Mechanotransduction is the process in which cells can convert extracellular mechanical stimuli into biochemical changes within a cell. While this a normal process for physiological development and function in many organ systems, tumour cells can exploit this process to promote tumour progression. Here we summarise the current state of knowledge of mechanotransduction in osteosarcoma (OSA), the most common primary bone tumour, referencing both human and canine models and other similar mesenchymal malignancies . Specifically, we discuss the mechanical properties of OSA cells, the pathways that these cells utilise to respond to external mechanical cues, and mechanotransduction-targeting strategies tested in OSA so far. We point out gaps in the literature and propose avenues to address them. Understanding how the physical microenvironment influences cell signalling and behaviour will lead to the improved design of strategies to target the mechanical vulnerabilities of OSA cells. The bone is a dynamic tissue that provides structural support to the human body throughout one’s life; as such, it must be able to undergo adaptive processes to maintain structural integrity during various mechanical stimuli, such as walking and running . The abiMechanotransduction is broadly defined as the ability of cells to convert mechanical stimuli from its surrounding environment into biochemical signals . DespiteInterestingly, the skeleton as a whole is believed to be subjected only to a minuscule amount of strain overall (approximately 0.04–0.3%) compared to the levels needed to elicit an effect on bone cells in vitro 1–10%) [% 10,15,,15,16. T2 (PGE2) and cyclooxygenase 2 (COX2), the levels of Ca2+, and secondary messengers adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP), through direct and indirect mechanisms. Fluid flow has been shown to directly increase the levels of COX2 mRNA and the release of PGE2 [2 acts in an autocrine manner by binding to EP2 receptors to increase both cAMP-dependent protein kinase A (PKA) signalling and phosphatidylinositol 3-kinase (PI3K)/Akt signalling [As the primary mechanosensor of the bone, the role of osteocytes appears to be two-fold: (1) increase the mechanical signal and (2) secrete signalling factors and other mediators to regulate the response of other bone cells (osteoblasts and osteoclasts). In response to mechanical stimuli, such as fluid flow, osteocytes have been shown to rapidly increase the release of prostaglandin E of PGE2 . This PGgnalling ,23. As agnalling ,25.2 release, another early response to mechanical signalling in osteocytes is the rapid increase in intracellular (cytoplasmic) Ca2+ levels. This increase in Ca2+ is mediated by various signalling molecules, pathways and cellular compartments and involves the activity of primary ciliary protein transient receptor potential vanilloid subfamily member 4 (TRPV4), the opening of T-type voltage-sensitive calcium channels, the controlled release and refilling of intracellular calcium stores in the endoplasmic reticulum (ER), P2 purinergic receptor (P2R)/phospholipase C (PLC)/inositol trisphosphate/ER pathway activation, sphingosine-1-phosphate signalling and the binding of matrix molecules to α3βv integrins [2+ in osteocytes is important for the release of ATP, which in turn modulates actin dynamics and extracellular vesicle release [2 result in actin contractions though non-muscle myosin and myosin light chain kinase (MLCK), leading to an increase in extracellular vesicle (EV) release. These vesicles contain receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OGN) and sclerostin, suggesting that its contents can regulate the bone formation process [Aside from PGEntegrins ,29,30,31 release ,33. In r2+ [vβ3 binding, as disruption of these adhesion sites prevented Ca2+ influxes. Mechanical loading on dendritic processes can lead to the opening of hemichannels on the cell body. This is dependent on glycocalyx that surrounds the dendritic processes. Abolishment of glycocalyx decreases the expression of α5 integrin and a subsequent decrease in hemichannel opening in osteocyte cell bodies upon mechanical stimulation [5 integrin to respond and transmit mechanical cues along the osteocyte.Not only are the dendritic process of osteocytes important for forming the junctions between cells, but they have shown to have roles in amplifying the mechanical signal. Thi and colleagues (2013) found that dendritic processes are very responsive to mechanical loading compared to the cell body and can rapidly increase the levels of intracellular Ca2+ . This inmulation . This suALP), osteocalcin (OCN) and collagen I (COLI) mRNA and protein levels [ALP and OCN mRNA expression [Although osteocytes are regarded as the mechanosensors of the bone, osteoblasts themselves have been widely demonstrated to be able to receive and respond to mechanical stimuli. One model proposed by Pavalko and colleagues (2003) suggests that osteoblasts activate gene transcription in response to mechanical loading through the formation of ‘mechanosomes’ . Upon men levels ,39. Simipression ,41. The Although mechanotransduction is a process that is often used by the skeletal system to maintain homeostasis, pathologies such as cancer, can exploit this process to aid in cancer progression . Osteosarcoma (OSA) is the most common primary cancer of the bone in both humans and canines. In humans, the disease most commonly affects adolescents, while middle-to-older aged canines are impacted ,43,44. HTreatment for both species includes the surgical removal of the tumour, either through limb amputation or limb-spare/limb-salvage surgery and adjuvant chemotherapy . The mosWhile osteosarcoma is a rare disease in humans, and is often considered an ‘orphan disease’, it affects canines at a higher  incidence rate . ResearcThe microenvironment that surrounds cancer cells, or tumour microenvironment, has been widely accepted to drive cancer progression. The microenvironment not only encompasses the classical signalling factors, but also includes the physical environment . ResearcThe increasing sophistication of molecular biology tools has allowed researchers to understand mechanobiology in much greater detail in the last two decades. The use of hydrogels, water-based crosslinked polymers that can be easily manipulated to varying stiffnesses (Young’s module), have permitted the assessment of how this physical parameter impacts mechanotransduction in relation to various ECM components, while 3D culture models have allowed for better mimicking of the architecture of the tissue of interest. For example, a 3D collagen scaffold model created by Liverani and colleagues (2019), allowed for the culture of breast cancer cells in an environment that mimics the hierarchically organised structure of extracellular collagen in actual tissue. Furthermore, this model permits the study of hypoxia effects on cell fate and metabolism, as tissue-like cellular structures are able to establish realistic oxygen gradients, which contributes to their overall phenotype . AnotherMechanical characterisation of mesenchymal stem cells, normal osteoblasts and OSA cells demonstrated that there are different mechanical profiles between and within cell types. The human OSA cell line MG63, for instance, appeared to be softer because of less organised cytoskeleton and had a rougher surface than its normal counterparts . These dStudies employing techniques that mimic the bone environment or different matrix stiffnesses allow for a better understanding of the pathways involved in transducing mechanical cues in OSA and the role of mechanotransduction in OSA.+ and CD133+ tumourspheres when the gel was 50 kPa in stiffness [Perhaps not surprising, OSA cells have been shown to respond best to mechanical cues when the substrate rigidity is closest to that of its origin tissue. OSA cells isolated directly from patient tissue were found to have a greater cell area, traction forces and increased survival when seeded on stiffer substrates (55 kPa), which approximates the reported Young’s modulus of collagenous bone, versus soft substrates (1 or 7 kPa) . Not onltiffness .OSA cells utilise a variety of mechanisms to respond to mechanical stress, such as enhancing expression and/or activity of molecules that mediate mechanical signalling. As mentioned previously, integrins are responsible for receiving external mechanical stimuli. MG63 OSA cells exposed to cyclic mechanical stimulation upregulate their integrin beta 1, pFAK and pERK protein levels. The increases in pFAK and pERK levels were abrogated with an integrin beta 1 blocking antibody, demonstrating that this integrin is a key mediator of OSA mechanotransduction . This caAside from transcription-dependent increases in the protein levels of several focal adhesion components, a study by Zheng and colleagues (2014) suggests that components of the ECM environment could be modified with mechanical stimuli as well as by similar or alternative mechanisms . TenasciThe ECM contains structural proteins, , glycoproteins  and proteoglycans, with collagen being most abundant . RNA anaProteomic analysis comparing primary and metastatic canine OSA cells under normoxic or hypoxic conditions found that P4HA1, PLOD1 and 2 and LOX were all increased by hypoxia, with a more dramatic effect seen in the metastatic cell line . ContrasIn summary, OSA cells can respond to mechanical cues from their microenvironment by increasing the expression of ECM proteins and focal adhesion complex proteins see . Based oThe Hippo pathway plays key roles in almost all organ systems—from embryonic development to adult tissue homeostasis and regeneration, as well as the development and advance of pathologies such as cancer ,109. CanAside from upstream kinases MST1/2 and LATS1/2, several other mediators and environmental factors have been demonstrated to modulate TAZ/YAP expression, localisation and activity (see recent review by Pocaterra and colleagues ). TAZ/YACYR61), connective tissue growth factor (CTGF), and cyclin D1 (CCND1). These findings are not solely limited to in vitro studies, as mice injected with ROCK-depleted OSA cells had a significantly decreased tumour volume and were unable to metastasise compared to mice injected with control OSA cells. Analysis of the tumour tissue isolated from mice injected with ROCK-depleted tumour cells demonstrated a decrease in total YAP immunolabeling, suggesting that modulation of YAP by ROCK might impact tumour growth and metastasis in OSA [Similar to what has been previously reported in the literature for osteoblasts differentiation , YAP nucs in OSA . Not onls in OSA . As ments in OSA . These rA similar rationale can be used to understand the results for a study by Molina and colleagues (2019), where MG63 cells were encapsulated within 3D meshes with varying concentrations of gelatin and poly (epsilon-caprolactone) (PCL), which mimics the porous structure of trabecular bone. Upon decreases in tensile moduli, YAP protein levels decrease, while TAZ levels stay consistent. Interestingly, the nuclear localisation of YAP and TAZ moderately increased as the tensile modulus decreases . These fAs TAZ/YAP have well documented roles in human cancer progression, it is not surprising that they have generated significant therapeutic interest. Several different strategies have been employed to disrupt TAZ/YAP signalling, either through targeting their upstream mediators, directly targeting TAZ/YAP and/or TAZ/YAP outputs Verteporfin, a small molecule benzoproyphyrin, is a light-activated drug currently used for the treatment of age-related macular degeneration. In the context of Hippo signalling, it is the only compound to date that has been shown to be able to bind to YAP. This prevents YAP binding to TEAD, and has been demonstrated to upregulate 14-3-3 binding proteins, thus causing YAP sequestration into the cytoplasm ,134. StuAn in vitro study completed by Ferraiuolo and colleagues (2018) found that agave extract decreased YAP and TAZ protein levels through enhancing protein degradation in human OSA cell lines. Agave also decreased TAZ and YAP mRNA levels and their known downstream gene targets, possibly through modulating the levels of NF-kappa B p65 and NF-kappa B p50 . NF-kappAs mentioned previously, many pathways contribute to the activation of TAZ/YAP, either in cooperation or independently of Hippo signalling. Some of these signalling pathways already have known or developed inhibitors that could be also used to directly, or indirectly, impact TAZ/YAP. Oku and colleagues (2015) screened small molecules on breast cancer cells and found that dasatinib, statins and pazopanib inhibited TEAD activity, and increased TAZ/YAP phosphorylation .Dasatinib is an FDA-approved small molecular inhibitor with demonstrated activity for several tyrosine kinases, such as Src family signalling, c-MET signalling and the ability to modulate the localisation of TAZ/YAP, and actin reorganisation ,140. ActPazopanib is a tyrosine kinase inhibitor of VEGF, platelet derived growth factor (PDGF) and stem cell factor (SCF)/c-KIT signalling, which is approved for the treatment of advanced soft tissue sarcoma. Pazopanib decreases the nuclear localisation of TAZ/YAP through enhancing its proteasome-mediated degradation . So far,Statins are a family of inhibitors of 3-hydroxy-methylglutaryl CoA reductase that are still under investigation for the treatment of various cancers and have shown to impair TAZ/YAP nuclear translocation . SimvastAlthough the aforementioned studies describing repurposed inhibitors provide interesting results, it is unclear if the effects are due to an inhibition of TAZ/YAP as it was not directly measured in the in vitro studies or in the clinical cases mentioned above. The original screening completed by Oku and colleagues (2015) was completed in breast cancer cells and needs to be completed in OSA to determine if the effects observed are TAZ/YAP-mediated, or simply due to the impact of the compounds on other signalling pathways. It will be also important to assess the signalling profiles of tumours in future OSA clinical trials, to address whether patient response correlates with TAZ/YAP activity. Drug repurposing is a beneficial avenue to explore as the safety profile of these compounds in humans are already known.Similar to TAZ/YAP, the subcellular localisation and activity of MRTFs have been demonstrated to be mediated by cytoskeletal dynamics. MRTFs are normally bound to G-actin. Upon focal adhesion assembly and ROCK-mediated actin polymerisation, G-actin is incorporated to make F-actin causing the dissociation of MRTFs from G-actin, allowing their translocation to the nucleus where they can bind to serum-response factors (SRF) ,152. ThiTo the best of our knowledge, little is known about MRTF-A/-B in OSA as our literature search only found one study conducted in human OSA. Dai and colleagues (2019) seeded MG63 human OSA cells on soft, medium, and rigid polyacrylamide hydrogels to determine the impact of rigidity on MRTF-A and epithelial-mesenchymal transition (EMT) protein levels and localisation. MRTF-A , vimentin, snail, fibronectin, and MMP9 protein levels were increased with increasing hydrogel stiffness. MG63 cells also had greater migration speed when seeded on a rigid hydrogel stiffness . These rThere are several small molecule inhibitors available that target the Rho/MRTF/SRF pathway, including CCG-1423, CCG-203971, and CCG-232601. Despite CCG-1423 having anti-invasive effects in prostate cancer cells, the compound was cytotoxic and had undesirable side effects in mice. This led to the creation of a 2nd generation inhibitor, CCG-203971 ,161, whi2) or S100P proteins binds to the FERM domain, allowing for its dissociation. This allows for the subsequent phosphorylation at threonine residue 567 (T567) which is mediated by protein kinase C (PKC) and ROCK; this phosphorylation is essential for ezrin’s activation and ability to bind to F-actin [Ezrin is a member of the ERM  family of proteins and has a unique protein structure enabling it to bind to both, the actin cytoskeleton and the plasma membrane ,165. Thr F-actin .Ezrin is highly expressed in sarcomas and was shown to associate with a shorter disease-free interval in both canine and human OSA patients ,171. A h2+ through calcium ion channel TRPV6. The increase in Ca2+ enhanced phosphorylation of Akt and ezrin, providing evidence that mechanosensitive microvilli could lead to an activation of ezrin [Although there are no studies to date that have directly investigated the role of ezrin in mechanotransduction in OSA cells, there are several lines of evidence to suggest that this relationship exists in other normal and cancerous cell types. Fluid shear stress (FFS) stimulation of human placenta cells promoted the formation of microvilli and an increase of internal Caof ezrin . Aside fof ezrin ,176. Aloof ezrin .Because of ezrin’s ability to modulate actin dynamics and promote metastasis in in vivo models of various cancers, small molecule inhibitors have been developed to target ezrin. Aside from ezrin itself being important for migration and metastasis, ezrin can modulate the levels and localisation of YAP in skin fibroblasts, as well as pancreatic and hepatocellular carcinoma cancer cells. The positive effect of ezrin on YAP can be direct, as seen in skin fibroblasts , or thro+p53fl/flpRBfl/fl) exhibited similar results, as the incidence of lung metastasis was significantly reduced with NSC305787 treatment, a compound that also reached the more favourable plasma concentrations as compared to NSC668394 [As the phosphorylated (active) form of ezrin has been associated with pro-migratory and pro-invasive behaviour in both canine and human OSA, small molecule inhibitors have been focused on disrupting the phosphorylation site of ezrin and its ability to bind to F-actin. A small molecule library screen to determine compounds that would directly bind to ezrin identified two molecules, NSC305787 and NSC668394. In vitro assays found that these compounds work by directly binding to ezrin to inhibit phosphorylation; although NSC305787 can inhibit PKC kinase activity at higher doses, this is not the case for NSC668394 . The invSC668394 .Because of the similarity in structure of NSC305787 to quinoline-based antimalarial treatments, several anti-malarial agents have been tested and were demonstrated to have anti-ezrin activity in a Medicines for Malaria Venture (MMV) screen. Several candidate compounds that were found in this screen  decreased cancer cell motility in vitro and substantially decreased lung metastatic growth in an ex vivo lung culture compared to NSC305787. Interestingly, all the compounds tested increased the expression of phosphorylated (T567) ezrin, the most modest being MMV667492, which caused no changes in total phospho-ezrin levels . This suAside from the most studied mechanotransducers, such as those mentioned in +) OSA stem cells and causes a fibrogenic reprogramming. This reprogramming creates a fibrotic-like environment and provides a survival advantage for lung metastasis growth, but not primary bone lesion growth [How does the mechanical bone and lung microenvironment change during osteosarcoma progression? Panciera and colleagues (2020) recently found that constitutive oncogenic signalling, specifically by Kirsten rat sarcoma viral oncogene homolog (K-RAS) or HER2, alone, is not enough to reprogram mammary cells into tumour-initiating cells but might additionally require a stiffer-than-normal mechanical microenvironment . Given tn growth . Althougn growth . It willDo mechanotransduction mechanisms vary in early stage as compared to advanced stage tumour cells? A majority of the research mentioned in this review only included studies that utilised primary OSA cell lines, which makes it difficult to understand any differences in how primary and metastatic OSA cells respond to mechanical cues. Future research could compare paired primary- and metastatic-derived OSA cells in microenvironments with varying stiffnesses to assess the mechanical signalling pathways that are activated and the differences, if any, in functional cell responses.Can we identify tumour mechanotransduction signatures to tailor therapy? Most studies that have explored mechanical signatures in epithelial cancer tissue include profiling the stiffness of cancer biopsy tissue ex vivo using indentation techniques ,195, or How do genomic aberrations in OSA impact mechanosignalling? The karyotype of OSA is notoriously complex; genomic analysis of OSA tumours from both human and canine samples show multiple copy number variations and structural variants of certain genes ,202. ThiCompared to epithelial malignancies, research on mechanical influences in osteosarcomagenesis and progression is still in its infancy. All the evidence discussed in this review is solely based on in vitro experiments utilising a limited number of cell lines and variable ex-vivo or animal models, making the results extremely difficult to extrapolate. There are a considerable number of unknowns that will need to be addressed in order to fully understand the mechanical landscape of OSA, some of which are briefly discussed below:While the role of cellular signalling pathways in OSA progression have been well investigated, the research on the role of mechanical signalling in OSA has been lacking. Preliminary research conducted to date has demonstrated that the mechanical properties of OSA cells changes with increasing malignancy and that OSA cells utilise several mechanisms  to respond to mechanical cues. Robust studies using clinically relevant mouse models and comparative oncology approach using canines as a model are imperative to enhance our understanding of mechanobiology and create suitable molecular targeted therapy for advanced OSA.


Owing to the rapid development and wide clinical application of direct acting antiviral (DAA) drugs in the treatment of hepatitis C virus (HCV) infection, the era of interferon-based therapy has almost come to an end. Cumulative studies show that DAA therapy renders high cure efficiency (>90%) and good safety profile, and may even bring some unexpected benefits to the patients. However, some issues of concern arise, one of which is the resistance mutation of HCV genome leading to failure of treatment. With the aim of providing some meaningful references for the treatment of chronic hepatitis C (CHC), this article summarizes the research progress on benefits of DAA accompanied by viral clearance in the treatment of chronic hepatitis and the drug resistance. Hepatitis C virus (HCV) infection is a global epidemic. According to the World Health Organization (WHO), approximately 71 million people are currently living with hepatitis C virus worldwide, which is the main cause of chronic hepatitis. The number of death from hepatitis C-related liver disease has exceeded 399 000 yearly As an infectious agent with high sequence variations, HCV is classified into seven genotypes (GT) and is further subdivided into nearly 100 subtypes to date. It is recognized that HCV genotypes have a regionally distinct global distribution: HCV GT1 (46.2%) and 3 (30.1%) dominate the global infections; GT 2, 4, and 6 are responsible for approximately 22.8% of HCV infections; GT 5 accounts for the remaining less than 1%; GT 7 has been identified so far in very few patients originating from Central Africa In the past two decades, the only standard treatment for patients with HCV infection is peg-interferon plus ribavirin (PegIFN/RBV) in 24 to 72 weeks, but only a limited proportion of patients can achieve a sustained virological response (SVR), defined as HCV RNA continues to be undetectable in serum at 12 weeks after completing treatment. What is worse, the interferon-based agent can cause many adverse effects in more than 10% of patients during and after the treatment In general, DAAs can be divided into three major classes based on their targets in HCV proteins: the nonstructural protein 3/4A (NS3/4A) protease inhibitors (PIs) that can inhibit HCV polyprotein processing; NS5A inhibitors, inhibiting viral replication and assembly; and NS5B polymerase inhibitors that can block HCV RNA replication Moreover, DAA combination therapies can achieve high SVR regardless of the HCV GTs and subtypes, even in patients with compensatory cirrhosis and decompensated liver disease.Surprisingly, numerous studies have shown that DAA can not only effectively and safely remove HCV, but also achieve some unexpected benefits when compared with interferon-based therapy, such as repairing liver function damage, recovery of metabolic impairment and restoration of immunity dysfunction , etc. caused by HCV infection, and the number of patients with adverse effects are much lower. However, despite the excellent antiviral potency and good compatibility of DAA therapy, the consequent challenges such as the association between DAA regimen and tumor genesis etc.In HCV-infected people, only a minority of those can spontaneously recover from HCV infection, relying on their strong immunity. More than 70% of people will gradually develop chronic hepatitis C virus infection, and a considerable proportion of patients eventually develop into liver fibrosis, cirrhosis, Liver function of more than two-thirds of patients will be damaged by HCV infection. Clinically, chronic HCV infection will contribute to obvious change of some related parameters: high concentration of transaminases , elevated bilirubin concentration, elevated serum globulin concentration, albumin concentration, and lower platelet count. In addition, liver stiffness values and Child-Pugh grading standards can be used to measure the severity of the related liver disease. These data have a positive or negative correlation with the severity of liver function damage.et al. have observed significant change of related parameters, which demonstrated improvement of liver function in a prospective study. In the study, they observed the evidently dropped aminotransferases (P < 0.0001), a progressive increase in serum albumin (P = 0.010), and a decrease in serum bilirubin (P = 0.011) as well as in gamma globulin (P = 0.0003) between baseline level and that after treatment In recent years, mounting clinical trials have manifested that successful DAA treatment can not only effectively remove HCV, but simultaneously repair liver function damage due to HCV infection in at least two-thirds of the patients Particularly, there are some studies that have evaluated global prognostic scores for patients with decompensated liver disease, such as the end-stage liver disease model (MELD), showing that the liver function of about 50% of patients has been improved after HCV was removed by DAA. Additionally, these improvements of liver function have been made in the short term et al. found that their liver function improvement after DAA treatment was often better than that of non-cirrhotic patients et al. also revealed that at least 85% of patients with cirrhosis had a decrease in liver stiffness that dropped by about 40%. However, there were still 10% of patients who had liver stiffness values enhanced, although the virus has been eradicated after DAA treatment Interestingly, with respect to patients who were diagnosed as liver fibrosis and cirrhosis, R. Flisiak et al. demonstrated that DAA treatment also improved liver function impairment in co-infected patients, and the degree of improvement in liver function was similar to that of patients with HCV mono-infection In view that most of these conclusions are based on the fact that the patient is mono-infected with HCV, we wonder whether the DAA treatment has the same effect of clearing HCV and repairing liver function damage for human immunodeficiency virus (HIV)/HCV co-infected patients, because it is well accepted that the HIV/HCV co-infection greatly increases the risk of developing advanced liver disease The association between HCV infection and dysregulation of metabolic processes has been observed since long ago. Furthermore, chronic HCV infection exerts a significant impact on the development of heart disease and stroke et al. showed that HCV replication can down- regulate the glucose transporter 2 expressions, which located on the surface of the cell, thereby affecting cellular uptake of glucose et al. found that HCV up-regulated hepatic glucose production via NS5A-mediated FoxO1-dependent pathway The hypothesis that HCV has a direct and important role in the regulation of glucose metabolism is supported by laboratorial investigations. Kasai D The complex association between HCV infection and dysregulation of lipid metabolism has also been observed in recent years. Numerous studies have shown that HCV is highly dependent on the host's lipid metabolism to create an environment more suitable for its replication. HCV associates with lipoproteins to form a structure called lipoviral particle (LPV), released by hepatocytes Because of the serious metabolic disorder and consecutive related diseases due to HCV infection, the question of whether the success of DAA treatment can repair the metabolic damage arises. Until now, progressing studies have shown that DAA treatment has some beneficial impacts on both glucose metabolism and lipid metabolism.Several studies indicated that DAA therapies indeed change the state of glucose metabolism in patients with the clearance of HCV. However, there is a certain contradiction in the results of these studies. A retrospective study carried out on 300 patients suggested that blood glucose levels significantly decreased in subjects with diabetes who achieved SVR. And most of the observed variation occurred early in time and in temporal concert with HCV clearance. Furthermore, the metabolic improvement was persistent with a reduction of average fasting glucose level for >1 year after the end of therapy et al. discovered that SVR is closely associated with the decline of fasting blood glucose levels in patients, and this decline will continue until 24 weeks after the completion of the treatment. However, the fasting blood glucose concentration will return to baseline level after 48 weeks et al. carried out on 384 HCV patients with T2D revealed that there was no significant difference in the concentration of HbA1c between untreated patients and those with treatment failure, whereas the variation of concentration of HbA1c in patients with successful DAA treatment experienced three stages: average HbA1c started off at roughly 7.7 and decreased significantly over time (P<0.001); then the concentration of HbA1c began to rise slowly (P=0.003); finally, HbA1c stabilized at an average level of 7.9  In contrast, Philip Weidner The contradictory of these results may be explained by the length of follow-up after the end of treatment as well as different characteristics of subjects employed. In summary, a conclusion can be drawn that DAA have a positive impact on glucose metabolism damage early in time after the completion of DAA treatment, but this beneficial effect will gradually disappear as the follow-up time is extended.et al. found that the level of serum total cholesterol (P<0.0001), low density lipid protein- C (P<0.0001), VLDL-C (P=0.0003) and triglyceride (P=0.0003) were greatly elevated in patients who had acquired SVR at the end of treatment and 1 year after the end of treatment, while HDL-C levels in serum were not significantly changed, comparing with the baseline level et al. They found out that post-treatment levels of cholesterol (n=36) and triglycerides (n=28) were also evaluated. Overall, a numerical increase was observed from baseline in both total cholesterol (P=0.06) and triglyceride levels (P=0.40). And patients with diabetes were noted to have an elevation in cholesterol after treatment , but no significant change in triglycerides  With respect to lipid metabolism impairment in patients, unlike the short-term beneficial impact of DAA therapies on glucose metabolism, numerous studies have supported the hypothesis that HCV clearance after therapy is associated with a significant improvement in the lipid profile. A study by Gilmar de Souza Lacerda Accordingly, these results support the conclusion that lipid metabolism damage caused by HCV can be completely reversed after DAA successfully removes the virus, which can improve the liver lipid degeneration and atherosclerosis To conclude, the question of whether DAA treatment can exert a beneficial impact on the metabolic impairment can be answered to date: DAA indeed can recover lipid metabolism damage, but only a short-term positive outcome on glucose metabolism damage.A great deal of studies have shown that HCV infection will induce the up-regulation of many genes involved in innate immunity characterized by up-regulation of interferon-stimulated genes (ISGs) expression Moreover, in addition to changing innate immune response, HCV infection also leads to changes in specific immune response. In patients with chronic HCV infection, T cell responses can only be detected at low levels Unlike interferon-based treatments, DAA treatment acts precisely on some critical steps of HCV replication, thereby preventing HCV replication. It plays a role in the treatment of CHC less dependent on the host's immune function. Hence, many researchers speculate that the dysfunction of CHC patient's immune system may be recovered partially or completely with the DAA clearing HCV.et al., interferon-free DAA treatment can trigger a global rearrangement of innate immune signals and inflammatory pathways in patients with chronic hepatitis C. They observed an obvious reduction in the transcription of the cytokine IL1β involved in innate immune activation, hepatic inflammation, and fibrosis parallel with the deregulated phosphorylation levels of NF-ΚB protein that is associated with the activation of downstream signaling and innate immune. In addition, the level of C-X-C motif chemokine (CXCL)-10 and CXCL 11 also decline rapidly during treatment acting as chemokines, which can guide the innate immune cells to accumulate in the inflammatory part. This phenomenon indicates a reduction of the levels of HCV RNA in peripheral blood and an amelioration of liver inflammation. In addition, the expression of innate immune signaling related molecules such as retinoic acid induced gene I (RIG-I), Signal transducing activator of transcription 1 (STAT1), and interferon regulatory factor 7 (IRF7) is down- regulated, therefore, the rapid dampening of innate immune activation following rapid viral clearance with IFN-free DAA therapy is independent of the treatment regimen utilizedTo date, there is an extensive body of evidence that innate immune and specific immune response damage can be recovered in subjects with HCV clearance by DAA treatment, and this restoration presents long-term effect. According to a study of Matthew A. Burchill et al. found out that the concentration of lymphocyte in peripheral blood was increased dramatically during the first two weeks of DAA treatment, but this upregulation did not last for the entire treatment process. The increase of the population of T lymphocytes in peripheral blood can be reflected as a combined effect of reduced intrahepatic migration of lymphocytes due to changes in chemotaxis and potential outflow of intrahepatic lymphocytes et al. speculated that the decrease of population of CD8+ T cells may be due to a decrease in HCV concentration, contributing to an intrahepatic cellular response to reduction in viral burden. In addition, the population of CD4+T cells also declined in the non-parenchymal region of liver, while the significant variation of frequencies of Kupffer cells associated with inflammation was not observed In fact, the repair of immune dysfunction stemmed from DAA therapies changes the expression of major immune-related molecules, and the number and phenotype of innate immune cells and specific immune cells are also changed. Eric G. Meissner et al. showed that the expression of depleted T cell markers was reduced during DAA treatment, and the proportion of exhausted phenotypes T cell was obviously declined ,but the proportion of HCV-specific CD8+ cells is correspondingly heightened. Because the exhausted T cells partially or completely lose their ability to secrete antiviral cytokines such as IFN-α, IFN-γ, and IL-2. However, after successful treatment, compared with baseline levels, the expression levels of these cytokines are increased in T cells, which mean a recovery to some extent in HCV-specific immune response. Thence, amplification in HCV-specific CD8+ T cell responses is a direct result of decreased expression of T cell depletion markers Furthermore, studies by Shikha Shrivastava et al. shown that patients co-infection with HIV/HCV lead to greater restoration of the immunologic outcomes to an extent that was greater in three-drug combination therapies than that observed in either two-drug regimensAdditionally, it is well documented that patients co-infected with HIV and HCV have higher levels of immune activation and impaired antigen-specific responses compared to patients mono-infected with HCVThus, available results all favored the conclusion that immune impairment due to HCV infection and HCV/HIV co-infection after the end of successful DAA treatment, which is characterized by the reduction in proportion of exhausted T cells and enhancement of virus-specific T cells response, and remarkable amelioration of liver inflammation, can be observed in the responders. Furthermore, lacking continuous IFN stimulation in the liver after clearance of the virus with DAA will likely also have a significantly beneficial effect on intrahepatic immune responses.Although DAA presents a high SVR for HCV-infected patients with the six major genotypes (HCV GT3 patients have a slightly lower SVR), it still confronts with the challenge of drug resistance, especially resistance-associated substitutions (RASs), which is the main reason of treatment failure. Drug resistance is an intrinsic and unavoidable problem in antiviral therapy because of the high adaptability of HCV and the failure to maintain a high pressure of inhibition 3 and 105 per copied base pair, as well as the high rate of viral replication (up to 1012 particles produced per day), leading to one mutation for every genome copied. As a consequence, HCV exists as a quasi-species population: a complex mixture of genetically distinct but closely related viral populations that constitutes a reservoir for the emergence of resistant strains It is well known that resistance mutations are favored by the lack of efficient proof-reading activity of HCV RNA-dependent RNA polymerase, which elevated to be between 10Encouraging clinical and retrospective study have shown that the prevalence of RASs in the gene regions of three non-structural proteins of HCV is not the same, and this may be due to their different roles in HCV life cycle, which determines the resistance barriers of the three non-structural proteins of HCV are different  and non-nucleoside inhibitors (NNIs). Because all NISs target the highly conserved active sites of polymerases, these inhibitors tend to be pan-generic In addition to these common resistance mutations, several prevalence studies have demonstrated that there are still some RASs pre-existing prior to the treatment within the viral population of an infected patient. Although not as common as the RASs merging under drug selective pressure, baseline resistance may also affect the outcome of DAA therapy.As epidemiological studies have shown that Q80K RAS in NS3 is associated with significantly lower SVR rates for treatment with SMV + peg-IFN-α + RBVVariants at positions 31, 54 and 93 are most common in NS5A, including L31M, Q54H and Y93H Variant L159F in NS5B was detected in 1% of the subjects with GT1 infection and was only observed to have a direct association with accumulated virological failure when patients were treated with SOF + RBV for short durations The prevalence of RASs is different among the major genotypes and subtypes of HCV, and it may due to various resistance barriers of them. For example, the most frequent RAS is Y93H in subjects with HCV GT3 and GT1b infection, but rarely observed in patients with HCV GT1a infection Therefore, it remains necessary to detect the HCV genotype and subtype of a patient. Because a correct HCV genotype/subtype determinate by baseline HCV sequencing can provide critical virological information for the detection of genetic variants that have a potential impact on therapy response, which can guide the selection of the optimal antiviral regimen for patients and decrease frequency of treatment failure.In addition, encouraging epidemiological studies have demonstrated that the prevalence of RAS is also related to geographical distribution, resulting in some of more frequent mutations in some places. Statistically, the overall prevalence of Q80K is 7.5%, decomposed into19.8% in GT1a, 0.5% in GT1b, and 18.2% in other or unknown GT1 subtypes In summary, the drug resistance of DAA is associated with a variety of factors, and RASs can be detected in all patients with treatment failure. Thus, the detection of RASs is critical for the success of treatment, including the RASs naturally occurring and selected out during therapy. Until now, the detection of drug resistance mainly relies on Sanger automated sequencing and second-generation sequencing (NGS), and NGS is an emerging technology with great potential in data analysis and data integrity Thus, the drug resistance of DAA is a complex and unavoidable problem, which may result in bad response to antiviral therapy and relapse in HCV infected patients. To date, with the aim of decreasing frequency of RASs, it is useful and common to select different classes DAAs for combination treatment. And a correct determination of HCV genotype and subtype and detection of pre-existing RASs in sequence remains important for guiding selection of most appropriate antiviral regimen.The combination, oral DAA (±RBV) therapies have become the clinical treatment of choice, which has revolutionized cure rate for patients with HCV infection. In addition to the excellent antiviral effect that more than 91% SVR can be achieved in patients, there are some unexpected effects of recovery in liver dysfunction, metabolic disorder and immune function damage in patients with clearance of HCV compared to IFN-based therapy. However, presence of baseline resistance and RASs occurring during antiviral treatment is critical and notable a challenge, which can be observed in all patients with treatment failure. Therefore, with the aim of reduction of frequency in RASs and non-responders, selection of DAA therapies should be taken with many factors into consideration, including correct determination of HCV genotype and subtype and detection of RAS in sequence. Thus, the exploitation of antiviral drugs suitable for the all main HCV genotypes, to minimize the incidence of resistance mutations in order to further improve the sustained viral response, remains a focus of future research and with a great pool of knowledge based on profound researches, more effective therapies are hoped to emerge.


The need for extended venous thromboembolism (VTE) treatment beyond 3 to 6 months is usually determined by balancing the risk of recurrence if treatment is stopped against the risk of bleeding from continuing treatment. The risk of recurrence, and in turn the decision to extend, can be determined through the nature of the index event. Patients with VTE provoked by surgery or trauma (major transient risk factors) are recommended to receive 3 months of anticoagulation therapy because their risk of recurrence is low, whereas patients with VTE provoked by a major persistent risk factor, such as cancer, or those considered to have “unprovoked” VTE, are recommended to receive an extended duration of therapy based on an established high risk of recurrence. Nonetheless, recent evidence and new guidance identify that this approach fails to consider patients with risk factors classed as minor transient  or minor persistent . Indeed, the risk of recurrence with respect to VTE provoked by minor persistent risk factors has been demonstrated to be not dissimilar to that of VTE without identifiable risk factors. This review provides an overview of the available data on the risk of recurrence according to the underlying cause of VTE, a critical evaluation of evidence from clinical studies on the available anticoagulants for extended VTE treatment, models of risk prediction for recurrent VTE and bleeding, and guidance on how to apply the evidence in practice. Venous thromboembolism (VTE), encompassing deep vein thrombosis (DVT) and acute pulmonary embolism (PE), affects approximately 1 to 2 in 1,000 individuals annually and is the third most common cause of vascular death after myocardial infarction and stroke.After a 3-month course of anticoagulant therapy, the need for extended treatment is usually determined by balancing the risk of recurrence if treatment is stopped against the risk of bleeding if treatment is continued.Previously, the need for extended anticoagulation beyond 3 months was often dichotomized depending on whether the index VTE event was considered “provoked” or “unprovoked.” Traditionally, VTE provoked by a major transient risk factor, such as surgery or trauma, only required a 3-month course of anticoagulation, whereas unprovoked VTE or VTE associated with a major persistent risk factor, such as cancer, required extended therapy beyond 3 months.Determining the risk of recurrence for patients with VTE provoked by major transient or major persistent risk factors is simple. More difficult is determining the risk for recurrence in patients with minor transient or minor persistent risk factors and in those with truly unprovoked VTE. The risk factors within each of these classifications are provided below, and a summary of the 1- and 5-year recurrence rates following discontinuation of anticoagulation therapy for each risk factor category is shown inSuch risk factors include major surgery and major trauma.Examples of major persistent risk factors include cancer and conditions such as antiphospholipid syndrome. Cancer is the most common major persistent risk factor. If anticoagulation is stopped after 3 months in patients with VTE in the setting of metastatic cancer, the annualized risk of recurrence is approximately 15% per year, with an unknown risk at 5 years because of the high mortality rate among such patients.Examples of minor persistent risk factors, including inflammatory bowel disease, congestive heart disease, obesity, and their associated risks of VTE recurrence are shown inExamples of minor transient risk factors, including immobilization, use of oral estrogen therapy, pregnancy, lower limb injury with impaired mobility, and minor surgery, and their respective risks of VTE recurrence are shown inVTE treatment has traditionally been divided into three phases: initial (first 5–10 days), long-term (up to 3 months), and extended (beyond 3 months). Direct oral anticoagulants (DOACs) are replacing warfarin for the initial, long-term, and extended treatment of VTE, with guidelines now giving preference to them over vitamin-K antagonists (VKAs). Evidence of the efficacy and safety of DOACs for extended VTE treatment comes from studies comparing them with placebo, acetylsalicylic acid (ASA), or warfarin. Studies with apixaban or rivaroxaban for extended VTE treatment indicate that their efficacy is maintained even if the dose of the drug is lowered. Lower dose regimens are likely to enhance the benefit–risk profile of DOACs, even further than full-dose regimens. Phase-III trial results , the findings of a phase-III study post hoc analysis, and real-world evidence evaluating the DOACs for extended VTE treatment are summarized inp < 0.001), without an increase in the incidence of major bleeding . The RRs of the composite endpoint of major or clinically relevant nonmajor (CRNM) bleeding with apixaban 5-mg bid and apixaban 2.5-mg bid were not significantly increased compared with placebo .In AMPLIFY-EXT, two doses of apixaban (5-mg twice daily [bid] and 2.5-mg bid) were compared with placebo in patients who had completed 6 to 12 months of anticoagulation therapy. Both doses were associated with a reduced risk of the primary outcome of recurrent VTE or death from any cause compared with placebo , and in RE-MEDY, dabigatran was noninferior to warfarin . The incidence of major bleeding was not significantly different with dabigatran versus warfarin or placebo . However, the incidence of the composite of major or CRNM bleeding with dabigatran was significantly lower than that with warfarin , but significantly higher than that with placebo .In two double-blind randomized trials, dabigatran 150-mg bid was compared with either warfarin (active control) or placebo in patients who had completed ≥3 months of anticoagulation therapy. In RE-SONATE, dabigatran significantly reduced the incidence of recurrent or fatal VTE or unexplained death versus placebo .p < 0.001), whereas the incidence of major bleeding was not significantly increased , but there was more CRNM bleeding with rivaroxaban than with placebo .EINSTEIN EXT compared rivaroxaban 20-mg once daily (od) with placebo for 6 or 12 months in patients who had completed 6 to 12 months of prior anticoagulation therapy. The incidence of recurrent VTE was significantly lower with rivaroxaban than with placebo . The incidence of major bleeding was similar in all groups .EINSTEIN CHOICE compared the efficacy and safety of the treatment with two doses of rivaroxaban  versus ASA (100-mg od) for secondary prevention in patients who had completed 6 to 12 months of anticoagulation. Both the high and the low doses of rivaroxaban regimen significantly reduced the incidence of recurrent symptomatic fatal or nonfatal VTE compared with ASA  of patients who received rivaroxaban, 5.5% (26/468) of patients who received ASA, and 8.2% (20/243) of patients who received placebo. The corresponding cumulative 1-year incidences for recurrent VTE were 2.0, 5.9, and 10.0%, respectively.In patients with VTE provoked by major transient risk factors, there were no recurrences in any treatment group. In contrast, the rates of recurrence in patients with VTE provoked by minor persistent or minor transient risk factors were not significantly lower than in patients with unprovoked VTE . Minor persistent risk factors included renal impairment, family history of VTE, and lower extremity paralysis or paresis; minor transient risk factors included travel >8 hours in duration, use of estrogen therapy and pregnancy, and leg injury with impaired mobility. These findings suggest that some patients with VTE provoked by minor persistent or minor transient risk factors should also be considered for extended anticoagulation (dependent on their risk of bleeding).Results from randomized controlled trials (RCTs) may not translate fully into clinical practice owing to differences in medication environments , patient populations (homogeneous vs. heterogeneous), and adherence to medication. Real-world evidence can help reinforce results from phase-III studies. The results from the pooled XALIA studies  and the SWIVTER, PREFER in VTE, GARFIELD-VTE, REMOTEV, Dresden non-VKA oral anticoagulant and RIETE registries have reported reductions in the incidence of recurrent VTE and major bleeding associated with DOACs compared with VKAs similar to those found in the RCTs. These real-world studies provide additional support for the efficacy and safety of anticoagulation in patients treated in routine practice to help inform clinical decision-making on extended anticoagulation.Guidelines recommend a minimum of a 3-month course of anticoagulation therapy for all patients with VTE.Estrogen use is considered a minor transient risk factor for VTE,Pregnancy has also been identified as a minor transient risk factor for VTE.Guidance from the International Society on Thrombosis and Haemostasis (ISTH) states that edoxaban and rivaroxaban are currently the only DOACs that have been compared with LMWH in RCTs in cancer populations.p < 0.001 for noninferiority) and was not associated with a significant increase in major bleeding .It is expected that recommendations in future updates to older guidelines on the duration of secondary prevention for VTE will also be amended in response to the latest evidence from the DOAC trials. For example, the recently published CARAVAGGIO study showed that apixaban 5-mg bid had noninferior efficacy to dalteparin for the treatment of cancer-associated VTE , chronic renal or hepatic disease, concomitant antiplatelet therapy or use of nonsteroidal anti-inflammatory drugs, other serious acute or chronic illness, and poor anticoagulation control.A predictive model of note is VTE-BLEED, a tool used to predict major bleeding during chronic anticoagulation for VTE.HAS-BLED  is another well-known bleeding risk score that was originally developed to estimate the risk of major bleeding in patients with atrial fibrillation. Evaluation of its predictive value for major bleeding risk has more recently been demonstrated in the first 6 months of anticoagulation therapy in patients with VTE but further adaptations and validation may be warranted.Risk factors and clinical presentation can provide guidance on extended anticoagulation for many patients with VTE but not all. Additional factors to consider when there is the choice of extending anticoagulation are D-dimer levels, clinical presentation , presence of postthrombotic syndrome or post-PE symptoms, patient preference, familial history of VTE, and thrombophilia testing (to uncover persistent risk factors). There are also some strategies that can be employed to help clinicians. Two such strategies are formal risk assessment models for recurrent VTE risk and risk of bleeding if anticoagulation is continued.The Vienna Prediction Model is based on the following three key parameters: (1) patient sex, (2) site of VTE, and (3) D-dimer level. This model allows prediction at 3 weeks and 3, 9, 15, and 24 months after stopping anticoagulation .2651The DASH  score predicts which patients are at low risk of VTE recurrence and, therefore, can stop anticoagulation after an appropriate 3- to 6-month period of treatment. The score is based on D-dimer levels, age, sex, and hormone treatment, and patients who score ≥2 should be considered at high risk of recurrent VTE and, therefore, eligible for extended VTE treatment. The DASH score has been externally validated and its utility in younger patients was confirmed; however, the risk of recurrence in patients >65 years of age is >5%, even in patients with low DASH scores . It sho2, and features of postthrombotic syndrome such as hyperpigmentation, edema, and redness  score operates on the principle that men with unprovoked VTE should continue anticoagulation treatment, whereas women with unprovoked VTE and a score ≥2 should continue. Risk factors considered are D-dimer levels ≥250 µg/mL on anticoagulant therapy, age ≥65 years, body mass index ≥30 kg/mredness .5455It should be noted that while all the VTE risk scores use D-dimer level as a variable, Men Continue and HERDOO2 measure it while patients are on anticoagulation, whereas the Vienna model and DASH score assess D-dimer levels after stopping treatment.Based on the latest guideline recommendations for patients with VTE and the latest evidence in the field, a stepwise approach should be considered, as outlined inThe benefit–risk profile of extending anticoagulation in the era of “low-dose” DOACs is different than it was in the VKA era, because of the improved safety profile and convenience associated with DOACs. When not opting for continued secondary prevention, thromboprophylaxis in high-risk situations, clinical vigilance, and timely diagnosis are valid alternatives for many patients .Guidance for the extension of anticoagulation beyond 3 months has traditionally been clear for patients with VTE provoked by a major transient risk factor  or a major persistent risk factor . However, debate has existed over whether anticoagulation should be extended in the case of minor persistent or minor transient risk factors. New evidence in the latter settings from clinical studies involving DOACs and updates to international guidelines, along with risk prediction tools, can help guide and inform clinical decisions on extending anticoagulation treatment in patients with such risk factors. Additional guidance on anticoagulation duration in special circumstances such as pregnancy and contact sports would also be of value in the future.


RECQL1, BLM, WRN, RECQL4, and RECQL5, are unique to mammals, as simpler eukaryotes and bacteria appear to have only one homolog, RecQ. The precise role of each of the five mammalian RecQ helicases remains to be determined. Whereas loss of function mutations of BLM, WRN, and RECQL4 in humans are associated with specific diseases, RECQL1 and RECQL5 have not yet been associated with specific disorders. Mice deficient in Recql5 are more likely to develop cancer, and human cells deficient in RECQL5 display chromosomal instability and elevated sister chromatid exchange events, similar to cells deficient in any of the other RecQ helicases. Recent studies support the hypothesis that RECQL5 can resolve intermediate DNA repair structures resulting from the collision of DNA transcription and replication machinery. In this review, we aim to summarize current knowledge regarding RECQL5 in the context of DNA repair, replication, and transcription to help uncover the role of RECQL5 in the maintenance of genome stability.Maintenance of genome stability is essential to prevent the accumulation of DNA mutations that can initiate oncogenesis and facilitate tumor progression. Studies of DNA repair genes have revealed a highly dynamic and redundant network of genes and proteins responsible for maintaining genome stability. Cancer cells are often deficient in DNA repair, and the resulting genome instability decreases their fitness but also allows for more rapid evolution under selective pressure. Of particular interest for genome stability are the RecQ class of helicases. Five genes in this class,  RecQ, whereas humans have five RecQ genes with a unique gene structure each, suggestive of functional divergence  responsible for interacting with DNA  domain, which together make up the catalytic core of the enzyme . Some mewith DNA . It is twith DNA .BLM, WRN, and RECQL4 are associated with specific diseases of marked premature aging and cancer predisposition such as Bloom Syndrome, Werner Syndrome, and Rothmund-Thompson Syndrome, respectively, whereas RECQL1 and RECQL5 remain to be associated with specific disorders  being the most predominant . Finally, strand extension of the invaded strand can occur either by synthesis-dependent strand annealing (SDSA) in the case of D-loop formation (step 3b) or through canonical DSB repair (DSBR) in the case of dHJ formation. Canonical DSBR occurs at the risk of forming hazardous crossover (CO) products where either sister chromatid or homologous chromosome donor molecules exchange strands of DNA between molecules  predominates during G1 because cells have yet to replicate their DNA and cannot access the redundancy of genetic material required as a template for faithful DNA repair by homologous recombination (HR) . There aolecules . SDSA prolecules . In canoolecules . In the olecules . When dHolecules  that canolecules . Alternaolecules . Efforts–/– BLM–/–RECQL5 double knockouts was the first evidence that these genes may have non-overlapping roles as well. It was later shown in vivo that RECQL5 is essential for this disruptive interaction with RAD51 and its ability to form D-loops  complex . Cells dAnother source of transcription-associated genome instability is the formation of R-loop structures at sites of active transcription during replication. The formation of ssDNA from negative supercoiling behind transcription allows RNA invasion, forming an R-loop and making it difficult for replication machinery to continue . RECQL5-These findings support a model of RECQL5 that intimately relates transcription to replication and serves to limit TRCs. There is evidence that it does so both proactively by either inhibiting transcript elongation near sites of replication or remodeling chromatin to dislodge RNAPII from DNA and retroactively by limiting RAD51-mediated replication fork reversal and promoting MUS81-EME1 cleavage and replication fork restart .It is clear that RECQL5 serves as an important regulator of DNA repair intermediate structures that may arise during DNA damage, replication stress, and transcriptional stress. This essential regulatory role of RECQL5 is further highlighted by the observed elevated RECQL5 expression and gene amplification in urothelial carcinoma of the bladder and in breast cancers . HoweverThe larger body of research on other RecQ helicases supports further studies of RECQL5 in parallel with other RecQ helicases. Given that loss of RECQL5 increases SCEs, it will be of interest to map the location of such events, as it was shown that BLM preferentially prevents SCE events near transcribed genes and G-quadruplex motifs . There mZH wrote the first draft of the manuscript. ZH and PL contributed to manuscript revision and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Waterlogging is one of the main abiotic stresses suffered by plants. Inhibition of aerobic respiration during waterlogging limits energy metabolism and restricts growth and a wide range of developmental processes, from seed germination to vegetative growth and further reproductive growth. Plants respond to waterlogging stress by regulating their morphological structure, energy metabolism, endogenous hormone biosynthesis, and signaling processes. In this updated review, we systematically summarize the changes in morphological structure, photosynthesis, respiration, reactive oxygen species damage, plant hormone synthesis, and signaling cascades after plants were subjected to waterlogging stress. Finally, we propose future challenges and research directions in this field. Plants achieve normal growth through the coordination of water absorption by the roots with transpiration from the leaves. Sufficient water is a prerequisite for normal growth of plants, but saturation of the soil water-holding capacity, or even super-saturation, easily leads to waterlogging stress. The inhibition of root respiration and accumulation of toxic substances during waterlogging stress have adverse effects not only on vegetative growth, but also on reproductive growth, eventually leading to yield loss or even complete harvest failure . Therefovia glycolysis and ethanol fermentation. However, prolonged duration of waterlogging and anaerobic respiration ultimately leads to the accumulation of toxic metabolites such as lactic acid, ethanol, and aldehydes, combined with an increases in reactive oxygen species (ROS), notably hydrogen peroxide, thus eventually leading to cell death and plant senescence  or other aeration tissues, rapid elongation of apical meristematic tissue, barriers to radial oxygen loss (ROL), and the formation of air films in the upper cuticle  Schott] after 8 days of waterlogging and found that the root tips turned blue, whereas no blue areas appeared in the middle sections of the roots. This indicated that O2 leakage was detected only near the root tip along the intercellular spaces of the rhizosphere, as the ROL barrier was formed in the middle of the root and prevented lateral losses (2 in the primordia of aerenchyma in rice (Oryza sativa) after 12 h of waterlogging , thus promoting internode elongation  seeds for 2, 4, 6, and 8 days to explore the changes in physiological and biochemical characteristics with time under waterlogging. The content of the photosynthetic pigments, chlorophyll A, B chlorophyll A + B, and carotenoids, was significantly lower in waterlogged seeds than in the unwaterlogged control; as the content of photosynthetic pigments decreased, the photosynthetic capacity also decreased.Photosynthetic pigments are the material basis of plant photosynthesis, and the change of pigment content and composition directly affects the photosynthetic rate. Gossypium hirsutum L.) leaves was down-regulated; a reduction in net photosynthetic rate of cotton was mainly caused by lower rubisco activity. Sucrose and starch are the main products of photosynthesis in most plants. Sucrose is the main transport carbohydrate from source to sink, a process that is very sensitive to waterlogging. The decreased photosynthetic rate, sucrose conversion rate, and initial rubisco activity directly reduced the boll weight of waterlogged cotton. The enzyme sucrose synthase is central to the metabolic breakdown of sucrose required for cellulose biosynthesis; increased gene expression and enzyme activity of sucrose synthase during waterlogging were associated with prolonging the period of rapid accumulation of seed fiber weight, tending to reduce the phenomenon of boll weight decline caused by waterlogging  by interfering with the electron transport chain, leading to inhibition of mitochondrial respiration  or via pyruvate decarboxylase (PDC) turning pyruvate into acetaldehyde, which is then reduced to ethanol by alcohol dehydrogenase (ADH)  . ADH andrlogging . Therefo soybean . The seeentation . The oveolerance . These rLDH significantly enhanced the PDC activity and hypoxia resistance of Arabidopsis, whereas LDH loss of function mutant ldh showed the opposite phenotype , hydroxyl radicals (⋅OH), and hydrogen peroxide (H2O2) have strong oxidizing activity that can lead to lipid peroxidation and delipidation of leaf membranes, oxidative damage to proteins, oxidative damage to DNA, and severe damage to cell membranes and organelles , as well as the regulation of hypoxia-induced downstream genes such as ERF73/HRE1 and ADH1 and the expression of genes encoding peroxidase and cytoplasmic P450. Subsequently, atrboh d and atrboh f and the double-mutant atrbohd/f. Both Atrboh D and Atrboh F play a role in hypoxia signal through the production of ROS, promoting the increase of Ca2+ and mediating hypoxia-induced expression of downstream genes, such as ADH1, PDC1, ERF73, MYB2, LDH, SUS1, SUS4, HsfA2, and HSP18.2, thus improving the tolerance of Arabidopsis to hypoxia stress. These findings provide new insights into the adaptation mechanism of Rboh gene regulation under waterlogging stress in plants.Although excessive ROS are harmful to plant cells, ROS can also act as signaling molecules in plant cells under stress. Plant NADPH oxidase is a key enzyme in the production of ROS and plays a vital role in ROS-mediated signal transduction. The expression of NADPH oxidase–related gene rlogging . Analysi2O2 is an essential signaling molecule involved in ET-induced epidermal cell death. The formation of aerenchyma in rice stems is controlled by H2O2, indicating that ROS play a key role in regulating various cell death processes in rice   . The H2Oo stress . In linevia complex signaling. Plant hormones, as important endogenous signals, play a central role in the mechanism of waterlogging tolerance , the direct precursor in the biosynthesis of ET, is produced in large quantities under the catalysis of ACC synthase (ACS), and the process can occur under hypoxic conditions. ACC is converted to ET under the catalysis of ACC oxidase (ACO), but the process needs the participation of OSolanum lycopersicum) seedlings, and 500 μM aminoethoxyvinylglycine (AVG), which inhibits ET biosynthesis, was sprayed on the above-ground parts daily. After 72 h of waterlogging, AR primordia became visible in the stem base of waterlogged plants that had not been treated with AVG, and these primordia elongated and generated a large number of ARs within 7 days. The number of ARs in tomato plants treated with the inhibitor AVG was significantly lower than in untreated plants  is an important transcription factor involved in plant responses to several different biotic and abiotic stresses. The ERF family genes specifically induce genes containing AGCCGCC elements and DRE/CRT cis-elements, activating or inhibiting the expression of downstream functional genes, and thereby mediating plant tolerance to various stresses  play an important role in ET signal transduction and plant responses to waterlogging  gene is another member of the ET-response factor ERF-VII family. Overexpression of Sub1A enhanced the transcription of ADH1 in transgenic rice and at the same time led to the enhanced ability to withstand waterlogging stress. Therefore, Sub1A gene could be the main determinant of submergence tolerance , can also regulate the waterlogging tolerance of rice. Overexpression of SK1/2 led to internode elongation and significantly improved the waterlogging tolerance of deepwater rice cultivars (The rice olerance . Interesultivars .Sub1A and SK1/2 have opposite functions in regulating rice growth in response to waterlogging. Sub1A negatively regulates the GA response by limiting the degradation of the DELLA family protein SLR1 (Slender Rice-1), which are GA signal inhibitors, and thereby inhibits the elongation of rice , and SD1 protein promotes the synthesis of GA, mainly GA4, thus promoting shoot elongation  has a main role in regulating stomata by adjusting the size of guard cells, thereby regulating the water potential in plants. Because of this, ABA is considered to be a key hormone in water stress responses .Abscisic acid is involved in the development of root aerenchyma under waterlogging. The ABA concentration in soybean hypocotyls decreased rapidly under waterlogging, falling by 50% within 24 h compared with plants without waterlogging. In addition, secondary aeration tissues appeared after 72 h. Exogenous 1 μM ABA treatment inhibited the cell development of aerenchyma, suggesting that the formation of secondary aerenchyma required a reduction in the concentration of the negative regulatory factor ABA .Solanum dulcamara, following the rapid down-regulation of ABA biosynthesis and up-regulation of ABA decomposition, the ABA concentration in the stem and AR primordia decreased sharply  gene AdPDC1 encoding pyruvate decarboxylase was significantly up-regulated under waterlogging, suggesting that the gene played an important role in the waterlogging response. ABA down-regulated the expression of AdPDC1 under waterlogging, whereas the overexpression of AdPDC1 in Arabidopsis inhibited seed germination and root elongation under ABA treatment, indicating that ABA might negatively regulate AdPDC1 under waterlogging  plays an important role in plant growth and development . ET prodOsPIN2, suggesting that NPA might inhibit the production of ARs through an effect on PIN , and treatment of rice with the transport inhibitor NPA decreased the expression of t on PIN . Similarransport .However, some studies found that waterlogging reduced the content of IAA in soybean plants. Waterlogging can cause a large amount of carbohydrate consumption in plants, leading to energy shortage. GAs are one of the essential plant hormones regulating growth and development. GAs regulate multiple processes in plant growth and development, mainly by controlling the size and number of cells .Arachis hypogaea) under waterlogging and found that spraying GA on the leaf surface could promote the growth of upper and underground parts of peanut plants and significantly increase the yield. Studies on different genotypes of soybean found that GA content in waterlogging-tolerant lines significantly increased under waterlogging, and GA content in waterlogging-resistant lines was significantly higher than that in waterlogging-sensitive lines . Huang eOs1, OsCPS2, OsKS2, OsKS5, OsKO2, OsKAO, Os13ox, OsGA20ox1, OsGA20ox2, OsGA20ox3, OsGA3ox1, OsGA3ox2) and signal transduction genes  also inhibited internode elongation  is a common phenolic compound in plants, which regulates the antioxidant mechanism of cells by inducing the expression of stress-related genes, thus enhancing the adaptability of plants to adverse conditions .Prunus persica L.) were subjected to waterlogging stress. Spraying exogenous SA on day 1 of waterlogging can significantly increase the activities of ethanol dehydrogenase, protective enzymes such as POD and CAT, and the content of proline in leaves and roots, thereby protecting leaves and root membranes from damage and stabilizing photosynthetic capacity of leaves as well as root activity. Together, these protective effects are conducive to the alleviation of waterlogging-induced stress  is a basic plant growth regulator that is known to be involved in the defense response produced by abiotic stress, but there are few studies on the relationship between JA and waterlogging tolerance .The interaction between JA and ET plays an important role in the formation and development of the root system and aerenchyma under waterlogging stress. Spraying methyl jasmonate on the leaves increased the content of ET . Thus, eBrassinosteroid (BR) is a naturally occurring steroid in plants. BR can induce resistance to a variety of biological and abiotic stresses, thus promoting plant growth and development .Exogenous 24-epi-brassinolide (EBR) promotes the transfer of carbohydrates from leaves to roots of cucumber seedlings under hypoxic stress, enhances the activity of glycolytic enzymes in the roots, and triggers the antioxidant enzymes activity and reduced ROS production, thus improving the resistance of the seedlings to hypoxic stress . EBR alsSub1A gene, an ET-response factor ERF-VII family member, has different regulatory effects on brassinolide biosynthetic gene expression and rice shoot elongation under waterlogging, compared with exogenous BR. Exogenous BR pretreatment can activate the tolerance mechanism in waterlogging-tolerant rice genotypes and inhibit shoot elongation under waterlogging. Compared to the LOES, higher expression of BR biosynthesis genes was observed in the Sub1A rice genotype, which triggered the endogenous BR level. The enhanced BR level induced the transcription of GA catabolism gene GA2ox7 resulting in reduced GA contents. At the same time, GA-mediated responses can be negatively regulated under submerged conditions by a DELLA family member, the GA-signal inhibitory factor SLR1 protein, so that the elongation of rice plants is inhibited (Sub1A genotype.The nhibited . TherefoSome selected recent studies on Phytohormones and plant growth regulator mediated waterlogging tolerance in plants are presented in Melatonin (MT) is a phytohormone and an excellent antioxidant molecule that augments plant growth under adverse conditions . The MT MbT5H1, MbAANAT3, and MbASMT9 increased the production of endogenous MT in the seedlings treated with MT . Also, the exogenous MT treatment not only increased the endogenous MT level but also stabilized the normal functioning of other biochemical and physiological parameters   . This memportant . The appor genes . TherefoAtACO5 gene in Arabidopsis triggered the ET production, and cell expansion activities resulted in enhanced tolerance against waterlogging stress  and ET biosynthesis gene (AtACO1), conferring resistance to waterlogging stress  improving cultivation management to reduce the direct damage to crops caused by waterlogging and (2) using modern molecular biology technology to discover the key genes regulating waterlogging tolerance and verify their functions.Building on existing research results and aiming to address identified problems, the following aspects should receive increased attention in future research on plant waterlogging tolerance:(1)Current studies focus mainly on the vegetative growth stage of plants under waterlogging stress. However, the molecular responses during seed germination, early seedling morphogenesis, and late reproductive growth under waterlogging stress are neglected topics that warrant further study.(2)Although a large number of related genes regulating plant waterlogging tolerance have been obtained by transcriptomics, proteomics, and other methods. However, most of them are preliminary study and required functional characterization.(3)There is a need to exploit additional genetic resources for waterlogging tolerance, using both isolated populations and natural populations to identify waterlogging tolerance–related genes.(4)The hormonal-induced waterlogging resistance has been studied extensively. Majority of the available studies mainly reported the effects of growth hormones on vegetative stages under waterlogging stress. Although studies are missing to investigate the role of these phytohormones when waterlogging stress happens at reproductive stages of the plant. Hormonal crosstalk under waterlogging stress in the early developmental stages of plant has been investigated and is presented in JP wrote the manuscript. RS, XX, and XC revised and finally approved the manuscript for publication. All the authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lymphedema is a complex disease caused by the accumulation of fluid in the tissues resulting from a dysfunctional or damaged lymphatic vasculature. In developed countries, lymphedema most commonly occurs as a result of cancer treatment. Initially, impaired lymph flow causes edema, but over time this results in inflammation, fibrotic and fatty tissue deposition, limited mobility, and bacterial infections that can lead to sepsis. While chronically impaired lymph flow is generally believed to be the instigating factor, little is known about what pathophysiological changes occur in the lymphatic vessels to inhibit lymph flow. Lymphatic vessels not only regulate lymph flow through a variety of physiologic mechanisms, but also respond to lymph flow itself. One of the fascinating ways that lymphatic vessels respond to flow is by growing bicuspid valves that close to prevent the backward movement of lymph. However, lymphatic valves have not been investigated in cancer-related lymphedema patients, even though the mutations that cause congenital lymphedema regulate genes involved in valve development. Here, we review current knowledge of the regulation of lymphatic function and development by lymph flow, including newly identified genetic regulators of lymphatic valves, and provide evidence for lymphatic valve involvement in cancer-related lymphedema. Insult to the lymphatic vasculature, be it through trauma, surgery, or infectious disease, is required for the development of secondary lymphedema. However, the gradual and unclear pathogenesis of cancer-related lymphedema (CRL) argues that the initial damage to the lymphatic vasculature is, by itself, not sufficient to cause overt swelling and fibrosis. Secondary lymphedema does not afflict all cancer patients, indicating that CRL patients may be predisposed to its development or that multiple hits are required to cause CRL as opposed to congenital lymphedema that is caused by single gene mutations. Curiously, secondary lymphedema does not occur immediately after cancer surgery, often taking up to several years to manifest, suggesting that there are likely several pathological processes that promote its development. Most agree that the first step in the development of secondary lymphedema is the impairment of lymph flow.Over the past decade, many studies have revealed how lymphatic vessels both regulate and are regulated by lymph flow. Two classifications of lymphatic vessels regulate lymph flow using different mechanisms that are relevant to understanding lymphedema . The firCancer-related lymphedema (CRL) is a progressive disease characterized by swelling, fibrosis, inflammation, and recurrent infections of the affected limb or tissues ,3. In deHowever, the pathophysiology of CRL remains poorly understood. The simple “stopcock hypothesis” that assumes surgery or trauma blocks lymph flow and leads to an accumulation of fluid and protein ,8 is ins+ T-cells or antibody depletion of the Th2 cytokines, IL4 and IL13, prevented the inflammation, fibrosis, and swelling in the mouse tail lymphedema model. The same approach was able to decrease the swelling that had already developed. Follow-up studies have confirmed the critical role of T-cells and the Th2 inflammatory response in other animal models of lymphedema [Based on the clinical features of lymphedema, it was recently hypothesized that surgery causes a reduction in lymph flow which initiates a feed forward cycle of inflammation, leading to tissue fibrosis that then inhibits lymphatic function and further reduces lymph flow . Using tmphedema ,12,13.4 (LTB4). At low doses, LTB4 was found to increase the expression of the lymphatic growth factor receptor, VEGFR3, whereas high doses similar to those found in chronic lymphedema inhibited both the VEGFR3 and Notch1 pathways [Confirming a crucial role for inflammation, it was recently discovered that the non-steroidal anti-inflammatory drug (NSAID) ketoprofen was able to reverse edema and fibrosis of experimental lymphedema in mice . Howeverpathways . A clinipathways . PatientFollowing axillary lymph node removal, it is known that lymph outflow from the arm is reduced ,17. BaseWhile these hypotheses are based on gradual, progressive processes that offer explanations for why BCRL does not develop immediately post-surgery, there are still several questions that remain. For example, how does reduced lymph flow initiate inflammation? Is lymphatic endothelium also involved in the pathogenesis of lymphedema? Are lymphatic valves involved in CRL pathogenesis? In the next sections, we will outline and propose a possible answer to these questions.The lymphatics play an important role in maintaining tissue fluid homeostasis by absorbing interstitial fluid in the form of lymph and returning it into the central circulation. Lymph formation involves movement of fluid from interstitial tissues into lymphatic capillaries and this requires a downhill pressure gradient between these compartments that is established by periodic external forces such as cardiac contractions, arterial pulsation, respiration, peristalsis of the gut, and skeletal muscle contractions . However, these external forces are not sufficient to transport lymph against a pressure gradient to the bloodstream in larger mammals, such as humans, where the force of gravity directly opposes the vertical transport of lymph.2O in humans. Due to the combination of lymphatic pump activity and external forces, the collecting lymphatic vessels experience constant oscillations in pressure and flow [The smooth muscle cells covering collecting lymphatic vessels, therefore, have an inherent contractile activity that enables them to actively regulate lymph flow. Lymphatic muscle cells circumferentially wrap collecting lymphatic vessels and exhibit regular, periodic, and forceful contractions that generate the pressure required to move lymph flow along the lymphatic vessel ,22. Reguand flow . Additioand flow .2, this is a poor indicator of the maximum shear stress that the endothelium experiences because lymph flow is oscillatory. Thus, the peak shear stress was between 2.5 and 12 dynes/cm2 [Lymph flow exerts a physical frictional force on the lymphatic endothelium called shear stress. Shear stress experienced by LECs lining the vessel can vary in magnitude and direction depending on local lymph flow dynamics. A study of mesenteric collecting lymphatic vessels in vivo described lymph flow as phasic with lymph flow velocity and shear stress being determined by the contraction cycle . While tynes/cm2 . Brief pynes/cm2 . While tynes/cm2 , which eynes/cm2  and is aThe physiological effect of shear stress on lymphatic function was demonstrated in a study wherein collecting lymphatic vessels were surgically removed from the mesentery and tied onto glass cannulas that controlled the pressure and flow through the vessel . Using tProx1 [Other studies have shown that lymphatic endothelium is highly sensitive to the direction and magnitude of lymph flow. In a mouse model of vascular malformation, redirection of blood flow through the lymphatic vasculature resulted in LECs losing the expression of the transcription factor responsible for conferring lymphatic fate, Prox1 . AdditioProx1 . TogetheProx1 .Prox1, Foxc2 and Gata2 to become specified to form valves [One of the most intensely studied roles of shear stress in the lymphatic vasculature has been the discovery that oscillatory shear stress drives the formation of lymphatic valves, which are crucial for maintaining forward lymph flow ,33. Valvm valves ,34. Aftem valves . Implicam valves . This wam valves . TherefoAfter their formation, lymphatic valve leaflets require constant lymph flow and shear stress signaling throughout life to escape cell death ,35,36. T2O). Therefore, the small pressures generated by pumping alone are insufficient to push lymph upwards through the legs, requiring lymphatic valves that prevent retrograde lymph flow and break up the pressure gradients to allow forward lymph flow. The same is true for the upper limbs, most of which hang below the level of the heart. Notably, BCRL patients have swelling of the upper limbs, while congenital lymphedema patients may have swelling in the lower and/or upper limbs. Over the past two decades, much has been discovered about the genetic regulators of lymphatic valve development, which will be discussed next.Physiologically, lymphatic valves open and close passively in response to pressure gradients exerted across the valve leaflets . If the The identification of gene mutations in patients presenting with congenital lymphedema has fueled the development of mouse models to gain insight into the genetic mechanisms underlying the development of lymphedema. Many of these studies have identified roles for these genes not only in lymphangiogenesis but also in valve formation and postnatal valve maintenance, thus supporting the assertion that valve defects likely underlie the initiation of congenital lymphedema and potentially CRL. Here, we review some of the genes that have been identified in congenital lymphedema patients and discuss their corresponding mouse models.FLT4, which encodes VEGFR3, have been linked to Milroy disease which is characterized by the onset of lymphedema in the lower limbs. Heterozygous missense mutations in the tyrosine kinase domain of the gene prevent the autophosphorylation of VEGFR3 which impairs its downstream signaling and affects lymphatic vessel development [Chy mouse strain that has impaired VEGFR3 signaling and is characterized by dermal lymphatic hypoplasia, lower limb swelling, and chylous ascites [4 signaling in CRL patients [The VEGFC/VEGFR3 signaling pathway is the central lymphangiogenic pathway that promotes lymphatic vessel growth from preexisting lymphatic vessels. During embryonic development, a subpopulation of blood endothelial cells in the cardinal vein begin to express the fate-determining transcription factor PROX1 to acquire a lymphatic identity. Due to a high concentration of VEGFC in the extracellular matrix outside the cardinal vein, these PROX1-positive cells begin to migrate and proliferate to form the lymphatic vasculature. This process is mediated by intracellular signaling events initiated upon binding of VEGFC to the tyrosine kinase receptor, VEGFR3, on the surface of LECs. Congenital mutations in elopment ,39. A si ascites . These spatients , which aPik3r1 and Pik3ca, which encode the regulatory and catalytic subunit of PI3K, respectively, caused defects in lymphatic sprouting and led to chylous ascites [Pik3r1 null mice failed to remodel into a mature lymphatic network and lacked lymphatic valves [Akt1−/− mice exhibited impaired LEC proliferation and remodeling defects as evidenced by reduced lymphatic capillary diameter, lack of lymphatic valves, and sparse smooth muscle coverage [The PI3K/Akt/mTOR pathway acts downstream of VEGFR3 activation to promote lymphangiogenesis ,42. Homo ascites ,44,45. Tcoverage . In the FOXC2 in patients with LD led to the development of +/−Foxc2 mice to study the pathogenesis of LD [FOXC2 gene contains a single exon, most mutations lead to a loss of the protein product and therefore global heterozygous mice are an accurate model for mutations in this gene [Foxc2 mice [Foxc2 gene were conditionally deleted from the lymphatic vasculature in postnatal mice, this led to a complete loss of lymphatic valves after many had already formed [FOXC2.Lymphedema distichiasis (LD) is an autosomal dominant disorder that is characterized by variable onset lymphedema in the lower limbs and the presence of an extra row of eyelashes  . Identifis of LD . Becausehis gene ,49. Lympxc2 mice ,49,50. Fy formed . This leGATA2 resulting in haploinsufficiency of the gene have been shown to underly Emberger syndrome [Prox1, Foxc2, Itga9, and Nfatc1 [Prox1 and Foxc2 locus and upregulating their expression in response to oscillatory shear stress [GATA2 that underlie Emberger syndrome produced mutant GATA2 proteins that were unable to bind enhancer elements in both the PROX1 and FOXC2 genes that were active only in the valve cells, and only these mutations were associated with lymphedema in patients. This study concluded that the GATA2 mutations abolishing enhancer-binding resulted in the loss of valves due to reduced expression of PROX1 and FOXC2, and strongly suggests that a loss of valves leads to lymphedema in human patients [Heterozygous germline mutations in syndrome ,52. Patisyndrome . GATA2 wd Nfatc1 . In a sur stress . Consistr stress . Additiopatients .CCBE1, FAT4, and ADAMTS3 underlie this syndrome and this has led to the development of mouse models to understand how mutations in these genes cause the lymphatic phenotypes associated with HS [Ccbe1−/− and Adamts3−/− embryos exhibited severe edema and were completely devoid of lymphatic vessels due to a failure of PROX1-positive LECs to migrate out from the cardinal vein [Fat4-deficient embryos, valve endothelial cells were unable to orient along the direction of flow due to impaired cell polarization and this affected cell migration and consequently valve leaflet formation [Generalized lymphatic dysplasia (GLD) presents as facial and full body edema along with some cases of pulmonary and intestinal lymphangiectasia, pleural effusion, chylothorax, and pericardial effusion . Henneka with HS ,56,57,58 with HS . The actnal vein ,61. Whilnal vein ,63. The ormation ,63.PIEZO1 [PIEZO1 in GLD patients prompted the investigation of the role of this ion channel in lymphatic development and function. In mice, it was later discovered that PIEZO1 was required for valve morphogenesis during embryonic development [Piezo1 caused the mice to develop pleural effusion and die within 2 weeks after birth, which was associated with an impairment in valve leaflet protrusion into the lumen leading to drastically fewer valves [The incidence of a distinct cohort of GLD patients exhibiting mild facial edema, no seizures, and normal intelligence led to whole exome sequencing studies that identified a new category of autosomal recessive GLD that was caused by mutations in the gene PIEZO1 ,65. PIEZelopment . This str valves . This str valves . A subser valves . These fITGA9 in fetuses with severe congenital chylothorax prompting further studies into the role of integrin-α9 in lymphatic vessel development [−/−Itga9 mice, which was responsible for leakage of lymph into the thoracic cavity and death of these mice postnatally [Integrin-α9 is a receptor on LECs that binds fibronectin-EIIIA (FN-EIIIA) that is a component of the extracellular matrix. Mice lacking integrin-α9 develop chylothorax and die of respiratory failure postnatally . In humaelopment . Integrielopment . The spetnatally . More imtnatally .RASA1. A small subset of patients harboring this mutation have also been found to exhibit lymphatic abnormalities such as lymphedema in the upper and lower extremities [RASA1 mutation revealed hyperplasia of lymphatic capillaries, abnormally dilated collecting vessels, and disrupted lymph flow [Rasa1 knockout mice, RASA1 was initially identified as a negative regulator of LEC proliferation, making it critical for preventing lymphatic vessel overgrowth in adult animals [Rasa1-deficient mice, it failed to explain the appearance of chylothorax. The same group later found that deletion of Rasa1 resulted in LEC apoptosis within valve leaflets, resulting in dysfunctional valves that were unable to seal tightly  against an adverse pressure gradient [Capillary malformation-arteriovenous malformation (CM-AVM) is a vascular disorder that is associated with mutations in the gene remities ,73, chylremities , chylothremities  and swel animals . Althouggradient . The lymgradient .In conclusion, a mutation in any one of the many genes reviewed here leads to valve defects in the corresponding genetic mouse model. The repeated finding of valve defects in mouse models of congenital lymphedema mutations strongly supports the idea that in humans with the same mutations, valve defects cause or significantly contribute to the lymphedema . Even geKnockout mouse models have revealed that many genes cooperate to orchestrate valve formation and maintenance, a potential contributor to the pathophysiology of lymphedema. As discussed in the preceding section, many of the genes that are mutated in congenital lymphedema patients are intracellular signaling molecules or transcription factors located in the cell nucleus of LECs. However, this fact highlights a glaring problem—how do intracellular proteins know when shear stress is altered or impaired? Are there proteins on the cell surface that transmit information about shear stress to proteins inside the cell? These questions are particularly relevant because the majority of pharmaceutical targets are comprised of ion channels and receptors on the cell surface.PIEZO1 were identified in human lymphedema patients, which encodes an ion channel that is sensitive to mechanical force. In the blood vasculature, others have identified a role for PIEZO1 in responding to shear stress and even differentiating between laminar and oscillatory shear stress [Piezo1 gene in mice leads to a severe reduction in the number of valves and pleural effusion [Recently, mutations in r stress ,84. In teffusion ,67. WhilPecam1−/− mice were reported to exhibit defective lymphatic valve formation in the embryo, numerous valves remained and these mutant mice survived to adulthood with no apparent defects [Cdh5 allele to enable the lymphatic-specific deletion of VE-cadherin to study its role in lymphatic valve development [So, what is the link between lymph flow and changes in gene transcription? To help address this question, we looked to how blood endothelial cells (BEC) respond to blood flow. In 2005, a protein complex was identified at cell-cell junctions that regulated endothelial cell signaling in response to shear stress . This me defects , indicatelopment .Embryonic deletion of VE-cadherin led to the complete absence of lymphatic valves, including the lymphovenous valves at the junction of the thoracic duct and subclavian vein . In addiPROX1 and FOXC2 [To gain insight into the mechanisms of how VE-cadherin regulated valve development, we focused on the well-known VE-cadherin binding partner, β-catenin, because it had been reported that β-catenin was required for lymphatic valve formation in the embryo by directly binding the promoters of nd FOXC2 . Using and FOXC2 . This fiThe canonical mechanotransduction signaling complex originally identified in BEC was shown to stimulate PI3K/AKT signaling instead of β-catenin . TherefoAlthough lymphatic valve incompetence is often cited as a characteristic of lymphedema in patients, this is based on indirect evidence using diagnostic imaging techniques that demonstrate retrograde lymph flow as an indicator of valve incompetency. No direct, quantitative evidence of valve defects—such as regression or defective morphology—has been shown in human patients with either congenital or cancer-related lymphedema. This is most likely due to the small size of lymphatic valves and the poor resolution of current clinical imaging modalities. However, lymphangiography has been used to show that in CRL patients, the lymphatic vessels become larger in diameter ,89, whicFoxc2, Gata2, Rasa1, Piezo1, Fat4, Itga9, and Akt1 (discussed above). While confirmation of valve defects in humans is currently lacking, isotope lymphoscintigraphy in lymphedema patients with mutations in FOXC2 demonstrate abnormally low tracer uptake and dermal backflow [FOXC2 have severe venous valve failure as measured by pathological venous reflux in several segments of veins in the lower limbs [Many of the numerous mouse models of congenital gene mutations exhibit lymphatic valve defects, including backflow , again ier limbs . Whilst Studies have identified several gene mutations  in the form of single nucleotide polymorphisms in CRL patients ,94,95,96To summarize, studies have shown that there is a loss of lymph flow after lymph node removal surgery in CRL patients ,7,17. LyThus, a goal for future investigation is to directly show whether human CRL patients have lymphatic valve regression or other valve defects. To accomplish this, there would need to be a way to visualize and image the lymphatic valves in vivo. Since near-infrared imaging has been used to map lymph flow in human lymphedema patients, perhaps this technique could be modified to label the valve leaflets without harming the patient. In lieu of this approach, mouse models of CRL should be employed to study whether lymph node removal affects lymphatic valve maintenance in vivo, and whether inflammation plays any role in valve regression. Genetic approaches to rescue valve regression could also be combined with animal models of CRL to implicate valve regression in this poorly understood disease.4 in CRL negatively regulate the expression of VEGFR3 [Our study demonstrating that constant VE-cadherin expression and signaling are required for the lifelong maintenance of lymphatic valves has potential consequences relevant to CRL . Becausef VEGFR3 , which if VEGFR3 ,101, andFinally, the order of events that lead to CRL need to be determined . Does in