Single-cell RNA sequencing unveils unique transcriptomic signatures of endothelial cells and role of ENO1 in response to disturbed flow.

Flow patterns exert significant effects on vascular endothelial cells (ECs) to lead to the focal nature of atherosclerosis. Using a step flow chamber to investigate the effects of disturbed shear (DS) and pulsatile shear (PS) on ECs in the same flow channel, we conducted single-cell RNA sequencing analyses to explore the distinct transcriptomic profiles regulated by DS vs. PS. Integrated analysis identified eight cell clusters and demonstrated that DS induces EC transition from atheroprotective to proatherogenic phenotypes. Using an automated cell type annotation algorithm (SingleR), we showed that DS promoted endothelial-to-mesenchymal transition (EndMT) by inducing the transcriptional phenotypes for inflammation, hypoxia responses, transforming growth factor-beta (TGF-ß) signaling, glycolysis, and fatty acid synthesis. Enolase 1 (ENO1), a key gene in glycolysis, was one of the top-ranked genes in the DS-induced EndMT cluster. Pseudotime trajectory analysis revealed that the kinetic expression of ENO1 was significantly associated with EndMT and that ENO1 silencing repressed the DS- and TGF-ß-induced EC inflammation and EndMT. Consistent with these findings, ENO1 was highly expressed in ECs at the inner curvature of the mouse aortic arch (which is exposed to DS) and atherosclerotic lesions, suggesting its proatherogenic role in vivo. In summary, we present a comprehensive single-cell atlas of ECs in response to different flow patterns within the same flow channel. Among the DS-regulated genes, ENO1 plays an important role in DS-induced EC inflammation and EndMT. These results provide insights into how hemodynamic forces regulate vascular endothelium in health and disease.

Epitranscriptional Regulation: From the Perspectives of Cardiovascular Bioengineering.

The central dogma of gene expression involves DNA transcription to RNA and RNA translation into protein. As key intermediaries and modifiers, RNAs undergo various forms of modifications such as methylation, pseudouridylation, deamination, and hydroxylation. These modifications, termed epitranscriptional regulations, lead to functional changes in RNAs. Recent studies have demonstrated crucial roles for RNA modifications in gene translation, DNA damage response, and cell fate regulation. Epitranscriptional modifications play an essential role in development, mechanosensing, atherogenesis, and regeneration in the cardiovascular (CV) system, and their elucidation is critically important to understanding the molecular mechanisms underlying CV physiology and pathophysiology. This review aims at providing biomedical engineers with an overview of the epitranscriptome landscape, related key concepts, recent findings in epitranscriptional regulations, and tools for epitranscriptome analysis. The potential applications of this important field in biomedical engineering research are discussed.

Longitudinal shear stress response in human endothelial cells to atheroprone and atheroprotective conditions.

The two main blood flow patterns, namely, pulsatile shear (PS) prevalent in straight segments of arteries and oscillatory shear (OS) observed at branch points, are associated with atheroprotective (healthy) and atheroprone (unhealthy) vascular phenotypes, respectively. The effects of blood flow-induced shear stress on endothelial cells (ECs) and vascular health have generally been studied using human umbilical vein endothelial cells (HUVECs). While there are a few studies comparing the differential roles of PS and OS across different types of ECs at a single time point, there is a paucity of studies comparing the temporal responses between different EC types. In the current study, we measured OS and PS transcriptomic responses in human aortic endothelial cells (HAECs) over 24 h and compared these temporal responses of HAECs with our previous findings on HUVECs. The measurements were made at 1, 4, and 24 h in order to capture the responses at early, mid, and late time points after shearing. The results indicate that the responses of HAECs and HUVECs are qualitatively similar for endothelial function-relevant genes and several important pathways with a few exceptions, thus demonstrating that HUVECs can be used as a model to investigate the effects of shear on arterial ECs, with consideration of the differences. Our findings show that HAECs exhibit an earlier response or faster kinetics as compared to HUVECs. The comparative analysis of HAECs and HUVECs presented here offers insights into the mechanisms of common and disparate shear stress responses across these two major endothelial cell types.

METTL3-dependent N6-methyladenosine RNA modification mediates the atherogenic inflammatory cascades in vascular endothelium.

Atherosclerosis is characterized by the plaque formation that restricts intraarterial blood flow. The disturbed blood flow with the associated oscillatory stress (OS) at the arterial curvatures and branch points can trigger endothelial activation and is one of the risk factors of atherosclerosis. Many studies reported the mechanotransduction related to OS and atherogenesis; however, the transcriptional and posttranscriptional regulatory mechanisms of atherosclerosis remain unclear. Herein, we investigated the role of N6-methyladenosine (m6A) RNA methylation in mechanotransduction in endothelial cells (ECs) because of its important role in epitranscriptome regulation. We have identified m6A methyltransferase METTL3 as a responsive hub to hemodynamic forces and atherogenic stimuli in ECs. OS led to an up-regulation of METTL3 expression, accompanied by m6A RNA hypermethylation, increased NF-κB p65 Ser536 phosphorylation, and enhanced monocyte adhesion. Knockdown of METTL3 abrogated this OS-induced m6A RNA hypermethylation and other manifestations, while METTL3 overexpression led to changes resembling the OS effects. RNA-sequencing and m6A-enhanced cross-linking and immunoprecipitation (eCLIP) experiments revealed NLRP1 and KLF4 as two hemodynamics-related downstream targets of METTL3-mediated hypermethylation. The METTL3-mediated RNA hypermethylation up-regulated NLRP1 transcript and down-regulated KLF4 transcript through YTHDF1 and YTHDF2 m6A reader proteins, respectively. In the in vivo atherosclerosis model, partial ligation of the carotid artery led to plaque formation and up-regulation of METTL3 and NLRP1, with down-regulation of KLF4; knockdown of METTL3 via repetitive shRNA administration prevented the atherogenic process, NLRP3 up-regulation, and KLF4 down-regulation. Collectively, we have demonstrated that METTL3 serves a central role in the atherogenesis induced by OS and disturbed blood flow.

Can patients contribute to enhancing the safety and effectiveness of test-result follow-up? Qualitative outcomes from a health consumer workshop.

BACKGROUND: Missed test-results and failure to follow-up test-results are major patient safety concerns. Strategies to improve test-results management have predominantly focused on clinician-based interventions, with patients principally involved in studies of test-result communication preferences, the impact of patient portals or experiences with reporting processes in primary care. OBJECTIVE: To identify consumer perspectives and experiences of the challenges they have faced with test-results management, through consumer participation in qualitative data analysis. DESIGN AND PARTICIPANTS: Volunteers (n = 10) were recruited to participate in a health consumer reference group workshop on test-results management. Prior to the workshop, consumers selected topics for discussion using a preference poll. During the workshop, consumers participated in qualitative data analysis of de-identified excerpts of previously collected interview data discussing hospital test-results management. Researchers (n = 5) guided consumers through the analytical process and discussion of themes. Discussions were audio-recorded and transcribed for qualitative analysis. RESULTS: Consumer-selected topics for discussion were 'Transitions of Care' and 'Access'. Consumer data analysis prompted broader discussion including lived experiences. Following the workshop, a second level of content analysis pinpointed issues with implications for patient safety highlighting that consumers were astutely aware of macrolevel 'Systems Factors' relating to 'Emergency Departments' and the health system, as well as microlevel 'Patient Factors' (eg patient preferences and circumstances) which impact a patient's understanding during the 'Communication' (clinician to patient/between clinicians) of test-results 'Information' (or lack thereof). CONCLUSIONS: Consumers identified the challenges patients experience with test-results management, and our findings highlight areas for potential improvement in patient safety. PATIENT OR PUBLIC CONTRIBUTION: Ten health consumer volunteers actively participated in the test-results management data analysis workshop conducted in this study. Two health consumers also volunteered to read and comment on the draft manuscript.

Evaluation of the accuracy of diagnostic coding for influenza compared to laboratory results: the availability of test results before hospital discharge facilitates improved coding accuracy.

BACKGROUND: Assessing the accuracy of diagnostic coding is essential to ensure the validity and reliability of administrative coded data. The aim of the study was to evaluate the accuracy of assigned International Classification of Diseases version 10-Australian Modification (ICD-10-AM) codes for influenza by comparing with patients' results of their polymerase chain reaction (PCR)-based laboratory tests. METHOD: A retrospective study was conducted across seven public hospitals in New South Wales, Australia. A total of 16,439 patients who were admitted and tested by either cartridge-based rapid PCR or batched multiplex PCR between January 2016 and December 2017 met the inclusion criteria. We calculated the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of ICD-10-AM coding using laboratory results as a gold standard. Separate analyses were conducted to determine whether the availability of test results at the time of hospital discharge influenced diagnostic coding accuracy. RESULTS: Laboratory results revealed 2759 positive influenza cases, while ICD-10-AM coding identified 2527 patients. Overall, 13.7% (n = 378) of test positive patients were not assigned an ICD-10-AM code for influenza. A further 5.8% (n = 146) patients with negative test results were incorrectly assigned an ICD-10-AM code for influenza. The sensitivity, specificity, PPV and NPV of ICD-10-AM coding were 93.1%; 98.9%; 94.5% and 98.6% respectively when test results were received before discharge and 32.7%; 99.2%; 87.8% and 89.8% respectively when test results were not available at discharge. The sensitivity of ICD-10-AM coding varied significantly across hospitals. The use of rapid PCR or hospitalisation during the influenza season were associated with greater coding accuracy. CONCLUSION: Although ICD-10-AM coding for influenza demonstrated high accuracy when laboratory results were received before discharge, its sensitivity was substantially lower for patients whose test results were not available at discharge. The timely availability of laboratory test results during the episode of care could contribute to improved coding accuracy.

COVID-19: protocol for observational studies utilizing near real-time electronic Australian general practice data to promote effective care and best-practice policy-a design thinking approach.

BACKGROUND: Health systems around the world have been forced to make choices about how to prioritize care, manage infection control and maintain reserve capacity for future disease outbreaks. Primary healthcare has moved into the front line as COVID-19 testing transitions from hospitals to multiple providers, where tracking testing behaviours can be fragmented and delayed. Pooled general practice data are a valuable resource which can be used to inform population and individual care decision-making. This project aims to examine the feasibility of using near real-time electronic general practice data to promote effective care and best-practice policy. METHODS: The project will utilize a design thinking approach involving all collaborators (primary health networks [PHNs], general practices, consumer groups, researchers, and digital health developers, pathology professionals) to enhance the development of meaningful and translational project outcomes. The project will be based on a series of observational studies utilizing near real-time electronic general practice data from a secure and comprehensive digital health platform [POpulation Level Analysis and Reporting (POLAR) general practice data warehouse]. The study will be carried out over 1.5 years (July 2020-December 2021) using data from over 450 general practices within three Victorian PHNs and Gippsland PHN, Eastern Melbourne PHN and South Eastern Melbourne PHN, supplemented by data from consenting general practices from two PHNs in New South Wales, Central and Eastern Sydney PHN and South Western Sydney PHN. DISCUSSION: The project will be developed using a design thinking approach, leading to the building of a meaningful near real-time COVID-19 geospatial reporting framework and dashboard for decision-makers at community, state and nationwide levels, to identify and monitor emerging trends and the impact of interventions/policy decisions. This will integrate timely evidence about the impact of the COVID-19 pandemic related to its diagnosis and treatment, and its impact across clinical, population and general practice levels.

Why is there variation in test ordering practices for patients presenting to the emergency department with undifferentiated chest pain? A qualitative study.

INTRODUCTION: Up to one-third of laboratory tests ordered in the ED for adults presenting with undifferentiated chest pain are generally not indicated by current Australian guidelines. This study set out to undertake a qualitative investigation of clinician perceptions to identify the reasons for variations in pathology requesting. METHODS: For this study, we draw on data from semistructured interviews (n=38) conducted in the EDs and laboratories across three hospitals as part of a larger study on the test result management process from test request to result follow-up. Thematic analysis was conducted to determine what aspects of the clinical routines and environment might contribute to variations in pathology requesting. Informed by the findings from the analysis, targeted questions were developed and further focus groups (n=5) were held with clinicians, hospital management and electronic medical record (eMR) analysts to investigate in more detail the reasons for requesting outside of guidelines. RESULTS: Participants cited four main reasons for ordering outside of guidelines. Clinicians requested tests outside of guidelines and the ED scope of practice to facilitate the patient journey along the broader continuum of care, including admission to hospital or transfer to another site. Clinicians were also faced with multiple and inconsistent guidelines regarding appropriate test selection. Limited access to in-house specialty and diagnostic services also influenced ordering patterns in smaller non-referral hospitals. Finally, certain features of the current electronic ordering framework within the eMR facilitated overordering and failed to impose any real restrictions on ordering inappropriately or outside of scope of practice. CONCLUSION: Beyond the standardisation of pathology requesting advice across electronic decision support, order sets and guidelines, attempts to address issues related to the appropriateness and variation of laboratory test ordering should consider local and systemic factors which also shape the ordering process.

Systems biology analysis of longitudinal functional response of endothelial cells to shear stress.

Blood flow and vascular shear stress patterns play a significant role in inducing and modulating physiological responses of endothelial cells (ECs). Pulsatile shear (PS) is associated with an atheroprotective endothelial phenotype, while oscillatory shear (OS) is associated with an atheroprone endothelial phenotype. Although mechanisms of endothelial shear response have been extensively studied, most studies focus on characterization of single molecular pathways, mainly at fixed time points after stress application. Here, we carried out a longitudinal time-series study to measure the transcriptome after the application of PS and OS. We performed systems analyses of transcriptional data of cultured human vascular ECs to elucidate the dynamics of endothelial responses in several functional pathways such as cell cycle, oxidative stress, and inflammation. By combining the temporal data on differentially expressed transcription factors and their targets with existing knowledge on relevant functional pathways, we infer the causal relationships between disparate endothelial functions through common transcriptional regulation mechanisms. Our study presents a comprehensive temporally longitudinal experimental study and mechanistic model of shear stress response. By comparing the relative endothelial expressions of genes between OS and PS, we provide insights and an integrated perspective into EC function in response to differential shear. This study has significant implications for the pathogenesis of vascular diseases.

Does health information technology improve acknowledgement of radiology results for discharged Emergency Department patients? A before and after study.

BACKGROUND: The inadequate follow-up of test results is a key patient safety concern, carrying severe consequences for care outcomes. Patients discharged from the emergency department are at particular risk of having test results pending at discharge due to their short lengths of stay, with many hospitals acknowledging that they do not have reliable systems for managing such results. Health information technology hold the potential to reducing errors in the test result management process. This study aimed to measure changes in the proportion of acknowledged radiology reports pre and post introduction of an electronic result acknowledgement system and to determine the proportion of reports with abnormal results, including clinically significant abnormal results requiring follow-up action. METHODS: A before and after study was conducted in the emergency department of a 450-bed metropolitan teaching hospital in Australia. All radiology reports for discharged patients for a one-month period before and after implementation of the electronic result acknowledgement system were reviewed to determine; i) those that reported abnormal results; ii) evidence of test result acknowledgement. All unacknowledged radiology results with an abnormal finding were assessed by an independent panel of two senior emergency physicians for clinical significance. RESULTS: Of 1654 radiology reports in the pre-implementation period 70.6% (n = 1167) had documented evidence of acknowledgement by a clinician. For reports with abnormal results, 71.6% (n = 396) were acknowledged. Of 157 unacknowledged abnormal radiology reports reviewed by an independent emergency physician panel, 34.4% (n = 54) were identified as clinically significant and 50% of these (n = 27) were deemed to carry a moderate likelihood of patient morbidity if not followed up. Electronic acknowledgement occurred for all radiology reports in the post period (n = 1423), representing a 30.4% (95% CI: 28.1-32.6%) increase in acknowledgement rate, and an increase of 28.4% (95% CI: 24.6-32.2%) for abnormal radiology results. CONCLUSIONS: The findings of this study demonstrate the potential of health information technology to improve the safety and effectiveness of the diagnostic process by increasing the rate of follow up of results pending at hospital discharge.

Managing Uncertainty During the Communication of Diagnostic Test Information Between Patients and Clinicians in Australian Emergency Care.

We sought (a) an inductive understanding of patient and clinician perspectives and experiences of the communication of diagnostic test information and (b) a normative understanding of the management of uncertainty that occurs during the clinical encounter in emergency care. Between 2016 and 2018, 58 interviews were conducted with patients and nursing, medical, and managerial staff. Interview data were sequentially analyzed through an inductive thematic analysis, then a normative theory of uncertainty management. Themes of "Ideals," "Service Efficiency," and "Managing Uncertainty" were inductively identified as influencing the communication of diagnostic test information. A normative theory of uncertainty management highlighted (a) how these themes reflected the interaction's sociocultural context, encapsulated various criteria by which clinicians and patients evaluated the appropriateness and effectiveness of their communication, and represented competing goals during the clinical encounter, and (b) how systemic tensions between themes accounted for when diagnostic test information communication occurred, was deferred or avoided.

The quality, safety and governance of telephone triage and advice services - an overview of evidence from systematic reviews.

BACKGROUND: Telephone triage and advice services (TTAS) are increasingly being implemented around the world. These services allow people to speak to a nurse or general practitioner over the telephone and receive assessment and healthcare advice. There is an existing body of research on the topic of TTAS, however the diffuseness of the evidence base makes it difficult to identify key lessons that are consistent across the literature. Systematic reviews represent the highest level of evidence synthesis. We aimed to undertake an overview of such reviews to determine the scope, consistency and generalisability of findings in relation to the governance, safety and quality of TTAS. METHODS: We searched PubMed, MEDLINE, EMBASE, CINAHL, Web of Science and the Cochrane Library for English language systematic reviews focused on key governance, quality and safety findings related to telephone based triage and advice services, published since 1990. The search was undertaken by three researchers who reached consensus on all included systematic reviews. An appraisal of the methodological quality of the systematic reviews was independently undertaken by two researchers using A Measurement Tool to Assess Systematic Reviews. RESULTS: Ten systematic reviews from a potential 291 results were selected for inclusion. TTAS was examined either alone, or as part of a primary care service model or intervention designed to improve primary care. Evidence of TTAS performance was reported across nine key indicators - access, appropriateness, compliance, patient satisfaction, cost, safety, health service utilisation, physician workload and clinical outcomes. Patient satisfaction with TTAS was generally high and there is some consistency of evidence of the ability of TTAS to reduce clinical workload. Measures of the safety of TTAS tended to show that there is no major difference between TTAS and traditional care. CONCLUSIONS: Taken as a whole, current evidence does not provide definitive answers to questions about the quality of care provided, access and equity of the service, its costs and outcomes. The available evidence also suggests that there are many interactional factors (e.g., relationship with other health service providers) which can impact on measures of performance, and also affect the external validity of the research findings.

How well do general practitioners manage laboratory test results for patients with diabetes mellitus and cardiovascular disease?: A systematic review.

PURPOSE: To evaluate how well general practitioners (GPs) manage and respond to laboratory results for patients with diabetes mellitus (DM) and cardiovascular disease (CVD). DATA SOURCES: MEDLINE, CINAHL, Embase, EBM reviews, ProQuest and Scopus. STUDY SELECTION: Peer-reviewed journal articles published between 2000 and 2015 that assessed GPs' management of laboratory results for patients with DM or CVD. DATA EXTRACTION: Study design and demographics, laboratory tests and key findings relating to GP management of laboratory results were extracted from studies. RESULTS OF DATA SYNTHESIS: Thirteen articles were included, comprising seven studies which utilized surveys, four observational studies, one cohort study and one randomized controlled trial. Findings indicate that GPs often overestimate the risk of complications associated with DM and CVD based on laboratory results and have unrealistically high expectations regarding the precision of laboratory tests. Considerable variation existed in the use of repeat testing for diagnostic confirmation and in GPs' identification of the difference between two consecutive results required to indicate a change in patient condition. GPs also often failed to initiate appropriate treatment for patients with DM and CVD based on laboratory results. Feedback to GPs about their test ordering patterns and educational messages on laboratory results improved clinical outcomes. CONCLUSION: Evidence about how well GPs manage results and its impact on patient outcomes remains weak and inconclusive. This review identified a number of areas where interventions could support GPs to improve the interpretation and management of laboratory test results, including feedback to GPs and educational messages on test result reports.

Bbof1 is required to maintain cilia orientation.

Multiciliate cells (MCCs) are highly specialized epithelial cells that employ hundreds of motile cilia to produce a vigorous directed flow in a variety of organ systems. The production of this flow requires the establishment of planar cell polarity (PCP) whereby MCCs align hundreds of beating cilia along a common planar axis. The planar axis of cilia in MCCs is known to be established via the PCP pathway and hydrodynamic cues, but the downstream steps required for cilia orientation remain poorly defined. Here, we describe a new component of cilia orientation, based on the phenotypic analysis of an uncharacterized coiled-coil protein, called bbof1. We show that the expression of bbof1 is induced during the early phases of MCC differentiation by the master regulator foxj1. MCC differentiation and ciliogenesis occurs normally in embryos where bbof1 activity is reduced, but cilia orientation is severely disrupted. We show that cilia in bbof1 mutants can still respond to patterning and hydrodynamic cues, but lack the ability to maintain their precise orientation. Misexpression of bbof1 promotes cilia alignment, even in the absence of flow or in embryos where microtubules and actin filaments are disrupted. Bbof1 appears to mediate cilia alignment by localizing to a polar structure adjacent to the basal body. Together, these results suggest that bbof1 is a basal body component required in MCCs to align and maintain cilia orientation in response to flow.

PATHOLOGY IMAGE OF THE MONTH: Sudden Cardiac Death Caused by an Uncommon Disease.

A 57-year-old female, found dead lying supine in bed, was transferred to the autopsy service for an unrestricted autopsy to be performed under the authorization by the coroner. Medical history was unknown. At the time of autopsy, an implantable cardioverter-defibrillator (ICD) was identified in the subcutaneous tissues of the left subclavicular chest, with distal leads terminating in a small amount of fibrous tissue within the right auricular appendage and along the medial wall of the right ventricle. The heart was enlarged at 430gm (312 ±78) and cross sections were notable for left ventricular hypertrophy at 1.9cm (1.0-1.5cm) and for dilatation of the right ventricular chamber on initial apical cross section. All cross sections, from cardiac apex to subvalvular base, showed broad patches of white-yellow myocardial discoloration, without obvious hemorrhage, along the free wall of the left ventricle, the free wall of the right ventricle, and within the anterior interventricular septum (Figure 1). Additional notable findings at autopsy included a vena caval filter devoid of thromboembolic material, a patent foramen ovale (0.7cm) and microscopic plexogenic arteriopathy, low grade, consistent with pulmonary hypertension within the intrapulmonary vasculature. Histology from the discolored patches of myocardium is seen in Figure 2. Special stains for microorgansims (periodic acid-Schiff, Gomori methanamine silver, and Fite) were all negative.

Histone deacetylase 3 unconventional splicing mediates endothelial-to-mesenchymal transition through transforming growth factor ß2.

Histone deacetylase 3 (HDAC3) plays a critical role in the maintenance of endothelial integrity and other physiological processes. In this study, we demonstrated that HDAC3 undergoes unconventional splicing during stem cell differentiation. Four different splicing variants have been identified, designated as HD3α, -ß, -γ, and -δ, respectively. HD3α was confirmed in stem cell differentiation by specific antibody against the sequences from intron 12. Immunofluorescence staining indicated that the HD3α isoform co-localized with CD31-positive or α-smooth muscle actin-positive cells at different developmental stages of mouse embryos. Overexpression of HD3α reprogrammed human aortic endothelial cells into mesenchymal cells featuring an endothelial-to-mesenchymal transition (EndMT) phenotype. HD3α directly interacts with HDAC3 and Akt1 and selectively activates transforming growth factor ß2 (TGFß2) secretion and cleavage. TGFß2 functioned as an autocrine and/or paracrine EndMT factor. The HD3α-induced EndMT was both PI3K/Akt- and TGFß2-dependent. This study provides the first evidence of the role of HDAC3 splicing in the maintenance of endothelial integrity.

Vascular endothelial cell growth-activated XBP1 splicing in endothelial cells is crucial for angiogenesis.

BACKGROUND: Vascular endothelial cell growth factor plays a pivotal role in angiogenesis via regulating endothelial cell proliferation. The X-box binding protein 1 (XBP1) is believed to be a signal transducer in the endoplasmic reticulum stress response. It is unknown whether there is crosstalk between vascular endothelial cell growth factor signaling and XBP1 pathway. METHODS AND RESULTS: We found that vascular endothelial cell growth factor induced the kinase insert domain receptor internalization and interaction through C-terminal domain with the unspliced XBP1 and the inositol requiring enzyme 1 α in the endoplasmic reticulum, leading to inositol requiring enzyme 1 α phosphorylation and XBP1 mRNA splicing, which was abolished by siRNA-mediated knockdown of kinase insert domain receptor. Spliced XBP1 regulated endothelial cell proliferation in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-dependent manner and modulated the cell size increase in a PI3K/Akt/GSK3ß/ß-catenin/E2F2-independent manner. Knockdown of XBP1 or inositol requiring enzyme 1 α decreased endothelial cell proliferation via suppression of Akt/GSK3ß phosphorylation, ß-catenin nuclear translocation, and E2F2 expression. Endothelial cell-specific knockout of XBP1 (XBP1ecko) in mice retarded the retinal vasculogenesis in the first 2 postnatal weeks and impaired the angiogenesis triggered by ischemia. Reconstitution of XBP1 by Ad-XBP1s gene transfer significantly improved angiogenesis in ischemic tissue in XBP1ecko mice. Transplantation of bone marrow from wild-type o XBP1ecko mice could also slightly improve the foot blood reperfusion in ischemic XBP1ecko mice. CONCLUSIONS: These results suggest that XBP1 can function via growth factor signaling pathways to regulate endothelial proliferation and angiogenesis.