In the past few decades, non-alcoholic fatty liver disease (NAFLD) and heart failure with preserved ejection fraction (HFpEF) have become the most common chronic liver disease and the main form of heart failure (HF), respectively. NAFLD is closely associated with HFpEF by sharing common risk factors and/or by boosting systemic inflammation, releasing other secretory factors, and having an expansion of epicardial adipose tissue (EAT). Therefore, the treatments of NAFLD may also affect the development and prognosis of HFpEF. However, no specific drugs for NAFLD have been approved by the Food and Drug Administration (FDA) and some non-specific treatments for NAFLD are applied in the clinic. Currently, the treatments of NAFLD can be divided into non-pharmacological and pharmacological treatments. Non-pharmacological treatments mainly include dietary intervention, weight loss by exercise, caloric restriction, and bariatric surgery. Pharmacological treatments mainly include administering statins, thiazolidinediones, glucagon-like peptide-1 receptor agonists, sodium-glucose cotransporter 2 inhibitors, and metformin. This review will mainly focus on analyzing how these treatments may affect the development and prognosis of HFpEF. Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases without specific treatments and covers the disease spectrum from simple steatosis to non-alcoholic steatohepatitis (NASH), which can evolve into end-stage liver diseases –3. With Theoretically, NAFLD is associated with HFpEF in two layers. On the one hand, NAFLD and HFpEF share common risk factors. Several documented risk factors of NAFLD, including age, male sex, obesity, and T2DM –13, are However, there are no specific drugs approved for the treatment of NAFLD yet, management of NAFLD is currently achieved by employing non-specific non-pharmacological and/or pharmacological treatments. Non-pharmacological treatments mainly include dietary intervention, weight loss by exercise, caloric restriction, and bariatric surgery. Pharmacological treatments mainly include administering statins, thiazolidinediones, glucagon-like peptide-1 receptor agonists, sodium-glucose cotransporter 2 inhibitors, and metformin. These non-pharmacological and pharmacological treatments can effectively treat the liver in NAFLD/NASH patients –26. InteThis review mainly focuses on overviewing the possible effects of current interventions available for NAFLD on the development and prognosis of HFpEF.NAFLD and HFpEF are both systemic diseases , 30. GroNAFLD is characterized by excessive fat accumulation in hepatocytes without or with lobular inflammation and fibrosis and affects more than a billion people worldwide today , 32. NAFHFpEF has been defined as a multisystem disorder with multiple pathophysiological abnormalities. In the pathophysiology of HFpEF, left ventricular diastolic dysfunction plays a crucial and indispensable role . In addiDuring the pathogenies of NAFLD, increased amounts of lipids accumulate in the liver and lead to oxidative stress, cell death of hepatocytes, recruitment of inflammatory cells, and increased secretion of inflammatory factors . The immInflammatory factors play a key role in linking NAFLD and HFpEF. Meanwhile, NAFLD could also promote the development of HFpEF through the effects of other secretory factors such as angiotensinogen, leptin, and hepatokines. Angiotensinogen is mainly secreted by the liver and is the only precursor of the renin-angiotensin system . In the In addition to systemic inflammation and the effects of other secretory factors, EAT also plays a significant role in the association between NAFLD and HFpEF. In addition to energy storage, adipose tissue is also an endocrine organ that can secrete many proinflammatory adipokines in chronic systemic disorders . CompareNAFLD may also be associated with the development of HFpEF through other pathophysiological processes such as metabolic abnormalities and oxidative stress. The overproduction of glucose and triglycerides in NAFLD would cause hyperglycemia and hyperglyceridemia . HyperglIn summary, accumulating studies have indicated that a close association is between NAFLD and HFpEF. Therefore, the treatments against NAFLD are also likely to exert beneficial or harmful effects on the development and prognosis of HFpEF.The above evidence indicates the close association between NAFLD and HFpEF. Moreover, accumulating studies also show that the progression of NAFLD may result in the occurrence of HFpEF, probably by preventing blood flow back to the heart, inducing changes in hemodynamics, and releasing secretory factors.About 25% of the total blood returns to the heart through the liver . When liIn summary, NAFLD can lead to the occurrence of HFpEF through the above-mentioned mechanism. In this aspect, more direct evidence is needed to confirm the causal relationship and to facilitate discovering the detailed molecular pathways. Employing the power of emerging frontier technologies, such as multi-omics technologies and single-cell technologies, may help us to achieve these goals.At present, there is no specific drug for the treatment of NAFLD. Instead, some non-specific non-pharmacological and pharmacological treatments are currently applied . Some ofNon-pharmacological treatment is the cornerstone of NAFLD treatment, which mainly includes dietary intervention and weight loss induced by exercise, caloric restriction, and bariatric surgery. These non-pharmacological approaches are effective in the treatment of NAFLD and also showed promising results in delaying the development of HFpEF.A large number of NAFLD patients overconsume fructose and saturated fatty acids and have reduced intake of dietary fiber and omega-3-rich food , 90. ObeThe Mediterranean diet, characterized by a large consumption of plant foods, consists of vegetables and fruits, fish, whole grains, legumes, etc. , 92. ThiExcessive calorie intake and lack of exercise, which lead to obesity and related comorbidities, play a vital role in the development of NAFLD and NASH . LifestyWeight loss caused by exercise and caloric restriction has a dose-dependent treatment effect on the liver histology of NAFLD patients. Patients with ≥ 5% of total body weight loss can have an improvement in hepatic steatosis, and patients with ≥ 7% of total body weight loss can have significant improvement in steatosis and NAFLD activity score . More im2 can meet the indications for bariatric surgery , a member of the nuclear hormone receptor superfamily, is mainly expressed in adipose tissue. When PPAR-γ is activated, it can enhance lipogenesis, insulin sensitivity, and adipocyte differentiation . HoweverPioglitazone, a thiazolidinedione, has been investigated in multiple trials. In a randomized, double-blind, placebo-controlled trial, 101 patients with NASH and prediabetes or T2DM received pioglitazone 45 mg/d or placebo for 18 months, followed by pioglitazone treatment for 18 months . PioglitHowever, thiazolidinediones can lead to sodium retention and weight gain through anti-natriuretic effects on human proximal tubular cells. Thiazolidinediones can increase the risk of HF, which may be HFpEF or HFrEF . NonetheGlucagon-like peptide-1 (GLP-1) is a hormone produced mainly by intestinal L cells and is an incretin. GLP-1, which is secreted in response to food digestion, stimulates insulin secretion by pancreatic β cells and inhibits glucagon secretion by α cells . GLP-1 aLiraglutide, a long-acting GLP-1 receptor agonist, has shown beneficial effects on NAFLD/NASH in clinical trials. In a multicenter, randomized, double-blind, placebo-controlled trial, liraglutide decreased the levels of liver enzymes and improved the NAFLD activity score which included liver ballooning and lobular inflammation. . FurtherIn summary, GLP-1 receptor agonists show encouraging results in NAFLD and may reduce the risk of HFpEF in patients by improving insulin sensitivity, anti-inflammation, and improving cardiac function. Therefore, GLP-1 receptor agonists deserve further investigation in patients with HFpEF.de novo lipogenesis (Sodium-glucose cotransporter 2 (SGLT2) inhibitors inhibit the reabsorption of glucose in the proximal tubule of the kidney. SGLT2 inhibitors increase the excretion of glucose, thereby reducing blood glucose and improving insulin resistance . The impogenesis , 159. ThEmpagliflozin, an SGLT2 inhibitor, is an effective oral antidiabetic drug and has been explored for the treatment of NAFLD. In a clinical trial, NAFLD participants were randomly assigned to empagliflozin (10 mg/day) (N = 43) or placebo (N = 47) for 24 weeks . The resMetformin, a biguanide drug, is a first-line antidiabetic medicine that can improve insulin sensitivity, and regulate the utilization of glucose by the liver. Moreover, metformin has additional effects on metabolism, such as improvements in hepatic gluconeogenesis and fatty acid metabolism , 169. GrIn a clinical trial, NAFLD patients were treated with metformin (2000 mg/day) for 48 weeks . Liver bIndividuals with NAFLD account for approximately a quarter of the world’s population. Individuals with NAFLD have a higher risk of CVD such as HFpEF. Common risk factors, thicker EAT, and release of secretory factors play an important role in the close association between NAFLD and HFpEF. Hence, the treatments of NAFLD, being able to control common risk factors, decrease the effects of secretory factors, or reduce EAT accumulation, should also have a therapeutic effect on HFpEF.This review summarizes the major non-pharmacologic and pharmacologic strategies currently applied in NAFLD and their impact on the development of HFpEF. In addition to showing encouraging results in NAFLD, non-pharmacologic therapies can also protect against obesity and diabetes, improve systemic inflammation, and decrease EAT accumulation. Non-pharmacologic therapies can even directly improve the condition of patients with HFpEF. Some drugs also show the potential effects of reducing the risk of HFpEF while showing a therapeutic effect on NAFLD, such as statins, GLP-1 receptor agonists, SGLT2 inhibitors, and metformin. However, some drugs have uncertain effects on the risk of HFpEF. Therefore, the impact of these drugs such as thiazolidinediones on the development of HFpEF needs to be rigorously assessed in subsequent clinical trials.Although there is a close relationship between NAFLD and HFpEF, the potential mechanism between NAFLD and HFpEF is still unclear and needs to be extensively studied. HFpEF is a systemic disease and the potential mechanisms of HFpEF itself have not been fully clarified so far. Based on a clearer understanding of the mechanism underlying HFpEF, it can be easier to deduce which NAFLD drugs have therapeutic effects on HFpEF. More importantly, uncovering the mechanism of HFpEF should also boost the development of new drugs for HFpEF. In addition, some drugs, such as GLP-1 receptor agonists, have shown encouraging results in preclinical studies. Therefore, authoritative clinical trial studies are needed to further verify whether these drugs have positive effects on HFpEF. Last but not least, as the mechanisms underlying both NAFLD and HFpEF are complex and may vary between different persons, personalized therapies should be applied to different populations.ZY and RT wrote the original manuscript. X-JZ, JC, HL, and Z-GS critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.


Arabidopsis PD proteome and have been shown to be present in primary PD at cytokinesis. It has been suggested that RTN proteins form protein complexes with proteins in the PD plasma membrane and desmotubule to stabilize the desmotubule constriction and regulate PD aperture. Viral movement proteins (vMPs) enable the transport of viruses through PD and can be ER‐integral membrane proteins or interact with the ER. Some vMPs can themselves constrict ER membranes or localize to RTN‐containing tubules; RTN proteins and vMPs could be functionally linked or potentially interact. Here we show that different vMPs are capable of interacting with RTN3 and RTN6 in a membrane yeast two‐hybrid assay, coimmunoprecipitation, and Förster resonance energy transfer measured by donor excited‐state fluorescence lifetime imaging microscopy. Furthermore, coexpression of the vMP CMV‐3a and RTN3 results in either the vMP or the RTN changing subcellular localization and reduces the ability of CMV‐3a to open PD, further indicating interactions between the two proteins.Plant reticulon (RTN) proteins are capable of constricting membranes and are vital for creating and maintaining tubules in the endoplasmic reticulum (ER), making them prime candidates for the formation of the desmotubule in plasmodesmata (PD). RTN3 and RTN6 have previously been detected in an  Different vMPs are capable of interacting with RTN3 and RTN6. Coexpression of the vMP CMV‐3a and RTN3 results in proteins changing subcellular localization and reduces the ability of CMV‐3a to open plasmodesmata. The plant endoplasmic reticulum (ER) is a multifaceted organelle with a variety of functions  . Examples include the remorin proteins REM1.2 and REM1.3 , as the nonmutated fragment can bind to Cub without other interacting proteins present; an empty vector (−) was used as a negative control Figure . In addiTo further test such interactions between RTNs and vMPs in vivo in a plant background, Förster resonance energy transfer (FRET) measured by donor excited‐state fluorescence lifetime imaging microscopy (FLIM) Becker,  was usedIn contrast to the mbSUS system, the in planta FRET‐FLIM analysis showed interactions of GFP‐PMTV‐TGB2 with mRFP‐RTN3 and mRFP‐RTN6. To clarify this discrepancy, GFP‐PMTV‐TGB2 was coexpressed with mRFP‐RTN3 or mRFP‐RTN6, and coimmunoprecipitation followed by western blotting was carried out Figure . Here, bArabidopsis) RTNs and vMPs in Arabidopsis thaliana, rather than a heterologous system, stable transgenic plants were generated expressing mRFP‐RTN3 together with CMV‐3a‐GFP, because cucumber mosaic virus can infect Arabidopsis. Expression of both proteins was observed in the hypocotyl and the root in approximately 60% of all cells in T1 plants 5 days after germination but could only be observed in fewer than 1% of the cells 10 days after germination and was not detectable in the T2 generation. Interestingly though, in all cells displaying both constructs the localization of either one or the other of the two proteins changed . However, in 32% of cells, CMV‐3a‐GFP additionally localized to puncta on the ER  as a negative control are shown. (a) The raw FRET‐FLIM data. (b) Pseudo‐coloured lifetime maps display the lifetime values for each point within the region of interest. (c) The distribution of lifetimes across the entire image with blue shades representing longer GFP fluorescence lifetimes than green ones. (d) Representative decay curves of a single point  with an optimal single exponential fit, where χ2 values from 0.9 to 1.2 are considered an excellent fit to the data pointsClick here for additional data file.Figure S2 FRET‐FLIM control with noninteracting endoplasmic reticulum (ER) proteins. Interactions were tested between RTN6 and RTN3, respectively, with the ER‐localized protein Annexin 4 (ANNAT4). (a) Example data for GFP‐RTN3 alone as a negative control or GFP‐RTN3 with mRFP‐ANNAT4. Average lifetimes ± SD are given in brackets. (b) Statistical analysis for GFP‐RTN3 and GFP‐RTN6 without and with mRFP‐ANNAT4. ANNAT4 shows no interaction with RTN3 or RTN6 (n.s.). Significance was analysed by the Kruskal–Wallis test Click here for additional data file.Figure S3 Coimmunoprecipitation assay to determine protein–protein interactions for PMTV‐TGB2 with RTN3 and RTN6. mRFP‐RTN3 or mRFP‐RTN6 was expressed in tobacco epidermal leaf cells alone or together with GFP‐PMTV‐TGB2. Coimmunoprecipitation was carried out using an anti‐GFP antibody linked to agarose . As a negative control mRFP‐RTN3 and mRFP‐RTN6 were incubated with GFP‐Trap beads without GFP‐PMTV‐TGB2. Proteins were separated by 12% SDS‐PAGE and probed with anti‐RFP antibodies (a) to detect the prey (RTN3/RTN6) as well as anti‐GFP antibodies (b) to show that the bait had been precipitated (PMTV‐TBG2). (c) The input for the prey mRFP‐RTN3/6Click here for additional data file.Figure S4 Localization of CMV‐3a and RTN3 individually and together in Arabidopsis cells. Stable Arabidopsis plants expressing CMV‐3a‐GFP or GFP‐RTN3 were created and the localization of both proteins throughout the cell was visualized. Representative example stacks together with 3D reconstructions are shown. CMV‐3a‐GFP alone localized to plasmodesmata (PD) and GFP‐RTN3 alone to the endoplasmic reticulum (ER). Slice depth is given. For comparison, a representative cell expressing both CMV‐3a and RTN3 is shown. Here CMV‐3a‐GFP was found in PD but also distributed in puncta over the ER. Size bars = 5 μmClick here for additional data file.Figure S5 Transient coexpression of CMV‐3a‐GFP and mRFP‐RTN3 in tobacco epidermal leaf cells. CMV‐3a‐GFP (green) was coexpressed in tobacco epidermal leaf cells with mRFP‐RTN3 (magenta) and the localization was analysed in n = 4 biological replicas with 10 cells each. Representative example images are shown in (a–f). In this system, CMV‐3a‐GFP localized to plasmodesmata (PD)  only but not the endoplasmic reticulum (ER) (c) in 68% of cells visualized. In 32% of cells, CMV‐3a‐GFP was additionally also localized in dots on the ER , labelling both the peripheral ER and some PD (f). Size bars = 5 μm for (a–d) and = 10 μm for (f)Click here for additional data file.Text S1 Materials and methods used in this workClick here for additional data file.


We hypothesized that blood flow, initiated to the lungs at birth, alters mechanical load on the PV and promotes ECM maturation. In the −/−Adamts5 PV, there was an increase in localization of the proteoglycan proteases ADAMTS1, MMP2, and MMP9 that correlated with reduced Versican (VCAN). At birth, Decorin (DCN), a Collagen I binding, small leucine-rich proteoglycan, exhibited complementary stratified localization to VCAN in the wild type at P0 but colocalized with VCAN in −/−Adamts5 PV; concomitant with the phenotypic rescue at P7, the PVs in −/−Adamts5 mice exhibited stratification of VCAN and DCN similar to wild type. This study indicates that increased mechanical forces on the PV at birth may activate ECM proteases to organize specialized ECM layers during cardiac valve maturation. Increased mechanical forces on developing cardiac valves drive formation of the highly organized extracellular matrix (ECM) providing tissue integrity and promoting cell behavior and signaling. However, the ability to investigate the response of cardiac valve cells to increased mechanical forces is challenging and remains poorly understood. The developmental window from birth (P0) to postnatal day 7 (P7) when biomechanical forces on the pulmonary valve (PV) are altered due to the initiation of blood flow to the lungs was evaluated in this study. Grossly enlarged PV, in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from postnatal day 0 (P0) to P7; the  Adult cardiac valves comprise a specialized, stratified extracellular matrix (ECM) that gives structural support to ensure unidirectional blood flow. In addition to imparting the mechanical properties of tissues, the ECM instructs cell behavior and signaling ,2. The rThe provisional ECM within endocardial cushions, the precursors of the cardiac valves, is rich in hyaluronic acid and the aggregating proteoglycan Versican (VCAN). This early ECM provides an environment conducive to cell migration and proliferation. As the embryo grows and mechanical forces are increased, the provisional ECM is remodeled by synthesis of fibrillar collagens, assembly of elastin, and proteolytic cleavage and clearance of VCAN . The mat−/−Adamts5) valve phenotype, indicating that cleavage and clearance of VCAN are essential for cardiac valve maturation [Transitioning of the ECM requires ECM proteases and their cleaved substrates which are components of the degradome, a relatively understudied subset of the ECM proteome. Activation of ECM proteases can elicit a transformative impact on the ECM architecture through proteolytic cleavage of their ECM substrates for bioassembly as well as activation, degradation, and clearance . Cleavagturation . There ituration ,25,26; hturation ,28,29,30−/−Adamts5 aortic valves (AV) did not exhibit significant morphological changes during this timeframe. Shortly after birth, the closure of the ductus arteriosus and foramen ovale routes blood to the lungs through the PV, thereby increasing its mechanical load. Coincident with birth, ECM proteoglycanases ADAMTS1, MMP2, and MMP9 exhibited increased expression profiles in the in the PVs of −/−Adamts5 mice, indicating a potential to compensate for the loss of ADAMTS5. These data indicated that ECM proteoglycanases may be amplified in response to increased mechanical load at birth to promote specialized ECM formation during postnatal cardiac valve maturation.This study focuses on our observation that the grossly enlarged PVs in mice deficient in the proteoglycan protease ADAMTS5, exhibited a transient phenotypic rescue from P0 to P7; the To quantify the cusps and hinge regions of the PV as well as the AV, histological sections over a depth of 30 μM were quantified for each heart; i.e., the widest portion of the anterior, right, and left cusps of the PV, and the right coronary, left coronary, and non coronary cusps of the AV were measured. The narrowest part of the hinge regions was also measured and quantified. Measurements were averaged then used for graphs and statistical analysis. Of note, hearts that exhibited a bicuspid phenotype or had other cardiac anomalies such as a ventricular septal defect were not utilized in this study. Amira™ 3D 2021.1  was used to generate three dimensional (3D) reconstructions from 5 μm-thick paraffin sections. For each P0 or P7 reconstruction, approximately 100–140 sections per valve were used. −/−Adamts5 and littermate controls . Digital images of Adamts5−/− and WT heart sections were acquired at identical confocal settings using the Leica TCS SP5 or SP8 AOBS Confocal Microscope System. Standard histological procedures were used . Decorin−/−Adamts5 (−/−), at timepoints, P0, P7 and 1 mo. When Anova assumptions of normality and equal variances could not be met, a Kruskal–Wallis test was used to compare groups. Adjusted p-values are indicated for each group comparison, with ⍺ = 0.05. Anova and Kruskal–Wallis tests revealed significant differences between groups. GraphPad was utilized to generate graphs. In graphs, each symbol represents data from a single mouse (n = 1) for evaluation of cardiac valve cusp and hinge regions. The colored bar height represents the mean of the measurements, with small bars above and below indicating the standard deviation. The animal numbers were assigned randomly prior to genotyping, and this served to blind the investigators until the grouping for statistical analysis. To ensure the fidelity of the −/−Adamts5 homozygous phenotype −/+Adamts5 het X −/+Adamts5 het matings were performed; this approach also generated −/−Adamts5 and +/+Adamts5 littermate controls. The number of mice utilized in this study represents a minimum of 10 different litters per timepoint to obtain an appropriate n for this study for each developmental stage analyzed. For quantification of the valve cusp and hinge width, data were analyzed using GraphPad Prism 9.1 (GraphPad Software Inc.). A one-way analysis of variance (Anova) or the nonparametric Kruskal–Wallis test was used to determine the differences between the groups wild type (+/+) and −/−Adamts5 mice at P0 (birth), P7, and 1 mo were analyzed. Histological sections and 3D reconstructions of the WT at P0 (n = 6) and P7 (n = 3) as well as −/−Adamts5 PV at P0 (n = 7) and P7 (n = 4) were generated and analyzed  and in adult mice (>6 mo) . In thisanalyzed . HistoloP0 to P7 . CompariP0 to P7 D, and P7−/− mice H highlig−/−Adamts5 at P0 (n = 7), P7 (n = 4), and 1 mo (n = 6) as well as WT mice at P0 (n = 6), P7 (n = 3), and 1 mo (n = 6), revealed that the PV exhibited a significant transient rescue of the enlarged phenotype and was indistinguishable from WT littermates at P7  were not significantly larger than WT (n = 7) at P0 or P7 . Of note the −/−Adamts5 hinge regions of the PV and AV also differed in their phenotypic progression , ). However, as postnatal development progressed to 1 mo, both the AV and PV cusps from −/−Adamts5 mice (n = 5) were significantly larger than WT . Quantification of the cusp and hinge regions of the PV and AV of es at P7 A,B. Alth PV cusp A or hing PV cusp B compareenlarged , indicatgression B,D. Ther−/−Adamts5 PV may be due to proteolytic cleavage and clearance of VCAN from proteoglycanases other than ADAMTS5 that were upregulated at birth in the −/−Adamts5 mice. We reasoned that the transient phenotypic correction of the −/−Adamts5 PV exhibited reduced ADAMTS1 localization compared to WT  Decorin (DCN), named because it decorates Collagen I, was evident in the venticularis of the cusps, as well as the anchor and hinge regions by P0 in both the WT and s5−/− PV A–F. The s5−/− PV A,B than amts5−/− D,E. In tn the WT G–L; i.e.−/−Adamts5 PV which correlated with the P7 phenotypic rescue of the enlarged −/−Adamts5 PV. The immunolocalization of DCN and VCAN indicated that the mature fibrosa and spongiosa layers, respectively, were distinct at P7 in both the WT and −/−Adamts5 mice were resolved to a sculpted appearance indistinguishable from WT at P7. This apparent phenotypic rescue of the −/−Adamts5 PV may be the result of increased ECM proteoglycanase activity in the VICs and endocardium after birth; of note, these proteoglycanases were reduced in the −/−Adamts5 PV compared to WT prior to birth. ADAMTS1, MMP2, and MMP9, that also cleave VCAN, may compensate for the loss of ADAMTS5 by decreasing VCAN levels thereby restoring normal PV morphology. The apparent increase in proteoglycanase activity at birth in the PV may be the result of increased blood flow to the lungs. It is likely that ADAMTS-VCAN-substrate interactions are activated by mechanotransducive pathways that drive cardiac valve maturation  with reduced proliferation of adjacent myocardial cells. Loss of endocardial expression of Adamts19 also perturbs shear stress signaling and leads to increased mesenchymal cells and proteoglycan deposition in cardiac valves [Adamts5 are upregulated in the −/−Adamts19 valvular endocardium [Adamts5 and Adamts19 are direct targets of the mechano-induced Klf2, but they are integrated within mechanosenstive pathways localized to the endocardium. Mechanosensors of shear force such as Krüppel-like factor 2 (Klf2/4) and endothelial nitrous oxide synthetase are expressed by endocardial cells of developing valves and their genetic perturbation in mice disrupts cardiac valve development ,34,35,36c valves . Klf2 toocardium . It is nDuring valve development, mechanical signals are transmitted from the endocardium to the underlying VICs, in part through incorporation of nitrous oxide. Mechanosensitive ion channels also regulate endothelial response to shear flow ,39. By mThe limitations of this study include the assumption that mechanical load changes on the PV when blood circulation to the lungs is initiated at birth. Proteoglycans are also regulated by transcription, not evaluated here. ADAMTS and MMP activity is inhibited by TIMP3 and not investigated in this study. While the known repertoire of substrates for the ADAMTS proteoglycanases is limited, our understanding of their role in development and disease will be significantly advanced as additional in vivo substrates are identified ,42,43.This study indicates that proteolytic cleavage of proteoglycans may be induced by altered mechanical load to promote ECM maturation during cardiac valve development. In the murine model of ADAMTS5 deficiency, loss of this protease results in enlarged cardiac valves that contain excess VCAN; the increased localization of other proteoglycanases in the maturing valve cusps corrects the morphological defect by P7 and correlates with ECM organization, specifically VCAN in the spongiosa and DCN in the Collagen I-rich fibrosa layer. This study indicates that proteoglycanase activity may be an essential downstream mechanism of mechanotransduction that is important for normal cardiac valve maturation. Since increases in proteoglycans are evident in diseased cardiac valve tissues, understanding mechanisms that reduce proteoglycan content is essential for developing effective therapeutic strategies to treat cardiac valve diseases.


PSA and F/T PSA retain significant diagnostic value for CSPCa in patients with TURP history. PI-RADS v2.1 is reliable for detecting peripheral-zone CSPCa but has limited diagnostic value when assessing transitional zone lesions.To explore the diagnostic value of the Prostate Imaging–Reporting and Data System version 2.1 (PI-RADS v2.1) for clinically significant prostate cancer (CSPCa) in patients with a history of transurethral resection of the prostate (TURP), we conducted a retrospective study of 102 patients who underwent systematic prostate biopsies with TURP history. ROC analyses and logistic regression analyses were performed to demonstrate the diagnostic value of PI-RADS v2.1 and other clinical characteristics, including PSA and free/total PSA (F/T PSA). Of 102 patients, 43 were diagnosed with CSPCa. In ROC analysis, PSA, F/T PSA, and PI-RADS v2.1 demonstrated significant diagnostic value in detecting CSPCa in our cohort (AUC 0.710 (95%CI 0.608–0.812), AUC 0.768 (95%CI 0.676–0.860), AUC 0.777 (95%CI 0.688–0.867), respectively). Further, PI-RADS v2.1 scores of the peripheral and transitional zones were analyzed separately. In ROC analysis, PI-RADS v2.1 remained valuable in identifying peripheral-zone CSPCa (AUC 0.780 (95%CI 0.665–0.854;  Prostate cancer (PCa) is one of the most common malignancies among men worldwide, leading to numerous cancer-related deaths ,2. To acPSA alone as a diagnostic biomarker is insufficient to distinguish PCa from benign prostatic diseases . StudiesBenign prostatic hyperplasia (BPH) is a progressive disease commonly seen in elderly men and is often addressed by a transurethral resection of the prostate (TURP). We came to notice a certain group of patients in clinical practice who had a surgical history of TURP due to BPH and were suspected of PCa during follow-up visits. On account of the removal of transitional zone tissue during the TURP and possible adenoma regrowth during the follow-up period, serum PSA or F/T PSA testing may be influenced by surgical history. Moreover, with no consensus established yet, whether mpMRI retains diagnostic value also remains uncertain, and patients are, in this case, assigned to prostate biopsies based mainly on clinicians’ judgment, leading to a certain number of unnecessary invasive procedures or delayed diagnoses. Given this situation, we hypothesize that PI-RADS v2.1 may be an effective tool in identifying patients suspected of PCa who require an immediate biopsy.Therefore, in this present study, we aim to investigate the diagnostic values of PI-RADS v2.1 scores for CSPCa in a cohort of patients with a history of TURP.In this retrospective study, consecutive patients who had undergone a 12-core transrectal ultrasound (TRUS)-guided prostate biopsy with previous TURP history at our department between October 2014 and August 2020 were recruited. PSA is reported to drop significantly within 3–6 months after TURP , while hAll mpMRI included T2W, DW, and DCE imaging sequences. Two experienced genitourinary radiologists blinded to the clinical details reviewed and reported readouts following the standards of PI-RADS v2.1 ,8. SinceAll patients underwent a 12-core transrectal ultrasound (TRUS)-guided transperineal prostate biopsy. The well-designed biopsy template covers the bilateral anterior TZ, posterior TZ, anterior horn of PZ, anterior lateral PZ, posterior lateral PZ, and posterior medial PZ. The biopsy template is shown in p < 0.05. We used the Mann–Whitney rank sum test for nonparametric variables and Fisher’s exact chi-square test for categorical variables. Receiver operating characteristic (ROC) analyses were performed using biopsy results as the gold standard to reflect the diagnostic performance of PSA, F/T PSA, and PI-RADS v2.1. ROC curves were compared using the DeLong test. The area under the curve (AUC) and Youden’s index were calculated. Logistic regression analyses were conducted to explore the predictive values of the variables, and the odds ratios (ORs) were computed to quantify the predictive ability of the factors.Statistical analyses were performed using SPSS version 26.0. Statistical significance was set as p = 0.000), higher PSA levels (p = 0.006), and lower F/T PSA (p = 0.000).A total of 102 patients were included in the study. The median age was 73.5 years , the median time after TURP was 8 years (IQR 4–10.25 years), the median PSA level was 12.3 ng/mL (IQR 7.41–19.26 ng/mL), and the median F/T PSA was 0.15 (IQR 0.11–0.20). In all, 56 patients were diagnosed with PCa, among which 43 patients had CSPCa, while a Gleason score of 3 + 3 = 6 was present in the remaining 13. ROC curves were constructed to determine the diagnostic value of PSA, F/T PSA, and PI-RADS v2.1 in our cohort . The areIn univariate logistic regression analysis, age, PSA, F/T PSA (<0.135 vs. ≥0.135), and PI-RADS v2.1(≥3 vs. <3) showed significant associations with the biopsy results. However, in multivariate logistic regression analysis, only age, F/T PSA, and PI-RADS v2.1 remained independent predictors for CSPCa .Among the 43 patients with CSPCa biopsy results, 31 had CSPCa detected from both PZ and TZ cores, 11 had CSPCa detected only in the PZ, and 1 had CSPCa detected only in the TZ. The PI-RADS v2.1 TZ score and the PI-RADS v2.1 PZ score were proposed and analyzed . There wp < 0.001)). At a cutoff value of ≥3, PI-RADS v2.1 PZ had the sensitivity, specificity, PPV, and NPV of 90.5%, 40.0%, 51.3%, and 85.7%, respectively. However, in the transitional zone, PI-RADS v2.1 TZ demonstrated no diagnostic value for CSPCa ).In ROC analysis , PI-RADSp = 0.002). As for the transitional zone, PI-RADS TZ scores of 2, 3, 4, and 5 showed no significant odds ratio compared to no suspected lesions by PI-RADS v2.1. PI-RADS TZ score ≥3 showed limited significance for predicting CSPCa .In univariate logistic regression analysis , compareBenign prostatic hyperplasia (BPH) is a condition with high prevalence among aged men; 50% of men with BPH develop LUTS that require medical intervention . TransurPSA levels can be affected by prostate volume or the presence of BPH or prostatitis. Aus et al.  reportedCatalona et al.  reportedPI-RADS v2.1 is a structured reporting system for standardizing the mpMRI results for detecting PCa. Our study revealed that PI-RADS v2.1 is a reliable tool for CSPCa detection in patients with a history of TURP. A cutoff at PI-RADS score ≥4 resulted in 88.4% of CSPCa cases found and 57.6% of unnecessary biopsies avoided. To further explore the value of PI-RADS V2.1, we reviewed the imaging scores of two prostatic zones separately. The results showed that PI-RADS V2.1 remains reliable in identifying CSPCa in the PZ. Rudolph et al.  reportedConcerning the TZ, the performance of CSPCa detection was limited with PI-RADS v2.1 scores. For lesions with a PI-RADS v2.1 score of 2, 3, 4, and 5, CSPCa detection rates were reported to be 7.4%, 8.3%, 40.0%, and 61.7%, respectively, in the general biopsy patient population , which dTo our knowledge, our study is the first study to probe into the diagnostic tests for CSPCa in patients with TURP history. This study has some limitations. First, it is a retrospective, single-center study with a relatively small sample size. Clinical characteristics of our cohort show great heterogeneity, especially in the time after TURP and PSA levels, which may reduce the representativeness of our study cohort. The small sample size limited the reliability of subgroup analysis. Studies ,29 have Our study reveals that PSA and F/T PSA retain significant diagnostic value for CSPCa in patients with TURP history. PI-RADS v2.1 is reliable for detecting CSPCa in the PZ but has limited diagnostic value when assessing TZ lesions.


However, its implication in type-2 diabetic nephropathy (T2DN) is still unexplored. Here, we detected progressive activation of Smad3 but reduction of Smad7 in db/db mice during T2DN development. Therefore, we optimized the dosage and the combination ratio of AANG to achieve a better rebalancing Smad3/Smad7 signaling for treatment of T2DN. Unexpectedly, preventive treatment with combined AANG from week 4 before the development of diabetes and T2DN effectively protected against the onset of T2DN. In contract, these inhibitory effects were lost when db/db mice received the late AANG treatment from 12-24 weeks. Surprisingly, preventive treatment with AANG ameliorated not only T2DN but also the primary disease type-2 diabetes (T2D) with relative normal levels of fasting blood glucose and HbA1c, and largely improving metabolic abnormalities especially on insulin insensitivity and glucose tolerance in db/db mice. Mechanistically, AANG effectively prevented both Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in the diabetic kidney in vivo and advanced glycation end-products (AGE) stimulated tubular epithelial mTEC cells in vitro. More importantly, we uncovered that preventive treatment with AANG effectively protected against diabetic-associated islet injury via restoring the β cell development in db/db mice. Taken together, we discovered that the early treatment with combined AANG can effectively protect against the development of T2D and T2DN via mechanism associated with protection against Smad3-depenedent islet injury.Diabetic nephropathy (DN) is a major cause of end-stage kidney disease, where TGF-β1/Smad signaling plays an important role in the disease progression. Our previous studies demonstrated a combination of Traditional Chinese Medicine derived Smad7 agonist Asiatic Acid (AA) and Smad3 inhibitor Naringenin (NG), AANG, effectively suppressed the progression of renal fibrosis  Diabetes mellitus affects 346 million people worldwide and 582.5 thousand Hong Kong population with 10-20% mortality due to kidney failure . Its onset and progression are multifactorial but largely due to the development of systemic insulin resistance and insufficient insulin secretion in vitroIn patients with T2DN, TGF-β/Smad3 signaling is highly activated, which is companied with the loss of its inhibitor Smad7, resulting in significant glomerular and tubulointerstitial fibrosis in vitro. Here, we successfully developed an AANG formula that can effectively rebalance renal TGF-β/Smad signaling and thus prevents the development of T2DN. Unexpectedly, we discovered that AANG protected db/db mice from diabetic islet loss via promoting β cell development, which markedly decelerated the development of type-2 diabetes (T2D), thereby preventing T2DN. Thus, AANG may represent as a novel, safe and effective strategy for T2D and T2DN prevention by effectively rebalancing TGF-β/Smad signaling.Traditional Chinese medicine (TCM) has been widely used for the treatment of diabetes and its complications in China. We have recently reported that Asiatic acid (AA) is a Smad7 agonist and Naringenin (NG) is a Smad3 inhibitor AA is a HLPC-purified product (95%) purchased from Guangxi Changzhou Natural Pharmaceutical Co. Ltd . NG is obtained from Shanxi Huike Botanical Development Co. Ltd  with the HLPC-purity of 98%. The db/db mice were treated with oral AA (30 or 300 mg/kg/day), NG (300 or 3000 mg/kg/day), or combination of AA and NG (AANG in 1:10 ratio) by daily food-intake from the pre-diabetic age of 4 to 24 weeks (preventive treatment) or in the established T2D and T2DN mice from the age of 12 to 24 weeks (intervention treatment). Untreated db/db and db/m mice served as control groups. Finally, all mice were sacrificed via injection of ketamine/xylene. All studies were approval by the Animal Experimentation Ethics Committee, the Chinese University of Hong Kong and the experimental methods were carried out in accordance with the approved guidelines.Blood glucose levels were measured by Accu-Chek glucose meter (Roche Diagnostics) after the mouse fasting for 6 hours as our previous study The glucose tolerance test (IPGTT) was performed as previously described Kidneys sections were fixed in 4% paraformaldehyde, stained with the Periodic Acid-Schiff (PAS) method. Immunohistochemistry was carried out by using a microwave-based antigen retrieval technique. The primary antibodies used in the current as previous described The mouse tubular epithelial cells (mTEC) were cultured in DMEM/F12 , supplemented with 10% FBS  and 1% antibiotic/antimycotic solution . The mouse mesangial cells (mMES)  was maintained in a 3:1 mixture of DMEM and Ham's F-12 medium containing 5% FBS, and 1% antibiotic/antimycotic solution in vitro following our previous procedure 4/well) were seeded on a 96-well plate and serial concentrations of AA, NG, or their combination with indicated concentration were added on the next day. After 24-hours of treatment, 30μl methyl-thiazoldiphenyl tetrazolium (MTT) (5 mg/ml) were added to each well and incubated for 2 h at 37°C. The MTT solution was then replaced by 100 μl dimethyl sulfoxide in each well and measured with a microtiter-plate reader (BioTek) at 540 nm and all data were calculated as percentage against the control.The MTT assay was used to determine the cytotoxicity of AANG on both mTES and mMES cells Protein from renal cortex and cultured renal cell lines was extracted using the radio immunoprecipitation assay (RIPA) lysis buffer. Western blot analysis was performed as pervious described -ΔCt method and is expressed as the mean ± S.E.M.Total RNA was extracted from the renal cortical tissues and cultured renal cell lines. Real-time PCR was carried out with machine  by using IQ SYBR Green Supermix reagent (Bio-Rad) 24-hours urinary samples of mice were collected in metabolic cages every 4 weeks from the age of 4 weeks to 24 weeks (for prevention study) and 12 weeks to 24 weeks (for intervention study). Urinary microalbumin was measured by competitive ELISA according to the manufacturer's instructions . Levels of blood and serum creatinine were determined accordingly with enzymatic method . Urinary albumin excretion was expressed as total urinary albumin/creatinine (μg/mg) as previously study. All measurements were performed as previously described Commercial kits Stanbio-ALT/SGPT Liqui-UV® Test and AST/SGOT Liqui-UV® Test from Stanbio Laboratory were used. QuantiChrom™ Lactate Dehydrogenase Kit (DLDH-100) used for LDH detection was purchased from BioAssay as previous studies Immunofluorescence staining was performed with 5 μm PLP-fixed frozen sections. Primary antibodies were incubated overnight, then followed by RHO-conjugated anti-mouse and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit secondary antibodies, respectively All the data are expressed as mean ± SEM. Statistical analyses were performed with one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison from GraphPad Prism 5.0 . In addition, a repeated analysis ANOVA analysis was used for albumin excretion, body weight, fasting blood glucose analysis.Figure 1). We first optimized AANG, the combined formula of AA and NG, specifically for targeting T2DN by conducting cytotoxicity screening assays in vitro and in vivo. The optimal ratio of AA and NG individually on cultured mTEC and mMES cells was determined by MTT assay. We identified that 20µM of AA and 200µM of NG were safe for either individual or combined usages, showing by their insignificant cytotoxicity on the epithelial mTEC and mesangial mMES cells at 24h in vitro . Therefore, we combined AA and NG in the ratio of 1:10 and tested the safety and therapeutic effect in vivo. The 10-weeks-old db/m and db/db mice were treated with AANG at the indicated dosages. Interestingly, compared to control db/db mice, high dose of AANG (300mg AA+3000mg NG) showed protective effects on diabetic liver injury according to their significant reduction of serum ALT and AST levels without alteration of serum LDH . Importantly, this dosage of AANG also showed to effectively protect db/db mice from the development of diabetes and T2DN by significantly lowering fasting blood glucose and microalbuminuria when compared to the untreated control as well as their monotherapy in vivo . Thus, we identified 300mgAA+3000mgNG as an optimal AANG formula for db/db mice.We have previously demonstrated that the combination treatment of AA and NG can effectively rebalance the Smad3 and Smad7 signaling in the acute kidney injury model Figure , preventive treatment with the combination of AA (300mg/kg/day) and NG (3000mg/kg/day) significantly protected db/db mice from the development of microalbuminuria. Surprisingly, we also detected that the provoked diabetic index HbA1c level in db/db mice was also markedly reduced in the preventive -treated db/db mice to the normal level as db/m mice . In addition, the metabolic abnormalities of db/db mice were significantly ameliorated by AANG preventive treatment (db/db+AANG) by dramatically improving the insulin sensitivity and glucose metabolism  and reduced glucose tolerance, body weight and fasting blood glucose levels . These findings unexpectedly uncovered a promising preventive effect of AANG on T2DN as well as its primary disease T2D.After determining the safe and effective dosage of AANG combination, we first examined whether AANG has preventive effect on T2D and T2DN. Since TCM can effectively protect against a number of inflammatory diseases Supplementary , the 12-weeks-treatment with the combination of AA (300mg/kg/day) and NG (3000mg/kg/day) from the age of week 12 was not able to improve the diabetic renal injury compared to their control group (db/db). No significant changes were also detected in the diabetic index HbA1c, insulin sensitivity (IPITT), body weight, and fasting blood glucose level in the AANG-treated db/db mice compared to their untreated db/db group (Supplementary ), although a significant improvement in glucose tolerance (IPGTT) was achieved in AANG-treated db/db mice (Supplementary ). By comparing with the clinical outcomes achieved in the preventive treatment, results from the intervention therapy suggested that AANG is more suitable for the early prevention rather than the late treatment for the established T2D and T2DN. Thus, the working mechanism of AANG for T2DN prevention was intensively elucidated in this study.In this study, we also examined the therapeutic potential of AANG on established T2DN by treating db/db mice with AANG from week 12 to week 24. According to Figure ). We thus examined the mechanisms of preventive versus intervention therapy with AANG on Smad3-mediated renal fibrosis. As shown in Figure , western blot analysis detected that Smad3 was highly activated in the kidney of db/db mice compared to db/m mice, which was markedly suppressed by the AANG preventive-treatment (P) but not by the intervention treatment (T). In contrast, AANG preventive treatment significantly enhanced renal Smad7 expression Figure C and D, resulting in inhibition of renal fibrosis by reducing collagen I and α-SMA expression in the diabetic kidney of db/db mice Figure A-D. These observations were also confirmed by real-time PCR at the mRNA levels. Interestingly, preventive treatment with AA, NG or AANG produced a differential effect on upregulation of renal Smad7 while inhibiting Smad3 mRNA expression .It is well established that inflammatory NF-kB signaling plays a promoting role in T2DN, which can be suppressed by Smad7 overexpression in vitro study in mTEC cells. In line with the findings from in vivo study , we demonstrated that AANG pre-treatment markedly upregulated Smad7 while significantly inhibiting AGE-induced Smad3 as well as NF-κB activation, thereby inhibiting collagen I and α-SMA expression in the mTEC in vitro . Taken together, these findings clearly demonstrated that AANG preventive treatment effectively inhibits Smad3-mediated renal fibrosis and NF-κB -driven renal inflammation, thereby protecting the kidney from the development of T2DN in db/db mice.To further examine the mechanisms by which AANG inhibits T2DN, we performed an Figure  preventive treatment with AA, NG, and AANG significantly blocked Erbb4-IR and LRNA9884 expression in the diabetic kidney. Interestingly, compared to monotherapy with AA or NG, combinational therapy with AANG produced a synergistic effect on the LRNA9884 suppression in db/db mice .We have also previously found that Smad3-mediated T2DN via a number of long noncoding RNAs (lncRNAs) including fibrotic Erbb4-IR Figures 4). We therefore further elucidated the underlying mechanism of AANG in preventing the development of T2D and T2DN. As shown in Figure , treatment with NG but not AA prevented the development of T2D in db/db mice with relative normal levels of HbA1c, fasting blood glucose, and serum insulin levels, which became more effective in db/db mice received AANG treatment. These observations are in line with the anti-diabetic effects found in the Smad3-KO db/db mice Supplementary Figure S2). It is known that db/db mice or islet cells lacking Smad3 are protected from T2D Supplementary Figure S3). Like the Smad3 KO-db/db mice, we uncovered that compared to the AA or NG monotherapy, preventive treatment with AANG also markedly blocked Smad3 activation in the diabetic pancreas, resulting in the protection of islet from the degenerative changes and the restoration of islet β cell mass and insulin production in db/db mice . Taken together, we discovered that the protection against Smad3-mediated islet deterioration may be a novel mechanism through which AANG treatment prevents T2D and T2DN.Previous studies have shown that Smad3 can target islet β cells to cause T2D in vitro and in vivo. Surprisingly, we further discovered that AANG could directly inhibit the progression of T2D by restoring β cell development in db/db mice via inhibiting the Smad3-dependent islet injury. Thus, AANG may represent as a novel and effective therapeutic for T2D and T2DN prevention.Diabetic nephropathy (DN) is a severe complication of T2D, and one of the major causes of ESRD, but effective therapies are still limited due to its unclear pathogenic mechanism. It is well documented that both NF-kB and Smad3 signaling pathways were elevated in T2D patients and experimental disease models The therapeutic effects of TCM have been well-documented for T2D and its complications Mechanically, we uncovered that the combinational treatment with AA and NG produced a better effect on rebalancing Smad3/Smad7 signaling in the diabetic kidney, thereby resulting in more effectively inhibiting Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. Indeed, the Smad signaling network, especially Smad3, has been shown to play a pathogenic role in renal fibrosis including T2DN In addition, we also identified a better inhibitory effect of AANG on NF-κB inflammatory pathway in the diabetic kidney due to its synergistic induction of Smad7 expression. It has been reported that AA can directly induce Smad7, and NG can also protect Smad7 from degradation by inhibiting Smad3-mediated Smurf2 mechanism It is possible that other signaling transducers such as the protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β) pathways may be also the therapeutic targets of AANG. Indeed, a recent study identified that inhibition of GSK3β is likely able to improve islet beta cell homeostasis in diabetic mice In conclusion, we identify a safe dosage and optimal combination ratio of AANG for preventive treatment of T2D and T2DN by effectively rebalancing Smad3/Smad7 signaling, thereby inhibiting TGF-β/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation. Unexpectedly, we also discover that treatment with AANG can protect islets from diabetic injury in db/db mice by promoting insulin-producing β cell development. Results from this study provide evidence and important clinical rationale for AANG as a novel and effective therapeutic strategy for diabetes and diabetic complications.Supplementary figures.Click here for additional data file.


Colorectal cancer (CRC) is one of the most lethal cancers of the digestive system. The tumor microenvironment (TME) plays a central role in the initiation and development of CRC. However, little is known about the modulation mechanism of the TME in CRC. In our study, we attempted to identify a biomarker related to the TME modulation that could serve as a potential prognostic biomarker for CRC. We identified differentially expressed genes between the ImmuneScore high/low and StromalScore high/low groups. Using univariate COX regression analysis and hub gene analysis (cytoHubba), SLC11A1 was identified as the only candidate gene for subsequent analysis. CIBERSORT, EPIC, MCPcounter, and immunogenic cell death were performed to evaluate the effect of SLC11A1 on the TME. We also collected samples and performed Real-time quantitative PCR to verify the expression levels of SLC11A1 in CRC and adjacent normal tissues. The IMvigor210 cohort, TIDE score, and immunophenoscore (IPS) were used to analyze the association between SLC11A1 and immunotherapy efficacy. SLC11A1 was highly expressed in CRC tissues compared with its expression in normal colorectal tissues and was associated with poor prognosis and advanced clinicopathological stages. Gene set enrichment analysis showed that TGF-β pathways, JAK-STAT pathways, and angiogenesis were significantly enriched in the high-SLC11A1 group. Single-cell analysis validated the correlation between SLC11A1 and the TME. Using CIBERSORT, EPIC, and MCPcounter algorithms, we found that there was more macrophage and fibroblast infiltration in the SLC11A1 high-expression group. Meanwhile, high-SLC11A1 patients had lower IPS scores, higher TIDE scores, and fewer immunotherapy benefits than those of low-SLC11A1 patients. In conclusion, SLC11A1 plays a crucial role in the TME and could serve as a potential biomarker for poor prognosis and immunotherapy efficacy in CRC. Colorectal cancer (CRC) is one of the most common gastrointestinal cancers worldwide, with high mortality rates . CurrentThe tumor microenvironment (TME), which is composed of tumor cells, immune cells, blood vessels, extracellular matrix, and fibroblasts, is considered to play a central role in tumorigenesis and tumor progression . TME maySLC11A1, also known as natural resistance-associated macrophage protein-1, is a member of the solute-carrier family. SLC11A1 was initially reported to fight several types of pathogens, and some studies have shown that SLC11A1 plays a role in innate immunity, autoimmune diseases, and infection . In tubeIn the present study, we applied the ESTIMATE  algorithm, hub gene analysis, and univariate COX regression and identified a TME-related factor in CRC, solute carrier family 11 member 1 (SLC11A1). The prognostic value of SLC11A1 has been evaluated in CRC and other digestive tract cancers. CIBERSORT, EPIC, MCPcounter, gene set enrichment analysis (GSEA), and single-cell analysis were used to further assess the potential effects of SLC11A1 in the TME and immunotherapy. Our findings provide a potential prognostic biomarker and may help in the individual selection of immunotherapy for patients with CRC.https://xenabrowser.net/datapages). Microarray data from GSE17536, including 177 patients, were downloaded from the GEO database  and rectal adenocarcinoma (READ) were collected from TCGA database using TCGAbiolinks  Supplem. This stdatabase . Moreovedatabase . In the A previous study has shown the application of ESTIMATE algorithm to microarray and RNA-sequencing data might help to clarify the role of the TME and provide novel insights into genomic alterations . ESTIMATp < 0.01. The overlapping DEGs in the StromalScore and ImmuneScore groups were considered TME-related genes. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analyses were performed using the DEGs associated with TME using the R package clusterProfiler  between patients with high and low ImmuneScores to identify genes associated with immune cells . MoreoveProfiler . The enrp < 0.05 were shown in the plot.We used the STRING database to construct a protein–protein interaction (PPI) network (confidence score > 0.9) based on the common DEGs in both the StromalScore and ImmuneScore groups . To idenThe expression differences between low-SLC11A1 and high-SLC11A1 groups were identified using DESeq2. To identify key pathways and biological processes associated with SLC11A1, GSEA was performed using KEGG and Hallmark gene sets as target sets.The Immuno-Oncology-Biological-Research (IOBR) R package integrates common algorithms for estimating the TME cells . The TMEhttps://tcia.at/home) to identify the relationship between IPS and SLC11A1 expression.A previous study on LUAD has shown that TIDE and IPS scores are valid scoring schemes to predict immunotherapy response. TIDE is one of the most effective methods for assessing the immune escape of tumors by analyzing their expression profiles . Higher GAPDH-F, 5′ GGAAGCTTGTCATCAATGGAAATC 3′; GAPDH-R 5′ TGATGACCCTTTTGGCTCCC 3′; SLC11A1-F 5′ GTCCGTCTCCTTTATCATCAACCT 3′; SLC11A1-R 5′ GAAGCCCTCCATCACGAACTG 3′.We collected 15 paired CRC tissues and adjacent normal colorectal tissues from the Liaoning Cancer Hospital. Total RNA was extracted from the tissue specimens using TRIzol reagent. Total RNA was treated with kits from Takara  to remove genomic DNA and to conduct reverse transcription. Real-time quantitative PCR (RT-qPCR) was performed using TB GREEN Premix Ex Taq (Takara). The primer sequences were: p-value < 0.05.All statistical analyses were conducted using R software (version 4.1.0). Kaplan–Meier (KM) curves were constructed to evaluate the relationship between OS rates and SLC11A1 expression. The survminer R package was used to identify the optimum cutoff point of SLC11A1 expression to classify CRC patients into low-SLC11A1 and high-SLC11A1 groups. The Wilcoxon test was used to compare differences between the two groups. The Kruskal–Wallis test was used to compare the differences between multiple groups. Univariate COX regression was performed to screen the prognostic factors of CRC using the survival R package. Statistical significance was set to p = 0.008), N stage (p = 0.017), and M stage (p = 0.002) (p = 0.032) and N stage (p = 0.046)  . Additio= 0.046) . However= 0.046) . These r), and 241 DEGs were considered TME-related genes. In addition, we analyzed the related functions and pathways of the genes using GO and KEGG enrichment analyses. The genes were mainly enriched in immune-related GO terms, such as chemokine activity, complement C3b binding, immunoglobulin binding, immune receptor activity, regulation of T cell adhesion, and leukocyte cell-cell adhesion   . We iden< 0.05) . We perf< 0.05) .p = 0.0036 and p = 0.0078, respectively)  . We alsoctively) . There wctively) . These rWe also investigated the prognostic value of SLC11A1 using pan-cancer data from TCGA. In other digestive tract cancers, such as STAD, PAAD, and LIHC, high expression of SLC11A1 was also associated with poor prognosis . Similarp < 0.01). APC showed a higher mutational frequency in the low-SLC11A1 group, and NER, PCI, DNAH11, RYR3, RYR2, SYME1, USH2A, RYR1, OBSCN, DNAH5, MUC16, ZFHX4, and CSM3 showed a higher mutational frequency in the high-SLC11A1 group   . Similar 0.0028) . Patient 0.0028) . We eval 0.0028) . Moreove 0.0028) . IPS, IPIn this study, we screened TME-related prognostic biomarkers for CRC patients. Through multiple bioinformatics analysis methods, we demonstrated that SLC11A1 could serve as a significant indicator in the TME for predicting prognosis and immunotherapy resistance in CRC. First, we calculated the ImmuneScore and StromalScore of TCGA-CRC patients using the ESTIMATE algorithm and found that the ImmuneScore and StromalScore were closely related to clinicopathological stages. To identify genes associated with TME, DEGs were identified between the ImmuneScore high/low groups and the StromalScore high/low groups. In total, 241 DEGs associated with TME were identified, and SLC11A1 was identified as a hub prognostic gene.We demonstrated that SLC11A1 was highly expressed in CRC tissues compared to that in normal tissues, and high-SLC11A1 patients had poor prognosis in CRC as well as other digestive cancers. Meanwhile, higher expression of SLC11A1 was associated with more fibroblast, CAF, and macrophage infiltration and less activated dendritic cell infiltration. Finally, our findings also revealed that increased SLC11A1 levels were correlated with decreased immunotherapy efficacy and poor prognosis in patients treated with immunotherapy. SLC11A1 was identified as a metabolism-related gene involved in predicting the prognosis of hepatocellular cancer . MutatioIn the TME, immune cell, blood vessel, and stromal components play crucial roles in the carcinogenesis and progression of cancer . AngiogeIn a data analysis of thyroid cancer, SLC11A1 is associated with macrophages and participates in the construction of a risk model for evaluating the prognosis of thyroid cancer patients . SLC11A1In various cancer types, immunosuppressive TME can inhibit the activity and anti-tumor ability of immunocytes . Immune in vitro or in vitro. Our research group is currently collecting more clinical samples from CRC patients. In future study, immunohistochemistry would be performed to further evaluate the expression levels and clinical values of SLC11A1 in CRC. We would use immunostaining to validate the relationship between SLC11A1 and immune cells, such as fibroblasts, cancer-associated fibroblasts (CAFs) and macrophages (M1 and M2). Further experimental studies are planned to explore roles of SLC11A1 in modulating CRC TME.Here, we propose SLC11A1 as a potential biomarker for prognosis and immunotherapy efficacy in patients with CRC. However, this study had some limitations. Independent CRC patients treated with immunotherapy are required, to evaluate the accuracy of SLC11A1 in predicting immunotherapy response. Although RT-qPCR was performed to examine the higher expression of SLC11A1 in CRC, other experimental methods are required to explore the specific functions of SLC11A1 In conclusion, we revealed that SLC11A1 was associated with poor prognosis and immunotherapy resistance in CRC for the first time. SLC11A1 may be a potential biomarker for predicting prognosis and immunotherapy efficacy in CRC.


Advances in tissue engineering have made possible the construction of organs and tissues with the use of biomaterials and cells. Three important elements are considered: a specific cell culture, an adequate environment, and a scaffold. The present study aimed to develop P3HB scaffolds by 3D printing and evaluate their biocompatibility with HaCaT epidermal cells, as a potential model that allows the formation of functional tissue. By using a method of extraction and purification with ethanol and acetone, a biopolymer having suitable properties for use as a tissue support was obtained. This polymer exhibited a higher molecular weight (1500 kDa) and lower contact angle (less than 90°) compared to the material obtained using the conventional method. The biocompatibility analysis reveals that the scaffold obtained using the ethanol–acetone method and produced by 3D printing without pores was not cytotoxic, did not self-degrade, and allowed high homogenous cell proliferation of HaCaT cells. In summary, it is possible to conclude that the P3HB scaffold obtained by 3D printing and a simplified extraction method is a suitable support for the homogeneous development of HaCaT keratinocyte cell lineage, which would allow the evaluation of this material to be used as a biomatrix for tissue engineering. Tissue engineering is the process of generating new tissues to replace diseased or injured ones. This technology uses biodegradable three-dimensional scaffolds to mimic the structure and functions of the natural extracellular matrix, in vitro tissue harvesting, and expansion methods ,2,3. TheBiomaterials, such as natural polymers , have frequently been used for tissue engineering applications, because they can have similar properties to the extracellular matrix, such as pseudoplastic behaviour, gelation capacity, water retention capacity, and biodegradability, among others ,6,7.Zoogloea ramigera, Ralstonia eutropha, Methylobacterium, Escherichia coli, Pseudomonas acidophilia, Pseudomonas oleovorans, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii [Poly-3-hydroxybutyrate (P3HB) is a natural polymer that belongs to the family of poly-3-hydroxyalkanoates (PHAs), composed of 3-hydroxy fatty acids that can be produced by bacteria, such as nelandii ,9. This nelandii ,11,12.3 kDa), which has better elastic properties [It is important to point out that P3HB with a molecular weight of less than 1000 kDa, produces a rigid and brittle material, due to its high degree of crystallinity (between 55 and 80%), and has faster thermal degradation than P3HB of high molecular weight , a P3HB overproducing mutant [The g mutant , was useThe culture medium used for the batch cultures contained sucrose and yeast extract (BD Bac-to) as the nitrogen and carbon sources in the concentrations previously described . Fed-batMicrobial growth to produce P3HB was evaluated through protein measurements by the Lowry method using bovine serum albumin as a standard . The totThis method was previously reported  and is bFor P3HB extraction using the hypochlorite-chloroform method, the system of two phases reported previously  was empl2SO4. Samples of 3–5 mg of the biomass or P3HB after extraction treatments, were hydrolysed with 1 mL of H2SO4 at 90 °C for 1 h. Hydrolysed samples were diluted (1/40 with Milli-Q water) and injected (20 μL) into HPLC equipment  with an Aminex HPX-87H column . The eluent was H2SO4 (5 mmol/L) at 50 °C with a flow rate of 0.65 mL/min. P3HB converted into crotonic acid was detected with a Diode array detector at 220 nm.Determination of P3HB was made by high-performance liquid chromatography  after its conversion into crotonic acid by acidic hydrolysis with HEquations (1) and (2) were used to estimate the recovery and purity yields.3 kDa. 15 mg of P3HB obtained using the different extraction methods were weighed and dissolved in 1 mL of chloroform for 2 or 3 h at 55 °C and 700 rpm in a thermoblock . Each sample was filtered through a 0.45 µm nylon membrane and placed in an HPLC vial. Empower software was used for sample processing and quantification.The P3HB molecular weight distribution was determined by gel permeation chromatography (GPC), using the SHODEX GPCK-806M column  . HPLC eq−1, with a resolution of 4 cm−1 [Infrared spectra were obtained with a Nicolet iS10 FTIR spectrometer from Thermo Scientific . P3HB samples obtained by the conventional method, as well as that obtained by the ethanol–acetone method were analysed, for each spectrum scans were performed between 4000 to 400 cmf 4 cm−1 .v/v) for 5 min in each solution. After washing, the P3HB scaffolds were stored in Falcon tubes under 50 mL of sterile PBS.Circular scaffold models with a diameter of 1 cm and a thickness of 100 µm were produced using design software (AutoCAD), and P3HB was isolated using the ethanol–acetone method. A 3D printer was fed with the P3HB filament at 40 mm/s. The nozzle was heated to 185 °C and the print bed was heated to 60 °C to generate the scaffolds. The printed P3HB scaffolds were sterilized using ethylene oxide and washed with sterile PBS-Antibiotic-Antimycotic 100× solutions . Images were taken at 15 kV at 300×, 1000× and 4000× magnifications. For this purpose, a variable pressure scanning electron microscope JEOL model 5900-LV  was used in order to avoid the sample coating by means of sputtering. The operational conditions used were a pressure of 20 Pa and an acceleration voltage of 15 kV; under these conditions, the charge of the samples was avoided because they are not conductive. The latest is based on the principle that the gas molecules in the chamber collide with the incident beam and with the electrons that leave the sample, these collisions become cations which are attracted by the negative charge of the sample and in that way the surface charge is neutralized. This is a good method for the analysis of non-conductive samples and avoids coating, especially if it is required to do microanalysis. It should be noted that we did not perform a porosity study, only surface morphology observation.®, New York, NY, USA) at 25× and processed with image analysis software to calculate the angle between the water droplet and the surface [The contact angle of the P3HB scaffolds was measured by the sessile drop technique at room temperature (25 °C), putting 1 μL of distilled water on the scaffold surface. Images were acquired using a digital microscope with a camera (MicroView surface .2 concentration of 5% in an Eppendorf cell culture incubator.Human HaCaT keratinocyte cells (ATCC: CSC-C8977H) were cultivated in culture medium DMEM-F12  supplemented with 10% Bovine Fetal Serum (BFS) at pH 7.2 ± 0.03. Cells were cultured and maintained at 37 °C with a CO5 cells/mL and incubated at 37 °C with a CO2 concentration of 5% for 5 days. Thereafter, bright field images of the scaffolds were taken using a microscope to verify that cell adhesion had occurred. Then, the scaffolds were washed twice with 1 mL of PBS and dyed with Hoesch, acridine orange, and propidium iodide to count the viable and non-viable cells to estimate cell proliferation by epifluorescence microscopy.Sterile scaffolds were carefully placed into 1 mL culture wells with a cell concentration of 5 × 10There are several methods reported in the literature regarding cell rupture based on halogenated and non-halogenated solvents for the extraction of P3HB from biomass. However, these studies showed that the use of compounds such as sodium hypochlorite and chloroform significantly affect the molecular characteristics of P3HB . For thiThe recovery percentage using the method of hypochlorite–chloroform was 30%, while, the extraction method with ethanol–acetone was 79% . Regardi3 kDa to satisfy the requirements for biomedical applications. In the present study, the molecular weight was affected by the extraction method used. For example, with the conventional method (hypochlorite–chloroform), a very low molecular weight was obtained (416 ± 68 kDa)  , Table 1P3HB is more soluble in chloroform than other organic solvents. Therefore, after recovery of the polymer using the hypochlorite–chloroform method, a material with high purity (99%) was obtained. This value is consistent with those reported in the literature, which ranges from 98 to 100%. In comparison, the material recovered using ethanol–acetone had a purity of 93%. As is shown below, this lower purity was due to the presence of cellular and solvent residues as evidenced by the FTIR analysis.3 (2974 cm−1), CH (2930 cm−1), C = O (1720 cm−1), and CH2 (1277 cm−1) characteristic of the P3HB polymer, which were observed in the materials recovered by the two extraction methods. However, for the P3HB obtained from the rupture and extraction method with ethanol–acetone, the OH groups (3283 cm−1), amides I (1647 cm−1), and amides II (1540 cm−1) were also present, in contrast to the polymer obtained from the extraction with chloroform.Analysing the FTIR spectra of P3HB recovered by the different extraction methods, the characteristic peaks of the chemical structure of poly-3-hydroxybutyrate were found. For the product obtained by rupture and extraction with hypochlorite–chloroform, the spectra showed the same characteristic peaks of P3HB as previously reported, and had the same purity of 99% . In contThe Young’s Modulus of the PH3B samples increased as a function of the molecular weight of the polymer . PreviouThe scaffolds generated by 3D printing from P3HB obtained by ethanol–acetone rupture and extraction showed surface roughness with protrusions  and presThe hydrophobicity of a surface can be determined by measuring the contact angle through the dispersion of a drop of water on the surface. The measurement of the contact angle on scaffolds generated in the present work by 3D printing using the P3HB obtained by the ethanol–acetone method yielded contact angles of 70° , indicatIn the case of P3HB, the low contact angle is attributed to the fact that the hydroxyl (OH) and carbonyl (COH) groups present in this structure, depending on the spatial conformation of the material, tend to take an additional hydrogen ion (hydrogen acceptors), which generates a strong hydrophilic effect, coupled with the roughness of the surface, which favours lower contact angles .On the other hand, the characterization of the grown HaCaT cells was carried out using a DMEM-F12 medium supplemented with 10% SFB to determine the kinetic parameters of cell growth prior to their use with the scaffolds. −1, with a doubling time of 32 h, values that coincide with those reported in the literature for the cell line of HaCaT keratinocytes [The specific growth rate of the cell line in the DMEM-F12 supplemented with 10% SFB was 0.022 hinocytes . The resA more detailed observation, based on nuclei staining under the HOESCH filter (nuclei of living cells fluoresce blue), indicated that cell proliferation was consistent in all cases, but in the case of the 3D-printed scaffold, the cell distribution depends on the size of the pores, and a more homogeneous cell carpet is generated. It is important to note that the pore size of the scaffold is a determinant to induce cell proliferation, and, depending on the application, such a parameter should be evaluated. The above correlates with scaffolds generated using various techniques that provide supports with smooth surface morphologies, with certain protrusions, relief and some micropores, in which some cell lines have been cultivated. In the case of the culture of HaCaT cells, osteoblasts, neuronal cells, and fibroblasts, it was found that, with P3HB scaffolds, the cell proliferation was higher than with other polymers or combinations of polymers ,37.It is important to point out that, despite the purity of the P3HB obtained using the ethanol-acetone method being only 93%, it was found that this material did not cause any type of toxicity to the cells, allowing the HaCaT cells to adhere to all three of the scaffolds evaluated. Bacterial residues, do not imply the presence of live bacteria or complete microorganisms. Therefore, we used biocompatibility studies to evaluate if such residues were toxic and the biocompatibility studies demonstrated that there was no toxic effect. In addition, heat and chemical sterilizations were carried out to ensure that the cell culture could be developed under sterile conditions.The novel ethanol–acetone extraction method used in this study produced P3HB with properties suitable for use as a scaffold in tissue engineering due to its high molecular weight. This kind of P3HB can be a material with better elastic properties for scaffold formation than low molecular weight P3HB ,13. The The results reveal that on the scaffolds made with P3HB recovered by the extraction method with ethanol–acetone and manufactured by the 3D printing technique without pores, a higher cell proliferation was obtained when compared to the control scaffold.This work shows that P3HB obtained by a simple ethanol–acetone extraction method works well as a biomaterial for the generation of scaffolds for application in epidermal tissue engineering. It also shows the influence of manufacturing parameters, such as the type of extraction or the method used for scaffold formation, on the properties of the obtained P3HB, and how they affect the proliferation of HaCaT cells, which are important model cells of human keratinocytes widely used in scientific research of skin tissue regeneration.In summary, our results show the feasibility of using P3HB supports for the growth of epidermal cells and, thus, as a biomatrix for tissue engineering. At the same time, it is recognised that more work should be done on the optimization of the scaffolding design and its mechanical and functional characterization.


Acquired middle ear cholesteatoma is a disease which promotes bone erosion resulting in potentially serious complications. The tumor necrosis factor alpha (TNF-α) is present in cholesteatoma and it is related to bone erosion, as shown by different authors. To understand the aggressiveness characteristics of cholesteatoma is necessary, however, to better address the presence and distribution of their receptors.To evaluate the expression of type 2 TNF-α receptor (TNF-R2) in fragments of cholesteatoma and correlate it to the degree of inflammation present.p ≤ 0.05).observational cross-sectional study, which analyzed 33 fragments of cholesteatomas through histological analysis and immunohistochemistry (using as primary antibody to TNF-R2 LabVision ® brand). The evaluation was performed by means of a qualitative and semi-quantitative agreement with the observed intensity. For statistical analysis we used the Fisher exact test and Spearman's correlation coefficient (considered statistically significant when The expression of TNF-R2 was present in all fragments, however a statistical analysis showed no correlation or association between inflammation and the expression of TNF-R2.TNF-R2 is present in cholesteatoma of the middle ear, however, its expression is not directly related to the degree of inflammation observed in patients with this disease. The middle ear acquired cholesteatoma was first described by, Curveilhier, in 182The cholesteatoma matrix has four distinct layers: basal, spinous, granulous and strata cornea, as well as the thin skin. The peri-matrix is characterized by the presence of a loose connective tissue made up of collagen and elastic fibers, fibroblasts and inflammatory cellsBecause of its capacity to cause bone erosion, present in 80% of the casesThe early 90's saw the publication of the first papers suggesting that TNF-α could be an important cytokine involved in the bone destruction processTNF-R2 has been described in different organs, e.g. the kidney, brain and ovary, being always associated with the inflammatory process because of the activation of the Kappa B nuclear factor (NF-kB)The two TNF-α receptors have similar activities. They are involved in the inflammatory process, being associated with bone erosion and they can trigger apoptosis (programmed cell death), the latter is especially associated with the TNF-R2. However, the mechanism responsible for establishing which effect will prevail is still not totally clearThe goal of the present study was to assess the expression of the type-2 tumor necrosis factor alpha receptor (TNF-R2), by means of using immunohistochemistry techniques in cholesteatoma fragments acquired from the middle ear and correlate them with the degree of inflammation present.This is a cross-sectional, observational study, approved by the Ethics Committee of the Medical School of Santa Casa de Misericórdia de São Paulo, where it was carried out. The material encompassed 68 cholesteatoma fragments taken from 68 patients submitted to ear surgery in order to remove the acquired cholesteatoma from the middle ear. The surgeries were done in the Department of Otorhinolaryngology of this Institution from August 2007 through March 2009. The disease diagnosis was based on the clinical history and physical exam of the patients. The surgical procedures were done by different otorhinolaryngologists.From this study we extracted those fragments which did not have an epithelium (matrix), visible upon light microscopy, dyed by hematoxylin-eosin (HE), and 35 fragments were excluded. This way, the total number of fragments used in this study was 33.After removing the cholesteatoma fragments, the material was fixed in 10% formaldehyde and processed by conventional histology techniques, with paraffin inclusion. For histology purposes, we considered the following patterns: atrophy (matrix with a thickness of a maximum of four layers of keratinocytes), acanthosis (matrix thickness increase in lieu of the spinous layer), basal layer hyperplasia , epithelial cone formation (matrix invaginations to inside the peri-matrix) and inflammatory process present in the peri-matrix. The assessment was carried out in qualitative (present or absent) and semi-quantitative fashions, grade varying between 0 and 3 according to the intensity observed .U+000AE, USA), in the title of 1:100. The secondary antibody used was the Max Polymer Detection System .For immunohistochemistry we used the primary TNF-R2 polyclonal primary antibody synthetized from rats  and semi-quantitative, graded between 0 and 3 according to the intensity of the color observed: absent = 0; weak = 1; moderatThe slides with cholesteatoma fragments were analyzed under a light microscope model Axioscope 40 (Carl Zeiss - Brazil), with a 10X eye piece and 10X, 20X and 40X magnification lens for histological evaluation and also for immunohistochemistry. All the slides were assessed by three independent observers, in a blinded way. The microscope was coupled to an Axiocam MRc 5 (Zeiss) camera in order to obtain the digital images of the material, using the Axiovision 4.8 software.After data collection we setup a computerized database which was submitted to statistical analysis, with the help of the Statistical Package for the Social Sciences (SPSS) version 13.0 software.We carried out a descriptive analysis, calculating minimum and maximum values, mean, median and standard deviation for the age variable and calculations of the absolute and relative frequencies for gender and for the histopathological and immunohistochemical variables.To study the association among the qualitative variables we used the Fisher's exact test. In order to check for the correlation among the quantitative variables we used the Spearman Coefficient.All the tests were carried out in a bicaudal way and the significance level adopted was 5%.We studied 33 cholesteatoma fragments from 33 patients submitted to ear surgery, 17 of them (51.5%) were men and 16 (48.5%) women. Minimum age was seven years, and the eldest patient was 70 years old, with mean age of 30 years, median of 31 years and standard deviation of 16.4. Of the 33 patients included in the study, two (6.1%) did not know how long it was between symptom onset and admission at the Department of Otorhinolaryngology for surgery. Of the 31 patients who reported it, 13 said they had had the disease for less than five years (41.9%), seven reported between six and ten years (22.6%), nine mentioned a time span between 11 and 20 years (29%) and, only two patients reported having had the disease for over 20 years (6.5%). Time between symptom onset and admission to surgery at the Department of otorhinolaryngology varied between one and 60 years, with a mean of 11.5, median of eight years and standard deviation of 11.6 years.In regards of the ear affected by the disease, four patients (12.1%) had chronic cholesteatomatous otitis media in both ears, and 29 (87.9%) had unilateral involvement; 15 (45.5%) in the right ear and 14 (42.4%) in their left ears.As far as clinical symptoms reported by the patients upon admission goes, 31 (93.9%) reported otorrhea, 30 (90.9%) reported hypoacusis and 27 (81.8%), reported dizziness spells.Ossicular chain involvement was reported in 28 patients (84.8%), who had erosion in one of the ossicles and, in five (15.2%) the ossicular chain was not seen involved during intraoperative assessment. Considering the ossicle involved, 23 patients (69.7%) had malleus erosion, 23 (69.7%) had incus erosion, and 18 (54.5%) had stapes erosion.Besides ossicular chain involvement, we noticed the presence of erosion in the semicircular canal, facial nerve exposure and also dura mater exposure. There was semicircular canal erosion in three patients (9.1%), facial nerve exposure in six (18.2%) and dura mater exposure in five (15.2%).Histological assessment found more than one histopathological pattern  simultaneously, in most of the cholesteatoma fragments analyzed.Because of the lack of a peri-matrix in one of the 33 fragments investigated, it was not possible to assess the intensity of the inflammation in that case. Of the 32 remaining cases, inflammation was absent in one (3.1%), weak in 14 (43.8%), moderate in 13 (40.6%) and severe in four (12.5%) . The infUpon immunohistochemical evaluation we observed a reaction from the anti-TNF-R2 antibody in the matrix cells in all the cholesteatoma fragments analyzed. This reaction was considered weak in 10 cases (30.3%), moderate in 18 (54.5%) and severe in five (15.2%) . Besidesp= 0.589). We also did not see positive and statistically significant correlation using the Spearman's correlation coefficient (p= 0.155).The statistical analysis did not show statistically significant association between the degree of inflammation present in the middle ear acquired cholesteatoma and the presence of TNF-R2 (The middle ear acquired cholesteatoma is a disease characterized by tympanic cavity invasion by an epithelium (matrix) which is different from the one usually found in the middle ear. Histologically, this epithelium is similar no normal skinHistological analysis also showed that the matrix may present with different histological patterns, such as atrophy, acanthosis, basal layer hyperplasia and epithelial cones. These patterns frequently coexist in the same fragment, as shown in the present study. These histological findings are in agreement with what has been published in the literatureThe evaluation of the peri-matrix from the cholesteatoma fragments reported inflammatory process present in 96.9% of the cases, in different severities. This fact is in agreement with reports from PereiraCholesteatomas usually cause ossicular chain erosion. In the present study, during surgery, we noticed that the ossicular chain was eroded 84.4% of the patients. These data corroborate literature data, which report ossicular chain erosion on 91.5% of the patientsIn the present study, the most affected ossicle was the incus, especially its long apophysis, and the malleus, both in 23 patients (69.7%). Stapes erosion happened in 18 patients (54.5%). These findings differ from the ones from TosThe middle ear acquired cholesteatoma is a disease which courses with an intensive infectious and inflammatory process. Such condition induces the release of different substances (cytokines and growth factors) which act together in synergism, resulting in the aggressive cholesteatoma characteristicsThere are two receptors for TNF-α, the TNF-R1 and the TNF-R2. The role of each one of the TNF-α receptors is still unclearThe goal of the present study was to investigate TNF-R2 expression by means of immunohistochemical techniques in middle ear acquired cholesteatoma fragments and correlate them to the degree of inflammation seen upon histology.In this study we assessed TNF-R2 expression by means of immunohistochemical techniques in middle ear acquired cholesteatoma fragments. TNF-R2 reaction was cytoplasmic and happened in all the keratinocytes of the matrix from analyzed fragments. There was also reaction in some peri-matrix inflammatory cells (lymphocytes and plasmocytes). Besides TNF-R2 expression, inflammation was also present.We carried out a statistical analysis in order to check whether or not there was any association or correlation between TNF-R2 expression and the degree of inflammation. However, in our study we did not see TNF-R2 involvement in the cholesteatoma's inflammation. In other words, the TNF-α receptor is present in the middle ear acquired cholesteatoma; nonetheless, its expression (number of receptors) is not associated with the degree of inflammation seen. Notwithstanding, we still need further studies, with larger cholesteatoma samples, in order to obtain a more reliable statistical analysis.In the present study, besides the cytoplasmic reaction (pattern) arising from the connection between the TNF-α and the TNF-R2, we also detected a significant nuclear expression in all the slides assessed, in epithelial cells of the matrix and in some cells of the peri-matrix inflammatory infiltrate (lymphocytes and plasmocytes). This reaction may indicate a crossed reaction with some cell nucleus proteins; however, this still needs further clarification in future studies.1) TNF-R2 is present in acquired middle ear cholesteatoma matrixes.2) It was not possible to correlate TNF-R2 expression with the degree of inflammation present in the acquired middle ear cholesteatoma.


Hypertension remains the leading risk factor for cardiovascular disease. Young females tend to be protected from hypertension compared with age-matched males. Although it has become increasingly clear that the immune system plays a key role in the development of hypertension in both sexes, few studies have examined how cytokines mediate hypertension in males versus females. We previously published that there are sex differences in the levels of the cytokine tumor necrosis factor (TNF)-α in spontaneously hypertensive rats (SHR). The goal of this study was to test the hypothesis that TNF-α inhibition with etanercept will lower BP in male and female SHR. However, as male SHR have a more pro-inflammatory status than female SHR, we further hypothesize that males will have a greater decrease in BP with TNF-α inhibition than females. Young adult male and female SHR were administered increasing doses of the TNF-α inhibitor etanercept or vehicle twice weekly for 31 days and BP was continuously measured via telemetry. Following treatment, kidneys and urine were collected and analyzed for markers of inflammation and injury. Despite significantly decreasing renal TNF-α levels, renal phospho-NFκB and urinary MCP-1 excretion, etanercept did not alter BP in either male or female SHR. Interestingly, treatment with etanercept increased urinary excretion of protein, creatinine and KIM-1 in both sexes. These results indicate that TNF-α does not contribute to sex differences in BP in SHR but may be vital in the maintenance of renal health. Etanercept treatment successfully decreased renal TNF-α levels in male and female SHR.Inhibition of TNF-α did not alter BP in SHR of either sex.Inhibition of TNF-α significantly elevated creatinine and KIM-1 levels in both male and female SHR, indicating TNF-α may be necessary for maintaining renal health. Although hypertension is a prominent risk factor for developing cardiovascular disease (CVD), the mechanisms underlying the development of hypertension remain poorly understood. Hypertension is a common medical condition characterized by an increase in blood pressure (BP). An increasing number of studies have found pro-inflammatory cytokines produced by immune cells to be important contributors to the development of essential hypertension , 2, but Young females have a lower prevalence of hypertension compared to age-matched males both clinically and in numerous experimental models of hypertension, include spontaneously hypertensive rats  (SHR). RThe pro-inflammatory cytokine TNF-α has been implicated in BP control in angiotensin II, Dahl salt-sensitive, DOCA-salt male rat models of hypertension –16, and Nine-week-old age-matched male and female SHR were received from Envigo Laboratories . Rats were housed in temperature- and humidity-controlled, light-cycled quarters and fed standard rat chow ad libitum. All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the approval of the Augusta University Institutional Animal Care and Use Committee. At 10 weeks of age, subsets of rats were implanted with intra-arterial telemeters for the measurement of mean arterial pressure (MAP) and allowed 5 days to recover before a baseline measurement was taken. At 12 weeks of age, rats began treatment with either the TNF-α inhibitor etanercept or vehicle via intraperitoneal injection twice weekly for 31 days. Based on previous studies in the literature, rats were treated with increasing doses of etanercept as follows: 0.4 mg/kg from days 1 to 12 , 0.8 mg/Cytokine levels were determined by ELISA in whole kidney homogenate. Levels of TACE (LSBio F22349), TGF-β (MyBioSource 260302), TNF-α (MyBioSource 2507393) and phospho-NFκB  were quantified in renal homogenates of etanercept-treated or vehicle control SHR and assayed according to the manufacturer’s instructions. Urinary MCP-1 excretion levels were determined by ELISA (BD Biosciences 555130).Creatinine and kidney injury molecule 1 (KIM-1) excretion were measured by ELISA  in terminal 24-h urine samples according to manufacturer instructions. Proteinuria was measured using a Bradford assay using bovine serum albumin as a standard. Albuminuria was measured by Nephrat II ELISA (Ethos Biosciences NR002).P < 0.05 was considered statistically significant. Renal cytokines and markers of renal injury were compared using two-way ANOVA. BP data were analyzed by two-way repeated-measures ANOVA. Analyses were performed using GraphPad Prism version 9.2 software .All data are expressed as means ± SE. For all comparisons, Psex < 0.0001, Ptreatment = 0.54, Pinteraction = 0.086). BP at the end 0.4 mg/kg dose of etanercept was 145 ± 1 mmHg in vehicle-treated males, 141 ± 2 mmHg in etanercept-treated males, 133 ± 4 mmHg in vehicle-treated females, and 135 ± 2 mmHg in etanercept-treated females . BP at the end 0.8 mg/kg dose of etanercept was 147 ± 1 mmHg in vehicle-treated males, 144 ± 1 mmHg in etanercept-treated males, 132 ± 1 mmHg in vehicle-treated females, and 130 ± 2 mmHg BP in etanercept-treated females . BP at the end of the 1.6 mg/kg dose of etanercept was 149 ± 1 mmHg in vehicle-treated males, 146 ± 2 mmHg in etanercept-treated males, mmHg in vehicle-treated females, and 133 ± 2 mmHg in etanercept-treated females . Males had a higher BP than females throughout the study (P < 0.05). Treatment with etanercept did not significantly lower BP in either male or female SHR. Indeed, BP of male SHR progressively increased over the course of the study in both vehicle control and etanercept-treated rats. BP of female SHR remained constant throughout the study regardless of treatment.To determine the relative contribution of TNF-α on BP control in male and female SHR, rats were treated with increasing doses of the TNF-α inhibitor etanercept or vehicle and BP was continuously measured by telemetry Fig. . BaselinPsex = 0.0023). Etanercept lowered TNF-α levels in both sexes (Ptreatment = 0.024), and the decrease was comparable in males and females (Pinteraction = 0.64). TNF-α induces the phosphorylation of NFκB leading to translocation to the nucleus and increased transcription of inflammatory cytokines, including MCP-1. Etanercept decreased NFκB activation in male, but not female SHR  and MCP-1 excretion in both sexes . Renal TACE levels were comparable between all groups . Males had higher renal TGF-β levels than females (Psex = 0.0209), but TGF-β was not impacted by treatment with etanercept .To confirm the effectiveness and specificity of etanercept to inhibit TNF-α, TNF-α, NFκB, TACE and TGF-β were measured in renal homogenates and MCP-1 was measured in urine from vehicle and etanercept-treated male and female SHR Fig. . Renal TPsex < 0.0001), albumin (Psex < 0.0001), and creatinine (Psex = 0.0002) than female SHR. KIM-1 excretion was comparable between the sexes (Psex = 0.1054). Etanercept treatment did not affect total protein excretion  or albumin excretion in either male or female SHR . In contrast, etanercept increased creatinine (Ptreatment < 0.0001) and KIM-1 excretion (Ptreatment < 0.0001) in both sexes to a comparable degree .Markers of renal injury were assessed by measuring urinary excretion of total protein, albumin, creatinine, and KIM-1, a marker of renal tubular injury Fig. . Male SHDespite an increasing number of studies implicating pro-inflammatory cytokines in the development of essential hypertension , 2, few While not intensively studied in animal models of hypertension, clinical studies have consistently found men to have higher circulating TNF-α levels compared to women , 30. In To assess the contribution of TNF-α on BP control in young, adult male and female SHR, rats were randomized to increasing doses of etanercept. Etanercept is a widely prescribed therapeutic which directly binds to TNF-α and prevents it from activating relevant cellular receptors . The dosTreatment with etanercept did not lower BP in young, adult SHR with established hypertension. There is some controversy in the literature regarding anti-hypertensive effects of etanercept. Etanercept, 1.25 mg/kg per day via subcutaneous osmotic minipump, delays the development of hypertension in male mice receiving angiotensin II plus a high-salt diet, although the effect is lost by day 12 of treatment . Renal iIn contrast to our findings in the current study, TNF-α blockade with infliximab , a mouse chimeric neutralizing antibody, was found to decrease BP in male SHR compared to SHR receiving vehicle control . HoweverTNF-α induces inflammation, in part, via activation of NFκB signaling leading to increases in pro-inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1). Therefore, the effectiveness and specificity of etanercept in the current study was confirmed by measuring decreases in TNF-α and MCP-1 with no effect on TACE or TGF-β. The impact of etanercept on immune cells and the source of TNF-α were not investigated in the current study. However, etanercept has been shown to decrease renal monocyte/macrophage infiltration in hypertension , and conDespite having no effect on BP, treatment with etanercept markedly increased urinary excretion of creatinine and KIM-1, suggesting a role for TNF-α in the control of renal tubular health and function in male and female SHR. Consistent with our finding, etanercept treatment has been associated with at least three clinical cases of acute kidney injury and subsequent renal damage –40. TNF-In summary, while we did not observe the hypothesized decrease in BP in SHR administered increasing dosages of etanercept, we found indications of renal damage in both male and female receiving etanercept treatment. Etanercept  is a common pharmaceutical approved for the treatment of rheumatoid arthritis. Therefore, even a slim probability for renal injury among a large patient population represents a risk of morbidity for a substantial number of patients. Further investigation is required to better understand how etanercept is increasing creatinine and KIM-1 excretion.