Rationale and design of the RAPID-WATER-FLOW trial: Radiolabeled perfusion to identify coronary artery disease using water to evaluate responses of myocardial FLOW.

OBJECTIVES: The objective of this study was to determine the diagnostic performance of 15O-water positron emission tomography (PET) myocardial perfusion imaging to detect coronary artery disease (CAD) using the truth-standard of invasive coronary angiography (ICA) with fractional flow reserve (FFR) or instantaneous wave-Free Ratio (iFR) or coronary computed tomography angiogram (CCTA). BACKGROUND: 15O-water has a very high first-pass extraction that allows accurate quantification of myocardial blood flow and detection of flow-limiting CAD. However, the need for an on-site cyclotron and lack of automated production at the point of care and relatively complex image analysis protocol has limited its clinical use to date. METHODS: The RAPID WATER FLOW study is an open-label, multicenter, prospective investigation of the accuracy of 15O-water PET to detect obstructive angiographic and physiologically significant stenosis in patients with suspected CAD. The study will include the use of an automated system for producing, dosing, and injecting 15O-water and enrolling approximately 215 individuals with suspected CAD at approximately 10 study sites in North America and Europe. The primary endpoint of the study is the diagnostic sensitivity and specificity of the 15O-water PET study using the truth-standard of ICA with FFR or iFR to determine flow-limiting stenosis, or CCTA to rule out CAD and incorporating a quantitative analytic platform developed for the 15O-water PET acquisitions. Sensitivity and specificity are to be considered positive if the lower bound of the 95% confidence interval is superior to the threshold of 60% for both, consistent with prior registration studies. Subgroup analyses include assessments of diagnostic sensitivity, specificity, and accuracy in female, obese, and diabetic individuals, as well as in those with multivessel disease. All enrolled individuals will be followed for adverse and serious adverse events for up to 32 hours after the index PET scan. The study will have >90% power (one-sided test, α = 0.025) to test the hypothesis that sensitivity and specificity of 15O-water PET are both >60%. CONCLUSIONS: The RAPID WATER FLOW study is a prospective, multicenter study to determine the diagnostic sensitivity and specificity of 15O-water PET as compared to ICA with FFR/iFR or CCTA. This study will introduce several novel aspects to imaging registration studies, including a more relevant truth standard incorporating invasive physiologic indexes, coronary CTA to qualify normal individuals for eligibility, and a more quantitative approach to image analysis than has been done in prior pivotal studies. CLINICAL TRIAL REGISTRATION INFORMATION: Clinical-Trials.gov (#NCT05134012).

Characteristics and key differences between patient populations receiving imaging modalities for coronary artery disease diagnosis in the US.

BACKGROUND: There are limited data on the impact of imaging modality selection for the assessment of coronary artery disease (CAD) risk on downstream resource utilisation. This study sought to identify differences between patient populations in the US undergoing stress echocardiography, single-photon emission computed tomography (SPECT) myocardial perfusion imaging (MPI), positron emission tomography (PET) MPI, and coronary computed tomography angiography (cCTA) for the assessment of CAD risk, and associated physician referral patterns. METHODS: Claims and electronic health records data for 2.5 million US patients who received stress echocardiography, cCTA, SPECT MPI or PET MPI between January 2016 and March 2018, from the Decision Resources Group Real-World Evidence US Data Repository, were analysed. Patients were stratified into suspected and existing CAD cohorts, and further stratified by pre-test risk and presence and recency of interventions or acute cardiac events (within 1-2 years pre-index test). Linear and logistic regression were used to compare numeric and categorical variables. RESULTS: Physicians were more likely to refer patients to standalone SPECT MPI (77%) and stress echocardiography (18%) than PET MPI (3%) and cCTA (2%). Overall, 43% of physicians referred more than 90% of their patients to standalone SPECT MPI. Just 3%, 1% and 1% of physicians referred more than 90% of their patients to stress echocardiography, PET MPI or cCTA. At the aggregated imaging level, patients who underwent stress echocardiography or cCTA had similar comorbidity profiles. Comorbidity profiles were also similar for patients who underwent SPECT MPI and PET MPI. CONCLUSION: Most patients underwent SPECT MPI at the index date, with very few undergoing PET MPI or cCTA. Patients who underwent cCTA at the index date were more likely to undergo additional imaging tests compared with those who underwent other imaging modalities. Further evidence is needed to understand factors influencing imaging test selection across patient populations.

Role of protein kinase Czeta in thrombin-induced RhoA activation and inter-endothelial gap formation of human dermal microvessel endothelial cell monolayers.

We studied the potential involvement of the Ca(2+)-independent atypical protein kinase C isoform PKCzeta in mediating the thrombin-induced increase in endothelial permeability. Studies were done using human dermal microvessel endothelial cells (HMEC), which we showed constitutively expressed PKCzeta. We quantified the patency of inter-endothelial junctions (IEJs) and endothelial barrier function by measuring transendothelial electrical resistance (TER) in confluent HMEC monolayers. In control monolayers, thrombin decreased TER by approximately 50%, indicating thrombin-dependent opening of IEJs. Thrombin also elicited increases in cytosolic Ca(2+) concentration [Ca(2+)](i), actin stress fiber formation, and myosin light chain (MLC) phosphorylation. Pan-PKC inhibitors, calphostin C and chelerythrine, abrogated these responses. Thrombin also decreased TER after depletion of conventional and novel Ca(2+)-dependent PKC isoforms using phorbol 12-myristate 13-acetate (PMA). In these PMA-treated cells, thrombin induced inter-endothelial gap formation, MLC phosphorylation, and actin stress fiber formation, but failed to increase [Ca(2+)](i). Inhibition of PKCzeta activation using the PKCzeta pseudosubstrate peptide (PSI), depletion of PKCzeta protein with siRNA, and competitive inhibition of PKCzeta activity using dominant-negative (dn) PKCzeta mutant all prevented the thrombin-induced decrease in TER and MLC phosphorylation. Expression of dn-PKCzeta also inhibited thrombin-induced RhoA activation. These findings reveal a novel Ca(2+)-independent, PKCzeta-dependent mechanism of thrombin-induced increase in endothelial permeability. The results raise the possibility that inhibition of PKCzeta may be a novel drug target for thrombin-induced inflammatory hyperpermeability.

Regulation of endothelial junctional permeability.

The endothelium is a semi-permeable barrier that regulates the flux of liquid and solutes, including plasma proteins, between the blood and surrounding tissue. The permeability of the vascular barrier can be modified in response to specific stimuli acting on endothelial cells. Transport across the endothelium can occur via two different pathways: through the endothelial cell (transcellular) or between adjacent cells, through interendothelial junctions (paracellular). This review focuses on the regulation of the paracellular pathway. The paracellular pathway is composed of adhesive junctions between endothelial cells, both tight junctions and adherens junctions. The actin cytoskeleton is bound to each junction and controls the integrity of each through actin remodeling. These interendothelial junctions can be disassembled or assembled to either increase or decrease paracellular permeability. Mediators, such as thrombin, TNF-alpha, and LPS, stimulate their respective receptor on endothelial cells to initiate signaling that increases cytosolic Ca2+ and activates myosin light chain kinase (MLCK), as well as monomeric GTPases RhoA, Rac1, and Cdc42. Ca2+ activation of MLCK and RhoA disrupts junctions, whereas Rac1 and Cdc42 promote junctional assembly. Increased endothelial permeability can be reversed with "barrier stabilizing agents," such as sphingosine-1-phosphate and cyclic adenosine monophosphate (cAMP). This review provides an overview of the mechanisms that regulate paracellular permeability.