Tong Lu

Angiogenic Function of Prostacyclin Biosynthesis in Human Endothelial Progenitor Cells

The role of prostaglandin production in the control of regenerative function of endothelial progenitor cells (EPCs) has not been studied. We hypothesized that activation of cyclooxygenase (COX) enzymatic activity and the subsequent production of prostacyclin (PGI2) is an important mechanism responsible for the regenerative function of EPCs. In the present study, we detected high levels of COX-1 protein expression and PGI2 biosynthesis in human EPCs outgrown from blood mononuclear cells. Expression of COX-2 protein was almost undetectable under basal conditions but significantly elevated after treatment with tumor necrosis factor-&agr;. Condition medium derived from EPCs hyperpolarized human coronary artery smooth muscle cells, similar to the effect of the PGI2 analog iloprost. The proliferation and in vitro tube formation by EPCs were inhibited by the COX inhibitor indomethacin or by genetic inactivation of COX-1 or PGI2 synthase with small interfering (si)RNA. Impaired tube formation and cell proliferation induced by inactivation of COX-1 were rescued by the treatment with iloprost or the selective peroxisome proliferator–activated receptor (PPAR)&dgr; agonist GW501516 but not by the selective PGI2 receptor agonist cicaprost. Downregulation of PPAR&dgr; by siRNA also reduced angiogenic capacity of EPCs. Iloprost failed to reverse PPAR&dgr; siRNA-induced impairment of angiogenesis. Furthermore, transfection of PGI2 synthase siRNA, COX-1 siRNA, or PPAR&dgr; siRNA into EPCs decreased the capillary formation in vivo after transplantation of human EPCs into the nude mice. These results suggest that activation of COX-1/PGI2/PPAR&dgr; pathway is an important mechanism underlying proangiogenic function of EPCs.

Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms

We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP‐sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild‐type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12‐EET. We found that 1 μm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12‐EET (5 μm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12‐EET (5 μm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 μmm‐iodobenzylguanidine or 5 μm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 μm 11,12‐EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.

Activation of vascular BK channels by docosahexaenoic acid is dependent on cytochrome P450 epoxygenase activity.

AIMSnn-3 Polyunsaturated fatty acids (PUFAs) are known to protect the cardiovascular system and improve blood pressure control. These important dietary constituents are converted into bioactive metabolites, but their role in regulation of the cardiovascular system is unclear. In particular, the functions of the cytochrome P450 (CYP) metabolites of n-3 PUFAs remain virtually unexplored. In this study, we examined the effects of docosahexaenoic acid (DHA) on the regulation of large-conductance calcium-activated potassium (BK) channel activities in coronary arterial smooth muscle cells.nnnMETHODS AND RESULTSnUsing whole-cell patch-clamp techniques, we found that DHA is a potent activator of BK currents in rat coronary arterial smooth muscle cells with an EC(50) of 0.23 ± 0.03 µM. This effect was abolished by pre-incubation with the CYP epoxygenase inhibitor, SKF525A (10 µM). The effects of DHA on the BK channels were reproduced by 16,17-epoxydocosapentaenoic acid (16,17-EpDPE) with an EC(50) of 19.7 ± 2.8 nM. The physiological role of the CYP metabolites of DHA was confirmed by measuring DHA-mediated vasodilatation in isolated rat coronary arteries. DHA dilated pressurized isolated coronary arteries in a dose-dependent manner, and the DHA effects were abolished after pre-treatment with SKF525A (10 µM) or with iberiotoxin (100 nM). In addition, 16,17-EpDPE directly produced coronary vasodilatation that was iberiotoxin sensitive.nnnCONCLUSIONnThese results suggest that DHA-mediated vasodilatation is mediated through CYP epoxygenase metabolites by activation of vascular BK channels.

Impaired Ca2+-Dependent Activation of Large-Conductance Ca2+-Activated K+ Channels in the Coronary Artery Smooth Muscle Cells of Zucker Diabetic Fatty Rats

The large-conductance Ca(2+)-activated K(+) (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca(2+) homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca(2+) sensitivity, including an increased free Ca(2+) concentration at half-maximal effect on channel activation, a reduced steepness of Ca(2+) dose-dependent curve, altered Ca(2+)-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel beta-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel beta(1)-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.

Activation of Peroxisome Proliferator-Activated Receptor–δ Enhances Regenerative Capacity of Human Endothelial Progenitor Cells by Stimulating Biosynthesis of Tetrahydrobiopterin

The mechanisms underlying the regenerative capacity of endothelial progenitor cells (EPCs) are not fully understood. We hypothesized that biosynthesis of tetrahydrobiopterin is an important mechanism responsible for the stimulatory effects of peroxisome proliferator-activated receptor–&dgr; (PPAR&dgr;) activation on regenerative function of human EPCs. Treatment of human EPCs with a selective PPAR&dgr; agonist GW501516 for 24 hours increased the levels of mRNA, protein, and enzymatic activity of GTP cyclohydrolase I (GTPCH I), as well as the production of tetrahydrobiopterin. The effects of GW501516 were mediated by suppression of PTEN expression, thereby increasing phosphorylation of AKT. The AKT signaling also mediated GW501516-induced phosphorylation of endothelial NO synthase. In addition, activation of PPAR&dgr; significantly enhanced proliferation of EPCs. This effect was abolished by the GTPCH I inhibitor, 2,4-diamino-6-hydroxypyrimidine, or genetic inactivation of GTPCH I with small interfering RNA but not by inhibition of endothelial NO synthase with NG-nitro-L-arginine methyl ester. Supplementation of NO did not reverse 2,4-diamino-6-hydroxypyrimidine-inhibited 5-bromodeoxyuridine incorporation. Furthermore, transplantation of human EPCs stimulated re-endothelialization in a mouse model of carotid artery injury. Pretreatment of EPCs with GW501516 significantly enhanced the ability of transplanted EPCs to repair denuded endothelium. GTPCH I-small interfering RNA transfection significantly inhibited in vivo regenerative capacity of EPCs stimulated with GW501516. Thus, in human EPCs, activation of PPAR&dgr; stimulates expression and activity of GTPCH I and biosynthesis of tetrahydrobiopterin via PTEN-AKT signaling pathway. This effect enhances the regenerative function of EPCs.

Hydrogen sulfide impairs shear stress-induced vasodilation in mouse coronary arteries

Hydrogen sulfide has emerged as an important endothelium-dependent vasodilator, but its role in shear stress-mediated dilation of coronary arteries is unclear. We examined the role of H2S on shear stress-mediated dilation of isolated mouse coronary arteries. In these vessels, Na2S produced concentration-dependent dilation, which was significantly inhibited by iberiotoxin and by 4-aminopyridine. In addition, BK and Kv currents in mouse coronary smooth muscle cells were directly activated by Na2S, suggesting that H2S produced vasodilation through BK and Kv channel activation. Using a pressure servo controller system, freshly isolated mouse coronary arteries were subjected to physiological levels of shear stress (1 to 25xa0dynes/cm2) and produced graded dilatory responses, but such effects were diminished in the presence of 100xa0μM Na2S. Pre-incubation with the cystathionine γ-lyase inhibitor, d,l-propargylglycine (PPG), resulted in a paradoxical augmentation of shear stress-mediated vasodilation. However, in the presence of L-NAME or in coronary arteries from eNOS knockout mice, PPG inhibited shear stress-mediated vasodilation, suggesting an interaction between NO and H2S signaling. Na2S inhibited eNOS activity in cultured mouse aortic endothelial cells and reduced the level of phospho-eNOS(serine 1177). These results suggest that both NO and H2S are important shear stress-mediated vasodilators in mouse coronary arteries but there is a complex interaction between these two signaling pathways that results in paradoxical vasoconstrictive effects of H2S through inhibition of NO generation.

Regulation of Large Conductance Ca2+-activated K+ (BK) Channel β1 Subunit Expression by Muscle RING Finger Protein 1 in Diabetic Vessels

Background: Impaired BK channel function in diabetic vessels is associated with decreased BK channel β1 subunit (BK-β1) expression. Results: Muscle RING finger protein 1 (MuRF1) physically interacts with BK-β1 and accelerates BK-β1 proteolysis. Conclusion: Increased MuRF1 expression is a novel mechanism underlying diabetic BK channelopathy and vasculopathy. Significance: MuRF1 is a potential therapeutic target of BK channel dysfunction and vascular complications in diabetes. The large conductance Ca2+-activated K+ (BK) channel, expressed abundantly in vascular smooth muscle cells (SMCs), is a key determinant of vascular tone. BK channel activity is tightly regulated by its accessory β1 subunit (BK-β1). However, BK channel function is impaired in diabetic vessels by increased ubiquitin/proteasome-dependent BK-β1 protein degradation. Muscle RING finger protein 1 (MuRF1), a muscle-specific ubiquitin ligase, is implicated in many cardiac and skeletal muscle diseases. However, the role of MuRF1 in the regulation of vascular BK channel and coronary function has not been examined. In this study, we hypothesized that MuRF1 participated in BK-β1 proteolysis, leading to the down-regulation of BK channel activation and impaired coronary function in diabetes. Combining patch clamp and molecular biological approaches, we found that MuRF1 expression was enhanced, accompanied by reduced BK-β1 expression, in high glucose-cultured human coronary SMCs and in diabetic vessels. Knockdown of MuRF1 by siRNA in cultured human SMCs attenuated BK-β1 ubiquitination and increased BK-β1 expression, whereas adenoviral expression of MuRF1 in mouse coronary arteries reduced BK-β1 expression and diminished BK channel-mediated vasodilation. Physical interaction between the N terminus of BK-β1 and the coiled-coil domain of MuRF1 was demonstrated by pulldown assay. Moreover, MuRF1 expression was regulated by NF-κB. Most importantly, pharmacological inhibition of proteasome and NF-κB activities preserved BK-β1 expression and BK-channel-mediated coronary vasodilation in diabetic mice. Hence, our results provide the first evidence that the up-regulation of NF-κB-dependent MuRF1 expression is a novel mechanism that leads to BK channelopathy and vasculopathy in diabetes.

Molecular determinants of cardiac KATP channel activation by epoxyeicosatrienoic acids

We have previously reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolites of arachidonic acid, are potent stereospecific activators of the cardiac KATP channel. The epoxide group in EET is critical for reducing channel sensitivity to ATP, thereby activating the channel. This study is to identify the molecular sites on the KATP channels for EET-mediated activation. We investigated the effects of EETs on Kir6.2ΔC26 with or without the coexpression of SUR2A and on Kir6.2 mutants of positively charged residues known to affect channel activity coexpressed with SUR2A in HEK293 cells. The ATP IC50 values were significantly increased in Kir6.2 R27A, R50A, K185A, and R201A but not in R16A, K47A, R54A, K67A, R192A, R195A, K207A, K222A, and R314A mutants. Similar to native cardiac KATP channel, 5 μm 11,12-EET increased the ATP IC50 by 9.6-fold in Kir6.2/SUR2A wild type and 8.4-fold in Kir6.2ΔC26. 8,9- and 14,15-EET regioisomers activated the Kir6.2 channel as potently as 11,12-EET. 8,9- and 11,12-EET failed to change the ATP sensitivity of Kir6.2 K185A, R195A, and R201A, whereas their effects were intact in the other mutants. 14,15-EET had a similar effect with K185A and R201A mutants, but instead of R195A, it failed to activate Kir6.2R192A. These results indicate that activation of Kir6.2 by EETs does not require the SUR2A subunit, and the region in the Kir6.2 C terminus from Lys-185 to Arg-201 plays a critical role in EET-mediated Kir6.2 channel activation. Based on computer modeling of the Kir6.2 structure, we infer that the EET-Kir6.2 interaction may allosterically change the ATP binding site on Kir6.2, reducing the channel sensitivity to ATP.

Down-regulation of the small conductance calcium-activated potassium channels in diabetic mouse atria.

Background: SK channels are implicated in atrial fibrillation (AF), and diabetes (DM) is a risk factor for AF. Results: Atrial SK2 and SK3 are significantly down-regulated from accelerated turnover in diabetic mice, resulting in action potential prolongation and arrhythmias. Conclusion: SK channel down-regulation may lead to arrhythmogenesis. Significance: SK channel down-regulation contributes to atrial electrophysiological dysfunction in DM. The small conductance Ca2+-activated K+ (SK) channels have recently been found to be expressed in the heart, and genome-wide association studies have shown that they are implicated in atrial fibrillation. Diabetes mellitus is an independent risk factor of atrial fibrillation, but the ionic mechanism underlying this relationship remains unclear. We hypothesized that SK channel function is abnormal in diabetes mellitus, leading to altered cardiac electrophysiology. We found that in streptozotocin-induced diabetic mice, the expression of SK2 and SK3 isoforms was down-regulated by 85 and 92%, respectively, whereas that of SK1 was not changed. SK currents from isolated diabetic mouse atrial myocytes were significantly reduced compared with controls. The resting potentials of isolated atrial preparations were similar between control and diabetic mice, but action potential durations were significantly prolonged in the diabetic atria. Exposure to apamin significantly prolonged action potential durations in control but not in diabetic atria. Production of reactive oxygen species was significantly increased in diabetic atria and in high glucose-cultured HL-1 cells, whereas exposure of HL-1 cells in normal glucose culture to H2O2 reduced the expression of SK2 and SK3. Tyrosine nitration in SK2 and SK3 was significantly increased by high glucose culture, leading to accelerated channel turnover. Treatment with Tiron prevented these changes. Our results suggest that increased oxidative stress in diabetes results in SK channel-associated electrical remodeling in diabetic atria and may promote arrhythmogenesis.

Muscle-Specific F-Box Only Proteins Facilitate BK Channel β 1 Subunit Downregulation in Vascular Smooth Muscle Cells of Diabetes Mellitus

Rationale: Activity of the large conductance Ca2+-activated K+ (BK) channels is profoundly modulated by its &bgr;1 subunit (BK-&bgr;1). However, BK-&bgr;1 expression is downregulated in diabetic vessels. The ubiquitin–proteasome system (UPS) is a major mechanism of intracellular protein degradation. Whether UPS participates in BK-&bgr;1 downregulation in diabetic vessels is unknown. Objective: We hypothesize that UPS facilitates vascular BK-&bgr;1 degradation in diabetes. Methods and Results: Using patch clamp and molecular biological approaches, we found that BK-&bgr;1–mediated channel activation and BK-&bgr;1 protein expression were reduced in aortas of streptozotocin-induced diabetic rats and in human coronary arterial smooth muscle cells (CASMCs) cultured in high glucose. This was accompanied by upregulation of F-box only protein (FBXO)-9 and FBXO-32 (atrogin-1), the key components of the Skp1-Cullin-F-box (SCF) type ubiquitin ligase complex. BK-&bgr;1 expression was suppressed by the FBXO activator doxorubicin but enhanced by FBXO-9 small interfering RNA or by the proteasome inhibitor MG-132. Cotransfection of atrogin-1 in HEK293 cells significantly reduced Flag-hSlo-&bgr;1 expression by 2.16-fold, compared with expression of Flag-hSlo-&bgr;1V146A (a mutant without the PDZ-binding motif). After cotransfection with atrogin-1, the ubiquitination of Flag-hSlo-&bgr;1 was increased by 1.91-fold, compared with that of hSlo-&bgr;1V146A, whereas cotransfection with atrogin-1&Dgr;F (a nonfunctional mutant without the F-box motif) had no effect. Moreover, inhibition of Akt signaling attenuated the phosphorylation of forkhead box O transcription factor (FOXO)-3a and enhanced atrogin-1 expression, which in turn suppressed BK-&bgr;1 protein levels in human CASMCs. Conclusions: Downregulation of vascular BK-&bgr;1 expression in diabetes and in high-glucose culture conditions was associated with FOXO-3a/FBXO-dependent increase in BK-&bgr;1 degradation.