Short-Term Disruption of TGFß Signaling in Adult Mice Renders the Aorta Vulnerable to Hypertension-Induced Dissection.

Hypertension and transient increases in blood pressure from extreme exertion are risk factors for aortic dissection in patients with age-related vascular degeneration or inherited connective tissue disorders. Yet, the common experimental model of angiotensin II-induced aortopathy in mice appears independent of high blood pressure as lesions do not occur in response to an alternative vasoconstrictor, norepinephrine, and are not prevented by co-treatment with a vasodilator, hydralazine. We investigated vasoconstrictor administration to adult mice 1 week after disruption of TGFß signaling in smooth muscle cells. Norepinephrine increased blood pressure and induced aortic dissection by 7 days and even within 30 minutes that was rescued by hydralazine; results were similar with angiotensin II. Changes in regulatory contractile molecule expression were not of pathological significance. Rather, reduced synthesis of extracellular matrix yielded a vulnerable aortic phenotype by decreasing medial collagen, most dynamically type XVIII, and impairing cell-matrix adhesion. We conclude that transient and sustained increases in blood pressure cause dissection in aortas rendered vulnerable by inhibition of TGFß-driven extracellular matrix production by smooth muscle cells. A corollary is that medial fibrosis, a frequent feature of medial degeneration, may afford some protection against aortic dissection.

Afadin-nectin forces its way to the front.

Force transmission at cell-cell junctions critically regulates embryogenesis, tissue homeostasis, and diseases including cancer. The cadherin-catenin linkage has been considered the keystone of junctional force transmission, but new findings challenge this paradigm, arguing instead that the nectin-afadin linkage plays the more important role in mature junctions in the intestinal epithelium.

Dysregulated cellular metabolism in atherosclerosis: mediators and therapeutic opportunities.

Accumulating evidence over the past decades has revealed an intricate relationship between dysregulation of cellular metabolism and the progression of atherosclerotic cardiovascular disease. However, an integrated understanding of dysregulated cellular metabolism in atherosclerotic cardiovascular disease and its potential value as a therapeutic target is missing. In this Review, we (1) summarize recent advances concerning the role of metabolic dysregulation during atherosclerosis progression in lesional cells, including endothelial cells, vascular smooth muscle cells, macrophages and T cells; (2) explore the complexity of metabolic cross-talk between these lesional cells; (3) highlight emerging technologies that promise to illuminate unknown aspects of metabolism in atherosclerosis; and (4) suggest strategies for targeting these underexplored metabolic alterations to mitigate atherosclerosis progression and stabilize rupture-prone atheromas with a potential new generation of cardiovascular therapeutics.

Mechanosensing through talin 1 contributes to tissue mechanical homeostasis.

It is widely believed that tissue mechanical properties, determined mainly by the extracellular matrix (ECM), are actively maintained. However, despite its broad importance to biology and medicine, tissue mechanical homeostasis is poorly understood. To explore this hypothesis, we developed mutations in the mechanosensitive protein talin1 that alter cellular sensing of ECM stiffness. Mutation of a novel mechanosensitive site between talin1 rod domain helix bundles 1 and 2 (R1 and R2) shifted cellular stiffness sensing curves, enabling cells to spread and exert tension on compliant substrates. Opening of the R1-R2 interface promotes binding of the ARP2/3 complex subunit ARPC5L, which mediates the altered stiffness sensing. Ascending aortas from mice bearing these mutations show increased compliance, less fibrillar collagen, and rupture at lower pressure. Together, these results demonstrate that cellular stiffness sensing regulates ECM mechanical properties. These data thus directly support the mechanical homeostasis hypothesis and identify a novel mechanosensitive interaction within talin that contributes to this mechanism.

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

A key feature of arteriogenesis is capillary-to-arterial endothelial cell fate transition. Although a number of studies in the past two decades suggested this process is driven by VEGF activation of Notch signaling, how arteriogenesis is regulated remains poorly understood. Here we report that arterial specification is mediated by fluid shear stress (FSS) independent of VEGFR2 signaling and that a decline in VEGFR2 signaling is required for arteriogenesis to fully take place. VEGF does not induce arterial fate in capillary ECs and, instead, counteracts FSS-driven capillary-to-arterial cell fate transition. Mechanistically, FSS-driven arterial program involves both Notch-dependent and Notch-independent events. Sox17 is the key mediator of the FSS-induced arterial specification and a target of VEGF-FSS competition. These findings suggest a new paradigm of VEGF-FSS crosstalk coordinating angiogenesis, arteriogenesis and capillary maintenance.

Gamma protocadherins in vascular endothelial cells inhibit Klf2/4 to promote atherosclerosis.

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide1. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2/4 anti-inflammatory program in endothelial cells (ECs)2-8. Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation9,10. We previously reported a whole genome CRISPR knockout screen11 that identified novel inducers of Klf2/4. Here we report suppressors of Klf2/4 and characterize one candidate, protocadherin gamma A9 (Pcdhga9), a member of the clustered protocadherin gene family12. Pcdhg deletion increases Klf2/4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2/4 by inhibiting the Notch pathway via physical interaction of cleaved Notch1 intracellular domain (NICD Val1744) with nuclear Pcdhg C-terminal constant domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis. This study identifies a novel fundamental mechanism of EC resilience and therapeutic target for treating inflammatory vascular disease.

Calcium flow at ER-TGN contact sites facilitates secretory cargo export.

Ca2+ influx into the trans-Golgi Network (TGN) promotes secretory cargo sorting by the Ca2+-ATPase SPCA1 and the luminal Ca2+ binding protein Cab45. Cab45 oligomerizes upon local Ca2+ influx, and Cab45 oligomers sequester and separate soluble secretory cargo from the bulk flow of proteins in the TGN. However, how this Ca2+ flux into the lumen of the TGN is achieved remains mysterious, as the cytosol has a nanomolar steady-state Ca2+ concentration. The TGN forms membrane contact sites (MCS) with the Endoplasmic Reticulum (ER), allowing protein-mediated exchange of molecular species such as lipids. Here, we show that the TGN export of secretory proteins requires the integrity of ER-TGN MCS and inositol 3 phosphate receptor (IP3R)-dependent Ca2+ fluxes in the MCS, suggesting Ca2+ transfer between these organelles. Using an MCS-targeted Ca2+ FRET sensor module, we measure the Ca2+ flow in these sites in real time. These data show that ER-TGN MCS facilitates the Ca2+ transfer required for Ca2+-dependent cargo sorting and export from the TGN, thus solving a fundamental question in cell biology.

Focal adhesion-derived liquid-liquid phase separations regulate mRNA translation.

Liquid-liquid phase separation (LLPS) has emerged as a major organizing principle in cells. Recent work showed that multiple components of integrin-mediated focal adhesions including p130Cas can form LLPS, which govern adhesion dynamics and related cell behaviors. In this study, we found that the focal adhesion protein p130Cas drives formation of structures with the characteristics of LLPS that bud from focal adhesions into the cytoplasm. Condensing concentrated cytoplasm around p130Cas-coated beads allowed their isolation, which were enriched in a subset of focal adhesion proteins, mRNAs and RNA binding proteins, including those implicated in inhibiting mRNA translation. Plating cells on very high concentrations of fibronectin to induce large focal adhesions inhibited message translation which required p130Cas and correlated with droplet formation. Photo-induction of p130Cas condensates using the Cry2 system also reduced translation. These results identify a novel regulatory mechanism in which high adhesion limits message translation via induction of p130Cas-dependent cytoplasmic LLPS. This mechanism may contribute to the quiescent state of very strongly adhesive myofibroblasts and senescent cells.

Intracellular tension sensor reveals mechanical anisotropy of the actin cytoskeleton.

The filamentous actin (F-actin) cytoskeleton is a composite material consisting of cortical actin and bundled F-actin stress fibers, which together mediate the mechanical behaviors of the cell, from cell division to cell migration. However, as mechanical forces are typically measured upon transmission to the extracellular matrix, the internal distribution of forces within the cytoskeleton is unknown. Likewise, how distinct F-actin architectures contribute to the generation and transmission of mechanical forces is unclear. Therefore, we have developed a molecular tension sensor that embeds into the F-actin cytoskeleton. Using this sensor, we measure tension within stress fibers and cortical actin, as the cell is subject to uniaxial stretch. We find that the mechanical response, as measured by FRET, depends on the direction of applied stretch relative to the cell's axis of alignment. When the cell is aligned parallel to the direction of the stretch, stress fibers and cortical actin both accumulate tension. By contrast, when aligned perpendicular to the direction of stretch, stress fibers relax tension while the cortex accumulates tension, indicating mechanical anisotropy within the cytoskeleton. We further show that myosin inhibition regulates this anisotropy. Thus, the mechanical anisotropy of the cell and the coordination between distinct F-actin architectures vary and depend upon applied load.

cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling.

Fluid shear stress (FSS) from blood flow is sensed by vascular endothelial cells (ECs) to determine vessel stability, remodeling and susceptibility to atherosclerosis and other inflammatory diseases but the regulatory networks that govern these behaviors are only partially understood. We used cSTAR, a powerful new computational method, to define EC transcriptomic states under low shear stress (LSS) that triggers vessel inward remodeling, physiological shear stress (PSS) that stabilizes vessels, high shear stress (HSS) that triggers outward remodeling, and oscillatory shear stress (OSS) that confers disease susceptibility, all in comparison to cells under static conditions (STAT). We combined these results with the LINCS database where EC transcriptomic responses to drug treatments to define a preliminary regulatory network in which the cyclin-dependent kinases CDK1/2 play a central role in promoting vessel stability. Experimental analysis showed that PSS induced a strong late G1 cell cycle arrest in which CDK2 was activated. EC deletion of CDK2 in mice resulted in inward artery remodeling and both pulmonary and systemic hypertension. These results validate use of cSTAR to determine EC state and in vivo vessel behavior, reveal unexpected features of EC phenotype under different FSS conditions, and identify CDK2 as a key element within the EC regulatory network that governs artery remodeling.

Fixation strength in arthroscopic labral repair of the hip: A head-to-head comparison of the biomechanical performance of a biocompatible vs. all-suture anchor in the setting of acetabuloplasty.

Much is known about the biomechanical performance of various types of suture anchors commonly used for labral fixation in the shoulder; however, similar studies in the hip are less common. We sought to compare all-suture and polyether ether ketone small-diameter anchors in the setting of labral repair during hip arthroscopy, with and without acetabuloplasty. We hypothesized that the biomechanical properties of the all-suture group when compared to polyether ether ketone anchors would be similar amongst native acetabula and significantly less following acetabuloplasty and that pullout forces would be reduced in the anterior and inferior regions of the acetabulum compared to the superior region. Bone density was measured in nine matched pairs of fresh-frozen cadaveric acetabula in the superior, anterosuperior, and anterior regions. Acetabuloplasty was performed in all three regions, while the contralateral acetabulum was left in situ as a control. Suture anchors were placed such that one each of two different types was placed within each region. Specimens were tested in cyclic fatigue and loaded to failure. The all-suture group had significantly higher cyclic displacement compared to the polyether ether ketone, but there was no significant difference in ultimate load, regardless of acetabuloplasty. Amongst all non-resected specimens, the lowest bone density was observed consistently in the inferior region. Our results indicate that, with or without acetabuloplasty, a small-diameter polyether ether ketone anchor appears to be more stable than an all-suture anchor, which needs to be set first.

SMAD4 maintains the fluid shear stress set point to protect against arterial-venous malformations.

Vascular networks form, remodel, and mature under the influence of both fluid shear stress (FSS) and soluble factors. Physiological FSS promotes and maintains vascular stability via synergy with bone morphogenic proteins 9 and 10 (BMP9 and BMP10). Conversely, mutation of the BMP receptors activin-like kinase 1 (ALK1), endoglin (ENG), or the downstream effector, SMAD family member 4 (SMAD4) leads to hereditary hemorrhagic telangiectasia (HHT), characterized by fragile and leaky arterial-venous malformations (AVMs). How endothelial cells (ECs) integrate FSS and BMP signals in vascular development and homeostasis and how mutations give rise to vascular malformations is not well understood. Here, we aimed to elucidate the mechanism of synergy between FSS and SMAD signaling in vascular stability and how disruption of this synergy leads to AVMs. We found that loss of Smad4 increased the sensitivity of ECs to flow by lowering the FSS set point, with resulting AVMs exhibiting features of excessive flow-mediated morphological responses. Mechanistically, loss of SMAD4 disinhibits flow-mediated KLF4-TIE2-PI3K/Akt signaling, leading to cell cycle progression-mediated loss of arterial identity due to KLF4-mediated repression of cyclin dependent Kinase (CDK) inhibitors CDKN2A and CDKN2B. Thus, AVMs caused by Smad4 deletion are characterized by chronic high flow remodeling with excessive EC proliferation and loss of arterial identity as triggering events.

FN (Fibronectin)-Integrin α5 Signaling Promotes Thoracic Aortic Aneurysm in a Mouse Model of Marfan Syndrome.

BACKGROUND: Marfan syndrome, caused by mutations in the gene for fibrillin-1, leads to thoracic aortic aneurysms (TAAs). Phenotypic modulation of vascular smooth muscle cells (SMCs) and ECM (extracellular matrix) remodeling are characteristic of both nonsyndromic and Marfan aneurysms. The ECM protein FN (fibronectin) is elevated in the tunica media of TAAs and amplifies inflammatory signaling in endothelial and SMCs through its main receptor, integrin α5ß1. We investigated the role of integrin α5-specific signals in Marfan mice in which the cytoplasmic domain of integrin α5 was replaced with that of integrin α2 (denoted α5/2 chimera). METHODS: We crossed α5/2 chimeric mice with Fbn1mgR/mgR mice (mgR model of Marfan syndrome) to evaluate the survival rate and pathogenesis of TAAs among wild-type, α5/2, mgR, and α5/2 mgR mice. Further biochemical and microscopic analysis of porcine and mouse aortic SMCs investigated molecular mechanisms by which FN affects SMCs and subsequent development of TAAs. RESULTS: FN was elevated in the thoracic aortas from Marfan patients, in nonsyndromic aneurysms, and in mgR mice. The α5/2 mutation greatly prolonged survival of Marfan mice, with improved elastic fiber integrity, mechanical properties, SMC density, and SMC contractile gene expression. Furthermore, plating of wild-type SMCs on FN decreased contractile gene expression and activated inflammatory pathways whereas α5/2 SMCs were resistant. These effects correlated with increased NF-kB activation in cultured SMCs and mgR aortas, which was alleviated by the α5/2 mutation or NF-kB inhibition. CONCLUSIONS: FN-integrin α5 signaling is a significant driver of TAA in the mgR mouse model. This pathway thus warrants further investigation as a therapeutic target.

TLN1 contains a cancer-associated cassette exon that alters talin-1 mechanosensitivity.

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-ß/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

Free-breathing Arterial Spin Labeling MRI for the Detection of Pulmonary Embolism.

Background Arterial spin labeling (ASL) MRI can be used to assess organ perfusion but has yet to be implemented for perfusion evaluation of the lung. Purpose To evaluate pseudo-continuous ASL (PCASL) MRI for the detection of acute pulmonary embolism (PE) and its potential as an alternative to CT pulmonary angiography (CTPA). Materials and Methods Between November 2020 and November 2021, 97 patients (median age, 61 years; 48 women) with suspected PE were enrolled in this prospective study. PCASL MRI was performed within a 72-hour period following CTPA under free-breathing conditions and included three orthogonal planes. The pulmonary trunk was labeled during systole, and the image was acquired during diastole of the subsequent cardiac cycle. Additionally, multisection, coronal, balanced, steady-state free-precession imaging was carried out. Two radiologists blindly assessed overall image quality, artifacts, and diagnostic confidence (five-point Likert scale, 5 = best). Patients were categorized as positive or negative for PE, and a lobe-wise assessment in PCASL MRI and CTPA was conducted. Sensitivity and specificity were calculated on a patient level with the final clinical diagnosis serving as the reference standard. Interchangeability between MRI and CTPA was also tested with use of an individual equivalence index (IEI). Results PCASL MRI was performed successfully in all patients with high scores for image quality, artifact, and diagnostic confidence (κ ≥ .74). Of the 97 patients, 38 were positive for PE. PCASL MRI depicted PE correctly in 35 of 38 patients with three false-positive and three false-negative findings, resulting in a sensitivity of 35 of 38 patients (92% [95% CI: 79, 98]) and a specificity of 56 of 59 patients (95% [95% CI: 86, 99]). Interchangeability analysis revealed an IEI of 2.6% (95% CI: 1.2, 3.8). Conclusion Free-breathing pseudo-continuous arterial spin labeling MRI depicted abnormal lung perfusion caused by acute pulmonary embolism and may be useful as a contrast material-free alternative to CT pulmonary angiography for selected patients. German Clinical Trials Register no. DRKS00023599 © RSNA, 2023.

Integrin αIIbß3 intermediates: From molecular dynamics to adhesion assembly.

The platelet integrin αIIbß3 undergoes long-range conformational transitions associated with its functional conversion from inactive (low-affinity) to active (high-affinity) during hemostasis. Although new conformations that are intermediate between the well-characterized bent and extended states have been identified, their molecular dynamic properties and functions in the assembly of adhesions remain largely unexplored. In this study, we evaluated the properties of intermediate conformations of integrin αIIbß3 and characterized their effects on the assembly of adhesions by combining all-atom simulations, principal component analysis, and mesoscale modeling. Our results show that in the low-affinity, bent conformation, the integrin ectodomain tends to pivot around the legs; in intermediate conformations, the headpiece becomes partially extended, away from the lower legs. In the fully open, active state, αIIbß3 is flexible, and the motions between headpiece and lower legs are accompanied by fluctuations of the transmembrane helices. At the mesoscale, bent integrins form only unstable adhesions, but intermediate or open conformations stabilize the adhesions. These studies reveal a mechanism by which small variations in ligand binding affinity and enhancement of the ligand-bound lifetime in the presence of actin retrograde flow stabilize αIIbß3 integrin adhesions.

Integrin Conformational Dynamics and Mechanotransduction.

The function of the integrin family of receptors as central mediators of cell-extracellular matrix (ECM) and cell-cell adhesion requires a remarkable convergence of interactions and influences. Integrins must be anchored to the cytoskeleton and bound to extracellular ligands in order to provide firm adhesion, with force transmission across this linkage conferring tissue integrity. Integrin affinity to ligands is highly regulated by cell signaling pathways, altering affinity constants by 1000-fold or more, via a series of long-range conformational transitions. In this review, we first summarize basic, well-known features of integrin conformational states and then focus on new information concerning the impact of mechanical forces on these states and interstate transitions. We also discuss how these effects may impact mechansensitive cell functions and identify unanswered questions for future studies.