Targeted B-domain deletion restores F8 function in human endothelial cells and mice.
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ABSTRACT:
SUBMITTER: Hu Z
PROVIDER: S-EPMC9207027 | biostudies-literature | 2022 Jun
REPOSITORIES: biostudies-literature
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Targeted B-domain deletion restores F8 function in human endothelial cells and mice.
Hu Zhiqing Z Li Zhuo Z Wu Yong Y Zhao Junya J Wu Lingqian L Zhou Miaojin M Liang Desheng D
Signal transduction and targeted therapy 20220620 1
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Project description:RationaleThe endoplasmic reticulum (ER) is a major intracellular Ca(2+) store in endothelial cells (ECs). The Ca(2+) concentration in the ER greatly contributes to the generation of Ca(2+) signals that regulate endothelial functions. Many proteins, including stromal interaction molecule 1/2 (STIM1/2), Orai1/2/3, and sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3), are involved in the ER Ca(2+) refilling after store depletion in ECs.ObjectiveThis study is designed to examine the role of Ca(2+) in the ER in coronary endothelial dysfunction in diabetes.Methods and resultsMouse coronary ECs (MCECs) isolated from diabetic mice exhibited (1) a significant decrease in the Ca(2+) mobilization from the ER when the cells were treated by SERCA inhibitor, and (2) significant downregulation of STIM1 and SERCA3 protein expression in comparison to the controls. Overexpression of STIM1 restored (1) the increase in cytosolic Ca(2+) concentration due to Ca(2+) leak from the ER in diabetic MCECs, (2) the Ca(2+) concentration in the ER, and (3) endothelium-dependent relaxation that was attenuated in diabetic coronary arteries.ConclusionsImpaired ER Ca(2+) refilling in diabetic MCECs, due to the decrease in STIM1 protein expression, attenuates endothelium-dependent relaxation in diabetic coronary arteries, while STIM1 overexpression has a beneficial and therapeutic effect on coronary endothelial dysfunction in diabetes.
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Project description:KCNE3 (MiRP2) forms heteromeric voltage-gated K+ channels with the skeletal muscle-expressed KCNC4 (Kv3.4) α subunit. KCNE3 was the first reported skeletal muscle K+ channel disease gene, but the requirement for KCNE3 in skeletal muscle has been questioned. Here, we confirmed KCNE3 transcript and protein expression in mouse skeletal muscle using Kcne3-/- tissue as a negative control. Whole-transcript microarray analysis (770,317 probes, interrogating 28,853 transcripts) findings were consistent with Kcne3 deletion increasing gastrocnemius oxidative metabolic gene expression and the proportion of type IIa fast-twitch oxidative muscle fibers, which was verified using immunofluorescence. The down-regulated transcript set overlapped with muscle unloading gene expression profiles (≥1.5-fold change; P < 0.05). Gastrocnemius K+ channel α subunit remodeling arising from Kcne3 deletion was highly specific, involving just 3 of 69 α subunit genes probed: known KCNE3 partners KCNC4 and KCNH2 (mERG) were down-regulated, and KCNK4 (TRAAK) was up-regulated (P < 0.05). Functionally, Kcne3-/- mice exhibited abnormal hind-limb clasping upon tail suspension (63% of Kcne3-/- mice ≥10-mo-old vs. 0% age-matched Kcne3+/+ littermates). Whereas 5 of 5 Kcne3+/+ mice exhibited the typical biphasic decline in contractile force with repetitive stimuli of hind-limb muscle, both in vivo and in vitro, this was absent in 6 of 6 Kcne3-/- mice tested. Finally, myoblasts isolated from Kcne3-/- mice exhibit faster-inactivating and smaller sustained outward currents than those from Kcne3+/+ mice. Thus, Kcne3 deletion impairs skeletal muscle function in mice.-King, E. C., Patel, V., Anand, M., Zhao, X., Crump, S. M., Hu, Z., Weisleder, N., Abbott, G. W. Targeted deletion of Kcne3 impairs skeletal muscle function in mice.
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Project description:Background and purposeAdipocyte fatty acid-binding protein (A-FABP) is up-regulated in regenerated endothelial cells and modulates inflammatory responses in macrophages. Endothelial dysfunction accompanying regeneration is accelerated by hyperlipidaemia. Here, we investigate the contribution of A-FABP to the pathogenesis of endothelial dysfunction in the aorta of apolipoprotein E-deficient (ApoE(-/-) ) mice and in cultured human endothelial cells.Experimental approachA-FABP was measured in aortae of ApoE(-/-) mice and human endothelial cells by RT-PCR, immunostaining and immunoblotting. Total and phosphorylated forms of endothelial nitric oxide synthase (eNOS) were measured by immunoblotting. Changes in isometric tension were measured in rings of mice aortaeKey resultsA-FABP was expressed in aortic endothelium of ApoE(-/-) mice aged 12 weeks and older, but not at 8 weeks or in C57 wild-type mice. Reduced endothelium-dependent relaxations to acetylcholine, UK14304 (selective ?(2) -adrenoceptor agonist) and A23187 (calcium ionophore) and decreased protein presence of phosphorylated and total eNOS were observed in aortae of 18 week-old ApoE(-/-) mice compared with age-matched controls. A 6 week treatment with the A-FABP inhibitor, BMS309403, started in 12 week-old mice, improved endothelial function, phosphorylated and total eNOS and reduced plasma triglyceride levels but did not affect endothelium-independent relaxations. The beneficial effect of BMS309403 on UK14304-induced relaxations was attenuated by Pertussis toxin. In cultured human microvascular endothelial cells, lipid-induced A-FABP expression was associated with reduced phosphorylated eNOS and NO production and was reversed by BMS309403.Conclusions and implicationsElevated expression of A-FABP in endothelial cells contributes to their dysfunction both in vivo and in vitro.
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Project description:Background and purposeThe effects of metformin, an antidiabetic agent that improves insulin sensitivity, on endothelial function have not been fully elucidated. This study was designed to assess the effect of metformin on impaired endothelial function, oxidative stress, inflammation and advanced glycation end products formation in type 2 diabetes mellitus.Experimental approachGoto-Kakizaki (GK) rats, an animal model of nonobese type 2 diabetes, fed with normal and high-fat diet during 4 months were treated with metformin for 4 weeks before evaluation. Systemic oxidative stress, endothelial function, insulin resistance, nitric oxide (NO) bioavailability, glycation and vascular oxidative stress were determined in the aortic rings of the different groups. A pro-inflammatory biomarker the chemokine CCL2 (monocyte chemoattractant protein-1) was also evaluated.Key resultsHigh-fat fed GK rats with hyperlipidaemia showed increased vascular and systemic oxidative stress and impaired endothelial-dependent vasodilatation. Metformin treatment significantly improved glycation, oxidative stress, CCL2 levels, NO bioavailability and insulin resistance and normalized endothelial function in aorta.Conclusion and implicationsMetformin restores endothelial function and significantly improves NO bioavailability, glycation and oxidative stress in normal and high-fat fed GK rats. This supports the concept of the central role of metformin as a first-line therapeutic to treat diabetic patients in order to protect against endothelial dysfunction associated with type 2 diabetes mellitus.
| S-EPMC3087142 | biostudies-literature
Project description:Aims/hypothesisDiabetic cardiomyopathy (DCM) is a serious and under-recognised complication of diabetes. The first sign is diastolic dysfunction, which progresses to heart failure. The pathophysiology of DCM is incompletely understood but microcirculatory changes are important. Endothelial glycocalyx (eGlx) plays multiple vital roles in the microcirculation, including in the regulation of vascular permeability, and is compromised in diabetes but has not previously been studied in the coronary microcirculation in diabetes. We hypothesised that eGlx damage in the coronary microcirculation contributes to increased microvascular permeability and hence to cardiac dysfunction.MethodsWe investigated eGlx damage and cardiomyopathy in mouse models of type 1 (streptozotocin-induced) and type 2 (db/db) diabetes. Cardiac dysfunction was determined by echocardiography. We obtained eGlx depth and coverage by transmission electron microscopy (TEM) on mouse hearts perfusion-fixed with glutaraldehyde and Alcian Blue. Perivascular oedema was assessed from TEM images by measuring the perivascular space area. Lectin-based fluorescence was developed to study eGlx in paraformaldehyde-fixed mouse and human tissues. The eGlx of human conditionally immortalised coronary microvascular endothelial cells (CMVECs) in culture was removed with eGlx-degrading enzymes before measurement of protein passage across the cell monolayer. The mechanism of eGlx damage in the diabetic heart was investigated by quantitative reverse transcription-PCR array and matrix metalloproteinase (MMP) activity assay. To directly demonstrate that eGlx damage disturbs cardiac function, isolated rat hearts were treated with enzymes in a Langendorff preparation. Angiopoietin 1 (Ang1) is known to restore eGlx and so was used to investigate whether eGlx restoration reverses diastolic dysfunction in mice with type 1 diabetes.ResultsIn a mouse model of type 1 diabetes, diastolic dysfunction (confirmed by echocardiography) was associated with loss of eGlx from CMVECs and the development of perivascular oedema, suggesting increased microvascular permeability. We confirmed in vitro that eGlx removal increases CMVEC monolayer permeability. We identified increased MMP activity as a potential mechanism of eGlx damage and we observed loss of syndecan 4 consistent with MMP activity. In a mouse model of type 2 diabetes we found a similar loss of eGlx preceding the development of diastolic dysfunction. We used isolated rat hearts to demonstrate that eGlx damage (induced by enzymes) is sufficient to disturb cardiac function. Ang1 restored eGlx and this was associated with reduced perivascular oedema and amelioration of the diastolic dysfunction seen in mice with type 1 diabetes.Conclusions/interpretationThe association of CMVEC glycocalyx damage with diastolic dysfunction in two diabetes models suggests that it may play a pathophysiological role and the enzyme studies confirm that eGlx damage is sufficient to impair cardiac function. Ang1 rapidly restores the CMVEC glycocalyx and improves diastolic function. Our work identifies CMVEC glycocalyx damage as a potential contributor to the development of DCM and therefore as a therapeutic target.
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