced with a P200 tip. After washing, medium was replaced with serum-free DMEM containing PDGF-BB (2-50ng/ml) and 100 μM hydroxyurea (Sigma). Wells were imaged at t0, and every 24h thereafter, using a Zeiss Axio Imager microscope. Wounds were measured and expressed as % relative to t0.

Statistics.
Randomisation of animals to treatment or genotype groups was introduced at the time of mini pump implantation (aneurysm); introduction of western diet (atherosclerosis) or harvest (baseline). Thereafter, tissues were processed and analysed by an independent observer whilst blinded to treatment or genotype. Statistical analyses were performed with GraphPad Prism Software. For the quantitative comparison of two groups, two-tailed unpaired Student's t-test was used to determine any significant differences, after assessing the requirements for a t-test using a Shapiro-Wilk test for normality and an F-Test to compare variances. Alternatively, a Mann-Whitney non-parametric test was used. For comparison of three groups or more, a one-      Mice lacking Tβ4 display more severe aneurysmal phenotypes, associated with accelerated VSMC phenotypic switching. Whole mount aortas from saline-or AngII-infused mice (A). White arrowheads indicate ascending and descending aortic aneurysm; red arrowheads indicate rupture. Incidence of dissection and rupture quantified per genotype in B. Verhoeff-van Gieson staining of abdominal aorta to visualize elastin integrity (C, breaks indicated with black arrowheads). Quantification of aortic diameter (D) and elastin grade (E). Immunofluorescence to assess medial layer morphology and VSMC phenotype at 5 (F) and 10 days' (G) AngII infusion. Data are presented as mean ± SD, with each data point representing an individual animal. Significance was calculated using one-Way ANOVA with Tukey's multiple comparison tests (D-E); ***: p≤0.001; ****p≤ 0.0001. Except in F (5 days), samples were harvested after 10 days' AngII. Scale bars: A: 2mm; C: 500µm; inset 100µm.  Surface biotinylation assays measure levels of LRP1 (C) and PDGFRβ (D) at the cell surface. LRP1-PDGFRβ complexes, indicated by proximity ligation assay (PLA) in primary aortic VSMCs, colocalising with endocytic compartments over a 60 minute time course (E-J). Shown at 30 minutes, co-localising with early endosomes (EEA1; white arrows, E) and late endosomes (Rab7; yellow arrows, E) and with recycling endosomes (TfR; white arrows, F) and lysosomes (LAMP-1; yellow arrows, F). Quantification of co-localisation in G-J. Data are presented as mean ± SEM; n=4 experiments (C-D); n=3 experiments (A-B; E-J). Significance was calculated using two-way ANOVA with Sidak's multiple comparison test (B; C-D; G-J).*p ≤ 0.05; **p≤0.01; ***: p≤0.001. Scale bars: E: 5µm (applies to all whole cell views in E-F); boxed areas 1 and 2 shown magnified to left, with scale 2µm (applies to all magnified views).