Outcomes of urgent/emergent transcatheter mitral valve repair (MitraClip): A single center experience.

OBJECTIVES: To describe the outcomes of urgent/emergent transcatheter edge-to-edge mitral valve repair (TMVr) and compare the clinical, echocardiographic, and procedural characteristics of survivors and nonsurvivors. BACKGROUND: TMVr is a treatment strategy for select patients with severe primary or secondary mitral regurgitation. However, knowledge regarding outcomes for urgent/emergent TMVr is limited. METHODS: All urgent or emergent TMVr procedures using MitraClip performed at the University of Washington Medical Center between January 2018 and March 2019 were identified and clinical, echocardiographic, hemodynamic, procedural, and outcomes data were obtained by chart review. Outcomes included all-cause mortality, hospital mortality, procedural success, periprocedural complications, and hospital readmission. RESULTS: Of the 20 patients who underwent urgent/emergent TMVr, eight were treated for cardiogenic shock (CS), four for acute decompensated heart failure (ADHF) with hypoxemic respiratory failure requiring mechanical ventilation, and eight for ADHF with failure of inpatient medical therapy. Mechanical circulatory support (MCS) was used in six patients; preceding TMVr in three patients and immediately post-TMVr in three patients. Overall, 30-day mortality and hospital readmission rates were 21 and 13%, respectively. Over a median 153 days (IQR 20-491) of follow-up, 10 patients (50%) died. Preprocedure CS, new or ongoing MCS post-TMVr, refractory respiratory failure post-TMVr, and acute kidney injury post-TMVr were associated with mortality. CONCLUSIONS: In a single-center retrospective analysis, urgent/emergent TMVr in high-risk patients with ADHF or CS was associated with high short-term mortality and periprocedural complications. Prospective studies are warranted to inform patient selection and periprocedural management for urgent/emergent TMVr.

Orbital Atherectomy for Treatment of Severely Calcified Coronary Artery Bifurcation Lesions: A Multicenter Analysis.

OBJECTIVE: This study evaluated the safety and efficacy of orbital atherectomy (OA) for the treatment of severely calcified coronary artery bifurcation lesions. BACKGROUND: Percutaneous coronary intervention (PCI) of severely calcified coronary artery lesions is associated with lower procedural success and higher rates of target lesion failure compared to non-calcified lesions. OA is an effective treatment for calcified coronary artery lesions prior to stent implantation. However, there is little data regarding the safety and efficacy of OA in patients with coronary artery bifurcation lesions. METHODS: Data were obtained from analysis of patients with severe coronary artery calcification who underwent OA and coronary stent implantation at ten high-volume institutions. Data were pooled and analyzed to assess peri-procedural outcomes and 30-day major adverse cardiac events (MACE). RESULTS: A total of 1156 patients were treated with OA and PCI. 363 lesions were at a coronary artery bifurcation. There were no statistically significant differences in baseline characteristics between the bifurcation and non-bifurcation groups. In the bifurcation group, treatment involved the left anterior descending artery and its branches more frequently and right coronary artery less frequently. After propensity score matching, the 30-day freedom from MACE was not statistically significant between the two groups. CONCLUSION: In this multicenter cohort analysis, patients with severely calcified coronary bifurcation lesions had low rates of MACE and target vessel revascularization at 30 days at rates comparable to non-bifurcation lesions. This analysis demonstrates that OA is safe and effective for complex coronary lesions at both bifurcation and non-bifurcation locations.

Temporal Trends in Percutaneous Coronary Intervention and Coronary Artery Bypass Grafting: Insights From the Washington Cardiac Care Outcomes Assessment Program.

Background Patient selection and outcomes for percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) have changed over the past decade. However, there is limited information on outcomes for both revascularization strategies in the same population. The study evaluated temporal changes in risk profile, procedural characteristics, and clinical outcomes for PCI- and CABG-treated patients. Methods and Results We analyzed all PCI and isolated CABG between 2005 and 2017 in nonfederal hospitals in Washington State. Descriptive analysis was performed to evaluate temporal changes in risk profile and, risk-adjusted in-hospital mortality. Over the study period, 178 474 PCI and 36 592 CABG procedures were performed. PCI and CABG volume decreased by 2.9% and 22.6%, respectively. Compared with 2005-2009, patients receiving either form of revascularization between 2014 and 2017 had a higher prevalence of comorbidities including diabetes mellitus and hypertension and dialysis. Presentation with ST-segment-elevation myocardial infarction (17% versus 20%) and cardiogenic shock (2.4% versus 3.4%) increased for patients with PCI compared with CABG. Conversely, clinical acuity decreased for patients receiving CABG over the study period. From 2005 to 2017, mean National Cardiovascular Data Registry CathPCI mortality score increased for patients treated with PCI (20.1 versus 22.4, P<0.0001) and decreased for patients treated with CABG (18.8 versus 17.8, P<0.0001). Adjusted observed/expected in-hospital mortality ratio increased for PCI (0.98 versus 1.19, P<0.0001) but decreased for CABG (1.21 versus 0.74, P<0.0001) over the study period. Conclusions Clinical acuity increased for patients treated with PCI rather than CABG. This resulted in an increase in adjusted observed/expected mortality ratio for patients undergoing PCI and a decrease for CABG. These shifts may reflect an increased use of PCI instead of CABG for patients considered to be at high surgical risk.

Outcomes of Emergency Transcatheter Aortic Valve Replacement.

OBJECTIVE: To identify outcomes of patients undergoing emergency transcatheter aortic valve replacement (TAVR) and determine predictors of in-hospital mortality. BACKGROUND: Emergency TAVR has emerged as a viable treatment strategy for patients with decompensated severe aortic stenosis and/or regurgitation; however, data on patients undergoing emergency TAVR are limited. METHODS: All emergency TAVR procedures were identified from a single tertiary academic center between January 2015 and August 2018. RESULTS: 31 patients underwent emergency TAVR due to cardiogenic shock (26 patients), electrical instability with incessant ventricular tachycardia (2 patients), severe refractory angina (2 patients), and decompensated heart failure with hypoxemic respiratory failure requiring mechanical ventilation (1 patient). Mechanical circulatory support (MCS) was used in 16 (51.6%). MCS initiation occurred immediately prior to TAVR in 10 patients and placed post-TAVR in 6 patients. 6 patients died before hospital discharge (in-hospital mortality 19.4%). 1-year and 2-year survival rates were 61.0% and 55.9%, respectively. Univariate predictors of in-hospital mortality were preprocedural pulmonary artery pulsatility index (PAPi) ≤1.8 (66.7% vs. 20.0%, p=0.01), intraprocedural cardiopulmonary resuscitation (CPR) (83.3% vs 4.0%, p ≤ 0.001), acute kidney injury post-TAVR (80.0% vs. 4.2%, p ≤ 0.001), initiation of dialysis post-TAVR (60.0% vs. 4.2%, p ≤ 0.001), and MCS initiation post-TAVR (50.0% vs. 12.0%, p=0.03). MCS initiation before TAVR was associated with improved survival compared with post-TAVR initiation. CONCLUSION: Emergency TAVR in extreme risk patients with acute decompensated heart failure or cardiogenic shock secondary to severe aortic valve disease is associated with high in-hospital mortality rates. Careful patient selection taking into account right heart function, assessed by PAPi, and early utilization of MCS may improve survival following emergency TAVR.

Management of Out-of-Hospital Cardiac Arrest Complicating Acute Coronary Syndromes.

PURPOSE OF THE REVIEW: Out-of-hospital cardiac arrest (OHCA) complicating acute coronary syndromes (ACS) continues to carry a high rate of morbidity and mortality despite significant advances in EMS and interventional cardiology services. In this review, we discuss an evidence-based approach to the initial care and management of patients with OHCA complicating ACS from the pre-hospital response and initial resuscitation strategy, to advanced therapies such as coronary angiography, targeted-temperature management, neuro-prognostication, and care of the post-arrest patient. RECENT FINDINGS: Early recognition of cardiac arrest and prompt initiation of bystander CPR are the most important factors associated with improved survival. A comprehensive and coordinated approach to in-hospital management, including PCI, targeted temperature management, critical care, and hemodynamic support represents a significant critical link in the chain of survival. OHCA complicated by ACS continues to be one of the most challenging disease states facing healthcare practitioners and maintains a high mortality rate despite substantial advancements in healthcare delivery. A comprehensive approach to in-hospital management and further exploration of novel interventions, including ECMO, may yield opportunities to optimize care and improve outcomes for cardiac arrest patients.

Management of Refractory Ventricular Fibrillation (Prehospital and Emergency Department).

Ventricular fibrillation is a life-threatening cardiac arrhythmia that leads to a loss of cardiac function and sudden cardiac death. In this review, we summarize therapeutic interventions and guidelines for providers managing patients with out-of-hospital cardiac arrest and refractory ventricular fibrillation in prehospital and emergency settings. Additionally, we review invasive management, including urgent coronary angiography, extracorporeal membrane oxygenation, and novel strategies for managing refractory ventricular fibrillation arrest. Although the majority of patients with refractory VF do not respond to conventional therapy, recent trials of novel strategies demonstrate encouraging results.

Concomitant Use of Antiplatelets and Anticoagulants in Patients with Coronary Heart Disease and Atrial Fibrillation: What Do Recent Clinical Trials Teach Us?

PURPOSE OF REVIEW: Coronary heart disease (CHD) and atrial fibrillation (AF) are among the most common cardiovascular diseases. A significant proportion of patients have both CHD and AF and are at increased risk for thrombotic complications. Current therapy for CHD and AF includes antiplatelet and anticoagulant medications, respectively. Patients with concurrent CHD and AF may be prescribed dual antiplatelet therapy (DAPT) in addition to anticoagulation, which increases their bleeding risk. Controversy remains on how to balance risks and benefits in patients with CHD and AF in which multiple antithrombotic therapies may be indicated. RECENT FINDINGS: We review clinical trials and current guidelines for antiplatelet and anticoagulant therapy in CHD and AF. Aspirin and P2Y12 inhibitors are the mainstay of antiplatelet therapy. Vitamin K antagonists (VKAs) are the most commonly used anticoagulant, although the use of non-VKA oral anticoagulants (NOACs) in patients with AF is increasing. Recent studies provide guidance on how to address antithrombotic therapies in patients with concomitant CHD and AF. To date, we have evidence that in patients with AF who undergo percutaneous coronary intervention (PCI), clopidogrel with VKA may be used safely without aspirin. Also, low-dose rivaroxaban in combination with either clopidogrel only or DAPT is as effective as the traditional regimen of triple therapy with VKA and DAPT with lower bleeding risk. Dabigatran with a P2Y12 inhibitor was also found to be safe with less bleeding compared to triple therapy with VKA and DAPT. Use of a single antiplatelet agent with anticoagulation has become a viable choice in patients with CHD and AF, but more clinical trial data is needed to confirm therapy and duration regimens.

Formulation and PEGylation optimization of the therapeutic PEGylated phenylalanine ammonia lyase for the treatment of phenylketonuria.

Phenylketonuria (PKU) is a genetic metabolic disease in which the decrease or loss of phenylalanine hydroxylase (PAH) activity results in elevated, neurotoxic levels of phenylalanine (Phe). Due to many obstacles, PAH enzyme replacement therapy is not currently an option. Treatment of PKU with an alternative enzyme, phenylalanine ammonia lyase (PAL), was first proposed in the 1970s. However, issues regarding immunogenicity, enzyme production and mode of delivery needed to be overcome. Through the evaluation of PAL enzymes from multiple species, three potential PAL enzymes from yeast and cyanobacteria were chosen for evaluation of their therapeutic potential. The addition of polyethylene glycol (PEG, MW = 20,000), at a particular ratio to modify the protein surface, attenuated immunogenicity in an animal model of PKU. All three PEGylated PAL candidates showed efficacy in a mouse model of PKU (BTBR Pahenu2) upon subcutaneous injection. However, only PEGylated Anabaena variabilis (Av) PAL-treated mice demonstrated sustained low Phe levels with weekly injection and was the only PAL evaluated that maintained full enzymatic activity upon PEGylation. A PEGylated recombinant double mutant version of AvPAL (Cys503Ser/Cys565Ser), rAvPAL-PEG, was selected for drug development based on its positive pharmacodynamic profile and favorable expression titers. PEGylation was shown to be critical for rAvPAL-PEG efficacy as under PEGylated rAvPAL had a lower pharmacodynamic effect. rAvPAL and rAvPAL-PEG had poor stability at 4°C. L-Phe and trans-cinnamate were identified as activity stabilizing excipients. rAvPAL-PEG is currently in Phase 3 clinical trials to assess efficacy in PKU patients.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death.

Neutral endopeptidase-resistant C-type natriuretic peptide variant represents a new therapeutic approach for treatment of fibroblast growth factor receptor 3-related dwarfism.

Achondroplasia (ACH), the most common form of human dwarfism, is caused by an activating autosomal dominant mutation in the fibroblast growth factor receptor-3 gene. Genetic overexpression of C-type natriuretic peptide (CNP), a positive regulator of endochondral bone growth, prevents dwarfism in mouse models of ACH. However, administration of exogenous CNP is compromised by its rapid clearance in vivo through receptor-mediated and proteolytic pathways. Using in vitro approaches, we developed modified variants of human CNP, resistant to proteolytic degradation by neutral endopeptidase, that retain the ability to stimulate signaling downstream of the CNP receptor, natriuretic peptide receptor B. The variants tested in vivo demonstrated significantly longer serum half-lives than native CNP. Subcutaneous administration of one of these CNP variants (BMN 111) resulted in correction of the dwarfism phenotype in a mouse model of ACH and overgrowth of the axial and appendicular skeletons in wild-type mice without observable changes in trabecular and cortical bone architecture. Moreover, significant growth plate widening that translated into accelerated bone growth, at hemodynamically tolerable doses, was observed in juvenile cynomolgus monkeys that had received daily subcutaneous administrations of BMN 111. BMN 111 was well tolerated and represents a promising new approach for treatment of patients with ACH.

Using the electronic health record to connect primary care patients to evidence-based telephonic tobacco quitline services: a closed-loop demonstration project.

Few smokers receive evidence-based tobacco treatment during healthcare visits. Electronic health records (EHRs) present an opportunity to efficiently identify and refer smokers to state tobacco quitlines. The purpose of this case study is to develop and evaluate a secure, closed-loop EHR referral system linking patients visiting healthcare clinics with a state tobacco quitline. A regional health system, EHR vendor, tobacco cessation telephone quitline vendor, and university research center collaborated to modify a health system's EHR to create an eReferral system. Modifications included the following: clinic workflow adjustments, EHR prompts, and return of treatment delivery information from the quitline to the patient's EHR. A markedly higher percentage of adult tobacco users were referred to the quitline using eReferral than using the previous paper fax referral (14 vs. 0.3 %). The eReferral system increased the referral of tobacco users to quitline treatment. This case study suggests the feasibility and effectiveness of a secure, closed-loop EHR-based eReferral system.

Erythrocyte-mediated delivery of phenylalanine ammonia lyase for the treatment of phenylketonuria in BTBR-Pah(enu2) mice.

Phenylketonuria (PKU) is an autosomal recessive genetic disease caused by defects in the phenylalanine hydroxylase gene. Preclinical and clinical investigations suggest that phenylalanine ammonia lyase (PAL) could be an effective alternative for the treatment of PKU. The aim of this study is to investigate if erythrocytes loaded with PAL may act as a safe delivery system able to overcome bioavailability issues and to provide, in vivo, a therapeutically relevant concentration of enzyme. Murine erythrocytes were loaded with recombinant PAL from Anabaena variabilis (rAvPAL) and their ability to perform as bioreactors was assessed in vivo in adult BTBR-Pah(enu2) mice, the genetic murine model of PKU. Three groups of mice were treated with a single i.v. injection of rAvPAL-RBCs at three different doses to select the most appropriate one for assessment of efficacy. Repeated administrations at 9-10 day-intervals of the selected dose for 10 weeks showed that the therapeutic effect was persistent and not affected by the generation of antibodies induced by the recombinant enzyme. This therapeutic approach deserves further in vivo evaluation either as a potential option for the treatment of PKU patients or as a possible model for the substitutive enzymatic treatment of other inherited metabolic disorders.

Defining success in graduate school.

We agree with the author that a quantitative analysis of the predictive nature of the metrics used in graduate student admissions is a worthy pursuit and value the sincere intentions behind the UCSF Tetrad study. However, these types of analyses would benefit from the same rigorous approaches that we employ in our other research endeavors. As UCSF Tetrad graduates with diverse careers in academia, medicine, industry, and publishing, we hope that the definition of success in graduate school can be as thoughtfully and scientifically examined as the measurements used to select the next young people to follow in our footsteps.

Evaluation of the therapeutic potential of a CNP analog in a Fgfr3 mouse model recapitulating achondroplasia.

Achondroplasia (ACH), the most common form of dwarfism, is an inherited autosomal-dominant chondrodysplasia caused by a gain-of-function mutation in fibroblast-growth-factor-receptor 3 (FGFR3). C-type natriuretic peptide (CNP) antagonizes FGFR3 downstream signaling by inhibiting the pathway of mitogen-activated protein kinase (MAPK). Here, we report the pharmacological activity of a 39 amino acid CNP analog (BMN 111) with an extended plasma half-life due to its resistance to neutral-endopeptidase (NEP) digestion. In ACH human growth-plate chondrocytes, we demonstrated a decrease in the phosphorylation of extracellular-signal-regulated kinases 1 and 2, confirming that this CNP analog inhibits fibroblast-growth-factor-mediated MAPK activation. Concomitantly, we analyzed the phenotype of Fgfr3(Y367C/+) mice and showed the presence of ACH-related clinical features in this mouse model. We found that in Fgfr3(Y367C/+) mice, treatment with this CNP analog led to a significant recovery of bone growth. We observed an increase in the axial and appendicular skeleton lengths, and improvements in dwarfism-related clinical features included flattening of the skull, reduced crossbite, straightening of the tibias and femurs, and correction of the growth-plate defect. Thus, our results provide the proof of concept that BMN 111, a NEP-resistant CNP analog, might benefit individuals with ACH and hypochondroplasia.

A novel method for the large-scale production of PG-CNP37, a C-type natriuretic peptide analogue.

Achondroplasia is the most common form of human dwarfism caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3), resulting in abnormal endochondral bone formation. C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth and represents a potential therapy for achondroplasia. We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from Escherichia coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. Approximately 0.5g of 95% pure, soluble and active PG-CNP37 peptide was produced from 1L of culture using this method and may represent a viable means for large-scale production of other therapeutic peptides.

Preclinical evaluation of multiple species of PEGylated recombinant phenylalanine ammonia lyase for the treatment of phenylketonuria.

Phenylketonuria (PKU) is a metabolic disorder, in which loss of phenylalanine hydroxylase activity results in neurotoxic levels of phenylalanine. We used the Pah(enu2/enu2) PKU mouse model in short- and long-term studies of enzyme substitution therapy with PEGylated phenylalanine ammonia lyase (PEG-PAL conjugates) from 4 different species. The most therapeutically effective PAL (Av, Anabaena variabilis) species was one without the highest specific activity, but with the highest stability; indicating the importance of protein stability in the development of effective protein therapeutics. A PEG-Av-p.C503S/p.C565S-PAL effectively lowered phenylalanine levels in both vascular space and brain tissue over a >90 day trial period, resulting in reduced manifestations associated with PKU, including reversal of PKU-associated hypopigmentation and enhanced animal health. Phenylalanine reduction occurred in a dose- and loading-dependent manner, and PEGylation reduced the neutralizing immune response to the enzyme. Human clinical trials with PEG-Av-p.C503S/p.C565S-PAL as a treatment for PKU are underway.

Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase.

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.

Decreased ceramide transport protein (CERT) function alters sphingomyelin production following UVB irradiation.

Increased cellular ceramide accounts in part for UVB irradiation-induced apoptosis in cultured human keratinocytes with concurrent increased glucosylceramide but not sphingomyelin generation in these cells. Given that conversion of ceramide to non-apoptotic metabolites such as sphingomyelin and glucosylceramide protects cells from ceramide-induced apoptosis, we hypothesized that failed up-regulation of sphingomyelin generation contributes to ceramide accumulation following UVB irradiation. Because both sphingomyelin synthase and glucosylceramide synthase activities were significantly decreased in UVB-irradiated keratinocytes, we investigated whether alteration(s) in the function of ceramide transport protein (or CERT) required for sphingomyelin synthesis occur(s) in UVB-irradiated cells. Fluorescently labeled N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-d-erythro-sphingosine (C(5)-DMB-ceramide) relocation to the Golgi was diminished after irradiation, consistent with decreased CERT function, whereas the CERT inhibitor N-(3-hydroxy-1-hydroxymethyl-3-phenylpropyl)dodecanamide (1R,3R isomer) (HPA-12) produced an equivalent effect. UVB irradiation also induced the rapid formation of a stable CERT homotrimer complex in keratinocytes as determined by Western immunoblot and mass spectrometry analyses, a finding replicated in HeLa, HEK293T, and HaCaT cells and in murine epidermis. Ceramide binding activity was decreased in recombinant CERT proteins containing the UVB-induced homotrimer. The middle region domain of the CERT protein was required for the homotrimer formation, whereas neither the pleckstrin homology (Golgi-binding) nor the START (ceramide-binding) domains were involved. Finally like UVB-treated keratinocytes, HPA-12 blockade of CERT function increased keratinocyte apoptosis, decreased sphingomyelin synthesis, and led to accumulation of ceramide. Thus, UVB-induced CERT homotrimer formation accounts, at least in part, for apoptosis and failed up-regulation of sphingomyelin synthesis following UVB irradiation, revealing that inactive CERT can attenuate a key metabolic protective mechanism against ceramide-induced apoptosis in keratinocytes.