Long Term Effect of High Glucose and Phosphate Levels on the OPG/RANK/RANKL/TRAIL System in the Progression of Vascular Calcification in rat Aortic Smooth Muscle Cells

Metformin exhibits diverse protective effects against diabetic complications, such as bone loss. Here, we investigated the effect of metformin on vascular calcification, another type 2 diabetes complication. In female rat aortic smooth muscle cells (RASMCs), we observed that metformin significantly alleviated β-glycerophosphate-induced Ca deposition and alkaline phosphatase activity, corresponding with reduced expression of some specific genes in osteoblast-like cells, including Runx2 and bone morphogenetic protein-2, and positive effects on α-actin expression, a specific marker of smooth muscle cells. Mechanistic analysis showed that phosphorylation levels of both AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) were increased with NO overproduction. After inhibition of either AMPK or eNOS with the pharmacologic inhibitors, compound C or Nω-Nitro-L-arginine methyl ester, NO production was lowered and metformin-meditated vascular protection against β-glycerophosphate-induced Ca deposition was removed. Our results support that metformin prevents vascular calcification via AMPK-eNOS-NO pathway.

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Abstract 560: Inflammation Converts Glucose Into A Deleterious Agent In Human Aortic Smooth Muscle Cells

Hypertension ◽

10.1161/hyp.64.suppl_1.560 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Carlos F Sanchez-Ferrer ◽

Concepción Peiró ◽

Tania Romacho ◽

Verónica Azcutia ◽

Laura Villalobos ◽

...

Keyword(s):

Smooth Muscle ◽

Smooth Muscle Cells ◽

Glucose Uptake ◽

High Glucose ◽

Inflammatory Responses ◽

Muscle Cells ◽

Pentose Phosphate ◽

Aortic Smooth Muscle Cells ◽

Aortic Smooth Muscle ◽

Excess Glucose

Although hyperglycemia is an independent risk factor for vascular diseases, the links between glucose metabolism and atherosclerosis still require elucidation. We have previously shown that vascular cells, which regulates the glucose entry, are not damaged by high glucose concentrations unless they are primed with an inflammatory stimulus like interleukin (IL)1β. We now analyze the mechanisms accounting for the synergism between high glucose and IL1β. Under high glucose conditions (22 mmol/L), cultured human aortic smooth muscle cells (HASMC) exhibited excess glucose uptake and consumption (from 4.2±0.6 to 7.5±0.7 pmol per cell/24 h) associated to increased GLUT1 transporters expression only when co-stimulated with 10 ng/mL IL1β. However, the simple excess entry of glucose was not deleterious in these cells, as the inhibition of mitochondrial respiration with 0.5 mmol/L sodium azide increased glucose uptake and consumption (from 6.0±0.1 to 13.2±0.8 pmol per cell/24 h) without triggering inflammatory responses, measured by NF-κB activation and iNOS expression. We found that, besides allowing glucose entry, IL1β enhances glucose-6-phosphate dehydrogenase (G6PD) expression by 3.6±1.0 fold and activates the pentose phosphate pathway (PPP) from 9.6±0.7 to 17.4±1.5 nmol/h.mg prot in HASMC submitted to high glucose, thus permitting some of the excess glucose to be metabolized by this route. This provides additional substrate for enhancing the NADPH oxidase enzymatic activity by from 472±30 to 785±41 RLUS/μg prot/min, producing superoxide anions that are required for the activation of NF-κB and iNOS. The higher the concentration of glucose the more the PPP pathway is activated, giving rise to an increased inflammatory condition which cannot be counterbalanced by the simultaneous regeneration of reduced glutathione. We conclude that IL1β transforms excess glucose into a deleterious agent in HASMC by increasing glucose uptake, which is diverted into the PPP, promoting the pro-oxidant conditions required for the exaggeration of inflammatory pathways. Interestingly, all these pathways were blocked with the IL1 receptor antagonist anakinra (1 μmol/L), suggesting this anti-inflammatory drug can be effective for preventing diabetic vasculopathy.

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Hydrogen Sulfide Prevents Elastin Loss and Attenuates Calcification Induced by High Glucose in Smooth Muscle Cells through Suppression of Stat3/Cathepsin S Signaling Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms20174202 ◽

2019 ◽

Vol 20 (17) ◽

pp. 4202 ◽

Cited By ~ 8

Author(s):

Ye-Bo Zhou ◽

Hong Zhou ◽

Li Li ◽

Ying Kang ◽

Xu Cao ◽

...

Keyword(s):

Smooth Muscle ◽

Hydrogen Sulfide ◽

Smooth Muscle Cells ◽

Vascular Calcification ◽

High Glucose ◽

Muscle Cells ◽

Calcium Deposition ◽

Cathepsin S ◽

Key Factor ◽

Factor Α

Vascular calcification can be enhanced by hyperglycemia. Elastin loss in tunica media promotes the osteogenic transformation of smooth muscle cells (SMCs) and involves arterial medial calcification (AMC) that is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes. Here, we tested whether hydrogen sulfide (H2S), an endogenous gaseous mediator, can prevent elastin loss and attenuate calcification induced by high glucose in SMCs. Calcification was induced by high glucose (4500 mg/L) in human aortic SMCs (HASMCs) under the condition of calcifying medium containing 10 mM β-glycerophosphate (β-GP). The experiments showed that NaHS (an H2S donor, 100 μM) mitigated the calcification of HASMCs treated with high glucose by decreasing calcium and phosphorus levels, calcium deposition and ALP activity and inhibited osteogenic transformation by increasing SMα-actin and SM22α, two phenotypic markers of smooth muscle cells, and decreasing core binding factor α-1 (Cbfα-1), a key factor in bone formation, protein expressions in HASMCs. Moreover, NaHS administration inhibited the activation of Stat3, cathepsin S (CAS) activity and its expression, but increased the level of elastin protein. Pharmacological inhibition or gene silencing Stat3 not only reversed elastin loss, but also attenuated CAS expression. Inhibition of CAS alleviated, while CAS overexpression exacerbated, elastin loss. Interestingly, overexpression of wild type (WT)-Stat3, but not its mutant C259S, elevated CAS protein expression and reduced elastin level. Moreover, NaHS induced S-sulfhydration in WT, but not in the C259S Stat3. These data suggest that H2S may directly regulate Cys259 residue in Stat3 and then impair its signaling function. Our data indicate that H2S may attenuate vascular calcification by upregulating elastin level through the inhibition of Stat3/CAS signaling.

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