Skeletal muscle is the most abundant tissue in the body and is required for numerous vital functions, including breathing and locomotion. Notably, deterioration of skeletal muscle mass is also highly correlated to mortality in patients suffering from chronic diseases . Numerous conditions can promote skeletal muscle wasting, including several chronic diseases, cancer chemotherapy, aging, and prolonged inactivity. Although the mechanisms responsible for this loss of muscle mass is multifactorial, mitochondrial dysfunction is predicted to be a major contributor to muscle wasting in various conditions. This systematic review will highlight the biochemical pathways that have been shown to link mitochondrial dysfunction to skeletal muscle wasting. Importantly, we will discuss the experimental evidence that connects mitochondrial dysfunction to muscle wasting in specific diseases , aging, cancer chemotherapy, and prolonged muscle inactivity . Finally, in hopes of stimulating future research, we conclude with a discussion of important future directions for research in the field of muscle wasting. In healthy adults, skeletal muscles comprise 40%–50% of total body mass; muscles provide several vital physiological functions and are required for both locomotion and breathing. Notably, muscle fibers are also an endocrine organ and play a key role in glucose homeostasis ,2. The lSkeletal muscle mass is regulated by the balance of the rates of protein synthesis and protein breakdown. A detailed discussion of the control of muscle protein synthesis and proteolysis is outside the scope of this review, and the reader is directed to comprehensive reviews for more information ,6,7. NonMuscle protein synthesis is controlled by the complex interplay between transcription and translational events . While mIncreased mechanical load on skeletal muscle fibers promotes an increase in muscle protein synthesis and results in fiber hypertrophy . In contSkeletal muscle protein degradation results from the coordinated action of four proteolytic systems: (1) autophagy; (2) the ubiquitin–proteasome system; (3) calpains; and (4) caspase-3. Numerous detailed reviews describing these proteolytic systems exist and, therefore, only a short synopsis is provided to highlight the role that ROS play in stimulating activation of specific proteases ,7,15. BrThe ubiquitin–proteasome system is comprised of a core proteasome subunit (20S) that provides an enclosed cavity where proteins are degraded. This 20S subunit is coupled with a regulatory complex (19S) connected to each end . CollectCalpains are calcium-activated proteases that selectively cleave target proteins ,20. CalpCaspase-3 is the fourth major proteolytic system found in muscle fibers. Caspase-3 can be activated via several interrelated signaling processes and, similar to calpain, oxidative stress is a prominent activator of caspase-3 . CaspaseTo summarize, skeletal muscle mass is regulated by the interplay between the rates of protein synthesis and rates of proteolysis. If follows that skeletal muscle atrophy occurs when the rate of proteolysis exceeds the rate of protein synthesis. Although numerous factors participate in the control of muscle protein synthesis and proteolysis, oxidative stress is a common factor that contributes to muscle atrophy by depressing protein synthesis and accelerating proteolysis . While sThe earliest suggestion that mitochondrial dysfunction contributes to skeletal muscle wasting was reported in 1964 . This stMitochondrial dysfunction can contribute to skeletal muscle wasting in at least three ways: (1) increased mitochondrial production of ROS; (2) mitochondrial release of proapoptotic factors; and (3) mitochondrial damage resulting in a reduced production of ATP via oxidative phosphorylation .The following examines increased mitochondrial ROS production. Numerous preclinical studies provide evidence that increased mitochondrial ROS emission accompanies muscle wasting in several conditions . For instance, it is well established that prolonged skeletal muscle inactivity is associated with increased mitochondrial ROS emissions ,30,31,32A chronic increase in mitochondrial ROS production can promote muscle wasting by inhibiting muscle protein synthesis and accelerating proteolysis . As mentIn addition to accelerating proteolysis, oxidative stress can also contribute to muscle wasting by depressing protein synthesis in skeletal muscle fibers. Indeed, numerous studies conclude that exposure of cells to oxidants depresses protein synthesis ,42,43. TNumerous factors, including cytosolic calcium levels and elevated mitochondrial ROS production, can result in permeabilization of the mitochondria outer membrane, resulting in the release of pro-apoptotic factors . For exaMany conditions that result in muscle wasting are associated with mitochondrial dysfunction and a compromised ability to produce ATP ,31,47,48Note that low energy levels in muscle fibers can also promote accelerated protein breakdown by influencing the AMPK/FOXO3 axis . BrieflyTo summarize, the mitochondrial dysfunction that occurs during muscle wasting can contribute to fiber atrophy in at least three ways: (1) increased ROS production; (2) release of proapoptotic factors; and (3) decreased oxidative phosphorylation capacity. Notably, it is likely that these three signaling pathways do not operate independently. Indeed, increased mitochondrial production of ROS can contribute to both increased release of proapoptotic factors from the mitochondria and the low energy stress-induced activation of AMPK, resulting in increased expression of select atrogenes . The remThe previous segments highlighted the cellular signaling networks that provide a mechanistic connection between mitochondrial dysfunction and skeletal muscle wasting. The final segments of this review will examine the evidence connecting mitochondrial dysfunction to skeletal muscle atrophy due to prolonged muscle inactivity, aging, treatment with chemotherapeutic drugs, and specific diseases known to foster muscle wasting. We begin with a discussion of the evidence linking mitochondrial dysfunction to inactivity-induced muscle atrophy.Prolonged skeletal muscle inactivity is associated with both muscle atrophy and a reduction in maximal muscle force production. Common clinical conditions resulting in muscle inactivity include prolonged mechanical ventilation, bedrest, and limb immobilization. For example, intensive care unit patients that are provided respiratory support via mechanical ventilation experience inactivity of both inspiratory muscles  and limb muscles. In contrast, limb immobilization and bed rest result in selective atrophy of the affected locomotor muscles. It is well known that disuse atrophy occurs due to both a decrease in muscle protein synthesis and increased proteolysis ,60,61,62The first evidence that oxidative stress contributes to inactivity-induced muscle wasting appeared over 30 years ago . Since tThe cellular sites for oxidant production in muscle fibers exposed to prolonged inactivity has received widespread investigation; these studies reveal that oxidants are produced from several sources, including NADPH oxidases, xanthine oxidase, and mitochondria ,48,69,70The mechanisms responsible for increased mitochondrial ROS emission within inactive muscle fibers have been widely debated, and several rival hypotheses exist  content . TherefoDisruption of the mitochondrial network is another common hallmark of mitochondrial dysfunction during catabolic conditions, such as inactivity and various other forms of muscle wasting . TheProlonged skeletal muscle inactivity is associated with calcium release from the sarcoplasmic reticulum, resulting in increased cytosolic levels of free calcium ; this elActivation of the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway has been shown to promote mitochondrial dysfunction, resulting in increased ROS production in skeletal muscle fibers ,87,88. FFinally, activation of NOX2 in muscle fibers results in increased mitochondrial ROS production . NOX2-inIn summary, five different mechanisms have been proposed to explain the link(s) between prolonged skeletal muscle inactivity and increased mitochondrial ROS emission, and it is feasible that several of these mechanisms may work in concert to promote increases in mitochondrial ROS production. Regardless of the mechanism(s) responsible for inactivity-induced mitochondrial dysfunction, convincing evidence exists that mitochondrial dysfunction results in increased mitochondrial ROS emission and is a key player in promoting inactivity-induced muscle wasting.Sarcopenia is defined as the age-related loss of skeletal muscle mass and function . ClinicaThe mechanisms responsible for sarcopenia are complex and likely involve several factors, including mitochondrial dysfunction, oxidative stress, satellite cell dysfunction, neurological deficiencies , chronic low-grade inflammation, and diminished anabolic signaling in muscle ,96,97,98Numerous studies confirm that sarcopenic muscles exhibit impaired mitochondrial fusion and fission (reviewed in ). For exAs discussed previously, it is widely reported that mitochondrial health in skeletal muscle fibers declines with age. The maintenance of mitochondrial health over the lifespan is dependent upon the removal of damaged mitochondria via mitophagy and mitochondrial biogenesis to replace these damaged mitochondria ,80. It fTo summarize, the etiology of sarcopenia is complex and likely involves the interaction of a variety of factors, including a decline in mitochondrial dysfunction. Indeed, accumulating evidence suggests that an age-related increase in mitochondrial dysfunction is a key contributor to sarcopenia.Doxorubicin (DOX) is an anthracycline antibiotic that is widely used as an antitumor agent in the treatment of human cancers. While DOX is highly effective in the treatment of numerous cancers , DOX is cytotoxic and promotes the rapid wasting of both cardiac and skeletal muscle fibers. Indeed, a common clinical risk of using this highly effective anticancer drug is the development of cardiomyopathy . TreatmeThe mechanism(s) responsible for DOX-induced cardiac and skeletal muscle wasting has been extensively studied and evidence reveals that DOX-induced toxicity to both cardiac and skeletal muscle fibers is driven by increased mitochondrial ROS production and resultant oxidative stress . ExSupport for the idea that increased mitochondrial ROS production is essential for DOX-induced atrophy of both cardiac and skeletal muscle fibers comes from several lines of evidence. For example, treatment of C2C12 myotubes with SS-31, a mitochondrial-targeted protective peptide, prevents DOX-induced myotube atrophy . SimilarTo summarize, although DOX is a highly effective chemotherapeutic agent against numerous solid tumors, treatment of cancer patients with DOX is limited by the drugs’ toxic effects on both cardiac and skeletal muscle. In this regard, compelling evidence reveals that treatment with DOX results in both mitochondrial dysfunction and increases in mitochondrial ROS emission within both cardiac and skeletal muscle fibers. Importantly, a growing number of investigations indicate that increases in mitochondrial production of ROS is a required trigger to promote DOX-induced muscle wasting.Cancer cachexia is characterized by the loss of skeletal muscle mass with or without the loss of fat mass. Clinically, cancer cachexia is defined as an involuntary loss of >5% of total body weight within 6 months or a body mass index (BMI) of <20 . It is eWhile significant progress has been made in our understanding of cancer cachexia in recent years, the complexities of various cancer pathologies and patient populations has mired the development of treatment options. Over 100 types of cancer have been identified with varying pathologies. This point is further complicated by the fact that similar cancer types can also manifest diverging signaling pathways. For instance, a recent report by Nosacka et al. demonstrated that cancerous xenografts from four different patients diagnosed with pancreatic ductal adenocarcinoma resulted in distinct physiological responses when transplanted in rodents . MoreoveFindings from both preclinical and clinical studies confirm that the hallmarks of cancer cachexia are decreased myofiber cross-sectional area, disrupted muscle ultrastructure, and increased fibrosis ,137,138.Reports from both preclinical and human studies reveal that mitochondrial morphology is disrupted with cancer cachexia, displaying a swollen phenotype ,144,145.With regard to the evidence that mitochondrial dysfunction contributes to cancer cachexia, several reports have provided compelling arguments for the involvement of mitochondrial dysfunction with cancer cachexia. Recently, Brown et al. provided evidence that mitochondrial dysfunction precedes the development of cachexia in rodent models of lung and colorectal cancer . FurtherAs discussed previously, chronic elevation in mitochondrial ROS emissions can elicit muscle wasting. Evidence from preclinical models show that mitochondrial ROS emissions increase with cancer cachexia and are accompanied by increased markers of oxidative stress ,150,151.A depressed ability for mitochondria to produce ATP may also contribute to cancer cachexia. In this regard, several studies have shown diminished mitochondrial complex activity and mitochondrial respiration ,149,153.While compelling evidence exists supporting the tenet that mitochondrial dysfunction contributes to various cancer types, the highly diverging physiological response between different cancer types warrants consideration and limits our current understanding. Evidence from preclinical models that mitochondrial dysfunction precedes cancer cachexia, and that mitochondrial targeted pharmacological agents attenuate cancer cachexia, supports this notion. Nonetheless, many cancer types exist with varying pathophysiology. Future research is required to delineate the precise role that mitochondria play in cancer cachexia.Sepsis is a pathological condition characterized by a systemic inflammatory response due to infection of microbial origin. Sepsis is a life threatening condition that can result in organ failure of one or more organ systems, and is estimated to affect more than 48 million patients worldwide . Among tWhile the factors that contribute to sepsis-induced muscle wasting are likely multifactorial, mitochondrial dysfunction has emerged as key contributor to this muscle wasting. The first evidence that mitochondrial dysfunction plays a major role in muscle wasting was reported by Brealey et al. . In thisSepsis has been well documented to result in a decreased capacity for mitochondria to produce ATP through oxidative phosphorylation in skeletal muscle. Preclinical and human studies have consistently shown that mitochondrial complex gene expression, protein abundance, and activity are decreased with sepsis ,171,172.While the cause(s) driving diminished mitochondrial respiratory capacity has not been fully elucidated, an increased production of free radicals has been purported to play an inhibitory role in mitochondrial respiration during sepsis . NotablyWhile other sources of ROS may play a role in triggering mitochondrial dysfunction during sepsis, mitochondria themselves are major contributors to ROS production in myofibers. Indeed, increased mitochondrial ROS emissions may contribute to sepsis-induced muscle wasting. Several reports have shown that muscle mitochondrial ROS emissions are increased with sepsis ,181,182.In regard to the direct evidence implicating mitochondrial dysfunction as a critical mediator of sepsis-induced muscle wasting, a recent report reveals that overexpression of parkin, a protein responsible for mitophagy , protects muscle against sepsis-induced wasting . MitochoLastly, it should also be noted that mitochondrial dysfunction may contribute to sepsis-induced myopathy by affecting muscle satellite cells. A recent report reveals that mitochondria in satellite cells become dysfunctional and that this dysfunction persists following recovery from sepsis . SkeletaThe prediction that mitochondrial dysfunction is a primary factor contributing to skeletal muscle atrophy originated in the mid-1900s. Nonetheless, specific evidence demonstrating that mitochondrial damage/dysfunction contributes to numerous forms of muscle wasting was not available until the early 2000s. Specifically, direct evidence connecting mitochondrial dysfunction to muscle wasting due to disease, aging, chemotherapy, and disuse muscle atrophy has steadily emerged over the past decade. Indeed, compelling support now exists that mitochondrial dysfunction contributes to muscle wasting in a variety of diseases (cancer and sepsis), aging, cancer chemotherapy, and muscle atrophy due to prolonged periods of muscle inactivity.While numerous reports link mitochondrial damage/dysfunction to muscle wasting, many questions remain unanswered. For example, limited information exists about the mechanisms responsible for the skeletal muscle mitochondrial dysfunction that occurs due to disease, aging, and during prolonged inactivity. Further, it remains unclear as to whether both subsarcolemmal and intermyofibrillar mitochondria become dysfunctional during conditions that promote muscle wasting.Additional research is also needed to identify specific therapeutic interventions that can protect against mitochondrial dysfunction and prevent muscle atrophy. For example, although a few studies suggest that specific mitochondrial-targeted peptides  can protect against muscle wasting during prolonged inactivity and in response to treatment with doxorubicin, additional studies are required to determine whether mitochondrial-targeted treatments are effective in preventing muscle wasting during long durations of muscle inactivity (weeks to months) and during prolonged treatment with chemotherapeutic drugs . Moreover, more experiments are needed to establish whether mitochondrial-directed compounds can prevent sepsis-induced muscle wasting. Similarly, while experiments indicate that treatment with mitochondrial-targeted peptides during late senescence do not prevent age-related muscle atrophy, it remains unclear whether an appropriate intervention can protect against sarcopenia if treatment begins early in life.Another important topic for future investigations relates to the observation that cancer-induced increases in skeletal muscle mitochondrial ROS emissions returns to baseline over time. This finding raises two key questions. First, what are the mechanism(s) responsible for this time-dependent fluctuation in cancer-induced increase mitochondrial ROS emission? Second, what is the significance of this fluctuation in mitochondrial ROS production in promoting sepsis-induced muscle atrophy?Finally, it remains unknown whether increased mitochondrial ROS production plays a key role in the regulation of fission and fusion in skeletal muscle mitochondria. Studies that address this and other unanswered questions are required to identify new treatments to prevent muscle wasting due to disease, doxorubicin, aging, and prolonged muscle disuse.


Chronic spinal cord injury (SCI) is a catastrophic condition associated with significant neurological deficit and social and financial burdens. It is currently being managed symptomatically with no real therapeutic strategies available. In recent years, a number of innovative regenerative strategies have emerged and have been continuously investigated in clinical trials. In addition, several more are coming down the translational pipeline. Among ongoing and completed trials are those reporting the use of mesenchymal stem cells, neural stem/progenitor cells, induced pluripotent stem cells, olfactory ensheathing cells, and Schwann cells. The advancements in stem cell technology, combined with the powerful neuroimaging modalities, can now accelerate the pathway of promising novel therapeutic strategies from bench to bedside. Various combinations of different molecular therapies have been combined with supportive scaffolds to facilitate favorable cell–material interactions. In this review, we summarized some of the most recent insights into the preclinical and clinical studies using stem cells and other supportive drugs to unlock the microenvironment in chronic SCI to treat patients with this condition. Successful future therapies will require these stem cells and other synergistic approaches to address the persistent barriers to regeneration, including glial scarring, loss of structural framework, and immunorejection. Spinal cord injuries (SCI) are a devastating event and can lead to physical, psychosocial, and vocational implications for patients and their family. In the United States, approximately 288,000 individuals are estimated to suffer from symptoms caused by SCI, and a recent survey showed the annual incidence of SCI to be approximately 54 cases per one million people ,2. WorldDespite this pressing need, one of the greatest challenges in developing an effective therapy for chronic SCI has been the inhibitory microenvironment of the injured spinal cord. After SCI, astrocytes activate, proliferate, and migrate to the perilesional region where they form a dense interwoven network of processes and deposit chondroitin sulfate proteoglycans (CSPGs) into the extracellular matrix. Dystrophic axons surround the injury epicenter and become trapped in the dense meshwork of scar tissue .https://www.clinicaltrials.gov/ (accessed on 1 June 2021)]. Among them, exogenous neural stem cell (NSC) therapies are particularly promising as these cells have the potential to differentiate into all three neuroglial lineages  to regenerate neural circuits, remyelinate denuded axons, and provide trophic support to endogenous cells  currently targeting chronic SCI in Cell-based translational therapies have been attempted using several types of stem cells to induce nerve–axon sprouting or to neutralize the growth inhibitor factors. We show the ongoing clinical trials  began a phase I safety trial (NCT01772810) at the University of California San Diego of NSI-566 NSC transplantation for chronic thoracic SCI in 2014 . Although the trial criteria only cover the subacute phase of cervical SCI, we purposely discuss the CL2020 trial because Muse cells are a very promising cell source for chronic SCI as well.In Japan, one of the recent topics in stem cell therapy for SCI is the clinical trial evaluating a Muse cell-based product (CL2020) [Life Science Institute, Inc. Available online: Muse cells are a novel type of non-tumorigenic pluripotent stem cells that can differentiate into various kinds of cells in the body . Muse ceIn this clinical trial, the target of treatment is subacute cervical SCI patients, and the aim is to study the efficacy and safety of intravenous administration of CL2020. Our institution is also participating in this trial.Yamanaka and his colleagues developed iPSCs ,45, whicSeveral papers showed the effectiveness of iPSCs-NSCs transplant intervention in rodent models of chronic SCI. Grafted iPSC-based cells introduced at the site of the chronic SCI led to the integration of material into the injured spinal cord, reduced cavitation, and supported survival of the graft cells, but did not result in a statistically significant improvement of locomotor recovery . TreatmeIn Japan, researchers are currently planning to launch a first in-human clinical study of an iPSC-based cell transplantation intervention for subacute SCI. In addition, as the next step, they plan to move to chronic SCI treatment using iPSCs-NSCs . SeveralOne case report described experimental treatment with the use of autologous adipose tissue-derived MSCs (ADSCs) in chronic posttraumatic SCI in a domestic ferret with paresis of the back legs . A pilotOlfactory ensheathing cells (OECs) are a type of ensheathing cell that possess the characteristics of both astrocytes and Schwann cells (SC); they originate from the olfactory substrate located at the first and second layer of the olfactory epithelium in the olfactory nerve and bulb . SeveralAs a candidate cell for SCI treatment, SCs possess many desirable properties that are known to promote regeneration and repair after CNS injury through a variety of possible mechanisms ,84. A prRecently, several combinatory treatments for chronic SCI with stem cells, biomaterial scaffolds, and others were reported in rodent models and clinically ,117,118.Poly(ε-caprolactone) electrospun nanofibers have good mechanical strength but also a prolonged biodegradation profile, making them unsuitable for neural implantation where remaining scaffolds may hamper tissue regeneration . In contFew bio-scaffold-based treatments showed robust therapeutic effects on promoting motor axons, especially CSTs regeneration across the injury epicenter ,122. AltSome researchers demonstrated that nanoscaffold technology platforms can be combined with other neurogenic drugs, as well as stem cell therapeutic efforts currently in development. Many researchers tend to agree that a multi-disciplinary approach is needed to solve chronic SCI repair. In this point of view, combinatory treatment of stem cells and biological scaffolds is quite an important approach to chronic SCI treatment in the future. We think that, in the future, stem cell therapy with the support of functionalized electrospun nerve scaffolds could be a promising therapy to cure nerve diseases in chronic SCI patients.Currently, numerous clinical and experimental studies have shown positive results in terms of functional improvement with stem cell treatment in chronic SCI. Various designs of chronic SCI trials need to be performed. However, human chronic SCI trials are not easily enforced because of some inherent limitations. First, comparison between treatment and control groups is difficult because of ethical aspects, and, in terms of safety and efficacy, the results of animal experiments cannot be directly applied to humans. Nevertheless, a lot of basic research and clinical trials of stem cell therapy have already been performed, and promising results have also been reported. We are convinced that stem cell therapy will provide the drastic treatment needed for chronic SCI patients in the near future.


Diagnosis of acute PE in pregnant women with haemodynamic instability is following the general integrated risk-adapted diagnostic algorithm and starts with bedside echocardiography to assess RV function. If RV dysfunction is identified, a prompt and immediate reperfusion without further imaging should be initiated. Although pregnancy is listed as a relative contraindication of systemic thrombolysis, in pregnant women with acute PE and haemodynamic instability thrombolysis must be considered. In those cases, other treatment strategies as surgical embolectomy or catheter-directed low-dose thromboylysis or percutaneous thrombectomy should be taken into consideration as well. A multidisciplinary team with experience of PE management in pregnancy should be consulted to reach consensus on the best treatment approach. Venous thromboembolism (VTE) is considered globally as the third most frequent acute cardiovascular syndrome and is an umbrella term for the clinical entities of acute pulmonary embolism (PE) and deep vein thrombosis (DVT) . For PE,Although an overall decreasing trend in PE-related mortality over the past two decades was observed in a recent analysis of vital registration data in Europe, more than 1% of all deaths in women aged 15–50 years are caused by PE , 4. VTE The management of acute PE during pregnancy is challenging since:symptoms of PE (particularly dyspnoea) as well as DVT  in pregnant women can in part be difficult to distinguish from “physiological” symptoms of pregnancy,lower threshold of PE suspicion,fewer publications on validation of PE diagnostic algorithms,potential concerns regarding the harm of radiations or iodine contrast exposure regarding PE diagnostics andlack of direct evidence from interventional trials regarding PE reperfusion treatment, notably systemic thrombolysis, surgical embolectomy or catheter-directed treatment options –11.Initial risk stratification is based on assessment of the patient's vital/haemodynamic parameters. In haemodynamically stable patients, significant progress has been made in the validation of clinical and biochemical criteria, which are generally considered to apply to pregnant patients as well . In contAim of this review is to provide a framework for the management of pregnancy- associated PE, especially focusing on critically ill patients.The diagnostic management of PE in pregnancy is particularly challenging due to the fact that pregnant women often have clinical symptoms, such as shortness of breath or tachycardia, which could point to the suspicion of PE, but can also be present as physiological changes during pregnancy . MoreoveAll patients with suspected PE and signs of haemodynamic compromise have a high-risk of death during the first hours and days . Thus, iIn contrast to pregnant women with haemodynamic instability, the diagnostic algorithm for normotensive pregnant women may occasionally vary from that used for patients without pregnancy. A pre-test clinical probability assessment along with high-sensitivity D-dimer testing as well as bilateral lower limb CUS are in the center of the diagnostic algorithm for normotensive pregnant women with suspected PE. If there is a high or intermediate pre-test probability, empirical heparin anticoagulation should be administered before diagnostic imaging is initiated . If therThe overall prevalence of confirmed PE among women is low (2 to 7%) and underlines the diagnostic challenges –18. BecaThe current ESC guidelines recommend to perform an X-ray in pregnant women with suspected PE. If the X-ray is normal, V/Q scan should be performed, due to the fact, that V/Q scan is associated with low fetal and maternal radiation exposure. If the X-ray is abnormal, showing, for example, pulmonary infiltrates, then CTPA should be performed directly , 17 Fig.International medical society guidelines address new evidence of diagnostic strategies in pregnant women with suspected PE , 20–25. Especially high-risk PE in pregnancy can be a devastating event with a high case-fatality rate up to 37% . In patiBleeding complications are reported as a common adverse event after thrombolytic treatment in 18 to 58% cases during pregnancy and in the post-partum period, respectively . MaternaIn the case of absolute contraindications, alternative treatment strategies such as surgical embolectomy or percutaneous low-dose thrombolysis (CDT) or thrombectomy should be considered  Table 1Table 1. An additional treatment option for pregnant women with absolute contraindications for anticoagulation could be the placement of an inferior vena cava (IVC) filter . Data onAnticoagulation remains the mainstay of treatment in pregnancy and must be administered to all patients with high-risk suspicion of PE and confirmed PE . Since hDiagnosis of acute PE in pregnant women with haemodynamic instabilityis following the general integrated risk-adapted diagnostic PE algorithm PE andstarts with bedside echocardiography to assess RV function. If RV dysfunction is identified, a prompt and immediate reperfusion without further imaging should be initiated.Although pregnancy is listed as a relative contraindication of systemic thrombolysis, in pregnant women with acute PE and haemodynamic instabilitysystemic thrombolysis must be considered andother treatment strategies as surgical embolectomy or catheter-directed low-dose thromboylysis or percutaneous thrombectomy should be taken into consideration as well.A multidisciplinary team with experience of PE management in pregnancy should be consulted to reach consensus on the best treatment approach.All authors: conception and design of the study, data collection, analysis of the data, interpretation of data, drafting of the manuscript and revising of the manuscript critically for important intellectual content, and final approval of the manuscript submitted.LH reports lecture/consultant fees from MSD and Janssen, outside the submitted work. SK reports institutional grants and personal lecture/consultant fees from Bayer AG, Daiichi-Sankyo, and Boston Scientific; institutional grants from Inari Medical; and personal lecture/consultant fees from Pfizer- Bristol-Myers Squibb and MSD, all outside the submitted work.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Adeno-associated virus (AAV) was first characterized as small “defective” contaminant particles in a simian adenovirus preparation in 1965. Since then, a recombinant platform of AAV (rAAV) has become one of the leading candidates for gene therapy applications resulting in two FDA-approved treatments for rare monogenic diseases and many more currently in various phases of the pharmaceutical development pipeline. Herein, we summarize rAAV approaches for the treatment of diverse types of cancers and highlight the natural anti-oncogenic effects of wild-type AAV (wtAAV), including interactions with the cellular host machinery, that are of relevance to enhance current treatment strategies for cancer. It was largely thought to be non-pathogenic as no adverse events were noted upon infection in cell culture or in several mammalian species inoculated with the virus ..®) and sDue to the heterogeneous nature of cancer cells both among and within individual tumors, pre-clinical rAAV cancer gene therapy approaches have widely varied in an effort to take advantage of an assortment of cancer-driving cell signaling networks. For example, several groups have attempted to overexpress tumor suppressors and/or DNA repair genes, such as p53, to enhance apoptosis in a variety of cell line and xenograft models including breast cancer and cervical cancer models ,25. AnotOther notable approaches using rAAV include the delivery of sequences that can alter/regulate gene expression specifically in cancer cells. These approaches include the expression of shRNA(s) that specifically target oncogenic driver genes such as FHL2 in a colorectal cancer model . AnotherAnother approach to rAAV cancer gene therapy has been to modify capsid proteins via rational design/directed evolution to enhance/restrict rAAV transduction of cancer cells. Several groups have attempted to rationally design capsid proteins fused to a ligand that binds a specific receptor on cancer cells, such as the designed ankyrin repeat proteins . Others One of the most popular approaches for cancer gene therapy is to express modulators of the immune system from rAAV to generate an immune response to a particular cancer. For example, several groups have attempted to deliver immune stimulating cytokines via rAAV to induce a tumor-specific host immune response. Ma et al. have shown in a pre-clinical model that rAAV expressing soluble TRAIL can induce apoptosis in liver metastases via hepatic portal vein injection in a mouse xenograft model of lymphoma, suggesting that rAAV–TRAIL may be able to treat liver metastases arising from hematogenous metastasis . Anotherrep or cap gene, it was determined that the rep mutant plasmid failed to suppress E1a/ras transformation. In contrast, a cap mutant plasmid or a plasmid that contained no viral origin of replication suppressed transformation as well as wt AAV2. rep mutant plasmids that expressed only Reps 52/40, spliced Reps 68/40, or a K446H mutant located in the purine binding site showed transformation suppression of varying levels. A plasmid containing ITRs, but no rep or cap sequences, did not suppress transformation, suggesting that the rep protein was required for suppressing the transformation of mouse NIH/3T3 fibroblasts by the adenovirus E1a and ras proteins, at least in a plasmid context [Although most approaches to cancer gene therapy have utilized rAAV strategies, wtAAV itself is reported to have inherent anti-tumorigenic properties that specifically disrupt cancer cell physiology. Much of the early work in this area was generated in the last century and focused on the ability of AAV to prevent viral transformation of mammalian cells upon co-infection with oncogenic viruses such as herpesvirus, bovine papillomavirus, and human papillomavirus ,45,46,47 context . Althoug context .Later reports have demonstrated that wtAAV can directly induce cell death in multiple types of cancer cell lines. Furthermore, the toxicity of wtAAV appears to be specific to cancer cells, as it does not seem to induce cell death in most primary or diploid cell lines. For example, more recent work has suggested that wtAAV2 can induce death in HPV-infected cells, while HPV negative keratinocytes remain unaffected . AAV2 apwtAAV’s tumoricidal effects are not limited to HPV-transformed cells. In fact, similar results were seen in multiple types of breast cancer cell lines including hormone receptor positive and triple negative breast cancer cell lines, suggesting that wtAAV exhibits a broader anti-tumorigenic effect that is not specific to a single cell line or class of cell lines . Again, Although the above studies demonstrate that Rep protein levels and viral replication are correlated with the tumoricidal effects of wtAAV, the mechanism(s) is/are not well defined. The Rep78 protein is thought to induce a prolonged S phase arrest via multiple mechanisms . Rep78 bIn addition to the anti-tumor effects of the Rep proteins, it has also been suggested that the AAV ITRs are capable of inducing death in tumor cells lacking p53 both in vitro and in vivo. Raj et al. have demonstrated that p53-negative tumor incidence and tumor growth were decreased in response to UV-irradiated AAV infection in a mouse xenograft model, suggesting that Rep expression is not required for the tumoricidal activity of wtAAV in cells deficient for p53 . FurtherConsistently, several epidemiological studies have suggested that natural AAV infection measured via seropositivity is inversely associated with cervical cancer development ,61,62. OIn addition to using rAAV vectors to treat multiple types of cancer, an orthogonal approach has been to combine rAAV gene therapy with currently available chemotherapeutics. Cytotoxic chemotherapies have been the clinical standard-of-care across a plethora of cancer types for decades. Although these cytotoxic chemotherapies can display effective responses, they are often associated with severe side-effects ,71. FurtSeveral pre-clinical studies have demonstrated that rAAV gene therapy in combination with cytotoxic chemotherapies enhances tumor cell killing in multiple tumor types including ovarian, gastric, colon, hepatocellular, and head and neck cancers . In an oThe above studies clearly demonstrate that rAAV-mediated gene therapy could enhance clinical efficacy when combined with the standard-of-care chemotherapy; however, the mechanism associated with such enhanced efficacy following the combinations remains to be elucidated. It is well established that chemotherapy induces genotoxic and cytotoxic stress in tumor cells. Although each of these effective combinations could be acting through disparate mechanisms as discussed above, increased rAAV-mediated transduction following the increased stress response with chemotherapy may contribute to the enhanced cytotoxicity . InteresThese studies should be pursued cautiously since similar mechanisms could also increase rAAV off-target tissue transduction resulting in increased rAAV-mediated toxicity; thus, approaches to limit rAAV-mediated transgene expression to tumor cells through optimizing the promoter, capsid, and/or delivery route should be explored. As previously noted, many of the synergistic effects of rAAV occurred with cytotoxic chemotherapies that target one or more cellular processes occurring in S phase; thus, elucidation of rAAV’s mechanistic interactions with these drugs in a cellular host context would likely lead to improved combination strategies for clinical applications. In summary, rAAV-mediated gene therapy could be explored as an avenue to enhance the efficacy and safety of the chemotherapeutic agents currently used in clinical settings.Cancer stem cells (CSCs) are a subpopulation of cancer cells residing within the solid tumor bulk (such as breast cancer or lung cancer) or hematological tumors (such as leukemia)  that havGene therapy has long been proposed as a promising approach for cancer treatment , as illuOne obvious strategy for CSC-targeted therapy is to target the CSC stemness features; this would require the use of AAV to mitigate the overexpression of the genes or perhaps miRs which play vital roles in maintaining the self-renewal capacity of CSCs. For example, one critical factor would be to identify an AAV capsid that could transduce the CSCs with high tropism and efficiency to confer CSC transduction specificity. Although the transduction efficiency of rAAV in different stem cells including embryonic stem cells (ESCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs) has been widely studied , to the In addition, previous reports have suggested that AAV vectors induce toxicity in some types of stem-like cells ,96,97. JNotably, it is reported that ESCs and CSCs share some common biomarkers, gene signatures, signaling pathways, and epigenetic regulators . This hiIn summary, AAV may be an excellent choice as an adjuvant therapy for multiple types of cancer. The genetic features of AAV combined with the cellular host response to AAV infection provide a unique opportunity to enhance the tumoricidal activity of cancer gene therapy vectors . Both th


Deciphering the evolutionary relationships of Chelicerata  has proven notoriously difficult, due to their ancient rapid radiation and the incidence of elevated evolutionary rates in several lineages. Although conflicting hypotheses prevail in morphological and molecular data sets alike, the monophyly of Arachnida is nearly universally accepted, despite historical lack of support in molecular data sets. Some phylotranscriptomic analyses have recovered arachnid monophyly, but these did not sample all living orders, whereas analyses including all orders have failed to recover Arachnida. To understand this conflict, we assembled a data set of 506 high-quality genomes and transcriptomes, sampling all living orders of Chelicerata with high occupancy and rigorous approaches to orthology inference. Our analyses consistently recovered the nested placement of horseshoe crabs within a paraphyletic Arachnida. This result was insensitive to variation in evolutionary rates of genes, complexity of the substitution models, and alternative algorithmic approaches to species tree inference. Investigation of sources of systematic bias showed that genes and sites that recover arachnid monophyly are enriched in noise and exhibit low information content. To test the impact of morphological data, we generated a 514-taxon morphological data matrix of extant and fossil Chelicerata, analyzed in tandem with the molecular matrix. Combined analyses recovered the clade Merostomata , but merostomates appeared nested within Arachnida. Our results suggest that morphological convergence resulting from adaptations to life in terrestrial habitats has driven the historical perception of arachnid monophyly, paralleling the history of numerous other invertebrate terrestrial groups. Chelicerates are a diverse group of arthropods that have played a major role as predators in ancient and recent ecosystems. United by the eponymous pincer-like appendages (the chelicerae/chelifores), chelicerates comprise the sister group to the remaining Arthropoda. The most familiar chelicerate orders are members of Arachnida, an assemblage of 12 terrestrial orders . Chelicerates also include two wholly marine clades—the sea spiders (Pycnogonida) and the horseshoe crabs (Xiphosura)—as well as considerable diversity of derived aquatic lineages within mites . The fosWhereas most higher-level phylogenetic relationships of arthropods have been resolved by the advent of phylogenomic approaches e.g., , the intA handful of phylogenomic matrices has recovered arachnid monophyly, attributing this result to 1) the use of slowly evolving  genes that are less prone to long-branch attraction (LBA) artifacts or 2) expanded taxonomic sampling . HoweverBeyond arachnid monophyly, internal relationships within Chelicerata are unstable across phylogenomic analyses, which is in part attributable to the incidence of multiple fast-evolving lineages that incur LBA artifacts, such as Acariformes, Parasitiformes (treated as single orders in this study), and Pseudoscorpiones. Well-resolved parts of the chelicerate phylogeny include the reciprocal monophyly of Pycnogonida and Euchelicerata (the remaining chelicerate orders), the monophyly and internal relationships of Tetrapulmonata , and the monophyly of each chelicerate order. More recently, phylogenomic analyses emphasizing dense taxonomic sampling, together with rare genomic changes, have supported the clade Panscorpiones (Scorpiones + Pseudoscorpiones), in turn sister group to Tetrapulmonata (forming the clade Arachnopulmonata)  fig. 1)fig. 1). Toward a comprehensive chelicerate phylogeny that can inform the debate on arachnid monophyly, we assembled a 506-taxon phylogenomic data set representing the major lineages of all extant chelicerate orders and densely representing species-rich groups, such as Araneae, Scorpiones, Pseudoscorpiones, and Opiliones. Our analyses examined sophisticated strategies to mitigate LBA, such as subsampling loci to minimize saturation, the use of infinite mixture site-heterogeneous models (CAT-GTR), and recently proposed recoding strategies in tandem with site-heterogeneous models applied to partitioned model analyses.A common feature of phylogenomic studies is the omission of morphological data in an analytical framework, a practice that has been argued to be detrimental to phylogenetic reconstruction . CombineHere, we show that analyses of molecular data sets alone, as well as combined analyses of morphology and molecules, consistently recover horseshoe crabs as nested within Arachnida. Interrogation of phylogenetic signal across loci showed that genes and sites supporting arachnid monophyly are more prone to systematic error than the remaining loci, suggesting that arachnid monophyly in molecular phylogenies reflects an analytical artifact.Γ4), 2) traditional partitioned-model maximum likelihood, and 3) gene tree summary (ASTRAL) approaches.We compiled 506 high-quality transcriptomes or genomes , sampling 24 outgroup and 482 chelicerate taxa  online. In all six analyses, we recovered the nested placement of Xiphosura within a paraphyletic Arachnida, with support  and with significance in tests of monophyly  online. In the case of Pseudoscorpiones, an external and independent phylogenetic data class informs the placement of this long-branched order. Specifically, a shared whole genome duplication unites the clade Arachnopulmonata, as evidenced by duplications of Hox clusters, systemic paralogy of developmental patterning genes, and enrichment of microRNA families  fig. 1)fig. 1). 2 (≥ 0.95); these loci were concatenated to form Matrix 3 and analyzed using the same approaches as Matrices 1 and 2. Analyses of Matrix 3 with partitioned models, site heterogeneous models, and ASTRAL all recovered the monophyly of Arachnopulmonata . To mitigate the impact of fast-evolving loci, we generated saturation plots for each locus and isolated a subset of 152 loci with high values for slope (≥ 0.4) and rta sensu  with maxta sensu b. Maximuta sensu d.Some of the most recalcitrant nodes in the tree of life that are impacted by LBA have been argued to be effectively resolved using analyses under the computationally intensive CAT-GTR infinite mixture model, as implemented in PhyloBayes-mpi . Examplemaxdiff) was driven by a soft polytomy at the base of Euchelicerata. We examined estimates both from combined chains and summary topologies resulting from each starting tree type , Panscorpiones (BS = 99%), and Arachnopulmonata (BS = 100%) c. The baTests of monophyly were performed using the approximately unbiased (AU) test . The difTraditional statistical measures of nodal support are prone to inflation in phylogenomic data sets. Gene and site concordance factors (gCF and sCF) have been shown to measure phylogenetic signal irrespective of data set size . We therTo examine whether the derived placement of Xiphosura stemmed from a systematic artifact, we explored phylogenetic signal and properties of genes and sites as a function of support for competing tree topologies . We founFurthermore, we discovered that genes supporting arachnid monophyly were shorter and exhibited fewer parsimony informative sites than genes supporting the unconstrained topology, across all matrices b. Short Flagellopantopus blocki and two exemplars of the extinct order Phalangiotarbida from codings in the literature, as well as all extant chelicerates in the molecular matrix. Errors previously entered in the character coding were corrected  were removed from this analysis. For the same reason, we excluded putative chelicerate stem groups of questionable and controversial placement for which molecular sequence data are inapplicable. Pycnogonida was used to root Euchelicerata.When analyzed by itself under equal weights parsimony, the morphological data set yielded little basal resolution a. A striMaximum likelihood analysis of the combined matrix c also reMolecular results that recover nontraditional groupings are often labeled as artifacts, especially when morphological patterns and long-held evolutionary scenarios come under question. Like the basal topology of groups like Metazoa, Aves, and Angiospermae, the basal topology of Euchelicerata has long defied stability in molecular data sets. Proposals to “correct” the tree and recover arachnid monophyly using molecular data sets have included restricting analyses to slowly evolving genes  , expansiDrosophila melanogaster proteome, we discovered that the cause of this noise was the widescale inclusion of paralogs in these data sets. Specifically, 29% (68/233) of loci in the Lozano-Fernández et al. Matrix A , and 41% (82/200) of loci in the Howard et al. 200-locus matrix  included paralogs, often from distantly related multigene families ? As previously shown, the matrices of families  online. families .Could the properties of orthologs that are able to recover arachnid monophyly inform the selection of “better” loci for chelicerate phylogenomics? To address this, we examined the distribution of phylogenetic signal in our data sets for genes and sites supporting arachnid monophyly, versus the unconstrained topology, using ΔGLS and ΔSLS approaches . Genes sThese analyses suggest that support for arachnid monophyly does not reflect hidden signal, so much as noise and error in the data sets that have putatively supported this grouping. We submit that the sum of our analyses, however counterintuitive, may reflect a phylogenetically accurate relationship—Xiphosura (and possibly the other merostomates) may simply constitute derived arachnids.As anticipated, several groups in our phylogeny reflected long root-to-tip distances, constituting lineages prone to LBA artifacts. The inclusion of Opilioacariformes, a slowly evolving group of Parasitiformes , was recPhylogenomic subsampling for slowly evolving genes did recover Panscorpiones within Arachnopulmonata b, a resuIntriguingly, Poecilophysidea and Cephalosomata were previously supported by a minority of Sanger-based phylogenetic analyses and were proposed on the basis of patterns of anterior sclerotization in these orders , potentiUnlike in other animal clades, the addition of morphological data to molecular partitions does not ameliorate the discordance with the traditional phylogeny of chelicerates; we found that combining morphological and molecular data sets using model-based approaches recovers Merostomata (the marine group that includes horseshoe crabs) as nested within Arachnida. The notion that morphological synapomorphies of Arachnida can outweigh the dissonance found in molecular data found no support in this study. Furthermore, only in combination with molecular data was morphology able to recover clades supported by rare genomic characters (Panscorpiones within Arachnopulmonata); by itself, morphology has never recovered this arrangement of Arachnopulmonata, either in our analysis or in any previous efforts.One caveat of our combined analysis is that outgroups like putative stem-groups of Chelicerata  were not included, as their phylogenetic position is controversial even in morphological data sets . The excThese outcomes suggest that morphological data partitions seeking to capture deep chelicerate relationships may feature far less robust phylogenetic signal than commonly portrayed, specifically in a total evidence framework. Concordantly, a recent paleontological study failed to recover even Tetrapulmonata , the onlAdmittedly, the scenario of a nested Xiphosura invites entrenched skepticism, particularly from adherents of paleontology. In addition to an extensive fossil record, horseshoe crabs exhibit an array of putatively plesiomorphic traits that are suggestive of a basally branching placement. The fossil record of merostomates is thought to represent a stepwise colonization of land via internalization of the book gill of these marine groups . Recent arguments in favor of arachnid monophyly  have thuHowever, a comparison with the history of mandibulate arthropod phylogeny offers compelling reasons to doubt the linearity of morphological evolutionary scenarios. Within Myriapoda, only one order of centipedes (Scutigeromorpha) has retained the faceted compound eye commonly found in extant pancrustaceans and mandibulate fossil outgroups, whereas all other myriapod groups bear ocelli  or are blind . This chSimilarly, discussions of the gnathobasic mouthparts of merostomates echo historical debates over the nature of the gnathobasic mandible of terrestrial mandibulates, as well as other correspondences of head appendages. It was previously thought that Hexapoda and Myriapoda constituted sister groups (the clade Tracheata), a relationship supported by their putatively shared gnathobasic mandible, appendage-free intercalary segment, uniramous appendages, and arrangement of the respiratory organs . The gradual overturning of this relationship by molecular phylogenies in favor of the Pancrustacea hypothesis revealed that striking morphological convergences could occur in distantly related taxa as a result of common selection pressures in terrestrial environments . In thisWaeringoscorpio; Convergent evolution of tracheal tubules in other terrestrial groups, such as Onychophora, Hexapoda, and Myriapoda, falsifies the interpretation that a lung-like organ is a necessary stepping-stone to the acquisition of tracheal tubules in chelicerates. The conventional and simplistic evolutionary transformation series of book gill to book lung to tracheal tubule is deeply undermined by the complexity of respiratory organ evolution in Chelicerata. This point is underscored by the recent discovery of a eurypterid with trabeculate respiratory organs well after the appearance of arachnids in the fossil record 340 Ma; , secondaAs for the putative musculoskeletal synapomorphies established for Arachnida , we submTaken together, morphological character systems that putatively support arachnid monophyly tend to exhibit high levels of homoplasy upon closer investigation, especially when examining their counterparts in Mandibulata. Given the remarkable morphological convergences exhibited by Hexapoda and Myriapoda, we postulate that parallel evolution in terrestrial arthropod groups may confound inferences of homology in morphological data sets. Although no morphological characters overtly support a closer relationship of Xiphosura to any subset of arachnid orders but see , 2019b, The nested placement of Xiphosura, together with the reconstruction of multiple terrestrialization events across a grade of arachnid diversity, must be treated as a valid competing hypothesis. Future efforts to integrate new phylogenetic data classes and rare genomic characters e.g.,  may offeAnalyses of molecular data and total evidence phylogenetic approaches do not support arachnid monophyly. The concept of Arachnida may reflect the intuitive idea that terrestrialization is rare or costly in evolutionary history. As revealed by the history of groups like mandibulate arthropods, nematodes, and pulmonate gastropods, terrestrialization has not only evolved many times independently within such taxa, but is also the cause of remarkable and misleading cases of morphological convergence. Even on relatively shallow timescales, repeated colonization of terrestrial environments has been substantiated in arthropod groups like terrestrial Decapoda . Morphological data sets, including the data set we generated, have consistently failed to recover this grouping (with or without the miniaturized Pseudoscorpiones). If morphological data sets can falter in the recovery of the only higher-level chelicerate groups robustly resolved by molecular data and genomic architecture, it stands to reason that phylogenetic signal in morphological data sets may not be sufficiently robust to adjudicate other nodes in chelicerate interordinal phylogeny. Given the history of erstwhile morphological groupings like Tracheata, Uniramia, Articulata, Polychaeta, Pulmonata, Opisthobranchia, Aschelminthes, and numerous others, we postulate that phylogenomic approaches to deep metazoan relationships should treat morphological interpretations with measured skepticism Future efforts to integrate morphology into the new phylogeny of Chelicerata may be aided by parametric tests for phylogenetic signal across anatomical character systems, with the goal of quantifying informativeness and assessing noise in anatomical partitions e.g., . ExploraDetails of the methods below are provided in the D.melanogaster proteome for annotation using the best hit. Sequences not matching the most common annotation were discarded. Separately, orthologs benchmarked using the BUSCO-Arthropoda v. 3 database were analyzed independently. The set of complete, single-copy BUSCOs > 100 amino acids in length was retained from each library.Taxon selection consisted of 24 outgroup and 482 ingroup terminals; these 506 transcriptomes and genomes  online sInitial sets of orthologs were filtered based on taxon decisiveness . We retar2Γ model, we selected 56 representative terminals from the slow-evolving data set (Matrix 2) such that major taxonomic groups  were run using a gene partitioning strategy implementing the best substitution models identified during gene tree reconstruction. Tree topologies were inferred using IQ-TREE, with nodal support estimated using ultrafast bootstrapping. Summary coalescent estimates of the species phylogenies were estimated from the individual gene trees using ASTRAL v 3.14.2  proved prohibitive for a data set of this size, with the C20 model demanding 1.1 Tb of RAM to compute site-specific model parameters  was estimated as the starting tree, followed by estimation of site frequencies and ML searches under the mixture model. Nodal support was estimated using ultrafast bootstrapping. Bayesian inference analysis was performed using PhyloBayes-mpi v. 1.8  is driven by a soft polytomy at the base of Euchelicerata.We computed maximum likelihood analyses with the posterior mean site frequency model  for Matri v. 1.8  and the 0 cycles  online. 0 cycles  and natiWe implemented a site heterogeneous mixture model approach to partitioned phylogenomics using the recently proposed RL2 strategy , which iggplot2 . Implied weighting was performed using a family of values of concavity.Model-fitting for the morphological data set was performed in IQ-TREE v. 1.6.10 from the data set initially partitioned based on the number of character states. Bayesian analyses using the same partitioning scheme were performed in MrBayes v 3.2.7a  using thWe additionally performed total evidence analyses using two recently published morphological matrices with differing representations of stem-group chelicerate taxa, , complemmsac021_Supplementary_DataClick here for additional data file.


The emerging links between cellular metabolism and epigenetics are an enthralling and timely research topic with significant implications for basic and translational research. In this editorial, we will discuss many key concepts that link a collection of review and original articles in the special issue titled ‘Metabolism and Epigenetics,’ which highlight the recent advances in our understanding of the connection between metabolism and epigenetics. We will only include a few citations, and we apologize to all our colleagues whose work we are not citing directly here. Individual review articles, to which we will refer in the text, contain references to their original work.Sanhueza-Salas et al., demonstrate that high glucose reduces SIRT6 levels leading to an increase in the expression of the pluripotent factor Sox9, which in turns promotes dedifferentiation of these cells conferring them with stem cell, proliferative and migratory properties. These results provide new clues on how metabolic inputs translate into epigenetic regulation of developmental pathways and regeneration.During development, epigenetic mechanisms determine which genes must be turned on or off and to what extent in specific cells. In this context, functional cooperation between metabolism and epigenetics is instrumental in controlling cell fate decisions . It is wLuo S et al., recently demonstrated that gestational diabetes mellitus (GDM) reduced the level of SAM metabolite in murine fetal brains, thereby altering hippocampal DNA methylation and the regulation of cognition-related genes. Similarly, chromatin de-modifying enzymes such as TETs and jumonji lysine demethylases (Jmj-KDMs) require alpha-ketoglutarate (α-KG) as a co-substrate for demethylation of DNA and histones respectively. Interestingly, several metabolites derived from central metabolic networks can also have an inhibitory impact on epigenetic machineries. For instance, TETs and Jmj-KDMs are inhibited by the TCA intermediates succinate and fumarate. Moreover, Sirtuins, a family of NAD+-dependent protein deacylases, serve as a link between energy status and gene expression regulation by sensing NAD+/NADH levels. Thus, some epigenetic factors can act as metabolic sensors. Furthermore, changes in catalytic activity and subcellular localization of metabolic enzymes can influence epigenetic changes and gene expression programs. This emerging topic has been elegantly discussed by Ruben Boon, where the author proposes, based on recent experimental evidences, that metabolic enzymes could co-localize with chromatin factors in phase-separated nuclear domains to locally produce metabolites required for their activity. Collectively, these connections clearly demonstrate that epigenetic processes are directly dependent on many core metabolic intermediates, and thus represents an innate mechanism that links nutritional status to gene expression.Epigenetic mechanisms include DNA methylation, histone modifications, chromatin organization, and RNA-mediated processes . MajoritYu et al., recently, showed that BMI1 epigenetically promotes testosterone production in the Leydig cells by inhibiting the p38MAPK pathway. Enhancer of zeste 2 (EZH2), Histone 3 lysine 4 trimethylating (H3K27me3) epigenetic factor, was shown to affect tumor cell metabolism, including carbohydrate metabolism, amino acid metabolism and lipid metabolism -induced obesity.The dysregulation of epigenetic mechanisms may cause several pathologies and contribute to metabolism-associated diseases such as obesity, diabetes, GDM, diabetic cardiomyopathy (DCM) and more (The articles presented in this Special Issue provide new experimental evidence on the dynamic interaction between the epigenome and the metabolome and, at the same time, put these findings into the context of emerging concepts in the field. We hope that the reader will find new provocative research hypothesis worth exploring, which will improve our understanding on the reciprocal regulation of epigenetics and metabolism in health and disease.


Lung cancer is still the major cause of cancer-related mortality around the globe. The interplay of permanent genetic and dynamic epigenetic changes leads to the onset and progression of lung cancer. The diagnosis is often made at an advanced stage when the prognosis is dismal and therapy choices are restricted. Epigenetic association with lung cancer has long been studied but with fewer success rates. Research is still progressing, and with an advanced understanding of human genomics, more and more information is being unveiled. In the last decade, epigenetics and particularly research on DNA methylation and histone modification have provided vital information to understand lung cancer pathogenesis better. As a result, stage-specific epigenetic modifications can be employed as strong and reliable tools for early lung cancer detection and patient prognosis monitoring. The information on epigenetic biomarkers for lung cancer is summarised in this review, which focuses on DNA methylation and histone modification, as well as its implications for early detection, diagnosis, prognostication, and treatments. Epigenetics, initially defined by C.H. Waddington as 'the cause-and-effect connections between genes and their products that give rise to the phenotype', involves understanding chromatin structure and its impact on gene function . HoweverLung cancer is the most common cancer in both men and women, and it continues to be the leading cause of cancer-related deaths globally, with an expected 2.2 million new cases and 1.8 million deaths in 2020 . In the The interplay of genetic, epigenetic, and environmental variables leads to the initiation and development of lung cancer. DNA undergoes multiple processes during transcription, including DNA methylation and histone acylation, which are disrupted in lung cancer, the biggest cause of cancer-related mortality globally. The major factors in the epigenomics of lung cancer include gene silencing induced by hypermethylation, chromosomal instability produced by hypomethylation, and translation remodeling mediated by histone deacylation. After exposure to various environmental risk factors such as smoking, medications, and chronic inflammation, these modifications can occur in specific nuclear locations and chromosomal domains . AccordiEarly detection of lung cancer can have a substantial impact on the disease prognosis; indeed, the survival rate can skyrocket. Many imaging and cytology-based procedures have been used in the endeavor to enhance early detection; however, none of them has shown to be particularly effective, either due to low sensitivity or the exorbitant expense they impose on public health systems ,9. The mTumour biomarkersBiomarkers, according to the National Cancer Institute, are biological molecules present in the blood, other body fluids, or tissues that signal whether a process, condition, or illness is normal or abnormal. Cancer biomarkers have improved the validity of risk assessment and early detection tactics and provided insight into disease progression and prognosis, all of which have proven crucial in developing targeted individualised cancer therapy. The discovery of new biomarkers to help in outcome prediction and tumour response is critical for optimising therapy efficacy and avoiding over-or under-treatment of lung cancer patients. Epigenetic biomarkers, particularly DNA methylation, histone modifications, and micro ribonucleic acid (RNA) expression, have unique features that make them promising prognostic indicators Figure . They arEpigenetics refers to the second layer of information encoded on the genome that governs genomic function and activity. Epigenetic alteration offers excellent potential as a cancer biomarker since it can be used to analyse specific, reversible changes in disease-relevant genes and monitor important events in carcinogenesis. Another critical application is predicting therapeutic response; predictive biomarkers assess the likely effectiveness of a specific treatment. Tumour biomarkers provide essential clinical information in selecting suitable treatment, leading to customised cancer therapy .DNA methylation as a tumour biomarkerDNA methylation changes in tumorigenesis have recently emerged as a viable prospective biomarker for cancer patients' early identification, prognosis, therapy response prediction, and recurrence monitoring.The chemical covalent alteration of cytosine in DNA is known as methylation, where a methyl group (CH3) is attached to the 5-C of the cytosine residue called 5’-cytosine-phosphate-guanine-3' (CpG) dinucleotides by DNA methyltransferases (DNMTs). The distribution of CpG dinucleotides in the genome is uneven, with CpG dinucleotides clustered in small areas known as CpG islands (CGIs). A methyl group is transferred from S-adenosyl-L-methionine to the 5'-cytosine carbon of CpG islands by three active DNMTs  -14. PromEpigenetic changes can be broadly characterised as focal hypermethylation and global hypomethylation exhibiting an abnormal methylation signature of tumour suppressor genes (TSGs) . HypermeDNA hypermethylation can be detected in lung cancer patients' bronchoscopic washings/brushings, aspirates from sputum, and blood (plasma and serum), all of which are less invasive and less painful than a tumour biopsy. These technologies might potentially help detect extremely early lung tumours or newly recurrent malignancies that would be undetected by conventional methods in the future . DNA metGenomic hypomethylation or a loss of methylation occurs throughout the whole genome, which generally occurs later in lung cancer development. Gene-specific hypermethylation, on the other hand, may occur early in cancer formation . HoweverHistone modificationsIt is well known that histone post-translation modification (PTM) regulates a wide range of crucial biological processes, most notably chromatin modification that promotes or inhibits the expression or repression of target genes. The majority of study has focused on acetylation, methylation, and phosphorylation. However, histone deacetylation, or the removal of acetyl groups by histone deacetylases (HDACs), plays a vital role in tumorigenesis. Histone acetyltransferases (HATs) are enzymes that add an acetyl group to the lysine residue on the N-terminal tails of histones. This mechanism is controlled by DNA methylation, which results in the accumulation of repressor proteins in CpG islands and post-translational modification of histones. Abnormal gene expression is induced by an imbalance between acetylation and deacetylation, which leads to cell proliferation, DNA replication, mitosis, and carcinogenesis.HDAC inhibitors are gaining popularity as new anti-cancer drugs due to their ability to kill cancer cells by autophagy, apoptosis, cellular necrosis, cell cycle arrest, production of reactive oxygen species (ROS), reduction of tumour angiogenesis, and immunomodulatory effects. They lower the total tumour cell apoptotic threshold by stimulating both death-receptor and intrinsic mitochondrial mechanisms. They increase the expression of pro-apoptotic genes involved in the death receptor pathway  as well as the intrinsic apoptotic pathway like Bax, Bak, and apoptotic peptidase activating factor 1 (APAF1) while decreasing the expression of pro-survival genes (BCL-2 and XIAP). They also initiate the intrinsic apoptotic pathway by selectively activating or inducing BH3-only proteins ,23.HDACEarly detection and diagnosisLung cancer mortality could be considerably decreased if the disease is detected earlier. However, only around 15% of lung tumours are localised at the time of diagnosis, with the vast majority presenting at an advanced stage . A diagnPrognosis and predictionTumour characteristics, including pathological subtype, nodal invasion, and metastasis, have traditionally been used to predict illness outcomes. Aside from these well-established prognostic factors, aberrant DNA methylation and microRNA expression have been widely researched and published in the literature due to the distinct properties that make them well-suited as prospective prognosis markers. The development of novel biomarkers to aid in predicting outcome and tumour response is crucial for improving therapeutic efficacy and preventing over-or under-treatment of lung cancer patients. TSG hypermethylation is essentially related to poor outcomes. Zhang et al. utilized methylation-explicit polymerase chain reaction (PCR) to explore the methylation status of 15 genes in 64 matched NSCLC and encompassing normal tissues. The analysts found that patients with at least four methylated genes simultaneously had a worse two-year progression-free survival . Lung caMiRNA methylation as a new biomarker in lung cancerMiRNAs are hypothesised to be novel epigenetic modifiers of gene expression that play critical roles in a wide range of biological processes. They are commonly released from tissues as exosomes or microvesicles after apoptosis and found in the bloodstream. MicroRNA levels in the blood have been connected to prognosis. Several studies have discovered a relationship between microRNA activity in blood or plasma and lung cancer outcomes . AlthougEpigenetics is the promising frontier of science entailed by copious reinforcing signals, embracing DNA methylation, micro RNAs, histone modifications, and chromatin remodelling. Epigenetics is essential in the aetiology of lung cancer and can be helpful as diagnostic and prognostic biomarkers. Large multicenter consortia are unavoidable to attain the statistical power needed for clinical biomarker validation and capitalise on DNA methylation's enormous promise to offer precise and sensitive diagnostic tools for early detection of lung cancer. The subject of DNA methylation biomarking is quickly growing, with promising discoveries that might have enormous implications for cancer patients in the future. Expanding our understanding of how epigenetic alterations contribute to lung cancer genesis and how they are being translated into clinically valuable markers and therapeutic targets will allow us to manage lung cancer effectively and, as a result, reduce the devastating global burden of this disease.


Community engagement is a critical component of translational research. Innovative educational approaches to support meaningful involvement of stakeholders in clinical research allows for bidirectional learning and greater engagement in translational efforts. Our Penn State Community-Engaged Research Core (CeRC) team has developed an innovative research curriculum for a variety of stakeholders, including patient partners, organizational representatives, and Community Health Workers (CHWs). This brief report will outline unique curricular approaches, guided by adult learning principles, to enhance stakeholder education and engagement in activities. Initial evidence of impact on learning is also reported. Engaging nontraditional partners  throughout the research process can further research priorities, enhance methodology, and accelerate translation to inform policy and practice and reduce disparities –3. This Growing evidence suggests a positive resultant impact of stakeholder engagement in research –3,8,9; hThe second gap is the utilization of stakeholders in effective research dissemination. Stakeholders representing community organizations, practice, or policy are well positioned to provide insight into research activities and can serve as conduits for results dissemination through their broad networks ,17. AddiDiverse stakeholders from multiple funded projects participated in our stakeholder educational programming to advance both their knowledge of PCOR and research engagement through project-tailored curriculum Table . To illuIn the second PCORI-funded project, PaTH to Health: Diabetes, patient partners, and other community stakeholders representing policy, practice, and professional organizations were engaged in educational trainings at key intervals throughout the 5-year project. Project stakeholders (n = 22) were recruited from prior participation on advisory boards, clinician referrals, or were strategically invited to participate, given their shared focus of improving diabetes outcomes.This unique educational portfolio developed by the core engagement team is classified into two categories: education about community engagement strategies to support engagement efforts; and specific training at key research phases within PCOR Fig. . All eduAlthough CHWs are uniquely poised to help address health problems created or exacerbated by public health crises, as well as engage minorities in research related to these resultant health disparities , they haEngagement activities in the PaTH to Health: Diabetes study has been described elsewhere ,17. ThisIn addition, partners attended Just-In-Time (JIT) trainings to address foundational knowledge gaps as the project entered key research phases, including research ethics, data extraction, data analysis, and results dissemination. Content focused on critical considerations in observational research, including how electronic health record (EHR) data can be used to answer research questions, the importance of data standardization and data security, and how study results can be shared with scientific and lay audiences. These interactive modules developed by project staff with senior research experience and adult education training included lay language, clear objectives, and opportunities for case analysis, group brainstorming, and debriefing . TraininThe AHeaD with CHWs in COVID-19 project team adapted Kirkpatrick’s Four Levels of Training Evaluation   to evaluIn the PaTH to Health: Diabetes study, stakeholders had the opportunity to participate in yearly quantitative and qualitative evaluations assessing the breadth of engagement activities, including education offerings, from the prior project period. This data helped elucidate the effectiveness of the research team’s involvement of stakeholders and of training and other engagement strategies as enhancements to stakeholder contributions to the project.All quantitative evaluation data were collected and managed using Research Electronic Data Capture (REDCap) hosted at the Penn State University College of Medicine.t-tests, there were statistically significant increases in reported knowledge of and confidence in virtual community engagement strategies, COVID-19 resources, motivational interviewing methods, and myth-busting techniques (Table n = 13) reported that they used the motivational interviewing techniques and community engagement strategies in their work . They also reported increased confidence in utilizing myth-busting strategies (mean 4.85 ± 0.38), an important skill given the multitude of misinformation creating barriers to health during a pandemic. Further, CHWs improved in their knowledge of PCOR concepts from a low–moderate pre to a moderate–high post-knowledge level.Roughly half of the training attendees in the AHeaD with CHWs in COVID-19 project completed the pre-training evaluation, though this was not mandatory (n = 51), and 29 participants completed the immediate post-training evaluation. We found evidence of impact on learning through increased community engagement reported by training participants. When comparing pre- and immediate post-training survey results (n = 15) using paired sample The PaTH to Health: Diabetes study also found evidence of impact on learning through enhanced engagement in research activities. For example, after partners attended a data extraction training to better understand how EHR data is used for research purposes while maintaining patient confidentiality, partners co-developed an interactive whiteboard video and dissemination brief on this topic. These patient-facing resources were helpful in mitigating the stigma associated with research by describing how health information can be used safely to answer important research questions. Similarly, after attending results dissemination training on the myriad ways to communicate research results to both scientific and lay audiences, patient partners assisted in the development of lay public dissemination briefs of published study manuscripts. Partners were helpful in pulling out key manuscript highlights and conveying these sections in lay-appropriate language, with visuals to display study findings. These one-pagers effectively translate scientific findings to a general audience, underscoring the important role partners can play in disseminating results to the broader community (see Appendix). Additionally, several stakeholders participated in scientific communications, including presenting updates at network meetings and serving as co-authors on study publications.Standard research training programs are lackluster in addressing the unique needs of stakeholders partnering in PCOR efforts. This report highlights the varying approaches to stakeholder education that may be appropriate depending on the type of stakeholder and level of engagement in research, underscoring it is not a one-size-fits-all approach. Given our limited evaluation data, further research is needed to understand how educational efforts continue to impact stakeholders post-training and if a more equipped stakeholder workforce facilitates enhanced community–researcher partnerships.


The structure of arterial networks is optimized to allow efficient flow delivery to metabolically active tissues. Optimization of flow delivery is a continuous process involving synchronization of the structure and function of the microcirculation with the upstream arterial network. Risk factors for ischemic cardiovascular diseases, such as diabetes mellitus and hyperlipidemia, adversely affect endothelial function, induce capillary regression, and disrupt the micro- to macrocirculation cross-talk. We provide evidence showing that this loss of synchronization reduces arterial collateral network recruitment upon arterial stenosis, and the long-term clinical outcome of current revascularization strategies in these patient cohorts. We describe mechanisms and signals contributing to synchronized growth of micro- and macrocirculation in development and upon ischemic challenges in the adult organism and identify potential therapeutic targets. We conclude that a long-term successful revascularization strategy should aim at both removing obstructions in the proximal part of the arterial tree and restoring “bottom-up” vascular communication. Arterial networks are tree-like hierarchically branched structures that distribute blood flow via resistance-sized arteries and arterioles of gradually decreasing diameter to the distal capillary network, which is the exchange area for oxygen and nutrients. The arterial branching architecture and lumen dimensions are optimized to allow for efficient blood flow distribution while minimizing transport costs . ContinuConspicuously, analysis of arterial growth and adaptation to exercise, and arterial development during embryogenesis and neonatal stages, revealed in striking similarity, that arteries obtain their structure through inward growth. This is according to a “bottom-up” principle involving substantial retrograde communication against the direction of blood flow ,4,5. TakThe relevance of the microcirculatory vessel compartment may not seem obvious for ischemic heart disease, one of the most prevalent factors reducing health in Western societies and beyond. Becoming apparent as acute myocardial infarction due to atherosclerotic plaque rupture or hibernating myocardium due to prolonged plaque progression, coronary artery disease is treated as large artery disease by interventional or surgical means. In that perspective, the relevance of the interdependence of arteries and capillaries is easily underestimated. However, cardiovascular and genetic risk factors, which are responsible for the majority of cardiac events, are also affecting microvascular integrity. For example, capillary rarefaction in organs such as the eye and the kidney precede and predict macro vessel obstruction . CapillaIf capillaries and arteries fall victim to cardiovascular risk factors, which options are biologically available to counteract this tendency? A look into vessel physiology is warranted. Oxygen delivery requires lumenized and perfused vessel segments, not blind-ending nonperfused angiogenic sprouts. It is therapeutically relevant to generate arterioles with a patent lumen that can carry blood flow. Titrating lumen diameter is a very important aspect. According to Poiseuille’s law, blood flow through a vessel is proportional to diameter to the power four . Hence aFocusing on the bottom of the “bottom-up” principle, lumen formation ex nihilo is not a trivial process, and intense investigation has proposed several models, such as fusion of intracellular vacuoles , cord hoConventional wisdom leaves a void here and suggests that the transition of a growing arteriole with a small diameter into a large caliber arteriole with a sizeable diameter occurs upon the blood flow driven change in a number of endothelial cells lining the arterial lumen ,22. An iAttempting to fill the void between formation of a capillary sprout and its growth and integration into a perfused microvascular network, we recently described an alternative, flow-independent model, involving the enlargement of arteriolar endothelial cells, which resulted in the formation of large diameter arterioles . EndotheThe role of vascular sFlt1 acting as a rheostat to control Vegf signaling strength in the context of arterial endothelial cell enlargement may explain some of the pro-arteriogenic effects observed with the Flt1 specific ligands Vegfb and Plgf ,29,30,31Deriving instructions from the observations in development and disease modeling in small and large animals, therapeutic angiogenesis has been applied before using protein and DNA wrapped in non-viral or viral vectors. Despite high pro-angiogenic potency and multiple clinical trials, VEGF-A has not been found to clearly improve ischemic heart function . Owing tAs outlined above, angiogenesis is essential to counteract capillary rarefaction, induced by three of five factors contributing most significantly to cardiovascular disease manifestations. However, evidence in preclinical and clinical studies revealed that stimulation of this process itself does not suffice to improve flow into an ischemic muscle area (reference), particularly in diabetic large animals . Thus, aLack of PDGF-B results in a hypercirculatory dilative heart and conductance vessel phenotype contrasted by capillary rarefaction , similarConversely, combining VEGF-A with maturation factors such as PDGF-B , a pericRecent observations from developing arterial networks during embryogenesis and neonatal stages indicate that arterial trees form in reverse order. Initial arterial endothelial cell differentiation occurs outside of arterial vessels, and these pre-artery endothelial cells then build trees by following a migratory path from smaller into larger arteries, a process guided by the forces imparted by blood flow . EndotheSeveral studies have shown that in the brain and hindlimb, pre-existing arterial collateral networks develop prior to birth during critical phases of embryonic development . In the At present several known angiogenic proteins significantly impact the formation of collaterals and the extent of pre-existing collateral networks in pia and skeletal muscle including VEGFA, VEGFR2, Dll4, Notch, ADAM10, ADAM17, Gja4, Gja5, and molecules implied in macrophage behavior including Egln1 and NFkB1 ,40,41,42Since the seminal work of Wolfgang Schaper, direct growth of conductance vessels seemed to be an overarching goal: build the large roads, and the small networks will follow. Arteriovenous shunts providing an unprecedented level of shear stress locally to the vessel wall proved to be the optimal stimulus, the growth potential of which none of the classical or un-canonical growth factors could match . Local iAre ischemia- and HIF1α-driven factors without alternative, when balanced angiogenesis/arteriogenesis is at stake? Recent observations by us and others have revealed that myogenic factors such as myocardin-related transcription factors (MRTFs), which in myocytes act as cofactors of SRF to keep up specific muscle protein stoichiometry, e.g., upon mechanosensing via the Rho-A pathway ,57,58. OOf note, the AAV-Tß4 approach in rabbits subjected to femoral artery excision improved arteriogenesis even when only locally applied to the ischemic lower limb. However, this was significantly less effective in increasing flow, when applied directly to the upper limb, i.e., the collateral growth area. Microvascular growth is signaling backward to collaterals to provide more flow, absent macrovessel obstruction. Since shear stress increase would be a likely candidate transmitting this growth signal, we applied L-NAME to inhibit its main effector, nitric oxide formation . Co-applRetrograde communication has long been recognized in the context of exercise-induced vascular adaptation. Arterial networks dynamically adapt arterial branching patterning, vessel number, and lumen dimensions to exercise-induced changes in organ metabolism. These functional and structural alterations in the arterial network allow precise fine-tuning of flow delivery with the new level of metabolic activity of the organ it innervates. Metabolically active tissue regions are typically close to or in direct contact with the distal parts of the arteriolar microcirculation and the capillary network. In the exercise scenario, the stimulus for arterial network remodeling originates from the change in metabolic status of active cells in tissues surrounding the distal part of the microcirculation. A local mismatch between flow delivery and the new metabolic demand triggers the release of vascular growth factors and vasodilators which promote angiogenesis and expansion of the capillary network. They furthermore reduce vascular resistance in the periphery of the network to attract flow cf. . The locThe lessons learned from exercise scenarios is that the initial trigger provoking the arterial network remodeling is caused by a change in resistance to flow at the pre-capillary to capillary level. Metabolic alterations induce the release of specific vascular growth factors , vasoactive metabolites , vasodilators (a.o. nitric oxide), as well as retrograde transfer—from the distal to proximal—of vasodilatory responses through electrical coupling in the vessel wall (connexins). Lower resistance in the distal areas will not only attract flow to this region but will also augment flow velocity and shear rates in the more proximal parts that feed the distal region. Increases in shear rates are by themselves a stimulus for dilation or—pending the duration of the stimulus—outward arterial remodeling. Hence the changes in the distal part are communicated to the proximal segments via changes in flow and translated into a gain of lumen dimension. Several risk factors for adverse vascular remodeling such as diabetes alter metabolism and expression of vascular growth factors. Thus they are most likely to affect retrograde communication efficiency.In summary, microvessel growth requires maturation by the attraction of mural cells to provide long-lasting, functional capillary beds. In addition to providing blood to previously ischemic tissue, microvessel networks are capable of backward signaling to enlarge collaterals via nitric oxide. This interdependence can be exploited therapeutically at various entry points. Whether currently investigated vectors, such as AAVs or AdVs, and delivery systems, such as intramuscular or locoregional applications, hold up to newer developments such as modified RNA-carrying lipid nanoparticles, remains to be seen. However, it appears safe to state that neglecting microvessel network formation and maturation may lead to incomplete and unbalanced vessel growth, which is unlikely to improve the function of ischemic muscle tissue.