The Association of Total Meat Intake with Cardio-Metabolic Disease Risk Factors and Measures of Sub-Clinical Atherosclerosis in an Urbanising Community of Southern India: A Cross-Sectional Analysis for the APCAPS Cohort.

AIM: Meat is commonly consumed in India; however, in comparison to Western settings, it is eaten in relatively lower quantities and with minimal processing. The association between meat intake and cardio-metabolic diseases (CMDs) and their risk factors in India is currently uncertain. We examined whether meat intake is associated with risk factors for CMDs and the measures of subclinical atherosclerosis in urbanising villages in southern India. METHODS: We conducted a cross-sectional analysis of 6012 adults (52.3% male) participating in the Andhra Pradesh Children and Parents' Study (APCAPS), which is a large prospective, intergenerational cohort study in Southern India that began with the long-term follow-up of the Hyderabad Nutrition Trial (1987-1990). We used cross-sectional data from the third wave of data collection conducted in 2010-2012, where total meat intake was assessed using 100-item, semi-quantitative validated food frequency questionnaires (FFQ). The FFQs were validated using multiple weighed 24 h dietary recalls. The main predictor, 'total meat intake', was calculated as the sum of chicken, red meat, and fish consumption. The risk factors for CMDs [systolic blood pressure (SBP), diastolic blood pressure (DBP), body mass index (BMI), waist circumference (WC), fasting glucose, total cholesterol, homeostasis model assessment insulin resistance (HOMA-IR), total cholesterol, low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol, triglycerides, and C-reactive protein] and measures of subclinical atherosclerosis [Carotid Intima-Media Thickness, Pulse Wave Velocity, and Augmentation Index] were assessed using standardised clinical procedures. Stratified by gender, the association of meat intake with the risk factors of CMDs and measures of subclinical atherosclerosis was examined using linear multilevel models with random intercept at the household level. RESULTS: The mean (SD) age of the male (n = 3128) and female participants (n = 2828) was 34.09 years (15.55) and 34.27 years (12.73), respectively. The median (IQR) intake of meat was 17.79 g/day (8.90, 30.26) in males and 8.90 g/day (4.15, 18.82) in females. In males, a 10 g increase in total meat intake/1000 Kcal/day was positively associated with DBP, BMI, WC, total cholesterol, LDL-C, and triglycerides, whereas in females, a 10 g increase in total meat intake/1000 Kcal/day was positively associated with SBP, DBP, fasting glucose, HOMA-IR, total cholesterol, LDL-C, and triglycerides. There was no relationship between meat consumption and measures of subclinical atherosclerosis. CONCLUSIONS: Meat intake had a linear positive association with CMD risk factors among the relatively younger Indian population who were consuming meat at lower levels compared to their European counterparts.

Interleukin 31 receptor α promotes smooth muscle cell contraction and airway hyperresponsiveness in asthma.

Asthma is a chronic inflammatory airway disease characterized by airway hyperresponsiveness (AHR), inflammation, and goblet cell hyperplasia. Multiple cytokines, including IFNγ, IL-4, and IL-13 are associated with asthma; however, the mechanisms underlying the effects of these cytokines remain unclear. Here, we report a significant increase in the expression of IL-31RA, but not its cognate ligand IL-31, in mouse models of allergic asthma. In support of this, IFNγ, IL-4, and IL-13 upregulated IL-31RA but not IL-31 in both human and mice primary airway smooth muscle cells (ASMC) isolated from the airways of murine and human lungs. Importantly, the loss of IL-31RA attenuated AHR but had no effect on inflammation and goblet cell hyperplasia in mice challenged with allergens or treated with IL-13 or IFNγ. We show that IL-31RA functions as a positive regulator of muscarinic acetylcholine receptor 3 expression, augmenting calcium levels and myosin light chain phosphorylation in human and murine ASMC. These findings identify a role for IL-31RA in AHR that is distinct from airway inflammation and goblet cell hyperplasia in asthma.

Nε-Carboxymethyl-Lysine Modification of Extracellular Matrix Proteins Augments Fibroblast Activation.

The extracellular matrix (ECM) is a dynamic complex protein network that provides structural integrity and plays an active role in shaping fibroblast behavior both in health and disease. Despite its essential functions, the impact of age-associated post-translational modifications on ECM-driven fibroblast activities such as proliferation, survival, fibroblast-to-myofibroblast transformation (FMT), and extracellular matrix production remains largely unknown. Nε-carboxymethyl-lysine (CML) is one of the well-characterized advanced glycation end-products (AGEs) that can occur on lysine residues within ECM proteins through non-enzymatic glycation. In this study, we determined the accumulation and the effects of the CML-modified ECM (CML-ECM) on fibroblast activation. Immunostainings and immunoblot analysis demonstrated significant increases in CML-AGE content in idiopathic pulmonary fibrosis (IPF) compared to age-matched healthy lungs. Gene expression analysis and fibroblast activation assays collectively implicate the ECM as a negative regulator of fibroblast activation. Notably, the CML modification of the ECM resulted in a significant decrease in its anti-fibrotic effects including proliferation, FMT, apoptosis, and ECM production. Together, the results of this study revealed an unexplored pathological role played by the CML-ECM on fibroblast activation, which has wide implications in IPF and other fibrotic diseases.

Obesity Associated with Prediabetes Increases the Risk of Breast Cancer Development and Progression-A Study on an Obese Rat Model with Impaired Glucose Tolerance.

Patients with comorbidities of obesity and diabetes are recognized to be at high risk of breast cancer development and face worse breast cancer outcomes. Though several reports showed the reinforced link between obesity, diabetes, and prediabetes with breast cancer, the underlying molecular mechanisms are still unknown. The present study aimed to investigate the underlying molecular link between increased risks of breast cancer due to coincident diabetes or obesity using a spontaneous obese rat model with impaired glucose tolerance (WNIN/GR-Ob rat). A single dose of solubilized DMBA suspension (40 mg/kg body weight) was orally administered to the animals at the age of 60 days to induce breast tumors. The tumor incidence, latency period, tumor frequency, and tumor volume were measured. Histology, immunohistochemistry, and immunoblotting were performed to evaluate the tumor morphology and expression levels of signal molecules. The development of mammary tumors in GR-Ob rats was characterized by early onset and shorter latency periods compared to control lean rats. While 62% of obese rats developed breast tumors, tumor development in lean rats was only 21%. Overexpression of ER, PR, Ki67, and p53 markers was observed in tumor tissues of obese rats in comparison with lean rats. The levels of the hallmarks of cell proliferation and angiogenesis involved in IGF-1/PI3K/Akt/GSK3ß/ß-catenin signaling pathway molecules were upregulated in obese rat breast tumors compared to lean rats. Furthermore, obesity with prediabetes is associated with changes in IGF-1 signaling and acts on PI3K/Akt/GSK3ß/ß-catenin signaling, which results in rapid cell proliferation and development of breast tumors in obese rats than the lean rats. These results indicate that tumor onset and development were faster in spontaneous obese rat models with impaired glucose tolerance than in their lean counterparts.

Prevalence of Vitamin B12 and Folate Deficiencies in Indian Children and Adolescents.

Deficiencies of vitamin B12 (B12) and folate (FA) are of particular interest due to their pleiotropic role in 1-carbon metabolism. In addition to adverse birth outcomes, deficiencies of B12 and FA, or an imbalance in FA/B12 status, are linked to metabolic disorders. Indian diets that are predominantly plant food-based could be deficient in these vitamins, but there are no national estimates of the prevalence of B12 and FA deficiency in Indian children and adolescents, nor their associations with age, sex and growth indicators. The recent Comprehensive National Nutrition Survey (CNNS-2016-18) provided estimates of the prevalence of B12 and FA deficiency at the national and state levels among preschool (1-4 years: 9976 and 11,004 children, respectively), school-age children (5-9 years: 12,156 and 14,125) and adolescents (10-19 years: 11,748 and 13,621). Serum B12 and erythrocyte FA were measured by the direct chemiluminescence method and their deficiency was defined using WHO cut-offs. The prevalence of B12 and FA deficiency was high among adolescents (31.0%, CI: 28.7-33.5 and 35.6%, CI: 33.1-8.2) compared to school-age (17.3%, CI: 15.4-19.3 and 27.6%, CI: 25.5-29.9) and preschool children (13.8%, CI: 11.7-16.2 and 22.8%, CI: 20.5-25.2, respectively). The prevalence of both B12 and FA deficiency was significantly higher by 8% and 5%, respectively, in adolescent boys compared to girls. There was no association between anthropometric undernutrition and B12 and FA deficiency. There was wide regional variation in the prevalence of B12 and FA deficiency, but no rural-urban differences were observed across all age groups. The national prevalence of B12 deficiency among preschool or school-age children was <20% (the cut-off that indicates a public health problem). However, FA deficiency in these age groups and both FA and B12 deficiencies in adolescents were >20%, warranting further investigation.

Combined prenatal to postnatal protein restriction augments protein quality control processes and proteolysis in the muscle of rat offspring.

Several human epidemiological and animal studies suggest that a maternal low-protein (MLP) diet affects skeletal muscle (SM) health in the offspring. However, effect of combined prenatal to postnatal protein restriction (chronic PR) and prenatal to perinatal protein restriction (PR) with postnatal rehabilitation maternal protein restriction (MPR) on protein quality control (PQC) processes and proteolysis in the offspring remains poorly understood. The current study explored the impact of chronic PR and MPR on SM protein degradation rates, chaperones, unfolded protein response (UPR), ubiquitin-proteasome system (UPS), autophagy, and apoptosis, in the adult offspring. Wistar rats were randomly assigned to a normal protein (NP; 20% casein), or low-protein (LP; 8% casein) isocaloric diets from 7 weeks prior to breeding through weaning. Offspring born to NP dams received the same diet (NP offspring) while a group of LP offspring remained on LP diet and another group was rehabilitated with NP diet (LPR offspring) from weaning for 16 weeks. LP offspring displayed lower body weight, lean mass, and myofiber cross-sectional area than NP. Furthermore, LP offspring demonstrated increased total protein degradation, urinary 3-methyl histidine, ER stress, autophagy, UPS components, proteasomal activity, muscle atrophy markers, and apoptosis-related proteins than NP. However, MPR showed little or no effect on muscle proteolysis, UPR, UPS, autophagy, apoptosis, and muscle atrophy in LPR offspring. These results indicate that exposure to chronic PR diets induces muscle atrophy and accelerates SM proteolysis via augmenting PQC processes in the offspring, while MPR shows little or no effect.

Protective effect of cinnamon on diabetic cardiomyopathy in nicotinamide-streptozotocin induced diabetic rat model.

There is an increase in the incidence and prevalence of type-2 diabetes and obesity which leads to the structural and functional changes in myocardium leading to a lethal complication called diabetic cardiomyopathy (DCM). In the present study, we investigated the preventive effect of cinnamon (3% of Cinnamomum zeylanicum bark powder in AIN-93 diet for 3 months) feeding on DCM and the concerned mechanisms in a rodent model. Experimental diabetes was induced by a single intraperitoneal injection of 40 mg/kg b.w streptozotocin (STZ), 15 min after the ip administration of 60 mg/kg b.w of nicotinamide (NA) in Wistar-NIN (WNIN) male rats. The oxidative stress parameters were investigated by assessing superoxide dismutase (SOD), glutathione-s-transferase (GST) enzyme activity, protein carbonyls and malondialdehyde (MDA) levels. The histopathology of myocardium was analyzed by H&E and Masson's trichrome staining, and scanning electron microscopy. The changes in diabetic rat heart involved the altered left ventricular parietal pericardium, structural changes in myocardial cells, enhanced oxidative stress. Masson's trichrome and H&E staining have shown increased fibrosis, and perinuclear vacuolization in NA-STZ induced diabetic rat myocardium. Cinnamon feeding prevented the oxidative stress and myocardial alterations in the heart of diabetic rats. Taken together, these results suggest that cinnamon can effectively prevent the metabolic and structural changes in NA-STZ induced diabetic cardiomyopathy.

Prevalence of vitamin A deficiency and dietary inadequacy in Indian school-age children and adolescents.

PURPOSE: There are no representative estimates of vitamin A deficiency (VAD) and risk of vitamin A (VA) dietary inadequacy in Indian children and adolescents. To evaluate, from national surveys, the prevalence of VAD measured by serum retinol concentrations (< 0.7 µmol/L or < 20 µg/dL), and the risk of VA dietary inadequacy and excess intake beyond the tolerable upper limit (TUL). METHODS: National and state-level VAD prevalence adjusted for inflammation was estimated in school-age children (5-9 years: 10,298) and adolescents (10-19 years: 9824) from the Comprehensive National Nutrition Survey (CNNS 2016-18). The risk of dietary inadequacy against age-specific average VA requirements, and excess intake against the TUL, was assessed from the National Sample Survey Office (NSSO 2014) data. RESULTS: Serum retinol concentrations increased with age (5-19 years) in both genders and were significantly lower in school-age children (1.02 µmol/L, CI: 1.01-1.03) compared to adolescents (1.13 µmol/L, CI 1.12-1.15). The inflammation-adjusted prevalence of VAD in school-age children and adolescents was 19.3% (CI 18.8-19.9) and 14.4% (CI 13.9-14.9) respectively, and this was > 20% in seven and four states for children and adolescents, respectively. The prevalence of VAD was significantly higher among children with lower socio-economic status. The risk of dietary VA inadequacy, from the NSSO survey, was 69 and 78% in children and adolescents, respectively. This risk reduced to 6 and 17% with VA fortified oil and milk intake, while the proportion of intakes exceeding the TUL became 6 and 0.5% in children and adolescents, respectively. CONCLUSIONS: The national prevalence of VAD in school-age children and adolescents in India was just less than 20%. The risk of dietary VA deficiency is likely to decline substantially with VA fortified food intake, but a risk of excessive intake also begins to appear; therefore, a careful assessment of the risk of hypervitaminosis A is required at these ages.

Chronic Effects of Maternal Low-Protein and Low-Quality Protein Diets on Body Composition, Glucose-Homeostasis and Metabolic Factors, Followed by Reversible Changes upon Rehabilitation in Adult Rat Offspring.

Several studies suggest that the maternal protein content and source can affect the offspring's health. However, the chronic impact of maternal quality and quantity protein restriction, and reversible changes upon rehabilitation, if any, in the offspring, remains elusive. This study examined the effects of maternal low-quality protein (LQP) and low-protein (LP) intake from preconception to post-weaning, followed by rehabilitation from weaning, on body composition, glucose-homeostasis, and metabolic factors in rat offspring. Wistar rats were exposed to normal protein (NP; 20% casein), LQP (20% wheat gluten) or LP (8% casein) isocaloric diets for 7 weeks before pregnancy until lactation. After weaning, the offspring were exposed to five diets: NP, LQP, LQPR (LQP rehabilitated with NP), LP, and LPR (LP rehabilitated with NP) for 16 weeks. Body composition, glucose-homeostasis, lipids, and plasma hormones were investigated. The LQP and LP offspring had lower bodyweight, fat and lean mass, insulin and HOMA-IR than the NP. The LQP offspring had higher cholesterol, T3 and T4, and lower triacylglycerides and glucose, while these were unaltered in LP compared to NP. The majority of the above outcomes were reversed upon rehabilitation. These results suggest that the chronic exposure of rats to maternal LQP and LP diets induced differential adverse effects by influencing body composition and metabolism, which were reversed upon rehabilitation.

Micronutrient intakes and status assessed by probability approach among the urban adult population of Hyderabad city in South India.

PURPOSE: To assess the dietary inadequacies of micronutrients and the associated factors among the apparently healthy urban adults. METHODS: This community-based cross-sectional study involved 300 urban adults (distributed into age groups: 21-40, 41-60, and > 60 years) residing in Hyderabad city, South India. Hemoglobin in whole blood, ferritin, folate, and vitamin B12 (B12) in plasma was estimated. Dietary intakes were assessed by three 24-h dietary recalls and calculated the probability of adequacy (PA) using estimated average requirement. RESULTS: The prevalence of anemia (30%), iron deficiency (ID, 23%), and iron deficiency anemia (IDA, 14.3%) was independent of age but higher in women. While folate deficiency (32.2%) was independent of age and gender, B12 deficiency (35.5%) varied by both age and gender. The PA of iron (89%) was higher, while that of folate, B12, and zinc (1-11%) were noticeably low. The mean PA (MPA) across the ten micronutrients was 38%, independent of age and gender, but associated with the educational status. Energy intake was a strong predictor of the MPA. Cereals and millets predominantly contributed to the intake of thiamine, niacin, zinc, and iron; green leafy vegetables and fruits to vitamins A, C, folate, and iron; animal foods to B12; and milk and milk products to calcium, vitamin A, riboflavin, and B12. The unadjusted and adjusted logistic regression models revealed that micronutrient inadequacy was associated with greater risk of IDA and folate deficiency. CONCLUSIONS: These results indicate a higher prevalence of micronutrient deficiencies among the healthy urban adults possibly due to the inadequacy of multiple micronutrients.

Circulating levels of Hsp27 in microvascular complications of diabetes: Prospects as a biomarker of diabetic nephropathy.

AIM: Heat shock protein 27 (Hsp27) is a small heat shock protein known to protect the cells from apoptosis under stress. In the present study, we determined the plasma Hsp27 levels in type 2 diabetes subjects without and with microvascular complications- diabetic retinopathy (DRe), diabetic nephropathy (DNe), and diabetic neuropathy (DNu) to understand if it could serve as a marker for these complications. METHODS: This is a hospital-based case-control study with 754 subjects including 247 controls, 195 subjects with diabetes, 123 with DRe, 80 with DNe and 109 with DNu. Plasma Hsp27 levels were measured by ELISA. RESULTS: The mean plasma Hsp27 was higher in the DNe group (631.5±355.2) compared to the control (496.55±308.54), diabetes (523.41±371.01), DRe (494.60±391.48) and DNu (455.21±319.74) groups with a p-value of 0.018. Receiver operating characteristic (ROC) curve analysis of Hsp27 in DNe group showed an area under the curve (AUC) of 0.617. Spearman correlation analysis shows a positive correlation of plasma Hsp27 with serum creatinine (p=0.053, r-value 0.083). Gender, age and BMI did not affect the plasma Hsp27 levels. CONCLUSION: The plasma Hsp27 levels in the DNe group are higher compared to the control and other complications, thereby it could be explored to be used as a potential biomarker of DNe.

Generating minicorneal organoids from human induced pluripotent stem cells.

Corneal epithelial stem cells residing within the annular limbal crypts regulate adult tissue homeostasis. Autologous limbal grafts and tissue-engineered corneal epithelial cell sheets have been widely used in the treatment of various ocular surface defects. In the case of bilateral limbal defects, pluripotent stem cell (PSC)-derived corneal epithelial cells are now being explored as an alternative to allogeneic limbal grafts. Here, we report an efficient method to generate complex three-dimensional corneal organoids from human PSCs. The eye field primordial clusters that emerged from differentiating PSCs developed into whole eyeball-like, self-organized, three-dimensional, miniature structures consisting of retinal primordia, corneal primordia, a primitive eyelid-like outer covering and ciliary margin zone-like adnexal tissues in a stepwise maturation process within 15 weeks. These minicorneal organoids recapitulate the early developmental events in vitro and display similar anatomical features and marker expression profiles to adult corneal tissues. They offer an alternative tissue source for regenerating different layers of the cornea and eliminate the need for complicated cell enrichment procedures.

Ubiquitin-proteasome system and ER stress in the retina of diabetic rats.

PURPOSE: Diabetic retinopathy (DR) is the most frequently occurring complication of diabetes. Alterations in ubiquitin-proteasome system (UPS) have been associated with several degenerative disorders. Hence, in this study, we investigated the status and role of UPS and ER stress in the retina of diabetic rats. METHODS: Diabetes was induced in rats by streptozotocin. Retinal markers, ER stress markers, components of UPS, ERAD, and autophagy were analyzed after 2- and 4-months of diabetes. Apoptosis was analyzed by TUNEL Assay. RESULTS: There were increased acellular capillaries and pericyte loss in diabetic rat retina. Decreased protein expression of UPS components - ubiquitin activating enzyme (E1), deubiquitinating enzymes (UCHL1 and UCHL5), SIAH1 (E3 ligase) and free ubiquitin were observed in the diabetic rats. Increased ER stress markers (ATF6, XBP1, and CHOP), decreased expression of HRD1, declined autophagy (LC3B) and increased apoptosis were observed in diabetic rats. Interestingly, treatment of diabetic rats with a chemical chaperone (4-PBA) restored the levels of DUBs and ameliorated ER stress-induced retinal cell death in type 1 diabetic rats. CONCLUSION: The declined UPS components: E1 and HRD1 in the retina of diabetic rats could elicit ER stress, and the prolonged ER stress may trigger CHOP-mediated neuronal apoptosis.

Impact of obesity with impaired glucose tolerance on retinal degeneration in a rat model of metabolic syndrome.

PURPOSE: Metabolic syndrome (MetS) is associated with several degenerative diseases, including retinal degeneration. Previously, we reported on progressive retinal degeneration in a spontaneous obese rat (WNIN/Ob) model. In this study, we investigated the additional effect of impaired glucose tolerance (IGT), an essential component of MetS, on retinal degeneration using the WNIN/GR-Ob rat model. METHODS: The retinal morphology and ultrastructure of WNIN/GR-Ob and age-matched littermate lean rats were studied by microscopy and immunohistochemistry. The retinal transcriptome of WNIN/GR-Ob was compared with the respective lean controls and with the WNIN/Ob model using microarray analysis. Expression of selected retinal marker genes was studied via real-time PCR. RESULTS: Progressive loss of photoreceptor cells was observed in WNIN/GR-Ob rats with an onset as early as 3 months. Similarly, thinning of the inner nuclear layer was observed from 6 months in these rats. Immunohistochemical analysis showed decreased levels of rhodopsin and postsynaptic density protein-95 (PSD-95) proteins and increased levels of glial fibrillary acidic protein (GFAP), vascular endothelial growth factor (VEGF), and calretinin in WNIN/GR-Ob rats compared with the age-matched lean controls, further supporting cellular stress/damage and retinal degeneration. The retinal transcriptome analysis indicated altered expression profiles in both the WNIN/GR-Ob and WNIN/Ob rat models compared to their respective lean controls; these pathways are associated with activation of pathways like cellular oxidative stress response, inflammation, apoptosis, and phototransduction, although the changes were more prominent in WNIN/GR-Ob than in WNIN/Ob animals. CONCLUSIONS: WNIN/GR-Ob rats with added glucose intolerance developed retinal degeneration similar to the parent line WNIN/Ob. The severity of retinal degeneration was greater in WNIN/GR-Ob rats compared to WNIN/Ob, suggesting a possible role for IGT in this model. Hence, the WNIN/GR-Ob model could be a valuable tool for investigating the impact of MetS on retinal degeneration pathology.

Dietary ginger improves glucose dysregulation in a long-term high-fat high-fructose fed prediabetic rat model.

The rapid increase in global diabetes burden with its associated morbidity and mortality is a major health concern for humans. Prediabetes is a condition which predispose a person not only to diabetes but also to the associated complications including morbidity even in the absence of an apparant hyperglycemia. However, appropriate dietary intervention may not only prevent but also improve one's condition as diet is the major contributor to such metabolic disorders. Here, we investigated the effect of dietary ginger (Zingiber officinale Roscoe) on the markers of insulin resistance and pathophysiology in a diet-induced prediabetic rat model. Male Sprague-Dawley (SD) rats were fed the following diets: control (5% groundnut oil + 65 % corn starch), high fat high fructose (HFHF; 25% beef tallow + 35 % fructose) and HFHF with 3 % ginger (HFHFG) for eight months. Plasma markers of insulin resistance, lipid profile, oral glucose tolerance (OGTT; 2nd and 5th month), intraperitoneal insulin tolerance (ITT), plasma total antioxidant capacity (TAC), liver histology and pancreatic immunohistochemistry (IHC) were examined. The impaired OGTT, ITT and insulin sensitivity indices with observed hyperinsulinemia and hypertriglyceridemia suggest that HFHF feeding resulted in prediabetes in rats. HFHF feeding also decreased insulin secretion in the pancreas, increased lipid accumulation in liver and total oxidants in plasma. The effects of HFHF feeding on glucose regulation, pathophysiology of pancreas and liver; total oxidative stress were improved by ginger feeding. The present study demonstrated thatlong-term HFHF feeding induces prediabetes in experimental rats while dietary ginger neutralizes the HFHF induced impairment in glucose regulation, dyslipidemia, and oxidative stress.

Gedunin abrogates aldose reductase, PI3K/Akt/mToR, and NF-κB signaling pathways to inhibit angiogenesis in a hamster model of oral carcinogenesis.

Aberrant activation of oncogenic signaling pathways plays a central role in tumor development and progression. The aim of this present study was to investigate the chemopreventive effects of the neem limonoid gedunin in the hamster model of oral cancer based on its ability to modulate aldose reductase (AR), phosphatidyl inositol-3-kinase (PI3K)/Akt, and nuclear factor kappa B (NF-κB) pathways to block angiogenesis. Administration of gedunin suppressed the development of HBP carcinomas by inhibiting PI3K/Akt and NF-κB pathways through the inactivation of Akt and inhibitory kappa B kinase (IKK), respectively. Immunoblot and molecular docking interactions revealed that inhibition of these signaling pathways may be mediated via inactivation of AR by gedunin. Gedunin blocked angiogenesis by downregulating the expression of miR-21 and the pro-angiogenic factors vascular endothelial growth factor and hypoxia inducible factor-1 alpha (HIF-1α). In conclusion, the results of the present study provide compelling evidence that gedunin prevents progression of hamster buccal pouch (HBP) carcinomas via inhibition of the kinases Akt, IKK, and AR, and the oncogenic transcription factors NF-κB and HIF-1α to block angiogenesis.

Ellagic acid inhibits VEGF/VEGFR2, PI3K/Akt and MAPK signaling cascades in the hamster cheek pouch carcinogenesis model.

BACKGROUND: Blocking vascular endothelial growth factor (VEGF) mediated tumor angiogenesis by phytochemicals has emerged as an attractive strategy for cancer prevention and therapy. METHODS: We investigated the anti-angiogenic effects of ellagic acid in a hamster model of oral oncogenesis by examining the transcript and protein expression of hypoxia-inducible factor-1alpha (HIF-1α), VEGF, VEGFR2, and the members of the PI3K/Akt and MAPK signaling cascades. Molecular docking studies and cell culture experiments with the endothelial cell line ECV304 were performed to delineate the mechanism by which ellagic acid regulates VEGF signaling. RESULTS: We found that ellagic acid significantly inhibits HIF-1α-induced VEGF/VEGFR2 signalling in the hamster buccal pouch by abrogating PI3K/Akt and MAPK signaling via downregulation of PI3K, PDK-1, p-Akt(ser473), mTOR, p-ERK, and p-JNK. Ellagic acid was also found to reduce the expression of histone deacetylases that could inhibit neovascularization. Analysis of the mechanism revealed that ellagic acid inhibits hypoxia-induced angiogenesis via suppression of HDAC-6 in ECV304 cells. Furthermore, knockdown of endogenous HDAC6 via small interfering RNA abrogated hypoxia-induced expression of HIF-1α and VEGF and blocked Akt activation. Molecular docking studies confirmed interaction of ellagic acid with upstream kinases that regulate angiogenic signaling. CONCLUSIONS: Taken together, these findings demonstrate that the anti-angiogenic activity of ellagic acid may be mediated by abrogation of hypoxia driven PI3K/Akt/mTOR, MAPK and VEGF/VEGFR2 signaling pathways involving suppression of HDAC6 and HIF-1α responses. GENERAL SIGNIFICANCE: Ellagic acid offers promise as a lead compound for anticancer therapeutics by virtue of its ability to inhibit key oncogenic signaling cascades and HDACs.

Effect of chronic hyperglycemia on crystallin levels in rat lens.

Crystallins are the major structural proteins in the vertebrate eye lens that contribute to lens transparency. Although cataract, including diabetic cataract, is thought to be a result of the accumulation of crystallins with various modifications, the effect of hyperglycemia on status of crystallin levels has not been investigated. This study evaluated the effect of chronic hyperglycemia on crystallin levels in diabetic cataractous rat lens. Diabetes was induced in rats by injecting streptozotocin and maintained on hyperglycemia for a period of 10weeks. At the end, levels of α-, ß-, γ-crystallins and phosphoforms of αB-crystallins (αBC) were analyzed by immunoblotting. Further, solubility of crystallins and phosphoforms of αBC was analyzed by detergent soluble assay. Chronic diabetes significantly decreased the protein levels of α-, ß- and αA-crystallins (αAC) in both soluble and insoluble fraction of lens. Whereas γ-crystallin levels were decreased and αBC levels were increased in lens soluble fraction with no change in insoluble fraction in diabetic rat lens. Although, diabetes activated the p38MAPK signaling cascade by increasing the p-p38MAPK in lens, the phosphoforms of αBC were decreased in soluble fraction with a concomitant increase in insoluble fraction of diabetic lens when compared to the controls. Moreover, diabetes strongly enhances the degradation of crystallinsand phosphoforms of αBC in lens. Taken together, the decreased levels of crystallins and insolubilization of phosphoforms of αBC under chronic hyperglycemia could be one of the underlying factors in the development of diabetic cataract.

Response of small heat shock proteins in diabetic rat retina.

PURPOSE: Small heat shock proteins (sHsps) have a critical role under stress conditions to maintain cellular homeostasis by their involvement in protein-folding and cytoprotection. The hyperglycemia in diabetes may impose cellular stress on the retina. Therefore, we investigated the expression of sHsps, phosphoregulation of αB-crystallin (αBC), and their localization in the diabetic rat retina. METHODS: Diabetes was induced in rats and maintained on hyperglycemia for a period of 12 weeks. The expression of sHsps, HSFs, and phosphorylated sHsps was analyzed by quantitative (q) RT-PCR and immunoblotting. The solubility of sHsps was analyzed by detergent solubility assay. Cellular localization of sHsps and phosphorylated αBCs was examined by immunohistochemistry. RESULTS: Of 10 sHsps, five sHsps were detected in the rat retina. Among those, increased expression for αA-crystallin (αAC), αBC, and Hsp22, and decreased expression for Hsp20 were seen in the diabetic retina, whereas Hsp27 mRNA levels were increased, while protein levels were decreased. While the expression of HSFs was either unaltered or decreased, expression of hypoxia inducible factor-1α (HIF-1α) was increased in the diabetic retina. The phosphorylation of αBC at Ser45 and Ser19 was increased in the retina of diabetic rats. However, phosphorylation of αBC at Ser59 was decreased in the soluble fraction with a concomitant increase in the insoluble fraction. Moreover, diabetes activated the p38MAPK signaling cascade by increasing the p-p38 MAPK in the retina. Further, diabetes induced the aggregation of Hsp27, αAC, αBC, and pS59-αBC in the retina. A strong immunoreactivity of Hsp27, αAC, αBC, and phosphorylated αBC was localized in different retinal layers of diabetic rats. CONCLUSIONS: The results indicate an upregulation of αAC, αBC, and Hsp22, but their solubility was compromised in the diabetic retina. There was increased phosphorylation at Ser59, Ser45, and Ser19 of αBC under diabetic conditions. Localization of sHsps and their phosphorylated forms was dispersed to many layers of the retina in diabetes. These results suggest that sHsps may be protecting the retinal neurons in chronic diabetes.