G.J. Dusting

Pharmacological response of human cardiomyocytes derived from virus-free induced pluripotent stem cells

AIMS Generation of human induced pluripotent stem cell (hiPSC) lines by reprogramming of fibroblast cells with virus-free methods offers unique opportunities for translational cardiovascular medicine. The aim of the study was to reprogramme fibroblast cells to hiPSCs and to study cardiomyogenic properties and ion channel characteristics of the virus-free hiPSC-derived cardiomyocytes. METHODS AND RESULTS The hiPSCs generated by episomal vectors generated teratomas in severe combined immunodeficient mice, readily formed embryoid bodies, and differentiated into cardiomyocytes with comparable efficiency to human embryonic stem cells. Temporal gene expression of these hiPSCs indicated that differentiation of cardiomyocytes was initiated by increasing expression of cardio/mesodermal markers followed by cardiac-specific transcription factors, structural, and ion channel genes. Furthermore, the cardiomyocytes showed characteristic cross-striations of sarcomeric proteins and expressed calcium-handling and ion channel proteins, confirming their cardiac ontogeny. Microelectrode array recordings established the electrotonic development of a functional syncytium that responded predictably to pharmacologically active drugs. The cardiomyocytes showed a chronotropic dose-response (0.1-10 µM) to isoprenaline and Bay K 8644. Furthermore, carbamycholine (5 µM) suppressed the response to isoprenaline, while verapamil (2.5 µM) blocked Bay K 8644-induced inotropic activity. Moreover, verapamil (1 µM) reduced the corrected field potential duration by 45%, tetrodotoxin (10 µM) shortened the minimal field potential by 40%, and E-4031 (50 nM) prolonged field repolarization. CONCLUSION Virus-free hiPSCs differentiate efficiently into cardiomyocytes with cardiac-specific molecular, structural, and functional properties that recapitulate the developmental ontogeny of cardiogenesis. These results, coupled with the potential to generate patient-specific hiPSC lines, hold great promise for the development of an in vitro platform for drug pharmacogenomics, disease modelling, and regenerative medicine.

Nitric oxide and sensory nerves are involved in the vasodilator response to acetylcholine but not calcitonin gene‐related peptide in rat skin micro vasculature

1 The contributions of sensory nerves and nitric oxide (NO) to vasodilator responses to acetylcholine (ACh) and calcitonin gene‐related peptide (CGRP) were examined in rat skin microvasculature with a laser Doppler flowmeter to monitor relative blood flow. 2 Perfusion of ACh (100 μm; for 30 min) over a blister base on the rat hind footpad elicited microvascular vasodilatation and this response was not sustained. CGRP (1 μm; 10 min perfusion) also elicited vasodilatation and this response was maintained even when CGRP was no longer in contact with the blister base. 3 The vasodilator response to ACh was significantly smaller in rats pretreated as neonates with capsaicin to destroy primary sensory afferents than it was in age‐matched controls. The vasodilator response to CGRP was unaffected by capsaicin pretreatment. 4 Selective inhibitors of NO synthase, NG‐nitro‐l‐arginine (l‐NOARG) and NG‐monomethyl‐l‐arginine (l‐NMMA) (both at 100 μm) attenuated the vasodilator response to ACh in control rats, but had no effect on the vasodilator response to CGRP. There was a significant l‐NOARG‐resistant component in control rats while in capsaicin‐treated rats the vasodilator response to ACh was virtually abolished by l‐NOARG. The inactive stereoisomer NG‐monomethyl‐d‐arginine (100 μm) did not affect the vasodilator response to ACh. 5 The efficacy of l‐NOARG and l‐NMMA as inhibitors of endothelium‐dependent responses was confirmed by use of an endothelium‐dependent vasodilator, the calcium ionophore A23187 (100 μm; 10 min perfusion). Vasodilatation to A23187 was strongly attenuated by both l‐NOARG and l‐NMMA. 6 These results suggest that sensory nerves and NO are both involved in the dilatation produced by ACh in rat skin microvasculature. A component of the vasodilator response elicited by ACh involves a direct action on the microvascular endothelium with subsequent generation of NO, while an additional component is elicited via activation of sensory nerves. The vasodilator mediator(s) released by ACh from sensory nerves acts largely independently of NO. 7 The vasodilator response to CGRP is independent of a prejunctional action on sensory nerves and of NO.

Effects of ageing on sensory nerve function in rat skin

Human studies have shown an age-related decrease in modulation of skin vascular reactivity by sensory nerves that correlates with a decline in wound repair efficacy. Using a vacuum-induced blister model in the rat hind footpad, we have investigated age-related changes in pre- and post-terminal activity of primary afferents involved in skin neurovascular function. Changes in local skin blood flow were monitored using a laser Doppler flowmeter. Pre-terminal stimulation was achieved by electrical stimulation of the distal end of the sciatic nerve (10 V, 15 Hz and 0.5 ms) in three groups of young, old and neonatally pretreated capsaicin rats (3, 24 and 3 months old, respectively). The effect of post-terminal stimulation, achieved using local perfusion of 1 microM substance P (SP) over the blister base, was examined in young (3 months old), mature (12 months old) and aged (24 months old) rats. In addition to changes in SP responsiveness, other post-terminal changes studied included changes in smooth muscle reactivity to sodium nitroprusside (SNP), which acts directly on smooth muscle and to endothelial cell function using N-nitro-L-arginine (L-NORAG), a selective inhibitor of nitric oxide synthesis and endothelium-dependent relaxation. Electrical stimulation of the sciatic nerve in young rats induced an increase in local blood flow (within 1 min) that was maintained during the stimulation period, while the capsaicin group and the old group showed a significantly increased latency and decreased amplitude of the response.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of receptors for platelet-activating factor on platelets, polymorphonuclear leukocytes and macrophages

1 We have compared the potency of the putative platelet‐activating factor (Paf) receptor antagonists (WEB 2086, L‐652,731 and BN 52021) against Paf‐induced aggregation of rabbit and guinea‐pig platelets, aggregation of rabbit polymorphonuclear leukocytes (PMNLs) and prostacyclin generation by guinea‐pig resident peritoneal macrophages. 2 On rabbit washed platelets and PMNLs WEB 2086, L‐652,731 and BN 52021 each antagonized competitively Paf‐induced aggregation. The rank order of potency was WEB 2086 ⋍ L‐652, 731 > BN 52021 and was the same for the two cell types. 3 The pA2 values for each of the three antagonists were similar on rabbit washed platelets and PMNLs. Moreover, the pA2 for WEB 2086 on rabbit platelets (7.58) did not differ significantly from that on guinea‐pig platelets (7.69). 4 On guinea‐pig resident peritoneal macrophages WEB 2086 was 10 fold less potent for receptors mediating increased generation of 6‐oxo‐prostaglandin F1α (6‐oxo‐PGF1α) than for those mediating platelet aggregation. 5 The potencies of L‐652,731 and BN 52021 were also markedly less (2 log units) for the macrophage receptors than for platelet or PMNL receptors and BN 52021 was more potent than L‐652, 731 in the macrophages. 6 WEB 2086 and L‐652,731 significantly reduced basal 6‐oxo‐PGF1α produced by macrophages, but none of the antagonists affected 6‐oxo‐PGF1α production during stimulation by A23187. 7 These data raise the possibility that there may be a Paf receptor‐subtype mediating prostacyclin generation in macrophages that is different from that on the platelet and PMNL. Hence, the potency of Paf antagonists against platelet aggregation would not be a good predictor of antagonist potency in disorders involving macrophages.

N-nitro l-arginine causes coronary vasoconstriction and inhibits endothelium-dependent vasodilatation in anaesthetized greyhounds

1 The effect of N‐nitro‐l‐arginine (l‐NNA), an inhibitor of nitric oxide biosynthesis, on large coronary artery diameter and coronary blood flow was examined in anaesthetized greyhounds. The effects of l‐NNA on the coronary vascular responses to acetylcholine (ACh), glyceryl trinitrate (GTN) and 5‐hydroxytryptamine (5‐HT) were also assessed. 2 l‐NNA (5 mg kg−1), infused into the left circumflex coronary artery, increased systemic mean arterial pressure and decreased the external diameter of the artery. Infusion of l‐NNA decreased coronary blood flow in 5 of the 7 dogs tested and increased mean coronary resistance but neither of these effects was statistically significant. There was no change in heart rate. 3 Intra‐arterial injection of both ACh (0.01–0.05 μg kg−1) and GTN (0.1–0.5μg kg−1) increased large coronary artery diameter and coronary blood flow. Coronary vascular responses to the endothelium‐dependent vasodilator ACh were significantly reduced by l‐NNA, whereas the responses to the endothelium‐independent vasodilator GTN were not significantly affected. 4 5‐HT (0.1 μg kg−1, injected into the left circumflex coronary artery) decreased coronary artery diameter but increased coronary blood flow. After the administration of l‐NNA the 5‐HT‐induced dilatation of the coronary resistance vessels was significantly attenuated whereas the constriction of the circumflex coronary artery was increased in 3 out of 3 dogs in which diameter could be measured, although the latter effect was not statistically significant. 5 These data indicate that l‐NNA causes coronary and systemic vasoconstriction and selectively inhibits endothelium‐dependent vasodilatation in the coronary circulation of the anaesthetized greyhound. Therefore endothelium‐derived NO has an important role in the regulation of coronary vascular tone in the large arteries and the resistance vessels.

Role of nitric oxide in the actions of substance P and other mediators of inflammation in rat skin microvasculature.

The role of nitric oxide in inflammatory responses to substance P and other mediators of inflammation was examined in rat skin microvasculature in a blister base raised on the hind footpad. Superfusion of substance P (1 microM) over the blister base caused an increase in plasma extravasation and a vasodilator response which was not maintained. N(G)-Nitro-L-arginine (100 microM), an inhibitor of nitric oxide biosynthesis, attenuated vasodilatation and plasma extravasation due to substance P. The inactive isomer N(G)-nitro-D-arginine was without effect. Neurokinin A (1 microM), 5-hydroxytryptamine (1 microM), ATP (50 microM) and vasoactive intestinal polypeptide (1 microM) elicited vasodilation, which for vasoactive intestinal polypeptide was maintained even after washout. 5-Hydroxytryptamine and neurokinin A, but not ATP or vasoactive intestinal polypeptide, significantly increased plasma extravasation. Vasodilatation to neurokinin A, 5-hydroxytryptamine and ATP, and the increase in plasma extravasation due to neurokinin A and 5-hydroxytryptamine were unaffected by N(G)-nitro-L-arginine (100 microM), whereas vasodilation due to vasoactive intestinal polypeptide was significantly attenuated. These findings suggest that in rat skin microvasculature in vivo, nitric oxide is involved in vasodilator responses due to substance P and vasoactive intestinal polypeptide, and plasma extravasation due to substance P, but does not contribute significantly to vasodilatation induced by neurokinin A, 5-hydroxytryptamine or ATP, or the plasma extravasation induced by neurokinin A or 5-hydroxytryptamine.

CGRP and nitric oxide of neuronal origin and their involvement in neurogenic vasodilatation in rat skin microvasculature

1 Sensory nerves are important for the initiation of neurogenic inflammation and tissue repair. Both calcitonin gene‐related peptide (CGRP) and nitric oxide (NO) have been implicated in neurogenic vasodilatation and inflammatory responses. 2 A blister model in the rat hind footpad was used as a site to induce neurogenic vasodilatation in response to antidromic electrical stimulation of the sciatic nerve. Blood flux was monitored with a laser Doppler flow monitor. 3 The quantitative contributions of CGRP and NO to vasodilatation were examined by use of the CGRP receptor antagonist CGRP8–37 and NO synthase inhibitors 7‐nitroindazole (7‐NI), 3‐bromo 7‐NI and NG‐nitro L‐arginine methyl ester (L‐NAME). The potential modulatory role of endothelin was examined by use of the ETA receptor antagonist BQ‐123. 4 CGRP8–37 (10 μM) was perfused over the blister base before nerve stimulation and continuously throughout the post‐stimulation period, resulting in a significant reduction (41%) in the blood flux vascular response. 5 Pretreatment with the specific neuronal NO synthase inhibitors, 7‐NI and 3‐bromo 7‐NI (10 mg kg−1, i.v.), and of the non‐specific L‐NAME (100 μM), resulted in significant inhibition of the blood flux response (36%, 72% and 57% decrease, respectively). In contrast, 7‐NI treatment in young rats pretreated with capsaicin had no further effect on the vascular response, suggesting that the source of NO is the sensory nerves. 6 BQ‐123 (10 μM) significantly enhanced the stimulation‐induced blood flux response (61% increase). When 7‐NI was co‐administered with either CGRP8–37 or BQ‐123, the drug actions were additive, suggesting that there was no interaction between NO and CGRP or endothelin. 7 These data suggest that both NO and CGRP participate in neurogenic vasodilatation in rat skin microvasculature and that this response is modulated by endogenous endothelin.

STIMULATION OF PROSTACYCLIN RELEASE FROM THE EPICARDIUM OF ANAESTHETIZED DOGS

1 The generation of prostanoids in the hearts of anaesthetized dogs was studied by irrigating in situ the epicardial surface with Krebs solution. Prostanoids were measured by direct bioassay on smooth muscles and by radioimmunoassay of 6‐oxo‐prostaglandin F1α (6‐oxo‐PGF1 α) and prostaglandin E2 (PGE2) in the epicardial irrigation fluid. 2 The epicardial irrigation fluid contained a prostacyclin‐like substance, as indicated by the bioassay tissues, and immunoreactive 6‐oxo‐PGF1α; PGE2‐like materials were also detected. By both methods the output of the prostacyclin‐like substance, which decreased with time of epicardial irrigation, was increased by manipulating the heart and by adding arachidonic acid (3 μg/ml), and decreased by adding indomethacin (1 μg/ml) to the irrigating fluid. 3 Bioassayed prostacyclin and immunoreactive 6‐oxo‐PGF1α in the epicardial irrigation fluid increased by about 3‐5ng/ml during and after infusion of isoprenaline (0.1 μg kg−1 min−1). The substance was not released by isoprenaline when indomethacin was added to the irrigating fluid, or when propranolol (0.5 mg/kg) was given intravenously. 4 Aortic constriction, bilateral carotid artery occlusion and intravenous angiotensin infusion all increased output of the prostacyclin‐like substance into the epicardial irrigation fluid. The output was abolished by treating the heart with indomethacin (10 mg/kg intravenously or 1 μg/ml epicardially). 5 The prostacyclin‐like substance was also released by all of the above stimuli after the parietal pericardium had been removed and replaced by a plastic sheet. 6 It is concluded that prostacyclin is continually released from tissues close to the epicardial surface and from the pericardium, and that prostacyclin generation increases when cardiac workload increases. Prostacyclin of epicardial or pericardial origin might therefore contribute to metabolic regulation of coronary blood flow.

NOX4 Is a Janus-Faced Reactive Oxygen Species Generating NADPH Oxidase

To the Editor: Hardly any chemical factor in biology serves only good or bad purposes in the body, and this is particularly true for some of the simplest biologically active molecules, the reactive oxygen species (ROS). As our understanding of their functions deepens, it is becoming apparent that ROS subserve both protective and damaging functions, depending on the actual reactive species, the amounts formed, and their subcellular locations.1–3 This Janus-faced role for ROS extends to their enzymatic sources and particularly applies to the only dedicated source of ROS, the NADPH oxidases. We were therefore surprised to read the article by Schroder et al in Circulation Research 4 with the assertive title: “NOX4 Is a Protective Reactive Oxygen Species Generating Vascular NADPH Oxidase,” which implies that the NOX4 isoform in blood vessels always serves a “protective” function. We need to consider whether or not NOX45 …

Requirement for endothelium‐derived nitric oxide in vasodilatation produced by stimulation of cholinergic nerves in rat hindquarters

1 We aimed to determine whether nitric oxide (NO) and/or the endothelium is involved in cholinergic neurogenic vasodilatation in the rat isolated hindquarters. 2 The abdominal aorta was cannulated for perfusion of the rat hindquarters with Krebs bicarbonate solution containing phenylephrine, to induce basal constrictor tone. In the presence of noradrenergic neurone blockade with guanethidine (200 mg kg−1, i.p.) electrical stimulation of peri‐aortic nerves induced frequency‐dependent decreases in hindquarters perfusion pressure, indicating vasodilatation. Both the endothelium‐dependent vasodilator, acetylcholine (ACh) and the endothelium‐independent vasodilator, sodium nitroprusside (SNP) induced dose‐dependent decreases in perfusion pressure. In each experiment, responses to either nerve stimulation, ACh or SNP were recorded before and after treatment with saline vehicle, atropine (1 μm), NG‐nitro‐l‐arginine (l‐NOARG, 100 μm), l‐arginine (1 mm), l‐arginine plus l‐NOARG, or 3–3 cholamidopropyl dimethylammonio 1‐propanesulphonate (CHAPS, 30 mg). Hindquarters dilatation after each treatment was expressed as a percentage of the control response. 3 Following treatment with saline, responses to nerve stimulation and ACh were 99 ± 9% and 107 ± 10% of control, respectively demonstrating the reproducibility of these responses. Nerve stimulation‐induced dilatation was abolished by atropine (0 ± 0% of control, P < 0.05) or reduced to 14 ± 10% of control by NO synthase inhibition with l‐NOARG (P < 0.05). Dilator responses to ACh were also abolished by atropine (0 ± 0% of control, P < 0.05) or inhibited by l‐NOARG (59 ± 10% of control, P < 0.05), indicating that the neurogenic dilatation is cholinergic and is mediated by NO. The administration of the NO precursor, l‐arginine, prevented the inhibitory effect of l‐NOARG on dilator responses to nerve stimulation and ACh (l‐arginine plus l‐NOARG: 89 ± 13% and 122 ± 24% of control, respectively). In addition CHAPS, which removes endothelial cells, inhibited responses to both nerve stimulation (0 ± 0% of control, P < 0.05) and ACh (33 ± 8% of control, P<0.05). In contrast, no treatment significantly reduced the vasodilator responses to SNP. 4 These observations suggest that cholinergic neurogenic vasodilatation in the rat isolated hindquarters requires the synthesis and release of NO from the endothelium.