Manganese Supplementation Reduces High Glucose-induced Monocyte Adhesion to Endothelial Cells and Endothelial Dysfunction in Zucker Diabetic Fatty Rats

Endothelial dysfunction is a hallmark of increased vascular inflammation, dyslipidemia, and the development of atherosclerosis in diabetes. Previous studies have reported lower levels of Mn²⁺ in the plasma and lymphocytes of diabetic patients and in the heart and aortic tissue of patients with atherosclerosis. This study examines the hypothesis that Mn²⁺ supplementation can reduce the markers/risk factors of endothelial dysfunction in type 2 diabetes. Human umbilical vein endothelial cells (HUVECs) were cultured with or without Mn²⁺ supplementation and then exposed to high glucose (HG, 25 mm) to mimic diabetic conditions. Mn²⁺ supplementation caused a reduction in monocyte adhesion to HUVECs treated with HG or MCP-1. Mn²⁺ also inhibited ROS levels, MCP-1 secretion, and ICAM-1 up-regulation in HUVECs treated with HG. Silencing studies using siRNA against MnSOD showed that similar results were observed in MnSOD knockdown HUVECs following Mn²⁺ supplementation, suggesting that the effect of manganese on monocyte adhesion to endothelial cells is mediated by ROS and ICAM-1, but not MnSOD. To validate the relevance of our findings in vivo, Zucker diabetic fatty rats were gavaged daily with water (placebo) or MnCl₂ (16 mg/kg of body weight) for 7 weeks. When compared with placebo, Mn²⁺-supplemented rats showed lower blood levels of ICAM-1 (17%, p < 0.04), cholesterol (25%, p < 0.05), and MCP-1 (28%, p = 0.25). These in vitro and in vivo studies demonstrate that Mn²⁺ supplementation can down-regulate ICAM-1 expression and ROS independently of MnSOD, leading to a decrease in monocyte adhesion to endothelial cells, and therefore can lower the risk of endothelial dysfunction in diabetes.     

Cyclophilin-A: a potential screening marker for vascular disease in type-2 diabetes

The pathophysiology of vascular disease in diabetes involves abnormalities in endothelial cells, vascular smooth muscle cells, and monocytes. The metabolic abnormalities that characterize diabetes, such as hyperglycemia, increased free fatty acids, and insulin resistance, each provoke molecular mechanisms that contribute to vascular dysfunction. Several molecules have been identified as risk markers, and have been studied to prevent progression of disease and long-term complications. Markers such as C-reactive protein and monocyte chemoattractant protein-1 are used to assess risk for adverse cardiac events, but elevated levels are possible due to the presence of other risk factors as part of the natural physiological defense mechanism. In this review we discuss potential of cyclophilin-A, a secreted oxidative-stress-induced immunophilin with diverse functions. We present evidence for a significant role of cyclophilin-A in the pathogenesis of atherosclerosis in diabetes, and its potential as a marker for vascular disease in type-2 diabetes.     

Abundance of TRPC6 protein in glomerular mesangial cells is decreased by ROS and PKC in diabetes

The present study was performed to investigate the underlying mechanism, particularly the roles of reactive oxygen species (ROS) and protein kinase C (PKC), in the diabetes-induced canonical transient receptor potential 6 (TRPC6) downregulation. We found that high glucose (HG) significantly reduced TRPC6 protein expression in cultured mesangial cells (MCs). TRPC6 protein was also significantly reduced in the glomeruli but not in the heart or aorta isolated from streptozotocin-induced diabetic rats. In the cultured MCs, H(2)O(2) suppressed TRPC6 protein expression in a dose- and time-dependent manner, which emulated the HG effect. Catalase as well as superoxide dismutase were able to prevent the inhibitory effect of HG on TRPC6. The antioxidant effect observed in cultured cells was also observed in diabetic rats treated with tempol for 2 wk, which exhibited a preservation of TRPC6 in the glomeruli. Specific knockdown of Nox4, a component of NADPH oxidase, increased TRPC6 protein expression. Furthermore, the PKC activator phorbol 12-myristate 13-acetate (PMA), but not its analog 4α-phorbol 12, 13-didecanoate (4α-PDD), suppressed TRPC6 expression, and this PMA effect was not affected by catalase. Moreover, G?6976, but not LY333531, attenuated the negative effect of HG on TRPC6 expression. G?6976 also inhibited H(2)O(2) effect on TRPC6. Furthermore, either knockdown of TRPC6 or HG treatment significantly decreased ANG II-stimulated MC contraction, and the HG-impaired MC contraction was rescued by overexpression of TRPC6. These results suggest that hyperglycemia in diabetes downregulated TRPC6 protein expression in MCs through a NADPH oxidase Nox4-ROS-PKC pathway, proving a mechanism for impaired MC contraction in diabetes.     

Prognostic Significance of CREB-Binding Protein and CD81 Expression in Primary High Grade Non-Muscle Invasive Bladder Cancer: Identification of Novel Biomarkers for Bladder Cancer Using Antibody Microarray

High-grade (HG) bladder cancers (BCs) are genetically unstable and have an unpredictable course. The identification of prognostic factors in HG non-muscle invasive BC (NMIBC) is crucial for improving patients’ quality of life and preventing BC-specific mortality. Here, we used an antibody microarray (AbM) to identify novel candidate biomarkers in primary HG NMIBC and validated the prognostic significance of the candidate biomarkers. Three pairs of tissue samples from primary HG NMIBC and normal urothelium were analyzed using an AbM kit containing 656 antibodies, and differentially expressed proteins were identified. Among the 42 upregulated and 14 downregulated proteins with statistical significance in BC tissues, CREB-binding protein and CD81 were selected as representative upregulated and downregulated candidate biomarkers, respectively. We then validated the expression of these candidate biomarkers in primary human urothelial cells and BC cell lines by western blotting and immunofluorescence assays, and the results were consistent with the AbM expression profiles. Additionally, Kaplan-Meier survival using immunohistochemical data from an independent primary HG NMIBC cohort comprising 113 patients showed that expression of the 2 biomarkers was significantly associated with recurrence-free and progression-free survival. In multivariate analysis, the 2 biomarkers remained significant predictors for recurrence-free survival. Taken together, our findings suggest that expression of CREB-binding protein and CD81 in BC tissue specimens may have prognostic value in patients with primary HG NMIBC.     

Baicalin,baicalein and wogonin inhibits high glucose-induced vascular inflammation in vitro and in vivo

Vascular inflammatory process has been suggested to play a key role in initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Thus, in this study, we attempted to determine whether three structurally related polyphenols found in the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, can suppress vascular inflammatory processes induced by high glucose (HG) in human umbilical vein endothelial cells (HUVECs) and mice. Data showed that HG induced markedly increased vascular permeability, monocyte adhesion, expressions of cell adhesion molecules (CAMs), formation of reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB. Remarkably, all of the above mentioned vascular inflammatory effects of HG were attenuated by pretreatment with baicalin, baicalein, and wogonin. Vascular inflammatory responses induced by HG are critical events underlying development of various diabetic complications, therefore, our results suggest that baicalin, baicalein, and wogonin may have significant therapeutic benefits against diabetic complications and atherosclerosis. [BMB Reports 2015; 48(9): 519-524]     

Endothelial TNF-α induction by Hsp60 secreted from THP-1 monocytes exposed to hyperglycaemic conditions

A non-resolving inflammation of the endothelium is recognised to be an important process leading to atherosclerosis. In diabetes, this process is thought to account for a significant number of cardiovascular disease-associated death and disability. However, the molecular mechanisms by which diabetes contributes to endothelial inflammation remain to be established. Whilst there is some evidence linking hyperglycaemia-induced reactive oxygen species (ROS) formation by the mitochondrial electron-transport chain to oxidative stress, cellular injury and apoptosis in the endothelium, a clear link to endothelium inflammation has not yet been established. The mitochondrial molecular stress protein Hsp60 is known to be secreted from mammalian cells and is capable of activating pro-inflammatory mediators on target cells expressing Toll-like receptors (TLRs). Hsp60 is also known to be elevated in serum of diabetes patients and has been shown to be upregulated by hyperglycaemic growth conditions in cultured human HeLa cells. This study shows that Hsp60 induced in human acute monocyte leukaemia cell line (THP-1) cells grown under hyperglycaemic conditions (25 mM glucose) was able to be secreted into growth media. Furthermore, the secretion of Hsp60 from THP-1 cells was able to be inhibited by 5,5-(N-N-dimethyl)-amiloride hydrochloride (DMA), an exosomal inhibitor. Interestingly, the conditioned media obtained from THP-1 cells grown in the presence of 25 mM glucose was able to induce the secretion of TNF-α in human vascular endothelium cell line (HUVEC). When conditioned media was immuno-depleted of Hsp60, there was a significant reduction in the release of TNF-α from the HUVEC cells. This suggests that a potential link may exist between hyperglycaemia-induced expression of Hsp60 in monocyte cells and vascular inflammation. Circulating levels of Hsp60 due to mitochondrial stress in diabetes patients could therefore be an important modulator of inflammation in endothelial cells and thus contribute to the increased incidences of atherosclerosis in diabetes mellitus.     

Glucose dysregulation promotes oncogenesis in human bladder cancer by regulating autophagy and YAP1/TAZ expression

Glucose dysregulation is strongly correlated with cancer development, and cancer is prevalent in patients with Type 2 diabetes (T2D). We aimed to elucidate the mechanism underlying autophagy in response to glucose dysregulation in human bladder cancer (BC). 220 BC patients were included in this retrospective study. The expression of YAP1, TAZ and AMPK, EMT-associated markers, and autophagy marker proteins was analysed by immunohistochemistry, western blotting, and quantitative real-time PCR (qPCR). Further, T24 and UMUC-3 BC cells were cultured in media with different glucose concentrations, and the expression of YAP1, TAZ, AMPK and EMT-associated markers, and autophagy marker proteins was analysed by western blotting and qPCR. Autophagy was observed by immunofluorescence and electron microscopy. BC cell viability was tested using MTT assays. A xenograft nude mouse model of diabetes was used to evaluate tumour growth, metastasis and survival. A poorer pathologic grade and tumour-node-metastasis stage were observed in patients with BC with comorbid T2D than in others with BC. YAP1 and TAZ were upregulated in BC samples from patients with T2D. Mechanistically, high glucose (HG) promoted BC progression both in vitro and in vivo and inhibited autophagy. Specifically, various autophagy marker proteins and AMPK were negatively regulated under HG conditions and correlated with YAP1 and TAZ expression. These results demonstrate that HG inhibits autophagy and promotes cancer development in BC. YAP1/TAZ/AMPK signalling plays a crucial role in regulating glucose dysregulation during autophagy. Targeting these effectors exhibits therapeutic significance and can serve as prognostic markers in BC patients with T2D.     

Accumulation of cyclophilin A isoforms in conditioned medium of irradiated breast cancer cells

Secreted proteins play a key role in cell signaling and communication. We recently showed that ionizing radiations induced a delayed cell death of breast cancer cells, mediated by the death receptor pathways through the expression of soluble forms of "death ligands." Using the same cell model, the objective of our work was the identification of diffusible factors, secreted following cell irradiation, potentially involved in cell death signaling. Differential proteomic analysis of conditioned media using 2DE resulted in detection of numerous spots that were significantly modulated following cell irradiation. The corresponding proteins were identified using MALDI-TOF MS and LC-MS/MS approaches. Interestingly, five isoforms of cyclophilin A were observed as increased in conditioned medium of irradiated cells. These isoforms differed in isoelectric points and in accumulation levels. An increase of cyclophilin A secretion was confirmed by Western blotting of conditioned media of irradiated or radiosentive mammary cells. These isoforms displayed an interesting pattern of protein maturation and post-translational modifications, including an alternating removal of N-terminal methionine, associated with a combination of acetylations and methylations. The role of the protein is discussed in relation with its potential involvement in the mechanisms of intercells relationships and radiosensitivity.     

Effects of SGLT2 Inhibition in Human Kidney Proximal Tubular Cells—Renoprotection in Diabetic Nephropathy?

Sodium/glucose cotransporter 2 (SGLT2) inhibitors are oral hypoglycemic agents used to treat patients with diabetes mellitus. SGLT2 inhibitors block reabsorption of filtered glucose by inhibiting SGLT2, the primary glucose transporter in the proximal tubular cell (PTC), leading to glycosuria and lowering of serum glucose. We examined the renoprotective effects of the SGLT2 inhibitor empagliflozin to determine whether blocking glucose entry into the kidney PTCs reduced the inflammatory and fibrotic responses of the cell to high glucose. We used an in vitro model of human PTCs. HK2 cells (human kidney PTC line) were exposed to control 5 mM, high glucose (HG) 30 mM or the profibrotic cytokine transforming growth factor beta (TGFβ1; 0.5 ng/ml) in the presence and absence of empagliflozin for up to 72 h. SGLT1 and 2 expression and various inflammatory/fibrotic markers were assessed. A chromatin immunoprecipitation assay was used to determine the binding of phosphorylated smad3 to the promoter region of the SGLT2 gene. Our data showed that TGFβ1 but not HG increased SGLT2 expression and this occurred via phosphorylated smad3. HG induced expression of Toll-like receptor-4, increased nuclear deoxyribonucleic acid binding for nuclear factor kappa B (NF-κB) and activator protein 1, induced collagen IV expression as well as interleukin-6 secretion all of which were attenuated with empagliflozin. Empagliflozin did not reduce high mobility group box protein 1 induced NF-κB suggesting that its effect is specifically related to a reduction in glycotoxicity. SGLT1 and GLUT2 expression was not significantly altered with HG or empagliflozin. In conclusion, empagliflozin reduces HG induced inflammatory and fibrotic markers by blocking glucose transport and did not induce a compensatory increase in SGLT1/GLUT2 expression. Although HG itself does not regulate SGLT2 expression in our model, TGFβ increases SGLT2 expression through phosphorylated smad3.     

Angiotensin II‐induced redox‐sensitive SGLT1 and 2 expression promotes high glucose‐induced endothelial cell senescence

High glucose (HG)‐induced endothelial senescence and dysfunction contribute to the increased cardiovascular risk in diabetes. Empagliflozin, a selective sodium glucose co‐transporter2 (SGLT2) inhibitor, reduced the risk of cardiovascular mortality in type 2 diabetic patients but the protective mechanism remains unclear. This study examines the role of SGLT2 in HG‐induced endothelial senescence and dysfunction. Porcine coronary artery cultured endothelial cells (ECs) or segments were exposed to HG (25 mmol/L) before determination of senescence‐associated beta‐galactosidase activity, protein level by Western blot and immunofluorescence staining, mRNA by RT‐PCR, nitric oxide (NO) by electron paramagnetic resonance, oxidative stress using dihydroethidium and glucose uptake using 2‐NBD‐glucose. HG increased ECs senescence markers and oxidative stress, down‐regulated eNOS expression and NO formation, and induced the expression of VCAM‐1, tissue factor, and the local angiotensin system, all these effects were prevented by empagliflozin. Empagliflozin and LX‐4211 (dual SGLT1/2 inhibitor) reduced glucose uptake stimulated by HG and H₂O₂ in ECs. HG increased SGLT1 and 2 protein levels in cultured ECs and native endothelium. Inhibition of the angiotensin system prevented HG‐induced ECs senescence and SGLT1 and 2 expression. Thus, HG‐induced ECs ageing is driven by the local angiotensin system via the redox‐sensitive up‐regulation of SGLT1 and 2, and, in turn, enhanced glucotoxicity.     

Identification of cyclophilin as the erythrocyte ciclosporin-binding protein

Previous studies on the distribution of circulating ciclosporin have shown that the majority of the drug is associated with erythrocytes. In order to investigate the nature of ciclosporin-erythrocyte binding, binding studies were performed on isolated erythrocytes. At therapeutic concentrations (approx. 0.5 microgram/ml in whole blood) greater than 90% of the erythrocyte associated ciclosporin was found in the cytosol. The cytosolic binding capacity was approximately (2-2.5).10(5) molecules of ciclosporin per cell. A lower affinity binding of the drug to the plasma membrane occurred only at higher ciclosporin concentrations. The ciclosporin-binding species was purified from erythrocyte cytosol using ciclosporin-Affigel affinity chromatography. This revealed a 16 kDa protein, similar in size to the ciclosporin-binding protein, cyclophilin, previously identified in lymphocyte cytosol. Immunochemical analysis using rabbit anti-bovine spleen cyclophilin antisera revealed that the erythrocyte ciclosporin-binding protein was either cyclophilin or a closely related protein. It is concluded that intracellular ciclosporin-binding within erythrocytes is mostly attributable to the presence of a single protein or protein family represented by cyclophilin. The presence of (2-2.5).10(5) copies of this binding protein within each erythrocyte is responsible for the ciclosporin found associated with erythrocytes.     

A novel secreted cyclophilin-like protein (SCYLP)

A novel cyclosporin A binding glycoprotein of 21 kDa was isolated from human milk by several steps of cation exchange chromatography. The corresponding gene was cloned from human T cells, expressed in Escherichia coli and the recombinant protein purified. The protein shares 58% amino acid identity with the cytosolic cyclophilin and is initially synthesized with a hydrophobic leader sequence. The cyclophilin-like protein has also peptidyl-prolyl cis/trans-isomerase activity, although less efficient, that is inhibited by cyclosporin A. The existence of a secreted form of cyclophilin-like protein in addition to the previously known cytosolic cyclophilin implies that these proteins act on different in vivo targets.