TRPC6 Expression Research Articles

Expression of pH-Sensitive TRPC4 in Common Skin Tumors.

TRPCs (transient receptor potential classical or cation channels) play a crucial role in tumor biology, especially in the Ca2+ homeostasis in cancer cells. TRPC4 is a pH-sensitive member of this family of proteins. As solid tumors exhibit an inversed pH-gradient with lowered extracellular and increased intracellular pH, both contributing to tumor progression, TRPC4 might be a signaling molecule in the altered tumor microenvironment. This is the first study to investigate the expression profiles of TRPC4 in common skin cancers such as basal cell carcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma (MM) and nevus cell nevi (NCN). We found that all SCCs, NCNs, and MMs show positive TRPC4-expression, while BCCs do only in about half of the analyzed samples. These data render TRPC4 an immunohistochemical marker to distinguish SCC and BCC, and this also gives rise to future studies investigating the role of TRPC4 in tumor progression, and especially metastasis as BCCs very rarely spread and are mostly negative for TRPC4.

The Beneficial Effect of the Blockade of Stim-Activated TRPC-ORAI Channels on Vascular Remodeling and Pulmonary Hypertension Induced byIntermittent Hypoxia Is Independent of Oxidative Stress.

Obstructive sleep apnea (OSA), a sleep breathing disorder featured by chronic intermittent hypoxia (CIH), is associated with pulmonary hypertension (PH). Rats exposed to CIH develop systemic and lung oxidative stress, pulmonary vascular remodeling, and PH and overexpress Stim-activated TRPC-ORAI channels (STOC) in the lung. Previously, we demonstrated that 2-aminoethyl-diphenylborinate (2-APB)-treatment, a STOC-blocker, prevents PH and the overexpression of STOC induced by CIH. However, 2-APB did not prevent systemic and pulmonary oxidative stress. Accordingly, we hypothesize that the contribution of STOC in the development of PH induced by CIH is independent of oxidative stress. We measured the correlation between right ventricular systolic pressure (RVSP) and lung malondialdehyde (MDA) with the gene expression of STOC and morphological parameters in the lung from control, CIH-treated, and 2-APB-treated rats. We found correlations between RVSP and increased medial layer and STOC pulmonary levels. 2-APB-treated rats showed a correlation between RVSP and the medial layer thickness, α-actin-ir, and STOC, whereas RVSP did not correlate with MDA levels in CIH and 2-APB-treated rats. CIH rats showed correlations between lung MDA levels and the gene expression of TRPC1 and TRPC4. These results suggest that STOC channels play a key role in developing CIH-induced PH that is independent from lung oxidative stress.

S-18-3: SGLT2 INHIBITOR CANAGLIFLOZIN ANTAGONIZES SALT-SENSITIVE HYPERTENSION THROUGH MODIFYING TRPC3 MEDIATED VASCULAR CALCIUM HANDLING

Objectiv: Salt-sensitive hypertension is highly prevalent and associated with cardiorenal damage. Large clinical trials have demonstrated that Sodium-glucose cotransporter 2 (SGLT2) inhibitors exert hypotensive effect and cardiorenal protective benefits in hypertensive patients with and without diabetes. However, the underlying mechanism remains elusive. Methods: Dahl salt-sensitive (SS) rats and salt-insensitive controls were fed with 8% high salt diet to establish salt-sensitive hypertensive model, some of them were treated with canagliflozin. The blood pressure, urinary sodium excretion, vascular function and remodeling were detected. Rat aortic proteomics and TRPC3 knockout mice were used to explain the mechanism of the effect of canagliflozin on salt-sensitive hypertension. Results: Canagliflozin treatment significantly reduced high salt induced hypertension and this effect was not totally dependent on urinary sodium excretion in SS hypertensive rats. Assay of vascular function and proteomics showed that canagliflozin significantly inhibited vascular cytoplasmic calcium increase and vasoconstriction in response to high salt diet. High salt intake increased vascular expression of TRPC3 in salt-sensitive rats, which could be alleviated by canagliflozin treatment. Overexpression of TRPC3 mimicked salt-induced vascular cytosolic calcium increase in vitro and knockout of TRPC3 erased the anti-hypertensive effect of canagliflozin. Mechanistically, high-salt-induced activation of sodium/calcium exchanger 1 (NCX1) reverse mode increased cytoplasmic calcium level and vasoconstriction, which required TRPC3, and this process could be blocked by canagliflozin. Conclusions: We define a previously unrecognized role of TRPC3/NCX1 mediated vascular calcium dysfunction in the development of high salt induced hypertension, which can be improved by canagliflozin treatment. This pathway is potentially a novel therapeutic target to antagonize salt sensitive hypertension.

METTL3 Promotes Endothelium-Mesenchymal Transition of Pulmonary Artery Endothelial Cells by Regulating TRPC6/Calcineurin/NFAT Signaling Pathways.

Endothelium-mesenchymal transition (EndMT) is a process of phenotypic and functional transition from activated endothelial cells to mesenchymal cells. Recently, EndMT has been proved to be one of the main pathological mechanisms of pulmonary artery hypertension (PAH). However, the molecular mechanism is not clear. Primary rat pulmonary arterial endothelial cells (rPAECs) were isolated from Sprague-Dawley rats and verified by CD31 immunofluorescence staining. rPAECs were exposed to hypoxic conditions to induce EndMT. RNA and protein levels in cells were detected by RT-qPCR and Western blot. The migration ability was verified by the transwell assay. The RIP experiment was used to test the m6A modification of TRPC6 mRNA and the binding relationship between TRPC6 and METTL3. Calcineurin/NFAT signaling was measured by using commercial kits. METTL3 was found to be highly expressed by hypoxia treatment in a time-dependent manner. Knockdown of METTL3 significantly suppressed cell migration, downregulated the levels of interstitial cell-related markers like α-SMA and vimentin, and increased the levels of endothelial cell markers including CD31 and VE-cadherin. Mechanistically, METTL3 increased TRPC6 expression by enhancing the m6A modification of TRPC6 mRNA, thus activating calcineurin/NFAT signaling. Our experiments showed that METTL3 silencing mediated the inhibitory roles in the hypoxia-mediated EndMT process, which were significantly reversed by TRPC6/calcineurin/NFAT signaling activation. Our results elucidated that METTL3 knockdown inhibited the hypoxia-mediated EndMT process by inactivating TRPC6/calcineurin/NFAT signaling.

Ginsenoside Rg1 treatment protects against cognitive dysfunction via inhibiting PLC–CN–NFAT1 signaling in T2DM mice

BackgroundAs a complication of Type II Diabetes Mellitus (T2DM), the etiology, pathogenesis, and treatment of cognitive dysfunction are still undefined. Recent studies demonstrated that Ginsenoside Rg1 (Rg1) has promising neuroprotective properties, but the effect and mechanism in diabetes-associated cognitive dysfunction (DACD) deserve further investigation. MethodsAfter establishing the T2DM model with a high-fat diet and STZ intraperitoneal injection, Rg1 was given for 8 weeks. The behavior alterations and neuronal lesions were judged using the open field test (OFT) and Morris water maze (MWM), as well as HE and Nissl staining. The protein or mRNA changes of NOX2, p-PLC, TRPC6, CN, NFAT1, APP, BACE1, NCSTN, and Aβ1-42 were investigated by immunoblot, immunofluorescence or qPCR. Commercial kits were used to evaluate the levels of IP3, DAG, and calcium ion (Ca2+) in brain tissues. ResultsRg1 therapy improved memory impairment and neuronal injury, decreased ROS, IP3, and DAG levels to revert Ca2+ overload, downregulated the expressions of p-PLC, TRPC6, CN, and NFAT1 nuclear translocation, and alleviated Aβ deposition in T2DM mice. In addition, Rg1 therapy elevated the expression of PSD95 and SYN in T2DM mice, which in turn improved synaptic dysfunction. ConclusionsRg1 therapy may improve neuronal injury and DACD via mediating PLC–CN–NFAT1 signal pathway to reduce Aβ generation in T2DM mice.

ANGPTL4 promotes nephrotic syndrome by downregulating podocyte expression of ACTN4 and podocin

Backgroundlipopolysaccharide (LPS) can induce nephrotic syndrome-like features such as massive proteinuria, hyperlipidemia, and fusion of glomerular podocytes with foot processes (FPs) in mice. Angiopoietin-like protein 4 (ANGPTL4) neutralized the negative charge of glomerular basement membrane charge and aggravated renal injury. The mechanism of ANGPTL4 aggravating podocyte injury has not been well clarified. In this study, we aimed to investigate the potential role of ANGPTL4 on podocyte FPs fusion and podocyte signal molecules. MethodsWe built angptl4 gene knocked out in C57BL6 mice using CRISPR/Cas9 technique. Nephrotic model was built by LPS in wild type and angptl4−/− mice. Expression of ACTN4, podocin and TRPC6 in the glomerulus were determined by immunohistochemistry. ResultsIn physical condition, the wild type and angptl4−/− mice showed no significant differences in biochemical indicators and kidney pathology. But in nephrotic condition, compared with wild type mice hyperlipidemia and proteinuria with the angptl4−/− mice was significantly relieved. Moreover, the degree of FPs fusion was notably improved in the nephrotic mice knocked out angptl4 gene. Expression of ACTN4 and podocin decreased drastically in the glomerulus of wild-type nephrotic mice. Different from wild-type, the ACTN4 and podocin expression showed slight weakening in angptl4−/− nephrotic mice. As transient receptor potential cation channel subfamily member, TRPC6 expression had no visible change in glomerulus of each group. ConclusionsANGPTL4 induces hyperlipidemia and podocyte injury in nephrotic mice, thereby promoting the formation of proteinuria. Its molecular mechanism may be related to ANGPTL4 down-regulating actin cytoskeletal regulatory signals ACTN4 and podocin.

Metformin prevents hypoxia-induced podocyte injury by regulating the ZEB2/TG2 axis.

Podocytes, a vital component of the glomerular filtration barrier, are vulnerable to various noxious stimuli, including Hypoxic. HIF1α that transduces hypoxic adaptations induces Transglutaminase 2 (TG2), which catalyses cross-linking of extracellular matrix proteins. In this study, we investigated the mechanism of regulation of TG2 by HIF1α. HIF1α was induced in podocytes by treating with FG4592 (Roxadustat) or hypoxia (1% oxygen) and in mice by treating with FG4592. Gene expression and protein analysis of ZEB2, TRPC6 and TG2 were performed in both experimental models. Histological and kidney function analyses were performed in mice. Data mining revealed co-expression of HIF1α, ZEB2, TRPC6 and TG2 in the chronic kidney diseases (CKD)-validated dataset. We observed elevated expression of ZEB2, TRPC6 and TG2 in FG4592-treated podocytes. Ectopic expression of ZEB2 resulted in high TRPC6 expression, elevated intracellular calcium levels and increased TG2 activity. Blocking the TRPC6 channel or inhibiting its expression partially attenuated FG4592-induced TG2 activity, whereas suppression of ZEB2 expression significantly abolished TG2 activity. Furthermore, we noticed the induction of the ZEB2/TRPC6/TG2 axis in podocytes in mice administered with FG-4592. Metformin ameliorated the HIF1α-induced podocyte injury and proteinuria in mice administered with FG-4592. This study demonstrates that HIF1α stimulates both TG2 expression and activity via ZEB2/TRPC6 axis, whereas abrogation of HIF1α by metformin prevented hypoxia-induced glomerular injury. Metformin could be explored to treat proteinuric diseases such as CKD, sleep apnea and renal Ischemia-reperfusion-injury, where hypoxia is considered a risk factor.

Tetrandrine Attenuates Podocyte Injury by Inhibiting TRPC6-Mediated RhoA/ROCK1 Pathway.

Tetrandrine (Tet), a compound found in a traditional Chinese medicine, presents the protective effect for kidney function. Our study is aimed at clarifying the efficacy and underlying mechanism of Tet on podocyte injury. In this study, podocyte injury was induced in rats with adriamycin (ADR), and MPC5 podocytes were constructed with TRPC6 overexpression. We found that Tet treatment reduced the levels of proteinuria, serum creatinine, and blood urea nitrogen and increased plasma albumin levels in ADR-induced rats. Tet reduced intracellular Ca2+ influx and apoptosis in MPC5 podocytes overexpressing TRPC6. Tet downregulated the expression of renal TRPC6, RhoA, and ROCK1 and upregulated the expression of synaptopodin; meanwhile, it reduced calcineurin activity in vivo and in vitro. In conclusion, Tet protects against podocyte by affecting TRPC6 and its downstream RhoA/ROCK1 signaling pathway.

Trpc5-regulated AMPKα/mTOR autophagy pathway is associated with glucose metabolism disorders in low birth weight mice under overnutrition

Previous studies have shown that low birth weight (LBW) individuals are at higher risk of glucose metabolism disorders compared with normal birth weight (NBW) individuals under overnutrition conditions, but the mechanism remains unclear. To explore the underlying mechanism of glucose metabolism disorders induced by LBW under overnutrition in adulthood, the prenatal malnutrition method was applied to ICR mice to establish the LBW mice model and high-fat diets were used to mimic overnutrition conditions. Then the mechanism was further explored on Hepg2 cells treated with nutritional deprivation plus palmitic acid. The results showed that LBW plus high-fat interventions will cause glucose metabolism disorders and inhibit autophagy flux in adulthood. Moreover, the expression of TRPC5-regulated AMPK/mTOR autophagy pathway was downregulated by LBW with high-fat interventions. Collectively, LBW plus high-fat intervention increased the risk of glucose metabolism disorders, which may be related to the alteration of TRPC5 expression level and its regulation of the AMPKα/mTOR autophagy pathway. This study may provide a fundamental basis for the molecular mechanism of glucose metabolism disorders induced by LBW with high-fat diets in adulthood and a new target for the treatment of metabolic diseases in LBW individuals.

Role of TRPC6 in periodontal tissue reconstruction mediated by appropriate stress

IntroductionThe basis of orthodontic tooth movement (OTM) is the reconstruction of periodontal tissue under stress. Increasing the speed of OTM has always been the focus of attention.ObjectivesPeriodontal ligament stem cells (PDLSCs) are direct effector cells of mechanical force, but the mechanism by which PDLSCs sense mechanical stimuli is unclear.MethodsHuman PDLSCs (hPDLSCs) were analyzed in the presence or absence of force loading with the Flexcell loading system in vitro. Then, periodontal tissues were analyzed after mechanical stimulation in vivo. In addition, cells in a confined microenvironment were analyzed to observe changes in the cytoskeleton and migration. Finally, TRPC6−/− mice were used to further verify the effect of TRPC6. After force application, the OTM distance, bone marrow density (BMD), TRPC6 and COL1 expression, and TRAP staining were evaluated in periodontal tissues.ResultsRNA sequencing (RNA-seq) and western blot analyses revealed that TRPC6 was important during mechanical force application to hPDLSCs. Appropriate mechanical force application also induced TRPC6 activation in the OTM model and the confined microenvironment. Under a slightly confined microenvironment, treatment with the TRPC6 inhibitor SKF96365 and TRPC6 knockout decreased the migration speed of hPDLSCs and mouse bone marrow mesenchymal stem cells (mBMSCs). In addition, TRPC6−/− mice showed lower OTM distances and reduced osteogenic and osteoclastic differentiation.ConclusionIn summary, TRPC6 activation in PDLSCs mediated by appropriate mechanical force application contributes to periodontal tissue reconstruction.Graphical abstractPDLSCs modulate periodontal tissue remodeling under appropriate mechanical stimulation through TRPC6; however, under excessive stress, alveolar bone and tooth roots are readily absorbed. Under this condition, environmental factors play a leading role, and the regulatory effect of TRPC6 is not obvious.

Mesenchymal stem cells derived from adipose accelerate the progression of colon cancer by inducing a MT-CAFs phenotype via TRPC3/NF-KB axis

BackgroundThere is increasing evidence that mesenchymal stem cells (MSCs) help shape the tumor microenvironment and promote tumor progression, and ion channels might play a critical role in this process. The objective of the present study was to explore the function and mechanism of MT-CAFs on progression of colon cancer.MethodsHere, a gene chip was used for a general analysis of gene expression changes in MSC-transformed CAF cells (MT-CAFs). Bioinformatic tool and western blot screened out the ion channel protein TRPC3 with significantly increased expression, and identify the function through two-photon microscope. The progression of cancer was detected via MTS, transwell and Wound Healing. ELISA deected the secretion of inflammation factors. TRPC3/NF-KB axis was identified by western blot and immunofluorescence.ResultsTRPC3 can caused calcium influx, which further activated the NF-KB signaling pathway. Knockdown or inhibition of TRPC3 in MSCs significantly reduced the activation of NF-KB, and decreased the growth, migration, and invasion of MT-CAFs. After TRPC3 knockdown, the ability of MT- CAFs to promote tumor migration and invasion was impaired. Conversely, the upregulation of TRPC3 expression in MT-CAFs had the opposite effect. In vivo, TRPC3 expressed on MSCs also contributed to the tumorigenesis and progression of cancer cells. In addition, the Oncomine and GEPIA databases showed that TRPC3 expression is higher in colon cancer tissues compared with normal colon tissues, and was positively correlated with the expression of the CAF genes alpha-smooth muscle (α-SMA/ACTA2) and fibroblast activation protein Alpha. The disease-free survival of patients with positive TRPC3 expression in MSCs was significantly shorter than those with negative expression.ConclusionsThese results indicate that TRPC3 expressed on MT-CAFs plays a critical role in tumor progression via the NF-KB signaling pathway, and is correlated with poor prognosis in colon cancer patients. Therefore, TRPC3 may be a novel therapeutic target for the treatment of colon cancer.

Effect of low androgen levels on transient receptor potential channels expression in rat penile corpus cavernosum tissue and its relationship with erectile function.

The exact mechanism by which testosterone deficiency causes ED has not yet been elucidated. TRPC is involved in the process of smooth muscle cell contraction and relaxation. The effect of androgens on TRPCs and their relationship with erectile function are currently unclear. Thirty male SD rats were randomly divided into six groups: control group, castration group, castration + testosterone (T) group (cast + T), control + transfection group (control + trans), control + empty transfection group and castration + transfection group (cast + trans). The transfection group rats were given with lentivirus (1 × 108 TU/mL, 15 μl) carrying the siRNA targeting TRPC4 gene in the rat penile cavernous tissue at 4 weeks after castration. The tests were performed at 5 weeks after castration. Comparing the cast group with the control, the ICPmax/MAP, p-eNOS/eNOS and NO levels in the rat penile tissue were significantly lower (p < 0.01) and the level of TRPC3, TRPC4 and TRPC6 in the rat penile tissue was significantly increased (p < 0.01). When the cast + trans group was compared to the cast group, ICPmax/MAP was markedly higher (p < 0.05), and the level of the TRPC4 was remarkably lower (p < 0.05). Low androgen levels might inhibit an erectile function through up-regulation of the expression of TRPC3, TRPC4 and TRPC6 in rat penile cavernous tissue. Inhibition the level of TRPC4 in rat penile tissue may improve the erectile function in low androgen levels.

DOT1L Methyltransferase Regulates Calcium Influx in Erythroid Progenitor Cells in Response to Erythropoietin.

Erythropoietin (EPO) signaling plays a vital role in erythropoiesis by regulating proliferation and lineage-specific differentiation of murine hematopoietic progenitor cells (HPCs). An important downstream response of EPO signaling is calcium (Ca2+) influx, which is regulated by transient receptor potential channel (TRPC) proteins, particularly TRPC2 and TRPC6. While EPO induces Ca2+ influx through TRPC2, TRPC6 inhibits the function of TRPC2. Thus, interactions between TRPC2 and TRPC6 regulate the rate of Ca2+ influx in EPO-induced erythropoiesis. In this study, we observed that the expression of TRPC6 in KIT-positive erythroid progenitor cells was regulated by DOT1L. DOT1L is a methyltransferase that plays an important role in many biological processes during embryonic development including early erythropoiesis. We previously reported that Dot1l knockout (Dot1lKO) HPCs in the yolk sac failed to develop properly, which resulted in lethal anemia. In this study, we detected a marked downregulation of Trpc6 gene expression in Dot1lKO progenitor cells in the yolk sac compared to the wild type (WT). The promoter and the proximal regions of the Trpc6 gene locus exhibited an enrichment of H3K79 methylation, which is mediated solely by DOT1L. However, the expression of Trpc2, the positive regulator of Ca2+ influx, remained unchanged, resulting in an increased TRPC2/TRPC6 ratio. As the loss of DOT1L decreased TRPC6, which inhibited Ca2+ influx by TRPC2, Dot1lKO HPCs in the yolk sac exhibited accelerated and sustained elevated levels of Ca2+ influx. Such heightened Ca2+ levels might have detrimental effects on the growth and proliferation of HPCs in response to EPO.

Protein detection and localization of the non-selective cation channel TRPC6 in the human heart

Due to longer lifespans in societies in industrialized countries, cardiovascular diseases are becoming increasingly important for medical research. It has already been shown that the cell membrane-bound, non-selective TRPC6 ion channel is important in the pathogenesis of heart diseases. Among other things, it is permeable to calcium ion, which plays a critical role in cardiac contraction and relaxation. The TRPC6 ion channel is a promising therapeutic target in the treatment of cardiovascular diseases. A deeper understanding of the physiological and pathophysiological role as well as the localization of TRPC6 in human cardiac tissue is the basis for new drug development. Although the TRPC6 channel has been detected in animal studies, at the mRNA level in humans, and sparse TRPC6 protein has been detected in humans, there are no systematic studies of TRPC6 protein detection in the human heart. For the first time, TRPC6 ion channel protein was detected histologically in human heart tissue from body donors in different structures, localizations, and histological layers - particularly in cardiomyocytes and intramuscular arterioles - by immunohistochemistry, just as TRPC6 expression has already been shown in animal models of the heart by other research groups. In the sense of the translational concept, this indicates a possible transferability of research results from animal models to humans.

TRPC6 Is Found in Distinct Compartments of the Human Kidney

In the context of renal proteinuric diseases, TRPC6 has been shown to play an important role in ultrafiltration associated with the slit diaphragm through the control of the intracellular Ca2+ concentration in the podocytes of glomeruli. However, to date, the properties of TRPC6 have been studied mainly in cell lines or in animal models. Therefore, the aim of the study presented here was to investigate the presence and distribution of TRPC6 in human kidneys in order to possibly verify the applicability of the results previously obtained in nonhuman experiments. For this purpose, kidneys from nine cadavers were prepared for immunohistochemical staining and were supplemented with a fresh human kidney obtained by nephrectomy. TRPC6 was detected in glomeruli and in the parietal epithelial cells of Bowman’s capsule. Larger amounts were detected in the tubular system and collecting ducts. In contrast to the peritubular capillary bed, which showed no immune reaction, the cortical resistance vessels showed mild TRPC6 staining. In conclusion, our studies on the expression of TRPC6 in human kidney tissue support the translational concept of the involvement of TRPC6 in various renal diseases and reveal new aspects of the distribution of TRPC6 in the human kidney.

Klotho Ameliorates Podocyte Injury through Targeting TRPC6 Channel in Diabetic Nephropathy.

Podocyte damage is vital for the etiopathogenesis of diabetic nephropathy (DN). Klotho (KL), a multifunctional protein, has been demonstrated to have renoprotective effects; nevertheless, the mechanism for protective effect has not been completely elucidated. Transient receptor potential cation channel subfamily C, member 6 (TRPC6), a potential target of KL, is implicated in glomerular pathophysiology. Here, we sought to determine whether KL could protect against podocyte injury through inhibiting TRPC6 in DN. We found that high glucose (HG) triggered podocyte injury as manifested by actin cytoskeleton damage along with the downregulation of KL and Synaptopodin and the upregulation of TRPC6. KL overexpression reversed HG-induced podocytes injury, whereas cotreatment with TRPC6 activator flufenamic acid (FFA) significantly abrogated the beneficial effects conferred by KL. Moreover, KL knockdown in podocytes resulted in actin cytoskeleton impairment, decreased Synaptopodin expression, and increased TRPC6 expression. In db/db mice, KL overexpression inhibited TRPC6 expression and attenuated diabetes-induced podocyte injury, which was accompanied by decreased albuminuria and ameliorated glomerulosclerosis. Our data provided novel mechanistic insights for KL against DN and highlighted TRPC6 as a new target for KL in podocytes to prevent DN.

The beneficial effect of chronic muscular exercise on muscle fragility is increased by Prox1 gene transfer in dystrophic mdx muscle.

Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated. Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice. Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation.

Sertoli cell transplantation attenuates microglial activation and inhibits TRPC6 expression in neuropathic pain induced by spinal cord injury

BackgroundCell therapy is a promising treatment method for relieving neuropathic pain caused by spinal cord injuries (SCI). Sertoli cells (SCs) are an attractive choice given their demonstrated secretion of growth factors and immunosuppressant effect. This study mechanistically characterizes the analgesic effect of SCs transplantation. MethodsThe clip compression SCI model was carried out on the T12-T13 level in male Wistar rats. One-week post-SCI, SCs were transplanted into the site of injury. Animals underwent Basso, Beattie, and Bresnahan locomotor scoring, mechanical allodynia, and thermal hyperalgesia on a weekly basis for a duration of six weeks. Histological examination of the spinal cord and molecular evaluation of Iba-1, P2Y4, TRPC6, and P-mTOR were performed. SCs survival, measured by anti-Müllerian hormone expression in the spinal cord. ResultsAnimals that received SCs transplantation showed improvement in motor function recovery and pain relief. Furthermore, a cavity was significantly decreased in the transplanted animals (p = 0.0024), the expression level of TRPC6 and caspase3 and the number of activated microglia decreased compared to the SCI animals, and p-mTOR and P2Y4R expression remarkably increased compared to the SCI group. ConclusionSCs transplantation produces an analgesic effect which may represent a promising treatment for SCI-induced chronic pain.

Crucial Role of Stromal Interaction Molecule-Activated TRPC-ORAI Channels in Vascular Remodeling and Pulmonary Hypertension Induced by Intermittent Hypoxia.

Obstructive sleep apnea (OSA), a sleep breathing disorder featured by chronic intermittent hypoxia (CIH), is associate with pulmonary hypertension. Rats exposed to CIH develop lung vascular remodeling and pulmonary hypertension, which paralleled the upregulation of stromal interaction molecule (STIM)-activated TRPC-ORAI Ca2+ channels (STOC) in the lung, suggesting that STOC participate in the pulmonary vascular alterations. Accordingly, to evaluate the role played by STOC in pulmonary hypertension we studied whether the STOC blocker 2-aminoethoxydiphenyl borate (2-APB) may prevent the vascular remodeling and the pulmonary hypertension induced by CIH in a rat model of OSA. We assessed the effects of 2-APB on right ventricular systolic pressure (RVSP), pulmonary vascular remodeling, α-actin and proliferation marker Ki-67 levels in pulmonary arterial smooth muscle cells (PASMC), mRNA levels of STOC subunits, and systemic and pulmonary oxidative stress (TBARS) in male Sprague-Dawley (200 g) rats exposed to CIH (5% O2, 12 times/h for 8h) for 28 days. At 14 days of CIH, osmotic pumps containing 2-APB (10 mg/kg/day) or its vehicle were implanted and rats were kept for 2 more weeks in CIH. Exposure to CIH for 28 days raised RVSP > 35 mm Hg, increased the medial layer thickness and the levels of α-actin and Ki-67 in PASMC, and increased the gene expression of TRPC1, TRPC4, TRPC6 and ORAI1 subunits. Treatment with 2-APB prevented the raise in RVSP and the increment of the medial layer thickness, as well as the increased levels of α-actin and Ki-67 in PASMC, and the increased gene expression of STOC subunits. In addition, 2-APB did not reduced the lung and systemic oxidative stress, suggesting that the effects of 2-APB on vascular remodeling and pulmonary hypertension are independent on the reduction of the oxidative stress. Thus, our results supported that STIM-activated TRPC-ORAI Ca2+ channels contributes to the lung vascular remodeling and pulmonary hypertension induced by CIH.

Tanreqing Injection Regulates Cell Function of Hypoxia-Induced Human Pulmonary Artery Smooth Muscle Cells (HPASMCs) through TRPC1/CX3CL1 Signaling Pathway.

Hypoxia-induced pulmonary arterial hypertension (HPAH) is due to hypoxia caused by vascular endothelial cell remolding and damage. Previous studies have suggested that CX3CL1 plays an important role in HPAH which is affected by oxidative stress. Ca2+ channel activation correlated with increasing NF-κB levels induced by ROS. Tanreqing injection (TRQ) is a traditional Chinese medicine (TCM) for acute upper respiratory tract infection and acute pneumonia. In the present study, we explored the effect of TRQ on human pulmonary artery smooth muscle cells (HPASMCs) undergoing hypoxia and feasible molecular mechanisms involved in. Cell proliferation was assayed using CCK8 kits. Immunofluorescence and western blotting along with ELISA assay were performed to investigate the effect of TRQ on hypoxia-induced ROS, Ca2+, hydroxyl free radicals, and the expression of Ca2+ channel protein TRPC1, CX3CR1, HIF-1α, NF-κBp65, and p-NF-κBp65 in HPASMCs. Human CX3CL1 and the inhibitor of TRPC1 as SKF96365 were used for further investigation. TRQ inhibited hypoxia-induced increasing cell adhesion, ROS, Ca2+, hydroxyl free radicals, CX3CR1, HIF-1α, NF-κBp65 activation, and even on TRPC1 expression in HPASMC which tended to be attenuated even reversed by CX3CL1. Our results suggested that TRQ might help to attenuate remodeling of HPASMC through inhibiting the ROS and TRPC1/CX3CL1 signaling pathway.