Antithrombin-III mitigates thrombin-mediated endothelial cell contraction and sickle red blood cell adhesion in microscale flow.

Individuals with sickle cell disease (SCD) have persistently elevated thrombin generation that results in a state of systemic hypercoagulability. Antithrombin-III (ATIII), an endogenous serine protease inhibitor, inhibits several enzymes in the coagulation cascade, including thrombin. Here, we utilize a biomimetic microfluidic device to model the morphology and adhesive properties of endothelial cells (ECs) activated by thrombin and examine the efficacy of ATIII in mitigating the adhesion of SCD patient-derived red blood cells (RBCs) and EC retraction. Microfluidic devices were fabricated, seeded with ECs, and incubated under physiological shear stress. Cells were then activated with thrombin with or without an ATIII pretreatment. Blood samples from subjects with normal haemoglobin (HbAA) and subjects with homozygous SCD (HbSS) were used to examine RBC adhesion to ECs. Endothelial cell surface adhesion molecule expression and confluency in response to thrombin and ATIII treatments were also evaluated. We found that ATIII pretreatment of ECs reduced HbSS RBC adhesion to thrombin-activated endothelium. Furthermore, ATIII mitigated cellular contraction and reduced surface expression of von Willebrand factor and vascular cell adhesion molecule-1 (VCAM-1) mediated by thrombin. Our findings suggest that, by attenuating thrombin-mediated EC damage and RBC adhesion to endothelium, ATIII may alleviate the thromboinflammatory manifestations of SCD.

Krüppel-Like Factors Orchestrate Endothelial Gene Expression Through Redundant and Non-Redundant Enhancer Networks.

Background Proper function of endothelial cells is critical for vascular integrity and organismal survival. Studies over the past 2 decades have identified 2 members of the KLF (Krüppel-like factor) family of proteins, KLF2 and KLF4, as nodal regulators of endothelial function. Strikingly, inducible postnatal deletion of both KLF2 and KLF4 resulted in widespread vascular leak, coagulopathy, and rapid death. Importantly, while transcriptomic studies revealed profound alterations in gene expression, the molecular mechanisms underlying these changes remain poorly understood. Here, we seek to determine mechanisms of KLF2 and KLF4 transcriptional control in multiple vascular beds to further understand their roles as critical endothelial regulators. Methods and Results We integrate chromatin occupancy and transcription studies from multiple transgenic mouse models to demonstrate that KLF2 and KLF4 have overlapping yet distinct binding patterns and transcriptional targets in heart and lung endothelium. Mechanistically, KLFs use open chromatin regions in promoters and enhancers and bind in context-specific patterns that govern transcription in microvasculature. Importantly, this occurs during homeostasis in vivo without additional exogenous stimuli. Conclusions Together, this work provides mechanistic insight behind the well-described transcriptional and functional heterogeneity seen in vascular populations, while also establishing tools into exploring microvascular endothelial dynamics in vivo.

Comparing Semiquantitative and Qualitative Methods of Vascular 18F-FDG PET Activity Measurement in Large-Vessel Vasculitis.

The study rationale was to assess the performance of qualitative and semiquantitative scoring methods for 18F-FDG PET assessment in large-vessel vasculitis. Methods: Patients with giant cell arteritis or Takayasu arteritis underwent independent clinical and imaging assessments within a prospective observational cohort. 18F-FDG PET/CT scans were interpreted for active vasculitis by central reader assessment. Arterial 18F-FDG uptake was scored by qualitative visual assessment using the PET vascular activity score (PETVAS) and by semiquantitative assessment using SUVs and target-to-background ratios (TBRs) relative to liver or blood activity. The performance of each scoring method was assessed by intrarater reliability using the intraclass correlation coefficient (ICC) and areas under the receiver-operating-characteristic curve, applying physician assessment of clinical disease activity and reader interpretation of vascular PET activity as independent reference standards. The Wilcoxon signed-rank test was used to analyze change in arterial 18F-FDG uptake over time. Results: Ninety-five patients (giant cell arteritis, 52; Takayasu arteritis, 43) contributed 212 18F-FDG PET studies. The ICC for semiquantitative evaluation (0.99 [range, 0.98-1.00]) was greater than the ICC for qualitative evaluation (0.82 [range, 0.56-0.93]). PETVAS and target-to-background ratio metrics were more strongly associated with reader interpretation of PET activity than SUV metrics. All assessment methods were significantly associated with physician assessment of clinical disease activity, but the semiquantitative metric liver tissue-to-background ratio (TBRLiver) achieved the highest area under the receiver-operating-characteristic curve (0.66). Significant but weak correlations with C-reactive protein were observed for SUV metrics (r = 0.19, P < 0.01) and TBRLiver (r = 0.20, P < 0.01) but not for PETVAS. In response to increased treatment in 56 patients, arterial 18F-FDG uptake was significantly reduced when measured by semiquantitative (TBRLiver, 1.31-1.23; 6.1% change; P < 0.0001) or qualitative (PETVAS, 22-18; P < 0.0001) methods. Semiquantitative metrics provided information complementary to qualitative evaluation in cases of severe vascular inflammation. Conclusion: Both qualitative and semiquantitative methods of measuring arterial 18F-FDG uptake are useful in assessing and monitoring vascular inflammation in large-vessel vasculitis. Compared with qualitative metrics, semiquantitative methods have superior reliability and better discriminate treatment response in cases of severe inflammation.