Senescent cell accumulation has been observed in age-associated diseases including cardiovascular diseases. Senescent cells lack proliferative capacity and secrete senescence-associated secretory phenotype (SASP) factors that may cause or worsen many cardiovascular diseases. Therapies targeting senescent cells, especially senolytic drugs that selectively induce senescent cell removal, have been shown to delay, prevent, alleviate, or treat multiple age-associated diseases in preclinical models. Some senolytic clinical trials have already been completed or are underway for a number of diseases and geriatric syndromes. Understanding how cellular senescence affects the various cell types in the cardiovascular system, such as endothelial cells, vascular smooth muscle cells, fibroblasts, immune cells, progenitor cells, and cardiomyocytes, is important to facilitate translation of senotherapeutics into clinical interventions. This review highlights: (1) the characteristics of senescent cells and their involvement in cardiovascular diseases, focusing on the aforementioned cardiovascular cell types, (2) evidence about senolytic drugs and other senotherapeutics, and (3) the future path and clinical potential of senotherapeutics for cardiovascular diseases. Age-related diseases, including cardiovascular diseases, are becoming more prevalent due to the increasing older population worldwide ,2. TheseSenescent cell accumulation is a fundamental aging process. Accumulating in vivo and in vitro evidence indicates that cellular senescence contributes to chronic disorders such as cardiovascular diseases ,6,7,8, tCIP1/WAF1 or p16INK4a/Rb, that induce cell cycle arrest in the G1 phase. Apart from telomeric dysfunction, DNA damage responses and subsequent cellular senescence can also be triggered by stimuli including oncogenes, radiation exposure, and chemotherapy [Human cells have limited replicative potential, as discovered by Hayflick and Moorehead in 1961 . They teotherapy . Cellulaotherapy ,42. Thesotherapy , excessiotherapy , and hypotherapy  contribuotherapy .Cellular senescence per se can contribute to tumor suppression. In response to DNA damage, cells enter cell cycle arrest, which suppresses uncontrolled proliferation of dysfunctional or cancerous cells. Cells that complete DNA repair can proliferate again. However, cells in which DNA repair has failed are eliminated by apoptosis. Those cells that escape such elimination become senescent and enter cell cycle arrest but are resistant to removal by apoptosis due to senescent cell anti-apoptotic pathways (SCAPs) . SenesceCIP1/WAF1 and p16INK4a/Rb, DNA damage-related factors such as gamma-H2AX (γ-H2AX), p38 mitogen-activated protein kinase (p38MAPK) activity [Markers that can be expressed by senescent cells include the cell cycle arrest proteins p53/p21activity ,53, teloactivity ,55, and activity . Howeveractivity . Pregnanactivity ,58. Inhiactivity ,60,61. Hactivity ,65,66,67activity ,69,70. Aactivity . This coactivity . These dWith senolytic clinical trials underway, there is a need for biomarkers, “gerodiagnostics”, for following the therapeutic effects of these drugs. These should preferably be measurable non-invasively in blood or other body fluids such as urine or saliva. Recent studies have proposed quantifying p16 expression of CD3-positive T cells in peripheral blood as a relatively non-invasive way to assess senescent cell burden ,74. PostCip1/Waf1 but not p16Ink4a was found [+Msr1+Ly6C-F4/80-Mac1+ [+PD-1+CD4+ T cells were identified as senescence-associated T (SAT) cells [The search for a single, common senescence marker for various senescent cell types has to date not been successful. Senescent cells often express different molecules depending on the initial trigger of senescence , the cell type that become senescent, how long the cells have been senescent, and the tissue microenvironment). One approach to detect senescent cells is to classify them by senescence markers, cell type, and localization, focusing on one sub-group of senescent cells at a time. A recent example of this approach was in studies of adipose tissue senescence induced by a high-fat diet, where upregulation of p21as found ,80. Rece80-Mac1+  which haT) cells  and a peT) cells . ClassifAccumulating evidence from in vitro and in vivo studies has revealed the molecular mechanisms behind senescent cell contribution to onset and progression of cardiovascular disorders. Here, we summarize the molecular patterns leading to cellular senescence and how various senescent cell types in the cardiovascular system can contribute to disease. , Table 2CIP1/WAF1 DNA damage response pathway, inducing proliferative arrest and cellular senescence. Chemotherapeutic drugs such as doxorubicin are also known to increase mitochondrial ROS [Endothelial cells are constantly exposed to circulating blood and pathogenic stimuli and flow/shear stress that predispose them to damage and induce senescence. Consequently, senescent cell burden is high in well-vascularized tissues with many endothelial cells . Endotherial ROS  and prodrial ROS .Senescent ECs exhibit functional abnormalities such as decreased expression of vasoprotective factors and increases in inflammatory cytokines and adhesion molecules. Senescent ECs also dysregulate blood flow and cause barrier dysfunction. They do not proliferate, impeding capacity to repair the blood vessel lumen. ECs produce the potent vasodilatory molecule nitric oxide (NO), which in addition to its vasodilatory effect mediated by VSMC relaxation, also reduces endothelial expression of adhesion molecules, improves barrier function, and prevents coagulation . FurtherINK4a and p21 CIP1/WAF1 [Vascular smooth muscle cells (VSMCs) are the main drivers of atherosclerosis, but senescent VSMCs appear not to be involved in initial plaque formation; rather, they may primarily lead to increased plaque size . AccumulIP1/WAF1 , and shoIP1/WAF1 . VSMC seIP1/WAF1 . One sigIP1/WAF1 . AccelerIP1/WAF1 . ROS havIP1/WAF1 . VSMCs aIP1/WAF1 . InhibitIP1/WAF1 . Other sIP1/WAF1 ,120, leaIP1/WAF1 . The monIP1/WAF1 . There aIP1/WAF1 . In miceIP1/WAF1 .As VSMCs senesce, they can lead to increased production of proinflammatory proteins such as IL-1α, IL-1β, IL-6, IL-8, IL-18, and TNF-α . These SInk4a expression increases with chronological age together with telomere attrition [Ink4a and p53/p21Cip1/Waf1 expression, increased SA-ßgal signaling, decreased cardiac troponin I phosphorylation, and decreased telomerase activity [As cardiomyocytes are terminally differentiated, post-mitotic cells in adult humans , senescttrition ,138. Thettrition  and hypettrition . Cardiomttrition . Cardiotactivity ,142, potactivity . Consistactivity .+-dependent sirtuins, FOXOs, and mammalian target of rapamycin (mTOR) play prominent roles in driving or inhibiting cardiomyocyte senescence. AMPK activation is reduced in aged myocardial tissues, and activation of AMPK improves mitochondrial dynamics, reduces ER stress, improves the function of cardiomyocytes, and represses cardiomyocyte senescence [Senescent cardiomyocytes produce SASP factors, including proinflammatory cytokines and chemokines, growth modulators, angiogenetic factors, and matrix metalloproteinases (MMPs) . Examplenescence ,150,151.nescence . IncreasImmune cells play a prominent role in CVDs, as demonstrated by a report showing that atherosclerotic plaques with high macrophage content were more vulnerable to rupture ,154,155.3 leukocytes per milligram of tissue in the steady state and 3380 ± 1279 CD45+ cells per cubic millimeter of tissue. Three-dimensional reconstructions of immunohistochemistry images detected CD45+ cells even within the healthy myocardium. Interestingly, cardiac muscle contained 12 times more leukocytes per milligram of tissue compared to skeletal muscle. Of all the leukocytes found in the healthy heart, only ∼13% were in direct contact with the bloodstream. Fundamental changes in cardiac leukocyte composition occur over time, affecting macrophages and T cells. The resident cardiac macrophage population (primarily CD206+ cells) significantly diminishes with aging, being replaced by granulocytes. Despite the reduction in macrophage numbers, aging does not affect the ratio of the F4/80+CD206+ and F4/80+CD206− subsets. Still, a slight increase in C-C motif chemokine receptor 2-(CCR2)-expressing macrophages was observed in the hearts of 12- to 15-month-old animals, suggesting possible macrophage replenishment [Myocardial cellular composition includes resident immune cells such as subsets of leukocytes encountered in the healthy heart. In a recent study, flow cytometry of cardiac tissue showed approximately 10nishment . Cardiacnishment . Macrophnishment . With inInk4a/SA-βgal-positive cells accumulating in aging mice [INK4a expression, display impaired cholesterol efflux, and exacerbate atherosclerosis [A recent study demonstrated that macrophages are the most common p16ing mice . In the clerosis ,161. Foaclerosis . There aclerosis . Oxidizeclerosis .+CD57+CD27−CD28null phenotype produce large amounts of IFN-γ and TNF-α and may promote inflammation in atherosclerotic disease [+CD28−CD8+ T cells and increased expression of CXCL11 has been noted in patients with hypertension compared to healthy controls, suggesting that immunosenescent cytotoxic CD8+ T cells are linked to hypertension [+ T cells producing interferon-gamma (IFN- γ) are found in high numbers in patients with acute coronary syndromes and may contribute to decline in myocardial function. Senescent CD8+ T cells have been connected to increased mortality six months after suffering a myocardial infarction [null CD4+ T cells were shown to accumulate in lymph nodes draining the heart of aged mice, and implanting these cells to young mice caused inflammation [+ T cells might infiltrate the heart and promote inflammation and an increased stress response, causing heart failure [+CD8+ T cells have been observed in patients with acute myocardial infarction (MI) in higher concentrations than controls, and their numbers correlate with post-MI cardiovascular mortality [Besides macrophages, senescent-like T cells may also be involved in the pathogenesis of chronic inflammatory diseases, including vascular diseases . In T ce disease . Further disease . It is s disease . Senesce disease . The res disease . During  disease . Senescertension . Senescefarction . In HIV-farction . IFN-γ-pammation ,174. It  failure . Senesceortality . PerhapsEndothelial progenitor cells (EPCs) from the bone marrow can participate in postnatal neovascularization and vascular repair ,177,178.Cardiac progenitor cells (CPC’s) can differentiate into cardiomyocytes, VSMCs, and ECs, and their myogenic potential is especially important since differentiated cardiomyocytes have low proliferative capacity. One characteristic of CPCs is expression of the protein c-kit; in the adult heart, only approximately 2% of the cells express this protein. Senescent CPCs accumulate in the heart right atrial appendage  and mostSenescent CPCs can produce proinflammatory and profibrotic SASP components such as IL-1ß, IL-6, IL-8, PAI-1, and MMP-3 . These pCardiac tissue obtained from nonaged (50- to 64-year-old) patients with type 2 diabetes mellitus (T2DM) and without DM (NDM) and post-infarct cardiomyopathy had higher ROS production in T2DM, which was associated with an increased number of senescent/dysfunctional T2DM-human CPCs with reduced proliferation, clonogenesis/spherogenesis, and myogenic differentiation versus NDM-CPCs in vitro. Moreover, T2DM-CPCs expressed a pathologic SASP .Further studies are needed to more fully elucidate the characteristics of senescent EPCs and CPCs and their role in CVD development. As of today, consensus about methods for identifying these cells and their function has not been reached. As discussed above, several groups have independently reported that senolytic administration improves CPC function and have suggested that progenitor cell senescence may become a target for future CVD interventions.Ink4a and p21Cip1/Waf1, were increased in post-myocardial infarction mouse hearts, and costaining of α-SMA with p53 or p16 supports the possibility that senescent myofibroblast numbers are increased in infarct-border regions [Ink4a, p21Cip1/Waf1, and SA-ßgal were higher in cardiac fibroblasts (CF), up to 20 times compared to sham models. These senescent cells accumulated within fibrotic areas [INK4a, p21CIP1/WAF1, and SA-ßgal positive cell populations. Another group found that heart tissue from patients undergoing ablation for atrial fibrillation (AF) exhibited increases in senescence markers co-localized with vimentin and α-SMA. Senescent fibroblasts were found to accumulate in the adventitial layer of blood vessels in the lungs of a pulmonary hypertension mouse model [Cellular senescence was originally discovered by L. Hayflick et al. using fibroblasts . In the  regions ,187. Senic areas . Inducedse model ,190. Anose model .When activated by injury, resident cardiac fibroblasts (CFs) may transition into being myofibroblasts with α-smooth muscle actin expression, possibly helping to attenuate injury due to increased production of extracellular matrix (ECM) components. During and after repair, these myofibroblasts may mature into matrifibroblasts or return to their initial state. With continuous stress, CFs can undergo apoptosis or develop a senescent-like state with increased cell cycle-arresting proteins and SASP expression. Senescent CFs and myofibroblasts have been suggested to drive pathologic fibrosis and can be identified through their increases in platelet-derived growth factor, vimentin, and α-smooth muscle actin co-localized with senescence markers. Senescent CFs may contribute to cardiomyocyte senescence through paracrine signals and extracellular matrix modulation (ECM) . SenesceThe senescent cells that accumulate with increasing age are damaged, dysfunctional cells without the proliferating capacity needed to repair tissue. These cells can promote chronic inflammation by producing SASP factors. The geroscience hypothesis is that targeting “hallmarks of aging”, such as cellular senescence, may delay, prevent, alleviate, or treat multiple chronic diseases . FurtherCIP1/WAF1), BCL-xL, mitochondrial pathways, and plasminogen-activated inhibitor-2 (PAI-2) [INK-ATTAC mice, from which highly Ink4ap16-expressing cells can be targeted for apoptosis using an agent, AP20187, that does not target cells without high expression of Ink4ap16 [INK-ATTAC mice it is those cells with high Ink4ap16 expression that are targeted, the effects of senolytics could differ from those in INK-ATTAC mice treated with AP20187.We tested if senescent cells with DNA damage and a pro-apoptotic SASP avoid undergoing apoptosis due to activation of senescent cell anti-apoptotic pathways (SCAPs) . Bioinfo (PAI-2) ,203,204. (PAI-2) ,206. D,  (PAI-2) . Quercet (PAI-2) . Combini (PAI-2) . Mouse mp16Ink4a . HoweverBCL-xL siRNA studies in the first article about senolytics, two groups subsequently found that the BCL-xL/BCL-2/BCL-w inhibitor, navitoclax (ABT263), also exhibits senolytic activity [Transplanting small numbers of senescent cells into middle-aged mice is sufficient to cause frailty, physical dysfunction, and premature onset of most or all of the diseases found in older non-transplanted mice . It was activity ,211. Manactivity , the flaactivity ,213, pipactivity , procyanactivity , FOXO4-ractivity , and sevactivity . Tissue-activity ,197,216.A new senolytic drug of interest is digoxin, a cardiac glycoside that has been used for many years for cardiac disease. Transplanted senescent cells are eliminated by digoxin administration in mouse models ,218. RepInk4a or p53-p21Cip1/Waf1 pathways would likely increase risk of cancer [Adipocyte-specific or EC-specific high-fat fed p53 knockout mice have improved metabolic function ,95,226. f cancer ,230,231,Ink4a/Arf locus activity. The same pathway is involved in IL-6 production and makes cells more receptive to OSKM factors [Another possible strategy is to reprogram cells using the Yamanaka factors, Oct4, Sox2, Klf4, and c-Myc (OSKM), a discovery for which the Nobel prize was awarded in 2012 . This ap factors . A probl factors ,239,240.+/NADH metabolism [Cip1/Waf1-Crep21 mice in which removal of those cells highly expressing Cip1/Waf1p21 can be induced. Inactivating the NF-κB pathway in cells with high Cip1/Waf1p21 expression yielded the same results as removing the cells, and both methods attenuated insulin resistance in obese mice [SASP inhibitors, “senomorphic” drugs, do not eliminate senescent cells directly. They actabolism . The effese mice ,80. Therese mice  and cognese mice , attenuaese mice ,247, andese mice . Rapamycese mice , cognitiese mice , immune ese mice , frailtyese mice , and posese mice . Ruxolitese mice  alleviatese mice  and fraiese mice . After tese mice ,256. A cSeveral preclinical studies have tested senotherapies, including senolytics for cardiovascular dysfunction and diseases . For exaIn atrial fibrillation (AF), the most common arrhythmia worldwide affecting aged individuals, pathological fibrosis is theorized to disrupt electrical conductance through cardiomyocyte gap junctions in the atrium ,264. SenINK4ap16 expression and SA-β-gal are positively correlated with fibrotic lesions [While fibrotic diseases are a promising target for senotherapies, there are a few reports that cellular senescence suppresses fibrosis. In the human atrium,  lesions . This fi lesions . While fIschemic heart disease, also known as coronary artery disease, causes myocardial necrosis leading to heart failure and other sequelae due to the low regenerative capacity of the adult heart. D+Q has been shown to activate approximately 10% of cardiac resident CPCs in aged mice  and in hINK4a positive CPCs being found in heart tissue biopsies from patients treated with doxorubicin and increased cardiac p53 expression in doxorubicin-treated mice [Advances in treatments have improved survival rates in cancer patients, and side effects of cancer treatments, particularly in the cardiovascular system, have become an important issue for cancer survivors ,277. Theted mice . RadiatiCIP1/WAF1 positive cells in patients with thoracic aortic aneurysms and dissections. Senescent VSMCs seem to cause the vessel wall to be weakened against hemodynamic pressure, contributing to aneurysm formation. D+Q reduces Ang II-induced abdominal aortic aneurysm (AAA) size in aged mice [In atherosclerotic diseases, senescent ECs, VSMCs, fibroblasts, and inflammatory cells cause or worsen plaque buildup and destabilize existing plaques. Senolytics such as navitoclax ,279, antged mice . UBX1967ged mice .Pulmonary arterial hypertension (PAH) is caused by thickening of the muscle  layer in pulmonary arteries and arterioles. PAH can lead to irreversible and detrimental cardiac changes as the right ventricle adapts to high pressure and medial thickening in the arteries also becomes irreversible over time. There is no cure for PAH so far; treatments can only alleviate symptoms, and in some cases, lung transplantation is necessary. A recent study has brought attention to senescent VSMC accumulation as a potential cause of irreversible PAH, and senolytic treatment successfully alleviated the condition in a monocrotaline- and shunt-induced PAH rat model . HoweverInk4a and p21Cip1/Waf1, in the ischemic limb along with reductions in factors such as VEGF-A, HIF1α, and PPARγ coactivator 1α. Targeting EC senescence using EC-p53 KO mice improved vasodilation and angiogenesis [Peripheral artery disease (PAD), also known as vascular claudication, is caused by narrowing of the arterial lumen due to atherosclerotic plaque buildup in the extremities. Critical limb ischemia is a serious consequence of PAD in which there is inadequate blood flow to the extremities, causing ischemic tissue damage that can eventually necessitate amputation in 10 to 40% of cases and may even cause death from necrosis and infection. In addition to atherosclerosis, reduced angiogenetic capacity to allow bypassing the narrowing vessels due to EC dysfunction and senescence may be an important contributor. In a murine model of induced limb ischemia, old mice exhibited increased senescence markers, such as p16ogenesis ,96. Advaogenesis ,288,289.ogenesis , suggestSystolic hypertension, characterized by high systolic blood pressure and normal diastolic blood pressure, is a common problem in the elderly . It is aThe aging lung is prone to low-grade chronic inflammation, with inflammatory SASP factors such as IL-6 being present at greater abundance in bronchoalveolar lavages from elderly compared to young patients. Factors such as cigarette smoke exposure and other inhaled toxins can drive senescence and increase release of SASP factors. Senescent cells can express many inflammatory and pro-fibrotic factors, such as MMPs, that disrupt tissues and the extracellular matrix and induce replacement of functioning organ tissue by fibrotic tissue. Senescent cell accumulation increases susceptibility to infections, disrupts alveolar gas exchange, and impairs repair after pulmonary injury. Senescent pulmonary cells secrete SASP factors that can accelerate malignancies ,296,297.Finally, preeclampsia, a hypertensive disorder that can occur during pregnancy, has been shown to be associated with pathological senescent endothelial cell and syncytiotrophoblast accumulation . IncreasIncreasing senescent cell burden in the cardiovascular system with aging parallels the increasing prevalence of cardiovascular diseases in elderly patients. Senescent cell accumulation can be detected in virtually every cardiovascular tissue and organ. Anti-senescent cell therapies, along with other strategies targeting the fundamental aging mechanisms, have potential to increase the healthy years of our lives. Besides pharmaceutical interventions, lifestyle modifications such as caloric restriction and exercise may enhance healthspan due to multifactorial effects, including but not limited to reduced senescent cell burden. According to The Unitary Theory of Fundamental Aging Mechanisms that builds on the Geroscience Hypothesis, senotherapies could have effects like those of these interventions because their effects on fundamental aging processes may interact. Cardiovascular diseases lead to high morbidity and mortality burden and are a primary target for strategies targeting fundamental aging mechanisms. Challenges include lack of long-term clinical studies evaluating the effects of systemic senescent cell elimination. Even in the case of senomorphics, while some drugs such as metformin appear to be well studied and safe, long-term effects on systemic and local tissue homeostasis are not fully understood. For example, some senomorphic compounds might be immunosuppressive. Cell type-specific senolytics might provide information about different forms of senescent cells and their contribution to cardiovascular diseases. Several dozen new senolytic agents have been discovered and are currently being tested extensively. When targeting senescent cells in vivo, it is important to recognize that cellular senescence is a mechanism that prevents tumorigenesis and is important during embryogenesis, possibly wound healing (although removing senescent cells appears to enhance healing of chronic wounds ,303, and


Arabidopsis thaliana.Plants, as sessile organisms, show a high degree of plasticity in their growth and development and have various strategies to cope with these alterations under continuously changing environments and unfavorable stress conditions. In particular, the floral transition from the vegetative and reproductive phases in the shoot apical meristem (SAM) is one of the most important developmental changes in plants. In addition, meristem regions, such as the SAM and root apical meristem (RAM), which continually generate new lateral organs throughout the plant life cycle, are important sites for developmental plasticity. Recent findings have shown that the prevailing type of alternative splicing (AS) in plants is intron retention (IR) unlike in animals; thus, AS is an important regulatory mechanism conferring plasticity for plant growth and development under various environmental conditions. Although eukaryotes exhibit some similarities in the composition and dynamics of their splicing machinery, plants have differences in the 3’ splicing characteristics governing AS. Here, we summarize recent findings on the roles of 3’ splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities in  Furthermore, the adenine-uridine (AU)-rich sequences throughout the whole length of the introns in plants are essential for recognizing the introns unlike in animals . De growth . These rAtSF1) has also been identified in Arabidopsis using forward and reverse genetic approaches [AtSF1 differentially binds to the BPS of different introns in FLM pre-mRNA in a temperature-dependent manner to regulate the production of the major functional FLM-β transcripts, thereby eventually affecting the temperature-responsive flowering  analyses revealed that the AtSF1-FLM module regulates temperature-dependent flowering by regulating the FLOWERING LOCUS T (FT) and LEAFY (LFY) expression in the leaf and shoot apex regions, respectively [AtSF1 result in developmental abnormalities, including plastochron length, dwarfism, and hypersensitivity to abscisic acid during seed germination, heat stress, and chloroplast development under cold stress [AtSF1 is essential for various developmental processes and abiotic stresses.In addition to the interaction between U2AF65 and U2AF35 for the recognition of the 3’ ss, splicing factor 1 (SF1) associated with U2AF65 recognizes the 3’ ss and binds to the BPS of introns [proaches ,40. A relowering , Table 1ectively . Furtherd stress ,41,42,43atu2af65a and atu2af65b mutants showed opposite flowering times [atu2af65a mutation compared to the atu2af65b mutation [AtSF1, AtU2AF35, and AtU2AF65) and RNA sequences is required.SF1, U2AF35, and U2AF65 function at the early stage of pre-mRNA splicing, during which it binds the BPS, the PPT, and the 3’ ss of the intron of the pre-mRNAs . Howeverng times , and themutation . Thus, fArabidopsis genome encodes 18 SR proteins that represent six different subfamilies, three of which  are orthologous to the animal SR proteins, whereas the other three are plant-specific and have structural features that are not found in the animal kingdom [AtSR30, AtSR34, AtSR34a, and AtSR34b), RSZ , SC (AtSC35), SCL , RS2Z (AtRS2Z32 and AtRS2Z33), and RS  subfamilies. For example, the members of the SCL subfamily, which are structurally related to the SC subfamily, have a unique N-terminal domain rich in charged amino acids. RS2Z members, resembling the RSZ subfamily, possess an additional zinc knuckle and a serine- and proline-rich acidic carboxyl-terminal domain [The first known plant splicing factors that regulate pre-mRNA splicing and spliceosome assembly were the SR family proteins . They afl domain . Althougl domain , the divAtSR40, AtRSZ22, AtSCL30, and others (AtSR45 and AtRSZ22a) interact with the cold-induced LAMMER KINASE AME3, and their loss-of-function mutants exhibit cold sensitivity [AtSC35 and other AtSCL proteins affect various developmental processes, such as the leaf and root morphology, flowering time, and silique phyllotaxy . These results suggest that AtSR45 regulates developmental plasticity without functional redundancy. and ABA , Table 1pre-mRNA . AtSR45 PSEUDO-RESPONSE REGULATOR 7 (PRR7) and PRR9, have also been reported in snw/ski interacting protein (skip)-1 mutant, a splicing factor that interacts physically with AtSR45 [SERRATED LEAVES AND EARLY FLOWERING (SEF), which eventually affects the H2A.Z enrichment at FLC, MADS AFFECTING FLOWERING 4 (MAF4), and MAF5 [Altered splicing patterns in the circadian clock genes, including h AtSR45 , suggesth AtSR45 . Interesh AtSR45 . Geneticand MAF5 .sme1 mutants, RNA-seq analysis showed that the FLC expression was severely reduced, which correlated with the early flowering phenotype. In addition, RNA expression analyses revealed that the transcripts of COOLAIR class I isoforms accumulated, whereas those of the COOLAIR class II isoforms were reduced in sme1 mutants. These results suggest that SME1 affects the pre-mRNA splicing of COOLAIR, thereby leading to the accumulation of FLC transcripts.The accumulation of the U1, U2, U4, and U5 small nuclear RNAs (snRNAs) in the spliceosome core complex is facilitated by another spliceosomal core component . Sm protSeveral recent studies have revealed that the plant SR proteins affect various developmental plasticities, including the flowering time, plant morphology, and abiotic stress. Considering the large number of SR proteins, distinct structural features in plant-specific subfamilies, and the interactions between them and other spliceosomal proteins , a singlDevelopmental plasticity is a critical process in plants because of their sessile properties and determines most developmental changes after embryogenesis during the entire lifespan. Therefore, the plastic properties of plants are required for their adaptive growth and development in response to unfavorable environmental conditions. In plants, primary meristems, including shoot apical meristems (SAMs) and root apical meristems (RAMs), contain undifferentiated stem cells and differentiating cells for lateral organ formation . Thus, tPCP, which is related to temperature-sensitive AS with SAM maintenance through a complex regulatory network , whereas the pcp-1 mutant grew similarly compared to wild-type plants at a normal temperature (23 °C). Interestingly, the PCP gene was shown to generate two transcript variants, PCP-α and PCP-β, via AS, and only the PCP-α isoform acted as an active form for appropriate plant growth at low ambient temperatures. Although the misregulation of the SAM maintenance genes, such as WUSCHEL (WUS) and CLAVATA3 (CLV3), was found in pcp-1 mutants at a low ambient temperature (16 °C), but not at 23 °C, neither the WUS nor CLV3 promoter-driven PCP expression rescues the pcp-1 mutant phenotypes, suggesting that the effect of PCP associated with temperature-sensitive AS indirectly affects SAM maintenance via regulating WUS and CLV3.Recent findings revealed the putative splicing regulator  network , Table 1PCP has also recently been characterized as SmE1 [sme1 mutants. Furthermore, the expression of SmE1 was increased at a low temperature (4 °C), indicating that SmE1 regulates the spliceosome activity depending on the environmental conditions. Interestingly, changes in the splicing events correlated with the developmental defects, such as smaller rosette leaves, early flowering, and short root growth, exhibited in sme1 mutants at a normal temperature (20 °C), suggesting that SmE1 regulates plant development via splicing activity. Although these two reports showed inconsistent growth phenotypes at normal temperatures owing to the different experimental conditions [Intriguingly,  as SmE1 , which e as SmE1  and inte as SmE1 ,65. Huer as SmE1  found thnditions ,59, theyskip mutants show severe developmental defects, such as failed shoot meristem formation, which was also observed in loss-of-function stm mutants, suggesting that SKIP, like STM, is required for SAM initiation and maintenance. In addition, SKIP-STM interactions have been shown to regulate downstream target genes, such as STM, Knotted-1-like 1 (KNAT1), CLV3, and GA2-oxidase 1 (GA2OX1), by binding to their promoters, supporting the transcriptional function of the SKIP-STM heterodimeric complex in SAM development. Interestingly, in plants, SKIP has previously been reported to interact with spliceosome components and to function in the circadian clock and salt stress responses by regulating AS [In addition to the negative feedback loop of the CLV3-WUS pathway , SHOOT Mating AS ,72, suggCLV3, WUS, and STM are insignificant [Several recent studies have raised the possibility of AS-mediated plastic development in SAM. However, because the splicing defects of the essential meristem maintenance genes such as nificant ,71, the RNA-directed DNA METHYLATION 16 (RDM16), which encodes a component of the U4/U6 snRNP complex involved in pre-mRNA splicing, increased the overall alterations in AS events in RNA-seq analysis and showed a short root phenotype [rdm16-4 mutant, a truncated RDM16 protein lacking the DUF1115 domain affected the AS of the root stem cell maintenance genes, such as PLETHORA1 (PLT1) and PLT2, and the cytokinin signaling genes, such as ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN5 (AHP5), ARABIDOPSIS RESPONSE REGULATOR1 (ARR1), ARR2, and ARR11, thereby leading to a disordered stem cell niche and reduced cytokinin response during root growth, respectively [rdm16 mutant is hypersensitive to salt and ABA with morphological defects [AS events have also been found to modulate the plastic development in root meristems , Table 1henotype ,74. In tectively , which iectively . Since Rectively , and the defects , these rlight-sensitive root-hair development 1 (lrh1), which exhibited enhanced root hair formation in response to light, whereas primary root elongation was inhibited. Interestingly, LRH1 is known to encode the p14 protein, a putative component of the SPLICING FACTOR 3b (SF3b) complex, for pre-mRNA splicing [lrh1 mutants [lrh1 mutant phenotype, these results suggest a possible link between AS and root growth for developmental plasticity in plants. However, as the genes involved in root hair development are not alternatively spliced in lrh1 mutants, a more direct mechanism by which AS affects root hair development should be further investigated.The formation of root hairs from epidermal cells expands the surface area of the root to absorb water and nutrients from the soil. Therefore, the dynamic morphogenesis of the root system, including root hairs, is important for plants that encounter various soil and environmental conditions ,79. A resplicing . In addisplicing . RNA-seq mutants . MoreoveThe studies demonstrate the involvement of AS in the development of SAM and RAM , Table 1Numerous studies have shown that the AS of the target pre-mRNAs regulated by splicing or splicing-related factors is a central mechanism of the plant growth, development, and abiotic stress responses, ensuring physiological and phenotypic plasticity and mediating the integration of various environmental cues. In this review, we focused on discussing the role of the 3’ splicing factors and their interacting partners in regulating the flowering time and other developmental plasticities, and the selected associated factors of the splicing machinery in the primary meristems.cis-elements, intron retention (IR), alternative 3’ ss selection, and alternative polyadenylation [Arabidopsis and crops provides the strategies to improve the plant productivity under unfavorable conditions.Although the mechanisms and factors underlying AS are largely conserved among eukaryotes, several plant-specific AS properties exist, including the less conserved nylation ,45,46,48nylation ,27. Thusnylation . Therefo


Vascular endothelial cells (ECs) that constitute the inner surface of blood vessels are essential for new vessel formation and organ homeostasis. ECs display remarkable phenotypic heterogeneity across different organs and the vascular tree during angiogenesis and homeostasis. Recent advances in single cell RNA sequencing (scRNA-seq) technologies have allowed a new understanding of EC heterogeneity in both mice and humans. In particular, scRNA-seq has identified new molecular signatures for arterial, venous and capillary ECs in different organs, as well as previously unrecognized specialized EC subtypes, such as the aerocytes localized in the alveolar capillaries of the lung. scRNA-seq has also revealed the gene expression profiles of specialized tissue-resident EC subtypes that are capable of clonal expansion and contribute to adult angiogenesis, a process of new vessel formation from the pre-existing vasculature. These specialized tissue-resident ECs have been identified in various different mouse tissues, including aortic endothelium, liver, heart, lung, skin, skeletal muscle, retina, choroid, and brain. Transcription factors and signaling pathways have also been identified in the specialized tissue-resident ECs that control angiogenesis. Furthermore, scRNA-seq has also documented responses of ECs in diseases such as cancer, age-related macular degeneration, Alzheimer’s disease, atherosclerosis, and myocardial infarction. These new findings revealed by scRNA-seq have the potential to provide new therapeutic targets for different diseases associated with blood vessels. In this article, we summarize recent advances in the understanding of the vascular endothelial cell heterogeneity and endothelial stem cells associated with angiogenesis and homeostasis in mice and humans, and we discuss future prospects for the application of scRNA-seq technology. Vascular endothelial cells (ECs) constitute the inner cellular lining of all blood vessels, including arteries, veins and capillaries, and are therefore essential for maintaining life. Dysfunction of ECs is associated with diverse pathologies, such as cancer, ischemic diseases, neurodegeneration, infection, and inflammation . HeterogEphrin B2, NRP1, Dll4, Alk1, Depp, Hey1, and Hey2, while veins express Eph B4, COUP−TFII, and NRP2 , an emerging and rapidly expanding technology that can profile individual cell transcriptomes . The scRRecent advances in scRNA-seq include the identification of new and rare EC subtypes, such as aerocytes , the hieThe first and probably the most important step in scRNA-seq is the acquisition of viable single-cell suspensions from the tissue of interest. Various methods have been reported, including organ perfusion, laser capture microdissection, selective attachment to culture dishes, manual sorting, magnetic-activated cell sorting (MACS), and fluorescence-activated cell sorting (FACS) . Among t+CD45− cells can be sorted by FACS. Recent studies have explored digestion methods for each tissue, including liver, heart, lung, kidney, brain, and retina  depends on the size, softness, and age of the target organ . Under-dd retina .Following sorting of the single-cell suspensions of ECs, each cell is captured for scRNA-seq . The droThe organ-specific heterogeneity of ECs is induced early during embryonic development and persists throughout adulthood to maintain diverse organ functions. Features of the surrounding tissue-specific microenvironment, such as neighboring cells, hemodynamics, organ-dependent growth factors, and metabolism, play an important role in maintaining this EC heterogeneity. Interestingly, each vascular EC subtype is characterized by distinct gene signatures associated with its respective role The lung has two different circulatory pathways. The pulmonary circulation participates in gas exchange, whereas the bronchial circulation delivers oxygen and nutrients to the bronchial walls. In the pulmonary circulation, the pulmonary arteries carry deoxygenated blood to the alveoli. The alveolar capillaries play an important role in blood gas exchange by adding oxygen from the alveoli to the blood and transferring carbon dioxide from the blood to the air in the alveoli. The pulmonary veins then return the newly oxygenated blood to the left atrium of the heart.Every alveolar capillary EC contains angiotensin-converting enzyme, whereas this enzyme is present in only 10% of systemic capillary ECs. The alveolar ECs are a major site of conversion of angiotensin I to the active vasoconstrictor form angiotensin II and therefore play a role in regulating vascular tone . The pulApln and/or Car4 and represented a unique subtype specialized for gas exchange and leukocyte trafficking in the alveoli  that can bind to the corresponding receptors (Kit and Aplnr) on gCaps. Conversely, gCap cells strongly express ligands (Edn1 and Vegfa) that can bind to the corresponding receptors (Ednrb and Kdr) on aerocytes. Both EC types were found to be conserved between mouse and human lungs but neither was identified in turtle or alligator lungs, indicating that these ECs may have arisen during the evolution of the mammalian lung , the pattern recognition receptors SPON2 and CLEC4E, and signaling mediators, such as CHRM2, EDA, PRKG, and S100A4 (HPGD), their lack of endothelial-specific Weibel-Palade bodies, and the absence of expression of genes such as PTPRB, CD93, THBD, ANO2, LIFR, CRIM1, CYYR1, PALMD, GNA14, MECOM and LEPR that are otherwise expressed in all other ECs. Conversely, human gCaps can be distinguished by their expression of genes related to innate immune responses , transcytosis of low-density lipoprotein cholesterol and other lipids , and expression of cytokine receptors . The ratio of aerocytes to gCap ECs is typically 0.48:1 in humans. Previously indistinguishable subtypes have also been identified among venous ECs, including pulmonary–venous ECs (COL15A1-negative) that are localized to the lung parenchyma and systemic–venous ECs (COL15A1-positive) that are localized to the bronchial vasculature and the visceral pleura , capillary ECs , other capillary ECs , and vein ECs   .BMX, SEMA3G, LTBP4, FBLN5, SERPINE2, NOS1, PDE3A, GJA5, GJA4, EFNB2, SOX17, DKK2, DLL4, and HEY1 (CA4 (orthologous to mouse Car4) and CYB5A related to gas exchange, PRX and SPARC that maintain structure, and AFF3 and MEIS1 that are related to transcription regulation. Capillary ECs were further divided into aerocytes and gCaps.In humans, arterial ECs were identified by their expression of and HEY1 . These gNR2F2 (COUP-TFII), VCAM1, ACKR1 and ACTN1, which are associated with transcription factors, diapedesis of leukocytes, transcytosis, and membrane proteins, respectively. Venous ECs contained two distinct populations, pulmonary-venous and systemic-venous ECs.Venous ECs were identified through their expression of the canonical transcription factor The kidney is critical for regulating the composition and volume of body fluids. The renal artery enters the kidney and divides into segmental arteries, followed by branching into interlobar arteries . The intTbx3, Pbx1, Gata5, and Prdm1, that are essential for the structure and function of glomerular ECs in mice . For example, Gja5, also known as connexin 40, controls renin release by regulating the communication between ECs and the granular cells in the JGA  afferent arteriolar ECs that express Edn1, Gja4, Alox12, Bmp4, Cldn5, Slc18a1, S1pr1, Kcnn4, and Cxcl12; 3) efferent arterioles that express Calca, Klf4, Slc6a6 and Cryab; 4) capillary ECs that strongly express ApoE; 5) capillary ECs that show low ApoE expression; 6) post-capillary venule ECs that express Kdr, Nr2f2, Tnxb and Jup; 7) vein ECs that express Nr2f2, CD9, Gas6 and Plvap; 8) capillary ECs with an angiogenic phenotype that express Gpihbp1, Col4a1, Esm1, Col4a2, Trp53i11, Aplnr, Apln, Plk2 and Fscn1; and 9) capillary ECs that exhibit an IFN-response phenotype and express Isg15 and Ifit family genes.In the renal cortex, peritubular capillaries provide oxygen and nutrients, while contributing to the reabsorption and secretion of solutes and water . Nine suSox17, Gja4 and Fbln5; 2) descending vasa recta ECs that express Slc14a1, Scin, Aqp1, Cldn5, Slc5a3 and Crip1; 3) ECs from the papillary portion of the descending vasa recta that express S100a4, S100a6, Fxyd5, Akr1b3, and Nrgn; 4) fenestrated capillary ECs that express Plpp3 and Cd36; 5) post-capillary venule ECs that express Kdr, Nr2f2, Tnxb and Jup; 6) vein-like ECs in the ascending vasa recta that express Gas6, Tek, and Fxyd6; 7) vein-like ECs in the papillary portion of the ascending vasa recta that express S100a6, Fxyd5, Akr1b3, Aldoa, Nrgn, Crip1, Gapdh and Ldha; 8) capillary ECs with an angiogenic phenotype that express Gpihbp1, Col4a1, Esm1, Col4a2, Trp53i11, Aplnr, Apln, Plk2 and Fscn1; 9) capillary ECs with an IFN-response phenotype that express Isg15 and Ifit family genes, and 10) venous ECs with an IFN-response phenotype that also express Isg15, and Ifit family genes.The renal medulla plays a role in urine concentration. Ten subtypes of medullary ECs have been identified, as follows: 1) arteriole ECs that express SEMA3G and CLDN5, ECs in the descending vasa recta that express KDR and PTPRB, and ECs in the ascending vasa recta that express PLVAP  and the dural venous sinuses that drain into the jugular vein.Mfsd2a), amino acids (Slc7A5 and Slc3A2), and glucose (Glut1/Slc2A1) (Foxq1) and Forkhead box F2 (Foxf2), have also been identified by scRNA-seq. Several genes, such as Pglyrp1, Lcn2, and Tmem100, are expressed specifically in brain ECs but not in other organs  protecting the brain from neurotoxicity. The characteristics of brain ECs include a lack of fenestration and the expression of unique tight junction complexes, ATP-binding cassette (ABC) transporters, and transporters involved in the transport of docosahexaenoic acid (/Slc2A1) . Recent /Slc2A1) . Brain Er organs .Piezo2, Scn1b, Kcne3, Kcna5, Pcdh17, Madcam1, Mcam, Clec1a, Sirpa, Cmklr1, Serpine1/Pai, Plaur, Adm, Angpt2, Smoc2, Pde4b, Ppm1j/Pp2c-zeta, NADPH oxidase organizer-1 (Noxo1), and Hecw2  between mice and humans . For exaThe retina has one of the highest metabolic demands of tissues that are responsible for vision. The retina is supplied by the central retinal artery derived from the ophthalmic artery and its branches . The humAnother radially-oriented capillary network, consisting of the radial peripapillary capillaries, is present near the optic disc . A capilSimilar to brain ECs, retinal ECs constitute a neurovascular unit that maintains the inner blood-retinal barrier (BRB) to protect the retina from neurotoxicity . TherefoPlaur, Angpt2, Lcp2, Cxcr4, Apln, and Kcne3, recent scRNA-seq has identified new marker genes, including Mcam, Lamb1, and Trp53i11 (in situ hybridization (ISH) in whole mount mouse retinas. Gene expression profiles of the tip cells have revealed that these are more closely related to arterial ECs than to venous ECs. Another study has shown that the tip cells that guide the deep retinal vasculature (D-tip cells) are distinct from the tip cells that guide the superficial retinal vascular plexus , which is involved in the hedgehog signaling pathway, as one of the transcriptional signatures that characterize choroidal ECs . ChoroidEln, Mgp, and Gkn3, arteriolar ECs that express Cxcl12, Aqp1, and Emcn, and shear stress-induced arterial ECs that express Pi16. The two choriocapillaris ECs include the outer choriocapillaris ECs characterized by fenestration (Plvap expression) and high VEGF-pathway signaling, and the inner choriocapillaris ECs characterized by low VEGF signaling (high Flt1). The four venous EC subtypes included large-vein ECs , shear stress–induced venous ECs , post-capillary venule ECs , and venule ECs (with a mixed choriocapillaris and post-capillary venule signature).In the mouse, scRNA-seq has revealed at least 3 ECs subtypes in the choroidal arteries, two in the choriocapillaris, and four in the choroidal veins . The thrDARC, adhesion protein MMRN1, and genes associated with leukocyte recruitment, including SELE, DARC, VCAM1, and CCL23  display multi-functional properties, including the regulation of hepatic circulation, filtration, endocytosis, antigen presentation, leukocyte recruitment, and regeneration of hepatocytes . The hepscRNA-seq has revealed enrichment of the Gata family in murine liver ECs . In lineClu, Plac8, and Lrg1), arterial sinusoid ECs , sinusoid ECs (that express Stab2 and Kdr), venous sinusoid ECs (that express Thbd), vein ECs , and proliferating ECs (that express Stmn1 and Cdkn1a)  . Sinusoi Cdkn1a)   . The peratocytes .scRNA-seq of human liver ECs has also revealed a zonation of the LESCs similar to that seen in the mouse liver . These cThe coronary arteries branch off from the ascending aorta to supply the heart. The left main coronary artery (LMCA) and the right coronary artery (RCA) are two primary coronary arteries. The LMCA branches into the left anterior descending artery (LAD) and the left circumflex (LCx) coronary arteries to supply the anterior portion of the left ventricle, the left atrium, and the lateral/posterior-lateral aspect of the left ventricle. The LAD further branches into the obtuse marginal artery (OMA), diagonal artery, and septal perforator artery. The RCA branches into the sinoatrial nodal artery to supply the sinoatrial node, the atrioventricular nodal artery to supply the atrioventricular node, and the right posterior descending artery (PDA) and right marginal artery. The PDA supplies the posterior one-third of the interventricular septum. The coronary arteries branch into intramuscular arteries that penetrate the myocardium perpendicularly to form arterioles and capillaries that supply the cardiomyocytes. The coronary veins return deoxygenated blood from the myocardium through the coronary sinus to the right atrium.Myl2), myosin regulatory light chain 3 (Myl3), ADP-ribosylhydrolase like 1 (Adprhl1), alpha 2-HS glycoprotein (Ahsg), aquaporin 7 (Aqp7), xin actin-binding repeat containing 2 (Xirp2), myoglobin (Mb), sodium-coupled nucleoside transporter (Slc28a2), Butyrophilin-like 9 (Btnl9), creatine kinase, leucine rich repeats and transmembrane domains 1 (Lrtm1), mitochondrial 2 (Ckmt2), Natriuretic Peptide Receptor 3 (NPR3), cytokine-like 1 (Cytl1), Cd36, and fatty acid-binding protein 4 (Fabp4)  . The uprFbln5, Hey1, and Mecom), arterial capillary ECs (that express Cxcl12 and Rbp7), capillary ECs , venous capillary ECs , vein ECs , interferon-responsive ECs , and angiogenic ECs   .HEY1, DKK2, and EFNB2), venous ECs (that express ACKR1 and NR2F2), capillary ECs (that express BTNL9 and CD36), lymphatic ECs (that express CCL21 and PROX1), and endocardial ECs (that express NRG3 and PCDH15)  . Heterog PCDH15) .Angiogenesis is the growth of new blood vessels from the existing vasculature and occurs under hypoxic and inflammatory conditions . Angioge+VE-cadherin+CD45−Hoechstlow, have been reported as vessel-resident VESCs responsible for angiogenesis (The reported cellular sources of new ECs during angiogenesis include bone marrow (BM)-derived circulating endothelial progenitor cells (EPCs), resident mature ECs in the pre-existing blood vessels that proliferate in a stochastic manner (random model), and specialized vascular endothelial stem cells (VESCs) that reside within the pre-existing blood vessels and proliferate in a hierarchical manner  . Recent ogenesis . Howeverogenesis . Howeverin vitro and can form three-dimensional functional blood vessels in vivo after transplantation, reflecting their stem cell properties. CD157-positive VESCs were not derived from the mesenchymal lineage or BM.Our group recently identified CD157, also known as BM stromal antigen-1 (Bst1), as a marker of VESCs in mouse blood vessels that proliferate in a hierarchical manner  Figure . These CMyc, ATF3, Fosl2, Sox7, and Foxp1, that are known to regulate cellular proliferation and differentiation  metabolism and catalyzes the conversion of NAD+ to cyclic ADP-ribose (cADPR) and NADP+ to nicotinic acid adenine dinucleotide phosphate (NAADP). NAD+ is important for the function of various stem cells -anchored membrane protein that is thought to act independently as a receptor and enzyme . As a reem cells . Therefoem cells ; 2022. T+CD31lowVEGFR2lowIL33+Sox9+Sox18+, PW1/Peg3, CD117 (c-kit), and CD133 , VE-cadherinnd CD133 . PotentiUnder normal physiological conditions, heterogeneous ECs in each organ have a specific role in organ homeostasis. However, in response to the development of cancer, the characteristics of these ECs change to control pathological angiogenesis, leukocyte infiltration, and vascular permeability due to hypoxia, various tumor-derived cytokines, reactive oxygen species, and epigenetic changes. The result is a transition to a more complex heterogeneity within the tumor blood vessels . In the CD36, CD68, MARCO, MSR1, CTSD, and CTSS, while activated capillaries express EC activation markers, such as CAV1.Recent scRNA-seq of human lung cancers has identified phenotypically heterogeneous tumor EC phenotypes, including artery ECs, activated postcapillary venule ECs, immature ECs, tip cell ECs, alveolar type I capillary ECs, alveolar type II capillary ECs, intermediate capillary ECs, scavenging capillary ECs, activated capillary ECs, and lymphatic ECs  Figure . Among tANGPT2, FSCN1, APLN, PGF, ADM, PLXND1, CXCR4, and PDGFB. Enrichment of novel transcription factors, such as TCF4, SOX4, and SMAD1, has also been identified in tumor tip ECs , but they also express genes related to basement-membrane breaching, such as Itga6, Itgb1, Itgb3, Mmp14, and Hspg2. They also play a role in the initiation of vessel sprouting by degrading the basement membrane to assist tip cells to lead the sprout.The tip EC is a conserved phenotype in both human and mouse tumors, and this may indicate the important role of sprouting angiogenesis for tumor growth in across species. Common tip EC markers include  tip ECs . In addi tip ECs . Breach One characteristic of tumor ECs is an increase in glycolytic metabolism, which is required to meet the great energetic requirement for sprouting angiogenesis. Although normal ECs show glycolytic activity , tumor EscRNA-seq has also aided in the exploration of the sensitivity of specific EC phenotypes to anti-VEGF therapy in a mouse lung tumor model. Anti-VEGF treatment of ECs did not affect the components of heterogeneous EC subtypes; however, the proportion of tip cells and breach cells significantly decreased, while the proportion of postcapillary venules significantly increased. These responses indicate that tip and breach ECs in the tumor were most sensitive to VEGF blockade and the postcapillary venules least sensitive . Tip celIn addition, ECs in post-capillary venules contain a subset of VESCs. These VESCs strongly express multidrug resistance genes, so they can survive and remain in the tumor to serve as EC-supplying cells even after treatment with antiangiogenic drugs . The smaAge-related macular degeneration (AMD) is a major cause of blindness in patients older than 50 years . Two typA recent comparative study of normal choroidal ECs and ECs associated with choroidal neovascularization revealed two metabolic angiogenic targets, Aldh18a1 and Sqle, that were associated with the growth of choroidal neovascularization. Silencing of Aldh18a1 and Sqle decreased the expression of genes involved in cell proliferation and DNA replication and reduced the size of the choroidal neovascularization . Thus, svia clonal expansion  is a life-threatening condition characterized by ischemic necrosis of cardiomyocytes resulting from acute coronary artery obstruction. Necrosis of cardiomyocytes triggers inflammatory cell infiltration, extracellular matrix (ECM) remodeling, angiogenesis, and fibroblast activation. The use of Confetti reporter mice showed that the cellular source of angiogenesis after MI was the proliferating ECs that originated from resident ECs xpansion . Some stxpansion . Howeverxpansion . FurtherCol1a1, Col3a1, and Serpine1, and the downregulation of endothelial marker genes, such as Cdh5, suggesting that ECs undergo the endothelial to mesenchymal transition (EndMT)  . However (EndMT) , its rolMyl2, Myl3, Mb, Tnnt2, Actc1, and Tnni3), indicating that cardiac ECs may switch on cardiomyogenic genes in response to MI (Plvap, which plays a role in EC proliferation, and may therefore represent a new therapeutic target for promotion of cardiac repair after MI (Another scRNA-seq analysis of murine cardiac ECs after MI showed that the EC clusters upregulated genes related to cardiac muscle morphogenesis (se to MI . The ECsafter MI .The introduction of scRNA-seq technology has greatly improved our understanding of the highly diverse vascular endothelial cell heterogeneity in the context of vascular formation, organ homeostasis, and disease. Unlike bulk RNA sequencing, which has been used to study gene expression patterns at the cell population level, the ability to generate gene expression profiles at the single-cell level by scRNA-seq has enabled the discovery of specific EC cell types that exert unique functions in health and disease, thereby improving the understanding of the numerous roles of ECs in various tissues. scRNA-seq has also provided insights into vascular structure–functional relationships, gene regulatory networks, the hierarchy of ECs within the vasculature, and the roles of activated EC subtypes during pathological angiogenesis. In this article, we have discussed the EC heterogeneity across the vascular tree, including arteries, arterioles, capillaries, post-capillary venules, and veins. However, further heterogeneity may also exist within the same vascular bed; for example, within the ECs that form arteries. Further advances in single-cell analysis will hopefully provide even more detailed information regarding EC heterogeneity and EC subtype-specific responses to disease. Various single-cell omics technologies will help advance this analysis. The association between specific EC subtypes and physiological or pathological phenomena will be identified in the near future. Furthermore, a greater understanding of endothelial heterogeneity is expected to reveal the diversity within EC populations, while also identifying cellular communication with other cell types in health and disease. This information will be valuable for elucidating vascular biological mechanisms and finding new therapeutic targets . More im


The balance between anabolism and catabolism is disrupted with aging, with the rate of anabolism being faster than that of catabolism. Therefore, mTOR, whose major function is to enhance anabolism and inhibit catabolism, has become a potential target of inhibition for anti-aging therapy. Interestingly, it was found that the downregulation of the mTOR signaling pathway had a lifespan-extending effect resembling calorie restriction. In addition, the mTOR signaling pathway promotes cell proliferation and has been regarded as a potential anti-cancer target. Rapamycin and rapalogs, such as everolimus, have proven to be effective in preventing certain tumor growth. Here, we reviewed the basic knowledge of mTOR signaling, including both mTORC1 and mTORC2. Then, for anti-aging, we cited a lot of evidence to discuss the role of targeting mTOR and its anti-aging mechanism. For cancer therapy, we also discussed the role of mTOR signaling in different types of cancers, including idiopathic pulmonary fibrosis, tumor immunity, etc. In short, we discussed the research progress and both the advantages and disadvantages of targeting mTOR in anti-aging and anti-cancer therapy. Hopefully, this review may promote more ideas to be generated for developing inhibitors of mTOR signaling to fight cancer and extend lifespan. The mammalian target of rapamycin  is a phosphatidylinositol 3 kinase-related protein kinase (PIKK) that controls cell growth, metabolism, survival, immune responses and so on. mTOR forms two complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 consists of six subunits A–D: mTORThe major functions of mTORC1 are to promote anabolism and to inhibit catabolism. mTORC1 promotes protein synthesis by phosphorylating p70 S6 kinase 1 (S6K1) and eIF4E-binding protein (4EBP). mTORC1 also promotes lipid synthesis via the sterol response element binding protein (SREBP) transcription factor. Meanwhile, mTORC1 can promote the synthesis of nucleotides by the ATF4-dependent expression of methylene-tetrahydrofolate dehydrogenase 2 (MTHFD2), and S6K1-dependent activated carbamoyl-phosphate synthetase (CAD). Additionally, mTORC1 can also promote a shift in glucose metabolism by increasing the translation of the transcription factor HIF1α. mTORC1 regulates cell growth and metabolism, while mTORC2 mainly controls proliferation and survival by phosphorylating certain members of the AGC family of protein kinases, including PKA, PKG and PKC. Compared with mTORC1, mTORC2 mainly acts as an effector of insulin/PI3K signaling. Vellai et al. first revealed the key role of mTOR in aging in 2003 [mtor+/− or mlst8+/−) exhibited reduced mTORC1 activity and prolonged lifespan but still maintained normal glucose tolerance and insulin sensitivity [s6k1 leads to increased lifespan and resistance to age-related diseases, such as bone diseases, immune system dysfunction, and a loss of insulin sensitivity [s6k1 induced gene expression patterns similar to those seen in caloric restriction (CR) or those with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is a conserved metabolic regulator in response to CR [tsc1-overexpressed female mice, but not male mice, was significantly increased. A modest increase in TSC1 overexpression can enhance overall health, especially cardiovascular health, and improve female survival [Apparently, the mTOR signaling pathway is a key anti-aging target, and there has been much supporting evidence. Mice with two hypomorphic (mTOR△/△) alleles, 129S1 and C57BL/6Ncr, expressed mTOR at approximately 25% of the wild-type levels and exhibited an approximately 20% increase in median survival through reduced mTORC1 and mTORC2 activity . Even thsitivity . In femator1△ or sch9△ failed to increase lifespan in the budding yeast Saccharomyces cerevisiae [Drosophila extended the lifespan through the downregulation of S6K activity and the upregulation of 4E-BP1 activity [Although it seems that the downregulation of mTOR (including both mTORC1 and mTORC2) can result in anti-aging, its mechanism is still not clear. In the above, we have mentioned that anti-aging through the inhibition of mTOR was similar to caloric restriction in some aspects. Kaeberlein et al. found that the dietary restriction of revisiae . This reactivity . In addiactivity , ULK2, Aactivity , and lysactivity . Lopez-Oactivity .C. elegans [Residual senescent cells contribute to aging-related diseases ; therefo elegans . Pan et  elegans . Further+) is a key contributor to aging-related metabolic decline. Tarrago et al. revealed that a thiazoloquin(az)olin(on)e CD38 inhibitor, 78c, increased NAD+ levels and activated pro-longevity and health span-related factors, including sirtuins, AMPK, and PARPs, and they observed the inhibition of pathways, including mTOR-S6K, ERK, and telomere-associated DNA damage [Recent studies have shown that a reduction in nicotinamide adenine dinucleotide , who had lower mTORC1 activity in liver, muscle, heart, and kidney tissues [ghr−/− mice than in littermate controls in all four tissues tested. However, a systemic or tissue-specific  lack of mTORC2 signaling impaired metabolic health and shortened lifespan in wild-type and long-lived mice.There is growing recognition that life-extending procedures may have sex-differential effects on survival. Harrison found that rapamycin led to an increase of 14% for females and 9% for males in extending lifespan . As ment tissues . SurprisSome evidence shows that the sustained activation of mTOR downregulates insulin signaling in insulin-responsive cells and reduces insulin secretion in beta cells, which contributes to insulin resistance. However, Vodenik et al. thought that mTOR also generates an inhibitory feedback loop on insulin receptor substrate (IRS) proteins . ChronicSASP is partially regulated by mTOR, possibly through the regulation of IL1A translation or MAPKAPK2 signaling, and is suppressed while using mTOR inhibitors ,51 or MACancer therapy by targeting mTORC1 signaling has been extensively studied over the past decades, and cancer therapy by targeting mTORC2 has received more and more focus recently. The situation in cancer therapy targeting mTOR is very complicated because the signal pathways always influence each other and coordinate with each other.Studies on the tuberous sclerosis complex, a well-known negative regulator of mTORC1, provided direct evidence for mTORC1 in tumorigenesis. Feng et al. demonstrated that p53, a tumor suppressor, regulates the mTOR pathway through AMPK activation and the TSC1/TSC2 complex and regulates autophagy . They coCancer cells proliferate faster than normal cells, which means that they need more energy, lipids, amino acids, nucleotides, and so on. mTORC1 regulates aerobic glycolysis by increasing the translation of hypoxia-inducible factor (HIF)-1α, a transcription factor that drives the expression of multiple glycolytic enzymes ,67. mTORpten+/− mice [rictor gene blocks mTORC2-dependent ductal branching and reduces mammary epithelial cell viability, invasion, and survival [Low ATP/high AMP levels activate AMP kinase (AMPK), an indirect mTORC1 inhibitor that acts by promoting the activity of the TSC1/TSC2 complex . The acc+/− mice . mTORC2 +/− mice . These rsurvival . In totasurvival . Morrisosurvival .The PI3K/Akt/mTOR axis is implicated in idiopathic pulmonary fibrosis (IPF), which is considered a risk factor for lung cancer . TGF-β1 Rapamycin exhibits antiangiogenic activity associated with the reduced production of vascular endothelial growth factor (VEGF) and significantly inhibits endothelial cell response to VEGF stimulation . The preIn tumor immunity, melanoma biospecimens after PD-1 treatment showed significantly reduced p-S6 expression compared with patient-matched pre-treatment biopsies, consistent with the findings in PD-1 antibody-treated melanoma cell lines . These dThese data seem to suggest that mTORC1 or mTORC2 inhibition has benefits for cancer therapy, and dual mTORC1 and mTORC2 inhibition is better than either one in many ways.In fact, targeting mTOR to treat cancer is also confusing in some ways. Pusapati et al. found that glycolysis-resistant ovarian cancer cells cultured with 2DG have upregulated mTORC1 activity and increased adipogenesis and nucleotide synthesis . This suPalm et al. found that protein macropinocytosiscan also serve as an essential amino acid source, and the activation of mTORC1 suppresses proliferation when cells depend on extracellular proteins as a source of amino acids . Recentlatg6/becn1 in 40–75% of human prostate, breast, and ovarian cancers [becn1 heterozygous mutant mice were prone to develop liver and lung tumors and lymphomas with prolonged latency [In tumor cells deficient in amino acids in culture and in poorly vascularized tumors in vivo, mTORC1 inhibition confers a growth advantage through the upregulation of macropinocytosis and the catabolism of phagocytic proteins . The dua cancers ,107,108. cancers . These h latency ,111. How latency ,114,115. latency . mTORC2 Rapalogs have some shortcomings in terms of cancer treatment efficacy. The inhibition of mTORC1 by rapamycin blocks the phosphorylation of S6K1, but the phosphorylation of 4EBP1 is weakly inhibited . The inhCellular senescence, an irreversible cell cycle exit, is traditionally viewed as an important tumor suppression mechanism . However


N = 11 adults).We determined whether cardiovascular (CV) measures show trait-like responses after repeated total sleep deprivation (TSD), baseline (BL) and recovery (REC) exposures in two long-duration studies , and three times at months 2, 4, and 8 in an 8-month study . Participants were not shift workers or exposed to TSD in their professions. During each experiment, various seated and standing CV measures were collected via echocardiography  or blood pressure monitor [systolic blood pressure (SBP)] after (1) two BL 8h time in bed (TIB) nights; (2) an acute TSD night; and (3) two REC 8–10 h TIB nights. Intraclass correlation coefficients (ICCs) assessed CV measure stability during BL, TSD, and REC and for the BL and REC average (BL + REC) across months 2, 4, and 8; Spearman’s rho assessed the relative rank of individuals’ CV responses across measures.A 5-day experiment was conducted twice at months 2 and 4 in a 4-month study (Seated BL (0.693–0.944), TSD (0.643–0.962) and REC (0.735–0.960) CV ICCs showed substantial to almost perfect stability and seated BL + REC CV ICCs (0.552–0.965) showed moderate to almost perfect stability across months 2, 4, and 8. Individuals also exhibited significant, consistent responses within seated CV measures during BL, TSD, and REC. Standing CV measures showed similar ICCs for BL, TSD, and REC and similar response consistency.This is the first demonstration of remarkably robust phenotypic stability of a number of CV measures in healthy adults during repeated TSD, BL and REC exposures across 2, 4, and 8 months, with significant consistency of responses within CV measures. The cardiovascular measures examined in our studies, including SV, HR, CI, LVET, SVRI, and SBP, are useful biomarkers that effectively track physiology consistently across long durations and repeated sleep deprivation and recovery. Chronic sleep deprivation is a prominent public health issue associated with numerous adverse health risks including cardiovascular (CV) disease, diabetes, cancer, obesity, morbidity, mortality, and Alzheimer’s disease . In respResearch has shown short-term and long-term stability in CV measures across repeated collections without sleep loss including HR , SV Row, CI/CO , LVET G, and SBPAlthough short-term and long-term stability of CV measures without sleep loss have been observed, only one study has examined CV stability during sleep loss: HR showed long-term stability (ICC: 0.790) after exposure to 26 h TSD twice across 2.5–15 months . To the In this study, for the first time, we sought to address several important gaps in prior research with the following aims: (1) To determine the stability of various seated and standing CV measures across long-duration time points during acute TSD ; (2) To compare the long-term stability of various seated and standing CV measures across BL  and across REC ; (3) To determine the stability of seated and standing CV measures across the average of BL and REC ; and (4) To examine the relative rank of individuals across seated and standing CV measures at BL, TSD, and REC (2 and 4 months). We hypothesized CV measures would show high ICCs during BL, TSD, and REC phases across 2, 4, and 8 months. We also predicted that CV measures would show robust stability across BL + REC . Finally, we hypothesized that in terms of relative rank, most CV measures would significantly relate to one another at BL, TSD, and REC.N = 6 healthy adults; 3 females; mean age ± SD, 34.3 ± 5.7 years; mean BMI ± SD, 22.5 ± 3.2 kg/m2) from March 2019-July 2019, and a similar 8-month study  from November 2021-July 2022 in the IMBP NEK facility (N = 6), American (N = 4), and Emirati (N = 1). Participants were screened thoroughly by the National Aeronautics and Space Administration (NASA). Inclusion/exclusion criteria required that the participants come from various cultures and nationalities, have no prior experience with space flight, and include both males and females; all participants passed a drug screen and a physical exam ensuring they were in excellent health with no history of neurological, CV, integumentary, musculoskeletal, or gastrointestinal problems, and passed a psychological assessment , located in the Institute of Biomedical Problems (IBMP) of the Russian Academy of Sciences, Moscow, Russia, is an isolation facility designed to conduct research studies examining the effects of spaceflight on behavioral health and performance . We condfacility . Particisessment . The stuDuring these NEK studies, a 5-day experimental protocol was conducted twice (at months 2 and 4) for the 4-month study, and a similar experimental protocol was conducted three times  for the 8-month study. The first two experiments (at months 2 and 4) occurred on the same days in the 4-month and 8-month studies. Participants received two nights of baseline sleep with 8-h time in bed (TIB) sleep opportunity . Baseline CV measure collection occurred between approximately 0700 h–1200 h after the B2 night. Following B2 daytime, participants experienced continued wakefulness for approximately 39 h of TSD. During the TSD nights, participants were ambulatory. They engaged in a number of tasks to maintain wakefulness throughout the night during the TSD protocol, including completing various types of cognitive and operational performance tasks, team communications and decision-making tasks, and questionnaires and surveys. Participants also conducted various routine maintenance and resupply tasks in the facility. The last meal ended latest by 2200 h, to ensure a 9-h fasting period prior to CV collections the next morning (see below). During the TSD nights, participants were also monitored continuously by wrist actigraphy and by outside observers to ensure adherence. During TSD, CV measure collection occurred between approximately 0700 h–1200 h. Recovery sleep opportunities were 10 h TIB  and 8 h TIB . CV measure collection then occurred between approximately 0700 h–1200 h during R2, after the second recovery night of 8 h TIB. All assessments were conducted at the same time of day (in the morning before eating). All participants fasted for approximately 9 h or longer prior to all collections to maintain consistency across the study and among participants. Wrist actigraphy  was used to measure and verify total sleep time during the 5-day experiments . ActigraDuring these repeated experiments, CV measures were collected via echocardiography or blood pressure monitor (for SBP) at three assessment time points: (1) after two baseline 8h time in bed (TIB) nights (BL); (2) after a night of acute TSD; and (3) after two recovery nights of 8–10 h TIB (REC). SBP, SV, HR, CI, LVET, and SVRI were collected under highly controlled conditions. For the NEK 4-month study, echocardiography data were collected in both seated and standing positions, while SBP was only collected in the seated position. For the NEK 8-month study, echocardiography and SBP measures were collected in both seated and standing positions. As noted above, all collections were completed between 0700 and 1200 h.Due to strict isolation conditions, one participant collected all cardiac ultrasound images on the other four or five participants during each study, and a second participant collected all cardiac ultrasound images on the primary collector during each study. All ultrasound operators were trained to collect ultrasound images and Doppler prior to the study and repeated identical collection procedures across each time point .Stroke volume was collected via ultrasound imaging [GE Vivid q ultrasound system ] in both seated and standing postures at all time points . Two-dimAnalysis of the digital data was conducted using Echo PAC PC (BT12) software . LVOT diameters were measured just proximal to the aortic valve leaflet insertion from three consecutive cine-loops at the maximum opening of the aortic valve. Five consecutive CW Doppler waveform profiles were traced to calculate the velocity time integral (VTI). The interval between each maximum peak on the Doppler spectral from the ascending aorta was used to calculate HR. The duration of each beat was measured to determine LVET for each SV. The VTI and LVET were then transferred from the Echo PAC software to Excel to calculate SV, HR, and CI using the following formulas:Any additional CW Doppler waveforms not included in the consecutive SV analysis were analyzed for HR in the seated and standing posture where available.Systolic blood pressure was recorded using an Omron BP791IT 10 series Plus Automatic Blood Pressure Monitor with ComFitTM Cuff  in a seated position (4-month and 8-month studies) and a standing position (8-month study only) on the non-dominant arm . ParticiN = 6) and the 8-month study (N = 5) were pooled together for analysis across months 2 and 4 (N = 11). The additional 8-month time point data were collected in a subset of the N = 11 participants (N = 5). Intraclass correlation coefficients (ICCs) and their 95% confidence intervals (CIs)  assessed the interindividual differences and intraindividual stability of seated and standing CV measures  at BL, TSD and REC across months 2, 4, and 8, and the average of BL and REC (BL + REC) across months 2, 4, and 8 . Paired t-tests assessed differences in total sleep time (TST) for each BL and each REC night between months 2 and 4 in the combined sample (N = 11). Repeated measures (RM) ANOVAs compared TST for each BL and each REC night across months 2, 4, and 8 in the 8-month subset (N = 5). Post hoc analyses with Bonferroni corrections compared TST for a specific night across months 2, 4, and 8 when there was a significant time effect . Any significant TST differences have been noted in P < 0.05 was considered significant. SBP was collected in a standing position only in the 8-month study subset (N = 5). For the 8-month study subset, N = 1 participant was not included in the standing BL or BL + REC analyses due to incomplete standing data during month 8 at BL (N = 4). Additionally, because SBP was not taken in the standing position in the 4-month study, there are no data for standing SBP or SVRI in the combined 4-month study and 8-month study.Data from the 4-month study , HR = 0.853 , CI = 0.944 , LVET = 0.821 , SBP = 0.887 , and SVRI = 0.887   . In explP = 0.001), HR = 0.767 , CI = 0.920 , and LVET = 0.891 . In exploratory analyses, we also examined ICCs for males and females separately for standing SV , HR , CI , and LVET .Similarly, standing BL ICCs were substantial to almost perfect across months 2 and 4 for SV = 0.909 , seated CV measures at BL showed substantial to almost perfect stability [except for SBP] while standing CV measures at BL (N = 4) demonstrated fair to almost perfect stability across months 2, 4, and 8 , HR = 0.962 , CI = 0.839 , LVET = 0.800 , SBP = 0.875 , and SVRI = 0.755   . In explP = 0.000), HR = 0.878 , CI = 0.954 , and LVET = 0.852 . In exploratory analyses, we also examined ICCs for males and females separately for standing SV , HR , CI , and LVET .Likewise, standing TSD ICCs were almost perfect across months 2 and 4 for SV = 0.958 , seated and standing CV measures at TSD showed substantial to almost perfect stability across months 2, 4, and 8 , HR = 0.735 , CI = 0.806 , LVET = 0.869 , SBP = 0.881 , and SVRI = 0.810   . In explP = 0.000), HR = 0.859 , CI = 0.918 , and LVET = 0.392 . In exploratory analyses, we also examined ICCs for males and females separately for standing SV , HR , CI , and LVET .Comparably, standing REC ICCs were almost perfect (except for LVET) across months 2 and 4 for SV = 0.952 , seated and standing CV measures at REC showed substantial to almost perfect stability across months 2, 4, and 8 , HR = 0.883 , CI = 0.935 , LVET = 0.874 , SBP = 0.943 , and SVRI = 0.898 . In exploratory analyses, we also examined ICCs for males and females separately for seated SV , HR , CI , LVET , SBP , and SVRI .Seated BL + REC ICCs were almost perfect across months 2 and 4 for SV = 0.961 , HR = 0.845 , CI = 0.955 , and LVET = 0.775 . In exploratory analyses, we also examined ICCs for males and females separately for standing SV , HR , CI , and LVET .Similarly, standing BL + REC ICCs were substantial to almost perfect across months 2 and 4 for SV = 0.965 , seated BL + REC ICCs were moderate to almost perfect across months 2, 4, and 8 for SV = 0.952 , HR = 0.870 , CI = 0.948 , LVET = 0.552 , SBP = 0.712 , and SVRI = 0.965 . Standing BL + REC ICCs in the 8-month study subset (N = 4) were substantial to almost perfect across months 2, 4, and 8 for SV = 0.952 , HR = 0.899 , CI = 0.894 , LVET = 0.819 , SBP = 0.759 , and SVRI = 0.965 .In the 8-month study subset (P = 0.021) and LVET . HR was negatively correlated with LVET  and CI was negatively correlated with SVRI . There were no other significant correlations. For standing measures during BL, SV was positively correlated with CI . There were no other significant correlations.Individuals also exhibited significant consistency of responses within seated and standing CV measures across months 2 and 4 during BL, TSD, and REC. For seated measures during BL , SV was P = 0.004). HR was negatively correlated with LVET  and CI was negatively correlated with SVRI . There were no other significant correlations. For standing measures during TSD, SV was negatively correlated with HR  and positively correlated with LVET , and HR was negatively correlated with LVET . There were no other significant correlations.For seated measures during TSD , SV was P = 0.008) and LVET , and negatively correlated with SVRI . CI was negatively correlated with SVRI . There were no other significant correlations. For standing measures during REC, SV was positively correlated with CI  and LVET  and CI was positively correlated with LVET . There were no other significant correlations.For seated measures during REC , SV was N = 12 adults, which found long-term HR stability (ICC = 0.790) after exposure to 26 h of TSD across 2.5–15 months  across months 2, 4, and 8. In addition, individuals demonstrated significant consistency of responses within seated CV measures across months 2 and 4 during BL, TSD, and REC: SV was positively correlated with CI and LVET while negatively correlated with SVRI, HR was negatively correlated with LVET, and CI was negatively correlated with SVRI. Standing CV measures showed similar ICCs and consistency of responses. For the first time, we demonstrate long-term robust phenotypic stability of CV measures in healthy adults during repeated BL, TSD, and REC exposures. Cardiovascular measures may serve as biomarkers given they can track cardiovascular physiology consistently across repeated acute sleep loss and recovery and across long duration. Our results demonstrated a higher seated HR ICC during TSD across 2 and 4 months (0.962) as well as across 2, 4, and 8 months (0.944) compared to a laboratory study of 5 months . The higThe stability of various CV measures at BL during fully rested conditions across months 2, 4, and 8 is also comparable to research on both short-term and long-term stability across repeated collections of CV measures that did not involve sleep deprivation . Our resOur findings are also comparable to past research that has examined the stability of various other measures across repeated exposures to sleep loss . Stable Of interest, the ICCs of the standing CV measures were within the ranges of the seated CV measures indicating that all participants were at equivalent baseline volume status in the two postural conditions. Standing ICCs at BL, TSD, and REC showed substantial to almost perfect stability for most CV measures, and BL + REC ICCs showed substantial to almost perfect stability across months 2, 4, and 8. Individuals also exhibited similar consistency of responses in both seated and standing CV measures, with a few minor exceptions across months 2 and 4 during BL, TSD, and REC, which may be due to sleep loss differentially affecting some variables and not others across postural positions. Of note, when comparing BL vs. REC, ICCs (not reported in the main results) for seated and standing CV measures maintained moderate to almost perfect stability at month 2, at month 4, and at month 8 (except for LVET). These results were within the ranges of seated and standing BL ICCs, REC ICCs, and BL + REC ICCs across months 2, 4, and 8.In exploratory analyses, we examined the stability of CV responses by sex at BL, TSD, and REC. For many of the CV measures across these phases, males and females showed similar stability; however, males were more stable on certain measures such as HR at BL, REC, and BL + REC and females were more stable on other measures including CI at BL, TSD, and REC, SVRI at TSD and REC, and SBP at TSD. These differences in CV stability may be due to fluctuations within the menstrual cycle , 1993 orIndividuals who are sleep deprived due to work obligations such as staying up late for shift work or lifestyle choices and who exhibit phenotypic vulnerability to the CV effects of sleep loss are at particularly heightened risk for developing CV and associated diseases. Examining the stability of CV indices after exposure to sleep deprivation is vital to the recommendation of mitigation strategies related to real-world settings such as transportation  and emerWhile definitive CV biomarkers for the prediction of vulnerability in sleep deprivation have yet to be discovered, genetic and omics  approaches have identified biomarkers to distinguish an individual’s response to TSD . This isThere are a few limitations to our studies. One limitation was the small sample size. In addition, because of the small sample size, the examination of potential sex differences in stability of CV measures can only be considered exploratory. Furthermore, it is difficult to generalize our findings to individuals with mood or sleep disorders, or with other medical conditions, since all participants in our studies were healthy adults who were not shift workers or exposed to TSD in their professions. We also note that physical and cognitive activity level, meal quality, quantity, and timing, as well as water intake were not strictly controlled. Our results may also not be generalizable to circumstances that do not involve isolation; notably, however, isolation was required for our experiments investigating hemodynamic changes in high-fidelity space analogs to simulate spaceflight conditions . MoreoveFor the first time, we demonstrate robust phenotypic stability of CV measures in healthy adults during BL, TSD, and REC across 2-month, 4-month, and 8-month time points in long-duration studies. We also demonstrate stability of other CV biomarkers in healthy adults never examined previously during sleep deprivation and recovery including SBP, SV, CI, LVET, and SVRI. In addition, individuals showed significant consistency of responses within CV measures in long-duration studies. Overall, our results herald the use of the CV biomarkers examined in our studies, including SV, HR, CI, LVET, SVRI, and SBP, and countermeasures for prediction and mitigation of this critical vulnerability in both fully rested and sleep deprivation conditions.The data generated and analyzed during the current study are available from the corresponding author upon reasonable request.The studies involving human participants were reviewed and approved by the Institutional Review Board of the NASA (4-month study and 8-month study) with primary oversight and also by the Institutional Review Boards of the University of Pennsylvania (4-month study) and Rush University Medical Center (8-month study). The participants provided their written informed consent to participate in this study.NG designed the overall study and provided the financial support. LP conducted the statistical analyses of the data. KR-G extracted and analyzed the echocardiography data. All authors prepared the manuscript and reviewed and approved the final manuscript.


LKB1 and/or KEAP1 mutations, along with the metabolic alterations in glycolysis and glutaminolysis observed in these cancer cells. Furthermore, ongoing trials targeting these metabolic alterations are discussed as potential approaches to overcome the extremely poor prognosis associated with this type of cancer.Currently, immune checkpoint inhibitors (ICIs) are widely considered the standard initial treatment for advanced non-small cell lung cancer (NSCLC) when there are no targetable driver oncogenic alternations. NSCLC tumors that have two alterations in tumor suppressor genes, such as liver kinase B1 (LKB1) and/or Kelch-like ECH-associated protein 1 (KEAP1), have been found to exhibit reduced responsiveness to these therapeutic strategies, as revealed by multiomics analyses identifying immunosuppressed phenotypes. Recent advancements in various biological approaches have gradually unveiled the molecular mechanisms underlying intrinsic reprogrammed metabolism in tumor cells, which contribute to the evasion of immune responses by the tumor. Notably, metabolic alterations in glycolysis and glutaminolysis have a significant impact on tumor aggressiveness and the remodeling of the tumor microenvironment. Since glucose and glutamine are essential for the proliferation and activation of effector T cells, heightened consumption of these nutrients by tumor cells results in immunosuppression and resistance to ICI therapies. This review provides a comprehensive summary of the clinical efficacies of current therapeutic strategies against NSCLC harboring  Currently, the standard first-line therapy for advanced NSCLC without targetable driver oncogenic alternations consists of multiple treatment regimens involving ICIs, either alone or in combination with platinum-based chemotherapy –8. PrediLKB1, encoded by serine/threonine kinase 11 (STK11), occur in approximately 20-25% of lung adenocarcinoma (LUAD), while inactivating mutations in KEAP1 are observed in approximately 10-15% of LUAD , have played a crucial role in identifying predictive biomarkers for ICI therapies and uncovering mechanisms of immune evasion in cancer , 12–15.  of LUAD –21. Sevend KEAP1 –24. NSCLotherapy . Howeverotherapy , 28, 29.otherapy , 18. Amootherapy , 31. Theotherapy –34.To understand how the inactivation of the two tumor suppressors leads to metabolic reprogramming of glycolysis and glutaminolysis, researchers have gradually uncovered the molecular mechanisms through various biological approaches. These metabolic alterations play a significant role in promoting tumor aggressiveness and reconstructing the TME to support tumor growth , 35, 36.22.1PD-L1 expression on cancer cells is regulated by various mechanisms, including inflammatory cytokines, mechanical signals, and tumor-intrinsic cell signaling. Consequently, there is heterogeneity in the PD-L1 expression levels across tumors –40, makiepidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), ROS proto-oncogene 1 (ROS1), B-Raf proto-oncogene (BRAF), and Ret proto-oncogene (RET) alterations, exhibiting over 50% antitumor response rates and long-term PFS  G12C mutation, following immunotherapy-based therapies   of 0.42 and 0.65, respectively, in KRAS mutation-positive and KRAS mutation wild-type populations  . Anotherus NSCLC . A subgrulations .EGFR mutant compared to those of wild-type , LKB1 (STK11), and KEAP1. These co-occurring patterns have significant biological implications and can influence tumor evolution and progression , while it was 1.8 months and 6.4 months for patients with KRAS/STK11 co-mutation (KL group). The underlying biological mechanism explaining the poor efficacy of ICIs in the KL group may be attributed to the immunosuppressive TME caused by LKB1 inactivation followed by STK11 mutation  (KRAS/TP53 co-mutation had a longer median PFS with ABCP compared to those with KRAS mutations and wild-type TP53 (14.3 months vs. 7.3 months)   (Supple months) . In the ild type   and 0.25 for those with KEAP1 mutation (n = 38) (STK11 mutation and 0.34 (95%CI: 0.14–0.83) for patients with KEAP1 mutation (STK11 mutation (n = 53) and 0.94 (95%CI: 0.33–3.35) for patients with KEAP1 mutation (n = 28)      and key glycolytic enzymes such as LDHA, hexokinases, and phosphofructokinase 1 (PFK1)  –82. Unde limited . Alongsi1 (PFK1) –88. MutaTumor cells that undergo rapid proliferation stimulate the formation of tumor blood vessels by releasing factors that promote angiogenesis. This process is crucial for acquiring nutrients and oxygen. However, the resulting vasculature is often immature and hyperpermeable, leading to the development of hypoxic regions within the tumor. These hypoxic areas create a barrier that hampers the infiltration of immune cells . MoreoveFurthermore, it is recognized that circulating lactate is transported into cells via MCT1 and used as an energy source and substrate for lipogenesis in certain cancer types . In an a3.2Glutamine (Gln) is a vital amino acid with significant roles in cellular functions, including energy and biomolecule synthesis, as well as ROS scavenging. Upon cellular uptake, Gln is converted into glutamate (Glu) by the enzyme glutaminase (GLS). It is further converted to α-ketoglutarate, which enters the TCA cycle, generating metabolic intermediates for lipid, nucleic acid, and protein synthesis. In the TME, both tumor cells and infiltrating immune cells have a high demand for Gln, similar to glucose. T-cell activation and proliferation heavily rely on Gln metabolism, and when Gln is insufficient in the TME, the high consumption by tumors can inhibit T-cell activity. Conversely, reduced Gln metabolism in tumors has shown to increase Gln utilization within the TME . In a mode novo pyrimidine synthesis. The CPS1 expression is transcriptionally regulated by LKB1 through AMPK, and cases with high CPS1 expression have been associated with poor prognosis, particularly in NSCLC with LKB1 inactivation , which promotes ROS detoxification . GSH, al3.3NFE2L2 gene, for degradation through ubiquitination. However, under stress conditions, KEAP1 activity is reduced, leading to increased transcription of NRF2 target genes. This activation of NRF2 signaling enhances antioxidant defense against ROS and regulates drug detoxification and immune response , with both alterations being mutually exclusive (The KEAP1-nuclear factor erythroid-derived 2-like 2 (NRF2) pathway plays a crucial role in regulating the cellular response to oxidative stress, and its signaling abnormalities have been observed in various cancer types, including NSCLC , 107. Inresponse , 107. Inxclusive . The conxclusive , 107. Fuxclusive –111.KEAP1-mutant NSCLC cell lines demonstrate sensitivity to GLS inhibition due to their high dependence on Gln uptake in the culture medium , KRAS-LKB1 (KL), and KRAS-KEAP1-LKB1 (KKL) mutant LUAD using genetically engineered mouse models . In KRASse model . Tumors se model . Tumors se model , 118. Inynthesis , 101. Exynthesis , 120. Beynthesis . Therefoynthesis . These f4LKB1 or KEAP1 inactivation compared to standard platinum doublet chemotherapies  and molecularly targeted therapies, recent fundamental research has uncovered that each driver gene alteration has a cancer-specific impact on the TME through metabolic reprogramming. Specifically, the alteration of glucose and glutamine (Gln) metabolism resulting from LKB1 or KEAP1 inactivation appears to play a significant role in diminishing the effectiveness of current immunotherapies by suppressing the activity of effector T cells. Ongoing clinical trials targeting glucose or Gln metabolism, as depicted in One therapeutic strategy being explored involves the addition of metformin, a commonly used medication for type 2 diabetes, to cytotoxic chemotherapy. Accumulating evidence supports the antitumor effects of metformin, as it enhances AMPK-mediated cell growth inhibition and cisplatin-induced apoptosis in LKB1-inactivated NSCLC , 136. InIn conclusion, high consumption of glycolysis and glutaminolysis in immune-resistant phenotype tumors, such as NSCLC with LKB1 and/or KEAP1 inactivation, not only contribute to tumor aggressiveness but also impede intratumor T-cell function. The presence of co-occurring mutations in NSCLC leads to distinct metabolic alterations that impact immune cells within TME. These differences in metabolic reprogramming may affect clinical efficacies of current ICI combination regimens and novel agents targeting metabolic enzymes. To develop new therapeutic strategies that target metabolic alterations in combination with ICI regimens for NSCLC with LKB1 and/or KEAP1 inactivation, further extensive analyses on a larger scale will be necessary.IT, JK, HI, SH, and MM were involved in the initial drafting of the manuscript, data collection, and analysis. They also contributed to the conceptualization of the study, reviewed the manuscript, and provided feedback and edits. All authors have read and given their approval for the final version of the manuscript. IT have demonstrated the dependency of CPS1, a metabolic enzyme, in cell growth, metabolism and prognosis in LKB1-inactivated lung adenocarcinomas. Furthermore, Serglycin secretion, which is a chondroitin sulfate proteoglycan involved in reprograming to an immunosuppressive TME, is epigenetically induced through nicotinamide N-methyltransferase-induced perturbation of methionine metabolism in TTF-1–negative lung adenocarcinoma. These results were published in J Natl Cancer Inst (2017) 109:1-9 and J Natl Cancer Inst (2022) 114:290-301.