Microvillar dynamic in renal tubular epithelial cells mediated by insulin/PLCγ signal pathway.

Significant cellular morphology changes in renal tubules were observed in diabetes patients and animal models. However, the interaction between insulin and tubular epithelial cells microvillar structure remains obscure. To understand microvillar dynamics, we used Scanning Ion Conductance Microscope to visualize microvillar in the living cell. Here, we found two layers of microvilli on the tubular epithelial cell surface: short compact microvilli and netlike long microvilli. Insulin treatment could increase microvilli length and density. This process was mediated by the PI3K/PLCγ signaling pathway, other than the PI3K/Arp2/3 signal pathway. In conclusion, our findings present a novel insulin signaling transduction mechanism, which contributes to understanding renal tubular epithelial cell microvilli dynamic regulation.

CRL4 antagonizes SCFFbxo7-mediated turnover of cereblon and BK channel to regulate learning and memory.

Intellectual disability (ID), one of the most common human developmental disorders, can be caused by genetic mutations in Cullin 4B (Cul4B) and cereblon (CRBN). CRBN is a substrate receptor for the Cul4A/B-DDB1 ubiquitin ligase (CRL4) and can target voltage- and calcium-activated BK channel for ER retention. Here we report that ID-associated CRL4CRBN mutations abolish the interaction of the BK channel with CRL4, and redirect the BK channel to the SCFFbxo7 ubiquitin ligase for proteasomal degradation. Glioma cell lines harbouring CRBN mutations record density-dependent decrease of BK currents, which can be restored by blocking Cullin ubiquitin ligase activity. Importantly, mice with neuron-specific deletion of DDB1 or CRBN express reduced BK protein levels in the brain, and exhibit similar impairment in learning and memory, a deficit that can be partially rescued by activating the BK channel. Our results reveal a competitive targeting of the BK channel by two ubiquitin ligases to achieve exquisite control of its stability, and support changes in neuronal excitability as a common pathogenic mechanism underlying CRL4CRBN-associated ID.

Modulation of Actin Filament Dynamics by Inward Rectifying of Potassium Channel Kir2.1.

Apart from its ion channel properties, the Kir2.1 channel has been found in tumors and cancer cells to facilitate cancer cell motility. It is assumed that Kir2.1 might be associated with cell actin filament dynamics. With the help of structured illumination microscopy (SIM), we show that Kir2.1 overexpression promotes actin filament dynamics, cell invasion, and adhesion. Mutated Kir2.1 channels, with impaired membrane expression, present much weaker actin regulatory effects, which indicates that precise Kir2.1 membrane localization is key to its actin filament remolding effect. It is found that Kir2.1 membrane expression and anchoring are associated with PIP2 affinity, and PIP2 depletion inhibits actin filament dynamics. We also report that membrane-expressed Kir2.1 regulates redistribution and phosphorylation of FLNA (filamin A), which may be the mechanism underlying Kir2.1 and actin filament dynamics. In conclusion, Kir2.1 membrane localization regulates cell actin filaments, and not the ion channel properties. These data indicate that Kir2.1 may have additional cellular functions distinct from the regulation of excitability, which provides new insight into the study of channel proteins.

Role of epithelial Na+ channels in endothelial function.

An increasing number of mechano-sensitive ion channels in endothelial cells have been identified in response to blood flow and hydrostatic pressure. However, how these channels respond to flow under different physiological and pathological conditions remains unknown. Our results show that epithelial Na(+) channels (ENaCs) colocalize with hemeoxygenase-1 (HO-1) and hemeoxygenase-2 (HO-2) within the caveolae on the apical membrane of endothelial cells and are sensitive to stretch pressure and shear stress. ENaCs exhibited low levels of activity until their physiological environment was changed; in this case, the upregulation of HO-1, which in turn facilitated heme degradation and hence increased the carbon monoxide (CO) generation. CO potently increased the bioactivity of ENaCs, releasing the channel from inhibition. Endothelial cells responded to shear stress by increasing the Na(+) influx rate. Elevation of intracellular Na(+) concentration hampered the transportation of l-arginine, resulting in impaired nitric oxide (NO) generation. Our data suggest that ENaCs that are endogenous to human endothelial cells are mechano-sensitive. Persistent activation of ENaCs could inevitably lead to endothelium dysfunction and even vascular diseases such as atherosclerosis.

Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension.

AIMS: Pulmonary arterial hypertension [1] is a proliferative disorder associated with enhanced proliferation and suppressed apoptosis of pulmonary artery smooth muscle cells (PASMCs). Reactive oxygen species (ROS) is implicated in the development of PAH and regulates the vascular tone and functions. However, which cellular signaling mechanisms are triggered by ROS in PAH is still unknown. Hence, here we wished to characterize the signaling mechanisms triggered by ROS. METHODS AND RESULTS: By Western blots, we showed that increased intracellular ROS caused inhibition of the glycolytic pyruvate kinase M2 (PKM2) activity through promoting the phosphorylation of PKM2. Monocrotaline (MCT)-induced rats developed severe PAH and right ventricular hypertrophy, with a significant increase in the P-PKM2 and decrease in pyruvate kinase activity which could be attenuated with the treatments of PKM2 activators, FBP and l-serine. The antioxidant NAC, apocynin and MnTBAP had the similar protective effects in the development of PAH. In vitro assays confirmed that inhibition of PKM2 activity could modulate the flux of glycolytic intermediates in support of cell proliferation through the increased pentose phosphate pathway (PPP). Increased ROS and decreased PKM2 activity also promoted the Cav1.2 expression and intracellular calcium. CONCLUSION: Our data provide new evidence that PKM2 makes a critical regulatory contribution to the PAHs for the first time. Decreased pyruvate kinase M2 activity confers additional advantages to rat PASMCs by allowing them to sustain anti-oxidant responses and thereby support cell survival in PAH. It may become a novel treatment strategy in PAH by using of PKM2 activators.

Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

[Impact of the CFTR chloride channel on the cytoskeleton of mouse Sertoli cells].

OBJECTIVE: To study the impact of the chloride channel dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) on the cytoskeleton of Sertoli cells in the mouse. METHODS: TM4 Sertoli cells were cultured and treated with CFTR(inh)-172 at the concentrations of 1, 5, 10 and 20 µmol/L for 48 hours. Then the cytotoxicity of CFT(inh)-172 was assessed by CCK-8 assay, the expressions of F-actin and Ac-tub in the TM4 Sertoli cells detected by immunofluorescence assay, and those of N-cadherin, vimentin and vinculin determined by qPCR. RESULTS: CFTR(inh)-172 produced cytotoxicity to the TM4 Sertoli cells at the concentration of 20 µmol/L. The expressions of F-actin and Ac-tub were decreased gradually in the TM4 Sertoli cells with the prolonging of treatment time and increasing concentration of CFTR(inh)-172 (P < 0.05). The results of qPCR showed that different concentrations of CFTR(inh)-172 worked no significant influence on the mRNA expressions of N-cadherin, vimentin and vinculin in the Sertoli cells. CONCLUSION: The CFTR chloride channel plays an important role in maintaining the normal cytoskeleton of Sertoli cells. The reduced function and expression of the CFTR chloride channel may affect the function of Sertoli cells and consequently spermatogenesis of the testis.

High glucose and interleukin 1ß-induced apoptosis in human umbilical vein endothelial cells involves in down-regulation of monocarboxylate transporter 4.

Hyperglycaemia and inflammatory can induce apoptosis in vascular endothelial cells, which contributes to the development of vascular complications in diabetes. Endothelial cells depend on glycolysis for their energy metabolism, and monocarboxylate transporters (MCTs) regulate intracellular pH by mediating the influx and efflux of lactate. Here, we evaluate the role of MCT4 in high glucose (HG) and interleukin 1ß (IL-1ß)-induced apoptosis in human umbilical vein endothelial cells (HUVECs). We demonstrate that aortic endothelium damage is severe in db/db mice by using scanning ion conductance microscopy (SICM). HG and IL-1ß decrease MCT4 and its location on plasma membrane, as well as increase lactic acid accumulation and apoptosis in HUVECs. Knockdown of MCT4 blocks lactate efflux to result in lactic acid accumulation and pH dropping, which is involved in triggering apoptosis in HUVECs.

Identification of microRNAs as novel biomarkers for detecting esophageal squamous cell carcinoma in Asians: a meta-analysis.

Accumulating evidence has suggested that microRNAs (miRNAs) may play potential role as ideal diagnostic indicators of esophageal squamous cell carcinoma (ESCC). However, previous studies have met discrepant results. Thus, we conducted this meta-analysis to assess the potential diagnostic value of miRNAs for ESCC. A systematic literature search was conducted in PubMed and other databases. The pooled sensitivity (SEN), specificity (SPE), positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR), and area under the curve (AUC) were calculated to evaluate the overall test performance. The Q statistic and the I(2) test were used to assess between-study heterogeneity. The potential sources of heterogeneity were further analyzed by subgroup analyses and meta-regression. Seventeen studies from eight articles, including 995 ESCC patients and 733 healthy controls, were included in this meta-analysis. The pooled SEN and SPE were 0.81 (95% confidence interval (CI) 0.76-0.85) and 0.83 (95 % CI 0.76-0.88), respectively. The pooled PLR was 4.6 (95% CI 3.3-6.5), NLR was 0.23 (95% CI 0.19-0.29), and DOR was 20 (95% CI 13-31). The pooled AUC was 0.91 (95% CI 0.88-0.93). Subgroup analyses indicated that blood-based miRNA assay displays better diagnostic accuracy than saliva-based miRNA assay. In summary, miRNA analysis may serve as novel noninvasive biomarkers for ESCC with excellent diagnostic characteristic. In addition, subgroup analysis suggested that blood-based assay yields better diagnostic characteristics than saliva-based assay. However, many issues should be managed before these findings can be translated into a clinically useful detection method for ESCC.

Docosahexaenoic acid attenuates hypoxic pulmonary vasoconstriction by activating the large conductance Ca2+-activated K+ currents in pulmonary artery smooth muscle cells.

BACKGROUND: The inhibition of potassium (K(+)) channels plays an important role in pulmonary circulation for its close relationship with hypoxic pulmonary vasoconstriction (HPV). Docosahexaenoic acid (DHA), a n-3 polyunsaturated fatty acid, is well known for its prevention and treatment of cardiovascular diseases. However the role which DHA plays in HPV remains unclear. Here, we tested the hypothesis that DHA contributes to pulmonary vascular tone by activating the large conductance Ca(2+)-activated K(+) (BKCa) channels via calcium sparks. METHODS AND RESULTS: Isolated resistance pulmonary artery preparation was used to study the vasomotor response to DHA. Pulmonary artery smooth muscle cells (PASMCs) were isolated from third- to fourth order branches of pulmonary arteries by collagenase digestion method. BKCa and the voltage-dependent potassium channel (Kv) currents in PASMCs were measured by the whole-cell patch-clamp technique. Fluo-8 was used as a fluorescence indicator for the real-time measurement of calcium dynamics in PASMCs. DHA dilated resistance pulmonary arteries in a dose-dependent manner in hypoxic or normoxic solution, and the effects of DHA were abolished after pre-treatment with heparin (100 µg/ml), a 1,4,5-triphosphate (IP3) receptor (IP3R) inhibitor or iberiotoxin (100 nmol/L), a specific inhibitor of BKCa channel. DHA activated BKCa channels in a dose-dependent manner, however, the activation induced by DHA was not seen in PASMCs pre-incubated with heparin. While the Kv currents decreased from 102.6 ± 5.4 to 36.5 ± 6.7 pA/pF by addition of 10 µmol/L DHA. DHA also caused calcium sparks in PASMCs. Moreover, hypoxia inhibited BKCa currents in PASMCs, but this inhibition was reversed by DHA. CONCLUSION: Our findings suggest that DHA is a novel BKCa opener in PASMCs, which may indicate a potential therapeutic role in HPV.

[Effects of early digoxin treatment on hypoxia-induced pulmonary artery hypertension].

OBJECTIVE: To explore the effects of digoxin on hypoxia-induced pulmonary artery hypertension (PAH) and the possible mechanisms. METHODS: A total of 48 Sprague-Dawley rats were randomly divided into 4 groups: normoxia control, normoxia+digoxin, hypoxia control and hypoxia+digoxin. The animals were exposed to chronic intermittent hypoxia (PO2: (10.5 ± 0.5) %, 8:00-16:00) or room air for 21 days.Each rat received a daily intraperitoneal injection of either digoxin (1.0 mg × kg⁻¹ × d⁻¹) or an equal volume of vehicle, starting at the first day of hypoxia or normoxia. At Day 21, mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy (RV/(LV+S)) and index of wall thickness of small pulmonary artery (WT% and WA%) among groups were compared. And in vitro the changes of pulmonary artery smooth muscle cells (PASMCs) proliferation were determined by methyl thiazolyl tetrazolium (MTT) assay. Migration assay was performed with a Transwell chamber.Real-time quantitative polymerase chain reaction (PCR) was performed to quantify the mRNA levels of smooth muscle cell phenotype markers such as smooth muscle-α-actin, calponin and smooth muscle 22α under normoxic or hypoxic conditions in the absence or presence of digoxin. And the protein expressions of matrix metalloproteinase (MMPs) were determined by Western blot. RESULTS: Digoxin treatment significantly lowered mPAP, reduced WT% and WA% and right ventricular hypertrophy compared with those of the hypoxic group (mPAP: (27.3 ± 2.7) vs (38.5 ± 2.3) mmHg (1 mmHg = 0.133 kPa); RV/(LV+S): (30.9 ± 3.3)% vs (42.8 ± 2.6)%, WT%: (21.7 ± 3.6)% vs (39.3 ± 2.0)%; WA%: (56.3 ± 4.7)% vs (79.5 ± 5.7)%, all P < 0.05). And in vitro, digoxin restored the hypoxia-induced inhibition of the expression of smooth muscle cell phenotype markers and prevented the hypoxia-induced activation of MMPs in PASMCs. CONCLUSION: Early digoxin therapy reduces pulmonary artery remodeling in hypoxia-induced PAH rat model and this effect is probably correlated with the inhibitions of proliferation, migration, phenotype switching and expression of MMPs induced by hypoxia in PASMCs.

[Effects of docosahexaenoic acid on hypoxia-induced pulmonary arterial hypertension].

OBJECTIVE: To investigate the effects of docosahexaenoic acid (DHA) on hypoxia-induced pulmonary arterial hypertension(PAH) and the mechanism. METHODS: PAH was induced by chronic intermittent hypoxia for 21 days in vivo. Forty male Sprague-Dawley rats were randomly divided into 4 groups (n = 10, each):a normal control group, DHA-treated groups in normoxia and hypoxia, and a PAH group. At the end of study, mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy and the index of wall thickness of small pulmonary artery (WT% and WA%) among groups were compared. The changes of pulmonary arterial smooth muscle cell (PASMC) proliferation were determined by MTT in vitro. Migration assay was performed using the Boyden chamber. Real-time quantitative PCR was performed to quantify mRNA levels of the smooth muscle cell phenotype markers SM-α-actin, calponin and SM 22α under normoxic or hypoxic conditions, in the absence or presence of DHA. RESULTS: DHA treatment significantly lowered mPAP [(22.7 ± 1.8) mmHg (1 mmHg = 0.133 kPa)], reduced thickening of small pulmonary artery wall [WT%:(21.6 ± 4.1)%, WA%: (52.0 ± 2.9)% ] and alleviated ventricular hypertrophy (34.2 ± 2.2) % compared to those of the hypoxic group (P < 0.05). DHA inhibited the proliferation, migration and phenotype switching of PASMCs induced by hypoxia in vitro. CONCLUSION: DHA therapy reduced mPAP in a rat model of hypoxia-induced PAH and this effect was linked with inhibition of pulmonary vascular remodelling.

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+.

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.

An oxygen-sensitive mechanism in regulation of epithelial sodium channel.

Epithelial sodium channels (ENaCs) are of immense importance, controlling Na(+) transport across epithelia and thus playing a central role in all aspects of fluid clearance as well as numerous other functions. Regulation of these channels is critical. Here, we show that haem, a regulator of Na(+) transport, directly influences ENaC activity, decreasing channel-open probability (but not unitary conductance) in inside-out patches (but not outside-out). Conversely, exposure to the protein in the presence of NADPH and at normoxic O(2) tension (requirements for activity of hemeoxygenase) increases channel activity. CO, a product of hemeoxygenase activity, activated ENaC in a manner similar to that of haem plus NADPH. However, under hypoxic conditions, inhibition of ENaC by haem occurred even in the presence of NADPH. These data demonstrate a potent, O(2)-sensitive mechanism for regulation of ENaC, in which hemeoxygenase acts as the O(2) sensor, its substrate and product inhibiting and stimulating (respectively) the activity of ENaC.

Universal Markers for hiPSCs Residue Detection.

BACKGROUND: Residual undifferentiated induced pluripotent stem cells (iPSCs) detection is essential for both Embryonic Stem Cells (ESCs) and iPSCs application in final cell therapy products. However, specific differentiated cells require specific genes for residual detection; identifying the suitable marker is costly and time-consuming. Thus, a universal marker for iPSCs residue detection for all three germline cells would greatly benefit PSC-derived cellular therapies. METHODS: Next-generation sequencing (NGS) was performed on total RNAs isolated from the iPSC cell lines and embryonic stem cells (H9), the top 30 expressed genes were selected as candidates. By analysis expression fold change comparing iPSC cells to the differentiated cells, seven genes were highly expressed in iPSCs but showed minimal background expression in differentiated cells. Tissue expression pattern of the candidate genes were explored in the Genotype-Tissue Expression (GTEx) project database, candidate genes were narrowed down to two genes. Spike-in experiments were performed to determine the detection limit and correlation with the number of iPSCs and gene expression by ddPCR. RESULTS: By next-generation sequencing (NGS), we identified two marker genes (ESRG and ZSCAN10) suitable for universal undifferentiated iPSC detection. Both ESRG and ZSCAN10 are highly expressed in iPSCs. ZSCAN10 is slightly expressed in the testis, pituitary, and cerebellum; ESRG is highly expressed in the vagina and scarcely expressed in the other tissues. Furthermore, the ddPCR method with a probe and primers for ESRG and ZSCAN10 detected a trace of undifferentiated hiPSCs to a spiked level of 0.0001%. CONCLUSIONS: These results suggest that targeting ESRG/ZSCAN10 transcripts is highly sensitive, quantitative, and could be broadly applied to quality control of almost all iPSC-derived cell therapy products.

Combined treatment with anti-PSMA CAR NK-92 cell and anti-PD-L1 monoclonal antibody enhances the antitumour efficacy against castration-resistant prostate cancer.

BACKGROUND: The chimeric antigen receptor NK-92 (CAR NK-92) cell targeting the prostate-specific membrane antigen (PSMA) has shown antitumour effects in castration-resistant prostate cancer (CRPC). However, the expression changes of programmed death ligand 1 (PD-L1) and its mechanisms on CAR NK-92 and CRPC cells and the effect of the anti-PD-L1 monoclonal antibody (mAb) on PD-L1 expressed on CAR NK-92 cells remain unknown. METHODS: Human dendritic cells and CD8+ T cells were acquired from blood samples of healthy donors and cocultured with C4-2 cells. Changes in PD-L1 expression were detected by flow cytometry. Differential gene expressions were investigated by RNA sequence analysis, while the regulation of PD-L1 molecular signaling was explored using western blotting. In vitro cytotoxicity was evaluated using the Cell Counting Kit-8 assay and the bioluminescent intensity (BLI) of green fluorescent protein-labelled C4-2 cells. CRPC growth in vivo was monitored using callipers and BLI in male NOD/SCID mice subcutaneously injected with C4-2 cells and treated intravenously with anti-PD-L1/PD-1 mAb, CAR NK-92 or cocultured CD8+ T cells. RESULTS: Significantly upregulated expression of PD-L1k was observed in cocultured C4-2 and CAR NK-92 cells. In addition, upregulation of PD-L1 expression was dependent on interferon-γ in C4-2 cells, while it was dependent on direct cell-to-cell interaction via the NK group 2 member D/ phosphatidylinositol 3-kinase/AKT pathway in CAR NK-92 cells. The anti-PD-L1 mAb directly acted on PD-L1 expressed on CAR NK-92 cells and augmented the cytotoxicity of CAR NK-92 cells against C4-2 and CRPC cells from one patient in vitro. Anti-PD-L1 mAb significantly enhanced the antitumour effect of CAR NK-92 cells against CRPC cells in vivo when compared to treatment with CAR NK-92 cells or combined with anti-PD-1 mAb in the absence or presence of cocultured CD8+ T cells. CONCLUSION: Combined treatment with CAR NK-92 and anti-PD-L1 mAb improved the antitumour efficacy against CRPC, which is of extraordinary translational value in the clinical treatment of CRPC.

The establishment of polypeptide PSMA-targeted chimeric antigen receptor-engineered natural killer cells for castration-resistant prostate cancer and the induction of ferroptosis-related cell death.

BACKGROUND: The mortality of castration-resistant prostate cancer (CRPC) is high due to lack of an effective treatment. Chimeric antigen receptor (CAR)-based therapy is a promising immunotherapeutic strategy. Here, we aimed to design a novel CAR-natural killer (NK) cells with a clinically significant tumoricidal effect on CRPC. METHODS: We constructed novel CAR-NK92MI cells with a CD244-based recombinant lentiviral vector. Different intracellular segments (CD244, NKG2D, or CD3ζ) were screened to identify the best candidate according to cell lysis assay and CD107a expression levels. To enhance the affinity of the CAR to the tumor antigen, we compared an antibody specific for prostate-specific membrane antigen (anti-PSMA) with PSMA-targeted polypeptide (p-PSMA), which was screened by phage display combinatorial library. Then, CAR-NK92MI cells with both a high affinity for PSMA and a strong tumoricidal capacity were generated. In addition, we verified their tumor-killing effect in vitro and in vivo. The release of cytokine by NK92MI cells was compared with that by CAR-NK92MI cells through flow cytometry and enzyme-linked immunosorbent assay. Moreover, ferroptosis-related cell death was explored as a possible underlying mechanism. RESULTS: Three different CAR intracellular regions CAR1 (CD244), CAR2 (CD244, NKG2D) and CAR3 (CD244, NKG2D, and CD3ζ) were constructed. CAR2 was chosen to confer a stronger tumoricidal ability on CAR-NK92MI cells. Compared with anti-PSMA, p-PSMA exhibited enhanced affinity for the tumor antigen. Thus, p-PSMA-CAR-NK92MI cells, which expressed CAR with a polypeptide-based antigen-binding region, an intracellular CD244 and a NKG2D costimulatory domain, were generated. They could selectively and successfully kill PSMA+ target cells and exhibited specific lysis rate of 73.19% for PSMA-positive C4-2 cells and 33.04% for PSMA-negative PC3 cells. Additionally, p-PSMA-CAR-NK92MI cells had significantly higher concentrations of IFN-γ, TNF-α and granzyme B than NK92MI cells. In a CRPC cancer xenograft model, p-PSMA-CAR-NK92MI cells significantly inhibited tumor growth and exerted a more consistent killing effect than NK92MI cells. Moreover, ferroptosis is a potential mechanism through which CAR-NK92MI cells attack cancer cells, and is triggered by IFN-γ. CONCLUSIONS: p-PSMA-CAR-NK92MI cells can effectively kill CRPCPSMA+ cells in vitro and in vivo. This strategy may provide additional treatment options for patients with CRPC.

Role of the epithelial sodium channel in salt-sensitive hypertension.

The epithelial sodium channel (ENaC) is a heteromeric channel composed of three similar but distinct subunits, α, ß and γ. This channel is an end-effector in the rennin-angiotensin-aldosterone system and resides in the apical plasma membrane of the renal cortical collecting ducts, where reabsorption of Na(+) through ENaC is the final renal adjustment step for Na(+) balance. Because of its regulation and function, the ENaC plays a critical role in modulating the homeostasis of Na(+) and thus chronic blood pressure. The development of most forms of hypertension requires an increase in Na(+) and water retention. The role of ENaC in developing high blood pressure is exemplified in the gain-of-function mutations in ENaC that cause Liddle's syndrome, a severe but rare form of inheritable hypertension. The evidence obtained from studies using animal models and in human patients indicates that improper Na(+) retention by the kidney elevates blood pressure and induces salt-sensitive hypertension.

[Expression of heme oxygenase enzyme in the testis tissue and azoospermia].

OBJECTIVE: To investigate the location of heme oxygenase (HO) enzyme in the human testis, and explore the correlation of the expression of HO enzyme with azoospermia by analyzing its different expression levels in the testes of nonobstructive azoospermia, obstructive azoospermia and normal men. METHODS: We detected the location of the cells expressing HO enzyme in the human testis tissue using immunohistochemistry, determined the mRNA and protein expression levels of HO-1 and HO-2 in the testes of azoospermia patients and normal healthy men by RT-fluorescence quantitative PCR (RT-FQ-PCR) and Western blot, and explored the correlation of HO expressions with the pathogenesis of azoospermia. RESULTS: HO-1 enzyme was expressed mainly in the Sertoli cells and HO-2 enzyme chiefly in the germ cells of the testis tissue. RT-FQ-PCR showed that the expression of HO-1 in the testis tissue was significantly lower in the nonobstructive azoospermia than in the normal and obstructive azoospermia groups (P < 0.05), with no significant difference between the latter two. Western blot revealed no obvious difference between the expression level of HO-1 protein and that of HO-1 mRNA. There were no differences in the expression level of HO-2 protein among the three groups. CONCLUSION: The expression level of HO enzyme is significantly decreased in the testis tissue of nonobstructive azoospermia patients, and the expression of HO-1 protein is consistent with that of HO-1 mRNA. As HO-1 protects the testis tissue against various stress injuries through its antioxidant, anti-inflammatory and anti-apoptotic effects, its decreased expression level may be correlated with spermatogenic dysfunction, and therefore considered as a possible mechanism of nonobstructive azoospermia.