Thoracic aortic aneurysms (TAA) are permanent and localized dilations of the aorta that predispose patients to a life-threatening risk of aortic dissection or rupture. The identification of pathogenic variants that cause hereditary forms of TAA has delineated fundamental molecular processes required to maintain aortic homeostasis. Vascular smooth muscle cells (VSMCs) elaborate and remodel the extracellular matrix (ECM) in response to mechanical and biochemical cues from their environment. Causal variants for hereditary forms of aneurysm compromise the function of gene products involved in the transmission or interpretation of these signals, initiating processes that eventually lead to degeneration and mechanical failure of the vessel. These include mutations that interfere with transduction of stimuli from the matrix to the actin–myosin cytoskeleton through integrins, and those that impair signaling pathways activated by transforming growth factor-β (TGF-β). In this review, we summarize the features of the healthy aortic wall, the major pathways involved in the modulation of VSMC phenotypes, and the basic molecular functions impaired by TAA-associated mutations. We also discuss how the heterogeneity and balance of adaptive and maladaptive responses to the initial genetic insult might contribute to disease. Aneurysms are permanent, localized dilatations of an artery greater than 50% of the normal diameter. They progressively dilate while remaining mostly asymptomatic until a life-threatening rupture and/or dissection occurs . ProphylThe healthy arterial wall is composed of three distinct layers: the tunica intima, the tunica media, and the tunica adventitia  Figure A. The prThe tunica media is the thickest portion of the arterial wall and the layer that endows the vessel with structural integrity and mechanical functionality ,17. It cThe external elastic lamina separates the media from the third and outermost layer of the arterial wall, the adventitia, which is comprised of fibroblasts and, according to some studies, progenitor cells with VSMC differentiation potential, all located within a collagen-rich matrix ,25,26,27Analysis of aneurysmal tissue obtained from both patients and mouse models reveals common end-point characteristics that are observed regardless of etiology ,32. AlthIn the healthy adult aorta, VSMCs are quiescent and primarily dedicated to regulation of vascular tone through their contractile function . This phMultiple signaling pathways are dysregulated in TAA in consequence of all these processes, including those activated by angiotensin II, insulin-like growth factor-1 (IGF-1), platelet-derived growth factor (PDGF), and TGF-β ,63,64,65Numerous environmental cues can modulate VMSC phenotypes. Although there is significant cross-regulation, these signals can be broadly classified as dependent on mechanical connections between the ECM and VSMCs, or as being primarily activated by cell receptors that bind soluble or cell-bound ligands. We will briefly summarize the basic components of these modulatory pathways by focusing on those elements that have been found to be directly involved in the pathogenesis of hereditary TAA B.VSMCs connect to the ECM via integrins and non-integrin matrix receptor molecules. Non-integrin receptors include CD44 , discoidin domain receptor tyrosine kinases (which are upregulated in response to injury and bind to collagen), and the receptor for advanced glycation end-products (RAGE), all of which are reviewed in references ,72.Integrins, a family of heterodimeric transmembrane receptors formed by the dimerization of an α and a β subunit, are the main transducers of mechanical cues between the ECM and VSMCs. Each heterodimeric combination of integrin molecules binds to specific ECM components ,74,75. U5β1 and αVβ3 connect the actin–myosin cytoskeleton to fibrillin-1 present in microfibrils [Integrin can transduce both biochemical and mechanical information to VSMCs and thus modulate their phenotype in response to the presence of specific ligands in the ECM and in response to shear stress, tension, or “stiffness” ,74,79. Iofibrils ,80. Microfibrils ,80 . The stofibrils ,81,82. Tofibrils ,84,85,862+) concentration, either through the opening of ion channels on the plasma membrane or by release from internal stores [2+ binds to and activates calmodulin, which in turn activates myosin light chain kinase (MLCK), the enzyme that phosphorylates myosin light regulatory chains and initiates myosin-dependent conversion of ATP into mechanical energy [2+ concentration [2+ concentration and by those that modulate the sensitivity of the actin–myosin unit to changes in Ca2+ concentration via post-translational modifications [In VSMCs, the actin–myosin unit is composed of antiparallel filaments of a smooth muscle specific form of actin  and myosin filaments that include dimers of a smooth muscle specific myosin heavy chain  . This stl stores ,89,90. Cl energy ,89,90. Mntration ,89,90. Tications ,89,90.In addition to controlling vascular tone, the contraction of the actin–myosin unit regulates the composition and function of focal adhesions, reinforcing the connections between anchoring proteins, integrins, and ECM, and promoting actin polymerization . AlthougMechanical stretch can directly activate several cell receptors, kinases, proteases, and other enzymes ,102,103.Growth factors, cytokines, and membrane-bound ligands bind to cell receptors expressed on VSMCs and activate signaling pathways that act on several transcription factors, regulating proliferation, synthesis of contractile proteins, deposition of new matrix components, and expression of matrix-degrading enzymes ,54. In t1Rs) activates signaling pathways that stimulate VSMC contraction within seconds, inducing vasoconstriction and regulating blood pressure [1R can promote vascular inflammation by the upregulation of adhesion molecules and leukocyte chemoattractants [1R effects are mediated by the transactivation of receptors for other growth factors including those for PDGF and epidermal growth factor (EGF) [1R signaling also increases the expression of other signaling molecules, including PDGF, transforming growth factor-β (TGF-β), and reactive oxygen species (ROS) [The renin–angiotensin system is a critical regulator of both physiological and pathological cardiovascular functions (comprehensively reviewed in reference ). Its prpressure . Longer pressure ,123. In ractants . Some ofor (EGF) ,126,127.es (ROS) ,129,130.1R signaling mediates the deposition of collagens in response to mechanical stimulation through a process that depends on increased expression of the TGF-β ligand [1R also potentiates TGF-β signaling by inducing the expression of metalloproteinase-2 and -9 (MMP-2 and MMP-9), which are proteolytic enzymes that can catalyze the conversion of inactive ECM-bound latent TGF-β into its bioactive form [1R signaling, which can be triggered through mechanical stretch, independently of angiotensin II [Agtr1a (the gene that in rodents codes for the major form of AT1R expressed in the thoracic aorta) via the ROS-dependent activation of nuclear factor kappa B (NF-kB); the de-repression of Agtr1a expression as a consequence of defective TGF-β signaling; and increased expression of angiotensin-converting enzyme, which is a positive regulator of the pathway [In VSMCs, ATβ ligand ,132,133.ive form ,135,136.ensin II ,139,140,ensin II . These i pathway ,142,143.TGF-β ligands  are secreted as inactive latent complexes that are activated by chemical (acidification), mechanical (integrins), and enzymatic (proteases) processes within the ECM . After cFocal adhesion-dependent tension modulates TGF-β signaling both negatively and positively . Focal aMyh11, Acta2, and Cnn1  [1R and Notch signaling [TGF-β signaling regulates many aspects of VSMC biology, including positive regulation of VSMC-specific transcripts such as ctively) ,157,158.ctively) ,161,162.ignaling ,163.Notch signaling is activated by engagement of a Notch receptor (Notch1 to Notch4) by one of its ligands (Jagged1/2 and Delta-like1/3/4 ligands) . The binActa2 and Pdgfbr (coding for PDGF-β receptor) [Adult VSMCs express Notch1, Notch2, and Notch3 receptors and the ligand Jagged-1. Notch2 and Notch3 appear to be the main receptors modulating phenotype and functions in VSMCs, but the activation of both Notch1 and Notch3 facilitates VSMC migration in vitro ,167,168.eceptor) ,170. Wheeceptor) ,175,176.Notch1, and inhibition of Notch1 decreases angiotensin-II dependent upregulation of Tgfb1 while promoting upregulation of MMP-2 and MMP-9 [Notch3 [Notch signaling interacts with both angiotensin-II and TGF-β signaling. In podocytes, stimulation with angiotensin II upregulates nd MMP-9 . Notch and MMP-9 ,178,179.nd MMP-9 ; in turn [Notch3 . The exp [Notch3 ,182,183.The identification of genetic variants that cause hereditary forms of aneurysm provides the opportunity to clearly define the molecular deficiencies that initiate this disease. Several recent reviews have summarized the current list of approximately 30 genes involved in the development of either syndromic or non-syndromic forms of TAA ,185,186 Mutations that cause TAA interfere with the function of genes that encode components of the ECM or proteins implicated in the transduction of either mechanical or biochemical signals in VSMCs ,185,186.FBN1 codes for fibrillin-1, the primary component of microfibrils, which surround and connect elastic fibers to cellular integrins [FBN1, as well as mutations affecting splicing, cause hereditary aneurysms associated with MFS [FBN2, the gene coding for fibrillin-2, cause congenital contractural arachnodactyly; although these mutations are associated with aortic dilatation and dissection in some patients, TAA is not a common phenotype, which is likely a consequence of the compensatory effect of fibrillin-1 [Several TAA-causing mutations are found in genes coding for structural components of elastic fibers or for proteins necessary for their assembly or maturation. ntegrins ,233. Hetwith MFS ,235,236.rillin-1 ,201FBN1 mutations result in defective synthesis, secretion, and/or the incorporation of fibrillin-1 in microfibrils [FBN1 have also been proposed to reduce the ability of fibrillin-1 to bind and sequester the latent TGF-β complex into the ECM, thus promoting its conversion into bioactive TGF-β and causing excess activation of this pathway [TAA-associated ofibrils ,237,238.ofibrils ,239. Mut pathway ,240,241. pathway ,243. Thi pathway ,245.LOX gene, which encodes lysyl oxidase, cause aneurysms in both patients and animal models [LOX mutations cause TAA [Heterozygous inactivating mutations in the l models ,198,246.l models ,248,249.l models ,198,246.l models ,251. Altl models ,253. It ause TAA ,255.EFEMP2, the gene coding for fibulin-4, a matrix glycoprotein. These mutations are predicted to interfere with fibulin-4-mediated enhancement of lysyl oxidase enzymatic activity and/or recruitment to immature elastin molecules [Phenotypes similar to those caused by deficiency in lysyl oxidase activity are observed as a consequence of homozygous loss-of-function variants in olecules ,256,257.olecules ,258,259.olecules ,261; thiMFAP5, which codes for microfibril-associated glycoprotein 2 (MAGP2) [Loss-of-function mutations in  (MAGP2) , also pr (MAGP2) ,263,264.BGN gene, which codes for the ECM protein biglycan, cause syndromic forms of TAA [FBN1 [X-linked inactivating mutations in the s of TAA . Biglycas of TAA ,267,268.s of TAA ,270. Sups of TAA ,272. ItsAA [FBN1 ,270.COL3A1 are associated with Ehlers–Danlos syndrome (EDS) [Mutations in the collagen gene me (EDS) ,194, whime (EDS) ,193,194.me (EDS) ,269,273.me (EDS) .ELN, the gene coding for tropoelastin, cause autosomal dominant Cutis Laxa (ADCL), which is a condition that is associated with aortic dilatation and rupture in approximately 30–50% of patients [Rare mutations in patients ,199,275.patients . These mpatients ,277,278.ELN, including deletion of the whole gene, cause supravalvular aortic stenosis and Williams Beuren Syndrome, but not aneurysm [-/-Eln mice, which completely lack elastin, die of obstructive arterial disease, not dissection, a few days after birth [-/-Lox mice, which lack lysyl oxidase, and which also die perinatally, but show large aortic aneurysms [Eln allele are hypertensive and have a paradoxically increased number of elastic lamellae, but they show no overt signs of degenerative vascular disease, and again, no aneurysm or dissection [FBLN5, the gene coding for fibulin-5, develop autosomal recessive Cutis Laxa type IA (ARCL1A). Deficiency in fibulin-5, a matrix protein that assists in the deposition of tropoelastin on microfibrils, results in impaired elastic fiber assembly and fragmentation of elastic laminae in the aorta, but there is no evidence of aneurysms or dissections in either patients or mouse models [Whereas dysfunctional elastin has been associated with TAA, elastin insufficiency associates with inherited obstructive arterial diseases; heterozygous loss-of-function mutations in aneurysm ,284,285.er birth . This isneurysms . More reneurysms . Mouse mssection ,288. Alte models ,291,292Taken together, this mutational repertoire strongly suggests that interference with the assembly and function of elastic lamellae, and therefore impairment of the elastin-contractile unit, is an initiating event in aneurysm pathogenesis ,293. HowACTA2, the gene coding for α-SMA, and in MYH11, which codes for SM-MHC [Mutations in genes involved in the structure or regulation of actomyosin filaments cause familial cases of TAA. These include mutations in r SM-MHC ,295,296.r SM-MHC ,295,296.MYLK, which codes for MLCK, reduce its kinase activity and thus impair contraction and promote relaxation [PRKG1, which codes for type I cGMP-dependent protein kinase (PKG-1), results in the constitutive activation of PKG-1, a cGMP-activated enzyme that promotes relaxation by phosphorylating and activating MLCP [TAA-causing mutations are also found in genes involved in the regulation of myosin-dependent contraction . TAA-asslaxation ,298,299.ing MLCP ,220.FLNA, which codes for filamin A, result in an increased risk of TAA, among other symptoms [X-linked loss-of-function mutations in the symptoms ,224,300.symptoms ,303,304.ARIH1, which codes for Ariadne drosophila homolog 1 (Ari-1), are found in individuals with aortic or cerebrovascular aneurysms [Rare variants in neurysms . Ari-1 ineurysms . The exineurysms ,112. Recneurysms . The defneurysms .The fact that mutations in genes that control the function of actin–myosin complexes cause TAA clearly points to the importance of force generation to the homeostasis of the aortic wall . TogetheTGFBR1, TGFBR2), transduction molecules (SMAD3 and SMAD2), and ligands (TGFB2 and TGFB3) cause TAA associated with LDS [LTBP3, the gene encoding latent TGF-β binding protein 3 (LTBP3), suffer from aneurysms and dissections of the thoracic aorta and other arteries [Heterozygous loss of function mutations in genes coding for positive regulators of TGF-β signaling, including receptors , SHF-derived VSMCs showed the expected deficit in responsiveness to TGF-β, whereas CNC-derived VSMCs retained normal signaling capacity [1R-dependent TGF-β ligand expression in SHF-derived VSMCs and an excessive activation of TGF-β signaling in surrounding CNC-derived VSMCs [Another model proposes that LDS mutations, which impair but do not fully abolish signaling, have an uneven effect on the signaling capacity of VSMCs of different embryonic lineages , and drive excess signaling thorough mechanisms that depend on this imbalance ,320. Thecapacity . This ased VSMCs ,320. Inted VSMCs ,323,324.1R-dependent secretion of TGF-β, thus causing increased signaling in neighboring CNC-VSMCs [Work conducted in induced pluripotent stem cells (iPS)-derived VSMCs has confirmed that lineage-of-origin modifies the effect of LDS-causing mutations, impairing signaling in SHF-like VSMCs derived from cardiovascular progenitors, while leaving the signaling capacity of CNC-derived VSMCs intact or increased . Only SHNC-VSMCs .BGN gene, which can function as a negative regulator of TGF-β signaling [SKI gene proposed to interfere with SKI’s ability to bind and inactivate the transcriptional activity of Smad proteins [Mutations in genes coding for negative regulators of the TGF-β pathway have been associated with syndromic forms of aneurysm. As discussed earlier, aneurysms can be caused by inactivating mutations in the ignaling ,271,272.proteins ,212,326.proteins . NOTCH1 predispose to hereditary forms of aneurysms that associate with aortic valve defects [Notch1 haploinsufficiency causes aortic dilation in mice of a permissive genetic background (129S6) and exacerbates aortic root pathology of MFS mouse models, independently of aortic valve defects [The perturbation of Notch signaling, a pathway that can both cooperate and antagonize TGF-β signaling during VSMC differentiation, increases susceptibility to aortic aneurysm ,175,176. defects ,216,328. defects . Althoug defects ,330. Not defects ,332.NOTCH1, ELN, ACTA2, FBN1, and LOX (discussed above), all of which, however, explain a very small portion of all BAV cases [ROBO4, which codes for Roundabout homolog 4 (ROBO4), have been identified in patients with BAV and aneurysm of the ascending aorta [Bicuspid aortic valve (BAV) is a common congenital defect that can be present in isolation or in association with other syndromic manifestations, and which associates with an increased risk of aortic aneurysm, frequently affecting the ascending aorta ,117,333.AV cases ,336,337.AV cases ,340,341,AV cases ,343,344,AV cases ,346, andAV cases . Recentlng aorta ; these mng aorta . Althougng aorta , and is ng aorta ,351,352.1R and TGF-β signaling in a specific subset of VSMCs [The discovery of mutations that cause hereditary forms of TAA has identified fundamental cellular and molecular process that are necessary to retain VSMC homeostasis and prevent medial degeneration. The preponderance of evidence supports a model whereby a defective assembly of connections between the ECM and VSMCs initiates the phenotypic transition of these cells from a quiescent, contractile phenotype to one that is highly synthetic and conducive to proteolytic degradation of the ECM ,53,353 . Experimof VSMCs ,244,354.1R and TGF-β signaling, further exacerbating ECM pathogenic remodeling [However, a loss of VSMC-elastic fibers connections does not appear to be sufficient to cause aneurysmal disease, given that elastin deficiency associates with obstructive arterial diseases and VSMC over-proliferation, but not aneurysm ,293. TAAmodeling ,261,354.modeling ,153.In this model, mutations in components of matrix proteins or enzymes necessary for their assembly/maturation would initiate disease by directly compromising the function of elastic fibers or other ECM structures to which integrins connect, whereas mutations in components and regulators of the actin–myosin cytoskeleton would do so by perturbing tension and/or myosin-dependent maturation of focal adhesions ,298,299.Mutations in components of signaling pathways that play a positive role in VSMC differentiation, such as TGF-β and Notch signaling, would initiate disease indirectly by interfering with the expression of components required for the assembly of functional focal adhesions. For example, TGF-β signaling controls the assembly of elastic fibers by regulating the expression of fibrillin-1, elastin, and lysyl oxidase; it also modulates the expression of proteins involved in focal adhesion and cytoskeletal rearrangement such as paxillin, Sm22 (Transgelin), and Lin11, Isl-1, and Mec-3 (LIM) domain-containing proteins CRP2 and zyxin ,364,365.Current therapeutic strategies focus on decreasing the rate of dilation, and the associated risk for acute aortic events, by reducing hemodynamic stress on the vessel wall with anti-hypertensive drugs, such as inhibitors of the β-adrenergic receptor . A betteWe have a limited understanding of the compensatory mechanisms activated in response to germline TAA-associated mutations. Feedback responses attempting to offset the negative consequences of a given genetic variant are active throughout prenatal and postnatal development and might significantly modify the structural, cellular, and molecular properties of the adult aorta. Mechanisms that are adaptive early, such as the activation of secondary pathways that compensate for the initial deficiency, might become maladaptive later on due to divergent effects on adult versus embryonic tissues, over-activation, or secondary activation of deleterious pathways. This might be especially true for mutations that impair signaling involved in morphogenesis, such as TGF-β and Notch signaling, given that the mitigation of defective signaling in these pathways is a condition necessary for the development of vascular structures and survival ,369,370.Loss of elastin and the increased deposition and crosslinking of collagen during aneurysm development translate into biomechanical changes that include reduced distensibility and increased stiffness of the aorta . ChangesPerhaps unintuitively, increased stiffness (resistance to deformation) can associate with decreased vessel strength (ability to withstand stress without breaking), with one study measuring an approximately 30% decrease in vessel strength accompanied by a 72% increase in stiffness in aneurysmal versus nonaneurysmal ascending aorta ,375. CorAlthough measures of distensibility and circumferential strain lose predictive power once aortic diameter is included in the analysis, a recent study of one hundred and seventeen MFS patients showed that measurements of longitudinal strain in the proximal aorta was a predictor of adverse aortic events (such as elective aortic root surgery or dissection), thus providing support to the notion that aortic stiffness could be considered for the stratification of patients based on risk ,391,392.These observations may suggest that increased collagen deposition and crosslinking is uniformly deleterious in TAA. However, other studies have shown that collagen deposition, especially in the adventitial layer, can be protective and part of beneficial “scar repair” mechanisms preventing transmural ruptures ,393,394.Taken together, these data suggest that the deposition of properly crosslinked collagen confers strength to the vessel, thus protecting it from mechanical failure. However, its effect might be highly dependent on the type and quality of collagen and the effect of ECM stiffness on VSMC phenotypes ,404,405.Tgfbr2 postnatally results in aortopathy and dissections as well as an exacerbation of pathology in mice with a pre-existing genetic predisposition to aortic aneurysm, suggesting that postnatal aortic VSMCs require a basal level of TGF-β signaling for homeostasis [Work performed in animal models clearly shows that TGF-β signaling is essential for aortic development and morphogenesis ,407. Addeostasis ,318,408.eostasis ,310,311.eostasis ,41.1R signaling, induction of protective factors such as nexin-1 and proteases inhibitors, and promotion of contractile proteins expression [Beneficial roles of TGF-β in TAA may include the suppression of ATpression ,412,413.pression ,416,417.pression ,427,428.Smad4 haploinsufficiency is deleterious in MFS, Smad2 deletion in CNC-derived VSMCs is beneficial in a mouse model of LDS [Accordingly, in contrast to the complete inactivation of TGF-β signaling in VSMCs, which unvaryingly enhances pathogenesis, the effect of partial TGF-β antagonism with neutralizing antibodies or by the inactivation of Smad proteins in selected cellular subsets is varied. Systemic TGF-β neutralization had no effect on angiotensin II-induced TAA , and it l of LDS ,432. Takl of LDS ,433.1R signaling, such as the angiotensin receptor blocker (ARB) losartan, invariably prevents the development of aneurysm in animal models; this beneficial effect associates with reduced levels of TGF-β ligand, pSmad2/3, and the expression of TGF-β target genes [Agtr1a (gene encoding AT1R in mice) prevents aortic root dilation in two different MFS mouse models [1R antagonism in mouse models of TAA have been attributed to its anti-hypertensive effects and to the inhibition of fibrotic, hypertrophic, and mitogenic responses activated by TGF-β and mitogen-activated protein kinase (MAPK) signaling [1R-dependent secretion of glycosaminoglycans, whose accumulation has deleterious effects on the aortic wall [1R antagonism in the treatment of aneurysm in MFS patients. Although the degree of efficacy varied, none of the studies could replicate the remarkable beneficial effects routinely achieved in pre-clinical models; in one trial, the rate of adverse events was higher in the losartan-treatment group than in those receiving a hemodynamically equivalent dose of anti-hypertensive medication [In contrast with direct TGF-β antagonism, treatment with antagonists of ATet genes ,435,436.e models ,438. Theignaling ,432,439.tic wall ,440. Basdication ,449,450.1R signaling might have protective effects in TAA, both directly and through the enhancement of any protective effects of TGF-β signaling [1R-dependent collagen deposition and maturation, directly or through TGF-β-dependent pathways, could recapitulate, in part the detrimental effect of lysyl oxidase inhibition with β-amino-propionitrile (BAPN), which causes dissection or rupture in a number of animal models [1R-dependent signaling could be beneficial through stimulation of VSMC contraction [1R signaling need to be considered, other reasons may account for different outcomes in clinical trials relative to mouse models [These results raised the possibility that ATignaling ,451. Forl models ,452,453.traction ,454. Alte models ,455.In mouse trials, losartan has been tested primarily in prevention rather than treatment of aneurysm, given that in most studies, the drug is initiated early, before overt disease is apparent; this is not the case for clinical trials, given that many patients have established aneurysms when treatment is started . Additio1R antagonism slows the progression of aortic root dilation and is not associated with a statistically significant difference in adverse aortic events [The relative low frequency of adverse aortic events  makes it difficult to perform clinical trials with sufficient power to assess the effect of treatment on these clinically relevant outcomes, and most trials rely on the measurement of aortic size or growth to assess efficacy. Despite the statistical limitations, the most recent metanalysis of available data showed that ATc events ,459. Addc events , showed c events . Althougc events ,455.Although the composition of the aorta appears grossly uniform across the length of the vessel, different segments vary in structural, mechanical, cellular, and developmental properties. This regional heterogeneity reflects distinct physiological requirements for each aortic segment and might underlie the uneven distribution of aneurysm risk observed in patients carrying TAA-causing mutations, which preferentially tend to develop disease in the proximal aorta .Heterogeneity along the length of the aorta has been reported in regard to the expression of gap junction proteins, MMPs, homeobox transcription factors, and embryological origin ,467,468.Lineage-dependent differences are retained in cultured VSMCs with respect to the expression of extracellular matrix proteins and contractile proteins, modulators of angiotensin II signaling, and responsiveness to TGF-β ,480,481.https://github.com/LI-Yan-Ming/Circulation.-2020-142-1374-1388. Their analysis shows that the upregulation or downregulation of several of these markers is cluster-specific, with some clusters showing increased and others showing decreased expression of ACTA2 (coding for αSMA) and TAGLN (coding for SM22 α) between control and TAA samples [Although we lack markers to identify different VSMC lineages in human samples, the heterogeneity of VSMC phenotypes in TAA has been highlighted by scRNA-Seq analysis of human aortic samples from controls and patients with non-syndromic forms of TAA. This study identified at least four subtypes of VSMCs, including a proliferating VSMC cluster that expresses both synthetic and contractile marker genes, which is a phenotype that does not align with the traditional binary understanding of either “contractile” or “synthetic” phenotypes . Althoug samples .Regional differences along the length of the aorta have been observed in regard to ECM composition, mechanical properties, and mechanical stresses ,384,498.Additional variation exists transmurally across the thickness of the vessel. Elastin fibers tend to organize circumferentially in the middle portion of the media while assuming a more longitudinal distribution in regions closer to the adventitia; they also manifest a “waviness gradient”, with inner layers of elastic lamellae being “wavier” than outer layers, which is a feature necessary to compensate for the extra circumferential stretch born by the inner surface of the vessel ,506. The1R  of a given lineage might contribute to rendering specific regions of the vessel, such as the proximal aorta, more susceptible to disease even when mutant gene products are expressed uniformly along the vessel. For example, SHF-derived VSMCs, which reside closest to the aortic root and are subject to the greatest longitudinal strain from the heartbeat, might rely more heavily on the activity of regulatory pathways that physiologically suppress the expression of mechanosensitive receptors such as ATignaling ). These ignaling .1R and TGF-β receptors, can have both adaptive and maladaptive consequences, depending on the cell types and phase of disease. Future areas of investigation include (a) human genetics efforts for the identification of new causative variants in genes not currently associated with TAA, which might offer fresh insights on the molecular defects that initiate aneurysm pathogenesis; (b) investigations of transcriptional and epigenetic changes at the single-cell level for each stage of disease using human samples, mouse models, and iPS-derived cell models, which may reveal the heterogenous and dynamic nature of responses to the primary genetic insult; and (c) the development of therapeutic strategies that target specific cellular subsets at specific phases of aortic remodeling and specifically promote the beneficial effects of compensatory responses while minimizing associated maladaptive consequences.Integrity of the aorta is maintained by a complex and dynamic network of mechanical and biochemical signals. Genetic variants that cause hereditary aneurysm disorders affect the ability of VSMCs to receive and interpret these signals, resulting in phenotypic modulation in these cells and maladaptive remodeling of the ECM, eventually leading to mechanical failure of the vessel wall. Delineation of the molecular functions affected by TAA-associated mutations has identified molecular deficiencies that initiate the pathogenic process. These include defects in components of the elastin-contractile unit and positive effectors of canonical TGF-β signaling. The mechanisms by which these primary deficiencies interact with other pathways regulating VSMC homeostasis to produce both adaptive and maladaptive outcomes is the subject of current investigation. Although current therapeutic strategies focus on reducing mechanical stress, the identification of adverse signaling events that participate in maladaptive responses to the primary genetic insult may offer new therapeutic opportunities. Current experimental evidence suggests that compensatory signaling responses, such as those downstream of AT


UA-pretreated human skin dermal fibroblast (HDF) cells were exposed to UVB radiation to evaluated cell viability, reactive oxygen species (ROS), mitochondrial membrane potential, lipid peroxidation, antioxidant status, DNA damage, proinflammatory response, apoptotic induction, and matrix metalloproteinase (MMP) alteration. The UA pretreatment of HDFs mitigated the UVB irradiation-induced cytotoxicity, ROS generation, and mitochondrial membrane potential alteration and lipid peroxidation, depletion of antioxidant status, DNA damage, and apoptotic induction. UA pretreatment of HDFs also attenuated the UVB-induced expression of inflammatory (TNF-α and NF-κB) and apoptotic  and MMPs (MMP-2 and MMP-9) and enhanced the Bcl-2 protein levels in 20 μM UA treatment, when compared to concentrations. Hence, these results revealed that UA has the potential to mitigate UVB-induced extracellular damage by interfering with the ROS-mediated apoptotic induction and photoaging senescence and thus is a potential therapeutic agent to protect the skin against UVB-irradiation induced photooxidative damage.Ultraviolet radiation is an environmental carcinogenic agent that enhances inflammation and immunological reactions in the exposed human skin cells leading to oxidative photoaging of the epidermal and dermal segment. In the present study, we investigated the protective role of ursolic acid (UA) against ultraviolet B (UVB) radiation- induced photoaging an  Ultraviolet radiation, predominantly UVB (290–320 nm wavelength), can be hazardous to human health by inducing cyclobutane pyrimidine dimer (CPD) formation, cellular damage, inflammation, and tissue photodamage . The UVBThe human skin contains predominantly collagen, elastin, proteoglycans, and fibronectin . CollageUVB irradiation leads to changes in the elastic-collagenous tissues of the skin by breaking down the core collagen fibrils, a major component of the extracellular matrix , 10. Thetriterpenoids represent an interesting class of active polyphenolic phytochemicals that protect the UV light-induced skin damage due to their wide spectrum of actions including anti-inflammatory, antioxidant, antimutagenic, and anticarcinogenic properties as well as modulation of enzyme activities [However, additional efforts are needed to prevent serious pathological consequences of UVB exposure. In particular, daily intake of dietary antioxidants or treatment of the skin with natural products containing antioxidant ingredients may be a useful strategy for the prevention of UVB irradiation-mediated skin damage . Furthertivities .Rosmarinus officinalis, Ocimum basilicum, Mentha spicata, and Veronica serpyllifolia. It is also present in fruits, such as Pyrus malus, Vaccinium oxycoccos, and Prunus domestica, some edible fruits, and food ingredients [in vitro.Ursolic acid UA;  is a lipredients , 19. UA redients –22. In tα, anti-NF-κB (p65), anti-p53, anti-Bax, anti-Bcl-2, anti-caspase-3, anti-MMP-2, anti-MMP-9, anti-β-actin, and goat anti-mouse IgG-HRP polyclonal antibody were also purchased from Sigma-Aldrich, St. Louis, USA. RNA isolation kit, cDNA synthesis kit, PCR primers, and PCR master mix were purchased from Qiagen, USA. Bovine serum albumin (BSA), RIPA buffer, low melting agarose (LMPA), normal melting agarose (NMPA), phosphate buffered saline (PBS) and reduced glutathione (GSH) were purchased from Merck, India. All other chemicals, gradient solvents, and other analytical grades were acquired from S.D Fine Chemical and Himedia, India.Ursolic acid (purity ≥ 98%), thiobarbituric acid (TBA), phenazinemethosulphate (PMS), nitroblue tetrazolium (NBT), 5,5-dithiobis 2-nitrobenzoic acid (DTNB), 3--2,5-diphenyl-2H tetrazolium bromide (MTT), 2′7′-diacetyl dichlorofluorescein (DCFH-DA), rhodamine-123, and nicotinamide adenine dinucleotide (NAD) were supplied by Sigma-Aldrich, St. Louis, USA. The mouse monoclonal antibodies anti-TNF-v/v fetal bovine serum, 1 μg/mL hydrocortisone, 10 ng/mL human epidermal growth factor, 3 ng/mL basic fibroblast growth factor, 10 μg/mL heparin, and 1% penicillin and streptomycin solution. Cell cultures were maintained in a humidified 95% O2 and 5% CO2 incubator at 37°C for 7-14 days until they reached 85% confluence. Then, the cells were subcultured and used in further experiments.HDF cells were purchased from Invitrogen Bioservices , India. MEM/F12 medium, Low Serum Growth Supplement  kit, trypsin/EDTA solution, and trypsin neutralizer solution were obtained from Casecade Biologics, Invitrogen cell culture, India. The primary HDF cells were propagated in MEM/F12 medium supplemented with the LSSG kit containing 2% μM) and kept incubated for 24 and 48 hours. Different concentrations of UA were added with culture media and incubated for the dose-dependent study. In the control group, DMSO (0.05%) alone was used. Similarly, cells were treated with various doses of UVB irradiation (5-100 mJ/cm2) and incubated at 24 hours. The cytotoxicity was measured by MTT assay in both the studies. LD50 values were calculated, and the optimum dose of UA and UVB irradiation was used for further studies.The 3--2,5-diphenyl-2H tetrazolium bromide (MTT) assay is a colorimetric nonradioactive assay for measuring the cell viability by increased metabolization of tetrazolium salt . 24 h fiμM of UA; Group 3: human skin dermal fibroblasts exposed to 40 mJ/cm2 UVB irradiation; Group 4: human skin dermal fibroblasts treated with 10 μM of UA plus 40 mJ/cm2 UVB irradiation; Group 5: human skin dermal fibroblasts treated with 20 μM of UA plus 40 mJ/cm2 UVB irradiation; and Group 6: human skin dermal fibroblasts treated with 40 μM of UA plus 40 mJ/cm2 UVB irradiation.Cultured human dermal fibroblasts were divided into six groups such as Group 1: control (nonirradiated and nontreated fibroblasts); Group 2: nonirradiated fibroblasts treated with 20 μM). Initially, the cell viability by trypan blue dye exclusion was carried out to confirm whether UA itself  caused any cell death. Before exposure to UVB light, the cells were washed thrice with PBS. There was no decrease in cell viability in control (non-UVB-irradiated fibroblasts) cells over the experimental periods.Before thirty minutes prior UVB irradiation, HDF cells in the above groups were pretreated with three doses of UA  was calculated using the following formula: T (min) = J (cm2) × 16.7/(mW/cm2). The output of UVB irradiation dose from the lamp was measured using a radiometer. The total UVB irradiation dose was exposed 0.05 to 0.4 J/cm2 at different times. Control samples were processed identically but were not UVB irradiated.For UVB irradiation, the cell culture medium was removed from the plates and the cells were washed with PBS thrice to ensure that components of the culture medium would not interfere with the irradiation procedure. The culture plates with cells in a thin layer of PBS were covered with a UV permeable membrane to prevent contamination. A battery of TL 20 W/20 fluorescent tubes served as the UVB source . The wavelength range was set at 290-320 nm and peaked at 312 nm and with an intensity of 2.2 mW/cm6 cells/mL were seeded into 96-well plates and pretreated with three different concentrations of UA . After 1 h incubation with/or without UA, the cells were exposed to UVB irradiation. Then, the cells were incubated at 37°C, 5% CO2 and 95% O2, for 24 h. MTT  was added, and the cells were further incubated for 4 h. 200 μL of DMSO was added in each well, and the plates were centrifuged at 1200 rpm for 5 minutes. The optical density of the cell supernatant was measured using a microplate reader at 540 nm.Cultured fibroblasts 1 × 10μM/mL DCFH-DA (dissolved in PBS) in 6-well plates for 15 minutes, followed by three washes with PBS. Fluorescence intensity was determined at 488/525 nm by a spectrofluorometer, and the images were captured by an Olympus BX53 fluorescence microscope.The intracellular ROS level was quantified by using 2′7′-diacetyl dichlorofluorescein diacetate (DCFH-DA) fluorescence probe for directly measuring the redox state of a cell . FibroblΔψm) have been evaluated by measuring rhodamine-123 fluorescence dissipate by a spectrofluorometer and microscopy [μL Rh-123 (10 mmol/L) was added to fibroblasts that were UA pretreated with/or without UVB irradiation in 6-well plates. Cells were incubated for 30 min, washed thrice with PBS, and viewed under a fluorescence microscope using blue filter (450-490 nm). Polarized mitochondria are marked by orange-red fluorescence, and depolarized mitochondria are marked by green fluorescence. The fluorescence intensity was quantified by a spectrofluorometer.Cationic dyes are incorporated electrophoretically into the mitochondrial matrix in response to the electric potential across the inner mitochondrial membrane during oxidative phosphorylation. The alterations of cell membrane potential  using a cell scraper. The cell suspension was collected and sonicated for 15 sec in 40 cycles. The homogenates were centrifuged at 12,000 g for 30 min at 4°C, and the supernatant was stored in separate aliquots at -80°C until biochemical analysis. The level of membrane lipid peroxidation was determined by analyzing TBA-reactive substance according to the protocol of Niehaus and Samuelsons . The pin2O2) and GSH for a specified time period. Then, the GSH content after the reaction was measured. The total GSH content was measured by the method of Ellman [The total superoxide dismutase (SOD) enzyme activity was quantified by the method of Kakkar et al. , based of Ellman . This meDNA damage was estimated by alkaline single-cell gel electrophoresis (comet assay) according to the method of Singh et al. . The expμg/mL) and EB (100 μg/mL) in PBS and incubated for 5 min; then, the excess unbinding dye was removed by washing with PBS. Stained cells were visualized under UV illumination using the 40x objective (BX53 Olympus fluorescence microscope), and the digitized images were captured. The apoptotic cells, with the shrunken, nuclear fragmentation, brightly fluorescent, apoptotic nuclei, were easily detected through their high fluorescence and condensed chromatin; ethidium bromide-positive nuclei were scored, and the percentage of apoptotic cells was calculated.Acridine orange/ethidium bromide (AO/EB) dual staining was performed to detect morphological changes upon apoptotic induction in UA-pretreated and UVB-irradiated cells . The celμL reaction for 1 h at 42°C. The reaction was stopped by heating for 10 min at 70°C. The PCR was performed on the resultant cDNA from each sample with specific primers for human MMP-2, MMP-9, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which severed as an internal control. PCR amplification was performed with a thermocycler . The 25 μL reaction mixtures consist of 2 μL cDNA, 1 μL forward and reverse primer, and 12.5 μL 2x PCR master mix. The annealing temperature and cycle for both MMP-2 and MMP-9 were 55°C and 40 cycles. At the end of amplification, the reaction mixture was heated for 10 minutes at 72°C and then cooled to 4°C. A 10 μL sample of each PCR product was separated by performing gel electrophoresis on 2% agarose containing ethidium bromide. The 2% agarose gel was analyzed under ultraviolet light against the DNA molecular length markers; the signal was detected using the Alpha Innotech imaging system .Total cellular RNA was extracted using Trizol reagent according to the manufacturer's instructions . One microgram of total RNA from each sample was annealed for 5 min at 70°C with 500 ng oligo (dT) and reverse transcribed to cDNA per 20 μg/well) were diluted with 1x lysis buffer, separated by SDS-PAGE, and transferred onto PVDF membranes . The primary antibodies including TNF-α (1 : 1000), NF-κB , p53 (1 : 1000), Bax (1 : 1000), Bcl-2 (1 : 1000), caspase-3 (1 : 1000), MMP-2 (1 : 1000), MMP-9 (1 : 1000), and β-actin (1: 2000) were added to the membranes and incubated at 4°C overnight. The membranes were washed three times for 5 minutes in Tris-buffered solution with Tween20 (TBST) and incubated with HRP-conjugated secondary antibody (1 : 5000) for 1 h at room temperature. Finally, membranes were washed three times for 5 minutes in TBST and ECL solution was added to develop the signals. The images were acquired by Image Station 2000R .Western blotting assay was performed as previously described . Brieflyn = 6) for determination; the significance was established at ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 levels compared to the control group.Statistical analysis was done using SPSS  version 16 software. The values were presented as mean ± standard deviation (M ± SD), and data were analyzed using one-way analysis of variance (ANOVA) and Duncan's Multiple Range Test (DMRT) to correlate the difference among the variables. All the values were expressed as means of six  increased in the “UA and UVB irradiation” (UA 10 μM+UVB 40 mJ/cm2 and UA 20 μM+UVB 40 mJ/cm2) groups compared to UVB-irradiated HDFs that did not receive UA pretreatment , they showed the absence of DCFDA staining, indicating reduced generation of ROS level (P = 0.01) in HDF cells  HDFs emitted green fluorescence suggesting an increased depolarization of Δψm (μM) prior to the UVB irradiation of the HDF cells enhanced membrane polarization significantly (P = 0.01) as showed by red fluorescence  prior to the exposure to UVB 40 mJ/cm2 radiation significantly decreased the levels of TBARS . Among the three different doses tested, 20 μM of UA showed maximum protection against UVB-induced oxidative lipid peroxidation in HDFs  increased both enzymatic  and nonenzymatic (GSH) antioxidant activities compared to the UVB-irradiated group. Among the three concentrations of UA, the 20 μM pretreatment greatly increased the intracellular antioxidant status  pretreatment with UVB radiation significant decreased the levels of DNA damage and increased the levels of head DNA in a concentration-dependent manner.The DNA was not affected in control and HDFs treated with UA 20  Figures , whereas control . The HDFl length  compared Figures . Our quaμM showed only green color suggesting the absence of apoptosis  compared to the control group  in the HDFs  whereas ethidium bromide is absorbed by apoptotic cells (red). The HDF cells treated with control and UA 20  Figures . Apoptotol group . The UA  Figures  whereas s μM+UVB .2) with or without pretreatment of UA . Cells were harvested at 4 h after UVB exposure, and cell lysates were prepared to determine the activation of TNF-α and NF-κB using western blot analysis. The expression of TNF-α and NF-κB was not affected in control cells and in cells treated with UA 20 μM (α and NF-κB was severely upregulated when the cells were exposed to UVB 40 mJ/cm2 (α and NF-κB in HDF cells exposed to UVB irradiation was significantly (P < 0.01) downregulated by pretreatment with UA at concentrations of 20 and 40 μM, compared to the UVB-irradiated group.HDF cells were exposed to UVB  treatment at concentrations of UA 20 and 40 μM significantly downregulated the p53, Bax, and caspase-3 and upregulated Bcl-2 in a dose-dependent manner   .μM UA  exposure increased the activation of both MMPs in HDFs, while UA pretreatment at concentrations of 20 and 40 μM significantly downregulated the intracellular MMP-2 and MMP-9 activation in the cells exposed to UVB .We examined the antiphotoaging properties of UA on the alteration of MMP-2 and MMP-9 induced by UVB irradiation Figures . The inh Figures . UVB  pretreatment decreased the UVB-induced ROS generation and consequently inhibited the mitochondrial membrane potential alteration in HDF cells. Previous studies reported that natural polyphenols such as quercetin, epicatechin, or green tea polyphenols conferred protection to skin cells from UV-induced oxidation damage through free radical scavenging ability [The free radical theory is one of the major mechanisms proposed to elucidate the aging process and has received particular attention in skin aging since the skin is constantly exposed to UVB radiation. Skin cell exposure to UVB radiation leads to release of ROS molecules. These free radicals are the most effective trigger of lipid peroxidation and oxidative DNA damage and primary proinflammatory responses . Corresp ability . Our pre ability .in vitro [In this study, the UVB irradiation-induced lipid peroxidation and antioxidant depletion were used as markers of oxidative damage, and these were significantly inhibited by the treatment with UA Figures . Elevatein vitro . We obsein vitro . Our stuin vitro . Thus, Uin vitro treatment of pomegranate fruit extract (PFE) was also effective at protecting human skin fibroblasts from cell death following UV exposure and this was related to reduced activation of the nuclear factor-kappa B (NF-κB), a downregulation of caspase-3, and an increased G0/G1 phase associated with DNA repair [Further, UVB radiation is directly absorbed by cellular DNA bases of the skin layer leading to the formation of DNA lesions, mainly cyclobutane pyrimidine dimers (CPDs) and pyrimidine-pyrimidone (6-4) photoproducts, resulting in mutagenesis . MoreoveA repair  Further,A repair .α and NF-κB plays key roles in regulating cellular senescence and photoaging [α in different cells [α and NF-κB expression, resulting in reduction of the photoaging process as shown in α-induced MMP activation and further reduces photoaging by suppressing p53 production through caspase-dependent cell signaling cascade activation (The intracellular signaling pathway downstream of TNF-otoaging . UVB irrnt cells , a proinnt cells . UVB radtivation .Studies have shown that naturally occurring polyphenols, specifically those present in fruits and vegetables, beverages, several herbs, and plants, with diverse pharmacological activities, are a promising class of phytochemicals that inhibit extracellular matrix degradation and cellular alteration . UA has Hence, the present study concludes that if the UVB radiation-induced, ROS-mediated DNA lesions are not repaired, these can initiate intracellular signaling and promote photooxidative damage in the skin. We found that UA pretreatment inhibits UVB irradiation-induced cytotoxicity, intracellular ROS generation, changes in mitochondrial membrane potential, lipid peroxidation, antioxidant depletion, DNA damage, and apoptotic induction. Furthermore, UA prevents UVB irradiation-induced apoptotic cell signaling and MMP alteration in HDFs. Besides its antioxidant properties, UA also protects against UVB radiation-induced photoaging process via MMP-mediated structural and extracellular matrix damage.


Breast cancer stem cells (BCSCs) are the minor population of breast cancer (BC) cells that exhibit several phenotypes such as migration, invasion, self-renewal, and chemotherapy as well as radiotherapy resistance. Recently, BCSCs have been more considerable due to their capacity for recurrence of tumors after treatment. Recognition of signaling pathways and molecular mechanisms involved in stemness phenotypes of BCSCs could be effective for discovering novel treatment strategies to target BCSCs. This review introduces BCSC markers, their roles in stemness phenotypes, and the dysregulated signaling pathways involved in BCSCs such as mitogen-activated protein (MAP) kinase, PI3K/Akt/nuclear factor kappa B (NFκB), TGF-β, hedgehog (Hh), Notch, Wnt/β-catenin, and Hippo pathway. In addition, this review presents recently discovered molecular mechanisms implicated in chemotherapy and radiotherapy resistance, migration, metastasis, and angiogenesis of BCSCs. Finally, we reviewed the role of microRNAs (miRNAs) in BCSCs as well as several other therapeutic strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs, which have been more considerable in the last decades. Approximately 25% of cancers in women and 15% of cancer deaths are caused by BC. About 0.8–1% of all BCs are due to male breast carcinoma, although the incidence of BC in men has increased recently. It has been estimated that the prevalence of BC in the world will reach 2.3 million by 2030 . The prevalence of BC is increasing; according to the recent report 2020), 279,100 new cases and 42,690 deaths of BC have been estimated in the United States , epithelial specific antigen (ESA), and aldehyde dehydrogenase (ALDH) activity in different types of BC. Unlike CD24, known as a differentiated BC cells marker, CD44 has been identified as a stemness marker on the surface of BCSCs. CD44 is bonded to hyaluronic acid (HA) and extracellular matrix proteins such as osteopontin (OPN) and matrix metalloprotease (MMP)  and closed  configurations. Closed configuration has a vital role in BC progression and CSC survival through activation of NFκβ, Akt, and focal adhesion kinase (FAK) signaling (BCSCs express variety of specific markers such as CD44se (MMP) . Severalse (MMP) . Howeveres of BC , 47, 48. markers . Frequen4−/CD24+ . ALDH ac markers , 51. ALD markers . To dete markers . A regul markers . The oth markers . A recenignaling .It has been reported that the use of radiation to destroy cancer cells after surgery may convert differentiated cancer cells to CSCs through the expression of CSC markers such as Oct4/Sox2/KLF4. Therefore, in some cancer cases, radiation is not recommended, as it can involve recurrence and metastasis . Hypoxiaε (PKCε) is mediated by hyaluronan and regulates human breast tumor cells and BCSC functions. Activated PKCε increases phosphorylation of Nanog, a stem cell marker. Phosphorylated Nanog is translocated into the nucleus and increases miR-21 expression and decreases tumor suppressor program cell death protein 4 (PDCD4) expression. Along with this process, inhibitors of apoptotic proteins (IAPs) and MD11 are upregulated and then leads to antiapoptosis and chemotherapy resistance in BCSCs , Notch, Wnt/β-catenin, and Hippo signaling have been implicated in stemness maintenance and regulation of self-renewal, metastasis, and therapeutic resistance into CSCs , 56–61. in BCSCs . The othin BCSCs . In addiin BCSCs   and HER2. In this way, activated ERK stimulates MAPK interacting kinase (MNK) signaling, and then, MNK activates XIAP, its downstream protein. XIAP is an apoptotic inhibitor protein that is upregulated in inflammatory and aggressive BC and links NFκβ signaling activity to MAPK signaling . On the Nanog and KIF4 via ZFP36L1 phosphorylation, which has been known as a Nanog and KIF4 mRNA binding protein -dependent manner. Chemotherapy in an HIF1-dependent manner regulates the expression of dual specificity phosphatase (DUSP9) and DUSP16. Reciprocally, increased expression of DUSP9 inhibits ERK and decreased expression of DUSP16, which activates p38 signaling pathway. ERK inhibition leads to increased expression of Nanog through decreased phosphorylation of FoxO3, and p38 activation stabilizes transcripts of stemness genes such as  protein   phenotype through induction of cell apoptosis. The role of PI3K/Akt in BCSCs may be mediated by HER2, which makes tumors more aggressive through unknown mechanism . HER2 dyOn the other hand, inflammation may show a critical role in the relapse of BC through the NFκB–IL-6 pathway, which is involved in BCSC characterization and recurrence of tumor progression. NFκB regulates interleukin-6 (IL-6) and helps in the maintenance of BCSC and conversion of non-CSC to CSC. In addition, some alterations in PI3K/mTOR signaling has been detected in triple negative BC. PI3K/mTOR inhibition through PI3K/mTOR inhibitor or TORC1 inhibitor increases mammosphere formation, a CSC enrichment model, and expression of CSC markers as well as fibroblast growth factor 1 (FGF1) and Notch1 expression in CSCs through increased mitochondrial metabolism . Hence, + to ER− transition is mediated by genetic and epigenetic changes. Proliferation and expansion of ER− CSCs could be driven by ER+ BC cells. The secretion of FGF in ER+ BC is through ER signaling, by induction of EGF receptor, Notch, and Wnt signaling through FGF paracrine manner in ER− CSCs (+ cells mediates binding of Receptor activator of NFκB (RANK) with its ligand, RANKL, as a paracrine signal transduction in PR-negative CSCs  and Has2 antisense (HAS2-AS) transcripts, high mobility group AT-Hook 2 (Hmga2), and EMT inducing transcription factors such as Snail, Snai1, Hmga2, and fibronectin 1 /interleukin-like EMT inducer (ILEI), an oncogenic protein. Interaction of ILEI with LIFR, its receptor, promotes cytokine receptor signaling through STAT3 activation to drive EMT and metastasis . Besidesnectin 1 . In addiof BCSCs .Wnt and Notch signaling pathways are other signaling pathways involved in cancer stem cell maintenance , 61. WntIt has been shown that there is an association between estrogen signaling and Notch signaling in ERα+ BC cells, which is mediated by Delta-like protein 1 (DLL1), Notch ligand. Upregulation of Notch signaling by overexpression of DLL1 promotes proliferation, migration, angiogenesis, and cancer stem cell phenotypes in ERα+ BC cells. This function for DLL1 is specific in ERα+ BC cells. Thus, it suggests that estrogen and estrogen signaling stabilizes DLL1 through inhibition of ubiquitylation and degradation of DLL1 .In BCSCs, activated Notch signaling by jagged1 leads to Akt phosphorylation and IκB kinase α (IKKα), NFκB, and Notch1 activation. Recruitment of IKKα, NFκB, and Notch1 to antiapoptotic gene cIAP-2 promoter increases expression of cIAP-2  Figure . Along w+ samples express high levels of vimentin and Snail. It suggests that the hedgehog signaling pathway and SHH/GLI1 axis are involved in EMT, migration, and invasion of BCSCs  are a class of proteins implicated in inflammatory signaling and tumorigenesis . TLRs arCAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) in BCSCs  such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein (VLDL) may regulate radiotherapy sensitivity. Decreasing cellular cholesterol through treatment with cholesterol-lowering agents such as HDL decreases radiotherapy resistance, whereas VLDL decreases radiotherapy sensitivity .EGFR and HER2, two growth factor receptors that have been overexpressed in BCSCs, have an important role in radiotherapy resistance of BCSCs. EGFR and HER2 inhibitors such as GW572016 may make BCSCs sensitive to radiotherapy as well as inhibit proliferation through inhibition of receptor phosphorylation . The theMetastasis has been described by two potential ability of BCSCs, migration, and invasion. As mentioned above, not only EMT is an important process in chemotherapy and radiotherapy resistance but also it is implicated in metastasis of BCSCs. Snail, a transcriptional repressor, is one of the most important inducer of EMT through repression of E-cadherin expression. In addition, the expression of snail maintains malignancy phenotypes of BCSCs such as metastasis. Therefore, it could be suggested that snail is a potential target to prevent metastasis of BCSCs . Along wExtracellular matrix protein 1 (ECM1) is an important prognosis marker for the invasion of breast cancer as well as migration and drug resistance. In addition, ECM1 controls the expression of genes involved in EMT and BCSC maintenance through induction of Mucin1 (MUC1). The cytoplasmic tail of MUC1 has a physical association with β-catenin that stabilizes and increases posttranslational expression of β-catenin. Therefore, altered expression of β-catenin changes the expression of genes related to BCSC phenotypes such as invasion and EMT progression .CD36, a member of the scavenger receptor family with binding to its receptor, thrombospondin-1 (TSP-1) plays an important role in metastasis of BCSCs through several pathways. CD36 binds to TSP-1 and TGF-β1 and promotes proliferation and migration of BCSCs. On the other hand, it can interact with NFκB and STAT3, which express genes related to carcinogenesis. In addition, CD36 has a main role in BCSC metastasis through uptaking fatty acids and regulating 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) enzyme and β-oxidation of fatty acids . FurtherThe role of CD44 in cancer progression, invasion, metastasis, and angiogenesis of BCSCs is remarkable. There are several ligands such as hyaluronic acid, osteopontin, and matrix metalloprotease that bond to CD44. Binding ligands to CD44 induces cleavage of CD44 at extracellular and transmembrane domain sites by membrane-type 1 matrix metalloprotease (MT1-MMP) and presenilin-1/g secretase, respectively. Then, the intracellular domain fragment of CD44 (CD44-ICD) displaces into the nucleus and activates expression of genes related to migration, invasion, metastasis, and angiogenesis of BCSCs .Cell survival mediated by hypoxic condition after radiotherapy is contributed to the angiogenesis of CSCs . In bothThe role of CD44 has been confirmed in angiogenesis as well as metastasis. CD44 binds to its ligand, hyaluronic acid, as well as VEGF through its binding domains and increases angiogenesis .miRNAs are introduced as one of the non-coding RNAs classes that suppress gene expression through inhibition of translation. Several studies suggest that several miRNAs regulate stemness phenotypes of BCSCs through targeting genes involved in signaling pathways. Substantial shreds of evidence support that resistance of BCSCs to chemotherapy and radiotherapy have been associated with metastasis and relapse of BC after treatment . TherefoViola odorata extract, a herbal medicine, decreases stemness potency and cell viability of BC-cell-derived mammospheres as well as induction of apoptosis , an active component of  pathway .p53, p21, p16, p15, BRCA1, and BRCA2, and reduced expression of ABCG2 through demethylation mechanism  and AD-01 (one of the its derivatives) are novel biological agents that have an effective role in inhibiting tumor growth initiation, angiogenesis, and metastasis of BC cells as well as BCSCs through targeting CD44 pathway and downregulating DLL4, a Notch ligand, and Notch4 , 119. Diechanism .Developing anti-inflammatory drugs and monoclonal antibodies could be useful for preventing proliferation, metastasis, and drug resistance of BCSCs. Aspirin (ASA) prodrug is one of the anti-inflammatory drugs that has shown antineoplastic activity without toxicity through targeting NFκB–IL6 pathway and cyclooxygenase (COX)/prostaglandin in BCSCs , 124. InPreviously, it has been shown that EGFR is a critical player in tumor initiation, metastasis, and drug resistance of BC and BCSCs. Therefore, implication of EGFR monoclonal antibody such as Cetuximab effectively could be suggested as a useful strategy to reduce BCSC population . BevacizRecently, nanoparticles have been an interesting approach for targeting cancer cells especially CSCs. Specific phenotypes of CSCs can be used for designing nanoparticles. Among nanomaterials, mesoporous silica particles have demonstrated efficient drug delivery in cancer therapy. It has been designed based on glycolytic phenotype and Notch activity of CSCs. Uptaking mesoporous silica nanoparticles accompanied with γ-secretase inhibitor by BCSCs targets Notch signaling pathway and eliminates stemness properties of BCSCs.miRNAs are the most attractive topics in BCSCs. They have been implicated in targeting BCSCs through several signaling pathways. +/CD24−, ALDH activity, and ESA or CD326 (EpCAM), CD133, CD166, Lgr5, CD47, and ABCG2 in different types of BCs. BC cells' conversion to BCSCs due to genetic and epigenetic changes leads to deregulation of several signal transduction pathways such as MAP kinase, PI3K/Akt/NFκB, TGF-β, Wnt/β-catenin, Notch, hedgehog, and Hippo signaling. Deregulation of these signaling pathways and other factors leads to several stemness phenotypes such as chemotherapy and radiotherapy resistance, EMT, invasion, migration, and angiogenesis. Additionally, the role of miRNAs in stemness features of BCSCs is remarkable. A variety of miRNAs upregulated in BCSCs are contributed to stemness phenotypes of BCSCs. Recently, implicating novel treatment strategies such as herbal medicine, biological agents, anti-inflammatory drugs, monoclonal antibodies, nanoparticles, and microRNAs that target different signaling pathways and other deregulated genes directly or indirectly could be effective for targeting BCSCs.BCSCs are one of the main reasons for tumor development and recurrence of BC after treatment. These cells have some specific markers: CD44SY and SS: conception, providing the data and design, manuscript writing. FS, MN, AG, and KG: conception and final approval of manuscript.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.


Introduction: Vestibular Evoked Myogenic Potential (VEMP) can be used to test central vestibular pathways from the midbrain to the lumbar spine, according to the muscle tested.Purpose: to compare the spinal cord alteration in individuals with HTLV-1-associated myelopathy (HAM) and with HTLV-1-asymptomatic infection using the VEMP recorded from different muscles.Methods: VEMP was recorded in 90 individuals of whom 30 had HAM, 30 were HTLV-1 asymptomatic carriers, and 30 negative controls. VEMP was recorded in the oculomotor muscle (oVEMP), testing the vestibulo-ocular reflex, and in the cervical muscle (cVEMP) and soleus muscle (sVEMP), testing the vestibulospinal reflex, respectively, in the cervical and in the lumbar spinal level. The type of stimulation was auditory for oVEMP and cVEMP, and galvanic for sVEMP. The compared variables were the latencies of the electrophysiological waves.Results: HTLV-1-asymptomatic group was similar to the controls regarding oVEMP (p = 0.461), but different regarding cVEMP (p < 0.001) and sVEMP (p < 0.001). HAM group has presented the worst latencies and was different from the HTLV-1-asymptomatic group in the VEMP of all the tested muscles (p < 0.001). The concomitant occurrence of VEMP alterations in the three recorded muscles of the same individual was found in 2 (6.7%) asymptomatic carriers and in 20 (66.7%) patients with HAM (p = 0.001). The analysis of VEMP alteration per group and per muscle has showed that, in HTLV-1-asymptomatic group, oVEMP was altered in 3 (10.0%) individuals, cVEMP in 10 (33.3%) and sVEMP in 13 (43.3%). In HAM group, oVEMP was altered in 23 (76.6%) individuals, cVEMP in 27 (90%), and sVEMP in 30 (100%).Conclusion: HTLV-1-neurological damage has followed an ascendant progression beginning at the lumbar spine in the stage of a clinically asymptomatic infection, whereas HAM has affected not only the spine, but also the midbrain. Human lymphotropic T-cell virus type 1 (HTLV-1) is widely disseminated worldwide, and it is estimated that 15 to 20 million people have been infected .Numerous diseases are correlated with HTLV-1 infection: uveitis, Sjogren's syndrome, infectious dermatitis, polymyositis, arthropathies, thyroiditis, polyneuropathies, lymphocytic alveolitis, cutaneous T-cell lymphoma, strongyloidiasis, scabies, leprosy, tuberculosis, and HTLV-1 associated myelopathy (HAM) –6.In HAM, the site of major involvement is the lower thoracic spine, although the entire neuro-axis can also be involved . AlteratPostural instability is a frequent clinical manifestation in HAM . The comVestibular Evoked Myogenic Potential (VEMP) is an electrophysiological test of a tri-synaptic reflex that evaluates the peripheral vestibular system and the central function related to the labyrinth connections. It is considered a test that evaluates the brainstem response , 17. TheIn oVEMP, the activation of the vestibulo-ocular reflex is presumed to follow the vestibular primary afferent, possibly medial longitudinal fasciculus, nucleus, and oculomotor nerves, including the mesencephalic connections . In cVEMTo better characterize the neurological disease associated with HTLV-1 infection, this study aims at comparing VEMP recorded from different muscles in patients with HAM and in HTLV-1 asymptomatic carriers, assessing the CNS at different levels.This research was conducted in accordance with the principles expressed in the Declaration of Helsinki and was approved by the Research Ethics Committee from Universidade Federal de Minas Gerais (COEP UFMG), logged under protocol number CAAE 92928518.3.0000.5149. This protocol number refers to a main project that includes subprojects of which the present study is one of them. All participants provided voluntary written consent and declared that they were aware of the study procedures and their choice to participate.This study was a comparative cross-sectional analysis. The oVEMP, the cVEMP, and the sVEMP were compared among individuals classified as definite HAM, HTLV-1-asymptomatic carriers, and healthy seronegative controls .* Power software 3.1.9.2  to achieve a power of 80% and a significance level of 5% based on the mean and standard deviation of the P13-N23 waves in the cVEMP response of patients with HAM and healthy controls  since 1997, in Belo Horizonte, Brazil. The GIPH evaluates the natural history, clinical manifestations, and epidemiological aspects of HTLV infection .Ninety participants of the GIPH cohort were invited to participate in this study. They consisted of 30 individuals with definite HAM, 30 with HTLV-1-asymptomatic infection, and a control group of 30 individuals not infected by HTLV-1 . The conThe classification of the participants infected by HTLV-1 regarding neurological impairment followed the Expanded Disability Status Scale (EDSS) , 26 and Individuals with a positive serology for the Human Immunodeficiency Virus (HIV), HTLV-2, or any other blood-tested disease were excluded, as well as an undetermined serology for HTLV-1 and a positive Venereal Disease Research Laboratory test. To avoid confusion factors related to the exam, we excluded individuals using metal prosthesis; with neurological diseases, neoplasms, otitis, and tympanic membrane perforation; with a history of cranio-encephalic trauma or otologic surgery, and peripheral vestibular disease; as well as individuals unable to perform cervical rotation and that were unable to remain in an orthostatic position.VEMP can be evoked by either auditory or electrical stimulus  , 21. TheThe oVEMP and cVEMP were performed simultaneously , using two channels. The stimuli were presented through ER 3A insertion phones (of brand Etymotic Research Inc.), with disposable foam ear tips. Tone burst stimuli at an intensity of 120 decibels, a normalized hearing level (dB nHL), were used. In this study, a bandpass filter of 10 to 1,500 Hertz (Hz) was used. To obtain each record, 100 stimuli were presented at a frequency of 500 Hz at a rate of four stimuli per second. The scan window was 50 milliseconds (ms). Each subject underwent at least two stimulations per side in order to verify the replication of the potential. The impedance values, which had to be below 5 kiloohms, were checked before each recording .For oVEMP recordings, the active electrode  on channel 1 was placed ~1 centimeter (cm) below the lower eyelid, and the reference electrode was placed distant approximately 1 cm from the active electrode. The ground electrode was placed on the forehead (Fpz). For cVEMP recording, the active electrode on channel 2 was placed on the opposite side of channel 1, on the anterior border of the sternocleidomastoid muscle in its upper third, while the reference electrode was placed in the sternal notch region .dx.doi.org/10.17504/protocols.io.zmzf476.The participants were instructed to sit on the chair and keep their heads rotated to the opposite side of the stimulated ear in order to contract the sternocleidomastoid muscle. We compared reflexes of approximately similar size, where the cVEMP asymmetry between sides was <34%. At the same time, the participant was instructed to look at a stationary target located on the wall in front of him/her and then immediately at a fixed point located above the target, which formed a vertical viewing angle of approximately 30° above the horizontal plane. The oVEMP and cVEMP protocols are available at The oVEMP trace is a biphasic wave. The two phases are characterized by a negative peak with an average latency of 10 milliseconds (ms) (N10), followed by a positive peak with an average latency of 15 ms (P15), which is known as N10–P15. The cVEMP trace consists also of a wave with two phases. The first peak is positive with an average latency of 13 ms (P13), followed by a negative peak with an average latency of 23 ms (N23), which it known as P13–N23 .Galvanic stimulation has been considered a tool to activate the vestibular system inducing both ocular and postural movements . The stiIn the present study, the galvanic vestibular stimulation (GVS) was characterized as a direct, monophasic, and rectangular current with an intensity of 2 mA and duration of 400 ms . The galvanic stimulus was offered at randomized intervals of 4–5 s and responses to 120 stimulations were measured. The bipolar current was applied on the mastoid processes using self-adhesive, circular surface electrodes .For transmastoid stimulation, the two current polarity settings were cathode left and anode right (CLAR) and cathode right and anode left (CRAL). The stimulation polarity was controlled by a computer. For each test, four sets of 30 stimuli were applied and distributed, resulting in 30 responses recorded from the left lower limb (15 CLAR and 15 CRAL stimuli) and 30 responses recorded from the right lower limb (15 CLAR and 15 CRAL stimuli). This procedure was repeated for each leg to ensure data replication.During the acquisitions, the subjects stood barefoot on a flat surface with their eyes closed, feet close together, and bodies leaning forward to contract the soleus muscle. To induce a stronger response, subjects were instructed to turn their heads approximately 90° to the side contralateral to the leg undergoing EMG response recording.The EMG response triggered by GVS was measured using self-adhesive electrodes . A pair of recording electrodes were placed bilaterally 5 cm below the popliteal fossa, which coincides with the position over the soleus muscles. Each pair was placed vertically distant 5 cm from each other. This distance can vary from 3 to 10 cm, according to the best recorded wave , 22. A rdx.doi.org/10.17504/protocols.io.nxbdfin.The EMG signals were measured, corrected, with a bandpass filter of 10 to 1,000 Hz, and digitized at a sampling frequency of 5,000 Hz. Data were recorded during 500 ms, starting 100 ms before GVS. The EMG responses to 15 consecutive stimuli associated with each polarity configuration  were averaged to produce the final traces. The sVEMP protocol is available at The EMG tracings were analyzed for the time of onset, in milliseconds, of the short latency response (SL), and the mean latency response (ML). Following the superimposition of traces with inverted polarity , the point where the traces diverged from the EMG baseline, which marked the onsets of SL and ML, could be visualized and measured by a cursor. The first trace divergence, which occurred at approximately 50 ms, marked the onset of the SL response. Following this, the traces returned to baseline and then diverged again. The second trace divergence, which occurred at approximately 100 ms, marked the onset of the ML response. The end of this response was defined as the point at which traces return to the baseline. To obtain a single value of the components of SL and ML, it was considered the worst response between the right and left sides .The parameters considered in the VEMP analysis are the latency and amplitude of the waves. However, the amplitude can vary according to age, muscular strength , and cocp ≤ 0.05).VEMP results were classified as normal and altered. The results with latency prolongation or no response were considered as altered. The VEMP latency prolongation was defined as a delay of 2.5 standard deviation (SD) when compared to the values of a normal control population, according to the American Society of Encephalography and Evoked Potentials' criteria for evoked potentials . The latThe receiver operating characteristic curve (ROC) was performed with the objective of identifying the sensitivity and specificity for each latency cutoff of oVEMP, cVEMP, and sVEMP, considering the neurological examination as the gold standard.p = 0.549) and age (p = 0.069).The general characteristics of the studied sample can be seen in p = 0.039; SL, p < 0.001), while the latency of the second components have remained similar between groups . The comparison between HTLV-1-asymptomatics and HAM has showed a delay in the first components for both groups , and the second components have not changed comparing to the controls. With regard to oVEMP, the prolonged latency was observed only in the HAM group.The comparison of oVEMP, cVEMP, and sVEMP among the groups regarding the delay of latencies of each peak of the biphasic waves is shown in p = 0.983), but it was different from the HAM group (p = 0.001).In To evaluate the use of VEMP tests in clinical practice, VEMP latencies were then compared to the neurological examination as the gold standard. We have constructed ROC curves to evaluate latency prolongation of N10-P15 for oVEMP, P13-N23 for cVEMP, and SL-ML for sVEMP . The betThe HAM diagnosis is based on a set of clinical criteria established by an international consortium in 2006 . HoweverMore recently, a longitudinal study based on eight years of follow-up has confirmed that the asymptomatic carrier has presented elevated morbidity related to HTLV-1, such as, autonomic changes, including alteration in bowel habits, urinary incontinence or urgency, erectile dysfunction, as well as motor disabilities . Thus, tRegarding oVEMP, it is assumed that the neural connections involved in EMG response are mesencephalic , 26, 41.Regarding cVEMP, as expected, EMG responses were worse in the HAM group when compared to the HTLV-1-asymptomatic group. These data confirm that HAM compromises the cervical spine, although the alterations have been worse in the thoracolumbar region , 43. TheThe analysis of sVEMP shows that, in HAM group, all the participants have had altered responses, with a higher frequency of absence of EMG waves . In the In sVEMP, the first component of the wave (SL) is assumed to result from a synchronous discharge of a common supraspinal structure, which means, the reticulospinal and the vestibulospinal tract, while the second component (ML) represents the polysynaptic synchrony , 22. In The In short, we have found that the neurological damage related to HTLV-1 follows an ascending progression since the subclinical stage. The image of the spinal atrophy in the advanced HAM confirms the ascendant damage as it shows that the spinal cord is thinner at the thoracolumbar region than at the cervical one . ConsideSchistosoma mansoni infection and represents 6% of non-traumatic transverse myelopathies in endemic areas , logged under protocol number CAAE 92928518.3.0000.5149. The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.TS: conceptualization, data curation, formal analysis, funding acquisition, methodology, project administration, resources, validation, visualization, writing-original draft, writing-review and editing. MR and LM: conceptualization, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, visualization, writing-review and editing. LL and JC: validation, visualization, writing-review and editing. RS: investigation and validation. DU: data curation, formal analysis, funding acquisition, methodology, project administration, resources, supervision, validation, visualization, writing-original draft, writing-review and editing.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.


Single-crystal X-ray analyses revealed two modes of Hhp ligation in these complexes: a monodentate coordination of carbonyl oxygen in all of them and an additional µ2-oxygen bridging coordination in the dinuclear complex 4. Three bridging molecules of Hhp span two nickel(II) ions in 4 with a 2.9802 (5) Å separation of the metal ions. Complex 3 is a chlorido-bridged nickel dimer with a planar Ni2(µ-Cl)2 framework. Hydrogen bonds and parallel stacking arrangements of the Hhp molecules govern the connectivity patterns in the crystals, resulting in 1D structures in 1 and 5 or 2D in 3. A single manganese compound [MnCl2(Hhp)4] (5), isostructural to 1, was isolated under the similar conditions. This is in contrast to four nickel(II) chloride complexes with Hhp. Thermal analyses proved the stability of complexes 1 and 3 in argon up to 145 °C and 100 °C, respectively. The decomposition of 1 and 3 yielded nickel in argon and nickel(II) oxide in air at 800 °C.Reactions of NiCl The diverse coordination modes of these ligands, the stabilization of complexes by hydrogen bonds and other weak intermolecular interactions result in various structural motifs for a plethora of compounds with interesting magnetic, catalytic, and biochemical properties. In contrast to the three binding modes of the neutral Hhp, nine ligation possibilities were described in the literature for hp− and derivatives, providing a huge structural diversity among the complexes of these ligands (Pyridin-2-one (Hhp), its anionic form  ions [− derivatives on the M–M bond lengths  in [ML4] ) was widely studied  and [Cu2(µ-HCO2)2(µ-hp)2(Hhp)2] [The similar nature of pyridonates and carboxylates was clearly demonstrated in dinuclear copper compounds displaying distinctive paddle-wheel structures. The hp2(Hhp)2] .− anion as a link between hetero metal ions in the 2.11 fashion was found in [MoW(mhp)4]·CH2Cl2 at the end of the 1970s 2+ were proposed in the perchlorates and tetrafluoroborates, the nitrates were either solely molecular [M(Hhp)4(NO3)2]  or molecular and ionic . Some Hhp complexes of manganese(II), cobalt(II), nickel(II), or copper(II) chloride were also prepared many years ago. The formula MCl2·Hhp was suggested for all four metals, but just two of them enabled the isolation of the additional adducts CoCl2·3Hhp and CuCl2·2Hhp [6]n+ (M = Fe(III), Co(II), Cu(II) [4(H2O)2]2+ [6](ClO4)2 [2(Hhp)3] and two dinuclear, [CuCl(µ2-Cl)(Hhp)]2, [CuCl2(Hhp)(µ2-Hhp)]2 [4X2]  were reported for both metal ions 2+  was late2-Hhp)]2 . The crytal ions ,38.d metals. In this way, they fulfill the coordination spheres of vanadium in acetylacetonate  and nucleic acid bases or their analogues   [4(Hhp)2(µ-Hhp)2(µ2-tfa)4] [µ-oxygen bridging coordination of Hhp to silver(I) ions creates chains in an ionic compound [Ag(Hhp)2(µ-Hhp)2]n(PF6)n  . In cont]n(PF6)n . Ag(I) i]n(PF6)n ,58. The ]n(PF6)n .We have continued our study of the first-row transition-metal chloride complexes with hydroxypyridines . The coo2.6H2O and Hhp strongly depend on the solvent and the molar ratio of the reactants (2(Hhp)4] (1) is achieved in methanol, [NiCl2(Hhp)(H2O)2]2·2Hhp (3) in tetrahydrofuran, dichloromethane, or acetone, and [Ni2Cl4(Hhp)5]·2MeCN (4) in acetonitrile. The formations of powder products 1 and 3 were proven by X-ray powder-diffraction measurements that correspond well to X-ray powder-diffraction patterns calculated from crystal structures. The formation of powder product 4 was confirmed by an IR spectrum. Powder product 1 was formed when a solution obtained in a reaction of NiCl2·6H2O and Hhp (molar ratio 1:4) in methanol was dried in vacuo. Reactions of NiCl2·6H2O and Hhp in tetrahydrofuran or acetonitrile resulted in suspensions, which were first left to settle and then filtrated. The precipitate obtained in acetonitrile was dried in vacuo yielding complex 4 and the precipitate in tetrahydrofuran dried in a desiccator, resulting in complex 3. Crystallization of powder product 1 or 3 from acetonitrile yields crystals 3 in the presence of moisture or crystals 4 in a closed water-free system. Crystals [Ni(Hhp)6]Cl2 (2) were grown from a solution of powder product 4 in acetonitrile in an aqua-free environment. The reaction time and the molar ratio of the reactants in acetonitrile control the isolation of the crystals 1 or 4 from filtrates in closed water-free systems 4] (5) was obtained only in a two-step reaction. In the first step the reaction of MnCl2·2H2O and chlorotrimethylsilane in THF resulted in [MnCl2(THF)x] as confirmed by an IR spectrum. Chlorotrimethylsilane was used to remove all the water in the coordination sphere of manganese(II) ions similar to the syntheses of manganese(II) complexes with diglyme (di(2-methoxyethyl) ether) or H2dhp (3-hydroxypyridin-2-one) 2 , only two Hhp molecules link the copper ions   (Ni–O 2.103(2) Å, Mn–O 2.204(3) Å, Ni–Cl 2.361(1) Å, Mn–Cl 2.506(1) Å) [The metal-to-donor atom distances are comparable in complexes on radii . In compH2dhp)4] ; a short06(1) Å) .2 (2.047(2) Å) than in the molecular 1 (2.057(4) Å). Relatively uniform M–O distances, as in 2, were found for the homoleptic ions [Co(Hhp)6]2+ (from 2.0695(15) Å to 2.1014(14) Å) [2dhp)6]2+ was not detected in a series of nickel chloride complexes with 3-hydroxypyridin-2-one , with L in the formula standing for Hhp, Hchp, or Hmhp [4X2] complexes  is the coordination of Hmhp subscribed to the hydroxyl oxygen atom due to the positions of the hydrogen atoms found from the differences in the Fourier synthesis. The reported ligation seems uncertain, because shorter C–O distances (1.264(3)–1.279(3) Å) also indicate double bonds in these [M(Hmhp)4(SCN)2] complexes and a coordination of carbonyl instead of hydroxyl oxygen  2+  [2(Hchp)4] (18.43°) [4(H2O)2](CLO4)2 compounds and resulted in different characteristics for these polymorphs  ,38,64,65 11.88°)  or Hchp 2(Hhp)4] molecules in 1 and 5 are linked by intermolecular hydrogen bonds into chains parallel to the a axis (R22(12) 2+—not only two as in 2—are engaged as donors of linear (2-center) intramolecular hydrogen bonds [6](ClO4)2] [6]2+ can be explained by the orientations of the aromatic ligands  [In complex en bonds . In cont(ClO4)2] . Two N–H ligands  shown by, 39.6°)  and more2 connect only a cation and two adjacent anions  and a hetero aromatic ring (soft base) plays the role of a CH-acceptor in a T-shaped C–H⋯ π interaction. The relevant parameters are H24⋯ centroid distance 2.89 Å, C24–H24⋯ Cg angle 152° and C24⋯ centroid distance 3.7334 Å [6]2+ and CoCl42− into chains was found in the cobalt complex [The intermolecular hydrogen bonds in t anions . The fou3.7334 Å . An alik3 ]2 (2.3443(5) Å, 2.4447(5) Å) [3 is the one to the carbonyl oxygen and a significant variation of the two Ni–O(water) distances is worth mentioning. The average Ni–Cl distance in 3 (2.403(2) Å) is a slightly longer and the Ni–O distance (2.081(4) Å) is shorter than the corresponding distances in [NiCl2(diglyme)]2 (2.378(2) Å), 2.104(4) Å) 2 [3 is similar to the nickel carbacylamidophosphate (3.419 Å) [2(diglyme)]2 (3.522 Å) 2 . The sep3.419 Å)  and a bi3.522 Å)  or in a n sphere . Shorterrization ,72,73. Br 3.65 Å ,75,76. Tramework ,71,72,743 displays intermolecular hydrogen bonds generating layers parallel to the ac plane (2(Hhp)(H2O)2]2 forms altogether twelve hydrogen bonds with six closer Hhp solvate molecules and two neighboring complex molecules. Complex molecules participate in building chains parallel to the a axis through the O2–H2A⋯ Cl1 hydrogen bonds (graph-set motif C22(10)[R22(8)]). Complex and solvate molecules are bound via O1–H1A⋯ O20 and N10–H10⋯ O20 (graph-set motif C42(18)[R42(16)]) into chains parallel to the c axis . T. T3  was chosen over Cc (no. 9) due to the better results obtained in the refinement. This selection imposes a two-fold rotation axis through the bridging O3=C31 carbonyl group. As a consequence, the asymmetric unit includes half of a complex molecule. According to the symmetry requirements, only half of the atom positions in the bridging Hhp ligated via O3 are independently determined; therefore, at one site a 50% occupancy by N3 and 50% by C32 was proposed.The two nickel ions in olecules . The two4 than in 1 or 3. A shorter and two longer Ni–O bonds in 4 are to the terminal and bridging ligands, respectively.A comparison reveals shorter Ni–Cl bond lengths in µ2-oxygen ligation of Hhp is well documented in the dinuclear copper(II) halides or acetate , a symmetrical one (V–O 2.0882(2) Å, 2.104(2)Å) and an unsymmetrical one (V–O 2.043(2) Å, 2.345(2) Å) [3(chp)6(EtOH)6] chp− anions 2+ or by four Hhp molecules and two chloride ions which are either in the trans, in [MCl2(Hhp)4] (1 and 5), or in the cis positions in [Ni2Cl4(Hhp)5]·2MeCN (4). Only one Hhp molecule together with three chloride ions in the fac and two water molecules in the cis positions render a coordination sphere of nickel in 3. Solvate Hhp molecules in 3 are due to multiple hydrogen bonds also firmly held in the structure, as shown by thermal analyses. The structural chemistry of nickel(II) and manganese(II) chloride complexes with pyridin-2-one (Hhp) is presented. The isolation of a single manganese and four nickel(II) complexes with Hhp under various conditions confirms the similar structural diversity of the nickel and copper complexes . Hhp is 6]2+ as nickel in 2 2 due to a trigonal bipyramidal environment of copper [4 is, to the best of our knowledge, the only example of a dinuclear nickel complex bridged by three neutral O-donor ligands.Copper(II) displays similar homoleptic complex ions [Cu(Hhp)kel in 2 ,54, but kel in 2 . Nickel(f copper . Complex3d metals are underway to gain a better understanding of the relationship of metal ions and the structural variety of these complexes.To summarize, the four isolated octahedral nickel complexes with Hhp prove that various coordination numbers of metal ions as observed in similar copper compounds are not a prerequisite for structural diversity. Syntheses and characterizations of various Hhp complexes with zinc or some early


Abnormal activation of wnt/β-catenin signaling and renin-angiotensin system is known to play a vital role in the development and progression of CKD. We hypothesized that abnormal expression of central wnt/β-catenin signaling and renin-angiotensin system  was tightly involved in CKD.We established sham-operated and 5/6 nephrectomized (5/6 NX) rat model and blocked the central wnt signaling by intracerebroventricular injection of adeno-associated virus vector which can overexpress target gene DKK1. The central and renal expression level of wnt/β-catenin signaling and RAS and renal injury were assessed.The expression levels of the main wnt/β-catenin signaling components in both brain and kidney of 5/6NX rats, such as wnt3a and active-β-catenin, were elevated obviously and the up-regulation were inhibited by central blockade of the wnt signaling. Furthermore, the expression of the major components of RAS in both brain and kidney in 5/6NX rats, such as angiotensinogen (AGT), angiotensin converting enzyme (ACE-1), angiotensin II AT1-receptor (AT1R), was significantly up-regulated and the up-regulated expression was inhibited by central blockade of the wnt singling. Notably, central blockade of the wnt signaling improved renal function as indicated by decreased serum creatinine and 24 h urinary protein, and attenuated interstitial fibrosis as indicated by reduced Sirius red staining and expression of Fibronectin, Collagen-I and α-SMA.These data suggest that the central WNT-RAS signaling plays a significant role in the development and progression of CKD. Wnt/β-catenin signaling plays an essential role in embryonic development, tissue homeostasis as well as organic injury and repairment –3. Its aIt is also well acknowledged that activation of intrarenal renin–angiotensin system (RAS) plays a pivotal role in the pathogenesis of chronic kidney disease (CKD), and the antagonists of RAS, such as angiotensin converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs), are widely used in the treatment of CKD , 16. TheIn recent years, Reno-Cerebral communication mechanism has been wildly studied and well established. The related mechanisms include the following: (1) Sympathetic afferent and efferent nerves serve as a bridge to connect kidney and brain, and (2) Signaling molecules, such as the components of RAS, directly bind to receptors distributed in the nucleus which lacks blood–brain barrier and result in cascade reactions. It has been well recognized that the hypothalamic paraventricular nucleus (PVN) is a vital integrative nucleus that expresses RAS and can markedly influence blood pressure and sympathetic nerve activity , 19. TheThus, we hypothesized that abnormal activation of cerebral WNT-RAS signaling contributes to the development and progression of CKD. To test our hypothesis, 5/6 nephrectomy (5/6 NX) rat model was established and the central wnt signaling was blocked by ICV AAV-DKK1.Five-week-old male Sprague–Dawley (SD) rats purchased from the Nanfang Hospital Animal Experiment Centre were maintained in a pathogen-free facility under temperature- and light-controlled conditions  and 55 ± 5% humidity for at least 1 week prior to the experiments. All animal experiments were approved by the Animals Experiment Ethics Committee of Southern Medical University, Guangzhou, China.10 vg. The process of intracerebroventricular injection was performed as we previously described [Five-sixths nephrectomy and sham operation were performed at 6 weeks of age as previously described . In brieescribed .Paraffin-embedded tissue samples were prepared as described . The braParaffin-embedded Sections (2 μm) of kidney were stained with periodic acid-Schiff (PAS), and Sirius red by routine protocol . ImmunohWestern blot was performed as previously described  using prSystolic blood pressure was monitored by the tail-cuff method. Serum creatinine concentration was measured with an automated chemistry analyzer . Twenty-four hour urine was collected by metabolic cage and 24 h urinary protein was measured using a Coomassie brilliant blue assay .All statistical data were expressed by mean ± SEM. The software of IBM SPSS statistics 23 was implied to analyze the data. Data comparison between groups was performed by one-way ANOVA and Student–Newman–Keuls (SNK) test. P < 0.05 was considered statistically significant.To investigate the function of central WNT-RAS signaling in development and progression of renal disease, this study targeted to block the wnt signaling by AAV-DKK1 which can over-express DKK1 gene in PVN in CKD rats. Double-staining immunofluorescence showed that the marker gene green fluorescent protein (GFP) only expressed in ICV AAV-DKK1 group, and DKK1 concentration is dramatically up-regulated compared to the control (ICV vector) group in PVN (Fig. Wnt/β-catenin signaling plays vital roles in central nervous system, such as development of central nervous system in embryonic period, regulation of nervous system disease and peripheral organ disease. To study the function of wnt/β-catenin signaling in PVN on renal disease, the expression levels of wnt3a and active-β-catenin were assessed by immunohistochemistry and western blot. The results showed that the key molecules of wnt/β-catenin signaling wnt3a and active-β-catenin significantly up-regulated in ICV vector+5/6NX group relative to sham group, but the up-regulation was inhibited in ICV DKK1+5/6NX group Fig. . These rImmunohistochemistry and western blot was performed to assess the expression levels of RAS components including AGT, ACE-1, and AT1. The results showed that AGT, ACE-1, and AT1 significantly up-regulated in PVN in ICV vector+5/6NX group compared with sham group, but the up-regulated expression was inhibited in ICV DKK1+5/6NX group Fig. . It is kThe 5/6NX model was successfully established and showed a progressive rise of systolic blood pressure, serum creatinine, 24 h urinary protein, and terrible loss of weight (Fig. Expression of intrarenal wnt3a and active-β-catenin, measured by both immunohistochemistry and western blot, was up-regulated significantly after 5/6NX operation, indicating activation of intrarenal wnt/β-catenin signaling in CKD (Fig. Our previous study has revealed that salt-induced strong co-activation and interlinkage of cerebral and intrarenal RAS contributes to renal fibrosis and proteinuria in CKD . Here, tThe pathologic changes of kidney tissue in 5/6NX rats were observed by PAS and Sirius red staining. The glomerulosclerosis index was significantly higher after 5/6NX. Central blockade of the wnt signaling by ICV AAV-DKK1 significantly improved glomerulosclerosis in 5/6NX rats detected by PAS Fig. a, c. In Abnormal activation of wnt/β-catenin signaling and RAS is known to play a vital role in the development and progression of CKD. It has been well recognized that the PVN is a vital integrative nucleus that expresses RAS and can markedly influence blood pressure and sympathetic nerve activity , 19. In The functions of central nervous system on renal disease have been reported, and the pathway of signal communication between central nervous system and kidney has been demonstrated , 19. TheWe have previously reported that the RAS signaling in PVN plays a significant influence to renal fibrosis , 19. In Interestingly, blockade of central wnt signaling by ICV AAV-DKK1 dramatically attenuated the renal pathological changes in 5/6NX rats, including glomerulosclerosis and interstitial fibrosis detected by PAS and Sirius red staining. Simultaneously, the expression of FN, collagen-I and α-SMA, the major ingredients of extracellular matrix, was evaluated by western blot, and the results showed that over-expression of these proteins in CKD were obviously alleviated by ICV AAV-DKK1. These data clearly showed that the development and progression of renal fibrosis were regulated by central WNT-RAS signaling. This is the first report illustrating that central WNT-RAS signaling makes a vital difference to renal fibrosis.Renal fibrosis is a common pathway of virtually all progressive kidney diseases in CKD patients and the major factor influencing prognosis , 25. The