Improved hygiene leading to reduced exposure to microorganisms has been implicated as one possible cause for the recent “epidemic” of chronic inflammatory diseases (CIDs) in industrialized countries. That is the essence of the hygiene hypothesis that argues that rising incidence of CIDs may be, at least in part, the result of lifestyle and environmental changes that have made us too “clean” for our own good, so causing changes in our microbiota. Apart from genetic makeup and exposure to environmental triggers, inappropriate increase in intestinal permeability (which may be influenced by the composition of the gut microbiota), a “hyper-belligerent” immune system responsible for the tolerance–immune response balance, and the composition of gut microbiome and its epigenetic influence on the host genomic expression have been identified as three additional elements in causing CIDs. During the past decade, a growing number of publications have focused on human genetics, the gut microbiome, and proteomics, suggesting that loss of mucosal barrier function, particularly in the gastrointestinal tract, may substantially affect antigen trafficking, ultimately influencing the close bidirectional interaction between gut microbiome and our immune system. This cross-talk is highly influential in shaping the host gut immune system function and ultimately shifting genetic predisposition to clinical outcome. This observation led to a re-visitation of the possible causes of CIDs epidemics, suggesting a key pathogenic role of gut permeability. Pre-clinical and clinical studies have shown that the zonulin family, a group of proteins modulating gut permeability, is implicated in a variety of CIDs, including autoimmune, infective, metabolic, and tumoral diseases. These data offer novel therapeutic targets for a variety of CIDs in which the zonulin pathway is implicated in their pathogenesis. This generated the hygiene hypothesis supporting the notion that we had made ourselves too clean for our own good and that people embracing a Western lifestyle would slowly die of CIDs instead of rapidly succumbing to infectious diseases as still is happening in developing countries.Twenty-five hundred years ago, when Hippocrates stated that “All disease begins in the gut”, he had an incredible intuition that only recently has been fully appreciated because of new insights into the pathogenesis of many chronic inflammatory diseases (CIDs) afflicting humankind. Until 30 years ago, when the Human Genome Project was still in its planning stage, the general hypothesis was that genetic predisposition and exposure to an environmental trigger were both necessary and sufficient to develop CIDs, including infectious, allergic, neuroinflammatory/neurodegenerative, autoimmune diseases, and cancer. However, the epidemiological observation showing a major surge of CIDs during the past four decades in the Western hemisphere coincident with the declining rate of infectious diseases was at odds with the gene/environment paradigm2) is not apparently visible, it plays a pivotal role through its dynamic interactions with a variety of factors coming from our surrounding environment, including microorganisms, nutrients, pollutants, and other materials. Intestinal permeability, together with luminal antigen (Ag) sampling by enterocytes via the transcellular pathway and dendritic cells, regulates molecular trafficking between the intestinal lumen and the submucosa, leading to either tolerance or immune response to non-self Ag5  as the first component of the TJ complex9 now being comprised of more than 150 proteins, including occludin10, claudins11, junctional adhesion molecules (JAMs)12, tricellulin13, and angulins14. However, despite major progress in our knowledge on the composition and function of the intercellular TJ, the mechanisms by which they are regulated are still incompletely understood. One of the major breakthroughs in understanding the role of gut permeability in health and disease has been the discovery of zonulin, the only physiologic intestinal permeability modulator described so far16. Therefore, this article will focus mainly on the body of literature published on zonulin as a biomarker of gut permeability to outline the pathogenic role of a leaky gut in a variety of CIDs. However, it should be pointed out that zonulin is not involved in all CIDs and that not all CIDs have been linked to increased gut permeability.What we learned when the Human Genome Project was completed is that we are genetically much more rudimentary than we had previously thought. The premise of “one gene, one protein, one disease” cannot explain the complexity of the balance between health and disease and, most definitively, the CIDs epidemics. Twenty-three thousand genes are insufficient to explain all the permutations of human pathophysiology, including if and when and why we develop diseases. Rather, it is the interplay between us as individuals and the environment in which we live that dictates our clinical destiny. This interplay is physically and mechanistically regulated by biological interfaces that divide our body from the surrounding environment. At about 8 to 9 meters in length, the human intestine provides the largest interface between our body and the outside world. Tightly packed single layers of epithelial cells cover the external surfaces of our intestinal mucosa and negotiate the interaction with the surrounding environment. Although this enormous mucosal interface , the precursor of HP2, was identified almost two decades ago21. Haptoglobins evolved from a complement-associated protein  that lost its protease function because of mutations in the catalytic domain to then acquire new functions, including the capability to modulate intercellular TJs18. The frequent zonulin polymorphisms secondary to high mutation rate during evolution led to a family of structurally and functionally related zonulins, including pre-HP2 and properdin, another member of the MASP family22.Zonulin is composed of a family of related proteins24. Zonulin secretion has been shown to be MyD88-dependent25 and is followed by an increase in gut permeability secondary to the disassembly of the protein ZO-1 from the tight junctional complex26. Gliadin triggers zonulin release through the CXCR3 receptor activated by its engagement to MyD88 with a subsequent increase in gut permeability27, suggesting that gluten is misinterpreted by the zonulin pathway as a potential harmful component of a microorganism. Taken together, these data suggest that the activation of the zonulin pathway may represent a defensive mechanism that “flushes out” microorganisms, contributing to the innate immune response of the host against changes in microbiome ecosystem, specifically bacterial colonization of the small intestine  or changes in its composition (dysbiosis) or both. These findings are in line with the growing evidence on the role of changes in gut microbiome composition and function in causing functional changes in gut permeability, with subsequent increased Ag trafficking and break of tolerance leading to CID in genetically susceptible individuals  and gluten, the protein causing celiac disease (CD), have been identified as the two most powerful triggersviduals .28. Similarly, mice lacking JAM-A, a key TJ structural component, or the muscle myosin IIA heavy chain (NM IIA) showed increased intestinal permeability but only low-grade colonic inflammation and normal epithelial architecture30. Finally, zonulin transgenic mice constitutively producing high levels of zonulin showed increased gut permeability but not pathologic phenotype31. Interestingly, zonulin transgenic, JAM-A−/−, and NM IIA−/− mice all showed increased susceptibility to chemical-induced colitis31. Together, these data suggest that gut permeability may contribute to the development of several CIDs provided that additional genetic components regulating immune response and an imbalanced microbiome are coexistent. Indeed, there is growing evidence that the additional and mutually influenced elements of the triad of gut permeability, immune system, and gut microbiome—together with genetic predisposition and exposure to environmental triggers—make the “perfect storm” for CIDs development.Modulation of intestinal permeability, including the activation of the zonulin pathway, is part of the physiological machinery to maintain mucosal homeostasis and therefore does not always translate in clinical pathological outcomes. Indeed, studies on transgenic mice with constitutive activation of myosin light-chain kinase (MLCK), an intracellular mediator of TJ disassembly, showed increased intestinal permeability without signs of overt disease32. Using different approaches, both we22 and others32 identified complement C3 as the top match protein identified by the antibodies used for the ELISA. However, complement C3 is most likely an unspecific product overshadowing the real targets. Indeed, the respective ELISA kits did not detect any complement C proteins obtained from different suppliers when tested under native and denatured conditions, as well as when C3 was spiked in serum22. Also, the same kit did not detect recombinant zonulin, mature HP1, or mature HP222. Given the additional mass spectrometric hits reported32, a few proteins stand out, although, without further validation, these data need to be interpreted with caution, since only more abundant proteins may be identified by mass spectrometric analysis, while the protein(s) of interest belonging to the zonulin family may be in low abundance in serum samples and therefore not identifiable with this approach. Keeping this in mind and the fact that the protein(s) of interest should be in the roughly 50-kDa range, combined Western blot analysis and ELISA test confirmed that the polyclonal antibodies raised against the zonulin-derived synthetic peptide AT1001 and used in the Immundiagnostik kit  detect properdin among other proteins22. However, when purified proteins/peptides, including the AT1001 peptide used to raise the polyclonal antibodies , were tested by ELISA, they were highly underestimated by the test. One possible explanation for these results is that zonulin as both pre-HP2 and properdin is not the main target detected by the ELISA; however, the fact that even AT1001 was underestimated seems to suggest that this hypothesis cannot entirely explain these results. Alternatively, it is possible that tertiary and quaternary (multimers) structure arrangements present in sera samples but not in recombinant proteins are necessary in order to properly detect any zonulin member by this ELISA. Given these results, it is likely that the commercially available ELISAs detect one or more members of the zonulin family that have not been discovered yet.Several groups, including ours, have been questioning what exactly the commercially available zonulin enzyme-linked immunosorbent assay (ELISA) kits measure33.With the appreciation that gut microbiome composition/function can affect intestinal permeability and vice versa and that loss of gut barrier function allows passage of endotoxin from gut lumen to systemic circulation, there are additional tools to be considered to monitor intestinal permeability. The presence of cytotoxic bacterial products in serum can be evaluated by using IgA/IgM responses to sonicated samples of common Gram-negative gut commensal bacteria, and assays of serum lipopolysaccharides (LPSs) and other bacterial toxins, including cytolethal distending toxin subunit B, provide good methods to screen for increased gut permeability in combination with IgM levels to zonulin and measuring gut dysbiosisZonulin has been implicated in many CIDs . Indepen34, the cellular senescence and apoptotic theory35, the immune system theory of aging36, and (pertinent to this article) the intestinal permeability and aging theory. Indeed, several reports in both animal models and humans link gut permeability to non-infective chronic inflammation and metabolic changes typical of the senescence process. In fruit fly, the increase in intestinal permeability leads to systemic metabolic defects and immune changes previously linked to aging and is the best predictor of imminent death, even more than the actual age of the insect96. In humans, it has been reported that zonulin serum concentration is higher in older adults, is positively associated with concentrations of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6), and intriguingly is negatively correlated with skeletal muscle strength and habitual physical activity37. These results suggest that zonulin-dependent gut permeability is associated with both systemic inflammation and two key indices of physical frailty associated with aging. These results have been corroborated by more recent data generated in disease-free ultra-centenarians showing lower levels of serum zonulin and endotoxemia as instigator of inflammation compared with young patients with acute myocardial infarction38, supporting the notion that, as in the fruit fly, gut permeability may impact lifespan expectations.Aging is the result of a constellation of cumulative changes that are deleterious, progressive, universal, and thus far irreversible. Aging damage can occur at the molecular , cellular, or organ level (or a combination of these). Recent scientific successes in rejuvenation and extending a lifespan of model animals give hope to achieve negligible senescence, reverse aging or at least significantly delay it. Many non-mutually excluding theories have been formulated to model the senescence process, including the free radical theoryCeliac disease. Celiac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten-containing grains in genetically susceptible individuals and can be reversed when gluten is eliminated from the diet. As mentioned above, indigestible fragments of gluten are able to bind CXCR3 and release zonulin27. CD has been used as a model disorder to study the effect of zonulin since its involvement in the development and pathogenesis of the disease has been well documented27. Even if gluten can trigger zonulin release in both healthy individuals and CD subjects, the amount and duration of zonulin produced are much higher in the latter group, leading to a significant increase in gut permeability, as shown by the capability of the zonulin inhibitor AT1001 (now named larazotide acetate) to prevent the zonulin permeating activity both inex vivo models44 and in a transgenic animal model of CD in which it prevented gluten-dependent inflammation and intestinal damage38. Larazotide acetate has been tested in patients with CD, showing good safety and efficacy in preventing gluten-dependent inflammation48, and is now in phase III clinical trial.Type 1 diabetes. Type 1 diabetes (T1D) is an autoimmune condition caused by the destruction of the insulin-producing β cells of the pancreas, and the pathogenesis of this disease is still not fully understood. Several studies, in both animal models and T1D patients, have shown increased intestinal permeability to precede the development of T1D98. In a recent elegant study, it was demonstrated that loss of gut barrier integrity was actually the causal factor for the microbiota-mediated T1D99 in susceptible mice, further supporting the critical role of the gut barrier–microbiome–immune system triad in the pathogenesis of CID. BioBreeding diabetes-prone rats, which spontaneously develop T1D, have increased small intestinal permeability which precedes the loss of tolerance to glucose by at least one month100. Oral administration of the zonulin blocker AT1001 in these rats corrected the gut barrier defect and reduced the incidence of diabetes, suggesting a mechanistic role of the zonulin-dependent gut barrier modulation in the pathogenesis of T1D91. The involvement of zonulin in T1D was confirmed in human studies showing that about 50% of patients with T1D have increased serum zonulin levels, some of them showing these changes in the pre-diabetic phase of the disease92. Interestingly, a subset (about 25%) of healthy first-degree relatives of patients with T1D also showed increased serum zonulin92. Similar data were generated in children at risk of T1D in which zonulin correlated with Glo-3A antibodies  in cases  but not in controls (at-risk children negative for auto-antibodies)93. Combined, these data suggest that zonulin may play a role in the pathogenesis of T1D in a subset of patients.Inflammatory bowel disease. Increased intestinal permeability has been shown to play a crucial role in the pathogenesis of inflammatory bowel diseases (IBDs)105. Arrietaet al., using the IL-10 knockout colitis mouse model, showed increased small intestinal permeability that preceded the onset of the overt colitis that can be ameliorated by oral treatment with zonulin inhibitor AT-100152. In humans, serum and fecal zonulin were found to be elevated in patients with active Crohn’s disease but not with ulcerative colitis50. In a more recent study, serum zonulin concentration was found to be higher in both diseases, and an inverse correlation was observed between serum zonulin concentration and disease duration51.Multiple sclerosis. In the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis (MS), zonulin-dependent increased intestinal permeability was shown during the pre-clinical phase of neurological symptoms, suggesting a role for zonulin in disease development69.70. Interestingly, baseline zonulin concentrations were associated with 1-year disease progression in progressive MS and closely mirror BBB breakdown in relapsing remitting MS. Considering these results, the authors concluded that zonulin may be responsible for the breakdown of both the intestinal barrier and the BBB in gut dysbiosis, thereby explaining how the gut–brain axis modulates neuroinflammation in MS70.It has been reported that patients affected by MS show increased permeability of both the blood–brain barrier (BBB) and the intestine. A recent report showed that zonulin concentrations were significantly higher in MS patients showing a compromised BBB as shown by magnetic resonance imagingAnkylosing spondylitis. Ankylosing spondylitis (AS) is an inflammatory, autoimmune condition that typically begins in young adulthood but often gets overlooked or is incorrectly diagnosed as pain from a previous injury or aging. It is an underdiagnosed form of arthritis that creates inflammation in the spinal joints and causes chronic back pain and stiffness. Dysbiosis has recently been demonstrated in patients with AS but its implications in the modulation of intestinal immune responses have never been studied. By analyzing ileal biopsies from patients with AS, Cicciaet al. showed that the presence of adherent and invasive bacteria in the gut of patients with AS with the bacterial scores significantly correlated with gut inflammation39. Impairment of the gut vascular barrier was also present in AS, accompanied by significant upregulation of zonulin, and was associated with high serum levels of LPS, LPS-binding protein, intestinal fatty acid–binding protein (iFABP), and zonulin39. Inin vitro studies, zonulin altered endothelial TJs while its epithelial release was modulated by isolated AS ileal bacteria. Furthermore, they provided evidence that bacterial products and zonulin influence monocyte behavior. Considering these results, the authors concluded that bacterial ileitis, increased zonulin expression, and damaged intestinal mucosal epithelial and endothelial barriers characterize the gut of patients with AS and are associated with increased blood levels of zonulin and bacterial products.Obesity. Obesity and its complications, including high cholesterol, type 2 diabetes (T2D), coronary heart disease, high blood pressure, and stroke, have been shown to be associated with chronic inflammation108 and frequently linked to alteration of the zonulin pathway, and more than 30 articles have been published on this topic. Several of these studies have shown elevated serum zonulin levels increased in obese versus non-obese subjects86, and there is evidence of a correlation between total bacteria and serum zonulin levels, suggesting that the gut microbiota may cause increased zonulin levels, and subsequent abnormal gut permeability to endotoxin and ultimately micro-inflammation has been reported in obesity106. A recent report also showed that zonulin serum correlates with total calorie, protein, carbohydrate, sodium, and vitamin B12 intake in obese women, andRuminococcaceae andFaecalibacterium were more abundant in the low-zonulin group, suggesting that butyrate-producing gut bacteria such asFaecalibacteria could decrease gut permeability by decreasing zonulin levels and lower inflammation87.Other metabolic disorders. Additional evidence suggests that zonulin is associated not only with obesity but also with its metabolic complications, including insulin resistance58, non-alcoholic fatty liver disease77, gestational diabetes55, hyperlipidemia61, and T2D95.Irritable bowel syndrome. Increased gut permeability has also been linked to the pathogenesis of irritable bowel syndrome (IBS)59. Specifically, patients with diarrhea-associated IBS showed increased serum zonulin levels60 and involvement of the protease-activated receptor 2 (PAR2)110, the zonulin target receptor23.Non-celiac gluten sensitivity. Non-celiac gluten sensitivity (NCGS) is a clinical entity triggered by gluten as in CD but without autoimmune enteropathy111. It has been shown that patients with NCGS may have increased serum zonulin levels and increased intestinal permeability following gluten exposure78.Environmental enteric dysfunction. Environmental enteric dysfunction (EED) is a chronic disease affecting mainly the proximal intestine. It is characterized by loss of barrier function, bacterial overgrowth in the small intestine, and low-grade intestinal inflammation leading to small intestinal villous atrophy that, in some aspects, resembles CD enteropathy. The potential developmental consequences of EE/EED can be devastating to the full physical and neurocognitive development in one third of the world’s children growing up in impoverished areas. It was recently reported that serum zonulin levels and other markers of barrier dysfunction were correlated with stunted growth in patients with EED53.There is growing evidence in the literature that Ag trafficking can also be involved in the immune component leading to the onset of a variety of cancers. Here are reported those conditions in which zonulin as a biomarker of epithelial and endothelial permeability has been associated with cancer.Glioma. Zonulin has also been shown to be involved brain tumors, mainly gliomas57. Increased zonulin expression of zonulin in gliomas correlated with the degree of malignancy and degradation of the BBB56.In vitro studies on a glioma cell line showed that zonulin was expressed in high amounts compared with non-glioma control cells57. Additionally, zonulin has been shown to induce transmigration of neuronal progenitor cells across the BBB57.Hepatocellular carcinoma. Hepatocellular carcinoma (HCC) globally ranks fifth for incidence and third for mortality among all malignant tumors. Although some progress has been made in exploring the pathological mechanisms and interventions of chronic liver diseases, there are still no effective biomarkers for the prediction and prevention of the progression of chronic liver diseases. Wanget al. recently reported that serum zonulin levels were significantly higher in patients with HCC compared with patients with liver cirrhosis or chronic hepatitis B or healthy subjects112. Moreover, the zonulin levels were increased in the advanced stage of liver cirrhosis and HCC.32.Loss of gut barrier function  with subsequent increased serum levels of microbiota-derived molecules  and activation of the immune system (increased cytokines production) leading to neuroinflammation has been described in many neuroimmune disorders, including chronic fatigue syndrome, autism spectrum disorder (ASD), major depressive disorders (MDDs), and schizophreniaAutism spectrum disorder. Increased serum zonulin that positively correlated with the Childhood Autism Rating Scale score has been reported in children with ASD41. In autism, as in other neuroinflammatory disorders, changes in zonulin-mediated gut permeability lead to pro-inflammatory status characterized by increased levels of mucosal pro-inflammatory cytokines  and decreased anti-inflammatory cytokines  detected mainly in ASD children experiencing gastrointestinal (GI) symptoms42.Schizophrenia. Increased plasma IgA/IgM responses to Gram-negative bacteria have been reported in deficit schizophrenia112 indicating leaky gut and gut dysbiosis. These results were confirmed more recently by Maeset al., who reported that the ratio of IgM to zonulin + occludin/talin + actin + viculin was significantly greater in patients with deficit schizophrenia than in those with non-deficit schizophrenia and higher in patients with schizophrenia than in controls and was significantly associated with increased IgA responses to Gram-negative bacteria88. IgM responses to zonulin were positively associated with schizophrenia (versus controls), whereas IgM to occludin was significantly associated with deficit schizophrenia (versus non-deficit schizophrenia and controls)88. The results show an upregulated paracellular pathway with breakdown of the tight and adherens junctions and increased bacterial translocation in deficit schizophrenia, suggesting their mechanistic role in causing neuroinflammation typical of the disease. These data were recently confirmed by our group89.Major depressive disorders. Gut dysbiosis consistent with pathophysiological gut metagenomic signatures  different when compared with normal controls has been described in MDD67. Parallel to these changes, subjects affected by MDD showed increased plasma levels of LPS, zonulin, and FABP267. Additionally, it has been demonstrated that, in patients with MDD, zonulin-mediated increased gut permeability causing increased bacterial translocation leads to marked alteration in circulating monocytes, with an expansion of the intermediate subset with increased frequency of IL-1β– and IL-6–producing cells. These changes are associated with a systemic pro-inflammatory state characterized by the enhanced serum TNF-α and IL-1β levels compared with those in the healthy controls68.Chronic fatigue syndrome or myalgic encephalomyelitis. Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is an illness characterized by profound and pervasive fatigue in addition to a heterogeneous constellation of symptoms. The etiology of this condition remains unknown; however, it has been suggested that enteric dysbiosis is implicated in the pathogenesis of CFS/ME49. In a systemic review, the microbiome composition of patients with CFS/ME was compared with that of healthy controls, showing statistically differences in some studies but with inconsistent findings in the studies considered49. Nevertheless, evidence of loss of barrier function in CFS/ME32 is consistent with the overall theme of mutual influence between gut dysbiosis and intestinal barrier function.Besides genetic predisposition and exposure to environmental triggers, the pathogenesis of a variety of CIDs seems to involve mutually influenced changes in gut permeability/Ag trafficking, immune activation, and changes in composition/function of the gut microbiome. Zonulin is a modulator of both epithelial and endothelial barrier functions and its role in health and disease remains an object of active research. Gut dysbiosis may cause the release of zonulin leading to the passage of luminal contents across the epithelial barrier causing the release of pro-inflammatory cytokines that themselves cause increased permeability establishing a vicious loop leading to massive influx of dietary and microbial Ags triggering the activation of T cells. Depending on the host genetic makeup, activated T cells may remain within the GI tract, causing CID of the gut , or migrate to several different organs to cause systemic CID. The effect of the zonulin inhibitor larazotide acetate in mitigating inflammation both in animal models and in human clinical trials not only confirms the pathogenic role of zonulin in many CIDs but also opens the possibility of targeting gut permeability in a variety of CIDs in which a pathogenic role for zonulin has been hypothesized or proven.Ag, antigen; AS, ankylosing spondylitis; ASD, autism spectrum disorder; BBB, blood–brain barrier; CD, celiac disease; CFS/ME, chronic fatigue syndrome/myalgic encephalomyelitis; CID, chronic inflammatory disease; EED, environmental enteric dysfunction; ELISA, enzyme-linked immunosorbent assay; GI, gastrointestinal; HCC, hepatocellular carcinoma; HP, haptoglobin; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; IL, interleukin; JAM, junctional adhesion molecule; LPS, lipopolysaccharide; MASP, mannose-binding lectin-associated serine protease; MDD, major depressive disorder; MS, multiple sclerosis; NCGS, non-celiac gluten sensitivity; T1D, type 1 diabetes; T2D, type 2 diabetes; TJ, tight junction; TNF-α, tumor necrosis factor alpha; ZO-1, zonula occludens 1


Extensive epidemiological and experimental evidence have shown that exposure to an adverse intrauterine environment as observed in offspring of pregnancies complicated by obesity or diabetes, can program susceptibility to metabolic, endocrine and cardiovascular disorders later in life. Although most studies have concentrated on the maternal environment, it is also becoming evident that paternal exposure to obesity or diabetes can result in the later development of metabolic disorders in the offspring. Such programmed effects might not be limited to the first directly exposed generation, but could be transmitted to subsequent generations. This suggests the existence of mechanisms by which metabolic changes in parental phenotype are transmissible to offspring. The mechanisms which underpin the transmission of the programmed effects across generations are still unclear. However, epigenetic regulation of transcription has emerged as a strong candidate for mediating the heritability of metabolic diseases. Here, we review the most relevant evidence from human and animal studies showing transmission of programming effects of obesity or diabetes across generations, and the current mechanisms underlying either maternal or paternal influences on the metabolic status of offspring. The prevalence of obesity and type 2 diabetes (T2D) worldwide has reached pandemic proportions. This is believed to be the result of complex interactions between an individual’s genome and environmental cues. Although single nucleotide polymorphisms at multiple genetic loci have been shown to be associated with T2D, for the majority of people with T2D, only a small proportion 5% to 10%) can be explained by genetic background 0% can be.The Developmental Origins of Health and Disease (DOHaD) concept has highlighted the crucial importance of the fetal and early postnatal environment in shaping long-term health , and a sObesity is a major risk factor for type 2 diabetes (T2D) development . Recent Thus, studies of siblings born before or after bariatric surgery to reduce maternal weight, have shown that those born prior to surgery have increased adiposity, increased blood pressure and reduced insulin sensitivity compared to those born after maternal surgery . On the The role of maternal T2D inheritance has also been recognized in a majority of epidemiological studies ,22. EarlExperimental studies support epidemiological observations and have provided strong evidence for transmission of the obese and diabetic phenotype from parent to offspring through non-genetic mechanisms. Numerous studies in rodents have investigated the effects of maternal obesity obtained in response to high-fat (HF) only, or high-fat/high-sugar diet, before and/or throughout pregnancy and during lactation . OvernutSince it is now well described that obesity or diabetes in pregnancy induces multiple phenotypic consequences in offspring, it is important to recognize the windows of exposure to the maternal insult that are responsible for conferring these effects .in utero exposure. When applied to the impact of maternal obesity (HF diet) [Both fetal and suckling periods have been shown to be critical time-windows in determining long-term metabolic heath. A large number of animal studies employing early weaning, cross-fostering, or nutritional manipulation during the lactation period only  support HF diet) , it was HF diet) . An expoHF diet) .Epigenomic information established during early development plays widespread roles in maintaining cell phenotypes across the mitotic generations throughout the lifetime of the organism. Some of this information (marks) has a potential to be influenced by environmental factors  and to get disrupted by being erased, reestablished or modified. Accordingly, they represent the best candidates underlying persistent effects of the fetal environment, later in life after termination of the challenge. It is known that the metabolic state of the organism directly influences epigenetic modifications, as DNA/histone methylation/acetylation rely upon substrates derived from intermediary metabolism such as S-adenosyl methionine, acetyl CoA, α-ketoglutarate, and nicotinamide adenine dinucleotide . For exaPdx1) gene was permanently reduced in beta cells and this contributed significantly to the diabetic phenotype reported in this model [Pdx1 is a key developmental transcription factor gene which regulates early pancreatic development and later differentiation and function of the beta cells [Pdx1 expression was epigenetically modulated by binding of mSin3A-HDAC complex to the Pdx1 promoter leading to histone deacetylation, followed by a reduction in H3K4 trimethylation and increase in H3K9 dimethylation in fetal and neonatal life [de novo DNA methylation leading to permanent reduction of Pdx1 gene expression in adulthood [Pdx1 gene expression have also been reported in islets of humans with T2D [Pgc1) and carnitine palmitoyl transferase I (Cpt1) in the same rats [Pdx1 expression and a return of the beta cell mass to normal level [Pdx1 expression. This study elegantly shows that an acute pharmacological intervention during a critical window may be sufficient to reverse aberrant epigenomic marks and prevent metabolic impairment in offspring.A number of animal models have been used to assess the role of epigenetics in pathogenesis of obesity and diabetes in the offspring ,64. Epigis model . Pdx1 ista cells . This retal life . Ultimatdulthood . Similarwith T2D . These fwith T2D . Additioame rats  and corrame rats . Howeveral level . ImportaNumerous animal studies have confirmed the detrimental effects of F0 maternal overnutrition/undernutrition exposure on F1 offspring’s glucose metabolism in adulthood. There are now also indications that glucose metabolism is altered in the F2 offspring as well as grand-offspring (F3) of in utero malnourished F1 females, even when the F1 and F2 females were well-nourished after weaning ,75. WhilAs a matter of fact, the intergenerational inheritance of disease risk may be mediated by various non-genomic mechanisms such as (1) persistence of maternal exposure to the external stressor, (2) indirect mechanisms associated with parental physiology , and (3)In some cases, developmentally programmed traits may simply be the result of persistent or replicated exposure during critical periods of development, generation after generation. It has been suggested that the history of severe socio-political disruptions and economic disadvantage suffered by minority populations, represents such recurring, multigenerational environmental insults (including chronic food insecurity) that may have led to developmentally programmed traits that contribute to the cardiometabolic alterations . Comparade novo in each new generation [Another potential mechanism for the transmission of programming effects is where programmed changes in the mother influence the development of her offspring during pregnancy. Thus, the phenotype is established neration . Maternaneration . By thisneration .There is now experimental evidence that maternally-induced non-genomic alterations can be transmitted through generations, and represents a vicious cycle that could lead to future generations becoming obese or diabetic without any exposure to suboptimal early nutrition.It has been reported that the peroxisome proliferator-activated receptor alpha and glucocorticoid receptor promoters are hypomethylated in the liver of protein-restricted female rats in both F1 and F2 generations issued from protein-restricted F0 female rats . To be cTo directly show that the maternally-triggered defects in the oocyte program metabolic disease in the offspring, Huypens et al.  used a mIt is well recognized that maternal obesity or diabetes can have a profound impact on the female fertility as evidenced by alterations of hormone levels, anovulation, and oocyte quality . Obese oAmong these lesions, mitochondrial inheritance is a candidate to explain the effect of maternal obesity on offspring health, since exposure of oocytes to an obese environment decreases mitochondrial DNA content  and funcAbnormal DNA methylation in oocytes  and poorFinally, similar to what is known for sperm ,98,99 . To know whether genes differentially methylated in the offspring are de novo methylated in the embryo or potentially inherited from sperm, Wei et al. analyzed the methylome in E3.5 blastocysts: Pik3ca showed higher methylation levels in F1-D blastocysts compared with F1-Control (F1-C) blastocysts, suggesting that this gene partially inherits methylated alleles from sperm. To sum-up, Wei’s results suggest that cytosine methylation status in diabetic sperm, strongly predisposes toward methylation in blastocysts, probably due to incomplete post-fertilization demethylation. Finally, Wei et al. found that several Differentially Methylated Regions (DMRs) distributed on different chromosomes in F1-D islets, were still significantly affected in F2-D islets, and that the methylation status of Pik3ca was also similar to that found in the F1 generation. This suggests that in this model, the epigenetic changes established in the F1 generation can be passed to the next generation (F2), despite the well-recognized genome-wide demethylation of the parental genomes during preimplantation embryo development. However, while these studies showed that altered DNA methylation signature of spermatozoa from HF male could be passed through the progenies, a recent study strongly indicated that sperm methylome was shaped by genetic and epigenetic variation but not by diet, thus disproving the idea that DNA methylation is the vector of epigenetic inheritance in this experimental HF model [Does paternal diabetes similarly alter the sperm methylome? A positive answer is brought about by the Wei et al. study . They obHF model .While many experimental studies focus on the epigenetic modifications involved in the inheritance of the newly acquired metabolic pathologies, few of them have been performed in humans. A recent study performed an epigenome profiling of sperm from lean and obese men . While sSperm DNA is mainly associated with highly basic proteins called protamines (protamine 1 and 2). The replacement of histone by protamine permits the typically condensed nuclear structure in mature spermatozoa, where DNA becomes organized in compacted units, similar to nucleosomes . At the There is recent compelling evidence that small noncoding RNAs (sncRNAs) play a crucial role in the intergenerational inheritance of the metabolic phenotype. sncRNAs can regulate DNA methylation, histone modifications, and mRNA transcription  and can Several studies have reported alterations of tRFs, piRNA or miRNAs expression profile in sperm from obese or diabetic rodents ,125,126.To assess the direct contribution of sperm cells in the transmission of obesity/diabetes to the next generation, the relevant method is to perform in vitro fertilization (IVF) of normal oocytes by sperm cells from obese or diabetic males, and to transfer the oocytes into surrogate mothers ,97. UsinCaenorhabditis elegans to humans [Paternal prediabetes could potentially affect the offspring’s metabolism via a number of different mechanisms. For example, paternal lifestyle changes can affect spermatogenesis and the composition of seminal fluid ,128. Stuo humans . Since pTo determine whether RNAs could be the vector of paternal traits inheritance, Grandjean et al.  performeTwo independent studies recently evaluated the potential contribution of sperm cleaved tRNA (tRFs) from obese male rodents to metabolic alterations of their offspring. Full-length tRNAs are associated with ribosomes during the synthesis of new proteins, but tRFs previously described in sperm  still exThe occurrence of tRFs in human sperm is yet to be determined, though they are present and functional in other human cell types .Sperm-borne miRNAs are important for embryo development, as evidenced by embryonic arrest in embryos deficient in sperm-derived miRNA . MoreoveIn the RNA analysis of sperm from F0-HF mice, Grandjean et al.  identifiThe combination of having both parents obese or diabetic on fecundity and offspring health, has received minimal attention. This information is not trivial, as the percentage of couples of reproductive age with both partners overweight/obese is increasing in most countries. To date, there have been few studies that have assessed the combined effects of maternal and paternal obesity on pregnancy and fetal health. Two studies in human assisted reproductive technology cohorts found no effect of maternal and paternal obesity on pregnancy establishment ,143. Howin utero exposure to maternal obesity. This may result from perturbed early embryo and fetal health, since reduced embryo developmental competency, reduced blastocyst cell numbers, impaired mitochondrial function, and alterations to active and repressive embryonic chromatin marks were found and were associated to aberrant placental gene expression and reduced fetal liver mtDNA copy numbers [Moreover, parental obesity in mice (HF diet) reduces fetal and placental weights without altering pregnancy establishment and was not dependent on an  numbers . These f numbers  (Figure It is clear that both the paternal and the maternal lineages are responsible for more than just their genetically encoded information. Several recent experimental studies have convincingly shown that obesity or diabetes in the father induces epigenetic alterations in sperm that can be inherited for several generations, and finally alters epigenetic marks in offspring somatic tissues. These epigenetic factors which are heritable, should be regarded as important as genetic factors in determining the risk for obesity and diabetes. Similarly, the passage of epigenetic information through the oocytes and originating from the obese or diabetic mother, is attractive and can explain the transmission of programmed effects to at least a second generation through the female lineage. However, it is unclear how often this might result in the induction of true oocyte epigenetic effects that are transmissible across several generations. Furthermore, one must not to forget alternative mechanisms for the intergenerational inheritance of the programmed phenotype via the maternal line, including maternal exposure to persisting environmental factors and maternal effects such as altered maternal physiology. Besides the inherited traits from parental obesity/diabetes extensively discussed in this review, epigenome modifications can also arise from a variety of environmental exposures including undernutrition, stress, physical activity and toxins, and they could separately or additively also contribute to the inheritance of the obese/diabetic phenotypes ,79.While our understanding of epigenetic contribution to the development of obesity and diabetes expands rapidly, there is much work to do to resolve the entire process. Are epigenetic germ cell modifications the result of direct effects on the germ cells or of effects induced in the soma being transmitted to the germ cells? How are such modifications protected from epigenetic reprogramming? Are DNA methylation, histone codes or ncRNAs involved? What makes one locus epigenetically stable and another environmentally susceptible? Do different mechanisms operate through oocytes and sperm cells? Do epigenetic germ cell modifications influence early preimplantation embryonic development, later stage embryonic growth, tissue-specific stem cells? What are the critical developmental periods? Are there differences depending on the tissue? Can interventions be targeted to specific epigenetic markers? Finally, the many limitations of the existing epigenetic studies do not allow to answer the crucial question whether epigenetics is a cause, a consequence or a correlation. Today, the relative importance of any or all of these issues in human populations is unknown. Thus, elucidation of these mechanisms and validation of interventions that improve health in men or women of reproductive age and potentially interrupt vicious cycles of disease risk transmission to their descendants, are critical public health and research issues. They bear the potential to halt the diabetes epidemic encountered throughout the world at present.


Methadone maintenance treatment (MMT) programs have been used worldwide to reduce the number of drug users and for HIV prevention; however, evidence of their effectiveness in mountainous areas is limited. This study aimed to identify changes in substance abuse and sexual practices among MMT patients after treatment in three Vietnamese mountainous provinces. A survey on risk behaviors was conducted among 300 drug users in six MMT clinics prior to and following one year of MMT. Cramér’s effect size of changes was extrapolated to justify the magnitude of the intervention’s effectiveness. A generalized estimation equation was used to find the factors associated with respondents’ substance use and sexual risk behavior. While drug-related risk behaviors were significantly reduced, alcohol and sex-related behaviors remained risk factors for HIV in this group. Additionally, condom use was common among participants at both time points, but not among those having sex with sex workers. Socio-economic characteristics of ethnic, education, occupation, as well as drug use history influenced the possibility of engaging in drug use and/or sexual risk behavior following treatment. Further emphasis on managing these among MMT patients is required, potentially by providing integrated services including smoking and drinking counseling and condom use promotion in accordance with MMT. HIV remains a significant global health burden, with 36.7 million people estimated to be HIV-positive in 2016, with over 2.1 million new infections reported globally in 2015 alone , though The effect of methadone maintenance treatment (MMT) on risk behaviors towards HIV/AIDS has been demonstrated in many studies. In China, Wang et al. showed that those engaged in drug- and sex-related risk behaviors including needle-sharing and unprotected sex were significantly less likely to have received MMT , while CAs it is estimated that up to 30% of HIV-positive patients in Vietnam are people who inject drugs (PWID) , the linIn addition, we have noticed a lack of studies focusing on the effectiveness of MMT on PWID and other populations vulnerable to HIV risks in the mountainous regions of Vietnam. The main reason for this literature gap may be the fact that attempts to conduct studies in mountainous regions are often faced with obstacles; for instance, the clinics that can potentially be used as study settings are often in remote areas and understaffed, the prospective participants live far from clinics and usually have low levels of education and willingness to respond to complex questions, which in turn lead to difficulties in obtaining data and monitoring respondents. Nonetheless, understanding how MMT benefits risk prevention towards HIV in mountainous areas is crucial in ensuring the effectiveness of nation-wide scale-up of the MMT program, especially since these areas may hold the biggest challenges in MMT implementation. The PWID in the mountainous areas of Northern Vietnam have been reported to account for over 30% of the total PWID in Vietnam, but the majority of these patients live remote from health facilities in general and MMT clinics in particular, indicating accessibility barriers . A studyWe conducted a before–after (pre–post) study with no control group from December 2014 to December 2015 in six clinics, including Provincial AIDS Centers in Dien Bien, Lai Chau, and Yen Bai City; and District Health Centers in Thanh Xuong, Phong Tho, and Nghia Lo, which satisfied the following criteria: (1) Placed in Dien Bien, Lai Chau, and Yen Bai provinces: (2) Currently providing MMT services; (3) Covering the provincial or district levels of the Vietnamese health system.A convenience sampling technique was adopted to recruit participants for the study. Selected participants were those who satisfied the following criteria: (1) Participating in an MMT service at one of the clinics included in the study (within 3 months) at the time of the study in 2014; (2) Agreeing to participate in research by giving written consent; (3) Having good physical and mental health to participate in the study; and (4) Having used the MMT service for 12 months. MMT patients were invited to enroll in the study when they initiated the treatment and after 12th-month of the MMT program. There were 300 patients participating in the MMT program at baseline, and 244 patients remaining after 12 months of treatment (81.3%). The 56 participants who were no longer attending their respective clinics dropped out of the study.Data were collected using face-to-face interviews with a structured questionnaire. The interviews were conducted by post-graduate students from Hanoi Medical University who had prior training in conducting community surveys. Information regarding participant socio-demographic characteristics as well as self-reported drug use status, alcohol use, smoking, and sexual behaviors was collected. This study utilized simple measures of outcomes in consideration of resource constraints . Nonetheless, the measures used were in line with comparable research conducted on the potential benefits of MMT implementation in Vietnam  and on tSocio-demographic characteristics of participants such as age, ethnicity, education, marital status, and occupation were also collected.Drug use behaviors included: (1) Using drugs (whether used or not); (2) Drug injection (whether conducted injection or not), and (3) Frequency of substance use per day in the past 30 days.Alcohol use and smoking behaviors included: (1) Currently smoke; (2) Using alcohol; (3) Frequency of alcohol use, and (4) Average alcohol consumption, all within the past 30 days.Sexual behaviors included: (1) Using condoms the last time they had sex; (2) Had sex with female sex workers; (3) Using condoms when having sex with female sex workers in the past month; (4) Had sex with female sex workers who inject drugs; (5) Had sex with male partner(s); and (6) Spouse/partner injected drugs within the last 30 days.p < 0.05. Effect size (ES), which indicates the magnitude of differences, was calculated to identify the effect of MMT programs. In this study, we applied the following formula to calculate Cramér's V as representing ES for categorical outcomes with more than two levels [We described the frequency (n) and prevalence (%) of variables at baseline and in follow-up data. The difference between before and after MMT intervention was assessed using Chi-square and Fisher's exact test. Statistical significance was set at o levels :∅c= X2N. We asked participants to provide written informed consent before starting interviews. Participants could withdraw from the study at any time. Patient information was coded and only used for research purposes.The socio-demographic characteristics of participants are described in Alcohol use and smoking behaviors among the participants are illustrated in Factors associated with substance abuse and sexual risk behavior among participants are presented in To our knowledge, this study is one of a few that explore the risk behaviors of MMT patients in the mountainous areas of Vietnam with regard to before and after 12 months of engaging in MMT service. In general, it was found that the majority of MMT patients were also smokers and frequent alcohol consumers who did not display significant sex-related risk behaviors. A significant reduction was found in drug use—especially in drug injection—following a 12-month MMT program, while smoking, alcohol assumption, and sex-related behaviors showed no significant changes. In addition, the socio-economic characteristics ethnicity, education, occupation, and drug use history were found to influence the possibility of engaging in drug use and/or sexual risk behavior following treatment.The findings of this study on drug use after 12 months of enrolment in an MMT program add to the literature supporting the effectiveness of MMT in reducing drug-related risk behaviors ,34. In aThe high prevalence of smoking and alcohol use among MMT patients found in this study is in line with the existing literature. Previous studies conducted in developed countries have indicated that cigarette use and alcohol consumption prevalence could be as high as 94% and 49%, respectively, among MMT patients . Within In addition, although most participants did not engage in risky sex-related behaviors, only half of them reported the use of condoms while having sex. Previous studies have also found relatively low condom use among drug users in Vietnam ,42,43. WOverall, this study provides insights into the effects of MMT on the risk behaviors of patients living in mountainous regions—a relatively under-researched area. The findings can serve as a reference point for policy makers and service providers when developing strategies for MMT implementation on national and regional levels. The need for the scaling-up of MMT programs as well as for providing additional counseling services targeting alcohol and cigarette use and HIV testing and counseling services at MMT clinics—which has been proved elsewhere ,44,45,46The most significant limitation of this study is the small sample size, as only 244 participants were included in the 12-month data. Over the course of the 12-month period, 56 patients withdrew from the study, and the exclusion of data regarding these dropouts may skew our results toward positive, improved risk behavior following MMT adherence. We suggest that further research on this topic have more frequent follow-ups with patients in order to reduce the drop-out rate and obtain less potential bias in results. The convenient sampling technique adopted when recruiting participants may also have undermined the representativeness of our study. Although efforts were made to ensure the participants’ anonymity, the lack of a control group and the reliance on self-reported data may have introduced a social desirability bias into our results due to cultural factors. Besides, despite having included participants across six provincial and district centers in an attempt to have a relatively good representative sample of drug users in the area of interest, using MMT patients to examine risk behaviors regarding HIV may have caused some skewing of the data towards lower risk. This is because these patients voluntarily came forward to better their health and therefore may have possibly already engaged in less risky behavior overall. Further studies accessing the hidden drug-using population in order to fully comprehend risk behavior are thus called for.Our data indicate that after 12 months of MMT, drug-related risk behavior for HIV was significantly reduced, but that alcohol-related and sex-related risk factors remained an issue. This information identifies aspects of MMT services that are lacking and areas where education and patient management are needed in order to reduce HIV risk even further. Given that HIV is a significant risk among PWID in Vietnam , the positive effects of MMT in lowering this risk are promising.


Type 2 diabetes mellitus (DM) is associated with an increased risk of cardiovascular disease (CVD). Atrial fibrillation (AF) and stroke are both forms of CVD that have major consequences in terms of disabilities and death among patients with diabetes; however, they are less present in the preoccupations of scientific researchers as a primary endpoint of clinical trials. Several publications have found DM to be associated with a higher risk for both AF and stroke; some of the main drugs used for glycemic control have been found to carry either increased, or decreased risks for AF or for stroke in DM patients. Given the risk for thromboembolic cerebrovascular events seen in AF patients, the question arises as to whether stroke and AF occurring with modified incidences in diabetic individuals under therapy with various classes of antihyperglycemic medications are interrelated and should be considered as a whole. At present, the medical literature lacks studies specifically designed to investigate a cause–effect relationship between the incidences of AF and stroke driven by different antidiabetic agents. In default of such proof, we reviewed the existing evidence correlating the major classes of glucose-controlling drugs with their associated risks for AF and stroke; however, supplementary proof is needed to explore a hypothetically causal relationship between these two, both of which display peculiar features in the setting of specific drug therapies for glycemic control. Cardiovascular disease (CVD) is the main cause of morbidity and mortality in type 2 diabetes patients. The increased cardiovascular risk seen in diabetic patients cannot be mitigated with a monofactorial intervention of plasma glucose control, requiring a multi-factorial control of all cardiovascular risk factors ,2. Some Among all potential clinical forms of diabetes-associated obstructive artery disease, cerebrovascular disease is a serious condition, inducing major disabilities and a shortened life span. In a large meta-analysis of 102 prospective studies, diabetes mellitus was associated with a 2.27-fold increase in the risk for ischemic stroke when compared with a non-diabetic status .Accumulating clinical evidence also seems to connect diabetes mellitus with an increased risk for atrial fibrillation (AF) . DiabeteIn a turning point in diabetes-related clinical research, several older or newer drugs used to control glycemic values in diabetic patients were recently shown—mostly in observational studies, post-hoc analyses of the major trials, or various meta-analyses—to display different levels of risk for either AF or stroke ,8. Such Recently published research has frequently depicted various classes of antihyperglycemic agents as being associated with modified levels of risk for either AF or stroke. Stroke episodes in AF patients frequently have a thromboembolic nature; hence, the question arises as to whether a specific risk for AF in one or the other of the antidiabetic drugs would reflect an accordingly modified risk for cerebral thromboembolism, and thus stroke. Unfortunately, the major clinical trials have not yet distinguished between the ischemic or hemorrhagic nature of stroke episodes, and least of all, between the atherothrombotic or thromboembolic etiology of ischemic strokes ,10. In tIn a case-control study on Taiwan registries, insulin therapy was associated with a higher risk of new-onset AF in diabetic patients than with other antihyperglycemic medications . Among pIn a cohort study on 645,710 Taiwan patients, monotherapy with metformin was associated with a 19% reduction in the risk of AF compared with the use of other antihyperglycemic medications during a 13-year follow-up. Metformin users had the lowest AF incidence rates in the first two years after diagnosis, but the protective effect tended to fade afterward . PossiblCurrent evidence suggests that metformin also has a protective effect against ischemic stroke, even though specific outcome studies analyzing a potential cause–effect relationship between the protective role of metformin against AF development and the rate of thromboembolic events are lacking in the medical literature. The results of the United Kingdom Prospective Diabetes Study (UKPDS) suggested that intensive blood glucose control with metformin, compared with the use of sulfonylureas or insulin, significantly reduced the risk of stroke . After aMost researchers analyzing the risk of AF development have considered SU therapy as only a control to report comparative AF outcomes of other antidiabetic medications. Among the few studies making an exception, the previously mentioned Taiwan case-control study found SUs to not be associated with an increased risk of new-onset AF .Research on the stroke risk associated with SU use generally precedes the publication of most studies using these drugs as an active comparator for other medications’ AF risk. This class of hypoglycemiant drugs acts on the SU receptor (SUR) unit of the ATP-sensitive potassium channels. In normal conditions, these ionic channels may play a protective role against neuronal ischemia. SUs were therefore feared by some authors to inhibit this neuroprotective mechanism, and thus to increase the risk of stroke ,20,21. IThiazolidinediones are insulin sensitizers acting primarily on the peroxisome proliferator-activated receptor (PPAR)-γ and, in the case of pioglitazone, also exerting a weak agonist activity on PPAR-α. Their action on these nuclear receptors is associated with anti-inflammatory and anti-oxidant benefits, potentially due to favorable effects on Transforming Growth Factor (TGF)-β, Tumor Necrosis Factor (TNF)-α, Atrial Natriuretic Peptide (ANP), superoxide dismutase (SOD), malonyldialdehyde, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits, or voltage-dependent calcium channels . ReportsThese conflicting features of TZDs led to research on their association with atrial fibrillation and stroke. In an observational study on 12,605 patients with insulin-naïve type 2 diabetes, the risk of developing AF was reduced by 31% after a five-year follow-up in patients treated with TZD . In anotSimilar to the case of metformin, no specific evidence links the potentially protective role of TZDs against AF and the effect of these drugs on the risk of cerebrovascular events. However, some data indicate a real possibility that TZDs have the ability to protect diabetic patients against stroke development. In another sub-analysis of the PROactive study, the risk for fatal or non-fatal stroke was significantly reduced with pioglitazone in type 2 diabetes patients with a history of previous stroke, but not in those without a history of cerebrovascular events . In the In a cohort study on 90,880 patients with type 2 diabetes previously treated with metformin as a first-line antihyperglycemic drug, the add-on of DPP-4 inhibitors (mostly sitagliptin) as a second-line therapy was found to be associated with a lower risk of AF development than the use of other drugs (mainly SUs) as the second antidiabetic medication . The useThese positive or neutral results on AF risk raised the question of potentially protective effects of DPP-4 inhibitors against stroke. In another longitudinal observational Taiwan study on 123,050 type 2 diabetes patients that were newly initiated on oral antidiabetic drugs, the use of DPP-4 inhibitors was associated with a lower risk for ischemic stroke compared with meglitinides or insulin; however, their risk for stroke was comparable to that observed in metformin users, and higher than the risk observed in patients treated with pioglitazone . None ofA side effect of GLP-1 receptor agonists includes a moderate increase in heart rate , which mHowever, aside from speculations about the risks of AF, GLP-1 receptor agonists are definitely not associated with a higher risk for stroke. All GLP-1 receptor agonists developed from the human GLP-1 backbone  are able to lower the risk for the composite outcome of major cardiovascular events  ,55,56,57SGLT-2 inhibitors exert their actions by inhibiting the active reabsorption performed by this specific co-transporter of sodium and glucose at the level of the proximal convoluted tubule. As a result, glucose, sodium, and water are lost in the final urine, lowering blood pressure and blood glucose levels, and creating a negative energy balance that induces weight loss. Based on these direct effects on multiple cardiovascular risk factors, but also on other adjunctive metabolic actions, SGLT-2 inhibitors seem able to lower the cardiovascular risk in diabetic patients. In the dedicated cardiovascular outcome trials, empagliflozin and canagliflozin were shown to reduce the progression to the composite outcome of major cardiovascular events ,59, wherCurrent evidence supports the existence of a relationship between diabetes mellitus and an increased risk for atrial fibrillation and stroke. In these high-risk patients, several reports linking antidiabetic medications to modified risks for atrial fibrillation, stroke, or both, have been published in the last years. The most relevant of these results are summarized in The cause–effect relationship between the modified risk for atrial fibrillation of these drugs and cerebrovascular disease due to thromboembolic events has not yet been analyzed in studies with dedicated outcomes. However, depicting the ability of some specific antihyperglycemic therapies in reducing the risks for both atrial fibrillation and stroke as completely separate mechanisms would mean allowing the existence of slightly too much coincidental evidence. Trials searching for a potentially causal triangular relationship between antidiabetic drugs, risks for atrial fibrillation, and cerebral thromboembolism are needed to fill in a gap in evidence, and to potentially supplement the adaptation of the recommendations of current guidelines to prevent the negative outcomes of cardiovascular disease in diabetic patients as much as possible.


The term “vulnerable plaque” is commonly used to refer to an atherosclerotic plaque that is prone to rupture and the formation of thrombosis, which can lead to several cardiovascular and cerebrovascular events. Coronary artery atherosclerosis has a wide variety of different phenotypes among patients who may have a substantially variable risk for plaque rupture and cardiovascular events. Mounting evidence has proposed three distinctive histopathological mechanisms: plaque rupture, plaque erosion and calcified nodules. Studies have demonstrated the characteristics of plaques with high vulnerability such as the presence of a thin fibrous cap, a necrotic lipid-rich core, abundant infiltrating macrophages and neovascularization. However, traditional coronary angiographic imaging fails to determine plaque vulnerability features, and its ability to individualize treatment strategies is limited. In recent decades, catheter-based intravascular ultrasound imaging (IVUS) modalities have been developed to identify vulnerable plaques and ultimately vulnerable patients. The aim is to individualize prediction, prevention and treatment of acute coronary events based on the identification of specific features of high-risk atherosclerotic plaques, and to identify the most appropriate interventional procedures for their treatment. In this context, the aim of this review is to discuss how personalized assessment of coronary atherosclerotic arteries can be achieved by intravascular ultrasound imaging focusing on vulnerable plaque detection. Cardiovascular diseases are the number one cause of death worldwide, and the most frequent is coronary artery disease (CAD). Acute coronary syndrome (ACS), a common complication of coronary artery disease, is a term used to describe a range of conditions where the blood supply to any part of the heart is suddenly reduced or blocked. Atherogenesis, the triggering factor of ACS, is a chronic and developing inflammatory condition in which atheromas (or “plaques”) are developed in the inner lining (the intima) of arteries. Over time, the plaques harden, narrow the lumen of the arteries and restrict the blood flow. Acute coronary syndrome is an umbrella term encompassing the following clinical disorders: ST-segment elevation myocardial infarction (STEMI), non-ST-segment elevation myocardial infarction (NSTEMI) and unstable angina ,4,5,6,7.The term “vulnerable” is defined as “susceptible to injury or susceptible to attack”, suggesting that an event has high conditional probability to occur in the future. The source of the majority of ACS cases is the erosion or rupture of an atherosclerotic plaque, which leads to the development of intraluminal thrombus that can further limit, or even block, the blood flow. Thus, the term “vulnerable plaque” is often used to denote a plaque prone to rupture ,10,11,12Nowadays, we are able to visualize detailed anatomical features of coronary arteries due to the evolution of high-resolution, intracoronary ultrasound imaging modalities, helping us to broaden our insight regarding the mechanisms responsible for atherogenesis and intravascular thrombus formation. Although several other intravascular modalities are now available for coronary artery assessment, such as optical coherence tomography (OCT), catheters for fractional flow reserve (FFR) measurement, near-infrared spectroscopy and so on, the present review aims to provide a brief overview assessing coronary atherosclerosis and plaque vulnerability by intravascular ultrasound (IVUS) imaging. Furthermore, we aim to highlight the contribution of IVUS in making the diagnosis of coronary atherosclerosis severity, and risk of coronary events, more precise by detecting plaque features that could not otherwise be assessed by traditional, routine angiographic examination (still the gold-standard method for coronary artery examination). Additionally, the significant advances in therapeutic precision introduced by the IVUS guidance of percutaneous coronary interventions (PCI) compared to angiographic-guided PCI are discussed.As mentioned above, acute coronary syndromes occur when atherosclerotic plaques rupture and occlusive thrombus is formed . AutopsyAutopsy studies have shown an inverse relationship between the thickness of the cap and the risk of plaque rupture, with 95% of plaque rupture occurring in cases with a cap thickness of <65 μm. In a study by Narula et al., the significance of various pathological features in the distinction of TCFA from the fixed atherosclerotic plaque was analyzed and it was shown that the most reliable discrimination criterion is the thickness of the fibrous cap . The exiCoronary arteries are likely to respond to the atherosclerotic process by dilatation or restriction of the arterial wall, defined as positive and negative remodeling, respectively. While vulnerable coronary artery plaques are associated with positive remodeling, stable plaques are most commonly related to negative remodeling of the vessel wall. Although by positive remodeling the vessel lumen may widen, continuous growth of the plaque into the interior of the lumen can induce neovascularization from small vessels originating from a network of vessels that irrigate the outside layer. It has been found that the development of these neovessels negatively affects plaque stability. They cause hemorrhages inside the plaque, which are due to the fact that these neoplastic vessels are fragile, with a lack of adequate supportive cells .Calcium deposition in atherosclerotic plaques is associated with all stages of atherosclerosis and may occur even in the initial phase of intima thickening. The presence of microcalcifications within the thin fibrous cap or a particular calcification pattern called “spotty calcification” can be an indicator of plaque vulnerability. In contrast, larger calcium deposits, which consist of the joining of smaller regions of microcalcification, are associated with greater plaque stability. Therefore, although the presence of microcalcification in the fibrous cap and spotty calcification have been related to plaque rupture, plaques composed primarily of large calcification deposits are considered to be more stable ,14,15,16Intravascular ultrasound is an invasive imaging technique using a catheter that allows real-time, longitudinal, cross-sectional evaluation of the vessel and lumen dimensions, as well as the distribution and morphology of atherosclerotic plaques. IVUS grayscale images are constructed by measuring the intensity and timing of reflected signals, with an axial analysis of 150–250 μm with a 45 mm penetration depth.Intravascular ultrasound uses a small-sized ultrasound probe placed at the end of a flexible (2.5–3.5 French) catheter, which is inserted into the coronary arteries. The standard operating frequencies of IVUS catheters are 20–60 MHz ,22. The In the classical intracoronary ultrasound based on grayscale, healthy coronary arteries display three distinct layers. Atherosclerotic plaque is formed in the first layer, the intima. The second layer is the middle tunica, which is opaque and consists of smooth muscle cells. The third layer distinguished in the IVUS consists of the acoustic surface between the outer elastic waist and the outside area . IVUS grUsing IVUS, a ruptured plaque is defined as the cavity formation in contact with the lumen of the vessel with a broken-surface fibrous cap . RuptureThe extent of coronary artery calcification correlates with the degree of atherosclerosis and future cardiovascular events. Calcium is a potent reflector of ultrasound and, consequently, ultrasound beams do not enter or even penetrate calcium. As a result, calcium casts a shadow over deeper arterial structures . The IVU2, or classified as TCFA based on VH–IVUS. Although VH–IVUS has been used in clinical research to identify vulnerable plaque characteristics, its ability to identify TCFA plaques and determine their quantitative characteristics remains challenging. An ex vivo study in pigs attempted to investigate the correlation between VH–IVUS and histological measurements of necrotic core size. The findings did not indicate a good correlation between VH–IVUS necrotic core measurements and histological findings [Virtual histology–intravascular ultrasound imaging (VH–IVUS) uses advanced technology to analyze the frequency spectrum of reflected sound waves, and thus it can provide a more detailed analysis of the plaque composition . Howeverfindings . This exCoronary angiography is the conventional method to assess the coronary anatomy and to search for stenotic lesions in the epicardial coronary vessels. Nonetheless, coronary angiography provides only a projection of the vessel lumen and cannot visualize the vascular wall or the detailed lumen geometry. The two-dimensional imaging of the coronary lumen, in addition to some technical features , can lead to inaccurate assessment of lesion severity. The evaluation of the hemodynamic significance of moderate coronary stenosis with 40% to 70% lumen reduction only using coronary angiography appears a difficult decision even for experienced interventional cardiologists . Conside2 has been proposed from several studies as a safe cut-off value to identify significant coronary lesions. An MLA ≥ 4 mm2, with a diagnostic accuracy of 89%, showed a clear correlation with a normal coronary flow reserve >2.0 [2 with an abnormal FFR < 0.75, with a specificity of 56% and a sensitivity of 92%, respectively [Fractional flow reserve (FFR) is an optional method for the hemodynamic evaluation of intermediate lesions . Nonetherve >2.0 . Anotherectively .2) to be correlated with an FFR < 0.75 [Nonetheless, assessment of the severity of a lesion dependent only on IVUS data could be erroneous and thus should be avoided. Numerous other factors, like lesion length, reference vessel diameters, area of the myocardium at risk and plaque burden should be co-evaluated when interpreting the acquired IVUS data. All this information (biomarkers) derived by IVUS may synthesize a more detailed and individualized “picture” of the atherosclerotic vessel leading to a more precise assessment of its vulnerability. Not surprisingly, studies on intermediate lesions in smaller vessels (reference diameter < 3.0 mm) showed a smaller MLA  and pathophysiologic factors (plaque burden) should be also taken into consideration [In clinical practice, an MLA ≥ 4 mmderation .2 as a cut-off for revascularization. After 2 years, only 8 patients (4.4%) from the deferred group (MLA > 6.0 mm2) underwent revascularization, none of them in the setting of acute coronary syndrome [2 should be considered as a criterion of a non-significant LMCA without need for intervention. Nevertheless, an MLA < 6 mm2 should not be exclusively regarded as an indication for revascularization without taking into account other clinical and angiographical parameters. In such a scenario, FFR or a noninvasive stress test should be performed [Anatomic factors  as well as technical factors  make the interpretation of left main disease angiographically difficult, thus necessitating the use of additional, more-precise diagnostic tools. Not surprisingly, angiographic assessment of the left main coronary artery (LMCA) shows the greatest interobserver variability . A multisyndrome . It shouerformed .Recently, the European Association of Percutaneous Cardiovascular Interventions published an updated expert consensus document on the clinical use of intracoronary imaging for guidance and optimization of coronary interventions . In the In the domain of bare metal stents (BMSs), several studies have demonstrated the clinical value of IVUS guidance for individualizing and optimizing the interventional result through reduction of restenosis and target vessel revascularization (TVR). In a meta-analysis of 2193 patients from seven randomized trials, an IVUS-guided interventional strategy in patients treated with BMSs reduced TVR by 13% compared with the group of angiography-guided PCI .Similar results have been noted in the era of drug-eluting stents (DESs) for IVUS-guided interventions of LMCAs as well as for non-left main disease. A large study of 1670 patients with LMCA lesions, treated with DESs, was undertaken to investigate the value of IVUS guidance in interventions. After a mean follow-up period of three years, the group of patients who underwent IVUS imaging showed significantly reduced incidence of major adverse cardiac events (MACE) and TVR compared to those who received angiographic guidance .Furthermore, the chronic total occlusions (CTO)–IVUS trial  and the Similarly, a meta-analysis of seven randomized controlled trials following 3192 patients treated with DESs confirmed the benefits of IVUS-guided intervention in regards to MACE, cardiovascular mortality and stent thrombosis after a follow-up of 12 to 24 months . HoweverRegarding the impact of the lesion complexity and the clinical setting under which IVUS was performed, a large observational study including 8583 patients demonstrated the most pronounced benefit of IVUS use in patients with ACS and complex lesions (ADAPT DES) . A similThe above findings were confirmed in the most recent meta-analysis of a total of 31 studies including of 17,882 patients. IVUS-guided PCI was associated with a significantly reduced risk of all-cause death, myocardial infarction, target lesion revascularization and stent thrombosis . NotablyPrior to intervention, IVUS can assess plaque composition and distribution and, according to this large amount of quantitative and qualitative information, the treatment strategy can be modified and individualized according to the specific patient’s characteristics and therefore the appropriate stent size and length can be selected. In cases of plaques with extensive calcification, a more aggressive therapy, like rotational atherectomy, may be preferred, whereas a lipid-rich plaque may require a less aggressive approach to avoid distal lipid embolization. Optimal stent sizing (stent diameter and length) plays a crucial role in early and long-term interventional results. Among various factors, stent under-expansion is a strong predictor of early stent thrombosis and restenosis ,62. Seve2 for non-left main lesions, 7 mm2 for distal left main disease and 8 mm2 for proximal left main disease should be targeted [After stent implantation, IVUS can identify correctable abnormalities, such as under-expansion, malposition and edge dissection. A minimum stent cross-sectional area of 5.5 mmtargeted ,66. Malptargeted . Howevertargeted . Large-etargeted .IVUS can be applied to investigate the mechanism of stent failure. The European Society for Cardiology (ESC) guidelines on myocardial revascularization recommended the performance of intracoronary imaging by IVUS in case of restenosis or stent thrombosis with a Class IIa recommendation . The mosBesides the classic applications of IVUS mainly targeting geometrical and structural features of atherosclerotic lesions, specific computational methods and algorithms have been developed that provide extra information concerning plaque vulnerability. Neovascularization (or neoangiogenesis) is a well-know process which might be more or less apparent in atherosclerotic lesions and is related with plaque progression and vulnerability. Combining contrast-enhanced IVUS imaging (using microbubbles as a contrast agent) and specific computational algorithms, it has been found that visualization and semi-quantification of the presence and density of vasa vasorum and neovessels within and around a coronary atherosclerotic lesion are feasible ,75,76. MThe rapid and emerging developments in biomedical engineering and computer science have a tremendous impact in medical imaging and particularly in intravascular modalities. More information and knowledge is continuously gained concerning not only atherosclerotic plaque phenotypes , but also about in vivo atherosclerotic processes and plaque progression. The plethora of information and the new computed structural, compositional and functional biomarkers using IVUS and IVUS-based modalities is valuable for the personalization of diagnosis and guidance of interventions  for the optimal management of patients with coronary artery disease. Nonetheless, more analyses of cost effectiveness remain to be conducted to further explore the use of IVUS modalities in clinical practice and determine those cases where IVUS will be most beneficial for patients’ outcomes.


Arteriogenesis is an intricate process in which increased shear stress in pre-existing arteriolar collaterals induces blood vessel expansion, mediated via endothelial cell activation, leukocyte recruitment and subsequent endothelial and smooth muscle cell proliferation. Extracellular RNA (eRNA), released from stressed cells or damaged tissue under pathological conditions, has recently been discovered to be liberated from endothelial cells in response to increased shear stress and to promote collateral growth. Until now, eRNA has been shown to enhance coagulation and inflammation by inducing cytokine release, leukocyte recruitment, and endothelial permeability, the latter being mediated by vascular endothelial growth factor (VEGF) signaling. In the context of arteriogenesis, however, eRNA has emerged as a transmitter of shear stress into endothelial activation, mediating the sterile inflammatory process essential for collateral remodeling, whereby the stimulatory effects of eRNA on the VEGF signaling axis seem to be pivotal. In addition, eRNA might influence subsequent steps of the arteriogenesis cascade as well. This article provides a comprehensive overview of the beneficial effects of eRNA during arteriogenesis, laying the foundation for further exploration of the connection between the damaging and non-damaging effects of eRNA in the context of cardiovascular occlusive diseases and of sterile inflammation. Cardiovascular diseases such as ischemic heart disease, stroke or peripheral arterial occlusive disease are a major public health burden, accounting for approximately 30% of deaths worldwide in 2017 [Extracellular RNA (eRNA) released upon increased fluid shear stress during arteriogenesis in vivo has recently been suggested to be this missing link by initiating the cascade of arteriogenesis through vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling . eRNA isThe initiating stimulus for collateral remodeling in arteriogenesis is increased arteriolar fluid shear stress as a result of the occlusion of the main supplying artery . In sharVEGF is a glycoprotein produced by a variety of cell types including leukocytes such as neutrophils or monocytes ,23,24,25165 and VEGF121) confirmed that eRNA directly interacts with VEGF, most likely via its heparin binding domain [VEGFR2 activation and signaling have been demonstrated to be involved in mediating the pro-inflammatory and permeability-enhancing effects of eRNA on endothelial cells. Binding studies of eRNA to different VEGF isoforms  formation in arteriogenesis . EndothevWF is an adhesive and prothrombotic glycoprotein, constitutively produced by endothelial cells and released from endothelial Weibel–Palade bodies upon cell stimulation. It forms large and ultralarge multimers and plays a key role in the tethering and adhesion of platelets to the injured vessel wall, particularly under fluid shear stress conditions ,58. In aWhile free platelets do not usually interact with vWF, they are able to transiently bind to vWF via their receptor glycoprotein Ibα (GPIbα) under high fluid shear stress, resulting in platelet activation and adhesion . GPIbα aThis important step during collateral remodeling might also be influenced indirectly by eRNA, marking another point where eRNA could play a role during arteriogenesis. In a murine model of arteriogenesis, RNase1 treatment decreased the formation of PNAs to a similar extent as was seen in mice which were either deficient in vWF or treated with a VEGFR2 inhibitor , suggestThe activation of platelets together with the secretion of highly reactive multimeric vWF from endothelial cells under inflammatory conditions during the process of arteriogenesis would provide an ideal setting for thrombus formation. Yet, such an outcome has never been observed, and it remains to be studied in which way local thrombus formation is inhibited during arteriogenesis. Following activation of endothelial cells and the initial release of vWF in a multimeric form to promote platelet adhesion under flow, the readily coagulable nature of these large vWF multimers needs to be weakened at a later phase to prevent the formation of microthrombi . vWF canFollowing PNA formation, the extravasation of neutrophils is mediated through urokinase-type plasminogen activator (uPA) , which, As previously described, eRNA released by endothelial cells upon cellular stress such as fluid shear stress has been attributed an essential role as the translator of shear stress into endothelial activation during the initial stages of collateral remodeling. However, eRNA is rapidly degraded by circulating RNases and could thus only unfold its actions over a short period of time . ArterioFurthermore, eRNA released by mast cells might also directly influence leukocyte adhesion, transmigration and cytokine/chemokine release such as of TNFα or MCP-1 during arteriogenesis and might thus amplify the budding local inflammatory response. TNFα is a key pro-inflammatory cytokine released by a variety of cell types including granulocytes, macrophages and smooth muscle cells and is able to enhance leukocyte adhesion, coagulation, and endothelial permeability ,86,87. IAt this point, it is interesting to note that eRNA administration in vitro was shown to prompt a shift in macrophage polarization towards the pro-inflammatory M1 phenotype with an upregulation of pro-inflammatory cytokines such as TNFα and a concurrent downregulation of anti-inflammatory cytokines . In the 165, NRP-1 and other hypoxia related factors such as HIF-1α was increased [The role of eRNA in other forms of vessel growth, namely vasculogenesis and angiogenesis, has not been studied in great detail so far. In vasculogenesis, tRNA and rRNA were found to stimulate the formation of new vessels and the leukocyte differentiation of embryonic bodies via increased VEGF signaling and ROS generation . VEGF sincreased . ROS forncreased , which increased . In angincreased ,110.The remodeling of pre-existing arteriolar collaterals during arteriogenesis is a complex process requiring the highly-coordinated interplay of different leukocytes to promote endothelial and smooth muscle cell proliferation and to ultimately establish perfusion. eRNA has been demonstrated to translate fluid shear stress into endothelial activation during arteriogenesis, further stimulating vWF release, PNA formation, mast cell degranulation and leukocyte recruitment, culminating in the beneficial process of arteriogenesis to promote perfusion recovery. In contrast to the beneficial role of eRNA in arteriogenesis, eRNA has been formerly established as a damaging or pathological factor in a variety of cardiovascular diseases based upon its modulation of endothelial cell and leukocyte function, whereby administration of RNase1 was demonstrated to serve as a tissue- and vessel-protective regimen. It remains to be established which particular molecular interactions and binding partners as well as which putative cellular receptors for eRNA appear to be responsible for its either adverse or beneficial functions in the cardiovascular system.