The βI domain promotes active β1 integrin clustering into mature adhesion sites

Modulation of integrin function is required in many physiological and pathological settings, such as angiogenesis and cancer. Integrin allosteric changes, clustering, and trafficking cooperate to regulate cell adhesion and motility on extracellular matrix proteins via mechanisms that are partly defined. By exploiting four monoclonal antibodies recognizing distinct conformational epitopes, we show that in endothelial cells (ECs), the extracellular βI domain, but not the hybrid or I-EGF2 domain of active β1 integrins, promotes their FAK-regulated clustering into tensin 1–containing fibrillar adhesions and impairs their endocytosis. In this regard, the βI domain–dependent clustering of active β1 integrins is necessary to favor fibronectin-elicited directional EC motility, which cannot be effectively promoted by β1 integrin conformational activation alone

Proteomic analysis of integrin-associated complexes from mesenchymal stem cells

PURPOSE: Multipotent mesenchymal stem cells (MSCs) have the capability to differentiate down adipocyte, osteocyte and chondrocyte lineages and as such offer a range of potential therapeutic applications. The composition and stiffness of the extracellular matrix (ECM) environment that surrounds cells dictates their transcriptional programme, thereby affecting stem cell lineage decision‐making. Cells sense force via linkages between themselves and their microenvironment, and this is transmitted by integrin receptors and associated adhesion signalling complexes. To identify regulators of MSC force sensing, we sought to catalogue MSC integrin‐associated adhesion complex composition. EXPERIMENTAL DESIGN: Adhesion complexes formed by MSCs plated on the ECM ligand fibronectin were isolated and characterised by MS. Identified proteins were interrogated by comparison to a literature‐based reference set of cell adhesion‐related components and using ontological and protein–protein interaction network analyses. RESULTS: Adhesion complex‐specific proteins in MSCs were identified that comprised predominantly cell adhesion‐related adaptors and actin cytoskeleton regulators. Furthermore, LIM domain‐containing proteins in MSC adhesion complexes were highlighted, which may act as force‐sensing components. CONCLUSION AND CLINICAL RELEVANCE: These data provide a valuable resource of information regarding the molecular connections that link integrins and adhesion signalling in MSCs, and as such may present novel opportunities for therapeutic intervention

Pancreatic ductal adenocarcinoma cells employ integrin α6β4 to form hemidesmosomes and regulate cell proliferation

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis due to its aggressive progression, late detection and lack of druggable driver mutations, which often combine to result in unsuitability for surgical intervention. Together with activating mutations of the small GTPase KRas, which are found in over 90% of PDAC tumours, a contributory factor for PDAC tumour progression is formation of a rigid extracellular matrix (ECM) and associated desmoplasia. This response leads to aberrant integrin signalling, and accelerated proliferation and invasion. To identify the integrin adhesion systems that operate in PDAC, we analysed a range of pancreatic ductal epithelial cell models using 2D, 3D and organoid culture systems. Proteomic analysis of isolated integrin receptor complexes from human pancreatic ductal epithelial (HPDE) cells predominantly identified integrin α6β4 and hemidesmosome components, rather than classical focal adhesion components. Electron microscopy, together with immunofluorescence, confirmed the formation of hemidesmosomes by HPDE cells, both in 2D and 3D culture systems. Similar results were obtained for the human PDAC cell line, SUIT-2. Analysis of HPDE cell secreted proteins and cell-derived matrices (CDM) demonstrated that HPDE cells secrete a range of laminin subunits and form a hemidesmosome-specific, laminin 332-enriched ECM. Expression of mutant KRas (G12V) did not affect hemidesmosome composition or formation by HPDE cells. Cell-ECM contacts formed by mouse and human PDAC organoids were also assessed by electron microscopy. Organoids generated from both the PDAC KPC mouse model and human patient-derived PDAC tissue displayed features of acinar-ductal cell polarity, and hemidesmosomes were visible proximal to prominent basement membranes. Furthermore, electron microscopy identified hemidesmosomes in normal human pancreas. Depletion of integrin β4 reduced cell proliferation in both SUIT-2 and HPDE cells, reduced the number of SUIT-2 cells in S-phase, and induced G1 cell cycle arrest, suggesting a requirement for α6β4-mediated adhesion for cell cycle progression and growth. Taken together, these data suggest that laminin-binding adhesion mechanisms in general, and hemidesmosome-mediated adhesion in particular, may be under-appreciated in the context of PDAC. Proteomic data are available via ProteomeXchange with the identifiers PXD027803, PXD027823 and PXD027827

Case Reports1. A Late Presentation of Loeys-Dietz Syndrome: Beware of TGFβ Receptor Mutations in Benign Joint Hypermobility

Background: Thoracic aortic aneurysms (TAA) and dissections are not uncommon causes of sudden death in young adults. Loeys-Dietz syndrome (LDS) is a rare, recently described, autosomal dominant, connective tissue disease characterized by aggressive arterial aneurysms, resulting from mutations in the transforming growth factor beta (TGFβ) receptor genes TGFBR1 and TGFBR2. Mean age at death is 26.1 years, most often due to aortic dissection. We report an unusually late presentation of LDS, diagnosed following elective surgery in a female with a long history of joint hypermobility. Methods: A 51-year-old Caucasian lady complained of chest pain and headache following a dural leak from spinal anaesthesia for an elective ankle arthroscopy. CT scan and echocardiography demonstrated a dilated aortic root and significant aortic regurgitation. MRA demonstrated aortic tortuosity, an infrarenal aortic aneurysm and aneurysms in the left renal and right internal mammary arteries. She underwent aortic root repair and aortic valve replacement. She had a background of long-standing joint pains secondary to hypermobility, easy bruising, unusual fracture susceptibility and mild bronchiectasis. She had one healthy child age 32, after which she suffered a uterine prolapse. Examination revealed mild Marfanoid features. Uvula, skin and ophthalmological examination was normal. Results: Fibrillin-1 testing for Marfan syndrome (MFS) was negative. Detection of a c.1270G > C (p.Gly424Arg) TGFBR2 mutation confirmed the diagnosis of LDS. Losartan was started for vascular protection. Conclusions: LDS is a severe inherited vasculopathy that usually presents in childhood. It is characterized by aortic root dilatation and ascending aneurysms. There is a higher risk of aortic dissection compared with MFS. Clinical features overlap with MFS and Ehlers Danlos syndrome Type IV, but differentiating dysmorphogenic features include ocular hypertelorism, bifid uvula and cleft palate. Echocardiography and MRA or CT scanning from head to pelvis is recommended to establish the extent of vascular involvement. Management involves early surgical intervention, including early valve-sparing aortic root replacement, genetic counselling and close monitoring in pregnancy. Despite being caused by loss of function mutations in either TGFβ receptor, paradoxical activation of TGFβ signalling is seen, suggesting that TGFβ antagonism may confer disease modifying effects similar to those observed in MFS. TGFβ antagonism can be achieved with angiotensin antagonists, such as Losartan, which is able to delay aortic aneurysm development in preclinical models and in patients with MFS. Our case emphasizes the importance of timely recognition of vasculopathy syndromes in patients with hypermobility and the need for early surgical intervention. It also highlights their heterogeneity and the potential for late presentation. Disclosures: The authors have declared no conflicts of interes

Global burden and strength of evidence for 88 risk factors in 204 countries and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Understanding the health consequences associated with exposure to risk factors is necessary to inform public health policy and practice. To systematically quantify the contributions of risk factor exposures to specific health outcomes, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 aims to provide comprehensive estimates of exposure levels, relative health risks, and attributable burden of disease for 88 risk factors in 204 countries and territories and 811 subnational locations, from 1990 to 2021. Methods: The GBD 2021 risk factor analysis used data from 54 561 total distinct sources to produce epidemiological estimates for 88 risk factors and their associated health outcomes for a total of 631 risk–outcome pairs. Pairs were included on the basis of data-driven determination of a risk–outcome association. Age-sex-location-year-specific estimates were generated at global, regional, and national levels. Our approach followed the comparative risk assessment framework predicated on a causal web of hierarchically organised, potentially combinative, modifiable risks. Relative risks (RRs) of a given outcome occurring as a function of risk factor exposure were estimated separately for each risk–outcome pair, and summary exposure values (SEVs), representing risk-weighted exposure prevalence, and theoretical minimum risk exposure levels (TMRELs) were estimated for each risk factor. These estimates were used to calculate the population attributable fraction (PAF; ie, the proportional change in health risk that would occur if exposure to a risk factor were reduced to the TMREL). The product of PAFs and disease burden associated with a given outcome, measured in disability-adjusted life-years (DALYs), yielded measures of attributable burden (ie, the proportion of total disease burden attributable to a particular risk factor or combination of risk factors). Adjustments for mediation were applied to account for relationships involving risk factors that act indirectly on outcomes via intermediate risks. Attributable burden estimates were stratified by Socio-demographic Index (SDI) quintile and presented as counts, age-standardised rates, and rankings. To complement estimates of RR and attributable burden, newly developed burden of proof risk function (BPRF) methods were applied to yield supplementary, conservative interpretations of risk–outcome associations based on the consistency of underlying evidence, accounting for unexplained heterogeneity between input data from different studies. Estimates reported represent the mean value across 500 draws from the estimate's distribution, with 95% uncertainty intervals (UIs) calculated as the 2·5th and 97·5th percentile values across the draws. Findings: Among the specific risk factors analysed for this study, particulate matter air pollution was the leading contributor to the global disease burden in 2021, contributing 8·0% (95% UI 6·7–9·4) of total DALYs, followed by high systolic blood pressure (SBP; 7·8% [6·4–9·2]), smoking (5·7% [4·7–6·8]), low birthweight and short gestation (5·6% [4·8–6·3]), and high fasting plasma glucose (FPG; 5·4% [4·8–6·0]). For younger demographics (ie, those aged 0–4 years and 5–14 years), risks such as low birthweight and short gestation and unsafe water, sanitation, and handwashing (WaSH) were among the leading risk factors, while for older age groups, metabolic risks such as high SBP, high body-mass index (BMI), high FPG, and high LDL cholesterol had a greater impact. From 2000 to 2021, there was an observable shift in global health challenges, marked by a decline in the number of all-age DALYs broadly attributable to behavioural risks (decrease of 20·7% [13·9–27·7]) and environmental and occupational risks (decrease of 22·0% [15·5–28·8]), coupled with a 49·4% (42·3–56·9) increase in DALYs attributable to metabolic risks, all reflecting ageing populations and changing lifestyles on a global scale. Age-standardised global DALY rates attributable to high BMI and high FPG rose considerably (15·7% [9·9–21·7] for high BMI and 7·9% [3·3–12·9] for high FPG) over this period, with exposure to these risks increasing annually at rates of 1·8% (1·6–1·9) for high BMI and 1·3% (1·1–1·5) for high FPG. By contrast, the global risk-attributable burden and exposure to many other risk factors declined, notably for risks such as child growth failure and unsafe water source, with age-standardised attributable DALYs decreasing by 71·5% (64·4–78·8) for child growth failure and 66·3% (60·2–72·0) for unsafe water source. We separated risk factors into three groups according to trajectory over time: those with a decreasing attributable burden, due largely to declining risk exposure (eg, diet high in trans-fat and household air pollution) but also to proportionally smaller child and youth populations (eg, child and maternal malnutrition); those for which the burden increased moderately in spite of declining risk exposure, due largely to population ageing (eg, smoking); and those for which the burden increased considerably due to both increasing risk exposure and population ageing (eg, ambient particulate matter air pollution, high BMI, high FPG, and high SBP). Interpretation: Substantial progress has been made in reducing the global disease burden attributable to a range of risk factors, particularly those related to maternal and child health, WaSH, and household air pollution. Maintaining efforts to minimise the impact of these risk factors, especially in low SDI locations, is necessary to sustain progress. Successes in moderating the smoking-related burden by reducing risk exposure highlight the need to advance policies that reduce exposure to other leading risk factors such as ambient particulate matter air pollution and high SBP. Troubling increases in high FPG, high BMI, and other risk factors related to obesity and metabolic syndrome indicate an urgent need to identify and implement interventions

Global incidence, prevalence, years lived with disability (YLDs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries in 204 countries and territories and 811 subnational locations, 1990–2021: a systematic analysis for the Global Burden of Disease Study 2021

Background: Detailed, comprehensive, and timely reporting on population health by underlying causes of disability and premature death is crucial to understanding and responding to complex patterns of disease and injury burden over time and across age groups, sexes, and locations. The availability of disease burden estimates can promote evidence-based interventions that enable public health researchers, policy makers, and other professionals to implement strategies that can mitigate diseases. It can also facilitate more rigorous monitoring of progress towards national and international health targets, such as the Sustainable Development Goals. For three decades, the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) has filled that need. A global network of collaborators contributed to the production of GBD 2021 by providing, reviewing, and analysing all available data. GBD estimates are updated routinely with additional data and refined analytical methods. GBD 2021 presents, for the first time, estimates of health loss due to the COVID-19 pandemic. Methods: The GBD 2021 disease and injury burden analysis estimated years lived with disability (YLDs), years of life lost (YLLs), disability-adjusted life-years (DALYs), and healthy life expectancy (HALE) for 371 diseases and injuries using 100 983 data sources. Data were extracted from vital registration systems, verbal autopsies, censuses, household surveys, disease-specific registries, health service contact data, and other sources. YLDs were calculated by multiplying cause-age-sex-location-year-specific prevalence of sequelae by their respective disability weights, for each disease and injury. YLLs were calculated by multiplying cause-age-sex-location-year-specific deaths by the standard life expectancy at the age that death occurred. DALYs were calculated by summing YLDs and YLLs. HALE estimates were produced using YLDs per capita and age-specific mortality rates by location, age, sex, year, and cause. 95% uncertainty intervals (UIs) were generated for all final estimates as the 2·5th and 97·5th percentiles values of 500 draws. Uncertainty was propagated at each step of the estimation process. Counts and age-standardised rates were calculated globally, for seven super-regions, 21 regions, 204 countries and territories (including 21 countries with subnational locations), and 811 subnational locations, from 1990 to 2021. Here we report data for 2010 to 2021 to highlight trends in disease burden over the past decade and through the first 2 years of the COVID-19 pandemic. Findings: Global DALYs increased from 2·63 billion (95% UI 2·44–2·85) in 2010 to 2·88 billion (2·64–3·15) in 2021 for all causes combined. Much of this increase in the number of DALYs was due to population growth and ageing, as indicated by a decrease in global age-standardised all-cause DALY rates of 14·2% (95% UI 10·7–17·3) between 2010 and 2019. Notably, however, this decrease in rates reversed during the first 2 years of the COVID-19 pandemic, with increases in global age-standardised all-cause DALY rates since 2019 of 4·1% (1·8–6·3) in 2020 and 7·2% (4·7–10·0) in 2021. In 2021, COVID-19 was the leading cause of DALYs globally (212·0 million [198·0–234·5] DALYs), followed by ischaemic heart disease (188·3 million [176·7–198·3]), neonatal disorders (186·3 million [162·3–214·9]), and stroke (160·4 million [148·0–171·7]). However, notable health gains were seen among other leading communicable, maternal, neonatal, and nutritional (CMNN) diseases. Globally between 2010 and 2021, the age-standardised DALY rates for HIV/AIDS decreased by 47·8% (43·3–51·7) and for diarrhoeal diseases decreased by 47·0% (39·9–52·9). Non-communicable diseases contributed 1·73 billion (95% UI 1·54–1·94) DALYs in 2021, with a decrease in age-standardised DALY rates since 2010 of 6·4% (95% UI 3·5–9·5). Between 2010 and 2021, among the 25 leading Level 3 causes, age-standardised DALY rates increased most substantially for anxiety disorders (16·7% [14·0–19·8]), depressive disorders (16·4% [11·9–21·3]), and diabetes (14·0% [10·0–17·4]). Age-standardised DALY rates due to injuries decreased globally by 24·0% (20·7–27·2) between 2010 and 2021, although improvements were not uniform across locations, ages, and sexes. Globally, HALE at birth improved slightly, from 61·3 years (58·6–63·6) in 2010 to 62·2 years (59·4–64·7) in 2021. However, despite this overall increase, HALE decreased by 2·2% (1·6–2·9) between 2019 and 2021. Interpretation: Putting the COVID-19 pandemic in the context of a mutually exclusive and collectively exhaustive list of causes of health loss is crucial to understanding its impact and ensuring that health funding and policy address needs at both local and global levels through cost-effective and evidence-based interventions. A global epidemiological transition remains underway. Our findings suggest that prioritising non-communicable disease prevention and treatment policies, as well as strengthening health systems, continues to be crucially important. The progress on reducing the burden of CMNN diseases must not stall; although global trends are improving, the burden of CMNN diseases remains unacceptably high. Evidence-based interventions will help save the lives of young children and mothers and improve the overall health and economic conditions of societies across the world. Governments and multilateral organisations should prioritise pandemic preparedness planning alongside efforts to reduce the burden of diseases and injuries that will strain resources in the coming decades. Funding: Bill & Melinda Gates Foundation

Definition of a consensus integrin adhesome and its dynamics during adhesion complex assembly and disassembly

Integrin receptor activation initiates the formation of integrin adhesion complexes (IACs) at the cell membrane that transduce adhesion-dependent signals to control a multitude of cellular functions. Proteomic analyses of isolated IACs have revealed an unanticipated molecular complexity; however, a global view of the consensus composition and dynamics of IACs is currently lacking. Here, we have integrated several IAC proteomes and generated a 2,412-protein integrin adhesome. Analysis of this dataset reveals the functional diversity of proteins in IACs and establishes a consensus adhesome of 60 proteins. The consensus adhesome likely represents a core cell adhesion machinery, centred around four axes comprising ILK-PINCH-kindlin, FAK-paxillin, talin-vinculin and α-actinin-zyxin-VASP, and includes underappreciated IAC components such as Rsu-1 and caldesmon. Proteomic quantification of IAC assembly and disassembly detailed the compositional dynamics of the core cell adhesion machinery. The definition of this consensus view of integrin adhesome components provides a resource for the research community

Linking integrin conformation to function

Integrins are αβ heterodimeric adhesion receptors that relay signals bidirectionally across the plasma membrane between the extracellular matrix and cell-surface ligands, and cytoskeletal and signalling effectors. The physical and chemical signals that are controlled by integrins are essential for intercellular communication and underpin all aspects of metazoan existence. To mediate such diverse functions, integrins exhibit structural diversity, flexibility and dynamism. Conformational changes, as opposed to surface expression or clustering, are central to the regulation of receptor function. In recent years, there has been intense interest in determining the three-dimensional structure of integrins, and analysing the shape changes that underpin the interconversion between functional states. Considering the central importance of the integrin signalling nexus, it is perhaps no surprise that obtaining this information has been difficult, and the answers gained so far have been complicated. In this Commentary, we pose some of the key remaining questions that surround integrin structure-function relationships and review the evidence that supports the current models