2′,5′-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation

Lee, Yi-Nan; Yeh, Hung‐I; Tian, Tin-Yi; Lu, Wenwei; Ko, Yousang; Tsai, Cheng‐Ho

doi:10.1016/s0300-483x(02)00289-5

Cited by 11 publications

(9 citation statements)

References 28 publications

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“…In the present study, we also used an alternative approach to assess intercellular coupling based on fluorescence recovery after photobleaching (FRAP) (18, 19). Briefly, wild‐type cells grown in monolayers were exposed to normoxia or hypoxia for 1 h, and then exposed to fresh normoxic DSM with 10 µg/ml 5(6)‐carboxyfluorescein diacetate dye at 37°C for 20 min.…”

Section: Methodsmentioning

confidence: 99%

Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild‐type but not connexin40 null mice in oxidant‐ and PKA‐dependent manner

Bolon

Ouellette

et al. 2005

FASEB j.

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Although electrical coupling along the arteriolar endothelium is central in arteriolar conducted response and in control of vascular resistance, little is known about the pathophysiological effect of hypoxia and reoxygenation (H/R) on this coupling. We examined this effect in a monolayer of cultured microvascular endothelial cells (ECs) derived from wild-type (WT) or connexin (Cx)40-/- mice (Cx40 is a key gap junction protein in ECs). To assess electrical coupling, we used a current injection technique and Bessel function model to compute the monolayer intercellular resistance. Hypoxia (0.1% O2, 1 h) followed by abrupt reoxygenation (5-90 min) reduced coupling (i.e., increased resistance) in WT but not in Cx40-/- monolayer. H/R increased superoxide production and reduced protein kinase A (PKA) activity in both monolayers. Activation of PKA by 8-bromo-cAMP prevented the reduction in coupling. Preloading of the WT monolayer with the antioxidant ascorbate prevented reductions in both PKA activity and cell coupling. Inhibition of PKA with 6-22 amide during normoxia mimicked the reduction in coupling. Finally, hypoxia followed by slow reoxygenation caused no change in superoxide level, PKA activity, or coupling. Using intravital microscopy, we assessed the physiological relevance of these findings in terms of KCl-induced conducted vasoconstriction in arterioles of WT mouse cremaster muscle in vivo. Ischemia (1 h) followed by abrupt reperfusion (15-30 min) reduced conduction. 8-bromo-cAMP prevented this reduction, while 6-22 amide mimicked this reduction in control nonischemic arterioles. We propose that abrupt reoxygenation reduces interendothelial electrical coupling via oxidant- and PKA-dependent signaling that targets Cx40. We suggest that this mechanism contributes to compromised arteriolar function after H/R.

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Section: Methodsmentioning

confidence: 99%

Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild‐type but not connexin40 null mice in oxidant‐ and PKA‐dependent manner

Bolon

Ouellette

et al. 2005

FASEB j.

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“…HUVEC were isolated as previously described. 28 Cells of passage 4 were seeded (200 or 800 cells/mm 2 ) onto the metallic sheets, which were placed at the center of a 35 mm tissue culture-treated polystyrene dish filled with 2 mL of culture medium (Medium 199 (GIBCO, New York, USA) containing fetal cattle serum (20%; Hyclone, Utah, USA), heparin (50 g/mL; Sigma, Missouri, USA), GlutaMAX (1%; GIBCO), penicillin (100 unit/mL; GIBCO), streptomycin (100 g/mL; GIBCO), MEM nonessential amine acids solution (1%; GIBCO), and endothelial cell growth factor (0.1 unit/mL; Sigma)). Forty eight hours later, the cells were examined by Western blotting, immunofluorescence microscopy, and scanning electron microscopy (SEM).…”

Section: Cell Culturementioning

confidence: 97%

Comparison of endothelial cells grown on different stent materials

Yeh

Tian

et al. 2005

J Biomedical Materials Res

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We compared the behavior of endothelial cells grown on stent materials. Human umbilical vein endothelial cells (HUVEC) were seeded (200 or 800 cells/mm(2)) onto different metallic sheets, including 316 stainless steel (low carbon; 316LSS), nitinol, and 316LSS coated with TiN or TiO(2). Cells seeded onto tissue culture-treated polystyrene dish coated with gelatin were used as controls. Forty-eight hours later, the cells were examined by Western blotting, immunofluorescence microscopy, and scanning electron microscopy (SEM). The results showed that for either seeding values, the levels of cellularity on TiN and TiO(2) are comparable or higher, and those on 316LSS and nitinol are lower compared to the controls (p < 0.05). SEM demonstrated that cells are well-attached on the metallic surface with various amount of cellular processes. In metals seeded with 800 cells/mm(2), Western blotting showed that the overlying cells expressed less amounts of endothelial nitric oxide synthase (eNOS), Von Willebrand factor (VWF), and connexin43 protein compared to the controls (p < 0.05). Immunofluorescence microscopy confirmed the results of immunoblotting. In conclusion, stent materials affect HUVEC's growth and protein expression profile. Down-regulation of eNOS, VWF, and connexin43 gap junctions is a common phenomenon in the cells growing on the examined metallic materials, suggesting the existence of endothelial dysfunction in the arterial segments containing the stents made of such materials.

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“…In addition, Cx43 has been documented to be important for cell migration and proliferation and in the progression of atherosclerosis 18,[22][23][24][25] In this protocol, we used TFM and MSM to investigate whether traction and intercellular stress generation within the confluent, endothelial monolayer would be impacted by the disruption of the endothelial gap junction Cx43. We disrupted Cx43 with 2,5dihydroxychalcone (chalcone), a molecule documented to inhibit Cx43 expression 26 . Chalcone was used to disrupt Cx43 instead of siRNA as chalcone has been reported previously by Lee et al to disrupt Cx43 expression 26 .…”

Section: Introductionmentioning

confidence: 99%

“…We disrupted Cx43 with 2,5dihydroxychalcone (chalcone), a molecule documented to inhibit Cx43 expression 26 . Chalcone was used to disrupt Cx43 instead of siRNA as chalcone has been reported previously by Lee et al to disrupt Cx43 expression 26 . In addition, we were particularly interested in chalcone's influence on the endothelium as it has also been reported to be an anti-inflammatory and anti-platelet compound that can potentially be used for the prevention and treatment of various vascular pathologies 26 .…”

Section: Introductionmentioning

confidence: 99%

“…Chalcone was used to disrupt Cx43 instead of siRNA as chalcone has been reported previously by Lee et al to disrupt Cx43 expression 26 . In addition, we were particularly interested in chalcone's influence on the endothelium as it has also been reported to be an anti-inflammatory and anti-platelet compound that can potentially be used for the prevention and treatment of various vascular pathologies 26 . Chalcone treatments were performed an hour after the experiment onset, chalcone-treated monolayers were imaged for a total of six hours, and image processing was performed with a custom-written MATLAB code to determine tractions and subsequently intercellular stresses.…”

Section: Introductionmentioning

confidence: 99%

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Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

Islam

Steward

2019

JoVE

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Endothelial cells have been established to generate intercellular stresses and tractions, but the role gap junctions play in endothelial intercellular stress and traction generation is currently unknown. Therefore, we present here a mechanics-based protocol to probe the influence of gap junction connexin 43 (Cx43) has on endothelial biomechanics by exposing confluent endothelial monolayers to a known Cx43 inhibitor 2,5-dihydroxychalcone (chalcone) and measuring the impact this inhibitor has on tractions and intercellular stresses. We present representative results, which show a decrease in both tractions and intercellular stresses under a high chalcone dosage (2 μg/mL) when compared to control. This protocol can be applied to not just Cx43, but also other gap junctions as well, assuming the appropriate inhibitor is used. We believe this protocol will be useful in the fields of cardiovascular and mechanobiology research.

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2′,5′-Dihydroxychalcone down-regulates endothelial connexin43 gap junctions and affects MAP kinase activation

Cited by 11 publications

References 28 publications

Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild‐type but not connexin40 null mice in oxidant‐ and PKA‐dependent manner

Abrupt reoxygenation following hypoxia reduces electrical coupling between endothelial cells of wild‐type but not connexin40 null mice in oxidant‐ and PKA‐dependent manner

Comparison of endothelial cells grown on different stent materials

Perturbing Endothelial Biomechanics via Connexin 43 Structural Disruption

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