Inhibition of the endoplasmic reticulum stress-associated IRE-1 pathway alleviates preterm birth.

BACKGROUND: Premature birth (PTB) remains a major global health concern due to its association with neonatal morbidity and mortality. The unfolded protein response (UPR) within the endoplasmic reticulum (ER) is tightly regulated by Inositol-requiring enzyme type 1 (IRE-1), a pivotal cellular modulator. This study seeks to elucidate the role of the ER stress (ERS)-related IRE-1 pathway in PTB. METHODS: Human placental trophoblast cells HTR8/Svneo were exposed to the ER-stress inducer tunicamycin (TM). The expression of IRE-1 and ERS-associated proteins ATF6, GRP78, and XBP-1 was assessed in placental tissues and TM-treated cells. Cellular viability, migration, invasion, and apoptosis were evaluated through a series of experimental assays. Additionally, various methods were employed to assess and verify the activation of autophagy, using the autophagy marker, microtubule-associated protein 1A/1B-light chain 3 (LC3). Additionally, TUDCA (an ERS inhibitor) was used to assess its potential to counteract the TM-induced cell effects. RESULTS: Elevated levels of ATF6, GRP78, and XBP-1 were observed in PTB tissues and cells. TM treatment substantially reduced cell viability, migration, and invasion while promoting apoptosis. Treatment with TUDCA (an ERS inhibitor) counteracted the effects of TM on the cells. Furthermore, we identified an overexpression of IRE-1 in PTB tissues and cells and its knockdown enhanced cell viability, migration, and invasion while suppressed apoptosis and autophagy under TM stimulation. Notably, IRE-1 was found to modulate the activity of the IRE-1/XBP1/CHOP signaling pathway in TM-treated cells. CONCLUSION: The upregulation of IRE-1 in PTB placental tissues is implicated in the pathogenesis of PTB. Importantly, inhibiting the ERS-associated IRE-1/XBP1/CHOP pathway may be a good strategy in mitigating PTB.

Non-coding RNA-mediated endothelial-to-mesenchymal transition in human diabetic cardiomyopathy, potential regulation by DNA methylation.

AIMS: Diabetic cardiomyopathy (DCM) is a major complication of diabetes and a risk factor for cardiovascular disease. Endothelial dysfunction is central to DCM, and endothelial-to-mesenchymal transition (EndMT) is a key form of endothelial dysfunction in diabetes. EndMT in DCM has been well-studied in model systems and has been found to be epigenetically regulated by non-coding RNAs (ncRNAs). However, EndMT in DCM and its associated epigenetic changes need further characterization in human patients. It is also not known if ncRNAs are affected by changes in DNA methylation in DCM. This study aims to confirm in human hearts, the findings from animal and cell studies, and potentially provide novel insight into interactions between DNA methylation and ncRNAs in EndMT in DCM. METHODS AND RESULTS: Heart tissues were collected from autopsy patients, fixed in formalin, and embedded in paraffin. Thin sections from paraffin-embedded tissues were used for histology and immunofluorescence analyses, where we confirmed that diabetic patients showed increased cardiac fibrosis that EndMT had occurred. Tissue curls from the paraffin-embedded tissues were used for RT-qPCR and methylation analyses. RT-qPCR quantitatively showed that EndMT occurs in the hearts of diabetics, and that EndMT in human hearts corresponded to changes in key ncRNAs. Methylation analysis showed that some of the EndMT-related ncRNAs were regulated by DNA promoter methylation, while others may be regulated through different epigenetic mechanisms. CONCLUSIONS: We show that EndMT is a relevant pathological process in human hearts during DCM, and that its occurrence coincides with changes in relevant ncRNAs. We further find that interplay between DNA methylation and certain ncRNAs involved in the regulation of EndMT may contribute to the observed changes in ncRNA expression. These findings reinforce the role of EndMT in patients afflicted with DCM and underscore the complexities and importance of the interactions between different facets of epigenetic regulation.

Distinguishing preeclampsia using the falling scaled slope (FSS) --- a novel photoplethysmographic morphological parameter.

BACKGROUND: Preeclampsia (PE) presence could lead to hemodynamic changes. Previous research suggested that morphological parameters based on photoplethysmographic pulse waves (PPGW) could help diagnose PE. AIM: To investigate the performance of a novel PPGPW-based parameter, falling scaled slope (FSS), in distinguishing PE. To investigate the advantages of the machine learning algorithm over the conventional statistical methods in the analysis. METHODS: Eighty-one pieces of PPGPW data were acquired for the study (PE, n = 44; normotensive, n = 37). The FSS values were calculated and used to construct a PE classifier using the K-nearest neighbors (KNN) algorithm. A predicted PE state varying from 0 to 1 was also calculated. The classifier's performance in distinguishing PE was evaluated using the ROC and AUC. A comparison was conducted with previously published PPGPW-based models. RESULT: Compared to the previous PPGPW-based parameters, FSS showed a better performance in distinguishing PE with an AUC value of 0.924, the best threshold of 0.498 could predict PE with a sensitivity of 84.1% and a specificity of 89.2%. As for the analysis method, training a classifier using the KNN algorithm had an advantage over the conventional statistical methods with the AUC values of 0.878 and 0.749, respectively. CONCLUSION: The result indicated that FSS might be an effective tool for identifying PE. Moreover, the machine learning algorithm could further help the data analysis and improve performance. [Figure: see text].

Efficacy and safety of HAIC alone vs. HAIC combined with lenvatinib for treatment of advanced hepatocellular carcinoma.

To investigate efficacy and safety of hepatic arterial infusion chemotherapy combined with lenvatinib (HAIC-Len) and HAIC alone for the treatment of advanced hepatocellular carcinoma (Ad-HCC). Totally 349 patients with Ad-HCC participated in the research from February 2018 to October 2020. On the basis of propensity score matching (PSM), 132 and 110 cases were assigned to the HAIC group and the HAIC-Len group, respectively, with a ratio of 1:1. Progression-free survival (PFS), overall survival (OS), and complications were compared between two groups. The Kaplan-Meier method and log-rank test were utilized to estimate cumulative OS and PFS. Additionally, uni- and multi-variate Cox regression models were employed to identify significant independent factors. The median follow-up period in this study was set to be 20.8 months. Following PSM, the one-, two- and three-year cumulative OS rates in the HAIC-Len and HAIC groups were 63.6%, 12.1%, and 3.0%, and 47.2%, 11.8%, and 2.7%, respectively, with a significant difference (P < 0.001). The first-three-year cumulative incidence rates PFS in the HAIC-Len and the HAIC groups were 15.2%, 1.5%, and ND, and 11.8%, 4.5%, and 3.6%, respectively, with no significant difference detected (P = 0.092). BMI (HR 0.709. 95% CI 0.549, 0.915. P = 0.008) and AST (HR 1.005. 95% CI 1.003, 1.007. P < 0.001) represented independent prognostic factors for OS. Additionally, the two groups exhibited no significant difference in the incidence rates of adverse events. HAIC-Len significantly improved survival outcomes of patients with Ad-HCC and demonstrated acceptable toxicity compared to HAIC alone.

Trajectories of maternal D-dimer are associated with the risk of developing adverse maternal and perinatal outcomes: A prospective birth cohort study.

OBJECTIVE: To assess the relationships of maternal D-dimer trajectories with the risk of developing adverse maternal and perinatal outcomes (AMPOs). METHODS: A prospective birth cohort study was conducted in China, and 7,095 women who had singleton birth were included. The latent class growth model was used to determine the maternal D-dimer trajectory. RESULTS: Three maternal D-dimer trajectories were identified: (1) slight increase (43.6%), (2) rapid rise (51.3%), (3) sustained high (5.1%). Compared to pregnant women with a slight increase in D-dimer trajectory, the risk of gestational diabetes mellitus, placenta previa, macrosomia, large for gestational age (LGA), and increased postpartum bleeding was significantly increased in those with a rapid rise trajectory (adjusted OR = 1.22, 2.00, 1.80, and 1.56, adjusted ß = 15.92 âˆ¼ 25.1 ml, respectively, P < 0.05), and women with a sustained high trajectory also demonstrated a relatively elevated risk of macrosomia and LGA (adjusted OR = 2.11 and 1.82, respectively, P < 0.05). While the odds of pregnancy-induced hypertension, low birth weight, and small for gestational age in pregnant women with the rapid rise D-dimer trajectory and fetal distress in those with sustained high trajectory exhibited a reduction (adjusted OR = 0.62, 0.38, 0.54, and 0.64, respectively, P < 0.05). CONCLUSION: This study highlights the influence of inappropriate maternal D-dimer trajectories on the risk of AMPOs.

Expressions of Serum lncRNAs in Diabetic Retinopathy - A Potential Diagnostic Tool.

With increasing incidence of diabetes worldwide, there is an ever-expanding number of patients with chronic diabetic complications such as diabetic retinopathy (DR), one of the leading causes of blindness in the working age population. Early screening for the onset and severity of DR is essential for timely intervention. With recent advancements in genomic technologies, epigenetic alterations in DR are beginning to unravel. Long non-coding RNAs (lncRNAs), which are key epigenetic mediators, have demonstrated implications in several (DR) related processes. Based on the previous research, we have developed a serum-based, multi-panel PCR test using 9 lncRNAs (ANRIL, MALAT1, WISPER, ZFAS1, H19, HOTAIR, HULC, MEG3, and MIAT) to identify and validate whether this panel could be used as a diagnostic and prognostic tool for DR. We initially used a cell culture model (human retinal endothelial cells) and confirmed that 25 mM glucose induces upregulations of ANRIL, HOTAIR, HULC, MALAT1, and ZFAS1, and downregulation of H19 compared to 5 mM glucose controls. Then as an initial proof-of-concept, we tested vitreous humor and serum samples from a small cohort of non-diabetic (N=10) and diabetic patients with proliferative retinopathy (PDR, N=11) and measured the levels of the 9 lncRNAs. Differential expressions of lncRNAs were found in the vitreous and serum of patients and showed significant correlations. We expanded our approach and assessed the same lncRNAs using samples from a larger cohort of diabetic (n= 59; M/F:44/15) and non-diabetic patients (n= 11; M/F:4/7). Significant increased lncRNA expressions of ANRIL, H19, HOTAIR, HULC, MIAT, WISPER and ZFAS1 were observed in the serum of diabetic patients (with varying stages of DR) compared to non-diabetics. No significant correlations were demonstrated between lncRNA expressions and creatinine or glycated hemoglobin (HbA1C) levels. Using ROC and further analyses, we identified distinct lncRNA phenotype combinations, which may be used to identify patients with DR. Data from this study indicate that a panel of serum lncRNAs may be used for a potential screening test for DR. Further large-scale studies are needed to validate this notion.

The Long Non-Coding RNA HOTAIR Is a Critical Epigenetic Mediator of Angiogenesis in Diabetic Retinopathy.

Purpose: Diabetic retinopathy (DR) remains a pressing issue worldwide. Abnormal angiogenesis is a distinct vascular lesion in DR, and research has established that vascular endothelial growth factor A (VEGF-A) is a primary mediator of such changes. However, limitations in current anti-VEGF therapies suggest that our understanding of molecular networks underlying ocular angiogenesis remains far from complete. Based on our long non-coding RNA (lncRNA) array analyses, HOX antisense intergenic RNA (HOTAIR) was identified as one of the top upregulated lncRNAs in high glucose-cultured human retinal endothelial cells (HRECs). Given the well-documented roles of HOTAIR in cancer, no studies have examined the epigenetic implications of HOTAIR in DR, and we investigated such relationships herein. Methods: We used HRECs exposed to various glucose concentrations and epigenetic modulators to examine HOTAIR, angiogenic, and DR-related molecular markers. Oxidative stress, angiogenesis, and mitochondrial dysfunction were assessed. Retinal tissues of diabetic rodents and the vitreous humor and serum of patients with proliferative DR were also investigated. Results: Hyperglycemia significantly augmented HOTAIR expression in HRECs and promoted angiogenesis, oxidative damage, and mitochondrial aberrations. Similarly, vitreous humor and serum from proliferative DR patients and retinas from diabetic animals demonstrated increased HOTAIR expression compared to non-diabetic controls. HOTAIR knockdown protected against glucose-induced increases of angiogenic and diabetes-associated molecules in the retina. Mechanistically, we showed that HOTAIR exerts its capabilities by preventing oxidative stress and modulating epigenetic pathways involving histone methylation, histone acetylation, DNA methylation, and transcription factors. Conclusions: Our findings suggest that HOTAIR is a critical lncRNA in the pathogenesis of DR and may potentially be important for diagnostic and therapeutic targeting.

Photoplethysmography-derived approximate entropy and sample entropy as measures of analgesia depth during propofol-remifentanil anesthesia.

The ability to monitor the physiological effect of the analgesic agent is of interest in clinical practice. Nonstationary changes would appear in photoplethysmography (PPG) during the analgesics-driven transition to analgesia. The present work studied the properties of nonlinear methods including approximate entropy (ApEn) and sample entropy (SampEn) derived from PPG responding to a nociceptive stimulus under various opioid concentrations. Forty patients with ASA I or II were randomized to receive one of the four possible remifentanil effect-compartment target concentrations (Ceremi) of 0, 1, 3, and 5 ng·ml-1 and a propofol effect-compartment target-controlled infusion to maintain the state entropy (SE) at 50 ± 10. Laryngeal mask airway (LMA) insertion was applied as a standard noxious stimulation. To optimize the performance of ApEn and SampEn, different coefficients were carefully evaluated. The monotonicity of ApEn and SampEn changing from low Ceremi to high Ceremi was assessed with prediction probabilities (PK). The result showed that low Ceremi (0 and 1 ng·ml-1) could be differentiated from high Ceremi (3 and 5 ng·ml-1) by ApEn and SampEn. Depending on the coefficient employed in algorithm: ApEn with k = 0.15 yielded the largest PK value (0.875) whereas SampEn gained its largest PK of 0.867 with k = 0.2. Thus, PPG-based ApEn and SampEn with appropriate k values have the potential to offer good quantification of analgesia depth under general anesthesia.

Effect of Laryngeal Mask Airway Insertion on Parameters Derived From Catacrotic Phase of Photoplethysmography Under Different Concentrations of Remifentanil.

BACKGROUND: Some parameters have been extracted from photoplethysmography (PPG) with a good relativity with nociception, but without encouraging results in qualifying the balance of nociception-anti-nociception (NAN). The features of PPG have not been thoroughly depicted and more prospective univariate parameters deserve to be explored. The aim of this study was to investigate the ability of parameters derived from catacrotic phase of PPG to grade the level of analgesia. METHODS: 45 patients with ASA I or II were randomized to receive a remifentanil effect-compartment target controlled infusion (Ceremi) of 0, 1, or 3 ng/ml, and a propofol effect-compartment target controlled infusion to maintain an acceptable level of hypnosis with state entropy (SE) at 40~60. Laryngeal mask airway (LMA) insertion was applied as a noxious stimulus. Five diastole-related parameters, namely diastolic interval (DI), diastolic slope (DS), the minimum slope during catacrotic phase (DSmin), the interval between DSmin and its nearest trough (DTI), and area difference ratio (ADR), were extracted. Pulse beat interval (PBI) was calculated as a reference parameter. RESULTS: LMA insertion elicited a significant variation in all parameters except ADR during Ceremi of 0 and 1 ng/ml. Compared to PBI (prediction probability ([Formula: see text]) = 0.796), the parameters of DI, DS, and DTI presented a better consistence with the level of anti-nociceptive medication, with [Formula: see text] of 0.825, 0.822, and 0.822 respectively. CONCLUSION: The features extracted from catacrotic phase of PPG, including DI, DS, and DTI, could provide a promising potential to qualify the balance of NAN.

Comparison of Three Normalization Methods in Monitoring Analgesic Depth with Photoplethysmographic Diastolic Interval.

The purpose of the present study was to investigate the ability of three different normalization methods, namely root mean square (RMS) value, mean value, and maximum which referred to pulse beat interval (PBI), based on photoplethysmographic diastolic interval (DI) in response to laryngeal mask airway (LMA) insertion under various remifentanil concentrations during general anesthesia. Sixty patients were randomly allocated to one of the four groups to receive a possible remifentanil effect-compartment target concentration (Ceremi) of 0, 1, 3, or 5 ng/ml, and an effect-compartment target controlled infusion of propofol to maintain the state entropy (SE) at 40~60. Three normalized measures DIRMS, DIMean, and DIPBI were compared with the DI values without normalization. Before LMA insertion, only DI showed a considerable correlation with remifentanil concentrations. DIRMS and DIMean performed better than DI in discriminating 'insufficient' concentrations (0 and 1 ng/ml) from 'sufficient' concentrations (3 and 5 ng/ml). DIRMS was superior to all other variables in grading analgesic depth after nociceptive event occurred with PK value of 0.836. These results demonstrate that the normalization using RMS value, compared to using mean value and maximum, seems to provide a more effective approach for signal pre-processing.

[Emotion Recognition Based on Multiple Physiological Signals].

Emotion is a series of reactions triggered by a specific object or situation that affects a person's physiological state and can, therefore, be identified by physiological signals. This paper proposes an emotion recognition model. Extracted the features of physiological signals such as photoplethysmography, galvanic skin response, respiration amplitude, and skin temperature. The SVM-RFE-CBR(Recursive Feature Elimination-Correlation Bias Reduction-Support Vector Machine) algorithm was performed to select features and support vector machines for classification. Finally, the model was implemented on the DEAP dataset for an emotion recognition experiment. In the rating scale of valence, arousal, and dominance, the accuracy rates of 73.5%, 81.3%, and 76.1% were obtained respectively. The result shows that emotional recognition can be effectively performed by combining a variety of physiological signals.

Ginsenoside metabolite 20(S)­protopanaxadiol promotes neural stem cell transition from a state of proliferation to differentiation by inducing autophagy and cell cycle arrest.

20(S)­Protopanaxadiol (PPD) is an active ginseng metabolite and is the final form of protopanaxadiol saponins metabolized by human intestinal microflora. The neuroprotective effects and mechanisms underlying PPD on neural stem cells (NSCs) are not completely understood. The aim of the present study was to assess the effects of PPD on the proliferation and differentiation of neural stem cells. In the present study, following treatment with different concentrations of PPD for 24 h, the percentage of BrdU­positive cells decreased significantly with increasing concentrations of PPD. Moreover, flow cytometric analysis results indicated that PPD treatment increased the proportion of cells in the G0/G1 and G2/M phase and decreased the proportion of cells in the S phase. The activation of autophagy, determined by an increased number of autophagic vacuoles and light chain 3 lipidation, was associated with an increase in the expression of the neuronal marker tubulin­ß3 following PPD treatment. PPD also partially rescued NSCs from the inhibitory effects of the autophagic inhibitor wortmannin, suggesting that the effect of PPD on NSC differentiation was associated with autophagy. Collectively, the results indicated that PPD promoted the transition of NSCs from a state of proliferation to differentiation through the induction of autophagy and cell cycle arrest. Therefore, the present study may provide a basis for the development of regenerative therapies based on ginsenoside, an approved and safe drug.

Glucose-induced oxidative stress and accelerated aging in endothelial cells are mediated by the depletion of mitochondrial SIRTs.

Diabetic complications cause significant morbidity and mortality. Dysfunction of vascular endothelial cells (ECs), caused by oxidative stress, is a main mechanism of cellular damage. Oxidative stress accelerates EC senescence and DNA damage. In this study, we examined the role of mitochondrial sirtuins (SIRTs) in glucose-induced oxidative stress, EC senescence, and their regulation by miRNAs. Human retinal microvascular endothelial cells (HRECs) were exposed to 5 mmol/L (normoglycemia; NG) or 25 mmol/L glucose (hyperglycemia; HG) with or without transfection of miRNA antagomirs (miRNA-1, miRNA-19b, and miRNA-320; specific SIRT-targeting miRNAs). Expressions of SIRT3, 4 and 5 and their targeting miRNAs were examined using qRT-PCR and ELISAs were used to study SIRT proteins. Cellular senescence was investigated using senescence-associated ß-gal stain; while, oxidative stress and mitochondrial alterations were examined using 8-OHdG staining and cytochrome B expressions, respectively. A streptozotocin-induced diabetic mouse model was also used and animal retinas and hearts were collected at 2 months of diabetes. In HRECs, HG downregulated the mRNAs of SIRTs, while SIRT-targeting miRNAs were upregulated. ELISA analyses confirmed such downregulation of SIRTs at the protein level. HG additionally caused early senescence, endothelial-to-mesenchymal transition and oxidative DNA damage in ECs. These changes were prevented by the transfection of specific miRNA antagomirs and by resveratrol. Retinal and cardiac tissues from diabetic mice also showed similar reductions of mitochondrial SIRTs. Collectively, these findings demonstrate a novel mechanism in which mitochondrial SIRTs regulate glucose-induced cellular aging through oxidative stress and how these SIRTs are regulated by specific miRNAs. Identifying such mechanisms may lead to the discovery of novel treatments for diabetic complications.

lncRNA H19 prevents endothelial-mesenchymal transition in diabetic retinopathy.

AIMS/HYPOTHESIS: The pathophysiology of diabetic retinopathy is linked to hyperglycaemia and its effect on retinal microvascular tissues. The resulting endothelial injury changes the endothelial cell phenotype to acquire mesenchymal properties (i.e. endothelial-mesenchymal transition [EndMT]). Such changes can be regulated by epigenetic mechanisms, including long non-coding RNAs (lncRNAs). lncRNA H19 may influence EndMT through TGF-ß. We investigated the role of H19 in regulating EndMT during diabetic retinopathy. METHODS: H19 was overexpressed or silenced in human retinal endothelial cells exposed to various glucose levels. The cells were examined for H19, endothelial and mesenchymal markers. We then expanded the study to retinal tissues in a mouse model of diabetic retinopathy and also examined vitreous humour samples from individuals with proliferative diabetic retinopathy. RESULTS: Expression of H19 was downregulated in high glucose conditions (25 mmol/l). H19 overexpression prevented glucose-induced EndMT. Such changes appear to involve TGF-ß through a Smad-independent mechanism. Diabetes caused downregulation of retinal H19. Using H19 knockout mice, we demonstrated similar EndMT in the retina. Examination of vitreous humour from individuals with proliferative diabetic retinopathy also reinforced the downregulation of H19 in diabetes. CONCLUSIONS/INTERPRETATION: We therefore concluded that H19 regulates EndMT in diabetic retinopathy through specific mechanisms. DATA AVAILABILITY: The results from our previous microarray can be found online using the GEO accession number GSE122189.

Photoplethysmography Response to Laryngeal Mask Airway Insertion during Propofol-Remifentanil Anethesia.

The present study was aimed to evaluate the abilities of photoplethysmography (PPG)-derived parameters, including sample entropy of PPG (SampEn), amplitude of PPG (PPGA), pulse beat interval (PBI) and diastolic interval (DI) extracted by nonlinear or linear methods, to monitor the balance between nociception and antinociception. 26 ASA I or II patients were randomized into one of the three groups to receive a remifentanil effect-compartment target controlled infusion (Ceremi) of 1, 3 and 5 ng/ml and an effect-compartment target controlled propofol infusion (Ceprop) to keep the state entropy (SE) at 50 (40~60). Laryngeal mask airway (LMA) insertion was applied as a noxious stimulus. The percentage of change in SampEn (△SampEn, AUC=0.896), PBI (△PBI, AUC=0.896) and DI (ΔDI, AUC=0.972), but not in PPGA (△PPGA, AUC=0.667), were statistically excellent in discriminating low Ceremi (1 ng/ml) from higher Ceremi (3 and 5 ng/ml). Additionally, the prediction probabilities (Pk) values of △SampEn, ΔPBI and ΔDI were high as well with 0.795, 0.754 and 0.813 for discriminating Ceremi. These results demonstrated that nonlinear and linear parameters of SampEn, PBI and DI had strong dependency on Ceremi in response to LMA insertion and could provide nociceptive information during propofol-remifentanil anesthesia. This indicated that PPG-derived parameters were potential to develop the clinical assessment of nociception-antinociception balance under general anesthesia.

Curcumin Analogs Reduce Stress and Inflammation Indices in Experimental Models of Diabetes.

Chronic inflammation and oxidative stress lead to a multitude of adverse cellular responses in target organs of chronic diabetic complications. Curcumin, a highly investigated phytochemical, has been shown to exhibit both anti-inflammatory and antioxidant activities. However, the clinical application of curcumin has been greatly limited due to a poor pharmacokinetic profile. To overcome these limitations, we have generated analogs of curcumin to enhance bioavailability and offer a preferable pharmacokinetic profile. Here, we explored the effects of two mono-carbonyl curcumin analogs, L2H21 and L50H46, in alleviating indices of inflammation and oxidative stress in cell culture and mouse model of diabetic complications. Our results show that L2H21 and L50H46 normalize inflammatory mediators (IL-6 and TNF-α), extracellular matrix proteins (FN and COL4α1), vasoactive factors (VEGF and ET-1) and a key transcriptional coactivator (p300) in cultured human retinal microvascular endothelial cells (HRECs) and dermal-derived microvascular endothelial cells (HMVECs) challenged with high levels of glucose. These curcumin analogs also reduced glucose-induced oxidative DNA damage as evidenced by 8-OHdG labeling. We further show that treatment of streptozotocin-induced diabetic mice with curcumin analogs prevents cardiac and renal dysfunction. The preservation of target tissue function was associated with normalization of pro-inflammatory cytokines and matrix proteins. Collectively, our results show that L2H21 and L50H46 offer the anti-inflammatory and antioxidant activities as has been reported for curcumin, and may provide a clinically applicable therapeutic option for the treatment of diabetic complications.

MALAT1: An Epigenetic Regulator of Inflammation in Diabetic Retinopathy.

Despite possessing limited protein-coding potential, long non-coding RNAs (lncRNAs) have been implicated in a myriad of pathologic conditions. Most well documented in cancer, one prominent intergenic lncRNA known as MALAT1 is notorious for its role in impacting epigenetic mechanisms. In this study, we established a novel epigenetic paradigm for MALAT in diabetic retinopathy (DR) by employing siRNA-mediated MALAT1 knockdown in human retinal endothelial cells (HRECs), a Malat1 knockout animal model, vitreous humor from diabetic patients, pharmacological inhibitors for histone and DNA methylation, RNA immunoprecipitation, western blotting, and a unique DNA methylation array to determine glucose-related alterations in MALAT1. Our findings indicated that MALAT1 is capable of impacting the expressions of inflammatory transcripts through its association with components of the PRC2 complex in diabetes. Furthermore, the vitreous humors from diabetic patients revealed increased expressions of MALAT1, TNF-α, and IL-6. Intriguingly, our DNA methylation array demonstrated that transient high glucose exposure in HRECs does not contribute to significant methylation alterations at CpG sites across the MALAT1 gene. However, global inhibition of DNA methyltransferases induced significant increases in MALAT1 and associated inflammatory transcripts in HRECs. Our findings collectively demonstrate the importance of MALAT1 in inflammation and epigenetic regulation in DR.

Endothelin-1 traps potently reduce pathologic markers back to basal levels in an in vitro model of diabetes.

BACKGROUND: Diabetes mellitus is a group of metabolic disorders in which there are high blood glucose levels over a prolonged period. Diabetes is one of many diseases associated with pathologically elevated levels of endothelin (ET)-1. We have recently proposed the development of ET-traps, which are an antibody - based fusion protein that potently bind and sequester pathologically elevated levels of endothelin-1. METHODS: We constructed ET-traps that were found to be very potent binders to ET-1, with a KD of 32.5ρM. We then treated human retinal microvascular endothelial cells (HRMECs), which are an in vitro model of glucose induced cellular damage, with 10 nM ET-1 or high glucose levels (25 mM). RESULTS: In this study, we investigated the effects of our ET-trap constructs on the expression levels of both collagen 4α1 and fibronectin, which are both important pathologic markers in diabetes. Treating HRMECs with 10 nM ET-1 or 25 mM glucose significantly induces the expression of the ECM proteins fibronectin and collagen 4α1, as is found in chronic diabetic complications; Incubation of the cells with the ET-traps significantly prevented the increased expression of fibronectin and collagen 4α1 back to basal levels. This was found with both mRNA and protein expression levels of the two ECM proteins. CONCLUSION: Our results provide the first evidence of the efficacy of ET-traps in reducing pathologic markers in an in vitro model (of diabetes). Further research is warranted to determine the efficacy of ET-traps as a therapeutic tool for diabetes, which is a major public health burden around the world.

Prevention of Diabetic Nephropathy by Modified Acidic Fibroblast Growth Factor.

BACKGROUND/AIMS: Oxidative stress (OS) contributes to all chronic diabetic complications, including diabetic nephropathy (DN). Acidic fibroblast growth factor (aFGF) has shown to confer protection from OS. However, it also has potent angiogenic activity. We hypothesized that a modified human aFGF (maFGF), with antioxidant properties but devoid of angiogenic activity, has preventative action in DN. METHODS: Streptozotocin-induced diabetic mice were treated with maFGF (intraperitoneally) daily for 1 or 6 months and were compared with untreated diabetic and non-diabetic controls. Microalbuminuria was assessed to determine functional damage. Renal cortical tissues were examined for multiple extracellular matrix proteins, vasoactive factors and OS markers. For mechanistic studies, immortalized mouse podocytes and human microvascular endothelial cells were exposed to high (25 mM) or low glucose (5 mM). OS, vasoactive factors, fibrosis and apoptosis-related gene expression were tested by real-time qPCR and Enzyme-Linked Immunosorbent Assay. Nitric oxide (NO) analyses were also performed. RESULTS: maFGF did not affect body weight and glycemia but prevented renal hypertrophy and functional changes in DN. It also prevented diabetes-induced DNA damage, nitrosative stress, vasoactive factors, angiotensinogen and endothelial NO synthase alterations. Although it failed to prevent transforming growth factor (TGF)-ß1 mRNA upregulation, it prevented fibronectin production. Similar results were obtained in vitro. Decreased NO production in vivo and in vitro was also prevented by maFGF. CONCLUSIONS: maFGF treatment prevents DN. This prevention probably involves NO production.