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Lung sections showing pulmonary arterioles in cross section (hematoxylin-eosin, magnification ). Scale bar 50 m.
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Citations
... The role of CaV3.1 in cell proliferation has been described in heart, lung and cancer cells [15,[25][26][27][28]. Human pulmonary artery myocytes express CaV3.1 and silencing of CaV3.1 inhibits serum-induced proliferation [25]. ...
... The role of CaV3.1 in cell proliferation has been described in heart, lung and cancer cells [15,[25][26][27][28]. Human pulmonary artery myocytes express CaV3.1 and silencing of CaV3.1 inhibits serum-induced proliferation [25]. This is consistent with studies in preadipocytes in primary culture. ...
Background
T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases.
Methods
In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-β1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177.
Results
We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-β1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death.
Conclusion
This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.
... cell cycle, increasing during the G1-S transition. [12][13][14][15][16][17] The presence of TTCCs in cancer cells was initially reported in cultured retinoblastoma Y79 cells. 18 In that study, the expression of Ca v 3.1 and Ca v 3.2 was reduced upon cell differentiation into neuronal or glial phenotypes, suggesting a role for TTCCs in cell cycle progression (but see [19][20][21][22] ). ...
Over the past 20 years, various studies have demonstrated a pivotal role of T-type calcium channels (TTCCs) in tumor progression. Cytotoxic effects of TTCC pharmacological blockers have been reported in vitro and in preclinical models. However, their roles in cancer physiology are only beginning to be understood. In this review, we discuss evidence for the signaling pathways and cellular processes stemming from TTCC activity, mainly inferred by inverse reasoning from pharmacological blocks and by gene silencing or channel activation. A thorough analysis indicates that drug-induced cytotoxicity is partially an off-target effect. Dissection of on/off-target activity is paramount to elucidate the physiological roles of TTCCs, and to deliver efficacious therapies suited to different cancer types and stages.
... The role of Ca V 3.1 in cell proliferation has been described in heart, lung and cancer cells [14,[23][24][25][26]. ...
... Human pulmonary artery myocytes express Ca V 3.1 and silencing of Ca V 3.1 inhibits serum-induced proliferation [23]. This is consistent with studies in preadipocytes in primary culture. ...
Background: T-type calcium channels (TTCC) are low voltage activated channels that are widely expressed in the heart, smooth muscle and neurons. They are known to impact on cell cycle progression in cancer and smooth muscle cells and more recently, have been implicated in rat and human mesangial cell proliferation. The aim of this study was to investigate the roles of the different isoforms of TTCC in mouse mesangial cells to establish which may be the best therapeutic target for treating mesangioproliferative kidney diseases.
Methods: In this study, we generated single and double knockout (SKO and DKO) clones of the TTCC isoforms CaV3.1 and CaV3.2 in mouse mesangial cells using CRISPR-cas9 gene editing. The downstream signals linked to this channel activity were studied by ERK1/2 phosphorylation assays in serum, PDGF and TGF-β1 stimulated cells. We also examined their proliferative responses in the presence of the TTCC inhibitors mibefradil and TH1177.
Results: We demonstrate a complete loss of ERK1/2 phosphorylation in response to multiple stimuli (serum, PDGF, TGF-β1) in CaV3.1 SKO clone, whereas the CaV3.2 SKO clone retained these phospho-ERK1/2 responses. Stimulated cell proliferation was not profoundly impacted in either SKO clone and both clones remained sensitive to non-selective TTCC blockers, suggesting a role for more than one TTCC isoform in cell cycle progression. Deletion of both the isoforms resulted in cell death.
Conclusion: This study confirms that TTCC are expressed in mouse mesangial cells and that they play a role in cell proliferation. Whereas the CaV3.1 isoform is required for stimulated phosphorylation of ERK1/2, the Ca V3.2 isoform is not. Our data also suggest that neither isoform is necessary for cell proliferation and that the anti-proliferative effects of mibefradil and TH1177 are not isoform-specific. These findings are consistent with data from in vivo rat mesangial proliferation Thy1 models and support the future use of genetic mouse models to test the therapeutic actions of TTCC inhibitors.
... Also, the inhibition of these channels mitigates the associated pathophysiology [125]. Ca 2+ influx through voltage-dependent Ca 2+ channels (VDCC, Ca v ) is implicated in the proliferation of PASMCs [126]. Pharmacological blockade and genomic interference of the Ca v 3.1 subtype have been proved to inhibit PASMC proliferation and the entry to cell cycle [127]. ...
Pulmonary hypertension (PH) is a progressive lung disease characterized by persistent pulmonary vasoconstriction. Another well-recognized characteristic of PH is the muscularization of peripheral pulmonary arteries. This pulmonary vasoremodeling manifests in medial hypertrophy/hyperplasia of smooth muscle cells (SMCs) with possible neointimal formation. The underlying molecular processes for these two major vascular responses remain not fully understood. On the other hand, a series of very recent studies have shown that the increased reactive oxygen species (ROS) seems to be an important player in mediating pulmonary vasoconstriction and vasoremodeling, thereby leading to PH. Mitochondria are a primary site for ROS production in pulmonary artery (PA) SMCs, which subsequently activate NADPH oxidase to induce further ROS generation, i.e., ROS-induced ROS generation. ROS control the activity of multiple ion channels to induce intracellular Ca2+ release and extracellular Ca2+ influx (ROS-induced Ca2+ release and influx) to cause PH. ROS and Ca2+ signaling may synergistically trigger an inflammatory cascade to implicate in PH. Accordingly, this paper explores the important roles of ROS, Ca2+, and inflammatory signaling in the development of PH, including their reciprocal interactions, key molecules, and possible therapeutic targets.
... By virtue of these distinctive properties, TTCC provide a key pathway for Ca 2+ entry near the resting membrane potential. The expression of TTCC is linked to the cell cycle, increasing during the G1-S transition (11) (12) (13). A number of reports associate the expression of Ca v 3.1 and Ca v 3.2 to the proliferation of cancer cells in vitro (14) (15) (16) (17). ...
T-type Ca 2+ channels (TTCC) have been identified as key regulators of cancer cell cycle and survival. In vivo studies in glioblastoma (GBM) murine xenografts have shown that drugs able to block TTCC in vitro (such as tetralol derivatives mibefradil/NNC-55-096, or different 3,4-dihydroquinazolines) slow tumor progression. However, currently available TTCC pharmacological blockers have limited selectivity for TTCC, and are unable to distinguish between TTCC isoforms. Here we analyzed the expression of TTCC transcripts in human GBM cells and show a prevalence of Ca v 3.1 mRNAs. Infection of GBM cells with lentiviral particles carrying shRNA against Ca v 3.1 resulted in GBM cell death by apoptosis. We generated a murine GBM xenograft via subcutaneous injection of U87-MG GBM cells and found that tumor size was reduced when Ca v 3.1 expression was silenced. Furthermore, we developed an in vitro model of temozolomide-resistant GBM that showed increased expression of Ca v 3.1 accompanied by activation of macroautophagy. We confirmed a positive correlation between Ca v 3.1 and autophagic markers in both GBM cultures and biopsies. Of note, Ca v 3.1 knockdown resulted in transcriptional downregulation of p62/SQSTM1 and deficient autophagy. Together, these data identify Ca v 3.1 channels as potential targets for slowing GBM progression and recurrence based on their role in regulating autophagy. Statement of significance: Findings identify Ca v 3.1 calcium channels as a molecular target to regulate autophagy and prevent progression and chemotherapeutic resistance in glioblastoma 3
... By virtue of these distinctive properties, TTCC provide a key pathway for Ca 2þ entry near the resting membrane potential. The expression of TTCC is linked to the cell cycle, increasing during the G 1 -S transition (11)(12)(13). A number of reports associate the expression of Ca v 3.1 and Ca v 3.2 to the proliferation of cancer cells in vitro (14)(15)(16)(17). ...
T-type Ca²⁺ channels (TTCC) have been identified as key regulators of cancer cell cycle and survival. In vivo studies in glioblastoma (GBM) murine xenografts have shown that drugs able to block TTCC in vitro (such as tetralol derivatives mibefradil/NNC-55-096, or different 3,4-dihydroquinazolines) slow tumor progression. However, currently available TTCC pharmacologic blockers have limited selectivity for TTCC and are unable to distinguish between TTCC isoforms. Here we analyzed the expression of TTCC transcripts in human GBM cells and show a prevalence of Cacna1g/Cav3.1 mRNAs. Infection of GBM cells with lentiviral particles carrying short hairpin RNA against Cav3.1 resulted in GBM cell death by apoptosis. We generated a murine GBM xenograft via subcutaneous injection of U87-MG GBM cells and found that tumor size was reduced when Cav3.1 expression was silenced. Furthermore, we developed an in vitro model of temozolomide-resistant GBM that showed increased expression of Cav3.1 accompanied by the activation of macroautophagy. We confirmed a positive correlation between Cav3.1 and autophagic markers in both GBM cultures and biopsies. Of note, Cav3.1 knockdown resulted in transcriptional downregulation of p62/SQSTM1 and deficient autophagy. Together, these data identify Cav3.1 channels as potential targets for slowing GBM progression and recurrence based on their role in regulating autophagy.
Significance: These findings identify Cav3.1 calcium channels as a molecular target to regulate autophagy and prevent progression and chemotherapeutic resistance in glioblastoma.
... Intracellular Ca 2+ [Ca 2+ ] i acts as a secondary messenger and its imbalance can greatly influence pathophysiology of PAH by inducing PASMC proliferation and P-EC instability 7 . Calcium channel blockers (CCBs), which are a class of drugs that avert entry of Ca 2+ into cardio myocytes, PASMC and P-EC, hence preclude contractility of heart and constriction of pulmonary arteries (PA) [8][9][10][11] . Previously, long acting CCBs (dihydropryamidine) like nifedipine, diltiazem, nicardipine, felodipine and amlodipine have been used in ameliorating effects of increased Ca 2+ insurgency into cytosol to treat pulmonary arterial remodelling 9,[12][13][14][15][16] . ...
p>Therapies to prevent onset and progression of pulmonary arterial pressure are not very effective yet. This study was designed to investigate the effects of a novel dihydropyrimidinone, ethyl 4-(4′-heptanoyloxyphenyl)-6-methyl-3,4-dihydropyrimidin-2-one-5-carboxylate (H-DHPM) on pathogenesis of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH). For the same purpose, rats were injected intraperitoneally (i.p.) a single dose (60 mg/kg) of MCT which led to development of PAH in 21 days. MCT insult caused high mortality, pulmonary vascular and parenchymal remodelling. Since the course of PAH pathogenesis is characterised by an early onset and progression phases, H-DHPM was administered i.p. at 30 mg/kg dosage in MCT pre-injected animals either from day 0 through day 21 or day 14 though day 21 of MCT injection in two separate treatment groups. H-DHPM significantly improved survival, prevented remodelling of pulmonary vasculature and parenchyma and subsequently ameliorated PAH pathogenesis. Moreover, we observed significant decrease in right ventricle hypertrophy, measured by wet weight of right ventricle (RV) divided by wet weight of left ventricle plus septum (LV+S), in H-DHPM treated groups as compared to MCT injected animals. These findings suggest H-DHPM not only prevented development of PAH but also treated the PAH pathogenesis in progressive phase. In conclusion, our data determines H-DHPM, might be a future drug for the prevention of PAH.</p
... By contrast, activation of Ca v 3.2 channels in coronary and cerebral smooth muscles cells induces a vasorelaxation [11,12]. Besides, in PH, Ca v 3.1 channels are involved in the remodeling of the vascular wall resulting in a reduction of the vessel lumen and thus leading to hypertension [6,13]. ...
In pulmonary arterial endothelial cells, Ca²⁺ channels and intracellular Ca²⁺concentration ([Ca²⁺]i) control the release of vasorelaxant factors such as nitric oxide and are involved in the regulation of pulmonary arterial blood pressure. The present study was undertaken to investigate the implication of T-type voltage-gated Ca²⁺channels (T-VGCCs, Cav3.1 channel) in the endothelium-dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Cav3.1-/- mice. Endothelial [Ca²⁺]i and NO production were measured, on whole vessels, with the fluo-4 and DAF-fm probes. Acetylcholine (ACh) induced a nitric oxide- and endothelium-dependent relaxation that was significantly reduced in pulmonary arteries from Cav3.1-/- compared to wild type mice as well as in the presence of T-VGCC inhibitors (NNC 55-0396 or mibefradil). ACh also increased endothelial [Ca²⁺]i and NO production that were both reduced in Cav3.1-/- compared to wild type mice or in the presence of T-VGCC inhibitors. Immunofluorescence labeling revealed the presence of Cav3.1 channels in endothelial cells that co-localized with endothelial nitric oxide synthase in arteries from wild type mice. TRPV4-, beta2 adrenergic- and nitric oxide donors (SNP)- mediated relaxation were not altered in Cav3.1-/- compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Cav3.1 channels activity. The present study thus demonstrates that T-VGCCs, mainly Cav3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca²⁺]i and ACh-mediated relaxation.
... In contrast, contractile SMCs are elongated, exhibit the shape of a spindle, express all of the contractile phenotypic markers that make up a functional contractile apparatus and respond to signals and stimuli that endorse cell contraction. SMCs with a contractile phenotype also predominantly express BKCa channels [62][63][64][65]and Ca V 1.2 (L-type voltage-gated Ca 2+ ) channels [66,67]. Therefore the differentiated MSCs derived in this work seem to mainly consist of the contractile phenotype, as they are spindle-shaped, express genes and proteins involved in SMC contraction including myogenic markers and functional ion channels, and contract when exposed to the K + agonist. ...
The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1-2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.
... Cette régulation s'effectue par la fixation du facteur inductible par l'hypoxie (HIF) sur un site élément de réponse à l'hypoxie qui est uniquement présent sur la séquence du gène du canal Cav3.2 336 . D'autre part, les canaux Cav3.1 sont impliqués dans la prolifération des CML en réponse à des agents proprolifératifs 337,338 . Un article en annexe de ma thèse présente des résultats quant à l'implication des canaux Cav3.1 dans le développement de l'HTP chez la souris. ...
Pulmonary hypertension (PH) is the main disease of the pulmonary circulation. This pathology ischaracterized by an increase of the intrapulmonary arterial (PA) pressure at rest (> 25 mmHg). This pressureexerts stretch forces on pulmonary arterial smooth muscle cells (PASMC). Stretch-activated channels (SAC)are present in PASMC and are able to transform a mechanical stimulus of stretch into a biological responseof contraction, a phenomenon called myogenic tone. Ca2+ is a second messenger that can be mobilizedfrom both the extracellular medium and intracellular Ca2+ stores. An increase of the intracellular Ca2+concentration ([Ca2+]i) leads to PASMC contraction. Using patch-clamp, microspectrofluorimetry,immunostainings and a pharmacological approach, we highlight Ca2+ signaling pathways induced by stretchin PASMC. Experiments were performed in normal rats and in two models of PH (chronically hypoxic ratsand monocrotaline rats). We showed that in normal rats a stretch induces a Ca2+ influx through SAC whichis amplified by (1) a plasma membrane hyperpolarization by BKCa channels and (2) a Ca2+ amplification bysubplasmalemnal ryanodine receptor 1 (RyR) of the sarcoplasmic reticulum (SR). Besides, mitochondria areinvolved in buffering cytoplasmic Ca2+. In PH rats, the Ca2+ influx by SAC and the Ca2+ release by RyR areenhanced due to a reorganization of intracellular Ca2+ stores. Furthermore, a functional associationbetween SR and caveolae conduce to a much greater amplification of the stretch-induced Ca2+ increase inPH rats. Finally, we showed that the mechanosensitive channel Piezo1 is expressed in PA. To conclude, thespatial organization of Ca2+ stores in PASMC is important for cell signaling and plays a casual role in PH.
