Physical Stimulation Methods Developed for In Vitro Neuronal Differentiation Studies of PC12 Cells: A Comprehensive Review.

PC12 cells, which are derived from rat adrenal pheochromocytoma cells, are widely used for the study of neuronal differentiation. NGF induces neuronal differentiation in PC12 cells by activating intracellular pathways via the TrkA receptor, which results in elongated neurites and neuron-like characteristics. Moreover, the differentiation requires both the ERK1/2 and p38 MAPK pathways. In addition to NGF, BMPs can also induce neuronal differentiation in PC12 cells. BMPs are part of the TGF-ß cytokine superfamily and activate signaling pathways such as p38 MAPK and Smad. However, the brief lifespan of NGF and BMPs may limit their effectiveness in living organisms. Although PC12 cells are used to study the effects of various physical stimuli on neuronal differentiation, the development of new methods and an understanding of the molecular mechanisms are ongoing. In this comprehensive review, we discuss the induction of neuronal differentiation in PC12 cells without relying on NGF, which is already established for electrical, electromagnetic, and thermal stimulation but poses a challenge for mechanical, ultrasound, and light stimulation. Furthermore, the mechanisms underlying neuronal differentiation induced by physical stimuli remain largely unknown. Elucidating these mechanisms holds promise for developing new methods for neural regeneration and advancing neuroregenerative medical technologies using neural stem cells.

SP600125 Enhances Temperature-Controlled Repeated Thermal Stimulation-Induced Neurite Outgrowth in PC12-P1F1 Cells.

This study evaluated the mechanism of temperature-controlled repeated thermal stimulation (TRTS)-mediated neuronal differentiation. We assessed the effect of SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, on neuronal differentiation of rat PC12-P1F1 cells, which can differentiate into neuron-like cells by exposure to TRTS or neurotrophic factors, including bone morphogenetic protein (BMP) 4. We evaluated neuritogenesis by incubating the cells under conditions of TRTS and/or SP600125. Cotreatment with SP600125 significantly enhanced TRTS-mediated neuritogenesis, whereas that with other selective mitogen-activated protein kinase (MAPK) inhibitors did not-e.g., extracellular signal-regulated kinase (ERK)1/2 inhibitor U0126, and p38 MAPK inhibitor SB203580. We tried to clarify the mechanism of SP600125 action by testing the effect of U0126 and the BMP receptor inhibitor LDN193189 on the SP600125-mediated enhancement of intracellular signaling. SP600125-enhanced TRTS-induced neuritogenesis was significantly inhibited by U0126 or LDN193189. Gene expression analysis revealed that TRTS significantly increased ß3-Tubulin, MKK3, and Smad7 gene expressions. Additionally, Smad6 and Smad7 gene expressions were substantially attenuated through SP600125 co-treatment during TRTS. Therefore, SP600125 may partly enhance TRTS-induced neuritogenesis by attenuating the negative feedback loop of BMP signaling. Further investigation of the mechanisms underlying the effect of SP600125 during TRTS-mediated neuritogenesis may contribute to the future development of regenerative neuromedicine.

Characterization of PC12 Cell Subclones with Different Sensitivities to Programmed Thermal Stimulation.

Neuritogenesis is the process underling nervous system regeneration; however, optimal extracellular signals that can promote neuronal regenerative activities require further investigation. Previously, we developed a novel method for inducing neuronal differentiation in rat PC12 cells using temperature-controlled repeated thermal stimulation (TRTS) with a heating plate. Based on neurogenic sensitivity to TRTS, PC12 cells were classified as either hyper- or hyposensitive. In this study, we aimed to investigate the mechanism of hyposensitivity by establishing two PC12-derived subclones according to TRTS sensitivity during differentiation: PC12-P1F1, a hypersensitive subclone, and PC12-P1D10, a hyposensitive subclone. To characterize these subclones, cell size and neuritogenesis were evaluated in subclones treated with nerve growth factor (NGF), bone morphogenetic protein (BMP), or various TRTS. No significant differences in cell size were observed among the parental cells and subclones. BMP4- or TRTS-induced neuritogenesis was increased in PC12-P1F1 cells compared to that in the parental cells, while no neuritogenesis was observed in PC12-P1D10 cells. In contrast, NGF-induced neuritogenesis was observed in all three cell lines. Furthermore, a BMP inhibitor, LDN-193189, considerably inhibited TRTS-induced neuritogenesis. These results suggest that the BMP pathway might be required for TRTS-induced neuritogenesis, demonstrating the useful aspects of these novel subclones for TRTS research.

Uremic serum residue decreases SN-38 sensitivity through suppression of organic anion transporter polypeptide 2B1 in LS-180 colon cancer cells.

Pharmacokinetics of SN-38 in patients with end-stage kidney disease (ESKD) is partially varied because of fluctuations in transporters expression and/or function by high protein bound-uremic toxins concentration. The fluctuations may induce variations in anticancer drugs sensitivity to cancer cells. We aimed to clarify the variations in sensitivity of SN-38 to cancer patients with ESKD and investigate this mechanism, by human colon cancer cells exposed to uremic serum residue. LS180 cells were exposed to normal or uremic serum residue (LS/NSR or LS/USR cells) for a month. IC50 values of SN-38 in LS/NSR or LS/USR cells were calculated from viability of each cells treated SN-38. mRNA expression and intracellular SN-38 accumulation was evaluated by RT-PCR and HPLC-fluorescence methods, respectively. The IC50 value in LS/USR cells was higher than that in LS/NSR cells. Organic anion transporter polypeptide (OATP) 2B1 mRNA expression was lower in LS/USR cells than in LS/NSR cells, and SN-38 accumulation in LS/USR cells was lower than that in LS/NSR cells. Only co-treatment baicalin, which is OATP2B1 inhibitor, almost negated the difference in SN-38 accumulation between LS/NSR and LS/USR. Anticancer effects of substrates of OATP2B1, such as SN-38, were reduced in ESKD patients at the same plasma substrate concentration.

Effects of Uremic Serum Residue on OATP1B1- and OATP1B3-Mediated Pravastatin Uptake in OATP-Expressing HEK293 Cells and Human Hepatocytes.

Patients with end-stage renal disease have increased plasma concentrations of statins, which is a risk factor for rhabdomyolysis, as well as elevated levels of uremic toxins (UTs). We investigated the effects of uremic serum residue and UTs on organic anion-transporting peptide (OATP1B1)- and OATP1B3-mediated pravastatin uptake. We evaluated the effects of normal serum residue with four UTs (hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furan propionate, indole-3-acetic acid, and 3-indoxyl sulfate) and uremic serum residue on pravastatin uptake by OATP1B1- or OATP1B3-expressing HEK293 cells. Furthermore, we assessed the contribution of each transporter using cryopreserved human hepatocytes. Uremic serum residue and UTs significantly inhibited OATP1B1-mediated pravastatin uptake. Uremic serum residue accelerated OATP1B3-mediated pravastatin uptake, while UTs had no effect. There was no difference in pravastatin uptake between uremic- and normal serum residue-treated hepatocytes. The results suggest that the effects of uremic serum on pravastatin hepatic uptake may be considered negligible in end-stage renal disease patients.

Acceleration of carboxylesterase-mediated activation of irinotecan to SN-38 by serum from patients with end-stage kidney disease.

PURPOSE: Pharmacokinetics and pharmacodynamics of irinotecan have been reported to be altered in cancer patients with end-stage kidney disease (ESKD). Carboxylesterase (CES) has an important role in metabolism of irinotecan to its active metabolite, SN-38, in human liver. The purpose of the present study was to investigate whether CES activity was altered in ESKD patients. METHODS: The present study investigated the effects of uremic serum, uremic toxins, and fatty acids on the hydrolysis of irinotecan and a typical CES substrate, p-nitrophenyl acetate (PNPA), in human liver microsomes. Normal and uremic serum samples were deproteinized by treatment with methanol were used in the present study. RESULTS: The present study showed that both normal and uremic serum significantly inhibited CES-mediated metabolism of both irinotecan and PNPA. The inhibition by uremic serum was weaker than that by normal serum, suggesting that CES activity may be higher in ESKD patients. Although four uremic toxins did not affect PNPA metabolism, arachidonic acid inhibited it. There was no difference in inhibitory effect of PNPA metabolism between both mixtures of seven fatty acids used at concentrations equivalent to those present in 10% normal or uremic serum. Interestingly, those mixtures had a more pronounced effect than either 10% normal or uremic serum. CONCLUSIONS: The present study showed that the inhibition of CES activity by uremic serum was weaker than that by normal serum, suggesting that an increase in maximum plasma concentration of SN-38 in cancer patients with ESKD can be attributed to an accelerated CES-mediated irinotecan hydrolysis.

Evaluation of Trace Elements in Augmentation of Statin-Induced Cytotoxicity in Uremic Serum-Exposed Human Rhabdomyosarcoma Cells.

Patients with end-stage kidney disease (ESKD) are at higher risk for rhabdomyolysis induced by statin than patients with normal kidney function. Previously, we showed that this increase in the severity of statin-induced rhabdomyolysis was partly due to uremic toxins. However, changes in the quantity of various trace elements in ESKD patients likely contribute as well. The purpose of this study is to determine the effect of trace elements on statin-induced toxicity in rhabdomyosarcoma cells exposed to uremic serum (US cells) for a long time. Cell viability, apoptosis, mRNA expression, and intracellular trace elements were assessed by viability assays, flow cytometry, real-time RT-PCR, and ICP-MS, respectively. US cells exhibited greater simvastatin-induced cytotoxicity than cells long-time exposed with normal serum (NS cells) (non-overlapping 95% confidence intervals). Intracellular levels of Mg, Mn, Cu, and Zn were significantly less in US cells compared to that in NS cells (p < 0.05 or 0.01). Pre-treatment with TPEN increased simvastatin-induced cytotoxicity and eliminated the distinction between both cells of simvastatin-induced cytotoxicity. These results suggest that Zn deficiencies may be involved in the increased risk for muscle complaints in ESKD patients. In conclusion, the increased severity of statin-induced rhabdomyolysis in ESKD patients may be partly due to trace elements deficiencies.

Cooperative inhibitory effects of uremic toxins and other serum components on OATP1B1-mediated transport of SN-38.

PURPOSE: Half-life of SN-38, an active metabolite of irinotecan, remarkably increases in patients with end-stage kidney disease (ESKD), even though SN-38 is excreted in bile. Uremic toxins (UTs), which accumulate in the serum of ESKD patients, were reported to inhibit organic anion-transporting polypeptide (OATP) 1B1-mediated uptake of SN-38; however, the relevance of this finding in a clinical setting is unknown. This study focused on cooperative effects of serum components and UTs on OATP1B1-mediated transport of SN-38. METHODS: Uptake of SN-38 by OATP1B1 was evaluated using cells stably expressing OATP1B1. Serum was obtained from > 400 ESKD patients undergoing hemodialysis. Deproteinized serum was combined with human serum albumin (HSA) to explore the effects of albumin-bound and unbound serum compounds. RESULTS: Uptake clearance of SN-38 in OATP1B1 cells decreased by 40% in the presence of uremic serum residue with albumin compared to that in the presence of normal serum residue. Additional UTs (3-carboxy-4-methyl-5-propyl-2-furanpropionic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) combined with normal serum residue in HSA decreased OATP1B1-mediated SN-38 transport by 32.1% compared to that in the presence of normal serum residue. The inhibitory effect of albumin-unbound fraction with UTs and normal serum residue was comparable to that of uremic serum residue, with an uptake decrease of 17.2% compared to that reported in the presence of normal serum residue. CONCLUSIONS: Hepatic uptake of SN-38 via OATP1B1 decreases in ESKD patients through cooperative inhibitory effects of UTs and serum components.

Uremic toxins enhance statin-induced cytotoxicity in differentiated human rhabdomyosarcoma cells.

The risk of myopathy and rhabdomyolysis is considerably increased in statin users with end-stage renal failure (ESRF). Uremic toxins, which accumulate in patients with ESRF, exert cytotoxic effects that are mediated by various mechanisms. Therefore, accumulation of uremic toxins might increase statin-induced cytotoxicity. The purpose of this study was to determine the effect of four uremic toxins-hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate-on statin-induced myopathy. Differentiated rhabdomyosarcoma cells were pre-treated with the uremic toxins for seven days, and then the cells were treated with pravastatin or simvastatin. Cell viability and apoptosis were assessed by viability assays and flow cytometry. Pre-treatment with uremic toxins increased statin- but not cisplatin-induced cytotoxicity (p < 0.05 vs. untreated). In addition, the pre-treatment increased statin-induced apoptosis, which is one of the cytotoxic factors (p < 0.05 vs. untreated). However, mevalonate, farnesol, and geranylgeraniol reversed the effects of uremic toxins and lowered statin-induced cytotoxicity (p < 0.05 vs. untreated). These results demonstrate that uremic toxins enhance statin-induced apoptosis and cytotoxicity. The mechanism underlying this effect might be associated with small G-protein geranylgeranylation. In conclusion, the increased severity of statin-induced rhabdomyolysis in patients with ESRF is likely due to the accumulation of uremic toxins.

Effects of decreased vitamin D and accumulated uremic toxin on human CYP3A4 activity in patients with end-stage renal disease.

In patients with end-stage renal disease, not only renal clearance but also hepatic clearance is known to be impaired. For instance, the concentration of erythromycin, a substrate of cytochrome P450 3A4 (CYP3A4), has been reported to be elevated in patients with end-stage renal disease. The purpose of this study is to elucidate the reason for the decrease in hepatic clearance in patients with end-stage renal disease. Deproteinized pooled sera were used to assess the effects of low-molecular-weight uremic toxins on CYP3A4 activity in human liver microsomes and human LS180 cells. Four uremic toxins (3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, hippuric acid, indole-3-acetic acid, and 3-indoxyl sulfate) present at high concentrations in uremic serum were also studied. Simultaneous treatment of uremic serum (less than 10%) or uremic toxins did not affect testosterone 6ß-hydroxylation in human liver microsomes. On the other hand, pretreatment of each serum activates CYP3A4 in LS180 cells, and the increased CYP3A4 activity in uremic serum-treated cells was smaller than normal serum-treated cells. In addition, CYP3A4 and CYP24A1 mRNA levels also increased in LS180 cells exposed to normal serum, and this effect was reduced in uremic serum-treated cells and in cells exposed to uremic serum added to normal serum. Furthermore, addition of 1,25-dihydroxyvitamin D to uremic serum partially restored the serum effect on CYP3A4 expression. The present study suggests that the decrease of 1,25-dihydroxyvitamin D and the accumulation of uremic toxins contributed to the decreased hepatic clearance of CYP3A4 substrates in patients with end-stage renal disease.

Influence of serum in hemodialysis patients on the expression of intestinal and hepatic transporters for the excretion of pravastatin.

It is known that the lipid-lowering agent pravastatin, which is not metabolized by cytochrome P450, is eliminated as an unchanged drug in bile and urine. It is interesting to note that the non-renal clearance of pravastatin in end-stage renal failure patients is decreased compared with that of healthy volunteers. This study investigated the influence of uremic serum and toxins on the transport mechanisms of pravastatin to elucidate the cause of decreased non-renal clearance in end-stage renal failure patients. Caco-2 and Hep3B cells were used as models of intestinal epithelial cells and hepatocytes respectively. Normal and uremic serum were deproteinized by treatment with methanol. 3-Carboxy-4-methyl-5propyl-2-furanpropanoic acid (CMPF), hippuric acid, indole-3-acetic acid, 3-indoxyl sulfate, and p-cresol were chosen as uremic toxins. Uremic serum-treated Caco-2 cells exhibited significantly increased accumulation of pravastatin and significantly decreased expression of MRP2 mRNA compared with normal serum-treated Caco-2 cells. In addition, the expression of MRP2 mRNA tended to decrease in cells treated with CMPF, indole-3-acetic acid, or 3-indoxyl sulfate. Uremic serum-treated Hep3B cells showed a significantly decreased initial uptake rate of pravastatin; furthermore, the expressions of OATP1B1 and OATP2B1 mRNA were decreased compared to normal serum-treated Hep3B cells. These results suggest that the decrease in the non-renal clearance of pravastatin in end-stage renal failure patients is partly induced by the downregulation of intestinal MRP2 and hepatic OATP1B1 and/or OATP2B1 by various uremic toxins in end-stage renal failure patients.

Myocardial ischemia due to compression of an unruptured thoracic aortic aneurysm in a patient with Marfan syndrome.

We report a 33-year-old woman who had a 60-mm thoracic aneurysm of the ascending aorta with Marfan syndrome and effort angina due to compression of the right coronary artery (RCA) by the aneurysm. Surgery was performed using the Bentall procedure and a coronary artery bypass graft to the RCA. Postoperatively, coronary angiography showed that the coronary flow of the RCA was restored by removing the aneurysmal compression. The patient was discharged without angina on postoperative day 21.

Repeat surgery for hemolysis 6 years after replacement of the ascending aorta for acute aortic dissection.

A 71-year-old woman underwent replacement of the ascending aorta for Type A aortic dissection. After 6 years, she suddenly developed severe hemolytic anemia, and a second operation for replacement of the ascending aorta was performed. Her hemolysis was thought to occur as follows: the proximal ascending aorta of the graft might have gradually expanded until it compressed the graft. The severe hemolysis was thought to be attributable to disturbance of blood flow by a jet of blood at the site of constriction or the reversed inner felt. Such a case as this is very unusual in that the second operation for hemolytic anemia occurred 6 years after the first surgery.