Yoshiko Tokutomi

Blockade of Kit signaling induces transdifferentiation of interstitial cells of Cajal to a smooth muscle phenotype

BACKGROUND & AIMS Interstitial cells of Cajal (ICC) serve as pacemaker cells and mediators of neurotransmission from the enteric nervous system to gastrointestinal muscles. ICC develop from mesenchymal cells that express c-Kit, and signaling via Kit receptors is necessary for normal development of ICC. We studied the fate of functionally developed ICC after blockade of Kit receptors to determine whether ICC undergo cell death or whether the phenotype of the cells is modified. The fate of undeveloped ICC was also investigated. METHODS Neutralizing, anti-Kit monoclonal antibody (ACK2) was administered to mice for 8 days after birth. ICC in the small intestine were examined by immunohistochemistry and electron microscopy. Occurrence of apoptosis was also assayed. RESULTS When Kit receptors were blocked, ICC nearly disappeared from the small intestine. Apoptosis was not detected in regions where ICC are normally distributed. Remaining Kit-immunopositive cells in the pacemaker region of the small intestine developed ultrastructural features similar to smooth muscle cells, including prominent filament bundles and expression of the muscle-specific intermediate filament protein, desmin, and smooth muscle myosin. ICC of the deep muscular plexus normally develop after birth in the mouse. Precursors of these cells remained in an undifferentiated state when Kit was blocked. CONCLUSIONS These data, along with previous studies showing that ICC in the pacemaker region of the small intestine and longitudinal muscle cells develop from the same Kit-immunopositive precursor cells, suggest inherent plasticity between the ICC and smooth muscle cells that is regulated by Kit-dependent cell signaling.

Rhythmic Cl− current and physiological roles of the intestinal c-kit-positive cells

Chronic injection of an anti-c-KIT receptor tyrosine kinase monoclonal antibody (ACK2) results in the disruption of the normal motility patterns of young BALB/c mice intestine. This effect is accompanied by a drastic decrease in the number of intestinal c-kit-expressing (c-kit+) cells when studied immuno-histochemically with the fluorescence-labelled antibody. In order to clarify the mechanism underlying the ACK2 action and the physiological roles of intestinal c -kit+ cells, we studied the excitability of intestinal c -kit+ cells in primary culture by use of the nystatin perforated-patch-clamp technique. Under voltageclamp at −40 mV, the majority of c -kif+cells tested (59/70) elicited rhythmic current waves with an amplitude and frequency of 263±24 pA and 2.30±0.25 cycles/min (mean±SEM), respectively. Intracellular perfusion of the c -kit+ cells with ethylenebis (okonitrilo) tetraacetate (EGTA) as well as a nominally Ca2+-free external solution or low holding voltage (<-60 mV) prevented the rhythmic current. The reversal potential of the rhythmic current was close to the equilibrium potential for Cl−(ECl) Moreover the rhythmic current was depressed by a Cl− channel blocker, 4-acetoamido-4-isothiocyanat-ostilbene-2,2′-disulphonic acid (SITS). The smooth muscle cells freshly dissociated from the same intestinal specimen revealed a Ca2+-activated K+current, as has been described in a variety of smooth muscle cells. Cultured smooth muscle cells from the ileum preparation lacked neither the Ca2+-activated K+nor rhythmic Cl− currents. Smooth muscle cells freshly dissociated from the same ileum preparation and those in culture showed no immunoreactivity with the labelled ACK2, which was consistent with our previous in situ study. Results provided direct evidence that the intestinal c -kit+ cells, but not the smooth muscle cells, possess a rhythmic Cl− current oscillation, suggesting their participation in pacemaker activity for the peristaltic gut movement.

Perindopril, a centrally active angiotensin-converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimer's disease

The purpose of this work was to test whether brain‐penetrating angiotensin‐converting enzyme (ACE) inhibitors (e.g., perindopril), as opposed to non‐brain‐penetrating ACE inhibitors (e.g., enalapril and imidapril), may reduce the cognitive decline and brain injury in Alzheimers disease (AD). We first compared the effect of perindopril, enalapril, and imidapril on cognitive impairment and brain injury in a mouse model of AD induced by intracerebroventricular (i.c.v.) injection of amyloid‐β (Aβ)1–40. Perindopril, with significant inhibition of hippocampal ACE, significantly prevented cognitive impairment in this AD mouse model. This beneficial effect was attributed to the suppression of microglia/astrocyte activation and the attenuation of oxidative stress caused by iNOS induction and extracellular superoxide dismutase down‐regulation. In contrast, neither enalapril nor imidapril prevented cognitive impairment and brain injury in this AD mouse. We next examined the protective effects of perindopril on cognitive impairment in PS2APP‐transgenic mice overexpressing Aβ in the brain. Perindopril, without affecting brain Aβ deposition, significantly suppressed the increase in hippocampal ACE activity and improved cognition in PS2APP‐transgenic mice, being associated with the suppression of hippocampal astrocyte activation and attenuation of superoxide. Our data demonstrated that the brain‐penetrating ACE inhibitor perindopril, as compared to non‐brain‐penetrating ACE inhibitors, protected against cognitive impairment and brain injury in experimental AD models.—Dong, Y. ‐F., Kataoka, K., Tokutomi, Y., Nako, H., Nakamura, T., Toyama, K., Sueta, D., Koibuchi, N., Yamamoto, E., Ogawa, H., Kim‐Mitsuyama, S. Perindopril, a centrally active angiotensin‐converting enzyme inhibitor, prevents cognitive impairment in mouse models of Alzheimers disease. FASEB J. 25, 2911–2920 (2011). www.fasebj.org

Pravastatin Enhances Beneficial Effects of Olmesartan on Vascular Injury of Salt-Sensitive Hypertensive Rats, via Pleiotropic Effects

Objective—This work was undertaken to investigate comparative effect of AT1 receptor blocker (ARB), 3-hydroxy-3-methylglutaryl (HMG) coenzymeA (CoA) reductase inhibitor (statin), and their combination on vascular injury of salt-sensitive hypertension. Methods and Results—Salt-loaded Dahl salt-sensitive hypertensive rats (DS rats) were treated with (1) vehicle, (2) hydralazine (5 mg/kg/d), (3) olmesartan (0.5 mg/kg/d), (4) pravastatin (100 mg/kg/d), and (5) combined olmesartan and pravastatin for 4 weeks. Olmesartan or pravastatin significantly and comparably improved vascular endothelium-dependent relaxation to acetylcholine, coronary arterial remodeling, and eNOS activity of DS rats. Olmesartan prevented vascular eNOS dimer disruption or the downregulation of dihydrofolate reductase (DHFR) more than pravastatin, whereas Akt phosphorylation was enhanced by pravastatin but not olmesartan, indicating differential pleiotropic effects between olmesartan and pravastatin. Add-on pravastatin significantly enhanced the improvement of vascular endothelial dysfunction and remodeling by olmesartan in DS rats. Moreover, pravastatin enhanced the increase in eNOS activity by olmesartan, being associated with additive effects of pravastatin on phosphorylation of Akt and eNOS. Conclusions—Olmesartan and pravastatin exerted beneficial vascular effects in salt-sensitive hypertension, via differential pleiotropic effects. Pravastatin enhanced vascular protective effects of olmesartan. Thus, the combination of ARB with statin may be the potential therapeutic strategy for vascular diseases of salt-sensitive hypertension.

Pioglitazone Exerts Protective Effects Against Stroke in Stroke-Prone Spontaneously Hypertensive Rats, Independently of Blood Pressure

Background and Purpose— Very recent subgroup analysis from the PROspective pioglitAzone Clinical Trial In macroVascular Events has shown that pioglitazone reduces the risk of recurrent stroke in type 2 diabetic patients. However, the underlying mechanism of stroke prevention by pioglitazone is unknown. Our aim was to examine the effect of pioglitazone on hypertension-based stroke in rats. Methods— Pioglitazone (1 mg · kg−1 · d−1) was orally administered to stroke-prone spontaneously hypertensive rats (SHRSP) to examine the effect on incidental stroke, cerebrovascular injury, brain inflammation, oxidative stress, and vascular endothelial dysfunction induced by hypertension. Results— Treatment of SHRSP with pioglitazone for 4 weeks, without affecting blood pressure and blood glucose values, improved vascular endothelial dysfunction (P<0.05), suppressed remodeling of the middle cerebral artery (P<0.05) and brain microvessels (P<0.05), and inhibited brain macrophage infiltration (P<0.05) and the upregulation of brain monocyte chemoattractant protein-1 and tumor necrosis factor-&agr; expression (P<0.01). Furthermore, pioglitazone treatment significantly delayed the onset of stroke signs and death in SHRSP (P<0.05). These beneficial effects of pioglitazone on cerebrovascular injury and stroke in SHRSP were associated with a reduction of brain and vascular superoxide via the inhibition of NADPH oxidase activity. Conclusions— Our work provides the first evidence that pioglitazone significantly protects against hypertension-induced cerebrovascular injury and stroke by improving vascular endothelial dysfunction, inhibiting brain inflammation, and reducing oxidative stress. These beneficial effects of pioglitazone were independent of blood pressure or blood sugar values. Thus, pioglitazone appears to be a potential therapeutic agent for stroke in type 2 diabetes with hypertension.

Excess Salt Causes Cerebral Neuronal Apoptosis and Inflammation in Stroke-Prone Hypertensive Rats Through Angiotensin II-Induced NADPH Oxidase Activation

Background and Purpose— The precise mechanism of salt-induced brain injury is unclear. We examined the detailed causative role of angiotensin II and NADPH oxidase in salt-accelerated brain injury of stroke-prone spontaneously hypertensive rats (SHRSP). Methods— We examined the effect of salt loading on brain reactive oxygen species (ROS), inflammation, and apoptosis in SHRSP. Salt-loaded SHRSP were given vehicle, valsartan (an angiotensin AT1 receptor blocker), or hydralazine to compare their efficacy on brain injury. We also examined the efficacy of apocynin (a NADPH oxidase inhibitor) on brain injury of salt-loaded SHRSP. Results— Cerebral NADPH oxidase activity and ROS in SHRSP were already increased at 1 week after salt loading followed by the significant increase in ED-1-positive cells and neuronal apoptosis. Thus, cerebral NADPH oxidase activation preceded cerebral inflammation and neuronal apoptosis. Despite comparable hypotensive effects between valsartan and hydralazine in salt-loaded SHRSP, valsartan reduced cerebral NADPH oxidase activity and ROS more than hydralazine being accompanied by more prevention of stroke by valsartan than hydralazine. Valsartan, but not hydralazine, prevented neuronal apoptosis, being associated with the suppression of apoptosis signal-regulating kinase 1 activation by valsartan. Moreover, cerebral inflammation was also prevented by valsartan more than hydralazine, being associated with more suppression of monocyte chemotactic protein-1 and tumor necrosis factor-&agr; expressions by valsartan. Thus, angiotensin II was directly involved in salt-induced neuronal NADPH oxidase activation, ROS, apoptosis, and inflammation in SHRSP. Apocynin attenuated the enhancement of ROS, cerebral inflammation, neuronal apoptosis, and apoptosis signal-regulating kinase 1 activation and prevented stroke in salt-loaded SHRSP, indicating the causative role of cerebral NADPH oxidase in salt-induced brain injury. Conclusion— We obtained the evidence that excess salt, through ROS produced by angiotensin II-activated NADPH oxidase, caused cerebral neuronal apoptosis and inflammation as well as stroke in SHRSP.

Enhancement of cardiac oxidative stress by tachycardia and its critical role in cardiac hypertrophy and fibrosis.

Objective To examine the mechanism and significance of tachycardia-induced cardiac damage, using azelnidipine, a relatively new dihydropyridine calcium channel blocker which does not increase heart rate. Methods Comparing azelnidipine and amlodipine, we examined the cardiac effects and the direct effects on a sinus node/atrial preparation in stroke-prone spontaneously hypertensive rats (spSHRs). By pacing the right atrium, we examined the effect of tachycardia per se on cardiac oxidative stress. Using apocynin, a reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, we investigated the role of oxidative stress in cardiac remodelling. Results Azelnidipine suppressed cardiac hypertrophy, fibrosis, NADPH oxidase and superoxide in spSHRs more potently than amlodipine, and was associated with lower heart rates than amlodipine. Azelnidipine caused a greater reduction than amlodipine in the beat rate of the sinus node/atrial preparation of spSHRs. A 10 or 20% increase in heart rate, independent of blood pressure or sympathetic nerve activity, significantly enhanced cardiac NADPH oxidase activity, superoxide and activated mitogen-activated protein kinases. Reduction of cardiac oxidative stress by apocynin led to the suppression of cardiac hypertrophy, inflammation and fibrosis in spSHRs, beyond its hypotensive effect. Conclusions Our work provided evidence that the increase in heart rate per se, independent of sympathetic nerve activity, enhances cardiac oxidative stress and activates mitogen-activated protein kinases, which seem to be responsible for cardiac remodelling. Azelnidipine, without causing an increase in heart rate, has the potential to be useful for the treatment of cardiac remodelling.

Beneficial Effects of Pioglitazone on Hypertensive Cardiovascular Injury Are Enhanced by Combination With Candesartan

The effect of pioglitazone, a peroxisome proliferator-activated receptor &ggr; agonist, on hypertensive cardiovascular injury is unknown. We examined the effect of pioglitazone on hypertensive cardiovascular injury and the significance of combination of pioglitazone with angiotensin type 1 receptor blocker. Stroke-prone spontaneously hypertensive rats (SHRSP) were orally given pioglitazone, candesartan, or combined pioglitazone and candesartan for 4 weeks to compare their effects on cardiovasucular injury. Pioglitazone, without lowering blood pressure, significantly suppressed cardiac inflammation and fibrosis and reduced vascular endothelial dysfunction, and these beneficial effects were associated with the reduction of superoxide by inhibition of cardiovascular NADPH oxidase. Thus, pioglitazone protects against hypertensive cardiovascular injury, by inhibiting reactive oxygen species (ROS). Combination of pioglitazone and candesartan suppressed cardiac hypertrophy, inflammation, and interstitial fibrosis of SHRSP to a greater extent than either monotherapy, and reduced vascular endothelial dysfunction of SHRSP more than either monotherapy. Furthermore, more beneficial effects of their combination on cardiovascular injury were associated with more reduction of NADPH oxidase–mediated cardiovascular ROS. To elucidate the underlying molecular mechanism, we examined cardiovascular NADPH oxidase subunits. Pioglitazone monotherapy significantly attenuated cardiovascular p22phox and Rac1 in SHRSP, whereas pioglitazone combined with candesartan more attenuated p22phox and significantly reduced Nox1. Thus, additive suppression of cardiovascular NADPH oxidase by the combination was attributed to its additive attenuation of p22phox and Nox1 protein levels. In conclusion, we showed that pioglitazone protected against hypertensive cardiovascular damage, and the combination of pioglitazone and candesartan exerted more beneficial effects on hypertensive cardiovascular injury by more suppressing ROS.

Aliskiren Enhances the Protective Effects of Valsartan Against Cardiovascular and Renal Injury in Endothelial Nitric Oxide Synthase–Deficient Mice

The protective effect of aliskiren, a direct renin inhibitor, against hypertensive cardiovascular and renal injury remains to be defined. This study was undertaken to examine the protective effects of the combination of aliskiren and valsartan, an angiotensin receptor blocker, against cardiovascular and renal injury. Endothelial NO synthase–deficient mice, subjected to cuff injury of femoral artery, were divided into 5 groups and were treated with the following: (1) vehicle; (2) aliskiren (25 mg/kg per day); (3) valsartan (8 mg/kg per day); (4) combined aliskiren (12.5 mg/kg per day) and valsartan (4 mg/kg per day); and (5) hydralazine (10 mg/kg per day) for 4 weeks. Aliskiren and valsartan alone markedly and similarly suppressed cardiac hypertrophy, inflammation and fibrosis, and coronary remodeling; prevented cuff injury–induced arterial intimal thickening; and reduced urinary albumin excretion, glomerular inflammation, and glomerulosclerosis in endothelial NO synthase–deficient mice. These beneficial effects of aliskiren and valsartan were associated with the significant attenuation of oxidative stress in these tissues. Hence, aliskiren and valsartan markedly exert the protective effects against cardiovascular and renal injury through the reduction of oxidative stress. Furthermore, compared with monotherapy with aliskiren or valsartan, the combination of a half dose of these drugs more greatly improved the above-mentioned cardiovascular and renal injuries of endothelial NO synthase–deficient mice, which were associated with greater attenuation of tissue oxidative stress by the combination therapy. Thus, the combination of aliskiren and valsartan exerts the synergistic organ-protective effects through synergistic attenuation of oxidative stress. The combination of aliskiren and valsartan seems to be a promising therapeutic strategy for hypertensive organ injury caused by endothelial NO synthase dysfunction.

Novel Mechanism and Role of Angiotensin II–Induced Vascular Endothelial Injury in Hypertensive Diastolic Heart Failure

Objective—The mechanism and role of angiotensin II–induced vascular endothelial injury is unclear. We examined the molecular mechanism of angiotensin (AII)-induced vascular endothelial injury and its significance for hypertensive diastolic heart failure. Methods and Results—We compared the effect of valsartan and amlodipine on Dahl salt-sensitive hypertensive rats (DS rats). Valsartan improved vascular endothelial dysfunction of DS rats more than amlodipine, by inhibiting endothelial apoptosis and eNOS uncoupling more. Moreover, valsartan inhibited vascular apoptosis signal-regulating kinase 1 (ASK1) more than amlodipine. Thus, AT1 receptor contributed to vascular endothelial apoptosis, eNOS uncoupling, and ASK1 activation of DS rats. Using ASK1−/− mice, we examined the causative role of ASK1 in endothelial apoptosis and eNOS uncoupling. AII infusion in wild-type mice markedly caused vascular endothelial apoptosis and eNOS uncoupling accompanied by vascular endothelial dysfunction, whereas these effects of AII were absent in ASK1−/− mice. Therefore, ASK1 participated in AII-induced vascular endothelial apoptosis and eNOS uncoupling. Using tetrahydrobiopterin, we found that eNOS uncoupling was involved in vascular endothelial dysfunction in DS rats with established diastolic heart failure. Conclusion—AII-induced vascular endothelial apoptosis and eNOS uncoupling were mediated by ASK1 and contributed to vascular injury in diastolic heart failure of salt-sensitive hypertension.