Endotoxin Induces Fibrosis in Vascular Endothelial Cells through a Mechanism Dependent on Transient Receptor Protein Melastatin 7 Activity

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial­to­mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca²⁺ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca²⁺ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca²⁺ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.     

Ubiquitin-specific protease 2 regulates Ang Ⅱ–induced cardiac fibroblasts activation by up-regulating cyclin D1 and stabilizing β-catenin in vitro

Cardiac fibrosis, featuring abnormally elevated extracellular matrix accumulation, decreases tissue compliance, impairs cardiac function and accelerates heart failure. Mounting evidence suggests that the ubiquitin proteasome pathway is involved in cardiac fibrosis. In the present study, ubiquitin-specific protease 2 (USP2) was identified as a novel therapeutic target in cardiac fibrosis. Indeed, USP2 expression was increased in angiotensin II–induced primary cardiac fibroblasts (CFs) from neonatal rats. In addition, USP2 inhibition suppressed CFs proliferation, collagen synthesis and cell cycle progression. Furthermore, USP2 interacted with β-catenin, thereby regulating its deubiquitination and stabilization in CFs. To sum up, these findings revealed that USP2 has a therapeutic potential for the treatment of cardiac fibrosis.     

Assessment of TRPM7 functions by drug-like small molecules

Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a plasma membrane ion channel linked to a cytosolic protein kinase domain. Genetic inactivation of this bi-functional protein revealed its crucial role in Ca²⁺ signalling, Mg²⁺ metabolism, immune responses, cell motility, proliferation and differentiation. Malfunctions of TRPM7 are associated with anoxic neuronal death, cardiac fibrosis, tumour progression and macrothrombocytopenia. Recently, several groups have identified small organic compounds acting as inhibitors or activators of the TRPM7 channel. In follow-up studies, the identified TRPM7 modulators were successfully used to uncover new cellular functions of TRPM7 in situ including a crucial role of TRPM7 in Ca²⁺ signaling and Ca²⁺ dependent cellular processes. Hence, TRPM7 has been defined as a promising drug target. Here, we summarize the progress in this quickly developing field.     

Differential mRNA Expression and Glucocorticoid-Mediated Regulation of TRPM6 and TRPM7 in the Heart and Kidney throughout Murine Pregnancy and Development

The transient receptor potential (TRP) channels TRPM6 and TRPM7 are critically involved in maintaining whole body and cellular Mg²⁺ homeostasis and ensuring the normal function of organs such as the heart and kidney. However, we do not know how the expression of TRPM6 and TPRM7 in these organs changes throughout fetal development and adult life, and whether this expression can be hormonally regulated. This study determined the ontogeny of TRPM6 and TRPM7 mRNA expression from mid-gestation through to adulthood in the mouse. In a second series of experiments, we examined how maternal administration of the glucocorticoids corticosterone and dexamethasone between embryonic days 12.5–15 affected TRPM6 and TRPM7 channel mRNA expression in the mother and fetus. Whilst renal TRPM7 expression was relatively constant throughout development, renal TRPM6 expression was markedly upregulated after birth. In contrast, cardiac TRPM7 expression was 2–4 fold higher in the fetus than in the adult. Surprisingly, TRPM6 expression was detected in the fetal heart (qPCR and in situ hybridization). Glucocorticoid administration during gestation increased fetal cardiac expression of both channels without affecting renal expression. In contrast, in the dam renal TRPM6 and TRPM7 expression was increased by glucocorticoids with no change in the cardiac channel expression. These data suggest that TRPM6 and TRPM7 channels are important in organogenesis, and that elevated maternal glucocorticoid levels can alter the expression of these channels. This suggests that perturbations in hormonal regulatory systems during pregnancy may adversely impact upon normal fetal development, at least in part by altering expression of TRPM channels.     

MicroRNA-24 regulates cardiac fibrosis after myocardial infarction

Cardiac fibrosis after myocardial infarction (MI) has been identified as a key factor in the development of heart failure. Although dysregulation of microRNA (miRNA) is involved in various pathophysiological processes in the heart, the role of miRNA in fibrosis regulation after MI is not clear. Previously we observed the correlation between fibrosis and the miR-24 expression in hypertrophic hearts, herein we assessed how miR-24 regulates fibrosis after MI. Using qRT-PCR, we showed that miR-24 was down-regulated in the MI heart; the change in miR-24 expression was closely related to extracellular matrix (ECM) remodelling. In vivo, miR-24 could improve heart function and attenuate fibrosis in the infarct border zone of the heart two weeks after MI through intramyocardial injection of Lentiviruses. Moreover, in vitro experiments suggested that up-regulation of miR-24 by synthetic miR-24 precursors could reduce fibrosis and also decrease the differentiation and migration of cardiac fibroblasts (CFs). TGF-β (a pathological mediator of fibrotic disease) increased miR-24 expression, overexpression of miR-24 reduced TGF-β secretion and Smad2/3 phosphorylation in CFs. By performing microarray analyses and bioinformatics analyses, we found furin to be a potential target for miR-24 in fibrosis (furin is a protease which controls latent TGF-β activation processing). Finally, we demonstrated that protein and mRNA levels of furin were regulated by miR-24 in CFs. These findings suggest that miR-24 has a critical role in CF function and cardiac fibrosis after MI through a furin-TGF-β pathway. Thus, miR-24 may be used as a target for treatment of MI and other fibrotic heart diseases.     

Cardiac Wnt5a and Wnt11 promote fibrosis by the crosstalk of FZD5 and EGFR signaling under pressure overload

Progressive cardiac fibrosis accelerates the development of heart failure. Here, we aimed to explore serum Wnt5a and Wnt11 levels in hypertension patients, the roles of Wnt5a and Wnt11 in cardiac fibrosis and potential mechanisms under pressure overload. The pressure overload mouse model was built by transverse aortic constriction (TAC). Cardiac fibrosis was analyzed by Masson’s staining. Serum Wnt5a or Wnt11 was elevated and associated with diastolic dysfunction in hypertension patients. TAC enhanced the expression and secretion of Wnt5a or Wnt11 from cardiomyocytes (CMs), cardiac fibroblasts (CFs), and cardiac microvascular endothelial cells (CMECs). Knockdown of Wnt5a and Wnt11 greatly improved cardiac fibrosis and function at 4 weeks after TAC. In vitro, shWnt5a or shWnt11 lentivirus transfection inhibited pro-fibrotic effects in CFs under mechanical stretch (MS). Similarly, conditional medium from stretched-CMs transfected with shWnt5a or shWnt11 lentivirus significantly suppressed the pro-fibrotic effects induced by conditional medium from stretched-CMs. These data suggested that CMs- or CFs-derived Wnt5a or Wnt11 showed a pro-fibrotic effect under pressure overload. In vitro, exogenous Wnt5a or Wnt11 activated ERK and p38 (fibrotic-related signaling) pathway, promoted the phosphorylation of EGFR, and increased the expression of Frizzled 5 (FZD5) in CFs. Inhibition or knockdown of EGFR greatly attenuated the increased FZD5, p-p38, and p-ERK levels, and the pro-fibrotic effect induced by Wnt5a or Wnt11 in CFs. Si-FZD5 transfection suppressed the increased p-EGFR level, and the fibrotic-related effects in CFs treated with Wnt5a or Wnt11. In conclusion, pressure overload enhances the secretion of Wnt5a or Wnt11 from CMs and CFs which promotes cardiac fibrosis by activation the crosstalk of FZD5 and EGFR. Thus, Wnt5a or Wnt11 may be a novel therapeutic target for the prevention of cardiac fibrosis under pressure overload.Subject terms: Heart failure, Translational research     

Matrix stiffness controls cardiac fibroblast activation through regulating YAP via AT1R

Cardiac fibrosis is a common pathway leading to heart failure and involves continued activation of cardiac fibroblasts (CFs) into myofibroblasts during myocardium damage, causing excessive deposition of the extracellular matrix (ECM) and thus increases matrix stiffness. Increasing evidence has shown that stiffened matrix plays an important role in promoting CF activation and cardiac fibrosis, and several signaling factors mediating CF mechanotransduction have been identified. However, the key molecules that perceive matrix stiffness to regulate CF activation remain to be further explored. Here, we detected significantly increased expression and nuclear localization of Yes-associated protein (YAP) in native fibrotic cardiac tissues. By using mechanically regulated in vitro cell culture models, we found that a stiff matrix-induced high expression and nuclear localization of YAP in CFs, accompanied by enhanced cell activation. We also demonstrated that YAP knockdown decreased fibrogenic response of CFs and that YAP overexpression promoted CF activation, indicating that YAP plays an important role in mediating matrix stiffness-induced CF activation. Further mechanistic studies revealed that the YAP pathway is an important signaling branch downstream of angiotensin II type 1 receptor in CF mechanotransduction. The findings help elucidate the mechanism of fibrotic mechanotransduction and may contribute to the development of new approaches for treating fibrotic diseases.     

LncRNA FAF inhibits fibrosis induced by angiotensinogen II via the TGFβ1-P-Smad2/3 signalling by targeting FGF9 in cardiac fibroblasts

The dysregulation of Long noncoding RNAs (lncRNAs) has been implicated in many cardiovascular diseases, including cardiac fibrosis. However, the functions and mechanisms of lncRNAs in cardiac fibroblasts (CFs) have not been fully elucidated. First, we observed a correlation between cardiac remodeling (CR) and lncRNA FAF (FGF9-associated factor, termed FAF) expression in the heart. In vitro, we found that the expression of lncRNA FAF was altered in CFs, whereas it behaved inconsistently in cardiomyocytes (CMs). Next, we investigated the effects of lncRNA FAF on angiotensinogen II (Ang II)-induced cardiac fibrosis in neonatal rat CFs and explored the mechanism underlying these effects. In this study, lncRNA FAF was enriched in CFs and was associated with cardiac fibrosis. Upregulation of lncRNA FAF significantly restrained Ang II-induced increases in cell proliferation, differentiation and collagen accumulation of CFs. Moreover, we found that the function of lncRNA FAF was mainly realized through Transforming growth factor β1 (TGFβ1) secretion and then downregulated phosphorylation of Smad2/3. Additional analysis revealed that Fibroblast growth factor 9 (FGF9) is a direct target of lncRNA FAF, as the overexpression of lncRNA FAF could increase the expression of FGF9 and knockdown of the FGF9 expression could attenuate the down-regulation of lncRNA FAF on TGFβ1-P-Smad2/3 pathway. Furthermore, knockdown of the FGF9 expression also abolished the inhibitory effect of FAF on fibrosis. In summary, we demonstrated that the overexpression of lncRNA FAF could inhibit fibrosis induced by Ang II via the TGFβ1-P-Smad2/3 signalling by targeting FGF9 in CFs.     

Poly(ADP-ribose) polymerase 1 induces cardiac fibrosis by mediating mammalian target of rapamycin activity

Cardiac fibrosis is involved in nearly all forms of heart diseases and is characterized by excessive deposition of extracellular matrix proteins by cardiac fibroblasts (CFs). We and others have reported the possibility of poly(ADP-ribose) polymerase 1 (PARP1), the founding subtype of the PARPs enzyme family, as a novel therapeutic target of heart diseases. The cardiac fibrotic induction of mammalian target of rapamycin (mTOR) is mainly due to collagen expression, Smad3- and p53/JNK-mediated apoptosis. However, the possible link between PARP1 and mTOR in the progression of cardiac fibrosis remains unclear. In this study, PARP1 protein expression, and the activity of mTOR and its three target substrates (p70 ribosomal S6 Kinase 1, eukaryotic initiation factor 4E­-binding protein 1, and UNC­51­like kinase 1) were augmented; meanwhile, the nicotinamide adenine dinucleotide (NAD) content was significantly reduced in the process of cardiac fibrosis in vivo and in vitro. Sprague-Dawley rats were intraperitoneally injected with 3-aminobenzamide (3AB) (20 mg/kg/d; a well-established PARP1 inhibitor) or rapamycin (Rapa; 1 mg/kg/d; used for mTOR inhibition) 7 days after abdominal aortic constriction (AAC) surgery for 6 weeks. Pretreatment of 3AB or Rapa both relieved AAC-caused cardiac fibrosis and heart dysfunction. Overexpression of PARP1 with adenovirus carrying PARP1 gene specifically transduced into the hearts via intramyocardial multipoint injection caused similar myocardial damage. In CFs, preincubation with PARP1 or mTOR inhibitors all blocked TGF-β1 induced cardiac fibrosis. PARP1 overexpression evoked cardiac fibrosis, which could be antagonized by mTOR inhibitors or NAD supplementation in CFs. These results provide novel and compelling evidence that PARP1 exacerbated cardiac fibrosis, which was partially attributed to NAD-dependent activation of mTOR.     

Serelaxin inhibits differentiation and fibrotic behaviors of cardiac fibroblasts by suppressing ALK-5/Smad2/3 signaling pathway

Serelaxin, a recombinant form of human relaxin-2, is currently regarded as a novel drug for treatment of acute heart failure. However, whether therapeutic effects of serelaxin are achieved by inhibiting cardiac fibrosis remains unclear. In this study, we investigate effects of serelaxin on inhibiting cardiac fibrosis. Cardiac fibroblasts (CFs) were isolated from the hearts of adult rats. Effects of serelaxin on differentiation of CFs towards myofibroblasts (MFs) and their fibrotic behaviors after induction with TGF-β1 were examined. Synthesis and degradation of collagens, secretion of IL-10, and expression of ALK-5 and p-Smad2/3 of TGF-β1-induced cells were assessed after treatment with serelaxin. Serelaxin inhibited differentiation of TGF-β1-induced CFs towards MFs, and reduced proliferation and migration of the induced cells. Moreover, serelaxin down-regulated expression of collagen I/III and TIMP-2, and up-regulated expression of MMP-2 and MMP-9 in the cells. After treatment with serelaxin, activity of MMP-2 and MMP-9 and secretion of IL-10 increased, expression of ALK-5 and the level of Smad2/3 phosphorylation was reduced significantly. These results suggest that serelaxin can inhibit differentiation of TGF-β1-induced CFs towards MFs, reduce production of collagens by suppressing ALK-5/Smad2/3 signaling pathway, and enhance extracellular matrix degradation by increasing MMP-2/TIMP-2 ratio and IL-10 secretion. Serelaxin may be a potential therapeutic drug for inhibiting cardiac fibrosis.     

MicroRNA‐503 promotes angiotensin II‐induced cardiac fibrosis by targeting Apelin‐13

Cardiac fibrosis is a major cause of heart failure. MicroRNAs (miRs) are important epigenetic regulators of cardiac function and cardiovascular diseases, including cardiac fibrosis. This study aimed to explore the role of miR‐503 and its mechanisms in regulating cardiac fibrosis. miR‐503 was found up‐regulated in the mouse LV tissues subjected to transverse aortic constriction (TAC) and in neonatal cardiac fibroblasts (CFs) cultured with Angiotension II. The role of miR‐503 in regulating CF cell proliferation and/or collagen production in mice neonatal CFs were determined using an MTT assay and RT‐PCR respectively. Forced expression of miR‐503 increased the cellular proliferation and collagen production in mice neonatal CFs. The effects were abrogated by cotransfection with AMO‐503 (a specific inhibitor of miR‐503). Injection of antagomiR‐503 elevated cardiac function and inhibited the expression of connective tissue growth factor (CTGF) and transforming growth factor (TGF)‐β in the TAC mice. Additional analysis revealed that Apelin‐13 is a direct target of miR‐503, as the overexpression of miR‐503 decreased the protein and mRNA expression levels of Apelin‐13. In the CFs with pre‐treatment of AngII, we transfected AMO‐503 into the cells treated with siRNA‐APLN. siRNA‐APLN abolished the effects of AMO‐503 on the production of collagen I and III and the expression of TGF‐β and CTGF. Furthermore, pre‐treatment of CFs with Apelin‐13 (1–100 nmol/l) inhibited angiotensin II‐mediated collagen production and activation of CTGF and TGF‐β. So we conclude that miR‐503 promotes cardiac fibrosis via miR‐503‐Apelin‐13‐TGF‐β‐CTGF‐collagen production pathway. Thus, miR‐503 is a promising therapeutic target for reducing cardiac fibrosis.     

Inhibition of cardiac fibroblast proliferation and myofibroblast differentiation by resveratrol

Cardiac fibroblasts (CFs) regulate myocardial remodeling by proliferating, differentiating, and secreting extracellular matrix proteins. Prolonged activation of CFs leads to cardiac fibrosis and reduced myocardial contractile function. Resveratrol (RES) exhibits a number of cardioprotective properties; however, the possibility that this compound affects CF function has not been considered. The current study tests whether RES directly influences the growth and proliferation of CFs and differentiation to the hypersecretory myofibroblast phenotype. Pretreatment of CFs with RES (5-25 microM) inhibited basal and ANG II-induced extracellular signal-regulated kinase (ERK) 1/2 and ERK kinase activation. This inhibition by RES reduced basal proliferation and blocked ANG II-induced growth and proliferation of CFs in a concentration-dependent manner, as measured by [(3)H]leucine and [(3)H]thymidine incorporation, respectively. RES pretreatment attenuated ERK phosphorylation when CFs were stimulated with 0.2 nM epidermal growth factor (EGF), a concentration at which EGF-induced ERK activation over basal was similar to the phosphorylation induced by 100 nM ANG II. Akt phosphorylation in CFs was unaffected by treatment with either 100 nM ANG II or 25 microM RES. Pretreatment of CFs with RES also reduced both ANG II- and transforming growth factor-beta-induced CF differentiation to the myofibroblast phenotype, indicated by a reduction in alpha-smooth muscle actin expression and stress fiber organization in CFs. This study identifies RES as an anti-fibrotic agent in the myocardium by limiting CF proliferation and differentiation, two critical steps in the pathogenesis of cardiac fibrosis.     

In vitro effects of pirfenidone on cardiac fibroblasts: proliferation, myofibroblast differentiation, migration and cytokine secretion

Cardiac fibroblasts (CFs) are the primary cell type responsible for cardiac fibrosis during pathological myocardial remodeling. Several studies have illustrated that pirfenidone (5-methyl-1-phenyl-2-[1H]-pyridone) attenuates cardiac fibrosis in different animal models. However, the effects of pirfenidone on cardiac fibroblast behavior have not been examined. In this study, we investigated whether pirfenidone directly modulates cardiac fibroblast behavior that is important in myocardial remodeling such as proliferation, myofibroblast differentiation, migration and cytokine secretion. Fibroblasts were isolated from neonatal rat hearts and bioassays were performed to determine the effects of pirfenidone on fibroblast function. We demonstrated that treatment of CFs with pirfenidone resulted in decreased proliferation, and attenuated fibroblast α-smooth muscle actin expression and collagen contractility. Boyden chamber assay illustrated that pirfenidone inhibited fibroblast migration ability, probably by decreasing the ratio of matrix metalloproteinase-9 to tissue inhibitor of metalloproteinase-1. Furthermore, pirfenidone attenuated the synthesis and secretion of transforming growth factor-β1 but elevated that of interleukin-10. These direct and pleiotropic effects of pirfenidone on cardiac fibroblasts point to its potential use in the treatment of adverse myocardial remodeling.     

TRPM7 and its role in neurodegenerative diseases

Calcium (Ca²⁺) and magnesium (Mg²⁺) ions have been shown to play an important role in regulating various neuronal functions. In the present review we focus on the emerging role of transient potential melastatin-7 (TRPM7) channel in not only regulating Ca²⁺ and Mg²⁺ homeostasis necessary for biological functions, but also how alterations in TRPM7 function/expression could induce neurodegeneration. Although eight TRPM channels have been identified, the channel properties, mode of activation, and physiological responses of various TRPM channels are quite distinct. Among the known 8 TRPM channels only TRPM6 and TRPM7 channels are highly permeable to both Ca²⁺ and Mg²⁺; however here we will only focus on TRPM7 as unlike TRPM6, TRPM7 channels are abundantly expressed in neuronal cells. Importantly, the discrepancy in TRPM7 channel function and expression leads to various neuronal diseases such as Alzheimer disease (AD) and Parkinson disease (PD). Further, it is emerging as a key factor in anoxic neuronal death and in other neurodegenerative disorders. Thus, by understanding the precise involvement of the TRPM7 channels in different neurodegenerative diseases and by understanding the factors that regulate TRPM7 channels, we could uncover new strategies in the future that could evolve as new drug therapeutic targets for effective treatment of these neurodegenerative diseases.     

Overexpression of peptidase inhibitor 16 attenuates angiotensin II–induced cardiac fibrosis via regulating HDAC1 of cardiac fibroblasts

Cardiac hypertrophy and fibrosis are the major causes of heart failure due to non‐ischaemia heart disease. To date, no specific therapy exists for cardiac fibrosis due to the largely unknown mechanisms of disease and lack of applicable therapeutic targets. In this study, we aimed to explore the role and associated mechanism of peptidase inhibitor 16 (PI16) in cardiac fibrosis induced by angiotensin II. In cardiac fibroblasts (CFs), overexpressed PI16 significantly inhibited CF proliferation and the levels of fibrosis‐associated proteins. Further analysis of epigenetic changes in CF revealed that overexpressed PI16 decreases the nuclear level of histone deacetylase 1 (HDAC1) after angiotensin II treatment, resulting in increased histone 3 acetylation in K18 and K27 lysine. However, overexpression of HDAC1 by an adenovirus vector in CFs reversed these changes. Echocardiography showed that PI16 transgenic (Tg) mice have smaller left ventricle mass than wild‐type mice. Histological analysis data showed that PI16 Tg mice demonstrated smaller cardiomyocyte size and less collagen deposition than wild‐type mice. The effects of PI16 on HDAC1 and histone 3 were also confirmed in PI16 Tg mice using immunostaining. Generally, PI16 is a HDAC1 regulator specifically in CFs, and PI16 overexpression prevents cardiac hypertrophy and fibrosis by inhibiting stress‐induced CF activation.     

Increase in Serum Ca2+/Mg2+ Ratio Promotes Proliferation of Prostate Cancer Cells by Activating TRPM7 Channels

TRPM7 is a novel magnesium-nucleotide-regulated metal current (MagNuM) channel that is regulated by serum Mg²⁺ concentrations. Changes in Mg²⁺ concentration have been shown to alter cell proliferation in various cells; however, the mechanism and the ion channel(s) involved have not yet been identified. Here we demonstrate that TRPM7 is expressed in control and prostate cancer cells. Supplementation of intracellular Mg-ATP or addition of external 2-aminoethoxydiphenyl borate inhibited MagNuM currents. Furthermore, silencing of TRPM7 inhibited whereas overexpression of TRPM7 increased endogenous MagNuM currents, suggesting that these currents are dependent on TRPM7. Importantly, although an increase in the serum Ca²⁺/Mg²⁺ ratio facilitated Ca²⁺ influx in both control and prostate cancer cells, a significantly higher Ca²⁺ influx was observed in prostate cancer cells. TRPM7 expression was also increased in cancer cells, but its expression was not dependent on the Ca²⁺/Mg²⁺ ratio per se. Additionally, an increase in the extracellular Ca²⁺/Mg²⁺ ratio led to a significant increase in cell proliferation of prostate cancer cells when compared with control cells. Consistent with these results, age-matched prostate cancer patients also showed a subsequent increase in the Ca²⁺/Mg²⁺ ratio and TRPM7 expression. Altogether, we provide evidence that the TRPM7 channel has an important role in prostate cancer and have identified that the Ca²⁺/Mg²⁺ ratio could be essential for the initiation/progression of prostate cancer.     

MicroRNA-101 protects cardiac fibroblasts from hypoxia-induced apoptosis via inhibition of the TGF-β signaling pathway

Cardiac fibroblasts (CFs) are the most numerous cells in the heart and are recognized primarily for their ability to maintain both the structural integrity and the physiological functions of the heart. The transforming growth factor beta (TGF-β) signaling pathway is reportedly involved in the modulation of CF functions, including apoptosis. Recent studies have indicated that microRNA-101 (miR-101) attenuates the TGF-β signaling pathway, either by inhibiting the expression of TGFβ1 or by targeting transforming growth factor-β receptor type I (TGFβRI). The present study aimed to determine whether miR-101 protects CFs from hypoxia-induced apoptosis and to investigate the mechanisms underlying its protective effects. The CCK-8 test, electron microscopy and TUNEL assay results demonstrated that miR-101a/b significantly inhibited hypoxia-induced CF apoptosis. The results of Western blotting, quantitative RT-PCR and immunofluorescence assays indicated that miR-101a dramatically inhibited the hypoxia-induced up-regulation of both TGFβRI and p-Smad 3 but not TGFβ1 in CFs. Additionally, miR-101a significantly reversed the hypoxia-induced up-regulation of Bax and Caspase-3, the down-regulation of Bcl-2 and the activation of Caspase-3 in CFs. Moreover, miR-101a markedly inhibited the intracellular Ca²⁺ ([Ca²⁺]_i) overload caused by hypoxia. Taken together, our results suggest that miR-101a protects CFs against hypoxia-induced apoptosis by inhibiting the TGF-β signaling pathway, which may be a potential therapeutic target for heart injury.