The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

In the adult, vascular smooth muscle cells (VSMC) are normally physiologically quiescent, arranged circumferentially in one or more layers within blood vessel walls. Remodelling of native VSMC to a proliferative state for vascular development, adaptation or repair is driven by platelet-derived growth factor (PDGF). A key effector downstream of PDGF receptors is store-operated calcium entry (SOCE) mediated through the plasma membrane calcium ion channel, ORAI1, which is activated by the endoplasmic reticulum (ER) calcium store sensor, stromal interaction molecule-1 (STIM1). This SOCE was shown to play fundamental roles in the pathological remodelling of VSMC. Exciting transgenic lineage-tracing studies have revealed that the contribution of the phenotypically-modulated VSMC in atherosclerotic plaque formation is more significant than previously appreciated, and growing evidence supports the relevance of ORAI1 signalling in this pathologic remodelling. ORAI1 has also emerged as an attractive potential therapeutic target as it is accessible to extracellular compound inhibition. This is further supported by the progression of several ORAI1 inhibitors into clinical trials. Here we discuss the current knowledge of ORAI1-mediated signalling in pathologic vascular remodelling, particularly in the settings of atherosclerotic cardiovascular diseases (CVDs) and neointimal hyperplasia, and the recent developments in our understanding of the mechanisms by which ORAI1 coordinates VSMC phenotypic remodelling, through the activation of key transcription factor, nuclear factor of activated T-cell (NFAT). In addition, we discuss advances in therapeutic strategies aimed at the ORAI1 target.

Sources of Funding, see page 214 BACKGROUND: Orai1 is a critical ion channel subunit, best recognized as a mediator of store-operated Ca 2+ entry (SOCE) in nonexcitable cells. SOCE has recently emerged as a key contributor of cardiac hypertrophy and heart failure but the relevance of Orai1 is still unclear. METHODS: To test the role of these Orai1 channels in the cardiac pathophysiology, a transgenic mouse was generated with cardiomyocytespecific expression of an ion pore-disruptive Orai1 R91W mutant (C-dnO1). Synthetic chemistry and channel screening strategies were used to develop 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline (hereafter referred to as JPIII), a small-molecule Orai1 channel inhibitor suitable for in vivo delivery. RESULTS: Adult mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and reduced ventricular function associated with increased Orai1 expression and Orai1-dependent SOCE (assessed by Mn 2+ influx). C-dnO1 mice displayed normal cardiac electromechanical function and cellular excitation-contraction coupling despite reduced Orai1-dependent SOCE. Five weeks after TAC, C-dnO1 mice were protected from systolic dysfunction (assessed by preserved left ventricular fractional shortening and ejection fraction) even if increased cardiac mass and prohypertrophic markers induction were observed. This is correlated with a protection from TAC-induced cellular Ca 2+ signaling alterations (increased SOCE, decreased [Ca 2+ ] i transients amplitude and decay rate, lower SR Ca 2+ load and depressed cellular contractility) and SERCA2a downregulation in ventricular cardiomyocytes from C-dnO1 mice, associated with blunted Pyk2 signaling. There was also less fibrosis in heart sections from C-dnO1 mice after TAC. Moreover, 3 weeks treatment with JPIII following 5 weeks of TAC confirmed the translational relevance of an Orai1 inhibition strategy during hypertrophic insult. CONCLUSIONS: The findings suggest a key role of cardiac Orai1 channels and the potential for Orai1 channel inhibitors as inotropic therapies for maintaining contractility reserve after hypertrophic stress.

INTRODUCTION: We aimed to identify high-risk nonalcoholic fatty liver disease (NAFLD) patients seen at the primary care and endocrinology practices and link them to gastrohepatology care. METHODS: Using the electronic health record, patients who either had the diagnosis of type 2 diabetes or had 2 of 3 other metabolic risk factors met criteria for inclusion in the study. Using noninvasive fibrosis tests (NITs) to identify high risk of fibrosis, patients who met the NIT prespecified criteria were referred to gastrohepatology for clinical assessment and transient elastography. RESULTS: From 7,555 patients initially screened, 1707 (22.6%) met the inclusion criteria, 716 (42%) agreed to enroll, and 184 (25.7%) met the prespecified NIT criteria and eligibility for linkage to GE-HEP where 103 patients (68 ± 9 years of age, 50% men, 56% white) agreed to undergo linkage assessments. Their NIT scores were APRI of 0.38 ± 0.24, FIB-4 of 1.98 ± 0.87, and NAFLD Fibrosis Score of 0.36 ± 1.03; 68 (66%) linked patients had controlled attenuation parameter >248 dB/m, 62 (60%) had liver stiffness <6 kPa, and 8 (8%) had liver stiffness >12 kPa. Liver stiffness for the overall group was 6.7 ± 4.2 kPa, controlled attenuation parameter 282 ± 64 dB/m, and FAST score 0.22 ± 0.22. Linked patients with presumed advanced fibrosis had significantly higher body mass index (36.4 ± 6.6 vs 31.2 ± 6.4 kg/m 2 , P = 0.025) and higher NIT scores (APRI 0.89 ± 0.52 vs 0.33 ± 0.14, FIB-4 3.21 ± 2.06 vs 1.88 ± 0.60, and NAFLD Fibrosis Score 1.58 ± 1.33 vs 0.25 ± 0.94). DISCUSSION: By applying a simple prespecified multistep algorithm using electronic health record with clinical risk factors and NITs followed by transient elastography, patients with nonalcoholic fatty liver disease seen in PCP and ENDO practices can be easily identified.

Objective The aim of this study was to investigate the reproducibility of anterior–posterior diameter (APdmax) and three-dimensional lumen volume (3DLV) measurements of abdominal aortic aneurysms (AAA) in a classical murine AAA model. We also compared the magnitude of change in the aortic size detected with each method of assessment. Methods Periadventitial application of porcine pancreatic elastase (PPE AAA) or sham surgery was performed in two cohorts of mice. Cohort 1 was used to assess for observer variability with the APdmax and 3DLV measurements. Cohort 2 highlighted the relationship between APdmax and 3DLV and changes in AAA detected. Results There was no significant observer variability detected with APdmax measurement. Similarly, no significant intraobserver variability was evident with 3DLV; however, a small but significant interobserver difference was present. APdmax and 3DLV measurements of PPE AAA significantly correlated. However, changes in the AAA morphology were detected earlier with 3DLV. Conclusion APdmax and 3DLV are both reliable methods for measuring an AAA. Both these methods correlate with each other. However, changes in AAA morphology were detected earlier with 3DLV, which is important to detect subtle but important changes to aortic geometry in a laboratory setting. 3DLV measurement of AAA is a simple, reproducible, and comprehensive method for assessing changes in disease morphology.