Background: Matrix metalloproteinase (MMP)-12 is highly expressed in abdominal aortic aneurysms and its elastolytic function has been implicated in the pathogenesis. This concept is challenged, however, by conflicting data. Here, we sought to revisit the role of MMP-12 in abdominal aortic aneurysm. Methods: Apoe ^-/- and Mmp12 ^-/- / Apoe ^-/- mice were infused with Ang II (angiotensin). Expression of neutrophil extracellular traps (NETs) markers and complement component 3 (C3) levels were evaluated by immunostaining in aortas of surviving animals. Plasma complement components were analyzed by immunoassay. The effects of a complement inhibitor, IgG-FH _1-5 (factor H-immunoglobulin G), and macrophage-specific MMP-12 deficiency on adverse aortic remodeling and death from rupture in Ang II–infused mice were determined. Results: Unexpectedly, death from aortic rupture was significantly higher in Mmp12 ^-/- / Apoe ^-/- mice. This associated with more neutrophils, citrullinated histone H3 and neutrophil elastase, markers of NETs, and C3 levels in Mmp12 ^-/- aortas. These findings were recapitulated in additional models of abdominal aortic aneurysm. MMP-12 deficiency also led to more pronounced elastic laminae degradation and reduced collagen integrity. Higher plasma C5a in Mmp12 ^-/- mice pointed to complement overactivation. Treatment with IgG-FH _1-5 decreased aortic wall NETosis and reduced adverse aortic remodeling and death from rupture in Ang II–infused Mmp12 ^-/- mice. Finally, macrophage-specific MMP-12 deficiency recapitulated the effects of global MMP-12 deficiency on complement deposition and NETosis, as well as adverse aortic remodeling and death from rupture in Ang II–infused mice. Conclusions: An MMP-12 deficiency/complement activation/NETosis pathway compromises aortic integrity, which predisposes to adverse vascular remodeling and abdominal aortic aneurysm rupture. Considering these new findings, the role of macrophage MMP-12 in vascular homeostasis demands re-evaluation of MMP-12 function in diverse settings.

Multidirectional or disturbed flow promotes endothelial dysfunction and is associated with early atherogenesis. Here we investigated the role of Wnt signalling in flow-mediated endothelial dysfunction. The expression of Frizzled-4 was higher in cultured human aortic endothelial cells (ECs) exposed to disturbed flow compared to that seen for undisturbed flow, obtained using an orbital shaker. Increased expression was also detected in regions of the porcine aortic arch exposed to disturbed flow. The increased Frizzled-4 expression in cultured ECs was abrogated following knockdown of R-spondin-3. Disturbed flow also increased the nuclear localisation and activation of β-catenin, an effect that was dependent on Frizzled-4 and R-spondin-3. Inhibition of β-catenin using the small-molecule inhibitor iCRT5 or knockdown of Frizzled-4 or R-spondin-3 resulted in reduced expression of pro-inflammatory genes in ECs exposed to disturbed flow, as did inhibition of WNT5A signalling. Inhibition of the canonical Wnt pathway had no effect. Inhibition of β-catenin also reduced endothelial paracellular permeability; this was associated with altered junctional and focal adhesion organisation and cytoskeletal remodelling. These data suggest the presence of an atypical Frizzled-4-β-catenin pathway that promotes endothelial dysfunction in response to disturbed flow.

Inflammation plays a major role in the pathogenesis of several vascular pathologies, including abdominal aortic aneurysm (AAA). Evaluating the role of inflammation in AAA pathobiology and potentially outcome in vivo requires non-invasive tools for high-resolution imaging. We investigated the feasibility of X-ray computed tomography (CT) imaging of phagocytic activity using nanoparticle contrast agents to predict AAA outcome.

Background: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of abdominal aortic aneurysm (AAA). Imaging aortic MMP activity, especially using positron emission tomography to access high sensitivity, quantitative data, could potentially improve AAA risk stratification. Here, we describe the design, synthesis, characterization, and evaluation in murine AAA and human aortic tissue of a first-in-class MMP-targeted positron emission tomography radioligand, ⁶⁴ Cu-RYM2. Methods: The broad spectrum MMP inhibitor, RYM2 was synthetized, and its potency as an MMP inhibitor was evaluated by a competitive inhibition assay. Toxicology studies were performed. Tracer biodistribution was evaluated in a murine model of AAA induced by angiotensin II infusion in Apolipoprotein E-deficient mice. ⁶⁴ Cu-RYM2 binding to normal and aneurysmal human aortic tissues was assessed by autoradiography. Results: RYM2 functioned as an MMP inhibitor with nanomolar affinities. Toxicology studies showed no adverse reaction in mice. Upon radiolabeling with Cu-64, the resulting tracer was stable in murine and human blood in vitro. Biodistribution and metabolite analysis in mice showed rapid renal clearance and acceptable in vivo stability. In vivo positron emission tomography/computed tomography in a murine model of AAA showed a specific aortic signal, which correlated with ex vivo measured MMP activity and Cd68 gene expression. ⁶⁴ Cu-RYM2 specifically bound to normal and aneurysmal human aortic tissues in correlation with MMP activity. Conclusions: ⁶⁴ Cu-RYM2 is a first-in-class MMP-targeted positron emission tomography tracer with favorable stability, biodistribution, performance in preclinical AAA, and importantly, specific binding to human tissues. These data set the stage for ⁶⁴ Cu-RYM2-based translational imaging studies of vessel wall MMP activity, and indirectly, inflammation, in AAA.

Haemodynamic wall shear stress varies from site to site within the arterial system and is thought to cause local variation in endothelial permeability to macromolecules. Our aim was to investigate mechanisms underlying the changes in paracellular permeability caused by different patterns of shear stress in long-term culture. We used the swirling well system and a substrate-binding tracer that permits visualisation of transport at the cellular level. Permeability increased in the centre of swirled wells, where flow is highly multidirectional, and decreased towards the edge, where flow is more uniaxial, compared to static controls. Overall, there was a reduction in permeability. There were also decreases in early- and late-stage apoptosis, proliferation and mitosis, and there were significant correlations between the first three and permeability when considering variation from the centre to the edge under flow. However, data from static controls did not fit the same relation, and a cell-by-cell analysis showed that <5% of uptake under shear was associated with each of these events. Nuclear translocation of NF-κB p65 increased and then decreased with the duration of applied shear, as did permeability, but the spatial correlation between them was not significant. Application of an NO synthase inhibitor abolished the overall decrease in permeability caused by chronic shear and the difference in permeability between the centre and the edge of the well. Hence, shear and paracellular permeability appear to be linked by NO synthesis and not by apoptosis, mitosis or inflammation. The effect was mediated by an increase in transport through tricellular junctions.

Background The neuropilin-like, Discoidin, CUB and LCCL domain containing 2 (DCBLD2) is a transmembrane protein with an unusually long signal sequence (SS) composed of N-terminal (N) and C-terminal (C) subdomains, separated by a transition (tra) subdomain. DCBLD2 interacts with VEGFR-2 and regulates VEGF-induced endothelial cell signaling, proliferation and migration, as well as angiogenesis. The exact mechanisms by which DCBLD2 interacts with VEGFR2 to modulate VEGF signaling remain unclear. Methods Searching for VEGFR2 interacting DCBLD2 domains, we generated various constructs containing different DCBLD2 domain combinations and conducted co-immunoprecipitation and signaling studies in HEK 293T and endothelial cells. Several peptides were synthesized based on the identified domain, and their effect on VEGF signaling was assessed in vitro in cell culture and in vivo using matrigel plug and corneal micropocket assays. The effect of the lead peptide was further evaluated using a murine hindlimb ischemia model. Results DCBLD2 SS interacted with VEGFR2 and promoted VEGF signaling. SS was not cleaved in the mature DCBLD2 and its hydrophobic transmembrane ‘traC’ segment, but not the ‘N’ subdomain, was involved in DCBLD2-VEGFR2 interaction. The smallest unit in DCBLD2 SS that interacts with VEGFR2 was the L5VL5 sequence. Even after the central valine was removed, the L10 sequence mimicked the DCBLD2 SS traC’s effect on VEGF-signaling, while shorter or longer poly-leucine sequences were less effective. Finally, a synthetic traC peptide enhanced VEGF signaling in vitro, promoted VEGF-induced angiogenesis in vivo, and improved blood flow recovery following hindlimb ischemia. Conclusion DCBLD2 SS along with its derivative peptides can promote VEGFR2 signaling and angiogenesis. Synthetic peptides based on DCBLD2 SS hold promise as therapeutic agents for regulating angiogenesis. Importantly these findings refine the traditional view of signal sequences as mere targeting elements, revealing a role in cellular signaling. This opens new avenues for research and therapeutic strategies.