JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.Osteoporosis and cardiovascular (CV) disease share common risk factors and pathophysiology. Low bone mineral density (BMD) and fractures appear to increase the risk for multiple CV diseases. Equally, prevalent CV disease appears to predispose to bone loss and increase fracture rates. This relationship has naturally provoked the hypothesis that stopping bone loss may result in some CV benefit. Secondary analyses of safety and adverse event data from many randomized controlled trials (RCTs) have attempted to clarify this putative association. Recently, the discontinuation of odanacatib  over stroke concerns and the imbalance in ischemic events in romosozumab‐treated  women compared to bisphosphonate‐treated women, has provided further justification to better characterize potential CV benefits and harms of osteoporosis medications. This review delves into the seminal, and other major RCTs of osteoporosis medications and, using both published data and additional information provided on trial registration pages, examines the evidence for CV safety and harms of these medications. Accepted and emerging “off‐target” effects are explored for validity, biological plausibility, and clinical importance. A brief research agenda is provided to stimulate the next wave of clinical development and CV understanding of osteoporosis medications. © 2021 The Authors.  Osteoporosis is a systemic skeletal disorder characterized by the loss of bone mass and deterioration in bone microarchitecture. These microstructural alterations combine to compromise bone strength leading to skeletal fragility and increased susceptibility of fracture.1 Recent health economic literature suggests that fractures pose a higher disease and cost burden than most cancers (except lung cancer) but do not attract as much public attention or institutional funding.Fractures are a devastating outcome. Recovery from a fracture is costly, takes many months and indeed some never truly recover and lose independence. Indeed, mortality and the development of comorbid disease, greatly increases after most types of fractures but particularly hip fractures.2 Rece Certainly, on a biological level, there appears to be bidirectionality between bone and vascular disease. Mechanisms underlying common aging, such as chronic inflammation as well as the decline in renal function predisposing to chronic kidney disease  are all thought to interact in the eventual co‐manifestation of osteoporosis and CV disease  disease may develop subsequently to type 2 diabetes mellitus. The association between CV disease and osteoporosis is of profound importance because CV disease is overrepresented as a cause of death in patients who have fractured; and fractures are common in patients with known CV disease. Atherosclerosis shares many risk factors with osteoporosis and is hypothesized to establish in the decades of life when the net flux of available calcium becomes deranged; “switching” from skeleton deposition to renal filtration and deposition in vessel walls and other soft tissues.4 Certse Table . Understanding what treatments can accomplish, in the widest possible sense, may impact the overall cost utility landscape of such medications rather than a narrow focus on bone health alone.Given this context, there is a natural case suggesting that perhaps improvements in skeletal health and fracture reduction may lead to nonskeletal benefit, particularly where CV diseases are concerned. Indeed, greater appreciation of the need for CV disease management alongside fracture risk is warranted across clinical specialties in order to do our best to improve the overall outcomes experienced by patients. By understanding these nonskeletal benefits and harms, we are doing our due diligence by the patient because treatment uptake is affected markedly by perceived risks of harmful albeit extremely rare side effects; most recently regarding prescription of bisphosphonates (BPs).12 Und This observation is not limited to women; data from the United States show that in men (and women), CV disease is exhibited by those who experience bone loss. We have also begun to appreciate the vascular‐bone relationship, of how vascular disease can lead to poorer skeletal outcome. In older women, atherosclerotic vascular disease (ASVD) including vascular calcification is highly prevalent and is associated with a higher risk of fractures. Though there is little awareness of this in daily clinical practice, patients with underlying stroke or ischemic heart disease go on to experience near double the number of fractures compared to those who are disease‐free.The scientific associations between the skeleton and heart and vessels are strong but has not been translated into a clinical message that bone protection may offer CV protection. For example, in women who experience bone loss over time, increases in systolic blood pressure were greatest in those who experienced the greater reduction in bone density.13 Thi Furthermore, another monoclonal antibody, against sclerostin, romosozumab, has been approved by the US Food and Drug Administration (FDA) with a black‐box warning for potential increases in stroke and myocardial infarction owing to an imbalance in adjudicated CV events in a large active‐comparator trial. In other words, both the patients and the prescriber will need to agree that any short‐term increase in cardiovascular morbidity is more than offset by long‐term prevention of serious fractures. This renewed interest in “off‐target” effects, particularly CV effects, of antifracture medications thus warrants further exploration. This concept is already applied in the diabetes setting where new glucose‐lowering medications must also have satisfactory results for both safety and efficacy in a CV outcome trial.Finally, there is a regulatory rationale to understand the CV effects of osteoporosis medications. The latest drugs off the pipeline have been subjected to intense CV scrutiny, which is of course appropriate given their prospective widespread use in an older population. Indeed, the cathepsin K monoclonal antibody, odanacatib, was discontinued by the sponsor (Merck) owing to concerns over increases rates of stroke compared to placebo 17 Fur This review is also aimed at the prescribing clinician with the additional intention to provide a foundation from which conversations about medication safety can be had with patients. The CV safety of antifracture medications needs to be explored in the context of patients with osteoporosis/low BMD having a high underlying vascular disease burden and so CV outcomes of osteoporosis RCTs may be confounded by indication. Furthermore, this review is also aimed at the interested scientist, as we explore knowledge gaps in the bone‐CV field.The purpose of this review is to provide a comprehensive overview of the antifracture medication prescribing landscape and examine the evidence for CV safety and harms of these medications. This review focuses on recent larger randomized controlled trials (RCTs) with reference to key historical and seminal trials in the development of some medications. Particular attention will be paid to studies conducted in postmenopausal women and older men with osteoporosis/low BMD and to nonhormonal treatments with proven antifracture efficacy. Further, this review will focus on the meaningful collective outcome of the disease processes leading to CV events as properly examining mechanistic pathways such as vascular calcification is challenging in the setting of large RCTs. A previous review on this topic was framed as practical guide for the new clinician.19 Thi Their main effects appear to be on inhibiting resorption by being taken up by osteoclasts and promoting their apoptosis, thereby tipping the remodeling balance in favor of greater deposition to resorption. The idea that BPs could have some potential CV effects stemmed from observations that etidronate  could delay the calcification process in the vasculature and outside the skeleton. Later generation, nitrogen‐containing BPs including oral alendronate, risedronate, and intravenous (iv) zoledronic acid have been shown to inhibit the farnesyl pyrophosphate synthase pathway in osteoclasts, which is a downstream step from the actions of hydroxymethylglutaryl–coenzyme A (HMG‐CoA) reductase inhibitors (statins) in the production of mevalonate in the cholesterol synthesis pathway. Given the robust literature surrounding the cardioprotective and mortality benefits of inhibiting this pathway, BPs too have been studied in this respect with mixed effects.BPs are pyrophosphate analogues that have high affinity for hydroxyapatite crystals in bone with a half‐life of several years. BPs have a long history, with the first clinical use noted in 1969.20 The In a meta‐analysis of 21 trials of BPs  versus placebo , BP treatment was associated with a nonsignificant 5% reduced relative risk (RR) of overall mortality . This finding was replicated in sensitivity analysis of zoledronic acid although with a larger effect size of 22% . Analysis of alendronate‐only studies  demonstrated a null effect . Interestingly, analysis of nitrogen‐containing BPs  trended toward significance . Furthermore, excluding zoledronic acid from analysis of nitrogen‐containing BPs marginally altered the effect estimate  suggestive of an oral BP–specific effect. These rates and effect estimates are mentioned in detail as the moderate to large effect sizes and upper limits of the confidence bounds  suggests that a clinically meaningful reduction in mortality cannot be excluded given the weight of evidence and results from observational cohorts. The meta‐analysis did not perform population specific sensitivity analyses  and thus these data indicate there may yet exist a certain population with osteoporosis who may experience a mortality benefit despite not necessarily being prescribed for this effect. Given the high CV burden in patients with osteoporosis, they may be interested in understanding these potential off‐target effects despite clinical suspicion that they are “too good to be true”. Finally, there appeared to be no mortality benefit for BPs in trials longer than 3 years, which was an interesting concept given the perceived risk reduction seen in the Active‐Controlled Fracture Study in Postmenopausal Women With Osteoporosis at High Risk (ARCH) trial occurred in the first 12 months and risk reductions appeared evident at approximately the 16‐month mark of the Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly–Recurrent Fracture Trial (HORIZON‐RFT) trial by Lyles and colleagues. This would point to some transient effect that could potentially be related to medication adherence  or represent a true, underappreciated clinical effect mediated through the skeleton. There is a complete paucity of head‐to‐head studies examining this and there is limited data exploring these effects in other agents to elucidate if any potential mortality benefit is mediated through skeletal effects.First, BPs appear to offer no mortality benefit as a class in women and men.24 In  This trial followed on from the clinical development of alendronate as an antifracture agent a decade earlier. The aim of the HORIZON study was to investigate if the more potent zoledronic acid had similar effects to oral alendronate. The intravenous nature of zoledronic acid was appealing to patients because it was required only once yearly rather than daily or weekly as per the oral formulation, which caused gastric problems. HORIZON enrolled over 7700 women aged between 65 and 89 years and randomly assigned to either 5 mg iv zoledronic acid or placebo both with additional daily calcium (1000 to 1500 mg) and vitamin D (400 to 1200 IU). There was an imbalance in overall adverse events, mostly attributable to postinfusion symptoms. Importantly, this seminal trial provided the first robust description for effects of BPs on atrial fibrillation. In those receiving iv zoledronic acid, there were more atrial fibrillation events  that did not reach statistical significance but was signal enough to prompt evaluation of serious atrial fibrillation. In this instance, the imbalance reached statistical significance . This finding motivated an FDA review of oral alendronate that showed 47 serious atrial fibrillation adverse events (1.5%) in alendronate‐treated and 31 (1.0%) in placebo‐treated during an average of 4 years , a finding that cannot rule out a clinically important effect. Reassuringly, there was no increased risk of all atrial fibrillation adverse events . The rationale for why BPs may provoke arrhythmia is uncertain. Some preclinical studies point to potential disruption of calcium handling dynamics in cardiomyocytes by BPs. Other important triggers initiating atrial fibrillation may arise from focally discharging cells located most commonly at the pulmonary vein ostia. These foci may lead to frequent atrial ectopy and paroxysms of atrial fibrillation. This effect is particularly relevant to the iv administration, which is clouded by flu‐like symptoms postinjection. BPs can also stimulate release of inflammatory cytokines, which are implicated in increasing the risk of atrial fibrillation but through mechanisms that have not been fully elucidated. These data were enough to include in atrial fibrillation as a rare but of uncertain causal origin side effect that clinicians should be aware of in current regulatory agency recommendations. More recent data cast doubt on this understanding. A recent trial of similar design to the HORIZON trial specifically enrolled 2000 women in the osteopenic range . In a secondary publication of prespecified safety data, Reid and colleagues demonstrated similar event rates for any atrial fibrillation 14.8  events per 1000 zoledronic acid treated and 15.6  events per 1000 placebo‐treated women. The crude event rates were much larger than HORIZON for both all atrial fibrillation events  and for number of women with at least one episode of atrial fibrillation . In comparing HORIZON with the more recent trial in patients with osteopenia, it may be argued that the women with osteopenia have a lesser underlying CV disease burden (given the shared underlying risk factors for both CV disease and osteoporosis), and thus the administration of zoledronic acid was not sufficient to provoke paroxysms. Cohort studies have demonstrated inconsistent findings regarding this pre‐presumptive association and thus continual investigation and post‐market monitoring is prudent.Despite no apparent effect of BPs as a class on overall mortality, cause‐specific effects have been explored in greater detail. One of the most well‐described effects has been the apparent increase in atrial fibrillation associated with iv zoledronic acid Table . This wa31 Thi osteopenic women experienced no increased rate of myocardial infarction, and indeed may even have experienced a reduced rate of infarcts . Survival analysis demonstrated a potential 40% improved survival in zoledronic acid–treated women  was shown, where the survival curves began to deviate at approximately 18 months. These data, although prespecified, were secondary endpoints and thus warrant interpretation with caution. This effect is another point of difference between the trial by Black and colleagues in women with osteoporosis. There were fewer myocardial infarction events in zoledronic acid–treated women (38 [1.0%] versus 45 [1.2%]), but this did not reach statistical significance . Similar to effects on overall mortality, these data may indicate that BP may exhibit some transient effect which is support by cohort data on medication adherence. Furthermore, given the potential action of BP on the cholesterol synthesis pathway, there is biological rationale to support an effect on ASVD. Further data are needed to understand if the background ASVD is important in determining CV effects on BPs or if indeed BPs have clinically significant effects in reversing ASVD as per statins. Abdominal aortic calcification (AAC) is recognized to be a surrogate indicator of ASVD and indeed predicts CV and non‐CV outcomes. In an exploratory substudy of the HORIZON‐PFT, progression of AAC was examined following 3 years of treatment. AAC progression (defined as a change in semiquantitative AAC score) was similar between treatment groups . This did not differ by baseline AAC score, nor did change in total hip  or femoral neck BMD  correlate with AAC change. This overall suggests against a link between the skeleton and calcification in the vasculature as a potential mechanism explaining CV benefits or harms of BPs. However, it should be noted that the semiquantitative AAC scale (0 to 8) may not be sensitive enough to detect changes over the follow‐up period and the intraobserver fidelity was moderate (0.62). Studies using more sensitive techniques and scoring systems may yet observe differences. Understanding subtle changes in AAC is important as it remains to be seen if an AAC score of 0 has similar prognostic power as a coronary artery calcium score of 0.Contrary to the history of zoledronic acid and atrial fibrillation, rates of myocardial infarction have not been shown to be increased with zoledronic acid treatment. In the abovementioned trial by Reid and colleagues,33 ost and Liberman and colleagues in the clinical development of alendronate, trials of other oral formulations have shown similar CV effects. In a trial of 1200 postmenopausal women younger than 85 years; oral risedronate either 2.5 mg/day or 5.0 mg/day did not increase the rate of CV events. Compared to placebo (38 events [9.3%]), there were fewer events in women treated with 2.5 mg  and the same event rate in women treated with 5.0 mg . When compared head‐to‐head there was a nonsignificant increased rate of events in those treated with the higher dose , which suggests against a dose‐response effect.Similar to the seminal trials by Black and colleagues37 and This double‐blind, placebo‐controlled trial enrolled 7808 postmenopausal women aged 60 to 90 years. Prespecified safety data included serious CV events, strokes, and coronary heart disease. Analysis of these secondary endpoints demonstrated no statistical difference in event rates between the treatment groups . This finding was consistent across baseline AAC status (score ≤6 versus score >6) and, importantly, renal function (estimated glomerular filtration rate ≤45 versus >45). Again, this supports a view that a mechanism independent of calcium handling may explain the benefits or harms of antiresorptive therapies.Denosumab is a fully human monoclonal antibody against the receptor activator of nuclear factor‐κB ligand (RANKL), which exerts potent antiresorptive effects. The largest trial conducted in the clinical development of denosumab was the Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months (FREEDOM) study.30 Thi reported a near balance of cardiac arrhythmias  and no reports of CV mortality, strokes, ischemic events, or other CV‐related endpoints. Abaloparatide is a peptide that selectively binds to the RG conformation of the PTH type‐1 receptor and is hypothesized to have greater skeletal effects than teriparatide. In the pivotal trial for its clinical development over 2400 postmenopausal women were randomized to either abaloparatide, teriparatide, or placebo for 18 months. In the safety analysis, there were similar events rates compared to placebo for the endpoints of myocardial infarction  and atrial fibrillation (0/820 [0.00%] versus 0/822 [0.00%]), which was similar to the experience with teriparatide two decades earlier. Interestingly, in the majority of trials, the direction of effect supports a notion of CV risk reduction, which indirectly supports the underlying theory of skeletal health being linked to CV health (Table Parathyroid hormone (PTH) stimulates bone resorption physiologically in order to maintain appropriate circulating calcium levels. However, the osteoclasts responsible for bone resorption release chemokines and other hormones that eventually promote the maturation of osteoblasts resulting in increases in bone formation. This apparent paradoxical effect is utilized clinically, where synthetic analogues of PTH such teriparatide  and abaloparatide  are the chief anabolic agents in the physician's armamentarium. In terms of CV safety, the key trial in the clinical development of teriparatide by Neer and colleagues in 2001,59 rep In contrast, in a similar trial of romosozumab against placebo in older men (n = 245), the BRIDGE trial, there was a nonstatistically significant imbalance in adjudicated serious CV events . This imbalance appeared to be driven by an increase in cardiac ischemic (3/163 (1.8%) versus 0/81 [0.00%]) and cerebrovascular events (3/163 [1.8%] versus 1/81 [1.2%]). This small signal was also evident in the active‐controlled trial in postmenopausal women, the ARCH trial. The comparator in this trial of over 4000 (~2000 in each arm) postmenopausal women was oral alendronate. The number of adjudicated serious CV events was higher in those treated with romosozumab for the first 12 months compared to alendronate . Again, this imbalance seemed to be driven by ischemic events as there were more myocardial infarctions  and strokes , though neither reached statistical significance. There was no imbalance in the number of CV deaths . Of note, participants in the ARCH trial included the highest proportion of patients with a previous history of CV disease 73%, compared to 66% and 65%, in Placebo‐Controlled Study Evaluating the Efficacy and Safety of Romosozumab in Treating Men With Osteoporosis (BRIDGE) and FRAME, respectively. Thus, it has been suggested that perhaps the drug treatment has exacerbated (or provoked) already underlying CV disease and thus a black box warning for romosozumab is in place for any patient who has experienced a major CV event. This position is not supported by recent a meta‐analysis, where romosozumab was not shown to increase the risk of a composite cardiovascular outcome of stroke, coronary artery disease, heart failure, and atrial fibrillation  or a three‐point major adverse cardiovascular event (MACE) outcome . However, the lower bounds of the CIs in these estimates cannot exclude a statistically or clinically important effect, that is to say the lower bounds of the CIs only just cross unity; therefore, the majority of the effect lies in the positive direct (namely more events meaning romosozumab is associated with relative harm). Interestingly, there was an increase in a four‐point MACE outcome . This suggests that assessing CV risks of romosozumab is limited by power (given that a four‐point outcome includes more events than a three‐point outcome). Therefore an event‐driven analysis and ongoing postmarketing analysis should be considered.Romosozumab is a monoclonal antibody that binds and inhibits sclerostin, with a dual effect of increasing bone formation and decreasing bone resorption. In a trial of romosozumab against placebo, the Fracture Study in Postmenopausal Women with Osteoporosis (FRAME) trial , there was no imbalance in the number of adjudicated serious CV events . Similarly, there was no imbalance in the number of adjudicated CV deaths .62 In  There exists much variation in analysis approach (any versus specific BP), included literature  and outcomes. Collectively, the message is unclear. In the case of drugs with extensive experience, such as BPs with over 50 years of clinical use, real‐world evidence can complement RCT evidence. There could be several reasons why many RCTs of BPs on CV outcomes have not provided firm conclusions. For example, the trials may be underpowered to detect an adequate number of events, the trial being too short (again a question of being event‐driven) and population characteristics (did the patients have too severe CV disease to benefit from mild risk reduction or too mild a disease so not enough events occur over follow‐up). Recent real‐world evidence for CV effects of BPs appear to indicate cardioprotection and possibly a signal toward differential atherogenic and arrhythmic effects. In a cohort of individuals undergoing bone density testing , oral BP  use was associated with a reduction in atrial fibrillation, heart failure, and stroke but not myocardial infarction. Small experimental studies in humans have emerged attempting to define the mechanism for this demonstrating acute QT effects on electrocardiography (ECG) following zoledronic acid infusion. However, this may just be a feature of the autonomic response to an infusion. Further to differential atherogenic and arrhythmic effects, other cohort studies indicate there may be differential oral and iv effects of BPs, with the iv zoledronic acid associated with greater harms than oral BPs. This is supported by a meta‐analysis of the pivotal RCTs for ibandronate, which showed that although ibandronate was deemed to be safe overall, there was a greater crude rate of atrial fibrillation events in patients receiving intravenous ibandronate (0.5%) compared to the oral formulation (0.3%).Meta‐analyses have proven largely unhelpful in clarifying the potential harmful and/or beneficial effects of bisphosphonates on cardiovascular outcomes.69 The In patients with osteoporosis, it was revealed that compared to placebo , there was no difference in rates of CV events . However, when compared to any BP control, there was a 46% increased risk of CV events in denosumab‐treated women . There was a doubling of risk when considering a five‐point MACE outcome . These findings mirror what was evidenced by the romosozumab trials—namely treatment arms experiencing elevated CV events in a BP‐controlled trial but not in a placebo‐controlled trial. This provides indirect evidence for cardioprotection by BPs. Furthermore, recent studies in postmenopausal women suggest that RANKL inhibition does not alter markers of ASVD, further supportive of the imbalance of events seen in the meta‐analysis being attributable to suppression of events in BP arms. Of note, the meta‐analysis did not perform sensitivity analyses for BP type to elucidate if effects were specific to oral or iv formulations, so direct comparisons to the zoledronic acid trials are limited. This is particularly relevant given that the MACE outcome only reached significance upon inclusion of atrial fibrillation an outcome for which zoledronic acid has perceived effects.The context of intense CV scrutiny of romosozumab and BPs was the scientific motivation to reanalyze the specific CV safety data of denosumab. Two previous meta‐analyses have reported on the CV safety of denosumab, but findings were limited, including omission of the pivotal trial in the clinical development of denosumab that reported CV safety.56 In  Heart rates, blood pressure, and adverse events presumed related to increased heart rates were recorded. Overall rates of treatment‐emergent adverse events were higher in participants receiving study medications as expected. However, treatment‐emergent adverse events related to elevated heart rates that lead to trial discontinuation were substantially more frequent in abaloparatide‐treated  and teriparatide‐treated  than placebo . By contrast, unadjudicated MACE was approximately half as frequent in abaloparatide  and teriparatide  compared to placebo , representing hazard reductions of 57%  in abaloparatide‐treated and 51%  in teriparatide‐treated. In a long‐term follow‐up to these participants, where all participants went on to receive alendronate from study months 19 to 42, there was no imbalance in events . These represent the most in‐depth, published data from RCT participants on direct cardiac effects of osteoporosis medications. Although hazard reductions were nonsignificant, given PTH analogues are anabolic, this trial provides indirect evidence to support the theory that bone health has an inverse relationship with cardiac health.At the request of the FDA, investigators of the “Abaloparatide Comparator Trial in Vertebral Endpoints” (ACTIVE) study performed a comprehensive post hoc cardiovascular safety study of trial participants.75 Hea and Fixen and Tunoa. Both reviews concluded romosozumab to be safe in this respect and warned against overinterpreting low event rates. There is biological rationale for why this may be the case. In a murine model of atherosclerosis, the SOST gene (which encodes sclerostin) was expressed in the aorta and upregulation of SOST conferred vasculoprotection. This finding supports the clinical experience of patients with sclerosteosis, a genetic mutation leading to the overexpression of the SOST gene and thus exhibit naturally high levels of sclerostin. These people exhibit marked bone deposition, but importantly, appear to be at no increased risk of CV disease. This would seem at odds with a uniquely designed genetic analysis of single nucleotide polymorphisms (SNPs) of the SOST gene associated with increased BMD from several large population databases. This analysis revealed at least one SNP that imposed an elevated CV risk, particularly for myocardial infarction and coronary revascularization. This finding would support the black‐box warning by the FDA. However, when considered in the context of myocardial infarction risk reduction in the zoledronate trial of osteopenic women, suggestive of cardioprotection by BPs, these data provide a compelling research justification an RCT with a vascular primary outcome. Presently, it is still uncomfortably uncertain if the romosozumab increases CV events or BPs suppress CV events.There has been substantial interest in understanding if sclerostin inhibition imposes CV risk since the publication of the ARCH trial Table . This wa76 and Equally, from a public health standpoint there is merit in looking for and confirming putative “off‐target” effects. Many osteoporosis medications, particularly alendronate, are currently available as a generic. Thus, if a genuine “off‐target” effect is established  and regulatory authorities grant an expanded indication, this could potentially save healthcare systems a significant amount of money. This is already happening, one example being ticagrelor. Originally marketed as an anti‐platelet agent for the treatment and prevention of stroke and coronary outcomes, the pivotal efficacy trial demonstrated reduced pulmonary events, and this has been followed up with studies demonstrating antimicrobial properties. The natural endpoint of repurposing is eventual incorporation into the approval process. There is precedent for this, as new agents for diabetes must now demonstrate safety as well as CV efficacy. One may argue that the links between bone disease and CV disease is just as strong as that between diabetes and CV disease. Thus, given the risk of CV outcomes imposed by osteoporosis and the high ASVD burden in this population, there can be an argument for this to also occur for new skeletal agents. The experience of romosozumab shows that the field seeks this evidence; and it would be wiser to have this data produced in the setting of a large, well‐designed RCT rather than relying on the eventual churn of reviews and subpar meta‐analyses. Such large, well‐designed RCTs are near prohibitively expensive and pharmaceutical companies are likely to resist subjecting their novel agents to this scrutiny. Thus, there is a role for well‐designed cohort studies to provide real‐world evidence and work in tandem with RCTs.In the big data age, it is becoming easier to investigate in a rapid and high throughout manner, “off‐target” effects of common medications, albeit not without the risk of confounding. There is a business case for this; most drugs have a finite lifespan under patent so the window for pharmaceutical companies to financially exploit their invention is limited. Repurposing of existing medications known to be safe by expanding the indication thus has enormous attraction. A recent success story of this has been the CV efficacy of sodium‐glucose co‐transporter 2 inhibitors in patients without diabetes.81 Equ This trial was included in summary estimates despite not reporting specific CV events in the primary publication nor are data available on the trial's registration page. Not reporting events does not necessarily mean zero events.The literature examining the potential CV harms or benefits of antifracture medications is now quite substantial. This is helped by more rigorous reporting, where registered trials are required to report adverse drug reactions (ADRs). The systematic collection of ADRs both in trials and population‐wide health systems through electronic medical records and claims databases means that the body of real‐world evidence is substantial. Generally, these findings have been complimentary to findings in RCTs. However, this has not led to firm conclusions. First, adverse event reporting from RCTs means that the majority of evidence on this topic is secondary and subject to bias and being underpowered. Meta‐analyses, in an attempt to overcome the issue of power, have largely proved unhelpful and are often poorly conducted. By way of example, a meta‐analysis of PTH‐analogues included the pivotal trial by Neer and colleagues.59 Thi Therefore, exploring other mechanisms such as potential electrophysical effects, antithrombotic effects, effects on autonomic function after iv infusion, or plaque stabilization effects may offer a window into the bone‐vascular axis. In conclusion, the clinical message should be that osteoporosis medications have a very good safety record and that there is a clear research need to study the potential CV benefits that may, perhaps surprisingly, accompany restoration of bone health.The overarching question of the CV safety for antifracture medications can only definitively be answered in an RCT. There is particular need for an RCT of BPs with a primary vascular endpoint in order to allay criticism that interpretation of safety data needs to be cautious. Further, such an RCT needs to be event‐driven to avoid overinterpretation of low event rates. There may be an ethical concern for conducting such a trial of “repurposing” a medication. Is it justified to apply a medication to examine an “off‐target” effect if proven treatments for those “off‐target” effects already exist? However, patients with osteoporosis have a high ASVD burden and likely other adverse risk markers so the balance of benefit and harm may fall marginally in favor of the trial. Overall, there is need to optimize CV management for improved skeletal outcomes. Furthermore, there is need to better understand the clinical and biological links between skeletal and CV disease because antifracture medications appear to have no effect on aortic calcification .58 TheAJR declares no conflicts of interest. BA declares research contracts with Novartis, Kyowa‐Kirin UK, and UCB with funds paid to the institution, and consulting or speakers fees from Amgen, UCB, Eli‐Lilly, Pharmacosmos, and Kyowa‐Kirin UK.https://publons.com/publon/10.1002/jbm4.10522.The peer review history for this article is available at


They demonstrate the safety and efficacy of this bacterial antimicrobial peptide (AMP) against various bacterial strains, describe its mode of action, and validate its use as systemic drug therapy against bacterial infections in mice. This study highlights human oral bacteria as a source of antimicrobial agents that need to be considered in the future to fight multidrug‐resistant bacteria.Multidrug resistance is one of the major public health issues the world is facing today. However, the World Health Organization (WHO) revealed recently that there has been little progress in the development of new antibiotics to tackle drug‐resistant infections. By mining the bacterial genome database, Zhu  et al (in this issue of EMBO Mol Med) that highlights human oral bacteria as a powerful source of antimicrobial agents to fight multidrug‐resistant bacteria.W. Liang and J. Diana discuss the study by Zhu  The abiEMBO Molecular Medicine, Zhu et al propose the use of human oral‐derived bacterial AMPs as safe and efficient antibiotics to treat multidrug‐resistant infections (Zhu et al, In this issue of in vitro the antibacterial potency of actinomycesin against various bacteria strains clinically relevant in human infections. Actinomycesin exhibited high activity against Streptococcus and Staphylococcus strains, including antibiotic‐resistant strains, whereas at similar concentrations, oral human AMPs such as defensins HNP1&2 or lysozyme C were not efficient. The authors, however, nicely demonstrated an antibacterial synergy between bacterial and human AMPs that was not due to physical interaction as demonstrated by bio‐affinity electrospray ionization mass spectrometry. They conducted an elegant series of experiments to demonstrate that the bactericidal activity of actinomycesin is mediated by the inhibition of bacterial cell wall synthesis, likely via the binding of the precursor molecule membrane‐anchored Lipid II. Importantly, this mechanism of action may explain the synergistic effect between actinomycesin and human AMPs. Indeed, lysozyme may facilitate actinomycesin access to Lipid II by partial hydrolysis of bacterial cell wall peptidoglycan, and HNP1&2 could facilitate the binding of actinomycesin by changing the conformation of Lipid II. Additional studies are required to confirm this stimulating hypothesis. Importantly, the authors show that, contrary to vertebrate AMPs, host molecules present in serum or pure DNA did not alter the microbicidal capacity of actinomycesin.First, using a classical inhibition‐zone assay, the authors demonstrated Since actinomycesin targets bacterial cell wall synthesis and does not disrupt bacterial membrane as many other AMPs, it could reduce the risk of toxicity on eukaryotic cells. The authors confirmed the low toxicity of actinomycesin on human kidney cell line and demonstrated that actinomycesin had no effect on ion channels expressed in human central nervous system and heart.et al evaluated the release of IL‐8 by human alveolar type II cell line treated by actinomycesin or by the human AMP LL‐37. They observed that even at high dose, actinomycesin induced only low level of IL‐8 compared with LL‐37. Using surface plasmon resonance analysis and bioinformatic prediction, the authors further showed that actinomycesin exhibited low immunogenicity and low allergenicity. However, follow‐up studies, including in vivo experiments, will be required to confirm the minimal impact of actinomycesin on the complex immune system of vertebrates.As mentioned above, the direct bactericidal activity of eucaryotic AMPs is reduced in physiological conditions. However, they exhibit either pro‐inflammatory or anti‐inflammatory activities that promote an efficient and controlled anti‐infectious immune response. Eucaryotic AMPs control the recruitment of various immune cell types at the site of infection, modulate the activity of neutrophils, enhance the antigen presentation ability of dendritic cells, but also induce regulatory mechanisms that terminate the immune response. While these properties are beneficial in infectious context, aberrant expression of vertebrate AMPs in sterile condition may be detrimental as shown in various autoimmune contexts (Liang & Diana, Streptococcus pneumoniae‐induced pneumonia and methicillin‐resistant Staphylococcus aureus‐induced peritonitis. In both infectious models, actinomycesin treatment showed high therapeutic efficacy, reducing bacterial load and improving mouse survival. Future studies will be required to evaluate the potential use of actinomycesin against other pathogenic bacteria and pathogens. As already performed with vertebrate AMPs, the sequences, and structures of bacterial defensins may allow the development of novel therapeutic synthetic peptides with optimized bactericidal capacity and reduced immunogenicity (Haney et al, Finally, the antibiotic potential of actinomycesin was evaluated in preclinical mouse models of et al. provides a proof of concept that mining the microbial genome is a promising approach to discover efficient AMPs for systemic therapy of bacterial infectious diseases.In conclusion, the study of Zhu


Fragaria vesca L.). This design allowed us to quantify the relative importance and interactive effects of herbivore‐ and pollinator‐mediated selection on nine traits related to plant defence and attraction. Our results showed that pollinators imposed stronger selection than herbivores on traits related to both direct and indirect  defence. However, conflicting selection was imposed on inflorescence density: a trait that appears to be shared by herbivores and pollinators as a host plant signal. However, in all cases, selection imposed by one agent depended largely on the presence or ecological effect of the other, suggesting that dynamic patterns of selection could be a common outcome of these interactions in natural populations. As a whole, our findings highlight the significance of plant‐herbivore‐pollinator interactions as potential drivers of evolutionary change, and reveal that pollinators likely play an underappreciated role as selective agents on direct and in direct plant defence.Tripartite interactions between plants, herbivores, and pollinators hold fitness consequences for most angiosperms. However, little is known on how plants evolve in response—and in particular what the net selective outcomes are for traits of shared relevance to pollinators and herbivores. In this study, we manipulated herbivory (“presence” and “absence” treatments) and pollination (“open” and “hand pollination” treatments) in a full factorial common‐garden experiment with woodland strawberry ( This study advances understanding of plant‐herbivore‐pollinator interactions in revealing how these interactions can influence the phenotypic evolution of plant defence and attractive traits.A full‐factorial manipulation of pollination and herbivory (in a common‐garden experiment approach with woodland strawberry) enabled our study to quantify and compare the relative strength, direction, and dynamics of selection by these agents.Herbivores have long been considered the primary drivers of defence trait evolution in plant populations. We offer a novel counter to this preconception, and provide the first empirical support demonstrating the importance of pollinators as selective agents on direct and indirect defence traits, which here even surpassed that of herbivores.We furthermore revealed that the net selective pressures arising from these interactions were highly dynamic, and depended largely on the presence or ecological effects of the other agent. These findings are important given there remains a lack of studies that simultaneously quantify pollinator‐ and herbivore‐mediated selection on the same trait.As a whole, our study highlights the possible evolutionary trajectories of defence and attractive traits in natural populations, and how pollinator and herbivore selective pressures are expected to interactively shape this evolution.To maximize reproductive success, most angiosperms must attract pollinating mutualists but evade herbivore antagonists Strauss . The plaBrassica rapa L.  and diffuse or nonadditive in nature  . We examined phenotypic selection on several chemical traits previously identified as markers of direct and indirect defence for this species  and North America  where it grows in diverse conditions from boreal to Mediterranean climates and in habitats ranging from disturbed forest margins to open grasslands . This oligophagous herbivore feeds on leaves and flowers of several Rosaceae plants  (Stenberg and Axelsson Cnephasia asseclana Denis and Schiffermüller (Tortricidae) and Ceramica pisi L. (Noctuidae)—was also observed on experimental plants. The former is a pest of cultivated strawberry  or received supplemental hand pollinations every about 5 days for the full flowering period. Only three plant genotypes were used as pollen donors to control for pollen quality effects. Pollen was applied using a fine‐hair paintbrush ensuring that no hand contact was made with plants during application. Herbivory was manipulated through the addition or removal of an ecologically important herbivore: the strawberry leaf beetle, Asecodes parviclava Thompson [Hymenoptera: Eulophidae]) on diets of varying genotypic composition. Several primary and secondary compounds were ultimately identified as markers associated with either direct or indirect defence  from measurements of plant length and width. Given their prior establishment for 1 year, plants were generally expected to have obtained their maximal size by the time traits were measured (mean plant size = 7.5 dm3 ± 3.0 SD). The latter three traits were based on flower counts (mean number of flowers per plant = 73.9 ± 30.3 SD). Frost‐damaged flowers showed a complete blackening of the receptacle at the center of the flower, and were hence hypothesized to negatively impact floral attractiveness to pollinators and florivores—possibly due to a lack of material for consumption . No visitation on frost‐damaged flowers was observed during the experiment, although visitation to nondamaged flowers on the same plant still occurred. Inflorescence density was quantified as the number of flowers per dm3. Conceptually, this trait differs from flower number in that it describes a different feature of relevance to pollinator or florivore attraction; that plants with many flowers could have these quite dispersed in the inflorescence, and vice versa. Bumblebees were also frequently seen to “walk” between flowers when the inflorescence was dense (pers. obs.), suggesting that this trait had the potential to be selected for independently of flower number. Phenotypic correlations between most of these traits were low , and one for berries that were picked dry . Berries were randomly selected across all genotypes and treatments to establish these equations. Only fertilized seeds were counted, which are easily visually differentiated from nonfertilized seeds . Although the multiple regression included only 27 genotypes for which complete trait data were available, we still considered these inferences to be robust given that (1) the full spectrum of genetic variation in defence was represented (see above and Table n = 27) or all (n = 81) genotypes were used  describe the strength of unique or direct selection acting on a trait (after controlling for inter‐trait correlations), whereas selection differentials (S) describe “total” selection , and herbivores on two (one defence‐related and one attractive) Fig. . PollinaHerbivores have long been considered the primary drivers of defence trait evolution in plant populations   table for the selection gradient model ran in Table S2.Table S4. Direction and strength of total selection on defence‐related traits and plant attractive traits in woodland strawberry (Fragaria vesca L.).Click here for additional data file.


Biopsy remains the gold‐standard measure for staging liver disease, both to inform prognosis and to assess the response to a given treatment. Semiquantitative scores such as the Ishak fibrosis score are used for evaluation. These scores are utilised in clinical trials, with the US Food and Drug Administration mandating particular scores as inclusion criteria for participants and using the change in score as evidence of treatment efficacy. There is an urgent need for improved, quantitative assessment of liver biopsies to detect small incremental changes in liver architecture over the course of a clinical trial. Artificial intelligence (AI) methods have been proposed as a way to increase the amount of information extracted from a biopsy and to potentially remove bias introduced by manual scoring. We have trained and evaluated an AI tool for measuring the amount of scarring in sections of picrosirius red‐stained liver. The AI methodology was compared with both manual scoring and widely available colour space thresholding. Four sequential sections from each case were stained on two separate occasions by two independent clinical laboratories using routine protocols to study the effect of inter‐ and intra‐laboratory staining variation on these tools. Finally, we compared these methods to second harmonic generation (SHG) imaging, a stain‐free quantitative measure of collagen. Although AI methods provided a modest improvement over simpler computer‐assisted measures, staining variation both within and between laboratories had a dramatic effect on quantitation, with manual assignment of scar proportion being the most consistent. Manual assessment also most strongly correlated with collagen measured by SHG. In conclusion, results suggest that computational measures of liver scarring from stained sections are compromised by inter‐ and intra‐laboratory staining. Stain‐free quantitative measurement using SHG avoids staining‐related variation and may prove more accurate in detecting small changes in scarring that may occur in therapeutic trials. Histological assessment of liver scarring is a pivotal endpoint for determining efficacy of potential anti‐fibrotic therapies in clinical development. Conventional scoring systems encompass broad architectural distribution of fibrosis rather than reflecting only the amount of scar deposition  and relyPicrosirius red (PSR) staining is established as the most reliable method for visualising fibrosis in a liver biopsy, shows concordance with other measures of collagen deposition , and mayThe relative ease and declining cost of both acquiring and storing whole‐slide images mean that there is now a significant amount of histological data available that can be mined by machine learning algorithms. As opposed to CPA and associated techniques, machine learning enables the characterisation of ‘sub‐visual’ features of a slide – information that would not be consciously captured by a pathologist or simple computational methods , 9. MachIn addition to artificial intelligence (AI) methods, stain‐free second harmonic generation (SHG) and two‐photon excited fluorescence (TPEF) microscopy have been proposed as tools to enable a more accurate and objective assessment of a liver biopsy that is not influenced by staining quality . SHG ligIn this exploratory study, we have compared the performance of an AI methodology with simple thresholding and manual assessment in quantifying scar proportion in PSR‐stained sections of liver, alongside a stain‐free method of scar quantification. For widespread application in large clinical trials or routine clinical practice, the ideal method of scar quantitation from stained sections must be robust to staining variation both between and within laboratories where sections must be stained daily rather than as study‐specific batches. We have used sequential sections from the same blocks, stained on two separate occasions at two independent National Health Service (NHS) clinical pathology laboratories. In the absence of a ‘ground‐truth’, the performance of different methods of scar quantitation has been evaluated by the consistency of derived metrics of scar amount across the set of sequential stained sections, testing both inter‐ and intra‐laboratory effects, i.e. an optimal method would produce the same ‘result’ from each of the four sections from the same block stained on two separate occasions in two independent NHS laboratories. Finally, the stain‐based measurement methods of scar quantification were compared to stain‐free SHG/TPEF imaging, which gives a similar readout of the amount of collagen on a slide but is not subject to bias relating to either laboratory protocols or stain interpretation. Specifically, the measurement of fibrosis‐related parameters in liver tissue by SHG is highly reproducible when the test–retest performance has been evaluated .The prevailing orthodoxy is that machine learning methods will provide a significant performance improvement over both simple colour space methods and human measurement, but we demonstrate significant challenges that must be overcome if AI methods are to be applied to histopathology in large multi‐centre studies and clinical practice.Anonymised unstained formalin‐fixed paraffin‐embedded sections from 20 cirrhotic explant livers  were provided after approval by the Lothian NRS Human Annotated Bioresource with permission granted under authority from the East of Scotland Research Ethics Service REC 1, reference 15/ES/0094.From each block, initial five adjacent 5 μm sections were cut for staining in Nottingham and Edinburgh. A single section from each case was PSR stained according to standard local protocols within two CPA UK‐accredited NHS pathology laboratories – Nottingham University Hospitals NHS Trust Queen's Medical Centre Pathology Department and NHS Lothian Department of Laboratory Medicine at the Royal Infirmary of Edinburgh see . To assehttps://rdmc.nottingham.ac.uk/handle/internal/9133).Stained sections were scanned using identical NanoZoomer scanners  at ×20 magnification. The raw scanned .ndpi whole‐slide images were split by ndpisplit  into ×5 All livers were cirrhotic, so the application of traditional ordinal scores of architecture provided no inter‐case discrimination. Instead, whole‐slide images of each section were stripped of all identifiers and randomly numbered. Four participants  provided their assessment of the percentage of tissue on each slide that was PSR positive, scoring each batch of 20 slides with a ‘washout’ period of at least 1 day in between different, randomly ordered batches.Each background cleaned tile was classified by two separate HSB colour space thresholds to calculate the number of pixels representing total tissue and the number of PSR‐positive pixels. The threshold values to determine PSR‐positive pixels were derived by selecting positive pixels within a representative tile and then testing on representative tiles from all cases, adjusting and iterating by‐eye until the most consistent thresholding was achieved. A single set of threshold values was applied to tiles from all stained image sets.Following pre‐processing described above, the Waikato Environment for Knowledge Analysis (WEKA) , an openIndividual WEKA classifiers (WEKA_i) specific to each single set of images were trained by using only tiles from that stained image set. For combination WEKA classifiers (WEKA_c1 and c2), classifiers were trained using tiles drawn from all stained image sets.SHG/TPEF imaging was carried out by Histoindex Pte Ltd (Singapore) using an unstained section from each of the 20 cases, at ×20 magnification. The raw SHG percentage (a measure of the amount of collagen) and qFibrosis, a score taking into account both the amount and distribution of collagen [The combination of metrics from four stained sections of the initial set from each case, Edinburgh 1 and 2 (E1 and E2) and Nottingham 1 and 2 (N1 and N2) from three different measurement approaches resulted in six pairs of observations used to compare the different methods , followed by staining of a second set 6 months later at both centres (July 2018), using the next sections from each FFPE block that were cut at the same time at the start of the study to allow evaluation of intra‐laboratory staining variation in a routine, real‐world clinical laboratory context. We observed substantial qualitative differences in the PSR colour and intensity in each batch stained, even when comparing slides stained at the same centre  and intra‐laboratory (ρ = 0.19) stain pairs  showed large differences between derived values of PSR percentage for each given case. The Spearman's rank correlation of values was poor both for inter‐laboratory  and inter‐laboratory stain sets  in isolation (WEKA_i). The classifiers trained on and then applied to each individual set of stained slides (WEKA_i) produced no increase in consistency compared with the simple colour space thresholding method. Spearman's correlation coefficients were similarly low for both intra‐  and inter‐laboratory (ρ = 0.29) stain sets  and applied to all cases. Unified training marginally increased the consistency of the classifier, with slightly increased Spearman's correlation coefficients for both intra‐  and inter‐laboratory (ρ = 0.37) stain sets  were used to further train an improved combined classifier (WEKA_c2). This targeted training led to further improvements in classifier consistency across all images, and increased Spearman's correlation coefficient for both intra‐  using each classifier applied to each set of stained images , it is evident that both inter‐ and intra‐ laboratory staining differences have a significant impact on classification Figures . Most imTo explore whether the duration between block sectioning and staining could account for some of the intra‐laboratory staining variation, a freshly cut set of sections from the same blocks were stained in Nottingham (rN3), where the PSR staining protocol remained unchanged, in February 2021. The derived scar proportion using the WEKA_c2 classifier from rN3 most closely correlated with the derived values from N1, suggesting that the duration of time between section preparation and staining was responsible for a proportion of the intra‐laboratory variation in PSR staining , repeating the process on renumbered image sets at least 24 h later. No prior training for the task was provided.Against all expectations, there was much greater consistency of given scar proportion across the stain sets for each observer alone compared with any computational method Figures  and 5, rHaving assessed an AI‐based method against existing methods of stain‐based measurement, we then compared these methods to commercially available, stain‐free SHG/TPEF imaging Figures  and 2. HIn 2015, a report commissioned by the UK government's Minister for Digital and Culture outlined potential benefits and opportunities of AI and machine learning tools, including how these could be applied to health care . In 2019In the research setting, large, multi‐centre trials using liver fibrosis as a primary efficacy endpoint currently rely on ordinal scores such as the non‐alcoholic fatty liver disease (NAFLD) activity score or Ishak fibrosis stage. Even when best practices are followed  , there iHowever, there are several challenges that machine learning‐based tools need to overcome if they are to be utilised in a clinical or research setting, including, but not limited to, a reliance on retrospective rather than prospective studies, the lack of standardisation to enable comparison between different AI tools, and the ‘AI‐chasm’, a term defining the gulf between reported accuracy measurements of a given machine learning tool during development and its actual diagnostic efficacy when used in the field . The AI‐As there is a lack of an established gold standard for measuring the amount of scarring in a liver biopsy, we have used consistency of the derived scar proportion across the four sets of stained sections as the metric to assess the performance of each method. Methods that are more robust to staining variation will produce more consistent values and give a tighter correlation between stain pairs.Our study demonstrates that a trained AI‐based method does increase consistency compared with simple colour space thresholding, an increase in performance that is enhanced by further training. As expected, this increase in consistency was higher between intra‐laboratory stain pairs, with protocol and environmental differences between laboratories more likely to produce significant changes in staining compared to reagent changes within a single laboratory. However, there was still considerable residual inconsistency in the calculated scar proportion and human observers were easily able to outperform these methods, despite the task putatively favouring computational methods. As observed for the computer‐based scoring methods, the human scores also showed a slightly higher consistency between intra‐laboratory pairs compared to inter‐laboratory pairs Figure .The age of a histological section is known to affect a variety of stains , therefoWe present this not to suggest that by‐eye estimations of scarring should be used but to highlight that staining variation is an inevitable factor in real‐world laboratories. Whilst iterative training will undoubtedly increase the consistency of methods used to assess scarring in stained sections and more sophisticated tools are in development to effectively allow ‘normalisation’ between multiple staining sites to attempt to account for the variation introduced , 29, staSHG/TPEF imaging has been proposed as a gold standard for the measurement of liver fibrosis , particuThe study is limited in the type of specimen and the stain assessed. Only sections from explant livers were used. Whilst this type of specimen in only encountered by laboratories at liver transplant centres, it was chosen because the available tissue for research was abundant with no risk of exhausting the blocks. Whilst the use of explants meant that the study was limited to cirrhotic livers, rather than representing the full spectrum of disease stage, there is no clear reason that the findings cannot be extrapolated to PSR‐stained sections with any amount of fibrosis. The examination of a set of cases where ‘gold‐standard’ ordinal scoring of fibrosis is unambiguously non‐informative  serves to illustrate the potential value for formal computational quantification. Finally, only PSR‐stained sections were examined. We would suggest that whilst the sources and extent of staining variation will vary depending on the specific stain, the susceptibility of computational methods of feature quantification in stained sections to such variation should always be evaluated where studies use anything other than self‐contained, single batch staining.In conclusion, we demonstrate that computational tools are not yet able to satisfactorily compensate for differences in tissue staining both between and within laboratories. The results here suggest that caution should be exercised when applying such methods to stain‐based quantification in histopathology, particularly in large multi‐centre studies, without applying extremely rigorous standardisation between staining centres.SA collected the data, analysed the data, and prepared and revised the manuscript. JIG obtained funding, collected the data, and prepared and revised the manuscript. DAD collected the data and revised the manuscript. ING obtained funding, designed the study, and revised the manuscript. JAF designed the study and revised the manuscript. TJK designed the study, collected the data, analysed the data, and prepared and revised the manuscript.Supplementary materials and methodsFigure S1. The effect of section ageClick here for additional data file.File S1. Script in ImageJ Macro Language to use in FIJIClick here for additional data file.File S2. Script in ImageJ Macro Language to use in FIJIClick here for additional data file.File S3. Excel file containing measurements using all stain‐based methods  and stain‐free methods (SHG % and qFibrosis score)Click here for additional data file.


Lung squamous cell carcinoma (LUSC), one of the main pathological types of lung cancer, has led to consequential socioeconomic burden. Ferroptosis is an iron‐dependent form of cell death process with potentials for therapeutic target in various kinds of tumors. However, whether ferroptosis‐related genes (FRGs) are associated with the prognosis of LUSC patients is still unclear. The aim of this study was to establish a FRGs‐based signature which could stratify patients with LUSC.The RNA sequencing profiles and corresponding clinical data of LUSC patients were retrieved from The Cancer Genome Atlas (TCGA) database and Gene Expression Omnibus (GEO) dataset. A FRG‐based signature was developed using the TCGA‐LUSC cohort and validated in the GEO cohort. Gene set enrichment analysis (GSEA) and analysis of immune cell characteristics were conducted to assess the relationship between FRGs and biological function or immune status. A nomogram based on selected clinical factors and the risk scores which were generated from the FRG‐based signature was developed using the TCGA cohort and validated in the GEO cohort.A set of 16 FRGs, significantly associated with overall survival (OS) in the TCGA cohort, was identified and could classify LUSC patients into two risk groups. Kaplan–Meier analysis illustrated that the survival rate of the high‐risk group was significantly lower than the low‐risk group. Assessment and external validation of the signature showed that the survival predictive performance of this signature was adequate. Additionally, multiple pathways and functions were enriched through GSEA and the analysis of immune cell characteristics showed significantly different abundances of immune cells among the two risk groups. Finally, a nomogram integrating the FRG‐based signature and selected clinical factors was also developed and assessed in both the TCGA and GEO cohort.This study indicated the association between the FRGs and prognosis of patients with LUSC. Targeting ferroptosis may serve as a novel potential therapeutic alternative for LUSC. We developed a nomogram to predict the overall survival of lung squamous cell carcinoma patients using the risk score calculated from the ferroptosis‐related gene‐based signature and the T stage. The nomogram showed satisfactory discrimination power, calibration, and clinical usefulness in both the TCGA and GEO cohort. Lung cancer poses a severe global health problem with burdening medical and socioeconomic consequences, with about 2 093 870 new patients diagnosed and leading to about 1 761 000 deaths worldwide annually.In recent years, considerable developments have been achieved in targeted therapies, and several effective molecular targets such as epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) have been identified.Ferroptosis is an iron‐dependent form of necrotic cell death that is driven by the lethal accumulation of lipid peroxidation products and reactive oxygen species (ROS).In this present study, we collected the mRNA expression profiles of LUSC from public databases and identified the differentially expressed genes (DEGs) of LUSC which were also categorized as ferroptosis‐related genes (FRGs). Then, functional enrichment analysis was performed to explore the underlying mechanisms. Moreover, a prognostic multigene signature was constructed with these selected FRGs. Finally, a FRGs and clinical factors‐based model was constructed to improve the prognostic evaluation of LUSC patients. This study explored the potential prognostic value of ferroptosis‐related genes in LUSC and developed a user‐friendly tool to assess the risk and prognosis for patients with LUSC.https://portal.gdc.cancer.gov) (TCGA cohort). The raw data of mRNA expression matrix and clinical information of 69 tumor samples were retrieved from the Gene Expression Omnibus (GEO) dataset (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE73403) (GEO cohort).Two patient cohorts were included in this study. RNA sequencing profiles and corresponding clinical data of 502 patients with LUSC patients, including 502 tumor samples and 49 normal samples, were downloaded from The Cancer Genome Atlas (http://www.zhounan.org/ferrdb/). We performed the following process to develop the prognostic signature. The differential analysis of the FRGs between LUSC and normal tissues were performed using the Wilcoxon test after within‐array replicate probes were replaced with their average via “limma” R package in the TCGA cohort.p‐value was adjusted with the false discovery rate (FDR).p < 0.05 were considered statistically significant. Subsequently, the least absolute shrinkage and selection operator (LASSO) regression algorithm was performed to establish the FRGs‐based signature.A total of 278 FRGs were gathered from the FerrDb database  and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of the FRGs among the high‐risk group and low‐risk group in the training set, which were selected according to the thresholds of |log2 (FC)| ≥ 1 and FDR < 0.05. The gene set enrichment analysis (GSEA) was conducted using the “clusterProfiler” R package. The pathways with Utilizing the “CIBERSORT” R package, the proportions of 22 tumor‐infiltrating immune cells from each patient in the TCGA cohort were determined. In brief, CIBERSORT was performed to analyze the relative expression levels of 547 genes in individual tissue samples based on their gene expression profiles, to predict the abundance of 22 types of immune cells in each patient.In this study, we developed a nomogram incorporating the risk score generated from the prognostic FRGs‐based signature and clinicopathological predictors in the training set using the “rms” R package. We then assessed the prognostic value of the nomogram through Kaplan–Meier survival analysis based on the high‐ and low‐risk groups stratified by the median value of the risk score, generated from the nomogram. The performance of the nomogram was also evaluated with respect to its discrimination and calibration in the training and validation set. The ROCs at 3‐ and 5‐year follow‐up were performed to assess the discrimination of the model, and calibration was evaluated by visualizing the discrepancy between actual probabilities and predicted probabilities using the calibration curves. In addition, the clinical usefulness of the nomogram was assessed by calculating the net benefits at different threshold probabilities in decision curve analysis (DCA). All these assessments were performed in both the TCGA cohort and the GEO cohort.https://www.r-project.org). Continuous variables were analyzed using the Student's t‐tests, U tests, or nonparametric rank‐sum tests. Categorical variables were analyzed using the Chi‐squared tests or Fisher's exact tests. The OS between different risk groups was compared using the Kaplan–Meier analysis with the log‐rank test. Univariate Cox regression analysis and multivariate Cox regression analysis were implemented to identify independent prognostic predictors of OS. All statistical tests were two‐tailed, and p < 0.05 were considered statistically significant.All statistical analyses were performed using the R statistical software, version 4.0.2  in the high‐risk group compared to the low‐risk group. In contrast, the abundances of naive B cells, CD8+ T cells, activated memory CD4+ T cells, follicular helper T cells, and M1 macrophages in the high‐risk group were markedly lower than the low‐risk group (p < 0.05)  cells, monocytes, activated dendritic cells, and neutrophils were significantly enriched  Figure .p < 0.05) in the multivariate analysis were included in the model. Forest plots were performed to visualize the p‐value, confidence interval (CI), and hazard ratio (HR) generated from Cox regression analyses , and was confirmed in the validation set  were identified. In the TCGA cohort, 111 ferroptosis‐related DEGs were identified between LUSC samples and normal samples. Then, univariate Cox regression analysis indicated that there were 20 genes associated with OS among the DEGs, and LASSO regression finally identified 16 FRGs  which were used to develop the ferroptosis‐related gene signature. The potential mechanism of these FRGs were diverse, for example, depletion of CP promoted erastin‐ and RSL3‐induced ferroptosis.To explore the potential mechanisms by which the FRGs‐based signature effectively stratifies LUSC patients, GSEA analysis demonstrated significant activity of multiple pathways mediated by FRGs incorporated in the signature. KEGG pathway analysis illustrated that FRGs were significantly enriched in the ferroptosis, PPAR signaling pathway, fluid shear stress, and atherosclerosis as well as NOD‐like receptor signaling pathway, with the exception of mitophagy in animals. Ferroptosis is recognized as a novel treatment strategy to inhibit tumor development in various tumors including lung cancer. Because of the association between ferroptosis regulators and chemotherapy resistance or immunotherapeutic effects, it is reasonable to utilize ferroptosis‐related drugs to assist tumor therapy. The FRGs identified in this study may serve as promising targets in treating patients with solid malignant tumors.When investigating the correlation of the FRGs‐based signature with the immune status of tumors, we found that the abundances of naive B cells, CD8+ T cells, activated memory CD4+ T cells, follicular helper T cells, and M1 macrophages were significantly lower in the high‐risk group compared to the low‐risk group, suggesting that the LUSC patients in the high‐risk group may have worse immune function and immune status. A previous study indicated that CD8+ T cells could induce ferroptosis in tumor cells, and the immune system can regulate ferroptosis susceptibility to suppress tumorigenesis.In the present study, we also developed a nomogram based on selected clinical factors and the risk scores which were generated from FRGs‐based signature to better predict prognosis in LUSC patients. The performance of this model was evaluated and externally validated. To our best knowledge, this has been the first study that developed FRGs‐based signature and nomogram to predict prognosis in LUSC. However, several limitations in our study should be considered. First, it was a retrospective study based on the data from public databases, some information may be unavailable. Second, the molecular mechanisms of LUSC could not be fully demonstrated without in vitro and in vivo experiments. Therefore, further research is urgently warranted to validate these findings.In conclusion, a signature based on ferroptosis‐related genes was constructed and provide potential biomarkers for prognostic prediction in LUSC. The treatment targeting ferroptosis might be a novel promising therapeutic alternative for patients with LUSC.The authors declare that they have no competing interests.


The main objective of this longitudinal study was to investigate the neural predictors of reading acquisition. For this purpose, we followed a sample of 54 children from the end of kindergarten to the end of second grade. Preliterate children were tested for visual symbol  and auditory language processing (spoken words) using a passive functional magnetic resonance imaging paradigm. To examine brain–behavior relationships, we also tested cognitive–linguistic prereading skills at kindergarten age and reading performance of 48 of the same children 2 years later. Face‐selective response in the bilateral fusiform gyrus was positively associated with rapid automatized naming (RAN). Response to both spoken and written words at preliterate age was negatively associated with RAN in the dorsal temporo‐parietal language system. Longitudinally, neural response to faces in the ventral stream predicted future reading fluency. Here, stronger neural activity in inferior and middle temporal gyri at kindergarten age was associated with higher reading performance. Our results suggest that interindividual differences in the neural system of language and reading affect literacy acquisition and thus might serve as a marker for successful reading acquisition in preliterate children. Longitudinal study examining reading acquisition from preliterate kindergarten age to the end of the second year of primary school. We used a passive fMRI paradigm to study the brain–behavior relationships of vision, language and reading. Preliterate response to print in the fusiform gyrus not only predicted word reading at the end of the second grade  and auditory language processing (spoken words) was obtained in preliterate children using a passive fMRI paradigm. Our research questions were threefold: First, we wanted to characterize visual and auditory language systems  in kindergarten children, which have previously been well described in adults and adolescents  approved the experimental procedures (AM042014).a, d), picture‐word matching of highly frequent words , syllable reading , decoding of phonotactically valid pseudowords  and word reading . Three children were excluded from the study as they were able to reliably decode syllables . At this point, three children from the original sample of 77 children withdrew their participation. Two children had to repeat the second grade and were retested 1 year later. Forty‐eight of these children had successfully completed the fMRI session at kindergarten age (T1). For these children, we were able to link the neural processing before onset of literacy to actual reading performance. All following results are based on the fMRI sample of 54  and 48  children. A summary of the demographic and psychometric data is given in Table 2.2Psychometric assessment was conducted at two distinct developmental time points: before literacy acquisition at the end of the last year of kindergarten (T1) and at the end of the second grade of primary school (T2).Raven's Colored Progressive Matrices , children were tested for non‐verbal intelligence and cognitive–linguistic skills associated with literacy. Non‐verbal intelligence was tested with Wechsler Intelligence Scale for Children , children completed a battery of standardized reading and spelling tests along with further psychometric assessments. Non‐verbal intelligence was retested with the 2.3N = 60) generated with MAC OSX voice Anna . Visual stimuli encompassed horizontal checkerboards (N = 15), houses (N = 60), faces (N = 60), and written words (N = 60). To match the visual angle across conditions, all stimuli were presented in gray scale and uniform size in the middle of the screen. Faces  were taken from the Karolinska Directed Emotional Faces  mono‐syllabic nouns of four to six letters and were taken from the childLex database, which provides age‐specific norms for children , in which the actual fMRI experiment was conducted. The experiment was divided into four runs, with the four visual and the auditory conditions presented block‐wise (15 stimuli per block). To ensure children's attention, a catch trial was performed at the beginning of each block (150 ms), in which a picture of the mascots of the project was shown and children were instructed to press a button using their middle or index finger to confirm its appearance. Average performance on the catch trials for the cohort of 54 kindergarten children was .67 . As indicated by the range of performance, the mean value was strongly influenced by a few children that did not manage to respond; they may possibly have been mentally overstrained by the highly new and demanding situation, or simply forgot to press the button. In any case, when asked afterwards whether they noticed the mascot, all of these children confirmed that they had. As such, the data from those children was kept for further analyses. Catch trials were followed by a jittered fixation cross (300–600 ms) and then replaced by the first stimulus. Visual stimuli were shown for 500 ms and auditory stimuli were presented for 500–600 ms. Each stimulus was followed by an inter‐stimulus interval of 200 ms (fixation cross). Three to five null‐events were included in each run to allow the blood‐oxygenation‐level dependent (BOLD) signal to return to baseline . Each run took about 136 s and was followed by a short break to improve compliance and maintain attention of the children  were acquired, resulting in a total of 272 axial volumes. Additionally, a T1‐weighted matched‐bandwidth high‐resolution anatomical scan with the same slice prescription as EPI was acquired . The standardized pictures were presented at the center of a white screen on dual display goggles  using Python 2.7 (Python Software Foundation). Auditory stimuli were presented via circumaural earphones . To attenuate scanner noise, child‐appropriate earplugs were provided, and the children's heads were padded with foam to improve their comfort and reduce motion artifacts.Imaging was performed using a 3.0 T Siemens Magnetom Tim Trio scanner  equipped with a 12‐channel head coil. In each of the four runs, 68 whole brain functional T2*weighted echoplanar (EPI) pulse sequences . Overall, less than 2.8% of the data was repaired. Tissue probability maps for native space components of the structural images were created according to an age‐matched pediatric template using the Template‐o‐Matic toolbox  were spatially smoothed with an 8 mm (FWHM) Gaussian kernel.FMRI data was preprocessed and analyzed using the SPM12 software package . Images were spatially realigned to the first volume by means of rigid body transformation, and unwarped. A confound of testing very young children with an fMRI paradigm is that head motion is highly correlated with age and further developmental characteristics . First, we examined the baseline contrast of each stimulus condition by entering the single‐subject maps into second level one‐sample t‐tests using the flexible factorial model of SPM. Similar to the results of Dehaene‐Lambertz et al. . This was most certainly due to perceived movement induced by the fast‐changing line orientations from trial to trial and checkerboards were discarded from further analysis. Consequently, the specific neural response to houses, faces and written words was extracted by subtracting the activation of the other two remaining visual conditions  and the differential contrast faces >  were entered into group level regression analyses. Here, results of the behavioral tests were entered as regressors to evaluate (A) the relationship between cognitive–linguistic prereading skills  and basic visual and auditory processing and (B) the power of preliterate neural processing to predict reading fluency after two years of formal reading instruction. Significant neural activation was inspected on the whole brain level with an initial cluster‐defining threshold of p < .001 (uncorrected) and a second family wise error (FWE‐)corrected cluster‐level extent threshold, measured in units of contiguous voxels (k), of pFWE < .05 corrected for multiple comparisons across the set of analyzed voxels  and p < .0125 for the longitudinal prediction of reading (4 fMRI contrasts × 1 reading skill) denoted as pcorr. For the sake of comparability with previous studies . Brain regions are reported according to the Montreal Neurological Institute (MNI) space brain atlas.In a next step, individual fixed‐effect models were computed using the default value of the high‐pass filter (128 s) which partitions out the confounding influence of physiological noise. Experimental conditions were entered into a general linear model (GLM) and motion parameters generated during realignment were included as regressors of no interest to control for overall motion effects. Basic contrast maps (target stimulus against null) were generated for each stimulus condition  analysis to capture more subtle effects of the brain–behavior relationship. Literature‐based ROI analysis was computed for the bilateral fusiform, MTG, and the STG; the left IOG, and the SPL. All ROIs were anatomically defined using the aal atlas of the wfupickatlas  and k > 10 voxels. To account for the number of regression models, we report the results at a stricter threshold of pcorr < .0005 for the concurrent brain–behavior analysis (1 fMRI contrast × 2 cognitive–linguistic skills). For the longitudinal prediction of reading fluency, no further correction was required (1 fMRI contrast × 1 reading skill). A figure of the ROIs and the results of the ROI analyses for all other contrasts is provided in the supplementary materials , Hmisc  and pFWE < .05. As predicted, houses and faces elicited a neural response in the ventral visual stream. Houses showed activation in the bilateral IOG reaching to the left occipital fusiform gyrus . Faces elicited left‐lateralized activation in the IOG . Written words showed a small cluster in the left OFuG to IOG . A large bilateral cluster in the occipital cortex was activated for houses >  . The faces >  contrast was associated with significant bilateral activation in the cuneus reaching to left lingual gyrus  and the supplementary motor cortex . The contrast written words >  did not yield any significant response.Figure spoken words activated the dorsal language stream associated with significant activation in the left STG . Detailed tables of all baseline and differential contrasts are provided in the supplementary materials : pcorr < .006 controlled for the number of regression models, and the standard threshold of pFWE < .05.Several regression models were computed to evaluate whether neural activation in response to visual symbols and spoken language was related to interindividual differences in PA and RAN  at kindergarten age explained interindividual variance in word and pseudoword reading . At pFWE the left precuneus, AG and SPL also yielded a significant association. None of the other contrasts tested predicted reading fluency .Next, we explored the predictive power of neural processing of visual symbols and spoken language before literacy acquisition on future reading fluency at 3.5written words > , we found a negative correlation for a small cluster in the left STG  and RAN. No further ROI yielded a significant brain–behavior relationship with RAN, PA or future reading fluency.Using more focal ROIs to examine the brain–behavior relationship for the differential contrast 4Literacy is a fundamental skill required in everyday life, academic education, and across the span of a career  have no special status as predictors of later reading ability prior to reading instruction. The initial stages of successful reading acquisition consist primarily of learning how to phonologically decode words Share, . Mere se5Due to the very young age of the children, we kept the fMRI tasks as short as possible at the expense of data points per subject. Similarly, we kept the number of stimulus conditions to a minimum to increase feasibility, and thus could not include control conditions for each stimulus. Additionally, we had to exclude checkerboards as a baseline condition from analysis due to atypically large activations. However, we believe that the relatively large number of participants and the rapid block design ensured stable and valid results. Likewise, motion artifacts are inevitable when scanning pediatric cohorts. Although we carefully checked the data and applied rather strict motion correction, a possible confound cannot be entirely ruled out. Although differences in brain sizes between children and adults are negligible by the age of six .Parents and children gave written informed consent to all parts of the study.AppendixS1. Supporting InformationClick here for additional data file.


Electrocatalysts for bifunctional oxygen reduction (ORR) and oxygen evolution reactions (OER) are commonly studied under hydrodynamic conditions, rendering the use of binders necessary to ensure the mechanical stability of the electrode films. The presence of a binder, however, may influence the properties of the materials under examination to an unknown extent. Herein, we investigate the impact of Nafion on a highly active ORR/OER catalyst consisting of MnFeNi oxide nanoparticles supported on multi‐walled carbon nanotubes. Electrochemical studies revealed that, in addition to enhancing the mechanical stability and particle connectivity, Nafion poses a major impact on the ORR selectivity, which correlates with a decrease in the valence state of Mn according to X‐ray absorption spectroscopy. These findings call for awareness regarding the use of electrode additives, since in some cases the extent of their impact on the properties of electrode films cannot be regarded as negligible. Binder's fee: The impact of Nafion on the properties of MnFeNi oxide nanoparticles as a highly active, bifunctional oxygen reduction/oxygen evolution reaction (ORR/OER) catalyst is investigated by voltammetric methods and X‐ray absorption spectroscopy. The ORR/OER activity of the catalyst increases in the presence of Nafion due to an improved particle contact, in addition to a binder‐induced Mn reduction that results in highly selective ORR sites towards the direct conversion of O2 to OH−. Electrocatalytic oxygen conversion–comprising the oxygen evolution (OER) and the oxygen reduction reaction (ORR)–is considered a major obstacle for the commercialization of green, regenerative energy conversion technologies, for example, reversible fuel cells or rechargeable metal‐air batteries, since both reactions suffer from sluggish kinetics.2,,As a case study, we investigate multiphase Mn, Fe and Ni oxide nanoparticles  being the active site for the OER,3, Fe‐L3 and Mn‐L3 edges are shown in Figure 3 spectra were shown by MnFeNiOx and MnFeNiOx(Nafion)  and after (MnFeNiOx(Nafion)) treating the catalyst in a Nafion‐containing water‐ethanol solution by sonication for 15 min (see sample preparation protocol in Supporting Information). The spectra obtained in the Ni‐L) Figure a, in the2+ species could be attributed to a Nafion‐induced chemical reduction, which has been observed previously with Mn‐containing complexes.3+, forming Mn2+ and Mn4+,2+ species due to a variation in the ratio Mn2+:Mn3+:Mn4+. To further understand the origin of the change in Mn oxidation state, two control experiments were conducted with MnFeNiOx treated in (1) a water‐ethanol mixture in the absence of Nafion (MnFeNiOx(WE)), and in (2) a solution containing cation‐exchanged Nafion (MnFeNiOx(Nafion‐Na+)), namely, after having replaced the H+ at the sulfonate groups with Na+ according to a previously reported procedure.+) (dark blue), thereby suggesting that the acidic proton in the binder does not play a major role in the observed Mn valence changes.The increase in the intensity of the spectral feature related to Mn2 and Mn2O3, for which the corresponding powders were treated in a water‐ethanol mixture in the absence (MnXOY(WE)) or presence (MnXOY(Nafion)) of the binder  was clearly seen with MnO2 (Mn4+) upon exposure to Nafion, whereas the spectra corresponding to Mn2O3 (Mn3+) did not display any substantial change, indicating that the valence changes are not related to disproportionation. We speculate that a strong chemical interaction between Mn4+ species in MnO2 and the electron donors in the binder takes place. Likely, this occurs similarly with Mn4+ species in MnFeNiOx, thus leading to an overall improvement in the ORR performance of the catalyst: on the one hand, the formation of Mn2+ species (from Mn4+) could favor the binding of *OOH intermediates according to recent DFT predictions,3+ atoms provide O2 absorption sites3+:Mn4+ ratio that facilitates the 4‐electron transfer pathway.[We further investigated the effect of Nafion on the XAS features of MnO2 to OH− predominantly via the transfer of 4 electrons. The impressive improvement in selectivity is attributed to a binder‐induced decrease in the oxidation state of Mn, as observed during XAS investigations, resulting in a more favorable Mn2+:Mn3+:Mn4+ ratio, and confirming that Mn plays a major role in the ORR performance of the trimetallic catalyst. Control experiments on MnFeNiOx and commercial Mn oxides indicated, on the one hand, that the valence changes observed are neither related to the acidic nature of Nafion, nor to disproportionation, and on the other hand, that Mn4+ species are susceptible to chemical reduction in the presence of Nafion. Although further studies are still required to fully reveal the extent of the chemical changes, as well as the variety of materials that may be susceptible to them, it is clear that Nafion cannot always be regarded as inert, and that awareness on its use is required for investigations where the intrinsic activity of a catalytic material is the main focus of the work.In summary, we investigated the influence of Nafion on the electrocatalytic properties of MnFeNiOx as a bifunctional ORR/OER catalyst. Besides the advantageous, yet expected, improvement of electrode film properties , a major benefit on the ORR selectivity of MnFeNiOx was revealed: while binder‐free MnFeNiOx displayed a preferred 2‐electron transfer pathway, in the presence of Nafion the composite electrode exhibited the reduction of OThe authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


To the Editor,We appreciate the thoughtful interest from Dr Cavagna and colleagues in the Empower Health program. We applied human‐centered design methodology when developing the model to ensure the application was locally and culturally appropriate.Availability and accessibility of essential drugs remain a challenge in low‐ and lower‐middle income countries and improving therapy access is an important role of the responsible health care system and governments.While Kenya and Ghana are considered middle income countries, the population served by the model was mostly low‐income and middle‐income segments and in mostly remote rural settings. Both are representative of many Sub‐Saharan countries with rural and urban populations that face similar challenges controlling hypertension. Note that smart phone ownership is not required in the model. Shared access via trained personnel or community health workers or location‐based set‐ups accommodate all patients including most patients who do not own a smartphone. Furthermore, the Empower Health model is currently available to over 20 000 patients in 2 countries, serving both public and private sectors and growing. We intend to continue to report program outcomes and have planned new randomized multi‐center prospective studies. Based on this design and ongoing expansion, the Empower Health model is relevant to address low blood pressure control rates in many other African countries.FK, CL, and DH are full time employees of Medtronic Inc; RS has received consulting and speaker fees from Medtronic; all other authors report no competing interests.HAO, GY, CM, and DAH drafted the manuscript all authors reviewed, edited, and contributed materially to the final document.