Enhanced chondrogenic differentiation of iPS cell-derived mesenchymal stem/stromal cells via neural crest cell induction for hyaline cartilage repair.

Background: To date, there is no effective long-lasting treatment for cartilage tissue repair. Primary chondrocytes and mesenchymal stem/stromal cells are the most commonly used cell sources in regenerative medicine. However, both cell types have limitations, such as dedifferentiation, donor morbidity, and limited expansion. Here, we report a stepwise differentiation method to generate matrix-rich cartilage spheroids from induced pluripotent stem cell-derived mesenchymal stem/stromal cells (iMSCs) via the induction of neural crest cells under xeno-free conditions. Methods: The genes and signaling pathways regulating the chondrogenic susceptibility of iMSCs generated under different conditions were studied. Enhanced chondrogenic differentiation was achieved using a combination of growth factors and small-molecule inducers. Results: We demonstrated that the use of a thienoindazole derivative, TD-198946, synergistically improves chondrogenesis in iMSCs. The proposed strategy produced controlled-size spheroids and increased cartilage extracellular matrix production with no signs of dedifferentiation, fibrotic cartilage formation, or hypertrophy in vivo. Conclusion: These findings provide a novel cell source for stem cell-based cartilage repair. Furthermore, since chondrogenic spheroids have the potential to fuse within a few days, they can be used as building blocks for biofabrication of larger cartilage tissues using technologies such as the Kenzan Bioprinting method.

Comparison of the cardioprotective and renoprotective effects of the L/N-type calcium channel blocker, cilnidipine, in adriamycin-treated spontaneously-hypertensive rats.

Cilnidipine is an L/N-type calcium channel blocker (CCB). The effects of cilnidipine on N-type channels give it unique organ-protective properties via the suppression of hyperactivity in the sympathetic nervous system (SNS) and renin-angiotensin-aldosterone system (RAAS). In the present study, we compared the effects of cilnidipine and amlodipine (an L-type CCB) on cardiac and renal functions in spontaneously-hypertensive rats injected with adriamycin (ADR). After the weekly administration of ADR for 3 weeks, spontaneously-hypertensive rats were orally administered cilnidipine (20 mg/kg per day), amlodipine (3 mg/kg per day), or vehicle once daily for 4 weeks. A control group received saline rather than ADR, followed by vehicle for 4 weeks. Cilnidipine and amlodipine produced similar reductions in blood pressure after 4 weeks. Cilnidipine ameliorated ADR-induced heart and kidney damage, whereas amlodipine slightly improved cardiac echocardiographic parameters, but did not protect against ADR-induced renal damage. Cilnidipine (but not amlodipine) suppressed the reflex SNS and RAAS hyperactivity caused by their antihypertensive effects. Furthermore, cilnidipine and amlodipine treatment decreased the urinary levels of adrenocortical hormones. The protective effects of cilnidipine against ADR-induced renal and cardiac dysfunction might be associated with its blockade of N-type calcium channels, in addition to its pleiotropic actions, which include the inhibition of the RAAS.

Voltage-gated calcium channels in the human adrenal and primary aldosteronism.

Calcium channel blockers can efficiently be used in the treatment of primary aldosteronism (PA) related hypertension, but details on the localization of calcium channel (CC) in the human adrenal and its disorders, including PA, have remained unclear. Therefore, in this study we analyzed the known α subunits of L-, N- and T-type CCs in 74 adrenocortical aldosterone-producing adenomas (APA) and 16 cortisol-producing adenomas (CPA) using quantitative RT-PCR (qPCR). We also examined the status of L-(CaV1.2, CaV1.3), N-(CaV2.2) and T-(CaV3.2) CC subunits in five non-pathological adrenals (NA), five idiopathic hyperaldosteronism (IHA) cases, and 50 APA using immunohistochemistry. After qPCR evaluation, only CaV1.2, CaV1.3, CaV2.2, and CaV3.2 mRNA levels could be detected in APA and CPA. Among those, only CaV3.2 mRNA levels were significantly correlated with plasma aldosterone levels (P=0.0031), CYP11B2 expression levels (P<0.0001) and the presence of KCNJ5 mutations (P=0.0019) in APA. The immunolocalization of CCs in NA and IHA was detected in the zona glomerulosa (ZG), with a predominance of CaV3.2 in APA. These findings suggest that different types of CC can be involved in calcium-related aldosterone biosynthesis.

Additive effects of cilnidipine, an L-/N-type calcium channel blocker, and an angiotensin II receptor blocker on reducing cardiorenal damage in Otsuka Long-Evans Tokushima Fatty rats with type 2 diabetes mellitus.

Cilnidipine (Cil), which is an L-/N-type calcium channel blocker (CCB), has been known to provide renal protection by decreasing the activity of the sympathetic nervous system (SNS) and the renin-angiotensin system. In this study, we compared the effects of the combination of Cil and amlodipine (Aml), which is an L-type CCB, with an angiotensin (Ang) II receptor blocker on diabetic cardiorenal damage in spontaneously type 2 diabetic rats. Seventeen-week-old Otsuka Long-Evans Tokushima Fatty rats were randomly assigned to receive Cil, Aml, valsartan (Val), Cil + Val, Aml + Val, or a vehicle (eight rats per group) for 22 weeks. Antihypertensive potencies were nearly equal among the CCB monotherapy groups and the combination therapy groups. The lowering of blood pressure by either treatment did not significantly affect the glycemic variables. However, exacerbations of renal and heart failure were significantly suppressed in rats administered Cil or Val, and additional suppression was observed in those administered Cil + Val. Although Val increased the renin-Ang system, Aml + Val treatment resulted in additional increases in these parameters, while Cil + Val did not show such effects. Furthermore, Cil increased the ratio of Ang-(1-7) to Ang-I, despite the fact that Val and Aml + Val decreased the Ang-(1-7) levels. These actions of Cil + Val might be due to their synergistic inhibitory effect on the activity of the SNS, and on aldosterone secretion through N-type calcium channel antagonism and Ang II receptor type 1 antagonism. Thus, Cil may inhibit the progression of cardiorenal disease in type 2 diabetes patients by acting as an N-type CCB and inhibiting the aldosterone secretion and SNS activation when these drugs were administered in combination with an Ang II receptor blocker.

Voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by angiotensin II in mice.

N-type voltage-dependent Ca(2+)channels (VDCCs), expressed predominantly in the nervous system, play pivotal roles in sympathetic regulation of the circulatory system. Although N-type VDCCs are also reportedly expressed in the vasculature, their pathophysiological role is obscure. We demonstrated that oxidative stress-related endothelial dysfunction induced by angiotensin (Ang) II is suppressed in mice lacking the N-type VDCC α1B subunit (Cav 2.2). Impairment of endothelium-dependent relaxation of the thoracic aorta observed following Ang II treatment in wild-type (WT) mice was significantly attenuated in the Ang II-treated Cav 2.2-deficient mice, despite the comparable increase of the blood pressure in the two groups of mice. The thoracic aorta of the Cav 2.2-deficient mice showed a smaller positive area of oxidative stress markers as compared to the WT mice. The Ang II-induced endothelial dysfunction was also suppressed by cilnidipine, an L/N-type VDCC blocker, but not by amlodipine, an L-type VDCC blocker; however, this unique effect of cilnidipine was completely abolished in the Cav 2.2-deficient mice. Furthermore, selective inhibition of N-type VDCCs by ω-conotoxin GVIA dramatically suppressed the production of reactive oxygen species (ROS) as well as agonist-induced Ca(2+) influx in the vascular endothelial cells. These results suggest that N-type VDCCs expressed in the vascular endothelial cells contribute to ROS production and endothelial dysfunction observed in Ang II-treated hypertensive mice.

Additive effects of cilnidipine and angiotensin II receptor blocker in preventing the progression of diabetic nephropathy in diabetic spontaneously hypertensive rats.

BACKGROUND: Cilnidipine (Cil) is an L/N-type calcium channel blocker (CCB) that is known to provide renal protection by decreasing the activity of the sympathetic nervous system and the renin-angiotensin system (RAS). However, very few studies have evaluated the renoprotective effects of Cil in hypertension complicated by diabetes mellitus. In this study, we compared the effects of cilnidipine and the L-type CCB, amlodipine (Aml), in combination with an angiotensin II receptor blocker (ARB) on diabetic nephropathy that developed as a result of inducing diabetes in hypertensive rats. METHODS: Diabetes was induced in 9-week-old male spontaneously hypertensive rats by intraperitoneally injecting them with streptozotocin (40 mg/kg twice) and the rats (8 per group) were randomly assigned to receive valsartan (Val), Cil + Val, Aml + Val, or vehicle for 8 weeks through a gastric tube. RESULTS: There were no significant differences in systolic blood pressure or plasma parameters between the two combination therapy groups. Blood pressure lowering by neither combination therapy significantly affected the glycemic variables. However, the increased glycogen levels in the kidney as a result of hyperglycemia were significantly suppressed in the groups that received combination therapy, and the increased proteinurea and glomerulosclerosis due to progression of the diabetic nephropathy were significantly suppressed in the Cil + Val group. In addition, a significant decrease in ED-1-positive cells was observed in the Cil + Val group alone. CONCLUSION: The results of this study suggested that the L/N-type CCB, cilnidipine, had additive antihypertensive and proteinuria-lowering effects when administered in combination with an ARB, even in type-1 diabetic rats, and that the L-type CCB, amlodipine, did not. Furthermore, combination therapy with cilnidipine and valsartan significantly reduced glycogen accumulation and ED-1-positive cell infiltration, suggesting that cilnidipine suppressed the excessive increase in the activity of the sympathetic nervous system and RAS through N-type calcium channel blockade.

Powerful vascular protection by combining cilnidipine with valsartan in stroke-prone, spontaneously hypertensive rats.

Cilnidipine is an L- and N-type calcium channel blocker (CCB), and amlodipine is an L-type CCB. Valsartan (10 mg kg(-1)), valsartan (10 mg kg(-1)) and amlodipine (1 mg kg(-1)), and valsartan (10 mg kg(-1)) and cilnidipine (1 mg kg(-1)) were administered once daily for 2 weeks to stroke-prone, spontaneously hypertensive rats (SHR-SPs). Blood pressure was significantly reduced by valsartan, and it was further reduced by the combination therapies. Vascular endothelial dysfunction was significantly attenuated in all therapeutic groups, and further significant attenuation was observed in the valsartan+cilnidipine-treated group, but not in the valsartan+amlodipine-treated group. Vascular nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit NOX1 gene expression was significantly attenuated in all therapeutic groups, and significantly greater attenuation was observed in the valsartan+cilnidipine-treated group than in the valsartan-treated group. Compared with the valsartan-treated group, the positive areas for 4-hydroxy-2-nonenal were significantly lower only in the valsartan+cilnidipine-treated group. Plasma renin activity was significantly augmented in the valsartan-treated group, and it was significantly attenuated in the valsartan+cilnidipine-treated group. A significant increase in the ratio of plasma angiotensin-(1-7) to angiotensin II was observed only in the valsartan+cilnidipine-treated group. Vascular angiotensin-converting enzyme (ACE) gene expression was significantly attenuated only in the valsartan+cilnidipine-treated group, but ACE2 gene expression was significantly higher in all of the therapeutic groups. Thus, valsartan and cilnidipine combination therapy might have a powerful protective effect in the vascular tissues via increases in the angiotensin-(1-7)/angiotensin II ratio in plasma.

Comparison of the effects of cilnidipine and amlodipine on cardiac remodeling and diastolic dysfunction in Dahl salt-sensitive rats.

OBJECTIVE: The L/N-type calcium channel blocker (CCB) cilnidipine suppresses sympathetic nerve activity and has a superior renoprotective effect compared with L-type CCBs such as amlodipine. The cardioprotective action of cilnidipine has remained largely uncharacterized, however. We have now investigated the effects of cilnidipine, in comparison with amlodipine, on cardiac pathophysiology in rats with salt-sensitive hypertension. METHODS: Dahl salt-sensitive rats fed a high-salt diet from 6 weeks of age were treated with vehicle (LVH group), amlodipine (3 mg/kg per day), or cilnidipine (3 mg/kg per day) from 7 to 11 weeks. RESULTS: The salt-induced increase in SBP apparent in LVH rats was attenuated to a similar extent by treatment with amlodipine or cilnidipine. The two drugs also similarly inhibited the development of left ventricular (LV) hypertrophy. However, cilnidipine attenuated the increase in relative wall thickness as well as ameliorated LV perivascular and interstitial fibrosis and diastolic dysfunction to a greater extent than did amlodipine. In addition, cilnidipine treatment was associated with greater inhibition of cardiac oxidative stress, inflammation, and renin-angiotensin system (RAS) gene expression. The decrease in cardiac norepinephrine content apparent in LVH rats was similarly inhibited by both drugs. CONCLUSIONS: Cilnidipine attenuated LV fibrosis and diastolic dysfunction as well as LV concentricity to a greater extent than did amlodipine in Dahl salt-sensitive rats. The superior cardioprotective action of cilnidipine is likely attributable, at least in part, to the greater antioxidant and anti-inflammatory effects associated with inhibition of cardiac RAS gene expression observed with this drug.

L/N-type calcium channel blocker suppresses reflex aldosterone production induced by antihypertensive action.

The L/N-type calcium channel blocker cilnidipine has been shown to suppress aldosterone production induced by angiotensin II (Ang II) in vitro. In addition, cilnidipine also suppresses the reflex tachycardia induced by its antihypertensive action in vivo. We investigated the effects of cilnidipine on the reflex aldosterone production induced by its antihypertensive action, to identify the differences in the effects of cilnidipine from those of the L-type calcium channel blocker nifedipine. Male SHR/Izm rats were anesthetized by intraperitoneal injection of pentobarbital sodium, and administered an intravenous infusion of saline supplemented or not with Ang II for 30 min. Blood pressure was monitored continuously in the femoral artery. Each of the calcium channel blockers under study was administered intravenously as a bolus through the femoral vein 1 min after the start of the Ang II infusion, and blood samples were collected 30 min after the start of the Ang II infusion. Following administration at nonhypotensive doses, all calcium channel blockers tended to decrease the plasma aldosterone. In particular, cilnidipine significantly suppressed the plasma aldosterone levels. On the other hand, under the condition of Ang II-induced hypertension, administration of a hypotensive dosage of cilnidipine showed no effect on the plasma aldosterone levels, whereas a hypotensive dosage of nifedipine significantly increased the plasma aldosterone levels. Our results suggest that the L/N-type calcium channel blocker cilnidipine reduces the plasma aldosterone level by suppressing the aldosterone production induced by reflex upregulation of the renin-angiotensin-aldosterone system associated with reduction of the blood pressure.

Measurements of cardiac ion channel subunits in the chronic atrioventricular block dog.

The chronic atrioventricular block (CAVB) dog has been widely used as an in vivo proarrhythmia model. mRNA levels of K(+) and Ca(2+) channels in the isolated ventricular tissues from normal and CAVB dogs were assayed using a real-time PCR. The mRNA levels of KvLQT1 and MiRP1 were significantly less in the CAVB heart compared with those in the intact heart, whereas no significant difference was detected in the mRNA levels of other K(+)- or Ca(2+)-channel subunits. Adaptation against chronic bradycardia-related pathophysiology may have decreased the mRNA levels of cardiac K(+) channels, which may partly explain the arrhythmogenic property of this model.

Effects of an N-type calcium antagonist on angiotensin II-renin feedback.

BACKGROUND: Interrupting the renin-angiotensin system (RAS) with an angiotensin II receptor blocker (ARB) has been found to induce RAS overactivation. In this study, we investigated the effect of 2 calcium channel blockers (CCBs), cilnidipine (L-/N-type CCB) and amlodipine (L-type CCB), on the RAS activation induced by an ARB in a strain of spontaneously hypertensive rats (SHR/Izm, 10 weeks of age). METHODS: Rats intravenously catheterized for blood collection were randomly divided into groups that were administered the vehicle, the ARB valsartan or valsartan combined with one of the 2 CCBs. Their blood and kidneys were collected 270 min after administration. RESULTS: Valsartan increased the plasma angiotensin II (Ang II) level in a dose-dependent manner. Cilnidipine suppressed the increase in plasma renin activity and plasma Ang II levels induced by valsartan, but amlodipine did not. Combined administration of cilnidipine, but not amlodipine, and valsartan significantly reduced the noradrenaline content in the renal cortex. CONCLUSIONS: The results of this study suggest that the suppressive effect of cilnidipine on the valsartan-induced increase in RAS activity can be partly explained by its sympatholytic action mediated by N-type calcium channel blockade, and that combined administration of cilnidipine and valsartan might provide a synergistic therapeutic effect.

Expression of N-type calcium channels in human adrenocortical cells and their contribution to corticosteroid synthesis.

The inhibition of aldosterone activity is a useful approach for preventing the progression of cardiovascular and renal diseases in hypertensive patients. Although the results of our previous in vivo study suggested that N-type calcium channels may have a role in regulating plasma aldosterone levels, the direct relationship between N-type calcium channels and aldosterone production in adrenocortical cells has not been examined. In this study, the analysis of quantitative reverse transcription-PCR, western blotting, and immunocytological staining indicated the possible presence of N-type calcium channels in human adrenocortical cells (H295R cell line). Patch clamp analysis indicated that omega-conotoxin GVIA (CnTX), an N-type calcium channel inhibitor, suppressed voltage-dependent barium currents. During steroidogenesis, CnTX significantly reduced the transient calcium signaling induced by angiotensin II (Ang II) and partially prevented Ang II-induced aldosterone and cortisol formation with no significant influence on CYP11B2 and CYP11B1 mRNA expression. In addition, in α1B calcium channel subunits, knockdown significantly decreased Ang II-induced aldosterone formation with increments in CYP11B2 mRNA expression. We also investigated the inhibitory activities of some types of dihydropyridine calcium channel blockers (CCBs; cilnidipine: L-/N-type CCB, efonidipine: L-/T-type CCB, and nifedipine: L-type CCB), and these agents showed a dose-dependent inhibition effect on Ang II-induced aldosterone and cortisol production. Furthermore, only cilnidipine failed to suppress CYP11B1 expression in H295R cells. These results suggest that N-type calcium channels have a significant role in transducing the Ang II signal for aldosterone (and cortisol) biosynthesis, which may explain the mechanism by which N-type calcium channels regulate plasma aldosterone levels.

The N-type and L-type calcium channel blocker cilnidipine suppresses renal injury in Dahl rats fed a high-salt diet.

The aims of the present study were to compare the effects of cilnidipine [L-type/N-type calcium channel blocker (CCB)] and amlodipine (L-type CCB) alone or in combination with the angiotensin II receptor blocker (ARB), valsartan, on blood pressure (BP), kidney function in Dahl salt-sensitive (DS) rats. DS rats fed a high-salt diet were divided into six groups; control (n = 13), two CCB (cilnidipine or amlodipine) groups at 1 mg/kg/day (n = 10), ARB (valsartan) at 10 mg/kg/day (n = 12), cilnidipine + valsartan (CV, n = 12), and amlodipine + valsartan (AV, n = 12). BPs were lower in the combination therapy groups than in those given either drug alone, but only CV inhibited the increase in urinary albumin excretion (UAE) and lowered the glomerular sclerosis score. In addition, AV elevated plasma renin activity and the angiotensin II concentration, and thus failed to inhibit increases in UAE and to lower glomerular sclerosis score. In conclusion, combination therapy with CCB and ARB decreases BP more effectively than either drug alone. When used in combination with valsartan, cilnidipine is more effective than amlodipine for preventing kidney injury.

Regulation of adrenal aldosterone production by serine protease prostasin.

A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells). Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC) inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

Different effects of L/N-type and L-type calcium channel blockers on the renin-angiotensin-aldosterone system in SHR/Izm.

BACKGROUND: The L/N-type calcium channel blocker (CCB) cilnidipine has been demonstrated to suppress sympathetic nerve activity. In the present study, we investigated the effects of cilnidipine on the renin-angiotensin-aldosterone system (RAAS) in SHR/Izm rats to confirm differences from the effects of L-type CCB. METHODS: Male SHR/Izm rats received vehicle, cilnidipine (0.3, 3 mg/kg) or amlodipine (0.3, 3 mg/kg) by gavage for systolic blood pressure (SBP) measurement. For biochemical analyses, the experiments were performed under anesthesia. RESULTS: Low doses of cilnidipine or amlodipine had no effect on SBP or RAAS parameters. A high dose of either drug produced similar effects on SBP levels. Although cilnidipine had no effect on plasma renin activity or the plasma angiotensin II level, amlodipine significantly increased these parameters as compared to levels in the vehicle group. The cilnidipine group had a significantly lower plasma aldosterone level and renal cortical tissue norepinephrine level than the vehicle group. CONCLUSIONS: Cilnidipine had effects different from those of L-type CCB on the RAAS in SHR/Izm rats. Our results indicate that suppression of RAAS hyperactivity by cilnidipine can be partly explained by its sympatholytic action.