Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells.

Bone morphogenetic protein 9 (BMP9) and BMP10 are circulating ligands that mediate endothelial cell (EC) protection via complexes of the type I receptor ALK1 and the type II receptors activin type-IIA receptor (ACTR-IIA) and bone morphogenetic type II receptor (BMPR-II). We previously demonstrated that BMP9 induces the expression of interleukin-6, interleukin-8 and E-selectin in ECs and might influence their interactions with monocytes and neutrophils. We asked whether BMP9 and BMP10 regulate the expression of chemokine (C-C motif) ligand 2 (CCL2), a key chemokine involved in monocyte-macrophage chemoattraction. Here, we show that BMP9 and BMP10 repress basal CCL2 expression and release from human pulmonary artery ECs and aortic ECs. The repression was dependent on ALK1 and co-dependent on ACTR-IIA and BMPR-II. Assessment of canonical Smad signalling indicated a reliance of this response on Smad4. Of note, Smad1/5 signalling contributed only at BMP9 concentrations similar to those in the circulation. In the context of inflammation, BMP9 did not alter the induction of CCL2 by TNF-α. As CCL2 promotes monocyte/macrophage chemotaxis and endothelial permeability, these data support the concept that BMP9 preserves basal endothelial integrity.

MicroRNA-542 Promotes Mitochondrial Dysfunction and SMAD Activity and Is Elevated in Intensive Care Unit-acquired Weakness.

RATIONALE: Loss of skeletal muscle mass and function is a common consequence of critical illness and a range of chronic diseases, but the mechanisms by which this occurs are unclear. OBJECTIVES: To identify microRNAs (miRNAs) that were increased in the quadriceps of patients with muscle wasting and to determine the molecular pathways by which they contributed to muscle dysfunction. METHODS: miRNA-542-3p/5p (miR-542-3p/5p) were quantified in the quadriceps of patients with chronic obstructive pulmonary disease and intensive care unit-acquired weakness (ICUAW). The effect of miR-542-3p/5p was determined on mitochondrial function and transforming growth factor-ß signaling in vitro and in vivo. MEASUREMENTS AND MAIN RESULTS: miR-542-3p/5p were elevated in patients with chronic obstructive pulmonary disease but more markedly in patients with ICUAW. In vitro, miR-542-3p suppressed the expression of the mitochondrial ribosomal protein MRPS10 and reduced 12S ribosomal RNA (rRNA) expression, suggesting mitochondrial ribosomal stress. miR-542-5p increased nuclear phospho-SMAD2/3 and suppressed expression of SMAD7, SMURF1, and PPP2CA, proteins that inhibit or reduce SMAD2/3 phosphorylation, suggesting that miR-542-5p increased transforming growth factor-ß signaling. In mice, miR-542 overexpression caused muscle wasting, and reduced mitochondrial function, 12S rRNA expression, and SMAD7 expression, consistent with the effects of the miRNAs in vitro. Similarly, in patients with ICUAW, the expression of 12S rRNA and of the inhibitors of SMAD2/3 phosphorylation were reduced, indicative of mitochondrial ribosomal stress and increased transforming growth factor-ß signaling. In patients undergoing aortic surgery, preoperative levels of miR-542-3p/5p were positively correlated with muscle loss after surgery. CONCLUSIONS: Elevated miR-542-3p/5p may cause muscle atrophy in intensive care unit patients through the promotion of mitochondrial dysfunction and activation of SMAD2/3 phosphorylation.

Deficient retinoid-driven angiogenesis may contribute to failure of adult human lung regeneration in emphysema.

BACKGROUND: Molecular pathways that regulate alveolar development and adult repair represent potential therapeutic targets for emphysema. Signalling via retinoic acid (RA), derived from vitamin A, is required for mammalian alveologenesis, and exogenous RA can induce alveolar regeneration in rodents. Little is known about RA signalling in the human lung and its potential role in lung disease. OBJECTIVES: To examine regulation of human alveolar epithelial and endothelial repair by RA, and characterise RA signalling in human emphysema. METHODS: The role of RA signalling in alveolar epithelial repair was investigated with a scratch assay using an alveolar cell line (A549) and primary human alveolar type 2 (AT2) cells from resected lung, and the role in angiogenesis using a tube formation assay with human lung microvascular endothelial cells (HLMVEC). Localisation of RA synthetic (RALDH-1) and degrading (cytochrome P450 subfamily 26 A1 (CYP26A1)) enzymes in human lung was determined by immunofluorescence. Regulation of RA pathway components was investigated in emphysematous and control human lung tissue by quantitative real-time PCR and Western analysis. RESULTS: RA stimulated HLMVEC angiogenesis in vitro; this was partially reproduced with a RAR-α agonist. RA induced mRNA expression of vascular endothelial growth factor A (VEGFA) and VEGFR2. RA did not modulate AT2 repair. CYP26A1 protein was identified in human lung microvasculature, whereas RALDH-1 partially co-localised with vimentin-positive fibroblasts. CYP26A1 mRNA and protein were increased in emphysema. CONCLUSIONS: RA regulates lung microvascular angiogenesis; the endothelium produces CYP26A1 which is increased in emphysema, possibly leading to reduced RA availability. These data highlight a role for RA in maintenance of the human pulmonary microvascular endothelium.

The pulmonary endothelium in acute respiratory distress syndrome: insights and therapeutic opportunities.

The pulmonary endothelium is a dynamic, metabolically active layer of squamous endothelial cells ideally placed to mediate key processes involved in lung homoeostasis. Many of these are disrupted in acute respiratory distress syndrome (ARDS), a syndrome with appreciable mortality and no effective pharmacotherapy. In this review, we consider the role of the pulmonary endothelium as a key modulator and orchestrator of ARDS, highlighting advances in our understanding of endothelial pathobiology and their implications for the development of endothelial-targeted therapeutics including cell-based therapies. We also discuss mechanisms to facilitate the translation of preclinical data into effective therapies including the application of biomarkers to phenotype patients with ARDS with a predominance of endothelial injury and emerging biotechnologies that could enhance delivery, discovery and testing of lung endothelial-specific therapeutics.

Systemic inflammation and oxidative stress post-lung resection: Effect of pretreatment with N-acetylcysteine.

BACKGROUND AND OBJECTIVE: N-acetylcysteine has been used to treat a variety of lung diseases, where is it thought to have an antioxidant effect. In a randomized placebo-controlled double-blind study, the effect of N-acetylcysteine on systemic inflammation and oxidative damage was examined in patients undergoing lung resection, a human model of acute lung injury. METHODS: Eligible adults were randomized to receive preoperative infusion of N-acetylcysteine (240 mg/kg over 12 h) or placebo. Plasma thiols, interleukin-6, 8-isoprostane, ischaemia-modified albumin, red blood cell glutathione and exhaled breath condensate pH were measured pre- and post-operatively as markers of local and systemic inflammation and oxidative stress. RESULTS: Patients undergoing lung resection and one-lung ventilation exhibited significant postoperative inflammation and oxidative damage. Postoperative plasma thiol concentration was significantly higher in the N-acetylcysteine-treated group. However, there was no significant difference in any of the measured biomarkers of inflammation or oxidative damage, or in clinical outcomes, between N-acetylcysteine and placebo groups. CONCLUSION: Preoperative administration of N-acetylcysteine did not attenuate postoperative systemic or pulmonary inflammation or oxidative damage after lung resection. CLINICAL TRIAL REGISTRATION: NCT00655928 at ClinicalTrials.gov.

MiR-181a: a potential biomarker of acute muscle wasting following elective high-risk cardiothoracic surgery.

INTRODUCTION: Acute muscle wasting in the critically ill is common and associated with significant morbidity and mortality. Although some aetiological factors are recognised and muscle wasting can be detected early with ultrasound, it not possible currently to predict in advance of muscle loss those who will develop muscle wasting. The ability to stratify the risk of muscle wasting associated with critical illness prior to it becoming clinically apparent would provide the opportunity to predict prognosis more accurately and to intervene at an early stage. MicroRNAs are small non-coding RNAs that modulate post-transcriptional regulation of translation, some are tissue specific and can be detected and quantified in plasma. We hypothesised that certain plasma microRNAs could be biomarkers of ICU acquired muscle weakness. METHODS: Plasma levels of selected microRNAs were measured in pre- and post-operative samples from a previously reported prospective observational study of 42 patients undergoing elective high-risk cardiothoracic surgery, 55% of whom developed muscle wasting. RESULTS: The rise in miR-181a was significantly higher on the second post-operative day in those who developed muscle wasting at 1 week compared to those who did not (p = 0.03). A rise in miR-181a of greater than 1.7 times baseline had 91% specificity and 56% sensitivity for subsequent muscle wasting. Other microRNAs did not show significant differences between the groups. CONCLUSION: Plasma miR-181a deserves further investigation as a potential biomarker of muscle wasting. Additionally, since mir-181a is involved in both regulation of inflammation and muscle regeneration and differentiation; our observation therefore also suggests directions for future research.

Methemoglobin-induced signaling and chemokine responses in human alveolar epithelial cells.

Diffuse alveolar hemorrhage is characterized by the presence of red blood cells and free hemoglobin in the alveoli and complicates a number of serious medical and surgical lung conditions including the pulmonary vasculitides and acute respiratory distress syndrome. In this study we investigated the hypothesis that exposure of human alveolar epithelial cells to hemoglobin and its breakdown products regulates chemokine release via iron- and oxidant-mediated activation of the transcription factor NF-κB. Methemoglobin alone stimulated the release of IL-8 and MCP-1 from A549 cells via activation of the NF-κB pathway; additionally, IL-8 required ERK activation and MCP-1 required JNK activation. Neither antioxidants nor iron chelators and knockdown of ferritin heavy and light chains affected these responses, indicating that iron and reactive oxygen species are not involved in the response of alveolar epithelial cells to methemoglobin. Incubation of primary cultures of human alveolar type 2 cells with methemoglobin resulted in a similar pattern of chemokine release and signaling pathway activation. In summary, we have shown for the first time that methemoglobin induced chemokine release from human lung epithelial cells independent of iron- and redox-mediated signaling involving the activation of the NF-κB and MAPK pathways. Decompartmentalization of hemoglobin may be a significant proinflammatory stimulus in a variety of lung diseases.

Effect of simvastatin on physiological and biological outcomes in patients undergoing esophagectomy: a randomized placebo-controlled trial.

OBJECTIVE: To test whether simvastatin improves physiological and biological outcomes in patients undergoing esophagectomy. BACKGROUND: One-lung ventilation during esophagectomy is associated with inflammation, alveolar epithelial and systemic endothelial injury, and the development of acute lung injury (ALI). Statins that modify many of the underlying processes are a potential therapy to prevent ALI. METHODS: We conducted a randomized double-blind placebo-controlled trial in patients undergoing esophagectomy. Patients received simvastatin 80 mg or placebo enterally for 4 days preoperatively and 7 days postoperatively. The primary end point was pulmonary dead space (Vd/Vt) at 6 hours after esophagectomy or before extubation. Inflammation was assessed by plasma cytokines and intraoperative exhaled breath condensate pH; alveolar type 1 epithelial injury was assessed by plasma receptor for advanced glycation end products and systemic endothelial injury by the urine albumin-creatinine ratio. RESULTS: Thirty-nine patients were randomized; 8 patients did not undergo surgery and were excluded. Fifteen patients received simvastatin and 16 received placebo. There was no difference in Vd/Vt or other physiological outcomes. Simvastatin resulted in a significant decrease in plasma MCP-1 on day 3 and reduced exhaled breath condensate acidification. Plasma receptor for advanced glycation end products was significantly lower in the simvastatin-treated group, as was the urine albumin-creatinine ratio on day 7 postsurgery. ALI developed in 4 patients in the placebo group and no patients in the simvastatin group although this difference was not statistically significant (P=0.1). CONCLUSIONS: In this proof of concept study, pretreatment with simvastatin in esophagectomy decreased biomarkers of inflammation as well as pulmonary epithelial and systemic endothelial injury.

Pulmonary venous hypertension and mechanical strain stimulate monocyte chemoattractant protein-1 release and structural remodelling of the lung in human and rodent chronic heart failure models.

INTRODUCTION: The burden of chronic heart failure (HF) is rising owing to an increased survivorship after myocardial infarction (MI). Pulmonary structural remodelling in patients with HF may protect against oedema while causing dyspnoea, the predominant symptom associated with HF. The cellular and molecular mechanisms underlying these processes in HF are poorly understood. We hypothesised that pulmonary venous hypertension (PVH) following MI provides a mechanical stimulus for structural remodelling of the lung via monocyte chemoattractant protein-1 (MCP-1). METHODS: Human lung microvascular endothelial cells (HLMVEC) and Ea.Hy 926 cells exposed to cyclic mechanical strain (CMS) in vitro were analysed for MCP-1 expression and activation of signalling intermediates. HF was induced in Sprague-Dawley rats 16 weeks after MI; a cohort was rescued with AAV9.SERCA2a gene therapy to reduce PVH. RESULTS: HLMVEC and Ea.Hy 926 cells exposed to CMS upregulated MCP-1 gene expression and protein release in an extracellular-signal-regulated kinase (ERK) 1/2 dependent manner. Supernatants from these experiments stimulated fibroblast (human fetal lung fibroblast -1) and pulmonary artery smooth muscle cell proliferation and differentiation. Total lung collagen, a marker of structural remodelling, and MCP-1 gene expression were increased in the lungs of rats with post-MI HF. SERCA2a gene therapy that attenuated PVH after MI was associated with lower levels of lung collagen and MCP-1 gene expression in the lung. CONCLUSIONS: Mechanical strain associated with PVH may stimulate pulmonary structural remodelling through ERK 1/2 dependent induction of MCP-1. These findings provide insights into the pathophysiology of lung remodelling in HF and highlight novel, potential therapeutic targets.

Sustained elevation of circulating growth and differentiation factor-15 and a dynamic imbalance in mediators of muscle homeostasis are associated with the development of acute muscle wasting following cardiac surgery.

OBJECTIVES: Acute muscle wasting in the critically ill is common and causes significant morbidity. In a novel human model of acute muscle wasting following cardiac surgery, known or potential circulating modulators of muscle mass--insulin-like growth factor-1, myostatin, and growth and differentiation factor-15--were measured over a week. It was hypothesized that patients who developed acute muscle wasting would show distinct patterns of change in these mediators. DESIGN: A prospective longitudinal observational study of high-risk elective cardiac surgical patients identifying, by ultrasound, those developing muscle wasting. SETTING: Tertiary cardiothoracic referral center: Royal Brompton Hospital, London, UK. PATIENTS: Forty-two patients undergoing elective high-risk cardiothoracic surgery. INTERVENTIONS: Circulating insulin-like growth factor-1, myostatin, and growth and differentiation factor-15 were assayed preoperatively and over the first week postoperatively. The ability of growth and differentiation factor-15 to cause muscle wasting in vitro was determined in C2C12 myotubes. MEASUREMENTS AND MAIN RESULTS: Of the 42 patients, 23 (55%) developed quadriceps atrophy. There was an acute decrease in insulin-like growth factor-1 and unexpectedly myostatin, known mediators of muscle hypertrophy and atrophy, respectively. By contrast, plasma growth and differentiation factor-15 concentrations increased in all patients. This increase in growth and differentiation factor-15 was sustained at day 7 in those who developed muscle wasting (day 7 compared with baseline, p<0.01), but recovered in the nonwasting group (p>0.05). Insulin-like growth factor-1 did not recover in those who developed muscle wasting (day 7 compared with baseline, p<0.01) but did in the nonwasting group (p>0.05). Finally, we demonstrated that growth and differentiation factor-15 caused atrophy of myotubes in vitro. CONCLUSION: These data support the hypothesis that acute muscle loss occurs as a result of an imbalance between drivers of muscle atrophy and hypertrophy. Growth and differentiation factor-15 is a potential novel factor associated with muscle atrophy, which may become a therapeutic target in patients with ICU acquired paresis and other forms of acute muscle wasting.

Pathophysiology of pulmonary hypertension in acute lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome are characterized by protein rich alveolar edema, reduced lung compliance, and acute severe hypoxemia. A degree of pulmonary hypertension (PH) is also characteristic, higher levels of which are associated with increased morbidity and mortality. The increase in right ventricular (RV) afterload causes RV dysfunction and failure in some patients, with associated adverse effects on oxygen delivery. Although the introduction of lung protective ventilation strategies has probably reduced the severity of PH in ALI, a recent invasive hemodynamic analysis suggests that even in the modern era, its presence remains clinically important. We therefore sought to summarize current knowledge of the pathophysiology of PH in ALI.

Influenza-related pneumonia.

Influenza-related pneumonia encompasses both primary viral pneumonia and secondary bacterial pneumonia, which may be difficult to differentiate clinically. A high index of suspicion, prompt initiation of antiviral and antibiotic therapy, and appropriate escalation to secondary/critical care are key to improving outcome.

Biomarkers of acute lung injury: worth their salt?

The validation of biomarkers has become a key goal of translational biomedical research. The purpose of this article is to discuss the role of biomarkers in the management of acute lung injury (ALI) and related research. Biomarkers should be sensitive and specific indicators of clinically important processes and should change in a relevant timeframe to affect recruitment to trials or clinical management. We do not believe that they necessarily need to reflect pathogenic processes. We critically examined current strategies used to identify biomarkers and which, owing to expedience, have been dominated by reanalysis of blood derived markers from large multicenter Phase 3 studies. Combining new and existing validated biomarkers with physiological and other data may add predictive power and facilitate the development of important aids to research and therapy.

Translational research: what does it mean, what has it delivered and what might it deliver?

PURPOSE OF REVIEW: In this article, we review recent developments in translational research in the fields of acute lung injury, acute kidney injury and sepsis with a focus on emerging biomarkers and outline future advances in the field. RECENT FINDINGS: There is currently a significant and unmet need for high quality translational research in critical care. The emergence of '-omics' technologies and sophisticated imaging techniques have resulted in a rapid growth of emerging biomarkers. Biomarkers would ideally provide early and reliable endpoints for proof of concept in clinical trials and inform clinical decision making through earlier and more precise diagnosis and risk stratification. SUMMARY: Despite significant investment in basic science and time-consuming clinical trials, the majority of pharmacological interventions developed for critical illness have yet to translate into measurable clinical benefit. Future validation and qualification of emerging biomarkers allied to advances in pharmacogenomic profiling have the potential to provide valuable clinical information while accurately phenotyping patients enrolled in future clinical trials.

Biomarkers of lung injury after one-lung ventilation for lung resection.

BACKGROUND AND OBJECTIVE: Acute lung injury contributes to the mortality of patients after lung resection and one-lung ventilation (OLV). The objective of this study was to characterise the effect of lung resection and OLV on proposed biomarkers of lung injury in exhaled breath condensate (EBC) and plasma. METHODS: In adults undergoing lung resection, EBC was collected before and at 30-min intervals during OLV. Inflammatory mediators were assayed in plasma samples taken preoperatively, immediately postoperatively and 24 h postoperatively. RESULTS: EBC pH decreased from 6.51 ± 0.43 preoperatively, to 6.17 ± 0.78 and 6.09 ± 0.83 at 30 and 60 min, respectively (mean ± SD, P = 0.034, n = 20). Plasma concentrations of the receptor for advanced glycation end-products, von Willebrand factor and interleukin-6 increased comparing preoperative and postoperative samples (all P < 0.001, n = 30). By contrast, levels of Krebs von den Lungen-6 and surfactant protein-D decreased (P < 0.001, n=30), and correlated inversely with the extent of lung resected. CONCLUSIONS: Lung resection and OLV was associated with a rapid reduction in EBC pH and differential changes in plasma biomarkers of lung injury. Further investigation of EBC pH as a marker of ventilator-induced lung injury is warranted.

Referral to an extracorporeal membrane oxygenation center and mortality among patients with severe 2009 influenza A(H1N1).

CONTEXT: Extracorporeal membrane oxygenation (ECMO) can support gas exchange in patients with severe acute respiratory distress syndrome (ARDS), but its role has remained controversial. ECMO was used to treat patients with ARDS during the 2009 influenza A(H1N1) pandemic. OBJECTIVE: To compare the hospital mortality of patients with H1N1-related ARDS referred, accepted, and transferred for ECMO with matched patients who were not referred for ECMO. DESIGN, SETTING, AND PATIENTS: A cohort study in which ECMO-referred patients were defined as all patients with H1N1-related ARDS who were referred, accepted, and transferred to 1 of the 4 adult ECMO centers in the United Kingdom during the H1N1 pandemic in winter 2009-2010. The ECMO-referred patients and the non-ECMO-referred patients were matched using data from a concurrent, longitudinal cohort study (Swine Flu Triage study) of critically ill patients with suspected or confirmed H1N1. Detailed demographic, physiological, and comorbidity data were used in 3 different matching techniques (individual matching, propensity score matching, and GenMatch matching). MAIN OUTCOME MEASURE: Survival to hospital discharge analyzed according to the intention-to-treat principle. RESULTS: Of 80 ECMO-referred patients, 69 received ECMO (86.3%) and 22 died (27.5%) prior to discharge from the hospital. From a pool of 1756 patients, there were 59 matched pairs of ECMO-referred patients and non-ECMO-referred patients identified using individual matching, 75 matched pairs identified using propensity score matching, and 75 matched pairs identified using GenMatch matching. The hospital mortality rate was 23.7% for ECMO-referred patients vs 52.5% for non-ECMO-referred patients (relative risk [RR], 0.45 [95% CI, 0.26-0.79]; P = .006) when individual matching was used; 24.0% vs 46.7%, respectively (RR, 0.51 [95% CI, 0.31-0.81]; P = .008) when propensity score matching was used; and 24.0% vs 50.7%, respectively (RR, 0.47 [95% CI, 0.31-0.72]; P = .001) when GenMatch matching was used. The results were robust to sensitivity analyses, including amending the inclusion criteria and restricting the location where the non-ECMO-referred patients were treated. CONCLUSION: For patients with H1N1-related ARDS, referral and transfer to an ECMO center was associated with lower hospital mortality compared with matched non-ECMO-referred patients.

Contemporary Management of Cardiogenic Shock: A RAND Appropriateness Panel Approach.

BACKGROUND: Current practice in cardiogenic shock is guided by expert opinion in guidelines and scientific statements from professional societies with limited high quality randomized trial data to inform optimal patient management. An international panel conducted a modified Delphi process with the intent of identifying aspects of cardiogenic shock care where there was uncertainty regarding optimal patient management. METHODS: An 18-person multidisciplinary panel comprising international experts was convened. A modified RAND/University of California Los Angeles appropriateness methodology was used. A survey comprising 70 statements was completed. Participants anonymously rated the appropriateness of each statement on a scale of 1 to 9: 1 to 3 inappropriate, 4 to 6 uncertain, and 7 to 9 appropriate. A summary of the results was discussed as a group, and the survey was iterated and completed again before final analysis. RESULTS: There was broad alignment with current international guidelines and consensus statements. Overall, 44 statements were rated as appropriate, 19 as uncertain, and 7 as inappropriate. There was no disagreement with a disagreement index <1 for all statements. Routine fluid administration was deemed to be inappropriate. Areas of uncertainty focused panel on pre-PCI interventions, the use of right heart catheterization to guide management, routine use of left ventricular unloading strategies, and markers of futility when considering escalation to mechanical circulatory support. CONCLUSIONS: While there was broad alignment with current guidance, an expert panel found several aspects of care where there was clinical equipoise, further highlighting the need for randomized controlled trials to better guide patient management and decision making in cardiogenic shock.

A stepwise approach to justify phase III randomized clinical trials and enhance the likelihood of a positive result.

Multiple time-consuming, expensive, and negative phase III clinical trials involving many thousands of patients have been undertaken in critical care and continue to be funded. The vast majority of these clinical trials are negative. Furthermore, phase III studies frequently fail to achieve their planned recruitment target. Often such studies are based on small pilot studies with inadequate phase II trial data and limited mechanistic data to provide a sound scientific rationale. The body of research required to justify undertaking a phase III trial in the critically ill population has not been defined adequately. In particular, guidance on the design of phase II studies for evaluating treatments in the critically ill population is needed. Research to inform critical care practice will progress more efficiently and effectively if this can be achieved. The following article presents a template on the minimum evidence required to justify phase III clinical trials in the critically ill population.

Human reliability analysis of bronchoscope-assisted percutaneous dilatational tracheostomy: implications for simulation-based education.

BACKGROUND: Teaching and assessing clinical procedures requires a clear delineation of the individual steps required to successfully complete the procedure. For decades, human reliability analysis (HRA) has been used to identify the steps required to complete technical procedures in higher risk industries. However, the use of HRA is uncommon in healthcare. HRA has great potential supporting simulation-based education (SBE) in two ways: (1) to support training through the identification of the steps required to complete a clinical procedure; and (2) to support assessment by providing a framework for evaluating performance of a clinical procedure. The goal of this study was to use HRA to identify the steps (and the risk associated with each of these steps) required to complete a bronchoscope-assisted percutaneous dilatational tracheostomy (BPDT). BPDT is a potentially high-risk minimally invasive procedure used to facilitate tracheostomy placement at the bedside or in the operating theatre. METHODS: The subgoals, or steps, required to complete the BPDT procedure were identified using hierarchical task analysis. The Systematic Human Error Reduction and Prediction Approach (SHERPA) was then used to identify potential human errors at each subgoal, the level of risk and how these potential errors could be prevented. RESULTS: The BPDT procedure was broken down into 395 subgoals, of which 18% were determined to be of high-risk. The most commonly identified remediation strategies for reducing the risk of the procedure included: checklist implementation and audit, statutory and mandatory training modules, simulation training, consultant involvement in all procedures, and fostering a safety-focused hospital culture. CONCLUSION: This study provides an approach for how to systematically identify the steps required to complete a clinical procedure for both training and assessment. An understanding of these steps is the foundation of SBE. HRA can identify 'a correct way' for teaching learners how to complete a technical procedure, and support teachers to give systematic and structured feedback on performance.