Tali Zitman-Gal

Vitamin D manipulates miR-181c, miR-20b and miR-15a in human umbilical vein endothelial cells exposed to a diabetic-like environment

BackgroundHigh blood and tissue concentrations of glucose and advanced glycation end-products are believed to play an important role in the development of vascular complications in patients with diabetes mellitus (DM) and chronic kidney disease. MicroRNAs (miRNA) are non-coding RNAs that regulate gene expression in a sequence specific manner. MiRNA are involved in various biological processes and become novel biomarkers, modulators and therapeutic targets for diseases such as cancer, atherosclerosis, and DM. Calcitriol (the active form of vitamin D) may inhibit endothelial proliferation, blunt angiogenesis, and be a cardioprotective agent. Calcitriol deficiency is a risk factor for DM and hypertension. The aim of this project was to study the miRNA microarray expression changes in human umbilical vein endothelial cells (HUVEC) treated in a diabetic-like environment with the addition of calcitriol.MethodsHUVEC were treated for 24xa0h with 200xa0μg/ml human serum albumin (HSA) and 100xa0mg/dl glucose (control group) or 200xa0μg/ml AGE-HSA, and 250xa0mg/dl glucose (diabetic-like environment), and physiological concentrations (10-10xa0mol/l) of calcitriol. miRNA microarray analysis and real time PCR to validate the miRNA expression profile and mRNA target gene expression were carried out.ResultsCompared to control, 31 mature human miRNA were differentially expressed in the presence of a diabetic-like environment. Addition of physiological concentrations of calcitriol revealed 39 differentially expressed mature human miRNA. MiR-181c, miR-15a, miR-20b, miR-411, miR-659, miR-126 and miR-510 were selected for further analysis because they are known to be modified in DM and in other biological disorders. The predicted targets of these miRNA (such as KLF6, KLF9, KLF10, TXNIP and IL8) correspond to molecular and biological processes such as immune and defense responses, signal transduction and regulation of RNA.ConclusionThis study identified novel miRNA in the field of diabetic vasculopathy and might provide new information about the effect of vitamin D on gene regulation induced by a diabetic-like environment. New gene targets that are part of the molecular mechanism and the therapeutic treatment in diabetic vasculopathy are highlighted.

Vitamin D receptor activation in a diabetic-like environment: potential role in the activity of the endothelial pro-inflammatory and thioredoxin pathways.

High blood and tissue concentrations of glucose and advanced glycation end products (AGEs) are thought to play an important role in the development of diabetic vascular complications. Thioredoxin interacting protein (TXNIP) is up-regulated in response to high levels of glucose and is an endogenous inhibitor of thioredoxin (TRX), and may play a contributory role in the occurrence of diabetic-related vascular diseases. Vitamin D inhibits endothelial proliferation and is a cardiovascular protective agent. The present study evaluated the impact of paricalcitol and calcitriol on the endothelial inflammatory and TXNIP pathways in cultured endothelial cells exposed to a diabetic-like environment. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated for 24h with 200 μg/ml AGE-HSA and 250 mg/dl glucose concentrations, with paricalcitol or calcitriol. IL6, IL8, NFκB (p50/p65), receptor of AGE (RAGE), TXNIP, and TRX expressions were evaluated at the levels of mRNA, protein, and TRX activity. Calcitriol and paricalcitol significantly down-regulated the markers involved in the inflammatory responses. Only paricalcitol induced a significant decrease in TXNIP mRNA and protein expressions. Neither paricalcitol nor calcitriol affected TRX reductase activity or TRX mRNA and protein expressions. Our findings indicate that in an endothelial diabetic-like environment, paricalcitol and calcitriol significantly decreased the expression of genes involved in the inflammatory pathway. In this in vitro study, it seems that the TRX antioxidant system was not involved. The different effects found between paricalcitol and calcitriol might reflect the selectivity of vitamin D receptor (VDR) activation.

Endothelial pro-atherosclerotic response to extracellular diabetic-like environment: Possible role of thioredoxin-interacting protein

BACKGROUND. High blood and tissue concentrations of glucose and advanced glycation end-products (AGEs) are thought to play an important role in the development of vascular diabetic complications. Therefore, the impact of extracellular AGEs and different glucose concentrations was evaluated by studying the gene expressions and the underlying cellular pathways involved in the development of inflammatory pro-atherosclerotic processes observed in cultured endothelial cells. METHODS. Fresh human umbilical vein cord endothelial cells (HUVEC) were treated in the presence of elevated extracellular glucose concentrations (5.5-28 mmol/l) with and without AGE-human serum albumin (HSA). Affymetrix GeneChip(R) Human Gene 1.0 ST arrays were used for gene expression analysis (total 20 chips). Genes of interest were further validated using real-time PCR and western blot techniques. RESULTS. Microarray analysis revealed significant changes in some gene expressions in the presence of the different stimuli, suggesting that different pathways are involved. Six genes were selected for validation as follows: thioredoxin-interacting protein (TXNIP), thioredoxin (TXN), nuclear factor of kappa B (NF-kappaB), interleukin 6 (IL6), interleukin 8 (IL8) and receptor of advanced glycation end-products (RAGE). Interestingly, it was found that the association of AGEs together with the highest pathophysiological concentration of glucose (28 mmol/l) diminished the expression of these specific genes, excluding TXN. CONCLUSIONS. In the present model that mimics a diabetic environment, the relatively short-term experimental conditions used showed an unexpected blunting action of AGEs in the presence of the highest glucose concentration (28 mmol/l). The interactive cellular pathways involved in these processes should be further investigated.

Vitamin D and Vascular Smooth Muscle Cells: Gene Modulation Following Exposure to a Diabetic-Like Environment

Background: Diabetes mellitus (DM) is one of the leading causes of chronic vascular disease, which can accelerate the development of cardiovascular and renal disorders. Vascular smooth muscle cells (VSMC) are involved in the occurrence of vascular atherosclerosis and arteriosclerosis. Calcitriol might inhibit endothelial proliferation, blunt angiogenesis, and serve as a cardioprotective agent. We evaluated the impact of a diabetic-like environment and calcitriol on VSMC gene and protein expression. Methods: VSMC were treated for 24 hours using 200 μg/ml AGE-HSA, 250 mg/dl glucose and 10-9 or 10-10 mol/l calcitriol. Microarray gene chip analysis, real time PCR, western blot and ELISA techniques were used to determine gene and protein expression. Results: A total of 2,693 genes were differentially expressed in a diabetic-like environment compared to control, with 1796 up-regulated and 897 down-regulated. These genes are responsible for functions involved in the regulation of metabolic processes, apoptosis and cell adhesion. Addition of calcitriol at physiological concentrations (10-10 mol/l) revealed 1,157 differentially expressed genes, with 673 up-regulated and 484 down-regulated. These genes significantly enriched functions such as regulation of small GTPase and RAS protein signal transduction, cell growth and extracellular matrix part. TXNIP, OPG, RANKL, KLF4, RANK and NFκB p50/p65 mRNA and protein expressions were further investigated. Conclusion: High throughput approach might lead to a better understanding of the pathways involved in the biological adaptation of VSMC exposed to diabetic-like conditions and eventually the possible beneficial effects of calcitriol treatment, which might delay the development of DM-related vascular complications.

Glucagon‐like peptide‐1 and vitamin D: anti‐inflammatory response in diabetic kidney disease in db/db mice and in cultured endothelial cells

Glucagon‐like peptide‐1 (GLP‐1) is a gut incretin hormone that stimulates insulin secretion and may affect the inflammatory pathways involved in diabetes mellitus. Calcitriol, an active form of vitamin D, plays an important role in renal, endothelial and cardiovascular protection. We evaluated the anti‐inflammatory and histologic effects of a GLP‐1 analogue (liraglutide) and of calcitriol in a db/db mouse diabetes model and in endothelial cells exposed to a diabetes‐like environment.

Kruppel-like factors in an endothelial and vascular smooth muscle cell coculture model: impact of a diabetic environment and vitamin D

Endothelial cells (EC) and vascular smooth muscle cells (VSMC) are involved in the development of local and diffuse vasculopathies by participating in inflammatory processes that can lead to uncontrolled vascular complications. Our aim was to study the possible interactions of EC and VSMC in an in vitro coculture model exposed to diabetic-like conditions and the effect of vitamin D on cellular pathways that might lead to an inflammatory response. EC and VSMC were isolated from different umbilical cords and stimulated in an in vitro coculture model in a diabetic-like environment and calcitriol for 24xa0h. Total RNA and protein were extracted from cells and analyzed for the expression of selected inflammatory-related markers. The EC-VSMC coculture in a diabetic-like environment induced the expression of inflammatory markers such as Kruppel-like factors, thioredoxin-interacting protein (TXNIP), IL-6, and IL-8. Addition of vitamin D to the EC-VSMC coculture induced selective changes in the inflammatory response. This model could lead to a better understanding of the interactions between EC and VSMC in the inflammatory processes involved in diabetes and emphasizes the role of vitamin D in the inflammatory response. The use of different donors strengthens the significance of our findings showing that genetic variations do not affect the impact of vitamin D on the expression of inflammatory-related proteins in our model.

High-density lipoproteins (HDL) composition and function in preeclampsia

PurposeTo evaluate (a) the properties of high-density lipoproteins (HDL)/cholesterol, which include apolipoprotein A-1 (ApoA1) and paraoxonase1 (PON1), both are negative predictors of cardiovascular risk and (b) HDL function, among women with preeclampsia (PE). PE is a multi-system disorder, characterized by onset of hypertension and proteinuria or other end-organ dysfunction in the second half of pregnancy. Preeclampsia is associated with increased risk for later cardiovascular disease. The inverse association between HDL, cholesterol levels and the risk of developing atherosclerotic cardiovascular disease is well-established.MethodsTwenty-five pregnant women [19 with PE and 6 with normal pregnancy (NP)] were recruited during admission for delivery. HDL was isolated from blood samples. PON1 activity and HDL were analyzed. An in vitro model of endothelial cells was used to evaluate the effect of HDL on the transcription response of vascular cell adhesion molecule-1 (VCAM-1) and endothelial nitric oxide synthase (eNOS) mRNA expression.ResultsPON1 activity (units/ml serum) was lower in the PE group compared to normal pregnancy (NP) (6.51u2009±u20090.73 vs. 9.98u2009±u20090.54; Pu2009=u20090.015). Increased ApoA1 was released from PE-HDL as compared to NP-HDL (3.54u2009±u20090.72 vs. 0.89u2009±u20090.35; Pu2009=u20090.01). PE-HDL exhibited increased VCAM-1 mRNA expression and decreased eNOS mRNA expression on TNF-α stimulated endothelial cells as compared to NP-HDL.ConclusionsHDL from women with PE reduced PON1 activity and increased ApoA1 release from HDL particles. This process was associated with increased HDL diameter, suggesting impaired HDL anti-oxidant activity. These changes might contribute to higher long-term cardiovascular risks among women with PE.