Suk Ying Tsang

Functional Integration of Electrically Active Cardiac Derivatives From Genetically Engineered Human Embryonic Stem Cells With Quiescent Recipient Ventricular Cardiomyocytes Insights Into the Development of Cell-Based Pacemakers

Background—Human embryonic stem cells (hESCs) derived from blastocysts can propagate indefinitely in culture while maintaining pluripotency, including the ability to differentiate into cardiomyocytes (CMs); therefore, hESCs may provide an unlimited source of human CMs for cell-based therapies. Although CMs can be derived from hESCs ex vivo, it remains uncertain whether a functional syncytium can be formed between donor and recipient cells after engraftment. Methods and Results—Using a combination of electrophysiological and imaging techniques, here we demonstrate that electrically active, donor CMs derived from hESCs that had been stably genetically engineered by a recombinant lentivirus can functionally integrate with otherwise-quiescent, recipient, ventricular CMs to induce rhythmic electrical and contractile activities in vitro. The integrated syncytium was responsive to the &bgr;-adrenergic agonist isoproterenol as well as to other pharmacological agents such as lidocaine and ZD7288. Similarly, a functional hESC-derived pacemaker could be implanted in the left ventricle in vivo. Detailed optical mapping of the epicardial surface of guinea pig hearts transplanted with hESC-derived CMs confirmed the successful spread of membrane depolarization from the site of injection to the surrounding myocardium. Conclusions—We conclude that electrically active, hESC-derived CMs are capable of actively pacing quiescent, recipient, ventricular CMs in vitro and ventricular myocardium in vivo. Our results may lead to an alternative or a supplemental method for correcting defects in cardiac impulse generation, such as cell-based pacemakers.

Visceral Periadventitial Adipose Tissue Regulates Arterial Tone of Mesenteric Arteries

Periadventitial adipose tissue produces vasoactive substances that influence vascular contraction. Earlier studies addressed this issue in aorta, a vessel that does not contribute to peripheral vascular resistance. We tested the hypothesis that periadventitial adipose tissue modulates contraction of smaller arteries more relevant to blood pressure regulation. We studied mesenteric artery rings surrounded by periadventitial adipose tissue from adult male Sprague-Dawley rats. The contractile response to serotonin, phenylephrine, and endothelin I was markedly reduced in intact vessels compared with vessels without periadventitial fat. The contractile response to U46619 or depolarizing high K+-containing solutions (60 mmol/L) was similar in vessels with and without periadventitial fat. The K+ channel opener cromakalim induced relaxation of vessels precontracted by serotonin but not by U46619 or high K+-containing solutions (60 mmol/L), suggesting that K+ channels are involved. The intracellular membrane potential of smooth muscle cells was more hyperpolarized in intact vessels than in vessels without periadventitial fat. Both the anticontractile effect and membrane hyperpolarization of periadventitial fat were abolished by inhibition of delayed-rectifier K+ (Kv) channels with 4-aminopyridine (2 mmol/L) or 3,4-diaminopyridine (1 mmol/L). Blocking other K+ channels with glibenclamide (3 &mgr;mol/L), apamin (1 &mgr;mol/L), iberiotoxin (100 nmol/L), tetraethylammonium ions (1 mmol/L), tetrapentylammonium ions (10 &mgr;mol/L), or Ba2+ (3 &mgr;mol/L) had no effect. Longitudinal removal of half the perivascular tissue reduced the anticontractile effect of fat by almost 50%, whereas removal of the endothelium had no effect. We suggest that visceral periadventitial adipose tissue controls mesenteric arterial tone by inducing vasorelaxation via Kv channel activation in vascular smooth muscle cells.

BIOLOGICAL PROPERTIES OF BAICALEIN IN CARDIOVASCULAR SYSTEM

The dried roots of Scutellaria baicalensis (S. baicalensis) Georgi (common name: Huangqin in China) have been widely employed for many centuries in traditional Chinese herbal medicine as popular antibacterial and antiviral agents. They are effective against staphylococci, cholera, dysentery, pneumococci and influenza virus. Baicalein, one of the major flavonoids contained in the dried roots, possesses a multitude of pharmacological activities. The glycoside of baicalein, baicalin is a potent anti-inflammatory and anti-tumor agent. This review describes the biological properties of baicalein (Table 1), which are associated with the prevention and treatment of cardiovascular diseases. Baicalein is a potent free radical scavenger and xanthine oxidase inhibitor, thus improving endothelial function and conferring cardiovascular protective actions against oxidative stress-induced cell injury. Baicalein lowers blood pressure in renin-dependent hypertension and the in vivo hypotensive effect may be partly attributed to its inhibition of lipoxygenase, resulting in reduced biosynthesis and release of arachidonic acid-derived vasoconstrictor products. On the other hand, baicalein enhances vasoconstricting sensitivity to receptor-dependent agonists such as noradrenaline, phenylephrine, serotonin, U46619 and vasopressin in isolated rat arteries. The in vitro effect is likely caused by inhibition of an endothelial nitric oxide-dependent mechanism. The anti-thrombotic, anti-proliferative and anti-mitogenic effects of the roots of S. baicalensis and baicalein are also reported. Baicalein inhibits thrombin-induced production of plasminogen activator inhibitor-1, and interleukin-1beta- and tumor necrosis factor-alpha-induced adhesion molecule expression in cultured human umbilical vein endothelial cells. The pharmacological findings have highlighted the therapeutic potentials of using plant-derived baicalein and its analogs for the treatment of arteriosclerosis and hypertension.

Electrophysiological Properties of Pluripotent Human and Mouse Embryonic Stem Cells

Pluripotent embryonic stem cells (ESCs) possess promising potential for cell‐based therapies, but their electrophysiological properties have not been characterized. Here we describe the presence of ionic currents in mouse (m) and human (h) ESCs and their physiological function. In mESCs, tetraethylammonium (TEA)–sensitive depolarization‐activated delayed rectifier K+ currents (IKDR) (8.6 ± 0.9 pA/pF at +40 mV; IC50 = 1.2 ± 0.3 mM), which contained components sensitive to 4‐aminopyridine (4‐AP) (IC50 = 0.5 ± 0.1 mM) and 100 nM Ca2+‐activated K+ current (IKCa) blocker iberiotoxin (IBTX),were detected in 52.3% of undifferentiated cells.IKDR was similarly present in hESCs (∼100%) but with an approximately sixfold higher current density (47.5 ± 7.9 pA/pF at +40 mV). When assayed by bromodeoxyurindine incorporation, application of TEA, 4‐AP, or IBTX significantly reduced the proliferation of mESCs and hESCs in a dose‐dependent manner (p < .05). A hyperpolarization‐activated inward current (Ih) (−2.2 ± 0.4 pA/pF at −120 mV) was detected in 23% of mESCs but not hESCs. Neither Nav nor Cav currents were detected in mESCs and hESCs. Microarray and reverse transcription–polymerase chain reaction analyses identified several candidate genes for the ionic currents discovered. Collectively, our results indicate that pluripotent ESCs functionally express several specialized ion channels and further highlight similarities and differences between the two species. Practical considerations for the therapeutic use of ESCs are discussed.

4-Aminopyridine-sensitive K+ channels contributes to NaHS-induced membrane hyperpolarization and relaxation in the rat coronary artery

The present study aimed at examining the role of potassium channels and endothelium in relaxations induced by sodium hydrogen sulphide (NaHS), which is the donor of gaseous hydrogen sulphide (H(2)S) and the effect of NaHS on endothelium-dependent relaxations in rat coronary arteries. Rat coronary arteries were suspended in a myograph for force measurement and changes of the membrane potential in arteries were determined by membrane potential-sensitive fluorescence dye. NaHS relaxed coronary arteries pre-contracted by U46619 and the relaxation was significantly less in high KCl-contracted rings. NaHS-induced relaxations were reduced by 4-aminopyridine (4-AP) but unaffected by glibenclamide, iberiotoxin, N(G)-nitro-L-arginine methyl ester, ODQ, indomethacin or by endothelium removal. The inhibitory effect of 4-AP was absent in NaHS-induced relaxations in high KCl-contracted rings. Addition of NaHS caused membrane hyperpolarization and this effect was inhibited by 4-AP but not by glibenclamide. NaHS causes endothelium-independent relaxations in rat coronary arteries partially through activation of 4-AP-sensitive potassium channel and ensuring hyperpolarization. Other potassium channels, Na(+)-K(+) pump or endothelium-derived relaxing factors play little role.

Urocortin‐induced endothelium‐dependent relaxation of rat coronary artery: role of nitric oxide and K+ channels

The mechanisms underlying the vasodilator response to urocortin are incompletely understood. The present study was designed to examine the role of endothelial nitric oxide and Ba2+‐sensitive K+ channels in the endothelium‐dependent component of urocortin‐induced relaxation in the rat left anterior descending coronary artery. Urocortin induced both endothelium‐dependent and ‐independent relaxation with respective pD2 of 8.64±0.03 and 7.90±0.10. Removal of endothelium reduced the relaxing potency of urocortin. In rings pretreated with 10−4 M NG‐nitro‐L‐arginine methyl ester, 10−5 M methylene blue or 10−5 M ODQ, the urocortin‐induced relaxation was similar to that observed in endothelium‐denuded rings. L‐Arginine (5×10−4 M) antagonized the effect of NG‐nitro‐L‐arginine methyl ester. The relaxant response to urocortin was reduced in endothelium‐intact rings preconstricted by 3.5×10−2 M K+ and abolished when extracellular K+ was raised to 5×10−2 M. Pretreatment with 10−4 M BaCl2 significantly inhibited urocortin‐induced relaxation. Combined treatment with 10−4 M BaCl2 plus 10−4 M NG‐nitro‐L‐arginine methyl ester did not cause further inhibition. In urocortin (10−8 M)‐relaxed rings, BaCl2 induced concentration‐dependent reversal in vessel tone. Tertiapin‐Q (10−6 M) also attenuated urocortin‐induced relaxation. In contrast, BaCl2 did not alter urocortin‐induced relaxation in endothelium‐denuded rings. In endothelium‐denuded rings, hydroxylamine‐ and nitroprusside‐induced relaxation was inhibited by 10−4 M BaCl2, but not by 10−6 M tertiapin‐Q. The endothelium of the coronary artery was moderately stained with the antiserum against urocortin. Taken together, the present results indicate that the urocortin‐induced endothelium‐dependent relaxation of rat coronary arteries is likely attributable to endothelial nitric oxide and subsequent activation of Ba2+‐ or tertiapin‐Q‐sensitive K+ channels. The urocortin‐induced endothelium‐dependent relaxation appears to be mediated by cyclic GMP‐dependent mechanisms.

CAG expansion induces nucleolar stress in polyglutamine diseases

The cell nucleus is a major site for polyglutamine (polyQ) toxicity, but the underlying mechanisms involved have yet been fully elucidated. Here, we report that mutant RNAs that carry an expanded CAG repeat (expanded CAG RNAs) induce apoptosis by activating the nucleolar stress pathway in both polyQ patients and transgenic animal disease models. We showed that expanded CAG RNAs interacted directly with nucleolin (NCL), a protein that regulates rRNA transcription. Such RNA–protein interaction deprived NCL of binding to upstream control element (UCE) of the rRNA promoter, which resulted in UCE DNA hypermethylation and subsequently perturbation of rRNA transcription. The down-regulation of rRNA transcription induced nucleolar stress and provoked apoptosis by promoting physical interaction between ribosomal proteins and MDM2. Consequently, p53 protein was found to be stabilized in cells and became concentrated in the mitochondria. Finally, we showed that mitochondrial p53 disrupted the interaction between the antiapoptotic protein, Bcl-xL, and the proapoptotic protein, Bak, which then caused cytochrome c release and caspase activation. Our work provides in vivo evidence that expanded CAG RNAs trigger nucleolar stress and induce apoptosis via p53 and describes a polyQ pathogenic mechanism that involves the nucleolus.

Apple polyphenols inhibit plasma CETP activity and reduce the ratio of non‐HDL to HDL cholesterol

Previous reports demonstrated that hypocholesterolemic activity of apple was associated with its pectin and fiber. This report was to investigate the effect of apple polyphenols (AP) on blood cholesterol level and gene expression of cholesterol-regulating enzymes in Golden Syrian hamsters maintained on a 0.1% cholesterol diet. It was found that dietary supplementation of 0.3 or 0.6% of AP did not affect plasma total cholesterol (TC), but it increased HDL cholesterol (HDL-C) and decreased non-HDL-C, thus leading to a lower ratio of non-HDL-C to HDL-C. Plasma total triacylglycerol (TG) level was also significantly reduced when hamsters were fed a diet supplemented with 0.6% AP. Western blot analysis did not find any effect of AP on sterol regulatory element-binding protein 2 (SREBP-2), LDL receptor (LDLR), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), and cholesterol-7alpha-hydroxylase (CYP7A). Most interesting was that supplementation of AP had no effect on protein abundance of plasma cholesteryl ester transport protein (CETP), but it suppressed plasma CETP activity. A series of in vitro assays confirmed that AP inhibited CETP in a dose dependent manner. It was concluded that AP favorably improved distribution of cholesterol in lipoproteins, most likely, by its inhibition on CETP activity.

Inhibition of Tumor-Induced Angiogenesis and Matrix-Metalloproteinase Expression in Confrontation Cultures of Embryoid Bodies and Tumor Spheroids by Plant Ingredients Used in Traditional Chinese Medicine

Tumor-induced angiogenesis is a prerequisite for excessive tumor growth. Blood vessels invade the tumor tissue after degradation of the extracellular matrix scaffold by matrix metalloproteinases (MMPs). Inhibition of MMPs has been therefore suggested to be a useful tool to abolish neoangiogenesis of solid tumors. In the present study, antioxidative plant ingredients used in traditional Chinese medicine were investigated for their capacity to down-regulate MMP expression and to inhibit angiogenesis in embryonic stem cell–derived embryoid bodies and tumor-induced angiogenesis in confrontation cultures consisting of embryoid bodies and multicellular DU-145 prostate tumor spheroids. Embryoid bodies transiently expressed MMP-1, MMP-2, and MMP-9 during the time of differentiation of capillary-like structures. In confrontation cultures, MMP expression was increased compared with control tumor spheroids and embryoid bodies cultivated separately. The increased expression of MMPs in confrontation cultures was a result of elevated levels of reactive oxygen species (ROS) upon confrontation culture and was totally abolished in the presence of the free radical scavenger vitamin E. Incubation of embryoid bodies with baicalein, epicatechin, berberine, and acteoside, which are herbal ingredients used in traditional Chinese medicine, significantly inhibited angiogenesis in embryoid bodies and decreased intracellular ROS levels. Tumor-induced angiogenesis in confrontation cultures was totally abolished in the presence of the free radical scavenger vitamin E. Because herbal ingredients down-regulated MMP expression, we conclude that ROS generated during confrontation culture induce the expression of MMPs that are necessary for endothelial cell invasion into the tumor tissue.

Roles of cyclic AMP and Ca2+-activated K+ channels in endothelium-independent relaxation by urocortin in the rat coronary artery

OBJECTIVE Urocortin possesses cardioprotective properties against the damaging effects of ischemia/reperfusion injury. Our previous study demonstrated that urocortin can induce both endothelium-dependent and -independent coronary relaxation. However, the mechanisms thereby urocortin triggers endothelium-independent relaxation have not been investigated. The present study aimed to examine the role of cyclic AMP and Ca(2+)-activated K(+) channels in the relaxant response to urocortin in the isolated endothelium-denuded rat left anterior descending coronary arteries. METHODS Changes in vessel tension were measured by using a force transducer built in a Multi Myograph System. RESULTS In 9,11-dideoxy-11alpha,9alpha-epoxy-methanoprostaglandin F(2alpha) (U46619)-contracted rings, urocortin-induced relaxation (pD(2): 8.40+/-0.04) was significantly reduced by cyclic AMP-dependent protein kinase (PKA) inhibitors, Rp-cAMPS triethylamine (Rp-cAMPS) and KT 5720. Treatment with the large-conductance Ca(2+)-activated K(+) channel blockers, iberiotoxin or tetraethylammonium ions (TEA(+)) attenuated urocortin-induced relaxation; this effect was abolished in the presence of 200 nmol/l KT 5720. In contrast, apamin (small-conductance Ca(2+)-activated K(+) channel blocker), glibenclamide (ATP-sensitive K(+) channel blocker), or BaCl(2) (inwardly rectifier K(+) channel blocker) had no effect. Urocortin-induced relaxation was reduced in rings contracted with increasing concentrations of extracellular K(+) (35 and 50 mmol/l). Treatment with TEA(+) or Rp-cAMPS inhibited the relaxant effect of urocortin in 35 mmol/l K(+)-contracted rings. Combined treatment with TEA(+) and Rp-cAMPS had no additional effect. Similarly, forskolin produced significantly less relaxant response in 50 mmol/l K(+)-contracted than U46619-contracted rings. Forskolin-induced relaxation was attenuated by pretreatment with 3 mmol/l TEA(+). CONCLUSION Urocortin relaxed the rat coronary artery in substantial part via activation of the vascular Ca(2+)-activated K(+) channels and this effect appears to be primarily mediated through PKA-dependent intracellular mechanisms.