Between 5 and 10 percent of fractures do not heal, a condition known as nonunion. In clinical practice, stable fracture fixation associated with autologous iliac crest bone graft placement is the gold standard for treatment. However, some recalcitrant nonunions do not resolve satisfactorily with this technique. For these cases, biological alternatives are sought based on the molecular mechanisms of bone healing, whose most recent findings are reviewed in this article. The pro-osteogenic efficacy of morin  has recently been reported, and the combined use of bone morphogenetic protein-9 (BMP9) and leptin might improve fracture healing. Inhibition with methyl-piperidino-pyrazole of estrogen receptor alpha signaling delays bone regeneration. Smoking causes a chondrogenic disorder, aberrant activity of the skeleton’s stem and progenitor cells, and an intense initial inflammatory response. Smoking cessation 4 weeks before surgery is therefore highly recommended. The delay in fracture consolidation in diabetic animals is related to BMP6 deficiency (35 kDa). The combination of bioceramics and expanded autologous human mesenchymal stem cells from bone marrow is a new and encouraging alternative for treating recalcitrant nonunions. It is estimated that between 5% and 10% of bone fractures do not heal properly . The cell-biomaterial association was performed in the surgical room prior to implantation  found in old fustic and osage orange trees) has pro-osteogenic capacity. In one study [Wan et al. observed that the natural compound morin -1β promotes the differentiation and osteogenic function of mouse bone marrow MSCs (MBMMSCs) through the bone morphogenetic protein/Smad (BMP/Smad) signaling pathway . MBMMSCsFactors that delay bone healing can be divided into local and systemic. The most important local factors are the following: inadequate bone reduction, unstable bone fixation, bone infection, and radiation. The most important systemic factors are the following: patient age , nutrition status . Smoking has a negative effect on bone healing. Steroids also can slow down the healing process. Systemic diseases such as hyperthyroidism and renal insufficiency delay fracture healing. Genetic diseases such as Marfan syndrome, Ehler–Danlos syndrome, osteogenesis imperfecta are among the factors affecting bone healing.Wu et al. evaluated the role of the estrogen receptor alpha (ERα) axis in bone consolidation and its possible mechanisms of action, demonstrating that inhibition of the ERα signaling delays bone regeneration. Female Institute of Cancer Research (ICR) mice were bred with a metaphyseal bone defect in the left femur and were administered methyl piperidino pyrazole (MPP), an ERα inhibitor, and bone consolidation was evaluated by microcomputer tomography. ERα placement with alkaline phosphatase (ALP) and ERα translocation into the mitochondria were determined, and the levels of ERα, ERβ, PECAM-1, VEGF, and β-actin were measured. The expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs was quantified. Angiogenesis was measured by immunohistochemistry. After surgery, the bone mass was increased in the bone-defect area in a time-dependent manner. Simultaneously, the ERα levels increased, correlating positively with bone consolidation. The administration of MPP decreased ERα levels and bone consolidation. Regarding the mechanism of action in bone consolidation, osteogenesis was improved; however, MPP attenuated osteoblast maturation. In parallel, the expression levels of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were increased in the injured zone. Treatment with MPP produced significant inhibition of the expression of ALP, Runx2, and osteocalcin genes, decreased translocation from ERα to the mitochondria, and expression of COX-1 and COX-2 genes related to mitochondrial energy production, and decreased levels of PECAM-1 and VEGF in the area of the experimentally created bone defects. The study demonstrated the role of the ERα axis in bone consolidation through the stimulation of energy production, osteoblast maturation, and angiogenesis .Smoking causes delayed union and/or nonunion of bone fractures. Unfortunately, orthopedic surgeons rarely delay surgery in patients who smoke nor do they suggest methods for patients to quit smoking. It is important to recommend smoking cessation methods such as transdermal patches, chewing gum, lozenges, inhalers, sprays, bupropion, and varenicline during the perioperative period. Smoking cessation in the perioperative period appears to be effective in reducing delayed union and nonunion rates of bone fractures, even if performed up to 4 weeks prior to the surgery .Hao et al. published a study in which they exposed three murine strains  to cigarette smoke for 3 months before performing a midshaft transverse femoral osteotomy. Using radiography, microcomputed tomography, and biomechanical tests, the authors evaluated fracture healing 4 weeks after the osteotomy. The radiographic study showed a significant decrease in the fracture healing capacity of 129 × 1/SvJ smoke-exposed mice. The microcomputed tomography results showed a delay in the remodeling of the fracture calluses in all three strains after exposure to cigarette smoke. The biomechanical tests showed a more significant deterioration of functional properties in the 129 × 1/SvJ mice than in the C57BL/6J and BALB/cJ mice after exposure to cigarette smoke. In other words, the 129 × 1/SvJ strain was the most suitable for simulating the smoke-induced deterioration of fracture healing. In the 129 × 1/SvJ mice, the authors investigated the molecular and cellular disorders of fracture healing caused by cigarette smoke using histology, flow cytometry, and multiplex cytokine/chemokine analysis. The histological analysis showed abnormal chondrogenesis due to cigarette smoke exposure. In addition, significant populations of repair cells, including skeletal stem cells and their subsequent progenitors, showed a decrease in post-injury expansion as a result of cigarette smoke exposure. Furthermore, the authors observed a significant increase in pro-inflammatory mediators and immune cell recruitment in fracture hematomas in the mice exposed to smoke. These results show the important cellular and molecular disorders that occur during fracture healing due to smoking, such as abnormal chondrogenesis, aberrant activity of skeletal stem and progenitor cells, and an intense initial inflammatory response . Table 3Transcriptomic and proteomic approaches have shown great potential in terms of bone regeneration because they offer new knowledge on the molecular physiological/pathological mechanisms that regulate bone consolidation ,76. TranUsing a diabetic rodent model, Guo et al. investigated the relationship between BMP6 and BMP9 and the effect of bone consolidation in diabetes. The authors analyzed the difference in size and calcification of the calluses of fractures and the mechanical resistance and expression of BMP6 and BMP9 in the calluses. The authors evaluated the consolidation of femoral fractures by quantifying callus size and calcification using X-rays, histological and histochemical imaging, the load capacity of the fractured bone, and the amount of BMP6 in the calluses and bones using western blotting. At the end of the second and fourth weeks after the fracture, the authors observed a significant increase in BMP6 in the calluses and fractured bones in both the nondiabetic and diabetic animals. However, the authors detected significantly lower levels of BMP6 (35 kDa) with smaller calcified callus sizes and low load-bearing capacity of bones with already fused fractures in the diabetic animals compared with the nondiabetic animals. A deterioration in BMP6 (35 kDa) maturation from its precursors could be the cause of the decrease in BMP6 in diabetic animals. Therefore, it seems that the delay in fracture healing in the diabetic animals is related to a BMP6 deficiency (35 kDa), which could be due to a defect in BMP6 maturation from its precursors to its functional format .Ambrosi et al. studied whether highly purified bona fide human skeletal stem cells (hSSCs) isolated from geriatric fractures showed intrinsic functional defects that would prevent them from healing. Using flow cytometry, the authors analyzed and isolated hSSCs from the calluses of 61 fractures from 5 different skeletal areas of patients aged 13 to 94 years to conduct functional and molecular studies. The authors observed that fracture-activated amplification of hSSC populations was comparable at all ages. However, the functional analysis of the isolated stem cells revealed that advanced age was significantly related to a reduction in osteochondrogenic potential but not to a decrease in clonogenicity in vitro. HSSCs derived from women displayed an exacerbated functional decline with age relative to those of older men. Transcriptomic comparisons showed downregulation of skeletogenic pathways such as WNT and upregulation of senescence-related pathways in younger versus older hSSCs. The loss of sirtuin1 expression played an important role in hSSC dysfunction, although reactivation by trans-resveratrol or a small molecular compound restored the differentiation potential in vitro. These findings demonstrate the age-related defects in purified hSSCs in geriatric fractures and could serve as a basis for further research into the functional mechanism and reversibility of skeletal stem cell aging in humans .Valiya Kambrath et al. published an improved method for the early-stage processing of fracture calluses and for the immunofluorescence labeling of sections to visualize the timing and spatial patterns of cellular and molecular events that regulate the healing of bone fractures. This method does not require prolonged decalcification, so the response time from sample collection to microscopy is short. Furthermore, the method preserves the structural integrity of the fragile callus, because it does not involve deparaffinization or harsh antigen retrieval methods. This method could be adapted for high-throughput screening of drugs that promote the healing of bone fractures .Haffner-Luntzer et al. published a new experimental mouse model to study the consolidation of metaphyseal fractures of the proximal femur. Their technique consisted of inserting a 24G needle into the femur in a closed fashion and subsequently performing an open Gigli 0.4-mm saw osteotomy of the proximal femur. The authors analyzed the fractured femurs by microcomputed tomography and histology 14 and 21 days after the surgery. All of the study animals showed normal limb loading and a physiological gait pattern in the first three days after the fracture. Robust endochondral ossification was observed during the fracture healing process, with high expression of late chondrocytes and early osteogenic markers on day 14. On day 21, all fractures had a bony bridging score of 3 or more, indicating successful healing. Callus volume decreased significantly from day 14 to day 21, while a high number of osteoclasts appeared in the fracture callus until day 21, indicating that callus remodeling had already begun on day 21. Thus, the authors developed a novel experimental model in mice that enables the consolidation of endochondral fractures of the proximal femur to be studied. This model could be useful for future research using transgenic animals to better understand the molecular mechanisms of consolidation in metaphyseal osteoporotic fractures .Co-culture systems allow us to explore interactions between cells. In addition, the replication of naturally occurring cells in multicellular tissues can help design reliable bone engineering models. However, there is still no optimal form of 3D co-culture of human bone cells that accurately reproduces the bone microenvironment. In vitro co-culture systems using human cells might, in the future, become a valid alternative to animal studies. In fact, we can create a mechanobiological environment using dynamic 3D co-culture models. In addition, the basic regenerative mechanisms of bone can be identified using new imaging modalities. Currently, in silico models provide data for adjusting the dynamic parameters applied to the culture systems, thereby better imitating the response of native tissues to scaffolds. Current in vivo models are taking into account various factors related to the healing site, such as the presence of macrophages, the angiogenic process, and the interaction between regenerative cells and immune counterparts .Bone tissue engineering appears promising, although its success often depends on a “smart” scaffold to host and guide bone formation through the precursors of bone cells. Bone homeostasis basically depends on osteoblasts and osteoclasts in a continuous cycle of bone resorption and formation. Studies on the periosteum are essential, given that it plays a crucial role in bone development and the process of fracture healing. The role of macrophages as central regulators during all phases of bone repair also needs further investigation. Research into the relationship between osteogenesis and angiogenesis is essential because they are intimately linked during bone growth and regeneration in bone modeling and during bone homeostasis in bone remodeling. The role of leptin as an enhancer of osteogenesis induced by BMP9 should be further explored through the cross-regulating BMP9 signaling by the JAK/STAT signaling pathway in MSCs. Lastly, the role of morin, which could be beneficial in the regeneration of bone defects, should also be further studied. A combination of autologous MSCs expanded from bone marrow and synthetic scaffolding  appear to be good options. A combination of bioceramics and expanded autologous hBM-MSCs is an encouraging new treatment option for recalcitrant nonunions in the clinical setting.


During neocortical development, many neuronally differentiating cells (neurons and intermediate progenitor cells) are generated at the apical/ventricular surface by the division of neural progenitor cells . Neurogenic cell delamination, in which these neuronally differentiating cells retract their apical processes and depart from the apical surface, is the first step of their migration. Since the microenvironment established by the apical endfeet is crucial for maintaining neuroepithelial (NE)/aRGs, proper timing of the detachment of the apical endfeet is critical for the quantitative control of neurogenesis in cerebral development. During delamination, the microtubule–actin–AJ (adherens junction) configuration at the apical endfeet shows dynamic changes, concurrent with the constriction of the AJ ring at the apical endfeet and downregulation of cadherin expression. This process is mediated by transcriptional suppression of AJ-related molecules and multiple cascades to regulate cell adhesion and cytoskeletal architecture in a posttranscriptional manner. Recent advances have added molecules to the latter category: the interphase centrosome protein AKNA affects microtubule dynamics to destabilize the microtubule–actin–AJ complex, and the microtubule-associated protein Lzts1 inhibits microtubule assembly and activates actomyosin systems at the apical endfeet of differentiating cells. Moreover, Lzts1 induces the oblique division of aRGs, and loss of Lzts1 reduces the generation of outer radial glia , another type of neural progenitor cell in the subventricular zone. These findings suggest that neurogenic cell delamination, and in some cases oRG generation, could be caused by a spectrum of interlinked mechanisms. The vertebrate central nervous system originates from the neuroepithelium lining the embryonic neural tube. Neuroepithelial (NE) cells have polarized morphology along the radial axis, spanning the apical surface to the basal side at the basement membrane, and behave as neural progenitor cells. In the early period of mammalian cerebral wall development, neural progenitor cells (NE cells) undergo symmetric, proliferative division to expand the progenitor pool . In the This review article briefly describes the subcellular architecture of the apical endfeet, which provides an environment for proper neurogenesis from aRGs, and then summarizes our current knowledge on the molecular mechanisms underlying delamination. This review further discusses the common features of neurogenic cell delamination and outer radial glial cell (oRG) generation. oRGs, also called basal radial glial cells (bRGs), are another type of undifferentiated neural progenitor cell with long radial fibers extending to the basal side, and their cell body exists in the SVZ, where they divide multiple times  polarity of aRGs is important for the maintenance of neural progenitor cells (or aRGs). Impaired AB polarity or apical protein complexes of aRGs induce cell cycle exit, precocious neuronal differentiation, and pathological delamination , fibroblast growth factor (FGF), Neuregulin, and Shh, from cerebrospinal fluid (CSF) filling with the ventricle  in neocortical development. For example, at the early developmental stage, during which NE cells undergo symmetric proliferative division, both daughter cells retain the apical endfeet , and if Cell delamination is the dynamic event with cytoskeletal remodeling of the apical microtubule–actin–AJ configuration  decreases Hes1 messenger RNA (mRNA) levels and produces an excess of IPs  cells during EMT, AKNA recruits the microtubule minus-end binding protein CAMSAP3    has been shown to control neuronal delamination. DSCAM starts to be expressed in differentiated neurons only before migration and locally suppresses the RapGEF2–Rap1–Cdh2 cascade at their apical endfeet to delaminate  cell divisions of aRGs  portion of the process during M phase (Kawaue et al., Lzts1 mRNA (Kawaguchi et al., In line with this, we found that Lzts1, a key molecule of neurogenic cell delamination, also induces oRG generation by the oblique division of aRGs (Kawaue et al., Overall, these observations suggest that, in the case of Lzts1, the oblique division that generates oRGs is controlled by a molecular mechanism similar to that of delamination in the context of the microtubule–AJ complex. Therefore, the junctional proteins would play critical roles both in maintaining epithelial structure at the apical endfeet (Zhang et al., Lzts1 function in oblique division may explain some of the diverse, contradictory conclusions of previous studies on spindle orientation and fate determinant (Lancaster and Knoblich, Unlike AKNA, which primarily affects microtubule dynamics (Camargo Ortega et al., In normal neocortical development, there are various cell departure patterns from the apical surface in the developing cerebral wall, as shown in In mice with lissencephalic brains, the number of oRGs is small, and their self-renewal potential in the SVZ is relatively limited (Wang et al., AK wrote and edited the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The placental methylation pattern is crucial for the regulation of genes involved in trophoblast invasion and placental development, both key events for fetal growth. We investigated LINE-1 methylation and methylome profiling using a methylation EPIC array and the targeted methylation sequencing of 154 normal, full-term pregnancies, stratified by birth weight percentiles. LINE-1 methylation showed evidence of a more pronounced hypomethylation in small neonates compared with normal and large for gestational age. Genome-wide methylation, performed in two subsets of pregnancies, showed very similar methylation profiles among cord blood samples while placentae from different pregnancies appeared very variable. A unique methylation profile emerged in each placenta, which could represent the sum of adjustments that the placenta made during the pregnancy to preserve the epigenetic homeostasis of the fetus. Investigations into the 1000 most variable sites between cord blood and the placenta showed that promoters and gene bodies that are hypermethylated in the placenta are associated with blood-specific functions, whereas those that are hypomethylated belong mainly to pathways involved in cancer. These features support the functional analogies between a placenta and cancer. Our results, which provide a comprehensive analysis of DNA methylation profiling in the human placenta, suggest that its peculiar dynamicity can be relevant for understanding placental plasticity in response to the environment. The placenta is the transient organ of primary importance during pregnancy, acting as a highly specialized interface intimately connecting mother and fetus. It ensures an adequate provision of nutrients and oxygen to the embryo as well as waste disposal and hormone production to support pregnancy and promote fetal growth. In order to allow the pregnancy to progress, even in presence of unfavorable conditions , the placenta can adapt dynamically. Several factors, including in utero environmental changes and adverse maternal conditions such as infections, metabolic changes, and poor nutrition or obesity can induce placental adaptability through fine regulation processes that are not fully understood ,2. DespiBeyond the presence of recognizable genetic defects, gene expression modifications in crucial windows of placental development could affect its functioning, thus modifying maternal and fetal physiology. These placental changes can be difficult to identify for different reasons: accessibility to placental cells during pregnancy, morphological heterogeneity and the complex cellular composition of the organ. Despite the difficulties in recognizing the genetic/epigenetic alterations arising during pregnancy, they can represent biological markers of a pregnancy outcome.Among the adverse factors, maternal undernutrition or poor nutrient intake due to a malfunction of placental transport can induce metabolic modifications in the fetus to maximize the limited nutrient resources and maintain survival. This process could be favored by epigenetic modifications to finely control the expression of genes crucial for fetal metabolic homeostasis. These signatures may persist after birth as an “epigenetic memory” of the metabolic adaptation during the prenatal life. A concept, in keeping with the DoHad hypothesis introduced by David Barker , links pEpigenetics regulates gene expression without altering the DNA sequence, by combining the actions of enzymes that allow or inhibit gene expression . It alsoThe development itself is an epigenetic process. This means that during fetal life the epigenome of the embryonic and extraembryonic compartments undergoes continuous change, which is essential for cell lineage differentiation, fetal and placental growth, and responses to external factors . The patThe DNA methylation landscape of the placenta is distinct from that of other tissues. The trophoblast and the mesenchyme core of the chorionic villi have a unique methylation profile compared with maternal decidua, fetal membranes and embryonic tissues . The preIn adult somatic tissues, DNA methylation is characterized by homogeneous distribution where most of the CpG sites show either high (> 90%) or low (<10%) methylation. In the placental DNA, however, the remarkable presence of PMDs results in a unique distribution of the methylation profile, characterized by peaks with partially methylated and highly methylated regions .The hypomethylation of different families of Retrotransposable Elements (REs) is more pronounced in the placenta compared to other tissues, and the specific methylation pattern depends on the REs family and the evolutionary age of the sequence ,18,19. FThe placental methylome undergoes dynamic changes during gestation ,20 not oMLLT1, PDE9A, ASAP2, and SLC20A2) in cord blood samples in relation to birth weight; on the other hand, increased methylation and lower expression of FOSL1 correlated with a high birth weight [A recent study, explored genome-wide DNA methylation in placental samples and identified 15 CpG sites with a methylation profile associated with birth weight . These sh weight ,36,37,38h weight .In this study we explored the methylome and LINE-1 methylation of embryonic (cord blood) and extraembryonic (placenta) tissues from 154 uncomplicated full-term pregnancies, stratified according to newborn weight percentile. The results were investigated by means of an ontology-driven approach and by comparing the results obtained by methylation microarrays and targeted sequencing.p-value < 0.001). The pregestational body mass index in the SGA group is on average 20.6 kg/m2, and in LGA group increases remarkably to 24.24 kg/m2. The placenta weight on average was 475 g in the SGA group and 741 g in the LGA group, respectively , 77 as “appropriate for gestational age” or AGA (>10th and <90th percentile), and 44 neonates as “large for gestational age” or LGA (≥90th percentile). This classification was based on the Italian infant growth charts developed by Bertino et al. . Regardiectively .An overview of experimental design is shown in For both methylation profiling array and targeted methylation sequencing approaches, samples were randomly selected across all pregnancies. We decided to perform these experiments on 15 pregnancies with different neonatal birth-weight  in order to avoid selecting a specific birth-weight category and to explore possible evident differences between low and high birth weight classes.We performed the analysis on maternal and cord blood and placenta samples of 154 pregnancies .−115). Notably, methylation levels in cord and maternal blood were very similar, thus suggesting that LINE-1 methylation pattern in blood is stable and does not change throughout life.As shown in p = 1.67 × 10−4) and LGA (p = 6.36 × 10−5) newborns. No differences were observed between AGA and LGA placentae  adjustment of the p-values for multiple testing, significant differences in methylation levels between cases and controls emerged at the site level. The list of probes that resulted in different methylation has been reported in the The analysis was carried out on cord blood and placental DNA from five pregnancies  using SuBy PCA we found that the main source of variability in the methylation levels was between the two tissues, allowing us to distinguish specific CpG methylation clusters  and confHierarchical clustering based on a CpG methylation score in cord blood and placenta  demonstrIn order to define the differentially methylated sites in cord blood and placenta, we selected the 1000 most variable sites and performed hierarchical clustering. We were thus able not only to distinguish between the two tissue clusters based on their opposite methylation patterns, but we also confirmed that the majority of differentially methylated sites are hypomethylated in the placenta B.p-value, to identify differences between the placenta and cord blood at the CpG site and at the region level, including tiling (5 kilo base pair regions), genes, promoters and CpG islands  software v.60467501 , we explored which genes are differentially methylated in the promoter and in the gene body. We found that 75 promoters, specifically methylated in the placenta, are involved in blood cell proliferation and immune cell migration and adhesion, as expected A. DifferConsidering the results for gene body methylation of the 1000 most differentially methylated loci in placenta, we identified 53 genes specifically methylated in the placenta, 12 of which also displayed hypermethylation at their promoters in the same tissue. We found that they are mainly involved in blood cell-specific biological functions such as lymphocyte and leukocyte proliferation and migration. Among the 1000 genes, 404 were specifically methylated in cord blood, and 111 of them displayed hypomethylation in both the promoter and gene body in the placenta. The most represented pathways of the 404 genes are involved in several cancers, especially skin tumors and neuroendocrine cancers .Considering the possible methylation differences in the birth weight groups, we used the RnBeads Differential Methylation module and investigated the differences in methylation among placental tissues. We did not find significant differences between SGA and LGA placentae, which could be explained by the small sample size as shown in IGF2 and H19 imprinted genes and master regulators of placental and fetal growth [Finally, we focused our attention on l growth ,42. TheyAlthough the comparison between two independent genome-wide techniques is biased because they consider different target regions, we tried to compare the data to identify regions with similar methylation status. Indeed, compared with an EPIC array, targeted methylation sequencing detects more CpG sites in coding regions and CpGs islands, whereas the proportion of CpG sites mapped on regulatory elements is comparable between the two techniques .We applied the pipeline for differential methylation and pathway analyses to the datasets derived from both techniques. Analyzing the 1000 most differentially methylated loci in placenta compared to cord blood, we identified by microarray and methyl-seq, respectively: 209 and 450 hypomethylated promoters, 56 of which were in common between the two techniques; 340 and 75 hypermethylated promoters, 58 of which were common to the two techniques; 224 and 404 hypomethylated gene bodies, 74 of which were shared in the two approaches; 212 and 53 hypermethylated gene bodies, 35 of which in the two approaches gave similar results see .Pathway analysis of these genomic loci returned similar results for data derived from microarray and targeted methylation sequencing, as both techniques identified an association with blood cell specific biological functions for loci that are hypermethylated in placenta, and an association with tumor-specific pathways for loci that are hypomethylated in the placenta .The key role of the placenta in supporting pregnancy and promoting fetal growth is made possible by the capacity of this organ to modify its epigenetics during pregnancy in response to external factors.This study explores the methylation signatures in the fetal and placental genome in uncomplicated full-term pregnancies with healthy newborns classified in three ranges of birth weight percentiles . As expected, we found that the maternal pregestational body mass index and placenta weight were strongly different in SGA, AGA and LGA groups. LINE-1 methylation analysis of the entire population (154 pregnancies) showed hypomethylation in placenta compared with both cord and maternal blood, confirming previous evidence ,18,19. ILess clear is a possible relationship of LINE-1 methylation and birth weight because of previous contradictory data ,33,34, wTo explore the placental methylome compared with cord blood in normal pregnancies and to define the main gene pathways of differentially methylated loci, we used a combined omics approach of methylation microarray and targeted sequencing by NGS.The exploratory analysis clearly showed a significant difference between placenta and cord blood epigenetic profiles, a result not completely unexpected since the two tissues are substantially different. Another important result emerged from this analysis: a significant difference in the epigenetic variability between the two tissues. In particular, cord blood methylation profiles were very similar and therefore clustered, whereas the placental cases were variable and widespread. These findings show the plasticity of the placenta in relation to the uniqueness of each pregnancy. From our study, there emerged a unique methylation profile in each placenta compared to the cord blood, which could represent the sum of adjustments that the placenta makes throughout the course of the pregnancy to preserve the epigenetic homeostasis of the fetus, thus contributing to the delivery of a healthy baby. On the other hand, it is known that the placenta is more prone to sustain genetic alterations. For example, mosaic trisomies confined in the placenta are much more tolerated than in the fetus ,46. SimiMethylation sequencing showed a global hypomethylation of placenta compared to cord blood, thus confirming our results on LINE-1 and those previously reported ,16. ThisThe differential methylation analysis confirmed that DNA methylation profiles of cord blood and placenta are different both at site and region levels. This is clearly shown in the volcano plots. Interestingly, both the array and sequencing experiments achieved comparable results.By restricting the analysis to the 1000 most variable sites between the two tissues, we could appreciate their opposite methylation patterns, thus highlighting the strong epigenetic differences of embryonic and extraembryonic compartments. We investigated the biological functions associated with these differentially methylated loci, by performing ontology pathway analysis on promoters and gene bodies. In particular, we found that promoters and gene bodies that are hypermethylated in the placenta and hypomethylated in cord blood are associated with blood cell specific functions such as proliferation and migration of immune cells, as would be expected for this tissue. However, promoters and gene bodies that are hypomethylated only in the placenta belong to six pathways involved in cancer and two related to ventricular myocytes.IGF2- and H19-imprinted genes, the master loci of fetal and placental growth, were variables among samples. There were no differences either between cord blood and placenta or among the samples, thus reinforcing the concept that genomic imprinting, once established, remains stable [In addition, we verified whether the methylation levels of s stable .In order to obtain relevant results we included only normal full-term pregnancies and found methylation NGS results in line with the EPIC array findings, despite the low number of cases. These analyses also highlighted significant overlapping differences between cord blood and the placenta at the most differentially methylated genome-wide sites. However, a point of weakness of our study is related to the small group size of neonates stratified for the birthweight analyzed with omics approaches; therefore, we could not find specific methylation-profiling related to birth weight. Nevertheless, a LINE-1 investigation carried out on the entire population revealed a peculiar hypomethylation associated with SGA phenotype.As with the other authors , we showTaken together, our findings confirmed the global hypomethylation of placental DNA including LINE-1, promoters, CpG islands, gene body, and tilings. They also showed that specifically activated pathways in the placenta are those involved in cancer. The placental heterogeneity of the methylation levels characterizing each pregnancy could explain its broader plasticity compared to the fetal compartment.Recruitment was carried out at the Unit of Obstetrics and Gynecology “L. Mangiagalli” . Participants were enrolled before the delivery either by cesarean section or vaginal birth. Inclusion criteria were: European origin of the mothers, singleton spontaneous pregnancies delivered full-term, spontaneous conception, absence of fetal malformations or known genetic abnormalities, and the absence of severe pregnancy complications . For each pregnancy, a medical history, clinical data and maternal and neonatal outcomes were recorded. The study protocol was approved by the local Ethical Committee , and written informed consent was obtained from all participants. This study was part of a broader project aimed at collecting 2000 normal pregnancies to create a biorepository of maternal and fetal tissues for present and future studies.For each pregnancy at delivery, we collected maternal peripheral blood and umbilical cord blood in EDTA tubes  and 1 cm³ of placental tissue taken from the fetal side in a cotyledon closed to cord insertion. All samples were frozen at −80 °C within 4 h of sampling and transferred in the repository Biobank of Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico.DNA was extracted from the cord blood, maternal peripheral blood and placenta tissue using the QIAsymphony  automated DNA extraction platform, according to the manufacturer’s instructions. DNA quality and concentration was assessed by NanoDrop and Qubit  analysis. DNA integrity and fragment size were assessed by TapeStation analysis . DNA bisulfite conversion was performed using the EZ DNA Methylation Gold Kit  for LINE-1 and methylation sequencing experiments, and the EZ DNA Methylation Kit  for the microarray.LINE-1 methylation was carried out on the placental, maternal and cord blood DNA of all cases  in orderLINE-1 methylation analyses were performed as previously described . In brieGenome wide methylation was carried out using the Infinium MethylationEPIC array  on both cord blood and placental DNAs from ten pregnancies (5 SGA and 5 LGA)  in orderTo evaluate the conversion efficiency and bsDNA integrity, a single-strand quantification of bsDNA was performed using NanoPhotometer Pearl .We used the RnBeads package (2.8.0) in the R environment (Version 4.0.0) and adopted all the available modules to carry out the quality control, normalization, exploratory analysis  and differential analysis of all the genomic regions .In the preprocessing phase 826,020 Sites of SNP-enriched probes were counted; 17,371 sites were removed because they were unreliable; and 2985 sites were context-specific. In addition, all the sex chromosome probes were removed .After the filtering procedures, 849,524 CpG sites were retained and all samples met the minimum criteria for being included in the study.The probes methylation values were normalized using the MINFI (1.36.0) package of the SWAN normalization method  and by tA combined p-value of the aggregated probes by regions  was calculated and used for the differential methylation analysis.n = 33,842); (ii) Promoter regions of Ensembl genes, version Ensembl Genes 75 ; (iii) CpG Islands, CpG island track of the UCSC Genome browser ; and iv) Tiling regions, non-overlapping tiling regions with a fixed window size of 5 kilobases defined over the whole genome .The outcome for the four regions was carried out as follows: (i) genes, represented with the Ensembl format version 75   using thBase calls and relative quality scores were computed from signal intensities during the sequencing run by the system’s Real Time Analysis (RTA) software. Sample de-multiplexing was performed by the Illumina bcl2fastq Conversion Software v2.2 resulting in sequence data in FASTQ format. Files corresponding to each of the four NextSeq550 lanes were merged with Samtools-merge.Subsequent steps were performed using the nf-core/methylseq v1.6dev Bismark  workflow. First, quality control (QC) was performed by FastQC. The trimming step was skipped. Then, Bismark-aligner was used to align paired-end reads to bisulfite-converted versions of the human genome (hg19), while Bismark-deduplicate estimated and removed the duplicate reads. Finally, methylation calls in CpG, CHG and CHH context were extracted by a Bismark-methylation_extractor.p-values on the site level were computed using the limma method and adjusted with False Discovery Rate (FDR). RnBeads also computes a combined rank, taking into consideration not only significance from p-values, but also the mean of the methylation differences observed for that site in the two conditions being compared as well as the mean methylation ratio. Methylation at region level  is computed as the mean of methylation values of sites falling into the region, while the combined p-value is obtained by the weighted inverse chi-square method.The resulting methylation data from Bismark was imported in the RnBeads v2.0.0 software for assay quality validation, CpG site filtering, sample clustering, and differential methylation analyses. During the RnBeads filtering step, CpG sites located in chromosomes other than 1–22  were removed, along with CpG sites mapping at annotated single nucleotide polymorphisms. Only sites with at least 5× coverage and no missing values in all analysed samples were conserved. We obtained 2,871,130 CpG sites with available methylation data across all 10 samples. This allowed us to drastically decrease the proportion of unreliable measurements (0% as estimated by RnBeads) while still maintaining a sufficient amount of reliable measurements (over 75%). During the Exploratory Analysis step, RnBeads performed hierarchical clustering of samples and Principal Component Analysis (PCA) based on methylation scores of the filtered CpG sites. For the Differential Methylation analysis,


Endosome maturation refers to the conversion of early endosomes (EEs) to late endosomes (LEs) for subsequent fusion with lysosomes. It is an incremental process that involves a combination of endosome fusion and fission and which occurs at contact sites between endosomes and the endoplasmic reticulum (ER), with knowledge of the underlying mechanisms having increased greatly in recent years. Protrudin is an ER-resident protein that was originally shown to regulate neurite formation by promoting endosome trafficking, whereas PDZD8 is a mammalian paralog of a subunit of the yeast ERMES  complex that possesses lipid transfer activity. A complex of protrudin and PDZD8 was recently found to promote endosome maturation by mediating lipid transfer at ER-endosome membrane contact sites. This review focuses on the roles of the protrudin-PDZD8 complex in tethering of endosomes to the ER, in mediating lipid transfer at such contact sites, and in regulating endosome dynamics, especially in neuronal cells. It also addresses the physiological contribution of endosome maturation mediated by this complex to neuronal polarity and integrity. Most intracellular organelles of eukaryotic cells communicate with the endoplasmic reticulum (ER) network through membrane contact sites (MCSs), at which the membranes of the ER and the interacting organelle come into close proximity and are tethered. MCSs are thus thought to function as intracellular synapses, where molecular information is exchanged.Neurons are polarized cells that consist of two distinct portions, the somatodendritic compartment and the axon. Trafficking of endosomes along the axon toward its terminus plays an important role in axonal outgrowth directed toward target cells as well as in neurotransmitter release. Protrudin was first identified as a protein that promotes neurite outgrowth through regulation of directional endosome trafficking . The proAxonopathy, a type of neurodegeneration, is caused by damage to the axon of neurons. The longest axons in the central nervous system of mammals are located in the corticospinal tract, which is the neural circuit responsible for voluntary movement. Hereditary spastic paraplegia (HSP) is an axonopathy in which upper motor neurons in the corticospinal tract undergo degeneration . Many HSIn this review, I summarize what is known of the mechanism responsible for regulation of endosome maturation by protrudin and its relation to the pathogenesis of neurological disease. We also address the role of PDZD8 (PDZ domain–containing protein 8), a protrudin-interacting protein, in the lipid transfer process underlying endosome maturation. Finally, we discuss the contribution of the protrudin-PDZD8 complex and its lipid transfer function to the maintenance of neuronal polarity and integrity.Protrudin was originally discovered as a protein of unknown function that interacts with FK506 binding protein 38 (FKBP38) . Forced Rab GTPases are master regulators and markers of organelle identity in the endocytic pathway . The traProtrudin also interacts with the microtubule-dependent motor protein KIF5, which mediates anterograde cargo trafficking along microtubules of axons in the plus-end direction . Indeed,VAP has also been identified as a protrudin binding protein , with thThe precursor mRNA for protrudin is alternatively spliced, resulting in the generation of mature transcripts for two different isoforms of protrudin, designated L (long) and S (short) . ProtrudEndosomes play an important role in fundamental cellular activities. A subset of EEs formed by endocytosis through invagination of the plasma membrane undergoes conversion to LEs. LEs contain multiple intraluminal vesicles (ILVs) that are derived from luminal invaginations of the LE membrane, and so they are also known as multivesicular bodies (MVBs) . EndosomThe membrane dynamics of endosome maturation are largely attributable to a combination of endosome fission and fusion . Such fiZFYVE27) are responsible for a subset of cases of HSP  superfamily of proteins that possess lipid transfer activity  fused to a form of PDZD8 lacking the TM domain [PDZD8(ΔTM)]. The mutant protein was thus unable to dock in the liposome membrane. This drawback was subsequently addressed by the addition of DGS-NTA(Ni), a conjugated phospholipid that binds the hexahistidine epitope tag, to the donor liposomes. Performance of the assay with His6-PDZD8(ΔTM) thus allowed association of the tagged PDZD8 protein with the liposome membrane. Under these conditions, PDZD8 also showed lipid extraction activity  but not with diacylglycerol . By analin vivo , given tin vivo . HoweverThe mechanism underlying lipid transfer at MCSs has been extensively studied, with multiple tethering factors and lipid transfer proteins having been identified, including those that function at MCSs between the ER and endosomes . VAP VA. Mutatio2, PIP2, and PIP3 P3 . Further3,4,5)P3 . However3,4,5)P3 , protrud3,4,5)P3 , mutatio3,4,5)P3 , and mut3,4,5)P3 .The amounts of endogenous protrudin and PDZD8 are higher in the brain than in other tissues, suggesting that the protrudin-PDZD8 complex may function selectively in the nervous system. Depletion of protrudin or PDZD8 with the use of small interfering RNAs (siRNAs) in mouse primary neurons induced abnormal enlargement of LEs, with the resulting vesicles thus being designated abnormal large vacuoles (ALVs) . This phNeurons depleted of PDZD8 showed a reduced axon length and increased somatodendritic area , reflectDefects in ER-endosome contacts induced by HSP-associated mutations of spastin or REEP1 result in lysosomal abnormalities in neurons . The facin vivo remain to be determined. In addition, the detailed mechanism underlying lipid transfer mediated by the protrudin-PDZD8 complex at ER-endosome MCSs, including the identity of the factor or factors responsible for lipid insertion, awaits further investigation.I have here focused on the role of protrudin at ER-endosome MCSs in endosome maturation in neurons. Protrudin regulates endosome dynamics as well as ER structure, especially in neuronal cells. Mutations of the protrudin gene in humans give rise to the axonopathy HSP as a result of a gain of toxic function. Ablation of the protrudin gene in mice, however, gives rise to psychiatric-like disorders as a result of a loss of function, suggesting that protrudin might play an indispensable role in normal neuronal development and behavior. Protrudin forms a complex with PDZD8 as well as interacts with VAP and Rab7 at ER-endosome contacts. PDZD8 is a mammalian paralog of the ERMES subunit Mmm1, which mediates lipid transfer between the ER and mitochondria. PDZD8 in association with protrudin similarly mediates lipid transfer from the ER to endosomes and thereby contributes to endosome maturation and maintenance of neuronal integrity. The types of lipids transferred by the protrudin-PDZD8 complex MS supervised the study and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Inherited thoracic aortopathies denote a group of congenital conditions that predispose to disease of the thoracic aorta. Aortic wall weakness and abnormal aortic hemodynamic profiles predispose these patients to dilatation of the thoracic aorta, which is generally silent but can precipitate aortic dissection or rupture with devastating and often fatal consequences. Current strategies to assess the future risk of aortic dissection or rupture are based primarily on monitoring aortic diameter. However, diameter alone is a poor predictor of risk, with many patients experiencing dissection or rupture below current intervention thresholds. Developing tools that improve the risk assessment of those with aortopathy is internationally regarded as a research priority. A robust understanding of the molecular pathways that lead to aortic wall weakness is required to identify biomarkers and therapeutic targets that could improve patient management. Here, we summarize the current understanding of the genetically determined mechanisms underlying inherited aortopathies and critically appraise the available blood biomarkers, imaging techniques, and therapeutic targets that have shown promise for improving the management of patients with these important and potentially fatal conditions. Consequently, although there are distinct similarities, it is prudent to consider thoracic and abdominal aortic disease processes separately. Here, we focus on the disease processes of thoracic aortopathy.The aorta is the largest artery in the body, beginning at the aortic valve of the heart and finishing at its bifurcation point in the pelvis. The aorta acts as a conduit for blood by connecting the heart to the major arteries, ensuring blood flow to all the vital organs of the body. Being the most proximal artery to the heart, the thoracic aorta, the portion of the aorta contained within the chest, withstands the highest pressure and wall stresses through >30 million pressure cycles a year. The thoracic aortic wall must therefore function as a robust biomechanical structure, loading and unloading the cardiac volume efficiently and reliably throughout life. To achieve this, the molecular and biomechanical environment of the aortic wall is monitored and stabilized through a complex interplay between vascular wall cells and the extracellular matrix. Unfortunately, this robust homeostatic process can be disturbed through a variety of disease processes, collectively described as thoracic aortopathies, in which the thoracic aortic wall becomes weak and vulnerable. Embryologically, the descending thoracic and abdominal aorta arises from the mesoderm, whereas the aortic root and ascending aorta are derived from the neural crest, leading to differences in gene expression and behavior of vascular smooth muscle cells in each region.8 Even with these guidelines and robust monitoring, many patients with inherited thoracic aortopathy still experience aortic dissection or rupture while still below the threshold diameter, whereas others may undergo surgery for thoracic aortic aneurysms that are unlikely to dissect or rupture.10 There is therefore universal agreement that better identification and risk stratification of thoracic aortopathy are urgently required. Indeed, a substantial body of research has been dedicated to exploring and developing novel solutions to this problem. This review summarizes the epidemiology and underlying pathological processes associated with inherited aortopathy, outlines current management strategies, and examines the evidence supporting promising therapeutic targets and emerging biomarkers for these high-risk aortopathies.Inherited thoracic aortopathies denote conditions in which abnormalities lead to thoracic aortic wall weakness or abnormal aortic hemodynamic profiles, predisposing patients to aortic dilatation, aneurysm formation, and acute aortic complications. Degradation of the components of the thoracic aortic wall can also occur as part of acquired processes, for example, associated with aging or high blood pressure, factors that potentially compound genetically influenced thoracic aortic wall weakness. Patients with inherited aortopathy are at substantially increased risk of acute aortic complications, including aortic dissection or rupture, carrying with it major mortality risk. Because the outcomes from elective surgery are superior to those associated with emergency repair once dissection has occurred, current international guidelines advocate a focus on recognizing those at risk early, regular monitoring with imaging, and prophylactically replacing the diseased aortic segment once a threshold diameter is reached Table .2–8 EvenThe thoracic aorta begins at the aortic valve, finishes as the aorta passes through the diaphragm, and is made up of 4 sections; the aortic root, which encompasses the coronary arteries that supply the heart; the tubular ascending aorta, which runs from the sinotubular junction to the origin of the brachiocephalic artery; the aortic arch, which contains the head and neck vessels and finishes just after the left subclavian artery; and the descending thoracic aorta, which ends at the level of the diaphragm Figure . Inherit11 However, concerns about increased risk of dissection occurring at lower gross diameters in smaller patients have led to wider use of aortic diameters adjusted for age, body surface area (aortic size index), or height (aortic height index), with aortic dilatation defined as an indexed aortic diameter z score >2.0 compared with reference populations.13 The terms “thoracic aortic aneurysm” and “aortopathy” are often used interchangeably with aortic dilatation, although thoracic aortic aneurysm refers to an increase in diameter of >50%, a measure not commonly used accurately, whereas aortopathy is a generic description of a pathological process affecting the aorta, which can be a useful descriptor of a diseased aorta regardless of its diameter.Aortic dilatation in adults has classically referred to increased absolute aortic diameters based on reference thresholds, typically >40 mm, although a range of thresholds have been proposed and referenced in research.The aortic wall is histologically made up of 3 sections Figure . The int14 Cross-linked elastin is formed primarily in the fetal and neonatal period and is insoluble with a half-life of 70 years, making it highly durable.15 Newly formed elastin in adults is of low quality, rendering any damage to elastic fibers largely irreversible. There is also a demonstrable relationship between the number of intact elastin layers and the pressure required to propagate dissection or cause aortic rupture.16 Elastic fibers are anchored to vascular smooth muscle cells via focal adhesions, as well as being supported by a scaffold of extracellular molecules, including fibrillin, integrins, and collagen. Vascular smooth muscle cells and fibroblasts also maintain the extracellular matrix environment by producing tropoelastin, collagens, and microfibrils, along with proteins that promote medial healing and remodeling, for example, matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs).The media is found superficially to the intima and is the most important contributor to biomechanical properties of the thoracic aortic wall. Specifically, the elastic fibers of the media, which are arranged in concentric layers and composed of extensively cross-linked tropoelastin, allow elastic fibers to be stretched and to recoil with minimal energy loss.17 Although aortic dissection represents a tear of the medial elastic fibers and preservation of the adventitial collagen, rupture denotes biomechanical failure of both adventitial collagen and medial elastic fibers.The outer section, the adventitia, is in communication with the nervous system and vasa vasorum, an external blood supply that also provides interaction with the immune system. The adventitia is composed primarily of fibroblasts that produce the fibrillar protein collagen. Unlike elastin, collagen has limited stretch or elasticity. However, it has a high resistance to biomechanical failure under peak stresses, meaning it plays an important role in protecting the aortic wall against rupture.Registry of Genetically Triggered Thoracic Aortic Aneurysms) results, include Marfan syndrome, Turner syndrome, Loeys-Dietz syndrome, and vascular Ehlers-Danlos syndrome.18 A number of other genetically mediated syndromes such as Shprintzen-Goldberg syndrome (SKI mutations) are associated with aortic dilatation, although in these conditions, progression to acute aortic events is exceptional or has not been reported  denotes a varied group of genetically mediated conditions that are associated with systemic features of disease, aortic dilatation, and acute aortic events. The major sHTADs, as published by the GenTAC Registry  describes a familial form of aortopathy and dissections at a young age but without systemic features. In the past decade, studies exploring the underlying genetic mechanisms of the nsHTAD groups have allowed a superior understanding of how gene mutations influence aortopathy risk.FBN1,which codes for a glycoprotein called fibrillin-1  and fibronectin, as well as enhancing elasticity through its microfibrillar structure , resulting in aortic aneurysm, arterial tortuosity, hypertelorism, and bifid uvula with a propensity toward arterial dissection or rupture at a young age.42 The systemic phenotype in Loeys-Dietz syndrome varies widely, with patients with more severe systemic phenotypes, particularly vertebral artery tortuosity, being more likely to experience aortic events.24 Initial descriptions of patients with TGFβR1 or TGFβR2 mutations suggested a particularly low life expectancy of 26 years without intervention. However, a contemporary large international study demonstrates that the majority of patients are surviving past 70 years, albeit with an enduring high dissection prevalence , or p38/extracellular signal-regulated kinase/c-Jun N-terminal kinase , the latter of which is thought to be detrimental to thoracic aortic aneurysm formation, although the precise mechanisms are still controversial  and, to a lesser extent, type 1 collagen  are associated with syndromic forms of aortopathy, and patients with such mutations are at increased risk of acute aortic events , but reports of dissection are very rare ; the structure and integrity of the aortic wall extracellular matrix, including FBN1 (described earlier); COL3A1 and lysyl oxidase (LOX); or those affecting the function of smooth muscle cells, including actin (ACTA2), myosin (MYH11), myosin light chain kinase (MYLK), and protein kinase cGMP-dependent 1 (PRKG1). Taken together, causative mutations can be found in only ≈20% of nsHTAD cases, leaving many without a genetic diagnosis.32 In probands with a confirmed mutation, many have mutations in FBN1 without meeting revised Ghent criteria to diagnose Marfan syndrome.47 Furthermore, up to 35% have mutations in SMAD3 and 14% have ACTA2 mutations, whereas pathological mutations in MYH11, MYLK, PRKG1, LOX,and other less well-depicted genes are limited to a smaller number of families.49 In a cohort of patients experiencing acute aortic events with confirmed pathological mutations, the average age at the time of the event was 41 years, with only 54% having aortic root aneurysms  and anchoring the elastic fibers of the extracellular matrix, which confers overall stability. The intracellular structure of vascular smooth muscle cells and tone generation are governed by smooth muscle cell–specific α-actin and myosin, the function of which can be disrupted in the vascular smooth muscle cell mutations, leading to increased cell death, decreased aortic wall tone, and reduced extracellular matrix stability  with a reasonable proportion in the highest risk category IV , depending on the condition and degree of prepregnancy aortic dilatation , aortic compliance can be assessed by the use of strain, the increase in the aortic wall length as a proportion of the baseline length. Strain can be measured in the circumferential and longitudinal directions, with distensibility representing circumferential strain divided by pulse pressure.64 Ex vivo biomechanical testing of dilated aortas demonstrates that increased stiffness appears to be attributable to higher collagen and lower elastin content, making aortic stiffness a proxy marker of wall remodeling, which may be apparent well before aortic diameters begin to increase.14 Consistently, the aortic stiffness increases with age, and a steep rise in stiffness is seen after 55 years of age, coinciding with the natural decreases in elastin. There is also a strong relationship between increasing diameter and increasing aortic stiffness. In Marfan syndrome, aortic stiffness is increased in those with either dilated or nondilated aortas. More important, reduced longitudinal aortic strain is associated with increased aortic event rates in Marfan syndrome after adjustment for aortic diameter.62 Larger studies are required to corroborate the role of aortic stiffness in identifying high-risk aortopathy after adjustment for dilatation in connective tissue disorders and bicuspid aortic valve.Aortopathy in Marfan syndrome, Loeys-Dietz syndrome, Turner syndrome, and nsHTAD is associated with increased aortic stiffness.In patients whose aorta is inherently weak or remodels abnormally, as with inherited aortopathies, increases in blood pressure, systemic vascular resistance, and aortic stiffness are detrimental and should be addressed. In the past decade, significant research effort has focused on improving our understanding of how medical therapies can affect outcomes through their influence on biomechanical and biological properties.65 The European Society of Cardiology/European Society of Hypertension 2018 guideline on the management of hypertension in aortic diseases recommends strict blood pressure control to <130/80 mm Hg.61 The evidence for a benefit for presumptive β-blocker therapy stems from a reduced rate of aortic dilatation associated with a reduction in blood pressure and acute aortic events in nonhypertensive patients with Marfan syndrome treated with propranolol compared with control subjects.66 Angiotensin receptor blockers, which target the AT1 receptor with a consequent reduction in TGFβ signaling, have also been investigated as a potential therapeutic target, given the promising results in animal models. Large randomized controlled trials conducted so far have not found an effect of angiotensin receptor blockers on clinical outcome compared with the protection provided by β-blocker therapy.69 There is, however, some recent promise for the ability of irbesartan to reduce aortic growth rate when added to current treatment.70 Although currently there is not enough evidence to recommend angiotensin receptor blocker therapy over β-blocker therapy, its use is reasonable when β-blocker therapy is poorly tolerated. Further elucidation of the mechanisms governing TGFβ activity in Marfan syndrome is required.Conventional therapies have focused on reducing aortic wall stress by lowering blood pressure and systemic vascular resistance. The 2017 American College of Cardiology/American Heart Association guideline for the treatment of hypertension recommends β-blockers as first-line treatment in all thoracic aneurysm disease.71 Similarly, fluoroquinolone use appears to be linked to aortic aneurysm formation and dissection in the general population, possibly through an imbalance of TIMPs and MMPs  was associated with a reduced event rate, although there was a small increase in brachial blood pressure, suggesting that blood pressure was not the mediator of aortic risk in these patients.74 The guidance on the medical management for other inherited thoracic aortopathy is extrapolated from studies on Marfan syndrome. Given the diverse mechanisms of aortic wall vulnerability in inherited aortopathies  and international consistency. Furthermore, because TGFβ1 is abundant throughout the body, whether plasma concentrations specifically reflect aortic wall activity and predict disease progression remains to be validated.As outlined, murine models of both Marfan syndrome and Loeys-Dietz syndrome have implicated TGFβ signaling, particularly the noncanonical pathway, as being detrimental Figure . Blood m79MMPs are a family of 23 separate molecules that share the characteristic feature of a zinc-binding catalytic area and are secreted by vascular smooth muscle cells, fibroblasts, and immune cells in response to chemical or biomechanical stress. The primary role of these MMPs is to cleave and digest extracellular proteins such as elastin, collagen, and integrins as part of aortic wall repair, remodeling, and homeostasis. Under pathological conditions, the unchecked activity of specific MMPs can be detrimental to the cardiovascular system by causing excess destruction of vascular wall components. Murine studies have specifically implicated MMP-2 and MMP-9 in thoracic aortic aneurysm formation, and they are the most promising targets for translation as markers and potential modulators of disease.80 As with TGFβ1, translation of MMPs into reliable biomarkers of aortic wall remodeling is attractive. However, blood MMP-9 and MMP-2 concentrations are sensitive to caffeine and the collection method (citrate deemed most stable) and require quick processing (within 2 hours) to prevent artifactual changes in quantification.82 Appropriately measured plasma MMP-2 concentrations have been linked to increased aortic diameter and repeatably linked to increased aortic stiffness, although no studies to date have made links to outcomes.84 Given their association with abnormal aortic function and the mechanism of action, MMPs have been a target for translation of medical therapy. Doxycycline, a generalized inhibitor of MMPs, can prevent aneurysm formation and preserve elastic fiber integrity and aortic biomechanics in animal models.85 However, when translated to human abdominal aortic aneurysms, 100 mg doxycycline over 18 months resulted in a small but significant increase, rather than decrease, in aortic growth rate compared with placebo.86 Although MMPs appear to play a role in aortic wall pathology, their precise role remains to be determined. Progress may be made by improving our understanding of how MMPs are moderated through their naturally occurring inhibitors.Efforts have been made to use MMP-2 and MMP-9 as prognostic biomarkers for thoracic aortic disease severity. Human induced pluripotent stem cell line study of patients with Marfan syndrome demonstrates increased MMP-9 levels compared with reference cell lines.TIMPs are a family of 4 naturally occurring and nonspecific MMP inhibitors found in a wide variety of biological systems, including the vasculature. They function by interacting with the active site of target MMPs and deactivating pro–MMP-2 and -9, working in concert to promote vascular homeostasis without excess destruction. Under pathological circumstances, MMP activity unchecked by TIMP is thought to promote abnormal remodeling.26 Moreover, because bicuspid aortic valve disease is male predominant, some have speculated that having 2 sets of X chromosomes is in some way protective against bicuspid aortic valve and aortopathy. Although TIMP1 may be implicated in inherited aortopathic disease, there is little evidence that plasma TIMP1 concentrations reflect gene copy number or mirror aortic pathological processes.87 Some researchers have instead focused on the ratio of MMPs to TIMPs, giving a picture of an overall balance between remodeling and destruction and finding increased ratios in those with bicuspid aortic valve, although these findings require further validation.88Turner syndrome is an interesting inherited thoracic aortopathy with a high incidence of bicuspid aortic valve that can be used to study TIMP1 as a mediator of thoracic aortopathy. The gene is located on the X chromosome, 1 copy of which is lost in many cases of Turner syndrome.Overall, the roles of TIMP1 as a mediator of remodeling make an attractive target for biomarker translation, particularly when coupled with MMP-2 and MMP-9 to give a representation of remodeling activity balance. However, as with many biomarkers, both MMPs and TIMPs are involved in many biological processes; therefore, blood concentrations may not be specific enough to represent thoracic aortic remodeling.89 Desmosine has very few other functions in humans, potentially making it a specific marker of aortic wall elastin breakdown.90 In abdominal aortic aneurysms, increasing plasma desmosine concentrations were associated with aortic diameter and an increasing likelihood of aortic events, after adjustment for diameter.91 Although the breakdown of elastin is not specific to a single inherited aortopathy, it represents an end point thought to correlate with biomechanical weakness and a tendency toward failure. Studies exploring the utility of plasma desmosine concentrations to predict dissection or rupture in thoracic aortopathy would be highly valuable.A promising development in biomarker research for thoracic aortopathies lies in plasma desmosine, a rare molecule that is necessary for cross-linking tropoelastin. Desmosine appears to increase with age and is elevated in patients with chronic obstructive pulmonary disease, reflecting elastin breakdown.Although a significant amount of research has focused on novel blood biomarkers, the evidence supporting their role as a complementary marker of aortopathy risk is relatively weak, with most targets being involved in many processes throughout the body. Therefore, blood concentrations are unlikely to reflect changes at the aortic wall level. In this regard, molecular imaging techniques, which allow the direct visualization of biological processes at specific anatomic locations, may prove beneficial.Advanced imaging techniques with specific tracers have the potential to localize and quantify (patho)biological processes occurring within the body. Molecular imaging with a radioactive molecule or a targeted smart contrast agent can be used to provide combined functional and structural imaging with a range of imaging techniques such as positron emission tomography, MRI, or computed tomography. The anatomic visualization of molecular activity allows organ-specific assessment of these processes, improving accuracy and specificity.92 In the murine model of Marfan syndrome, in vivo ESMA MRI quantification demonstrated reduced elastin compared with wild-type mice, a finding corroborated on histology  is now being investigated as a way of highlighting the aortic wall, with reduced uptake associated with decreased elastin content and aortic wall disease.y Figure .93 The E95 Because the presence of medial microcalcification represents ongoing pathological processes, noninvasive detection and quantification could be a valuable marker of aortic wall disease. 18F-sodium fluoride is a molecular tracer that binds to hydroxyapatite, thus allowing noninvasive detection and quantification of aortic wall microcalcification.96 A recent study investigating the utility of 18F-sodium fluoride to predict clinical events in abdominal aortic aneurysms found that patients in the highest tertile of aneurysmal 18F-sodium fluoride uptake had the quickest growth rate and an increased incidence of surgical repair or rupture.97 Given the role that medial microcalcification plays in many common end points of pathological change of ascending aortopathy  and the promising results toward abdominal aortic aneurysms prognostication, it would appear to be an ideal candidate for translation in inherited thoracic aortopathy.Vascular medial microcalcification is the pathological accumulation of hydroxyapatite crystals within the media of large arteries and has been strongly linked to adverse cardiovascular outcome. It occurs through multiple mechanisms that are separate from the intimal calcification that occurs in in atherosclerosis. Vascular medial microcalcification is understood to occur via 3 primary mechanisms: calcium deposition on damaged elastin, excess intracellular calcium released by stressed smooth muscle cells into vesicles or through cell death, or pathological osteogenic phenotype switching by vascular smooth muscle cells.18F-fluorodeoxyglucose is a positron emission tomography radiotracer that nonspecifically highlights areas of high glucose metabolism, commonly found in areas with a high concentration of inflammatory cells or metabolically active cells. In abdominal aortic aneurysms, high 18F-fluorodeoxyglucose positron emission tomography imaging has been linked to increased cardiovascular events in general, as well as correlating with increased inflammation and MMPs in abdominal aortic aneurysms.98 However, at present there are no studies directly assessing the relationship between 18F-fluorodeoxyglucose uptake in the thoracic aorta and clinical outcomes focused on those with inherited aortopathy.Unlike abdominal aortic aneurysms, the inflammatory process in thoracic aortopathy is less pronounced, with only weak associations between immune system activation and thoracic aneurysm formation. Indeed, there is some association with T cell–mediated medial degeneration, but this appears to be a late manifestation of the thoracic aneurysm formation process. 99mTc-RP805 correlated with aneurysm size and predicted rupture or aneurysm formation.99 Although promising and relevant to ascending aortopathy, there are no reported applications in inherited thoracic aortopathy. Similar to MMP radiotracers, preclinical agents are being developed for visualizing collagen  and Wellcome Trust (WT103782AIA to Dr Newby).Dr Fletcher has received a 1-year salary as part of a Siemens imaging fellowship. The other authors report no conflicts.


Extracellular vesicles are heterogeneous populations of naturally occurring secreted small vesicles. EVs function as signaling platforms to facilitate intracellular communication, which indicates the physiological or pathophysiological conditions of cells or tissues. Considering that EVs can be isolated from most body fluids and that molecular constituents could be reprogrammed according to the physiological status of the secreting cells, EVs are regarded as novel diagnostic and prognostic biomarkers for many diseases. The ability to protect encapsulated molecules from degradation in body fluids suggests the potential of EVs as biological medicines or drug delivery systems. This article focuses on the EV-associated biomarkers and therapeutic approaches in autoimmune diseases. Extracellular vesicles (EVs) , membranReleased EVs involve in intercellular communication and cellular function regulation under normal physiological conditions, while reprogrammed EVs cargo can lead to an immune response and contribute to the development of diseases under pathological conditions . VariousThis review focuses on the EV-associated biomarkers and potential applications of EVs in autoimmune diseases.Autoimmune diseases, characterized by self-immune responses, are one of the leading causes of morbidity and mortality among chronic diseases . ImbalanPrimary Sjögren’s syndrome, a chronic female‐dominant autoimmune disorder influencing approximately 1% of the general population and 3% of people older than 50 years , is charOne previous study reported that although the levels of MPs in pSS patients with high or low disease severity were higher than health controls, those in pSS patients with high disease severity were lower than those in patients with low disease severityTable  22].The.The22].TSystemic lupus erythematosus, a systemic autoimmune disease influencing multiple organs simultaneously with poor quality of life and substantial mortality, is characterized by the presence of autoreactive T cells and hyperactive B cells that produce autoantibodies forming immune complex deposits , 32.Proven to increase adhesion molecule expressions, chemokine productions, and structural alterations in macrovascular and microvascular endothelial cells, which can lead to endothelial alterations and tissue leukocyte infiltration , 33, MPsMany studies have shown increased total MPs levels in the plasma of SLE patients compared with those of healthy controls , 34–38. EV-associated biomarkers have been intensively studied in other autoimmune diseases Table . Oral liBehçet’s syndrome (BS), a multisystem inflammatory disorder involving venous and arterial vessels , is charType 1 diabetes mellitus (T1DM), a disorder caused by an autoimmune response against insulin-producing β cells in the pancreatic islets, is the most severe form of diabetes mellitus. A recent study indicated that EVs play an important role to transfer autoantigen peptides from insulin-producing β cells in the pathogenesis of T1DM . Study hIn addition to the promising use as biomarkers, EVs have been suggested as potential therapeutic approaches which can be divided into four categories: (i) utilizing EVs to transfer the natural cargo of EVs to induce immunosuppressive or immunostimulatory effects, including antimicrobial effects, anti-inflammatory effects, and antitumor effects or utilizing EVs as an alternative to mesenchymal stem cell transplantation; (ii) utilizing bioengineering techniques to modify EVs as nanocarriers for drug delivery systems to deliver specific nucleic acids , proteins, and therapeutic agents to target cells or tissues; (iii) utilizing EVs to induce tissue regeneration and tissue repair; and (iv) utilizing EVs as novel vaccines in the treatment of tumors or infections Fig.  20].Fig.Fig20].FAccumulating evidence supports that EVs involve in intercellular communication inducing immunostimulation and immunosuppression, and EVs are promising biomarkers or therapeutic approaches for autoimmune diseases. In this review, we provided evidence for the biomarker potential of EVs in several autoimmune diseases and summarized the potential use of EVs in therapies. However, both basic and applied studies of EVs are still in the early stages, and the poor understanding of the underlying mechanisms hinders the clinical translation of EVs. Obviously, extensive studies of EVs are necessary before application for the clinical diagnosis, prognosis and therapy of autoimmune diseases can be performed, including (i) studies on the separation and purification of EVs; (ii) studies providing an intensive understanding of EVs biogenesis and targeting; (iii) studies providing an intensive understanding of the mechanism by which EVs induce immunostimulation and immunosuppression; (iv) studies assessing the effect and reliability of EVs as nanodrugs or drug delivery systems in vivo; and (v) studies on clinical applications. Despite the challenges and difficulties remaining before EVs can be clinically applied, their biological and physiological characteristics have shown the great potential of EVs as biomarkers and therapeutic tools. In conclusion, intensive study of the biological functions and mechanisms of EVs could help to identify potential biomarkers and facilitate the clinical translation of EVs.