Search

Here, we review the highlights of cardiovascular basic science published in 2021 and early 2022 on behalf of the European Society of Cardiology Council for Basic Cardiovascular Science. We begin with non-coding RNAs which have emerged as central regulators cardiovascular biology, and then discuss how technological developments in single-cell ‘omics are providing new insights into cardiovascular development, inflammation, and disease. We also review recent discoveries on the biology of extracellular vesicles in driving either protective or pathogenic responses. The Nobel Prize in Physiology or Medicine 2021 recognized the importance of the molecular basis of mechanosensing and here we review breakthroughs in cardiovascular sensing of mechanical force. We also summarize discoveries in the field of atherosclerosis including the role of clonal haematopoiesis of indeterminate potential, and new mechanisms of crosstalk between hyperglycaemia, lipid mediators, and inflammation. The past 12 months also witnessed major advances in the field of cardiac arrhythmia including new mechanisms of fibrillation. We also focus on inducible pluripotent stem cell technology which has demonstrated disease causality for several genetic polymorphisms in long-QT syndrome and aortic valve disease, paving the way for personalized medicine approaches. Finally, the cardiovascular community has continued to better understand COVID-19 with significant advancement in our knowledge of cardiovascular tropism, molecular markers, the mechanism of vaccine-induced thrombotic complications and new anti-viral therapies that protect the cardiovascular system.

Ischaemic heart disease is a global healthcare challenge with high morbidity and mortality. Early revascularisation in acute myocardial infarction has improved survival, however, limited regenerative capacity and microvascular dysfunction often lead to impaired function and the development of heart failure. New mechanistic insights are required to identify robust targets for the development of novel strategies to promote regeneration. Single cell RNA sequencing (scRNA-seq) has enabled profiling and analysis of the transcriptomes of individual cells at high resolution. Applications of scRNA-seq have generated single cell atlases for multiple species, revealed distinct cellular compositions for different regions of the heart, and defined multiple mechanisms involved in myocardial injury-induced regeneration. In this review, we summarise findings from studies of healthy and injured hearts in multiple species and spanning different developmental stages. Based on this transformative technology, we propose a multi-species, multi-omics, meta-analysis framework to drive the discovery of new targets to promote cardiovascular regeneration.

Myocardial infarction remains the leading cause of heart failure. The adult human heart lacks the capacity to undergo endogenous regeneration. New blood vessel growth is integral to regenerative medicine necessitating a comprehensive understanding of the pathways that regulate vascular regeneration. We sought to define the transcriptomic dynamics of coronary endothelial cells following ischaemic injuries in the developing and adult mouse and human heart and to identify new mechanistic insights and targets for cardiovascular regeneration.We carried out a comprehensive meta-analysis of integrated single cell RNA-sequencing data of coronary vascular endothelial cells from the developing and adult mouse and human heart spanning healthy and acute and chronic ischaemic cardiac disease. We identified species-conserved gene regulatory pathways aligned to endogenous neovascularisation. We annotated injury-associated temporal shifts of the endothelial transcriptome and validated four genes: VEGF-C, KLF4, EGR1 and ZFP36. Moreover, we showed that ZFP36 regulates human coronary endothelial cell proliferation and defined that VEGF-C administration in vivo enhances clonal expansion of the cardiac vasculature post-myocardial infarction. Finally, we constructed a coronary endothelial cell meta-atlas, CrescENDO, to empower future in-depth research to target pathways associated with coronary neovascularisation.We present a high-resolution single cell meta-atlas of healthy and injured coronary endothelial cells in the mouse and human heart, revealing a suite of novel targets with great potential to promote vascular regeneration, and providing a rich resource for therapeutic development.Myocardial infarction is the leading cause of heart failure, a condition with high morbidity and mortality. Vascular regeneration is vital to tissue survival and functional restoration while a comprehensive understanding of the underpinning mechanisms is lacking. We show that the meta-analysis of single cell RNA-sequencing data excels at deciphering multi-faceted vascular responses conserved between mouse and human following ischaemic injury. We envision that these data will inform future strategies and accelerate therapeutic development for promoting vascular regeneration in the ischaemic heart.

Isolation of high quality cardiac endothelial cells is a prerequisite for successful bulk and single cell sequencing for RNA (scRNA-seq). We describe a protocol using both enzymatic and mechanical dissociation and fluorescence-activated cell sorting (FACS) to isolate endothelial cells from larval and adult zebrafish hearts and from healthy and ischemic adult mouse hearts. Endothelial cells with high viability and purity can be obtained using this method for downstream transcriptional analyses applications.

AimsEndothelial cell (EC) dysfunction drives the initiation and pathogenesis of pulmonary arterial hypertension (PAH). We aimed to characterize EC dynamics in PAH at single-cell resolution.Methods and resultsWe carried out single-cell RNA sequencing (scRNA-seq) of lung ECs isolated from an EC lineage-tracing mouse model in Control and SU5416/hypoxia-induced PAH conditions. EC populations corresponding to distinct lung vessel types, including two discrete capillary populations, were identified in both Control and PAH mice. Differential gene expression analysis revealed global PAH-induced EC changes that were confirmed by bulk RNA-seq. This included upregulation of the major histocompatibility complex class II pathway, supporting a role for ECs in the inflammatory response in PAH. We also identified a PAH response specific to the second capillary EC population including upregulation of genes involved in cell death, cell motility, and angiogenesis. Interestingly, four genes with genetic variants associated with PAH were dysregulated in mouse ECs in PAH. To compare relevance across PAH models and species, we performed a detailed analysis of EC heterogeneity and response to PAH in rats and humans through whole-lung PAH scRNA-seq datasets, revealing that 51% of up-regulated mouse genes were also up-regulated in rat or human PAH. We identified promising new candidates to target endothelial dysfunction including CD74, the knockdown of which regulates EC proliferation and barrier integrity in vitro. Finally, with an in silico cell ordering approach, we identified zonation-dependent changes across the arteriovenous axis in mouse PAH and showed upregulation of the Serine/threonine-protein kinase Sgk1 at the junction between the macro- and microvasculature.ConclusionThis study uncovers PAH-induced EC transcriptomic changes at a high resolution, revealing novel targets for potential therapeutic candidate development.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells., Graphical Abstract

While humans lack sufficient capacity to undergo cardiac regeneration following injury, zebrafish can fully recover from a range of cardiac insults. Over the past two decades, our understanding of the complexities of both the independent and co-ordinated injury responses by multiple cardiac tissues during zebrafish heart regeneration has increased exponentially. Although cardiomyocyte regeneration forms the cornerstone of the reparative process in the injured zebrafish heart, recent studies have shown that this is dependent on prior neovascularization and lymphangiogenesis, which in turn require epicardial, endocardial, and inflammatory cell signalling within an extracellular milieu that is optimized for regeneration. Indeed, it is the amalgamation of multiple regenerative systems and gene regulatory patterns that drives the much-heralded success of the adult zebrafish response to cardiac injury. Increasing evidence supports the emerging paradigm that developmental transcriptional programmes are re-activated during adult tissue regeneration, including in the heart, and the zebrafish represents an optimal model organism to explore this concept. In this review, we summarize recent advances from the zebrafish cardiovascular research community with novel insight into the mechanisms associated with endogenous cardiovascular repair and regeneration, which may be of benefit to inform future strategies for patients with cardiovascular disease.

Purpose Endothelial dysfunction is central to the pathogenesis of acute coronary syndrome. The study of diseased endothelium is very challenging due to inherent difficulties in isolating endothelial cells from the coronary vascular bed. We sought to isolate and characterise coronary endothelial cells from patients undergoing thrombectomy for myocardial infarction to develop a patient-specific in vitro model of endothelial dysfunction. Methods In a prospective cohort study, 49 patients underwent percutaneous coronary intervention with thrombus aspiration. Specimens were cultured, and coronary endothelial outgrowth (CEO) cells were isolated. CEO cells, endothelial cells isolated from peripheral blood, explanted coronary arteries, and umbilical veins were phenotyped and assessed functionally in vitro and in vivo. Results CEO cells were obtained from 27/37 (73%) atherothrombotic specimens and gave rise to cells with cobblestone morphology expressing CD146 (94 ± 6%), CD31 (87 ± 14%), and von Willebrand factor (100 ± 1%). Proliferation of CEO cells was impaired compared to both coronary artery and umbilical vein endothelial cells (population doubling time, 2.5 ± 1.0 versus 1.6 ± 0.3 and 1.2 ± 0.3 days, respectively). Cell migration was also reduced compared to umbilical vein endothelial cells (29 ± 20% versus 85±19%). Importantly, unlike control endothelial cells, dysfunctional CEO cells did not incorporate into new vessels or promote angiogenesis in vivo. Conclusions CEO cells can be reliably isolated and cultured from thrombectomy specimens in patients with acute coronary syndrome. Compared to controls, patient-derived coronary endothelial cells had impaired capacity to proliferate, migrate, and contribute to angiogenesis. CEO cells could be used to identify novel therapeutic targets to enhance endothelial function and prevent acute coronary syndromes.

Cardiac injury leads to the loss of cardiomyocytes which are rapidly replaced by proliferation of surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronised live imaging, RNA sequencing and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterise macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche triggering the expansion of the epicardium which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signalling. Identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

The human transcriptome comprises a complex network of coding and non-coding RNAs implicated in a myriad of biological functions. Non-coding RNAs exhibit highly organized spatial and temporal expression patterns and are emerging as critical regulators of differentiation, homeostasis, and pathological states, including in the cardiovascular system. This review defines the current knowledge gaps, unmet methodological needs, and describes the challenges in dissecting and understanding the role and regulation of the non-coding transcriptome in cardiovascular disease. These challenges include poor annotation of the non-coding genome, determination of the cellular distribution of transcripts, assessment of the role of RNA processing and identification of cell-type specific changes in cardiovascular physiology and disease. We highlight similarities and differences in the hurdles associated with the analysis of the non-coding and protein-coding transcriptomes. In addition, we discuss how the lack of consensus and absence of standardized methods affect reproducibility of data. These shortcomings should be defeated in order to make significant scientific progress and foster the development of clinically applicable non-coding RNA-based therapeutic strategies to lessen the burden of cardiovascular disease. ispartof: CARDIOVASCULAR RESEARCH vol:118 issue:4 pages:1004-1019 ispartof: location:England status: published

Ischaemic heart disease remains a leading global cause of death. Restoring blood perfusion in the peri-infarct border region may limit further infarct expansion and promote cardiac regeneration. However, the pathways driving endogenous vascular regeneration following myocardial infarction (MI) remain poorly understood. We aimed to investigate the origin, clonal dynamics and transcriptional profiles of resident coronary endothelial cells (EC) in the post-ischaemic adult mouse heart and to apply single cell RNA-sequencing (scRNAseq) to identify and validate novel targets with a potential role in neovasculogenesis following MI. MI was induced in an EC-specific multispectral lineage-tracing mouse, ‘Pdgfb-iCreERT2-R26R-Brainbow2.1’ by permanent ligation of the left anterior descending coronary artery. Blood vessel formation via clonal proliferation by resident coronary vascular EC was significantly upregulated in the ischaemic border at 7 days post-MI, compared to the healthy heart (Pdgfb-Confetti+ EC per clone = 4.0 ± 2.1 versus 10.3 ± 10.6, P

Autophagy is an essential cellular quality control process that has emerged as a critical one for vascular homeostasis. Here, we show that trichoplein (TCHP) links autophagy with endothelial cell (EC) function. TCHP localizes to centriolar satellites, where it binds and stabilizes PCM1. Loss of TCHP leads to delocalization and proteasome‐dependent degradation of PCM1, further resulting in degradation of PCM1's binding partner GABARAP. Autophagic flux under basal conditions is impaired in THCP‐depleted ECs, and SQSTM1/p62 (p62) accumulates. We further show that TCHP promotes autophagosome maturation and efficient clearance of p62 within lysosomes, without affecting their degradative capacity. Reduced TCHP and high p62 levels are detected in primary ECs from patients with coronary artery disease. This phenotype correlates with impaired EC function and can be ameliorated by NF‐κB inhibition. Moreover, Tchp knock‐out mice accumulate of p62 in the heart and cardiac vessels correlating with reduced cardiac vascularization. Taken together, our data reveal that TCHP regulates endothelial cell function via an autophagy‐mediated mechanism., The interaction of TCHP with PCM1 regulates basal autophagy through controlling GABARAP stability. Loss of TCHP function results in impaired autophagy, the accumulation of SQSTM1/p62 and endothelial dysfunction.

Aims Long non-coding RNAs (lncRNAs) play functional roles in physiology and disease, yet understanding of their contribution to endothelial cell (EC) function is incomplete. We identified lncRNAs regulated during EC differentiation and investigated the role of LINC00961 and its encoded micropeptide SPAAR in EC function. Methods and results Deep sequencing of human embryonic stem cell differentiation to ECs was combined with ENCODE RNA-seq data from vascular cells, identifying 278 endothelial enriched genes, including 6 lncRNAs. Expression of LINC00961, first annotated as a lncRNA but reassigned as a protein coding gene for the SPAAR micropeptide, was increased during the differentiation and was EC-enriched. LINC00961 transcript depletion significantly reduced EC adhesion, tube formation, migration, proliferation, and barrier integrity in primary ECs. Overexpression of the SPAAR open reading frame increased tubule formation, however overexpression of the full length transcript did not, despite production of SPAAR. Further, overexpression of an ATG mutant of the full length transcript reduced network formation, suggesting a bona fide non-coding RNA function of the transcript with opposing effects to SPAAR. As the LINC00961 locus is conserved in mouse, we generated a LINC00961 locus knockout (KO) mouse that underwent hind limb ischaemia to investigate the angiogenic role of this locus in vivo. In agreement with in vitro data, KO animals had a reduced capillary density in the ischaemic adductor muscle after 7 days. Finally, to characterise LINC00961 and SPAAR independent functions in ECs, we performed pull-downs of both molecules and identified protein binding partners. LINC00961 RNA binds the G-actin sequestering protein thymosin beta-4x (Tβ4) and Tβ4 depletion phenocopied the overexpression of the ATG mutant. SPAAR binding partners included the actin binding protein, SYNE1. Conclusion The LINC00961 locus regulates EC function in vitro and in vivo. The gene produces two molecules with opposing effects on angiogenesis: SPAAR and LINC00961. Translational perspective Treatment of ischemic conditions remains a major cardiovascular health burden. Identification of genes and non-coding RNAs that regulate the function of the vascular endothelium is important to understand and evolve potential new strategies that might enhance vascular regeneration. Here, we describe and dissect the functional importance of a micropeptide-encoding RNA transcript in the vascular endothelium, and demonstrate that both the RNA and the peptide regulate endothelial biology. Modulation of this axis may be a novel approach to regulate angiogenesis.

Aims: A better understanding of the pathways that regulate regeneration of the coronary vasculature is important for future strategies to treat patients with heart disease. We investigated (i) the clonal dynamics of endothelial cells (EC) associated with neovascularization in the ischemic border region (ii) transcriptional signatures of regenerative EC in the ischemic heart using single cell RNA-sequencing (iii) the functional relevance of selected targets. Methods: MI was induced in ‘EC-Confetti’ mice by coronary artery ligation. EC clonal proliferation was quantified or hearts dissociated for scRNAseq. Immunofluorescence staining for targets identified by scRNAseq was performed on cardiac tissue from patients with ischemic heart disease. EC proliferation was assessed in vitro following siRNA gene silencing. Results: EC-Confetti mice express YFP, RFP, GFP, or CFP specifically in EC. Fluorophores are inherited by EC progeny following proliferation, allowing quantitative clonal analysis. Clonal proliferation was significantly increased in the infarct border at 7 days post-MI compared to healthy hearts ( P P =0.002) and mouse ( P =0.002) hearts, compared to healthy myocardium. siRNA gene silencing of Plvap significantly inhibited EC proliferation ( P = 0.0038), strong evidence that Plvap can directly modulate EC function. Conclusions: Generation of new blood vessels following ischemic injury in the mouse heart is predominantly mediated by clonal proliferation of resident EC. We present a gene expression atlas of resident cardiac EC, and the transcriptional hierarchy underpinning endogenous vascular repair following MI. This resource identifies novel targets, including Plvap, that may augment myocardial perfusion post-MI, and inform future design of strategies aimed at promoting vascular perfusion in ischemic heart disease.

Autophagy is an essential cellular quality control process that emerged critical for vascular homeostasis. Here we describe, the role for Trichoplein (TCHP) protein in linking autophagy with endothelial cells (ECs) function. The depletion of TCHP in ECs impairs migration and sprouting. TCHP directly binds PCM1, to regulate degradation of GABARAP, thus leading to a defective autophagy. Mechanistically, TCHP is indispensable for autophagosome maturation and its depletion resulted in the accumulation of SQSTM1/p62 (p62) and unfolded protein aggregates in ECs. The latter process is coupled to TCHP-mediated NF-kB activation. Of note, low levels of TCHP and high p62 levels were detected in primary ECs from patients with coronary artery disease. In addition, Tchp knock-out mice showed accumulation of p62 in the heart and cardiac vessels and reduced cardiac vascularization. Here, we reveal an autophagy-mediated mechanism for TCHP down-regulation, which poses a plausible target for regulation of endothelial function.

Long non-coding RNAs (lncRNAs) have structural and functional roles in development and disease. We have previously shown that the LINC00961/SPAAR (small regulatory polypeptide of amino acid response) locus regulates endothelial cell function, and that both the lncRNA and micropeptide counter-regulate angiogenesis. To assess human cardiac cell SPAAR expression, we mined a publicly available scRNSeq dataset and confirmed LINC00961 locus expression and hypoxic response in a murine endothelial cell line. We investigated post-natal growth and development, basal cardiac function, the cardiac functional response, and tissue-specific response to myocardial infarction. To investigate the influence of the LINC00961/SPAAR locus on longitudinal growth, cardiac function, and response to myocardial infarction, we used a novel CRISPR/Cas9 locus knockout mouse line. Data mining suggested that SPAAR is predominantly expressed in human cardiac endothelial cells and fibroblasts, while murine LINC00961 expression is hypoxia-responsive in mouse endothelial cells. LINC00961–/– mice displayed a sex-specific delay in longitudinal growth and development, smaller left ventricular systolic and diastolic areas and volumes, and greater risk area following myocardial infarction compared with wildtype littermates. These data suggest the LINC00961/SPAAR locus contributes to cardiac endothelial cell and fibroblast function and hypoxic response, growth and development, and basal cardiovascular function in adulthood.

The annual image competition, Reflections of Research, provides a glimpse into the cutting-edge heart and circulatory research funded by the British Heart Foundation

BACKGROUND: Endothelial dysfunction is central to the pathogenesis of coronary artery disease, but the role of local and circulating endothelial progenitor cells in maintaining vascular health is poorly understood. We hypothesised that impaired local and circulating vascular repair mechanisms predispose to endothelial dysfunction and the premature onset of coronary artery disease.METHODS AND RESULTS: Patients with premature coronary artery disease (n = 16) and healthy age- and sex-matched controls (n = 16) underwent venous occlusion plethysmography with intra-arterial infusion of acetylcholine and sodium nitroprusside. Numbers of circulating endothelial progenitor cells were directly quantified in whole blood by flow cytometry. Endothelial cells were isolated from the blood vessel wall and from peripheral blood mononuclear cells, and expanded in vitro for phenotypic and functional characterisation and analysis of microRNA expression levels. A dose-dependent increase in forearm blood flow (p CONCLUSION: Local vessel wall derived endothelial cells, rather than circulating endothelial progenitor cells and their progeny, are impaired in patients with vascular dysfunction and premature coronary artery disease.

Background: Rates of myocardial infarction in firefighters are increased during fire suppression duties, and are likely to reflect a combination of factors including extreme physical exertion and heat exposure. We assessed the effects of simulated fire suppression on measures of cardiovascular health in healthy firefighters. Methods: In an open-label randomized crossover study, 19 healthy firefighters (age, 41±7 years; 16 males) performed a standardized training exercise in a fire simulation facility or light duties for 20 minutes. After each exposure, ex vivo thrombus formation, fibrinolysis, platelet activation, and forearm blood flow in response to intra-arterial infusions of endothelial-dependent and -independent vasodilators were measured. Results: After fire simulation training, core temperature increased (1.0±0.1°C) and weight reduced (0.46±0.14 kg, P P P =0.03). There was a dose-dependent increase in forearm blood flow with all vasodilators ( P P =0.01) and sodium nitroprusside ( P =0.004). This was associated with a rise in fibrinolytic capacity, asymptomatic myocardial ischemia, and an increase in plasma cardiac troponin I concentrations (1.4 [0.8–2.5] versus 3.0 [1.7–6.4] ng/L, P =0.010). Conclusions: Exposure to extreme heat and physical exertion during fire suppression activates platelets, increases thrombus formation, impairs vascular function, and promotes myocardial ischemia and injury in healthy firefighters. Our findings provide pathogenic mechanisms to explain the association between fire suppression activity and acute myocardial infarction in firefighters. Clinical Trial Registration: URL: http://www.clinicaltrials.gov . Unique identifier: NCT01812317.

Objective Innate immune responses to pulmonary resection may be critical in the pathogenesis of important postoperative pulmonary complications and potentially longer-term survival. We sought to compare innate immunity of patients undergoing major pulmonary resection for bronchogenic carcinoma via video-assisted thoracoscopic surgery (VATS) and thoracotomy. Methods Bronchoalveolar lavage was conducted in the contralateral lung before staging bronchoscopy and mediastinoscopy and immediately after lung resection. Blood and exhaled nitric oxide were sampled preoperatively and at 6, 24, and 48 hours postoperatively. Results Forty patients were included (26 VATS and 14 thoracotomy). There was a lower systemic cytokine response from lung resection undertaken by VATS compared with thoracotomy [interleukin 6 (IL-6), analysis of variance (ANOVA) P = 0.026; IL-8, ANOVA P = 0.018; and IL-10, ANOVA P = 0.047]. The VATS patients had higher perioperative serum albumin levels (ANOVA P = 0.001). Lower levels of IL-10 were produced by lipopolysaccharide-stimulated blood monocytes from the VATS patients compared with the thoracotomy patients at 6 hours postoperatively (geometric mean ratio, 1.16; 95% confidence interval, 1.08–1.33; P = 0.011). No statistically significant differences in the neutrophil phagocytic capacity, overall leukocyte count, or differential leukocyte count were found between the surgical groups (ANOVA P > 0.05). No statistically significant differences in bronchoalveolar lavage fluid parameters were found. Exhaled nitric oxide levels fell postoperatively, which reached statistical significance at 48 hours (geometric mean ratio, 1.2; 95% confidence interval, 1.02–1.46; P = 0.029). There were no significant differences found between the surgical groups (ANOVA P = 0.331). Conclusions Overall, a trend toward greater proinflammatory and anti-inflammatory responses is seen with lung resection performed via thoracotomy compared with VATS.

The pathogenesis of chronic obstructive pulmonary disease is not fully understood. The objective of this study was to compare circulating endothelial progenitor cells in patients with chronic obstructive pulmonary disease to age, sex, and cigarette smoking matched healthy controls. Patients with chronic obstructive pulmonary disease ( n = 37) and healthy controls ( n = 19) were matched by age, sex, and smoking status. Circulating hematopoietic progenitor cells (CD34+ or CD133+ mononuclear cells) and endothelial progenitor cells (CD34+KDR+ or CD34+CD133+KDR+ mononuclear cells) were quantified by flow cytometry. Endothelial cell-colony forming units from peripheral blood mononuclear cells were quantified in vitro and phenotypic analysis carried out using immunocytochemistry. Patients with chronic obstructive pulmonary disease had more circulating mononuclear cells compared with controls (8.4 ± 0.6 vs. 5.9 ± 0.4 × 109 cells/l; P = 0.02). CD34+ hematopoietic progenitor cells were reduced as a proportion of mononuclear cells in patients compared with controls (0.99 ± 0.12 vs. 1.9 ± 0.12%; P = 0.02); however, there were no differences in the absolute number of CD34+, CD34+KDR+, or CD34+CD133+KDR+ cells ( P > 0.05 for all). Endothelial cell-colony forming units were increased in patients with chronic obstructive pulmonary disease compared with controls (13.7 ± 5.2 vs. 2.7 ± 0.9 colonies; P = 0.048). In contrast to previous studies, the number of circulating progenitor cells was not reduced in patients with chronic obstructive pulmonary disease compared with carefully matched controls. It seems unlikely that circulating endothelial progenitor cells or failure of angiogenesis plays a central role in the development of emphysema.

Rationale: Depletion of monocytes reduces LPS-induced lung inflammation in mice, suggesting monocytes as potential therapeutic targets in acute lung injury.Objectives: To investigate whether depletion of circulating blood monocytes has beneficial effects on markers of systemic and pulmonary inflammation in a human model of acute lung inflammation.Methods: A total of 30 healthy volunteers were enrolled in a randomized controlled trial. Volunteers inhaled LPS at baseline, and were randomized to receive active mononuclear cell depletion by leukapheresis, or sham leukapheresis, in a double-blind fashion (15 volunteers per group). Serial blood counts were measured, bronchoalveolar lavage (BAL) was performed at 9 hours, and [18F]fluorodeoxyglucose positron emission tomography at 24 hours. The primary endpoint was the increment in circulating neutrophils at 8 hours.Measurements and Main Results: As expected, inhalation of LPS induced neutrophilia and an up-regulation of inflammatory mediators in the blood and lu...

Background and Objectives There is increasing evidence that monocytes play a key role in the pathogenesis of acute lung inflammation. Mononuclear cell (MNC) leukapheresis can be used to remove large numbers of monocytes from circulating blood; however, the detailed characteristics of monocyte subpopulations removed by MNC leukapheresis, and the biological effects on the lung, remain incompletely defined. Material and Methods Six healthy male volunteers underwent MNC leukapheresis of four total blood volumes. Blood was collected at 0, 2, 4, 6, 8 and 24 h; bronchoscopy with bronchoalveolar lavage (BAL) was performed at 8–9 h. Multiparameter flow cytometry was used to identify subpopulations of monocytes in blood and monocyte-like cells in BAL fluid. Results A median of 5·57 × 109 monocytes were retrieved. Blood monocyte counts indicated that the circulating blood monocyte pool was actively replenished during leukapheresis and subsequently contained a greater proportion of classical (CD14++ CD16−) monocytes. A particular subpopulation of monocyte-like cells, reminiscent of classical monocytes, was also prominent in BAL fluid after leukapheresis. Conclusion Mononuclear cell leukapheresis was safe. The greater proportion of classical monocytes present in blood after MNC leukapheresis may be clinically significant. MNC leukapheresis also appears to affect the proportion of monocyte-like cells in the lung; however, we found no evidence that leukapheresis has a clinically important pro-inflammatory effect in the human lung.

The co-ordinated recruitment of monocyte subpopulations, neutrophils and regulatory T-cells (Tregs) during the early stages of human acute lung inflammation remains poorly understood. We therefore performed a detailed characterisation of these lineages in the blood and lungs in a model of human acute lung inflammation. Healthy volunteers inhaled lipopolysaccharide (LPS) or saline (n=6 for each group). Blood was collected at 0, 2, 4, 6 and 8 h and bronchoscopy with bronchoalveolar lavage (BAL) performed at 8 h. Multiparameter flow cytometry was used to characterise monocyte subpopulations, neutrophils and Tregs in the blood and lung. Inhalation of LPS was associated with significant blood and BAL fluid neutrophilia. Blood populations of monocyte subpopulations and Tregs were unaltered by LPS. In contrast, LPS induced an accumulation of a pulmonary monocyte-like cell (PMLC) population, which was further subdivided into "inducible" CD14(++)CD16(-) and "resident" CD14(++)CD16(+) subsets. Inducible PMLCs were significantly increased following LPS inhalation (p=0.0046), whereas resident PMLCs were unchanged. In addition, we noted a significant decrease in Tregs in BAL fluid with LPS inhalation (p=0.027). The early stages of LPS-induced inflammation in humans is characterised by pulmonary accumulation of a novel inducible monocyte-like subpopulation, accompanied by significant changes in both neutrophil and Treg numbers.

beta3-Integrin is a cell surface adhesion and signalling molecule important in the regulation of tumour angiogenesis. Mice with a global deficiency in beta3-integrin show increased pathological angiogenesis, most likely due to increased vascular endothelial growth factor receptor 2 expression on beta3-null endothelial cells. Here we transplanted beta3-null bone marrow (BM) into wild-type (WT) mice to dissect the role of BM beta3-integrin deficiency in pathological angiogenesis. Mice transplanted with beta3-null bone marrow show significantly enhanced angiogenesis in subcutaneous B16F0 melanoma and Lewis lung carcinoma (LLC) cell models and in B16F0 melanoma lung metastasis when compared with tumours grown in mice transplanted with WT bone marrow. The effect of bone marrow beta3-integrin deficiency was also assessed in the RIPTAg mouse model of pancreatic tumour growth. Again, angiogenesis in mice lacking BM beta3-integrin was enhanced. However, tumour weight between the groups was not significantly altered, suggesting that the enhanced blood vessel density in the mice transplanted with beta3-null bone marrow was not functional. Indeed, we demonstrate that in mice transplanted with beta3-null bone marrow a significant proportion of tumour blood vessels are non-functional when compared with tumour blood vessels in WT-transplanted controls. Furthermore, beta3-null-transplanted mice showed an increased angiogenic response to VEGF in vivo when compared with WT-transplanted animals. BM beta3-integrin deficiency affects the mobilization of progenitor cells to the peripheral circulation. We show that VEGF-induced mobilization of endothelial progenitor cells is enhanced in mice transplanted with beta3-null bone marrow when compared with WT-transplanted controls, suggesting a possible mechanism underlying the increased blood vessel density seen in beta3-null-transplanted mice. In conclusion, although BM beta3-integrin is not required for pathological angiogenesis, our studies demonstrate a role for BM beta3-integrin in VEGF-induced mobilization of bone marrow-derived cells to the peripheral circulation and for the functionality of those vessels in which BM-derived cells become incorporated.

There is a growing realization that bone marrow-derived cells (BMDCs) are a potential therapy for many diseases including ischemic heart disease, arterial stenosis and osteogenesis imperfecta. On the other hand, the fact that BMDCs may also contribute to fibrosis in many solid organs as well as to fibrosis surrounding tumours suggests that BMDCs are also involved in disease progression. This review focuses on the contribution of bone marrow cells to organ and tumour fibrosis, noting the utility of BMDCs as a potential new portal through which to direct anti-tumour therapies. Conversely, bone marrow cell therapy has been claimed to reduce fibrosis in some organs, highlighting a seemingly beneficial as opposed to a detrimental effect of BMDCs on organ fibrosis.

CD133 (prominin-1) was the first in a class of novel pentaspan membrane proteins to be identified in both humans and mice, and was originally classified as a marker of primitive haematopoietic and neural stem cells. Due to the highly restricted expression of CD133 family molecules on plasma membrane protrusions of epithelial and other cell types, in association with membrane cholesterol, a role in the organization of plasma membrane topology has also recently been assigned to this family. Studies have now confirmed the utility of CD133 as a marker of haematopoietic stem cells for human allogeneic transplantation. In addition, CD133 represents a marker of tumour-initiating cells in a number of human cancers, and therefore it may be possible to develop future therapies towards targeting cancer stem cells via this marker. The development of such therapies will be aided by a clearer understanding of the molecular mechanisms and signalling pathways that regulate the behaviour of CD133-expressing cells, and new data outlining the role of Wnt, Notch, and bone morphogenetic protein (BMP) signalling in CD133(+) cancer stem cell regulation are discussed within.

Despite the increasing importance of long noncoding RNA in physiology and disease, their role in endothelial biology remains poorly understood. Growing evidence has highlighted them to be essential regulators of human embryonic stem cell differentiation. SENCR, a vascular-enriched long noncoding RNA, overlaps the Friend Leukemia Integration virus 1 (FLI1) gene, a regulator of endothelial development. Therefore, we wanted to test the hypothesis that SENCR may contribute to mesodermal and endothelial commitment as well as in endothelial function. We thus developed new differentiation protocols allowing generation of endothelial cells from human embryonic stem cells using both directed and hemogenic routes. The expression of SENCR was markedly regulated during endothelial commitment using both protocols. SENCR did not control the pluripotency of pluripotent cells; however its overexpression significantly potentiated early mesodermal and endothelial commitment. In human umbilical endothelial cell (HUVEC), SENCR induced proliferation, migration, and angiogenesis. SENCR expression was altered in vascular tissue and cells derived from patients with critical limb ischemia and premature coronary artery disease compared to controls. Here, we showed that SENCR contributes to the regulation of endothelial differentiation from pluripotent cells and controls the angiogenic capacity of HUVEC. These data give novel insight into the regulatory processes involved in endothelial development and function.

The participation of circulating precursor cells in the development of experimental pulmonary fibrosing lesions in mice has been recently demonstrated.This study analyzes whether circulating, bone marrow-derived, fibroblastic precursor cells contribute to the development of fibrosing lesions in human lungs, especially bronchiolitis obliterans.The occurrence of in situ microchimerism in bronchiolitis obliterans lesions of human lung allografts (n = 12) as well as of autologous lung tissue from patients post-bone marrow transplantation (n = 2) was analyzed using laser-assisted microdissection after immunohistochemical labeling of leukocytes followed by short tandem repeat-polymerase chain reaction-based genotyping. Combined immunofluorescence and fluorescence in situ hybridization for sex chromosomes was performed for independent confirmation in cases with appropriate sex mismatch (n = 2).The bronchiolitis obliterans lesions of all 12 lung transplant patients contained considerable numbers of recipient-derived fibroblasts (mean, 32%). The fibrosing pulmonary lesions of the two bone marrow-transplanted patients also displayed clear in situ microchimerism. The in situ detection methodology confirmed these results, although to a lower degree (6-16%).These data clearly demonstrate the involvement of circulating fibroblastic precursor cells in the development of human fibrosing lung lesions and provide evidence that these cells are most probably bone marrow derived. These results may open new venues regarding the prevention of fibrosis in lung transplants and potentially in other organs.

The authors have previously reported the derivation of colonic subepithelial myofibroblasts (SEMFs) in both humans and mice from bone marrow (BM). In the pathogenesis of inflammatory bowel disease (IBD), such as Crohn's disease and ulcerative colitis, colonic SEMFs mediate several types of inflammatory response. In the present study, interleukin (IL)-10−/− mice were used as a model of IBD to investigate the involvement of BM-derived cells in the inflamed mucosa. Male whole BM [either C57/BL10 (wild type: WT) or IL-10−/− donor mice] was used to perform bone marrow transplantation (BMT) into both WT and IL-10−/− female mice. Tissue samples were evaluated by immunohistochemistry for α-smooth muscle actin expression and by in situ hybridization using a Y-chromosome-specific probe to track the donor-derived colonic SEMFs. The mucosal expression of mRNA for pro-inflammatory cytokines was analysed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, mRNA expression of matrix metalloproteinase (MMP)-7 and osteopontin in the inflamed mucosa was assessed using in situ hybridization. Body weights and histological scores showed that IL-10−/− mice that received WT BM had an improved course of colitis, decreased mucosal pro-inflammatory mRNA expression, and up to 30% of their SEMFs were of BM origin. Conversely, IL-10−/− mice receiving IL-10−/− BM progressed to extensive colitis, and Y probe analysis revealed that up to 45% of colonic SEMFs were of BM origin. WT mice receiving IL-10−/− or WT BM had no signs of colonic inflammation. The expression of MMP-7 and osteopontin was up-regulated in the inflamed mucosa. In conclusion, IL-10−/− mice displayed ameliorated disease activity after WT BMT, whilst colitis was not induced in WT mice by IL-10−/− BMT. The contribution of BM-derived cells to colonic SEMFs was significantly increased in the inflamed mucosa compared with non-inflamed mucosa. Copyright © 2006 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Stem cells are primitive cells located in a specialised mesenchymal “niche” that lack expression of any definitive markers of lineage commitment and are therefore difficult to define and identify. Stem cells maintain their capacity for limitless self replication throughout the lifetime of their host, and can also divide to produce daughter cells, committed to the formation of every adult cell lineage within their tissue of origin. The stem cells of the gastrointestinal tract remain unidentified which has led to many conflicting hypotheses as to their precise nature and function. For example, the numbers and location of stem cells in the intestinal crypts and gastric glands have never been conclusively proven and, consequently, the clonal origins of these structures under normal circumstances and in neoplasia are clouded issues. The morphological events of gastrointestinal carcinoma formation are hotly debated, with two main conflicting hypotheses of the mechanisms of expansion of a mutated stem cell clone. However, with the emergence of the molecular pathways governing gastrointestinal stem cell function, and the identification of putative intestinal molecular stem cell markers, such as Musashi-1, comes a clearer insight into the properties of the gastrointestinal stem cell. Adult stem cells from several tissues can leave their niche and engraft into extraneous tissues, including the gastrointestinal mucosa and underlying mesenchyme, and transform to produce adult cell lineages common to these foreign environments. This process is optimal when the requirement for regeneration is enhanced (that is, in diseased or damaged tissue) and indeed, the contribution of transplanted bone marrow stem cells to intestinal myofibroblasts is significantly upregulated in colitis. However, adult stem cell plasticity has recently been disparaged by reports suggesting that stem cells spontaneously fuse with indigenous adult cells to form a diploid cell with an aberrant karyotype, and it is important to investigate if bone marrow …

Myofibroblasts are ubiquitous cells with features of both fibroblasts and smooth muscle cells. We suggest that the bone marrow can contribute to myofibroblast populations in a variety of tissues and that this is exacerbated by injury. To assess this, female mice were transplanted with male bone marrow and the male cells were tracked throughout the body and identified as myofibroblasts. Skin wounding and paracetamol administration were used to assess whether myofibroblast engraftment was modulated by damage. Following radiation injury, a proportion of myofibroblasts in the lung, stomach, esophagus, skin, kidney, and adrenal capsule were bone-marrow derived. In the lung, there was significantly greater engraftment following paracetamol administration (17% versus 41% p < 0.005). Bone-marrow-derived fibroblasts were also found. We suggest that bone marrow contributes to a circulating population of cells and, in the context of injury, these cells are recruited and contribute to tissue repair.

Objectives The primary aim of this prospective study was to perform a comprehensive serial characterisation of monocyte and neutrophil function, circulating monocyte subsets, and bronchoalveolar lavage (BAL) fluid after lung resection. A secondary aim was to perform a pilot, hypothesis-generating evaluation of whether innate immune parameters were associated with postoperative pneumonia. Methods Forty patients undergoing lung resection were studied in detail. Blood monocytes and neutrophils were isolated preoperatively and at 6, 24 and 48 h postoperatively. BAL was performed preoperatively and immediately postoperatively. Monocyte subsets, monocyte responsiveness to lipopolysaccharide (LPS) and neutrophil phagocytic capacity were quantified at all time points. Differential cell count, protein and cytokine concentrations were measured in BAL. Pneumonia evaluation at 72 h was assessed using predefined criteria. Results After surgery, circulating subsets of classical and intermediate monocytes increased significantly. LPS-induced release of proinflammatory cytokines from monocytes increased significantly and by 48 h a more proinflammatory profile was found. Neutrophil phagocytosis demonstrated a small but significant fall. Factors associated with postoperative pneumonia were: increased release of specific proinflammatory and anti-inflammatory cytokines from monocytes; preoperative neutrophilia; and preoperative BAL cell count. Conclusions We conclude that postoperative lung inflammation is associated with specific changes in the cellular innate immune response, a better understanding of which may improve patient selection and prediction of complications in the future.

Turnover of the epithelial cell lineages within the gastrointestinal tract is a constant process, occurring every two to seven days under normal homeostasis and at increased rates after damage. This process is regulated by multipotent stem cells, which generate all gastrointestinal epithelial cell lineages and can regenerate whole intestinal crypts and gastric glands. The stem cells of the gastrointestinal tract are as yet undefined, although it is generally agreed that they are located within a ’niche’ in the intestinal crypts and gastric glands. Studies of allophenic, tetraparental chimeric mice and targeted stem cell mutations suggest that a single stem cell undergoes an asymmetrical division to produce an identical daughter cell, thus replicating itself, and a committed progenitor cell, which further differentiates into an adult epithelial cell type. The discovery of stem cell plasticity in many tissues, including the ability of transplanted bone marrow to transdifferentiate into intestinal subepithelial myofibroblasts, provides a potential use of bone marrow cells to deliver therapeutic genes to damaged tissues, for example, in the treatment of mesenchymal diseases in the gastrointestinal tract, such as fibrosis and Crohn’s disease. Studies are beginning to identify the molecular pathways that regulate stem cell proliferation and differentiation into adult gastrointestinal cell lineages, such as the Wnt and Notch–Delta signaling pathways, and to discover the importance of mesenchymal–epithelial interactions in normal gastrointestinal epithelium and in development and disease. Finally, despite some dispute, a strong case can be made for intestinal neoplasia arising as a result of a series of mutations in stem cells. The mechanism and direction of the spread of this mutated clone in the gastrointestinal mucosa is hotly disputed, and central to this argument is the position and nature of the gastrointestinal stem cell.

Expression of the cell adhesion molecule E-cadherin in the developing mouse has been investigated by immunocytochemistry and disaggregated organ culture. The principal aims were firstly, to determine whether E-cadherin is expressed in the indifferent gonad and if so with which cell population(s) it is associated. Secondly, to investigate the pattern of expression in the mesonephros, especially in relation to ventral mesonephric tubule cells, which contribute to the somatic cell population of the gonad. Thirdly, to discover whether there are any sex differences in expression of E-cadherin in differentiating gonads. Germ cells in the indifferent gonad showed strong immunoreactivity whereas somatic cells were unstained unless their membranes were in contact with those of germ cells. Positive staining for E-cadherin was found in epithelial cells of the mesonephric duct and tubules. Staining was weak at the ventral margins of the ventral mesonephric tubules. At later stages, germ cell immunoreactivity could be correlated with stages of ovarian differentiation, being reduced or absent between germ cells at 16 days post coitum, when ovigerous cords become dissociated as a prelude to follicle formation. Stronger staining reappeared briefly at 17 days post coitum, the time of follicular cell attachment to oocytes, before waning again 2 days later. Similarly, immunoreactivity in the differentiating testis was initially restricted to the germ cell population but pre-Sertoli cells were strongly positive between 16 and 19 days post coitum. The most striking sex difference was seen in the somatic cell population, where Leydig cells of the testis became strongly positive for E-cadherin from 17 days post coitum onwards. At this time, unlike controls, dissociated cells from gonads of either sex were unable to reform their initial contacts when cultured in the presence of the antibody to E-cadherin, confirming its functional importance.