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STIM1 restores coronary endothelial function in type 1 diabetic mice.

ABSTRACT: RATIONALE:The 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. OBJECTIVE:This study is designed to examine the role of Ca(2+) in the ER in coronary endothelial dysfunction in diabetes. METHODS AND RESULTS:Mouse 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. CONCLUSIONS:Impaired 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.

SUBMITTER: Estrada IA

PROVIDER: S-EPMC3627729 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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STIM1 restores coronary endothelial function in type 1 diabetic mice.

Estrada Irene A IA Donthamsetty Reshma R Debski Patryk P Zhou Meng-Hua MH Zhang Shenyuan L SL Yuan Jason X-J JX Han Wenlong W Makino Ayako A

Circulation research 20120814 9

<h4>Rationale</h4>The 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.<h4>Objective</h4>This study is designed to ...[more]

PMID: 22896585

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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.

Project description:Damaged endothelial progenitor cells (EPCs) are associated with poor prognosis in diabetic myocardial infarction (DMI). Our previous studies revealed that an impaired Sonic hedgehog (Shh) pathway contributes to insufficient function in diabetic EPCs; however, the roles of the Shh pathway in diabetic EPC apoptosis under basal and hypoxic/ischemic conditions remain unknown. Therefore, the present study investigated whether Shh revitalized diabetic EPCs and consequently improved the deteriorative status of DMI. For this purpose, streptozotocin injection was used in male C57/BL6 mice to induce type‑1 diabetes, and diabetic EPCs were isolated from the bone marrow. Apoptosis, cell function, and protein expression were investigated in EPCs in vitro. Mouse hearts were injected with adenovirus Shh‑modified diabetic EPCs (DM‑EPCShh) or control DM‑EPCNull immediately after coronary artery ligation in vivo. Cardiac function, capillary numbers, fibrosis, and cell apoptosis were then detected. First, the in vitro results demonstrated that the apoptosis of diabetic EPCs was reduced following treatment with Shh protein for 24 h, under normal or hypoxic conditions. BMI1 proto‑oncogene (Bmi1), an antiapoptotic protein found in several cells, was reduced in diabetic EPCs under normal or hypoxic conditions, but was upregulated after Shh protein stimulation. When Bmi1‑siRNA was administered, the antiapoptotic effect of Shh protein was significantly reversed. In addition, p53, a Bmi1‑targeted gene, was demonstrated to mediate the antiapoptotic effect of the Shh/Bmi1 pathway in diabetic EPCs. The Shh/Bmi1/p53 axis also enhanced the diabetic EPC function. In vivo, Shh‑modified diabetic EPCs exhibited increased EPC retention and decreased apoptosis at 3 days post‑DMI. At 14 days post‑DMI, these cells presented enhanced capillary density, reduced myocardial fibrosis and improved cardiac function. In conclusion, the present results demonstrated that the Shh pathway restored diabetic EPCs through the Shh/Bmi1/p53 axis, suppressed myocardial apoptosis and improved myocardial angiogenesis, thus reducing cardiac fibrosis and finally restoring myocardial repair and cardiac function in DMI. Thus, the Shh pathway may serve as a potential target for autologous cell therapy in diabetic myocardial ischemia.

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STIM1 restores coronary endothelial function in type 1 diabetic mice.

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STIM1 restores coronary endothelial function in type 1 diabetic mice.

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